An enzyme immunoassay for rat growth hormone - Applications to the study of growth hormone variants

NASA Technical Reports Server (NTRS)

Farrington, Marianne A.; Hymer, W. C.

1987-01-01

A sensitive and specific competitive enzyme immunoassay for rat growth hormone (GH) is described and its use in the detection of GH variants is demonstrated. In the present assay, soluble GH and GH adsorbed to a solid-phase support compete for monkey anti-GH antibody binding sites. The immobilized antibody-GH complex is detected and quantified using goat antimonkey immunoglobin G covalently conjugated to horseradish peroxidase. It is noted that the assay can be performed in 27 hours and that sensitivities in the range of 0.19 to 25 ng can be obtained in the region of 10 to 90 percent binding.

GWAS4D: multidimensional analysis of context-specific regulatory variant for human complex diseases and traits.

PubMed

Huang, Dandan; Yi, Xianfu; Zhang, Shijie; Zheng, Zhanye; Wang, Panwen; Xuan, Chenghao; Sham, Pak Chung; Wang, Junwen; Li, Mulin Jun

2018-05-16

Genome-wide association studies have generated over thousands of susceptibility loci for many human complex traits, and yet for most of these associations the true causal variants remain unknown. Tissue/cell type-specific prediction and prioritization of non-coding regulatory variants will facilitate the identification of causal variants and underlying pathogenic mechanisms for particular complex diseases and traits. By leveraging recent large-scale functional genomics/epigenomics data, we develop an intuitive web server, GWAS4D (http://mulinlab.tmu.edu.cn/gwas4d or http://mulinlab.org/gwas4d), that systematically evaluates GWAS signals and identifies context-specific regulatory variants. The updated web server includes six major features: (i) updates the regulatory variant prioritization method with our new algorithm; (ii) incorporates 127 tissue/cell type-specific epigenomes data; (iii) integrates motifs of 1480 transcriptional regulators from 13 public resources; (iv) uniformly processes Hi-C data and generates significant interactions at 5 kb resolution across 60 tissues/cell types; (v) adds comprehensive non-coding variant functional annotations; (vi) equips a highly interactive visualization function for SNP-target interaction. Using a GWAS fine-mapped set for 161 coronary artery disease risk loci, we demonstrate that GWAS4D is able to efficiently prioritize disease-causal regulatory variants.

H3.Y discriminates between HIRA and DAXX chaperone complexes and reveals unexpected insights into human DAXX-H3.3-H4 binding and deposition requirements

PubMed Central

Zink, Lisa-Maria; Delbarre, Erwan; Eberl, H. Christian; Keilhauer, Eva C.; Bönisch, Clemens; Pünzeler, Sebastian; Bartkuhn, Marek; Collas, Philippe; Mann, Matthias

2017-01-01

Abstract Histone chaperones prevent promiscuous histone interactions before chromatin assembly. They guarantee faithful deposition of canonical histones and functionally specialized histone variants into chromatin in a spatial- and temporally-restricted manner. Here, we identify the binding partners of the primate-specific and H3.3-related histone variant H3.Y using several quantitative mass spectrometry approaches, and biochemical and cell biological assays. We find the HIRA, but not the DAXX/ATRX, complex to recognize H3.Y, explaining its presence in transcriptionally active euchromatic regions. Accordingly, H3.Y nucleosomes are enriched in the transcription-promoting FACT complex and depleted of repressive post-translational histone modifications. H3.Y mutational gain-of-function screens reveal an unexpected combinatorial amino acid sequence requirement for histone H3.3 interaction with DAXX but not HIRA, and for H3.3 recruitment to PML nuclear bodies. We demonstrate the importance and necessity of specific H3.3 core and C-terminal amino acids in discriminating between distinct chaperone complexes. Further, chromatin immunoprecipitation sequencing experiments reveal that in contrast to euchromatic HIRA-dependent deposition sites, human DAXX/ATRX-dependent regions of histone H3 variant incorporation are enriched in heterochromatic H3K9me3 and simple repeat sequences. These data demonstrate that H3.Y's unique amino acids allow a functional distinction between HIRA and DAXX binding and its consequent deposition into open chromatin. PMID:28334823

Electrostatic control of DNA intersegmental translocation by the ETS transcription factor ETV6.

PubMed

Vo, Tam; Wang, Shuo; Poon, Gregory M K; Wilson, W David

2017-08-11

To find their DNA target sites in complex solution environments containing excess heterogeneous DNA, sequence-specific DNA-binding proteins execute various translocation mechanisms known collectively as facilitated diffusion. For proteins harboring a single DNA contact surface, long-range translocation occurs by jumping between widely spaced DNA segments. We have configured biosensor-based surface plasmon resonance to directly measure the affinity and kinetics of this intersegmental jumping by the ETS-family transcription factor ETS variant 6 (ETV6). To isolate intersegmental target binding in a functionally defined manner, we pre-equilibrated ETV6 with excess salmon sperm DNA, a heterogeneous polymer, before exposing the nonspecifically bound protein to immobilized oligomeric DNA harboring a high-affinity ETV6 site. In this way, the mechanism of ETV6-target association could be toggled electrostatically through varying NaCl concentration in the bulk solution. Direct measurements of association and dissociation kinetics of the site-specific complex indicated that 1) freely diffusive binding by ETV6 proceeds through a nonspecific-like intermediate, 2) intersegmental jumping is rate-limited by dissociation from the nonspecific polymer, and 3) dissociation of the specific complex is independent of the history of complex formation. These results show that target searches by proteins with an ETS domain, such as ETV6, whose single DNA-binding domain cannot contact both source and destination sites simultaneously, are nonetheless strongly modulated by intersegmental jumping in heterogeneous site environments. Our findings establish biosensors as a general technique for directly and specifically measuring target site search by DNA-binding proteins via intersegmental translocation. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Parental origin of sequence variants associated with complex diseases.

PubMed

Kong, Augustine; Steinthorsdottir, Valgerdur; Masson, Gisli; Thorleifsson, Gudmar; Sulem, Patrick; Besenbacher, Soren; Jonasdottir, Aslaug; Sigurdsson, Asgeir; Kristinsson, Kari Th; Jonasdottir, Adalbjorg; Frigge, Michael L; Gylfason, Arnaldur; Olason, Pall I; Gudjonsson, Sigurjon A; Sverrisson, Sverrir; Stacey, Simon N; Sigurgeirsson, Bardur; Benediktsdottir, Kristrun R; Sigurdsson, Helgi; Jonsson, Thorvaldur; Benediktsson, Rafn; Olafsson, Jon H; Johannsson, Oskar Th; Hreidarsson, Astradur B; Sigurdsson, Gunnar; Ferguson-Smith, Anne C; Gudbjartsson, Daniel F; Thorsteinsdottir, Unnur; Stefansson, Kari

2009-12-17

Effects of susceptibility variants may depend on from which parent they are inherited. Although many associations between sequence variants and human traits have been discovered through genome-wide associations, the impact of parental origin has largely been ignored. Here we show that for 38,167 Icelanders genotyped using single nucleotide polymorphism (SNP) chips, the parental origin of most alleles can be determined. For this we used a combination of genealogy and long-range phasing. We then focused on SNPs that associate with diseases and are within 500 kilobases of known imprinted genes. Seven independent SNP associations were examined. Five-one with breast cancer, one with basal-cell carcinoma and three with type 2 diabetes-have parental-origin-specific associations. These variants are located in two genomic regions, 11p15 and 7q32, each harbouring a cluster of imprinted genes. Furthermore, we observed a novel association between the SNP rs2334499 at 11p15 and type 2 diabetes. Here the allele that confers risk when paternally inherited is protective when maternally transmitted. We identified a differentially methylated CTCF-binding site at 11p15 and demonstrated correlation of rs2334499 with decreased methylation of that site.

SvABA: genome-wide detection of structural variants and indels by local assembly.

PubMed

Wala, Jeremiah A; Bandopadhayay, Pratiti; Greenwald, Noah F; O'Rourke, Ryan; Sharpe, Ted; Stewart, Chip; Schumacher, Steve; Li, Yilong; Weischenfeldt, Joachim; Yao, Xiaotong; Nusbaum, Chad; Campbell, Peter; Getz, Gad; Meyerson, Matthew; Zhang, Cheng-Zhong; Imielinski, Marcin; Beroukhim, Rameen

2018-04-01

Structural variants (SVs), including small insertion and deletion variants (indels), are challenging to detect through standard alignment-based variant calling methods. Sequence assembly offers a powerful approach to identifying SVs, but is difficult to apply at scale genome-wide for SV detection due to its computational complexity and the difficulty of extracting SVs from assembly contigs. We describe SvABA, an efficient and accurate method for detecting SVs from short-read sequencing data using genome-wide local assembly with low memory and computing requirements. We evaluated SvABA's performance on the NA12878 human genome and in simulated and real cancer genomes. SvABA demonstrates superior sensitivity and specificity across a large spectrum of SVs and substantially improves detection performance for variants in the 20-300 bp range, compared with existing methods. SvABA also identifies complex somatic rearrangements with chains of short (<1000 bp) templated-sequence insertions copied from distant genomic regions. We applied SvABA to 344 cancer genomes from 11 cancer types and found that short templated-sequence insertions occur in ∼4% of all somatic rearrangements. Finally, we demonstrate that SvABA can identify sites of viral integration and cancer driver alterations containing medium-sized (50-300 bp) SVs. © 2018 Wala et al.; Published by Cold Spring Harbor Laboratory Press.

Diff-seq: A high throughput sequencing-based mismatch detection assay for DNA variant enrichment and discovery

PubMed Central

Karas, Vlad O; Sinnott-Armstrong, Nicholas A; Varghese, Vici; Shafer, Robert W; Greenleaf, William J; Sherlock, Gavin

2018-01-01

Abstract Much of the within species genetic variation is in the form of single nucleotide polymorphisms (SNPs), typically detected by whole genome sequencing (WGS) or microarray-based technologies. However, WGS produces mostly uninformative reads that perfectly match the reference, while microarrays require genome-specific reagents. We have developed Diff-seq, a sequencing-based mismatch detection assay for SNP discovery without the requirement for specialized nucleic-acid reagents. Diff-seq leverages the Surveyor endonuclease to cleave mismatched DNA molecules that are generated after cross-annealing of a complex pool of DNA fragments. Sequencing libraries enriched for Surveyor-cleaved molecules result in increased coverage at the variant sites. Diff-seq detected all mismatches present in an initial test substrate, with specific enrichment dependent on the identity and context of the variation. Application to viral sequences resulted in increased observation of variant alleles in a biologically relevant context. Diff-Seq has the potential to increase the sensitivity and efficiency of high-throughput sequencing in the detection of variation. PMID:29361139

Structural determinants of phosphoinositide selectivity in splice variants of Grp1 family PH domains

PubMed Central

Cronin, Thomas C; DiNitto, Jonathan P; Czech, Michael P; Lambright, David G

2004-01-01

The pleckstrin homology (PH) domains of the homologous proteins Grp1 (general receptor for phosphoinositides), ARNO (Arf nucleotide binding site opener), and Cytohesin-1 bind phosphatidylinositol (PtdIns) 3,4,5-trisphosphate with unusually high selectivity. Remarkably, splice variants that differ only by the insertion of a single glycine residue in the β1/β2 loop exhibit dual specificity for PtdIns(3,4,5)P3 and PtdIns(4,5)P2. The structural basis for this dramatic specificity switch is not apparent from the known modes of phosphoinositide recognition. Here, we report crystal structures for dual specificity variants of the Grp1 and ARNO PH domains in either the unliganded form or in complex with the head groups of PtdIns(4,5)P2 and PtdIns(3,4,5)P3. Loss of contacts with the β1/β2 loop with no significant change in head group orientation accounts for the significant decrease in PtdIns(3,4,5)P3 affinity observed for the dual specificity variants. Conversely, a small increase rather than decrease in affinity for PtdIns(4,5)P2 is explained by a novel binding mode, in which the glycine insertion alleviates unfavorable interactions with the β1/β2 loop. These observations are supported by a systematic mutational analysis of the determinants of phosphoinositide recognition. PMID:15359279

Crystal structure of CotA laccase complexed with 2,2-azinobis-(3-ethylbenzothiazoline-6-sulfonate) at a novel binding site

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Zhongchuan; Xie, Tian; Key Laboratory of Environmental Microbiology of Sichuan Province, Chengdu 610041, People’s Republic of

2016-03-24

The crystal structure of CotA complexed with 2,2-azinobis-(3-ethylbenzothiazoline-6-sulfonate) in a hole motif has been solved; this novel binding site could be a potential structure-based target for protein engineering of CotA laccase. The CotA laccase from Bacillus subtilis is an abundant component of the spore outer coat and has been characterized as a typical laccase. The crystal structure of CotA complexed with 2,2-azinobis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS) in a hole motif has been solved. The novel binding site was about 26 Å away from the T1 binding pocket. Comparison with known structures of other laccases revealed that the hole is a specific feature ofmore » CotA. The key residues Arg476 and Ser360 were directly bound to ABTS. Site-directed mutagenesis studies revealed that the residues Arg146, Arg429 and Arg476, which are located at the bottom of the novel binding site, are essential for the oxidation of ABTS and syringaldazine. Specially, a Thr480Phe variant was identified to be almost 3.5 times more specific for ABTS than for syringaldazine compared with the wild type. These results suggest this novel binding site for ABTS could be a potential target for protein engineering of CotA laccases.« less

Phenotype of CNTNAP1: a study of patients demonstrating a specific severe congenital hypomyelinating neuropathy with survival beyond infancy.

PubMed

Low, K J; Stals, K; Caswell, R; Wakeling, M; Clayton-Smith, J; Donaldson, A; Foulds, N; Norman, A; Splitt, M; Urankar, K; Vijayakumar, K; Majumdar, A; Study, Ddd; Ellard, S; Smithson, S F

2018-06-01

CHN is genetically heterogeneous and its genetic basis is difficult to determine on features alone. CNTNAP1 encodes CASPR, integral in the paranodal junction high molecular mass complex. Nineteen individuals with biallelic variants have been described in association with severe congenital hypomyelinating neuropathy, respiratory compromise, profound intellectual disability and death within the first year. We report 7 additional patients ascertained through exome sequencing. We identified 9 novel CNTNAP1 variants in 6 families: three missense variants, four nonsense variants, one frameshift variant and one splice site variant. Significant polyhydramnios occurred in 6/7 pregnancies. Severe respiratory compromise was seen in 6/7 (tracheostomy in 5). A complex neurological phenotype was seen in all patients who had marked brain hypomyelination/demyelination and profound developmental delay. Additional neurological findings included cranial nerve compromise: orobulbar dysfunction in 5/7, facial nerve weakness in 4/7 and vocal cord paresis in 5/7. Dystonia occurred in 2/7 patients and limb contractures in 5/7. All had severe gastroesophageal reflux, and a gastrostomy was required in 5/7. In contrast to most previous reports, only one patient died in the first year of life. Protein modelling was performed for all detected CNTNAP1 variants. We propose a genotype-phenotype correlation, whereby hypomorphic missense variants partially ameliorate the phenotype, prolonging survival. This study suggests that biallelic variants in CNTNAP1 cause a distinct recognisable syndrome, which is not caused by other genes associated with CHN. Neonates presenting with this phenotype will benefit from early genetic definition to inform clinical management and enable essential genetic counselling for their families.

Phonetic spelling filter for keyword selection in drug mention mining from social media.

PubMed

Pimpalkhute, Pranoti; Patki, Apurv; Nikfarjam, Azadeh; Gonzalez, Graciela

2014-01-01

Social media postings are rich in information that often remain hidden and inaccessible for automatic extraction due to inherent limitations of the site's APIs, which mostly limit access via specific keyword-based searches (and limit both the number of keywords and the number of postings that are returned). When mining social media for drug mentions, one of the first problems to solve is how to derive a list of variants of the drug name (common misspellings) that can capture a sufficient number of postings. We present here an approach that filters the potential variants based on the intuition that, faced with the task of writing an unfamiliar, complex word (the drug name), users will tend to revert to phonetic spelling, and we thus give preference to variants that reflect the phonemes of the correct spelling. The algorithm allowed us to capture 50.4 - 56.0 % of the user comments using only about 18% of the variants.

A Xylenol Orange-Based Screening Assay for the Substrate Specificity of Flavin-Dependent para-Phenol Oxidases.

PubMed

Ewing, Tom A; van Noord, Aster; Paul, Caroline E; van Berkel, Willem J H

2018-01-14

Vanillyl alcohol oxidase (VAO) and eugenol oxidase (EUGO) are flavin-dependent enzymes that catalyse the oxidation of para -substituted phenols. This makes them potentially interesting biocatalysts for the conversion of lignin-derived aromatic monomers to value-added compounds. To facilitate their biocatalytic exploitation, it is important to develop methods by which variants of the enzymes can be rapidly screened for increased activity towards substrates of interest. Here, we present the development of a screening assay for the substrate specificity of para -phenol oxidases based on the detection of hydrogen peroxide using the ferric-xylenol orange complex method. The assay was used to screen the activity of VAO and EUGO towards a set of twenty-four potential substrates. This led to the identification of 4-cyclopentylphenol as a new substrate of VAO and EUGO and 4-cyclohexylphenol as a new substrate of VAO. Screening of a small library of VAO and EUGO active-site variants for alterations in their substrate specificity led to the identification of a VAO variant (T457Q) with increased activity towards vanillyl alcohol (4-hydroxy-3-methoxybenzyl alcohol) and a EUGO variant (V436I) with increased activity towards chavicol (4-allylphenol) and 4-cyclopentylphenol. This assay provides a quick and efficient method to screen the substrate specificity of para -phenol oxidases, facilitating the enzyme engineering of known para- phenol oxidases and the evaluation of the substrate specificity of novel para -phenol oxidases.

Molecular Imprint of Exposure to Naturally Occurring Genetic Variants of Human Cytomegalovirus on the T cell Repertoire

NASA Astrophysics Data System (ADS)

Smith, Corey; Gras, Stephanie; Brennan, Rebekah M.; Bird, Nicola L.; Valkenburg, Sophie A.; Twist, Kelly-Anne; Burrows, Jacqueline M.; Miles, John J.; Chambers, Daniel; Bell, Scott; Campbell, Scott; Kedzierska, Katherine; Burrows, Scott R.; Rossjohn, Jamie; Khanna, Rajiv

2014-02-01

Exposure to naturally occurring variants of herpesviruses in clinical settings can have a dramatic impact on anti-viral immunity. Here we have evaluated the molecular imprint of variant peptide-MHC complexes on the T-cell repertoire during human cytomegalovirus (CMV) infection and demonstrate that primary co-infection with genetic variants of CMV was coincident with development of strain-specific T-cell immunity followed by emergence of cross-reactive virus-specific T-cells. Cross-reactive CMV-specific T cells exhibited a highly conserved public T cell repertoire, while T cells directed towards specific genetic variants displayed oligoclonal repertoires, unique to each individual. T cell recognition foot-print and pMHC-I structural analyses revealed that the cross-reactive T cells accommodate alterations in the pMHC complex with a broader foot-print focussing on the core of the peptide epitope. These findings provide novel molecular insight into how infection with naturally occurring genetic variants of persistent human herpesviruses imprints on the evolution of the anti-viral T-cell repertoire.

Structural studies of human histone deacetylase 8 and its site-specific variants complexed with substrate and inhibitors.

PubMed

Dowling, Daniel P; Gantt, Stephanie L; Gattis, Samuel G; Fierke, Carol A; Christianson, David W

2008-12-23

Metal-dependent histone deacetylases (HDACs) require Zn(2+) or Fe(2+) to regulate the acetylation of lysine residues in histones and other proteins in eukaryotic cells. Isozyme HDAC8 is perhaps the archetypical member of the class I HDAC family and serves as a paradigm for studying structure-function relationships. Here, we report the structures of HDAC8 complexes with trichostatin A and 3-(1-methyl-4-phenylacetyl-1H-2-pyrrolyl)-N-hydroxy-2-propenamide (APHA) in a new crystal form. The structure of the APHA complex reveals that the hydroxamate CO group accepts a hydrogen bond from Y306 but does not coordinate to Zn(2+) with favorable geometry, perhaps due to the constraints of its extended pi system. Additionally, since APHA binds to only two of the three protein molecules in the asymmetric unit of this complex, the structure of the third monomer represents the first structure of HDAC8 in the unliganded state. Comparison of unliganded and liganded structures illustrates ligand-induced conformational changes in the L2 loop that likely accompany substrate binding and catalysis. Furthermore, these structures, along with those of the D101N, D101E, D101A, and D101L variants, support the proposal that D101 is critical for the function of the L2 loop. However, amino acid substitutions for D101 can also trigger conformational changes of Y111 and W141 that perturb the substrate binding site. Finally, the structure of H143A HDAC8 complexed with an intact acetylated tetrapeptide substrate molecule confirms the importance of D101 for substrate binding and reveals how Y306 and the active site zinc ion together bind and activate the scissile amide linkage of acetyllysine.

Studies on the regulation of the human E1 subunit of the 2-oxoglutarate dehydrogenase complex, including the identification of a novel calcium-binding site.

PubMed

Armstrong, Craig T; Anderson, J L Ross; Denton, Richard M

2014-04-15

The regulation of the 2-oxoglutarate dehydrogenase complex is central to intramitochondrial energy metabolism. In the present study, the active full-length E1 subunit of the human complex has been expressed and shown to be regulated by Ca2+, adenine nucleotides and NADH, with NADH exerting a major influence on the K0.5 value for Ca2+. We investigated two potential Ca2+-binding sites on E1, which we term site 1 (D114ADLD) and site 2 (E139SDLD). Comparison of sequences from vertebrates with those from Ca2+-insensitive non-vertebrate complexes suggest that site 1 may be the more important. Consistent with this view, a mutated form of E1, D114A, shows a 6-fold decrease in sensitivity for Ca2+, whereas variant ∆site1 (in which the sequence of site 1 is replaced by A114AALA) exhibits an almost complete loss of Ca2+ activation. Variant ∆site2 (in which the sequence is replaced with A139SALA) shows no measurable change in Ca2+ sensitivity. We conclude that site 1, but not site 2, forms part of a regulatory Ca2+-binding site, which is distinct from other previously described Ca2+-binding sites.

A new detection method for the K variant of butyrylcholinesterase based on PCR primer introduced restriction analysis (PCR-PIRA).

PubMed Central

Shibuta, K; Abe, M; Suzuki, T

1994-01-01

The K variant of human butyrylcholinesterase is caused by a G/A transition in the butyrylcholinesterase gene, which neither creates nor destroys any restriction site. In an attempt to detect the K variant both simply and rapidly, we developed a two step method of "PCR primer introduced restriction analysis" (PCR-PIRA). The first step was used to introduce a new Fun4HI site into the normal allele for a screening test, while the second step was performed to create a new MaeIII site on the variant allele for a specific test. This method thus enabled us to distinguish clearly the K variant from the normal allele, and also showed that the frequency of the K variant allele is 0.164 in the Japanese population. Images PMID:7966197

Dimerization site 2 of the bacterial DNA-binding protein H-NS is required for gene silencing and stiffened nucleoprotein filament formation.

PubMed

Yamanaka, Yuki; Winardhi, Ricksen S; Yamauchi, Erika; Nishiyama, So-Ichiro; Sowa, Yoshiyuki; Yan, Jie; Kawagishi, Ikuro; Ishihama, Akira; Yamamoto, Kaneyoshi

2018-06-15

The bacterial nucleoid-associated protein H-NS is a DNA-binding protein, playing a major role in gene regulation. To regulate transcription, H-NS silences genes, including horizontally acquired foreign genes. Escherichia coli H-NS is 137 residues long and consists of two discrete and independent structural domains: an N-terminal oligomerization domain and a C-terminal DNA-binding domain, joined by a flexible linker. The N-terminal oligomerization domain is composed of two dimerization sites, dimerization sites 1 and 2, which are both required for H-NS oligomerization, but the exact role of dimerization site 2 in gene silencing is unclear. To this end, we constructed a whole set of single amino acid substitution variants spanning residues 2 to 137. Using a well-characterized H-NS target, the slp promoter of the glutamic acid-dependent acid resistance (GAD) cluster promoters, we screened for any variants defective in gene silencing. Focusing on the function of dimerization site 2, we analyzed four variants, I70C/I70A and L75C/L75A, which all could actively bind DNA but are defective in gene silencing. Atomic force microscopy analysis of DNA-H-NS complexes revealed that all of these four variants formed condensed complexes on DNA, whereas WT H-NS formed rigid and extended nucleoprotein filaments, a conformation required for gene silencing. Single-molecule stretching experiments confirmed that the four variants had lost the ability to form stiffened filaments. We conclude that dimerization site 2 of H-NS plays a key role in the formation of rigid H-NS nucleoprotein filament structures required for gene silencing. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Specificity and non-specificity in RNA–protein interactions

PubMed Central

Jankowsky, Eckhard; Harris, Michael E.

2016-01-01

Gene expression is regulated by complex networks of interactions between RNAs and proteins. Proteins that interact with RNA have been traditionally viewed as either specific or non-specific; specific proteins interact preferentially with defined RNA sequence or structure motifs, whereas non-specific proteins interact with RNA sites devoid of such characteristics. Recent studies indicate that the binary “specific vs. non-specific” classification is insufficient to describe the full spectrum of RNA–protein interactions. Here, we review new methods that enable quantitative measurements of protein binding to large numbers of RNA variants, and the concepts aimed as describing resulting binding spectra: affinity distributions, comprehensive binding models and free energy landscapes. We discuss how these new methodologies and associated concepts enable work towards inclusive, quantitative models for specific and non-specific RNA–protein interactions. PMID:26285679

Enrichment of colorectal cancer associations in functional regions: Insight for using epigenomics data in the analysis of whole genome sequence-imputed GWAS data.

PubMed

Bien, Stephanie A; Auer, Paul L; Harrison, Tabitha A; Qu, Conghui; Connolly, Charles M; Greenside, Peyton G; Chen, Sai; Berndt, Sonja I; Bézieau, Stéphane; Kang, Hyun M; Huyghe, Jeroen; Brenner, Hermann; Casey, Graham; Chan, Andrew T; Hopper, John L; Banbury, Barbara L; Chang-Claude, Jenny; Chanock, Stephen J; Haile, Robert W; Hoffmeister, Michael; Fuchsberger, Christian; Jenkins, Mark A; Leal, Suzanne M; Lemire, Mathieu; Newcomb, Polly A; Gallinger, Steven; Potter, John D; Schoen, Robert E; Slattery, Martha L; Smith, Joshua D; Le Marchand, Loic; White, Emily; Zanke, Brent W; Abeçasis, Goncalo R; Carlson, Christopher S; Peters, Ulrike; Nickerson, Deborah A; Kundaje, Anshul; Hsu, Li

2017-01-01

The evaluation of less frequent genetic variants and their effect on complex disease pose new challenges for genomic research. To investigate whether epigenetic data can be used to inform aggregate rare-variant association methods (RVAM), we assessed whether variants more significantly associated with colorectal cancer (CRC) were preferentially located in non-coding regulatory regions, and whether enrichment was specific to colorectal tissues. Active regulatory elements (ARE) were mapped using data from 127 tissues and cell-types from NIH Roadmap Epigenomics and Encyclopedia of DNA Elements (ENCODE) projects. We investigated whether CRC association p-values were more significant for common variants inside versus outside AREs, or 2) inside colorectal (CR) AREs versus AREs of other tissues and cell-types. We employed an integrative epigenomic RVAM for variants with allele frequency <1%. Gene sets were defined as ARE variants within 200 kilobases of a transcription start site (TSS) using either CR ARE or ARE from non-digestive tissues. CRC-set association p-values were used to evaluate enrichment of less frequent variant associations in CR ARE versus non-digestive ARE. ARE from 126/127 tissues and cell-types were significantly enriched for stronger CRC-variant associations. Strongest enrichment was observed for digestive tissues and immune cell types. CR-specific ARE were also enriched for stronger CRC-variant associations compared to ARE combined across non-digestive tissues (p-value = 9.6 × 10-4). Additionally, we found enrichment of stronger CRC association p-values for rare variant sets of CR ARE compared to non-digestive ARE (p-value = 0.029). Integrative epigenomic RVAM may enable discovery of less frequent variants associated with CRC, and ARE of digestive and immune tissues are most informative. Although distance-based aggregation of less frequent variants in CR ARE surrounding TSS showed modest enrichment, future association studies would likely benefit from joint analysis of transcriptomes and epigenomes to better link regulatory variation with target genes.

Converting Transaldolase into Aldolase through Swapping of the Multifunctional Acid-Base Catalyst: Common and Divergent Catalytic Principles in F6P Aldolase and Transaldolase.

PubMed

Sautner, Viktor; Friedrich, Mascha Miriam; Lehwess-Litzmann, Anja; Tittmann, Kai

2015-07-28

Transaldolase (TAL) and fructose-6-phosphate aldolase (FSA) both belong to the class I aldolase family and share a high degree of structural similarity and sequence identity. The molecular basis of the different reaction specificities (transferase vs aldolase) has remained enigmatic. A notable difference between the active sites is the presence of either a TAL-specific Glu (Gln in FSA) or a FSA-specific Tyr (Phe in TAL). Both residues seem to have analoguous multifunctional catalytic roles but are positioned at different faces of the substrate locale. We have engineered a TAL double variant (Glu to Gln and Phe to Tyr) with an active site resembling that of FSA. This variant indeed exhibits aldolase activity as its main activity with a catalytic efficiency even larger than that of authentic FSA, while TAL activity is greatly impaired. Structural analysis of this variant in complex with the dihydroxyacetone Schiff base formed upon substrate cleavage identifies the introduced Tyr (genuine in FSA) to catalyze protonation of the central carbanion-enamine intermediate as a key determinant of the aldolase reaction. Our studies pinpoint that the Glu in TAL and the Tyr in FSA, although located at different positions at the active site, similarly act as bona fide acid-base catalysts in numerous catalytic steps, including substrate binding, dehydration of the carbinolamine, and substrate cleavage. We propose that the different spatial positions of the multifunctional Glu in TAL and of the corresponding multifunctional Tyr in FSA relative to the substrate locale are critically controlling reaction specificity through either unfavorable (TAL) or favorable (FSA) geometry of proton transfer onto the common carbanion-enamine intermediate. The presence of both potential acid-base residues, Glu and Tyr, in the active site of TAL has deleterious effects on substrate binding and cleavage, most likely resulting from a differently organized H-bonding network. Large-scale motions of the protein associated with opening and closing of the active site that seem to bear relevance for catalysis are observed as covalent intermediates are exclusively observed in the "closed" conformation of the active site. Pre-steady-state kinetics are used to monitor catalytic processes and structural transitions and to refine the kinetic framework of TAL catalysis.

Structural and Biochemical Characterization of a Copper-Binding Mutant of the Organomercurial Lyase MerB: Insight into the Key Role of the Active Site Aspartic Acid in Hg-Carbon Bond Cleavage and Metal Binding Specificity.

PubMed

Wahba, Haytham M; Lecoq, Lauriane; Stevenson, Michael; Mansour, Ahmed; Cappadocia, Laurent; Lafrance-Vanasse, Julien; Wilkinson, Kevin J; Sygusch, Jurgen; Wilcox, Dean E; Omichinski, James G

2016-02-23

In bacterial resistance to mercury, the organomercurial lyase (MerB) plays a key role in the detoxification pathway through its ability to cleave Hg-carbon bonds. Two cysteines (C96 and C159; Escherichia coli MerB numbering) and an aspartic acid (D99) have been identified as the key catalytic residues, and these three residues are conserved in all but four known MerB variants, where the aspartic acid is replaced with a serine. To understand the role of the active site serine, we characterized the structure and metal binding properties of an E. coli MerB mutant with a serine substituted for D99 (MerB D99S) as well as one of the native MerB variants containing a serine residue in the active site (Bacillus megaterium MerB2). Surprisingly, the MerB D99S protein copurified with a bound metal that was determined to be Cu(II) from UV-vis absorption, inductively coupled plasma mass spectrometry, nuclear magnetic resonance, and electron paramagnetic resonance studies. X-ray structural studies revealed that the Cu(II) is bound to the active site cysteine residues of MerB D99S, but that it is displaced following the addition of either an organomercurial substrate or an ionic mercury product. In contrast, the B. megaterium MerB2 protein does not copurify with copper, but the structure of the B. megaterium MerB2-Hg complex is highly similar to the structure of the MerB D99S-Hg complexes. These results demonstrate that the active site aspartic acid is crucial for both the enzymatic activity and metal binding specificity of MerB proteins and suggest a possible functional relationship between MerB and its only known structural homologue, the copper-binding protein NosL.

Engineering a switch-on peptide to ricin A chain for increasing its specificity towards HIV-infected cells.

PubMed

Au, Ka-Yee; Wang, Rui-Rui; Wong, Yuen-Ting; Wong, Kam-Bo; Zheng, Yong-Tang; Shaw, Pang-Chui

2014-03-01

Ricin is a type II ribosome-inactivating protein (RIP) that potently inactivates eukaryotic ribosomes by removing a specific adenine residue at the conserved α-sarcin/ricin loop of 28S ribosomal RNA (rRNA). Here, we try to increase the specificity of the enzymatically active ricin A chain (RTA) towards human immunodeficiency virus type 1 (HIV-1) by adding a loop with HIV protease recognition site to RTA. HIV-specific RTA variants were constructed by inserting a peptide with HIV-protease recognition site either internally or at the C-terminal region of wild type RTA. Cleavability of variants by viral protease was tested in vitro and in HIV-infected cells. The production of viral p24 antigen and syncytium in the presence of C-terminal variants was measured to examine the anti-HIV activities of the variants. C-terminal RTA variants were specifically cleaved by HIV-1 protease both in vitro and in HIV-infected cells. Upon proteolysis, the processed variants showed enhanced antiviral effect with low cytotoxicity towards uninfected cells. RTA variants with HIV protease recognition sequence engineered at the C-terminus were cleaved and the products mediated specific inhibitory effect towards HIV replication. Current cocktail treatment of HIV infection fails to eradicate the virus from patients. Here we illustrate the feasibility of targeting an RIP towards HIV-infected cells by incorporation of HIV protease cleavage sequence. This approach may be generalized to other RIPs and is promising in drug design for combating HIV. Copyright © 2013 Elsevier B.V. All rights reserved.

The Structures of the C185S and C185A Mutants of Sulfite Oxidase Reveal Rearrangement of the Active Site

DOE Office of Scientific and Technical Information (OSTI.GOV)

Qiu, James A.; Wilson, Heather L.; Pushie, M. Jake

Sulfite oxidase (SO) catalyzes the physiologically critical conversion of sulfite to sulfate. Enzymatic activity is dependent on the presence of the metal molybdenum complexed with a pyranopterin-dithiolene cofactor termed molybdopterin. Comparison of the amino acid sequences of SOs from a variety of sources has identified a single conserved Cys residue essential for catalytic activity. The crystal structure of chicken liver sulfite oxidase indicated that this residue, Cys185 in chicken SO, coordinates the Mo atom in the active site. To improve our understanding of the role of this residue in the catalytic mechanism of sulfite oxidase, serine and alanine variants atmore » position 185 of recombinant chicken SO were generated. Spectroscopic and kinetic studies indicate that neither variant is capable of sulfite oxidation. The crystal structure of the C185S variant was determined to 1.9 {angstrom} resolution and to 2.4 {angstrom} resolution in the presence of sulfite, and the C185A variant to 2.8 {angstrom} resolution. The structures of the C185S and C185A variants revealed that neither the Ser or Ala side chains appeared to closely interact with the Mo atom and that a third oxo group replaced the usual cysteine sulfur ligand at the Mo center, confirming earlier extended X-ray absorption fine structure spectroscopy (EXAFS) work on the human C207S mutant. An unexpected result was that in the C185S variant, in the absence of sulfite, the active site residue Tyr322 became disordered as did the loop region flanking it. In the C185S variant crystallized in the presence of sulfite, the Tyr322 residue relocalized to the active site. The C185A variant structure also indicated the presence of a third oxygen ligand; however, Tyr322 remained in the active site. EXAFS studies of the Mo coordination environment indicate the Mo atom is in the oxidized Mo{sup VI} state in both the C185S and C185A variants of chicken SO and show the expected trioxodithiolene active site. Density functional theory calculations of the trioxo form of the cofactor reasonably reproducd the Mo=O distances of the complex; however, the calculated Mo-S distances were slightly longer than either crystallographic or EXAFS measurements. Taken together, these results indicate that the active sites of the C185S and C185A variants are essentially catalytically inactive, the crystal structures of C185S and C185A variants contain a fully oxidized, trioxo form of the cofactor, and Tyr322 can undergo a conformational change that is relevant to the reaction mechanism. Additional DFT calculations demonstrated that such methods can reasonably reproduce the geometry and bond lengths of the active site.« less

Engineering peptide ligase specificity by proteomic identification of ligation sites.

PubMed

Weeks, Amy M; Wells, James A

2018-01-01

Enzyme-catalyzed peptide ligation is a powerful tool for site-specific protein bioconjugation, but stringent enzyme-substrate specificity limits its utility. We developed an approach for comprehensively characterizing peptide ligase specificity for N termini using proteome-derived peptide libraries. We used this strategy to characterize the ligation efficiency for >25,000 enzyme-substrate pairs in the context of the engineered peptide ligase subtiligase and identified a family of 72 mutant subtiligases with activity toward N-terminal sequences that were previously recalcitrant to modification. We applied these mutants individually for site-specific bioconjugation of purified proteins, including antibodies, and in algorithmically selected combinations for sequencing of the cellular N terminome with reduced sequence bias. We also developed a web application to enable algorithmic selection of the most efficient subtiligase variant(s) for bioconjugation to user-defined sequences. Our methods provide a new toolbox of enzymes for site-specific protein modification and a general approach for rapidly defining and engineering peptide ligase specificity.

The Centromere: Chromatin Foundation for the Kinetochore Machinery

PubMed Central

Fukagawa, Tatsuo; Earnshaw, William C.

2014-01-01

Since discovery of the centromere-specific histone H3 variant CENP-A, centromeres have come to be defined as chromatin structures that establish the assembly site for the complex kinetochore machinery. In most organisms, centromere activity is defined epigenetically, rather than by specific DNA sequences. In this review, we describe selected classic work and recent progress in studies of centromeric chromatin with a focus on vertebrates. We consider possible roles for repetitive DNA sequences found at most centromeres, chromatin factors and modifications that assemble and activate CENP-A chromatin for kinetochore assembly, plus the use of artificial chromosomes and kinetochores to study centromere function. PMID:25203206

A Non-Degenerate Code of Deleterious Variants in Mendelian Loci Contributes to Complex Disease Risk

PubMed Central

Blair, David R.; Lyttle, Christopher S.; Mortensen, Jonathan M.; Bearden, Charles F.; Jensen, Anders Boeck; Khiabanian, Hossein; Melamed, Rachel; Rabadan, Raul; Bernstam, Elmer V.; Brunak, Søren; Jensen, Lars Juhl; Nicolae, Dan; Shah, Nigam H.; Grossman, Robert L.; Cox, Nancy J.; White, Kevin P.; Rzhetsky, Andrey

2013-01-01

Summary Whereas countless highly penetrant variants have been associated with Mendelian disorders, the genetic etiologies underlying complex diseases remain largely unresolved. Here, we examine the extent to which Mendelian variation contributes to complex disease risk by mining the medical records of over 110 million patients. We detect thousands of associations between Mendelian and complex diseases, revealing a non-degenerate, phenotypic code that links each complex disorder to a unique collection of Mendelian loci. Using genome-wide association results, we demonstrate that common variants associated with complex diseases are enriched in the genes indicated by this “Mendelian code.” Finally, we detect hundreds of comorbidity associations among Mendelian disorders, and we use probabilistic genetic modeling to demonstrate that Mendelian variants likely contribute non-additively to the risk for a subset of complex diseases. Overall, this study illustrates a complementary approach for mapping complex disease loci and provides unique predictions concerning the etiologies of specific diseases. PMID:24074861

Expansion of Protein Farnesyltransferase Specificity Using “Tunable” Active Site Interactions

PubMed Central

Hougland, James L.; Gangopadhyay, Soumyashree A.; Fierke, Carol A.

2012-01-01

Post-translational modifications play essential roles in regulating protein structure and function. Protein farnesyltransferase (FTase) catalyzes the biologically relevant lipidation of up to several hundred cellular proteins. Site-directed mutagenesis of FTase coupled with peptide selectivity measurements demonstrates that molecular recognition is determined by a combination of multiple interactions. Targeted randomization of these interactions yields FTase variants with altered and, in some cases, bio-orthogonal selectivity. We demonstrate that FTase specificity can be “tuned” using a small number of active site contacts that play essential roles in discriminating against non-substrates in the wild-type enzyme. This tunable selectivity extends in vivo, with FTase variants enabling the creation of bioengineered parallel prenylation pathways with altered substrate selectivity within a cell. Engineered FTase variants provide a novel avenue for probing both the selectivity of prenylation pathway enzymes and the effects of prenylation pathway modifications on the cellular function of a protein. PMID:22992747

Structure of insoluble immune complexes as studied by spectroturbidimetry and dynamic light scattering

NASA Astrophysics Data System (ADS)

Khlebtsov, Boris N.; Burygin, Gennadii L.; Matora, Larisa Y.; Shchyogolev, Sergei Y.; Khlebtsov, Nikolai G.

2004-07-01

We describe two variants of a method for determining the average composition of insoluble immune complex particles (IICP). The first variant is based on measuring the specific turbidity (the turbidity per unit mass concentration of the dispersed substance) and the average size of IICP determined from dynamic light scattering (DLS). In the second variant, the wavelength exponent (i.e., the slope of the logarithmic turbidity spectrum) is used in combination with specific turbidity measurements. Both variants allow the average biopolymer volume fraction to be determined in terms of the average refractive index of IICP. The method is exemplified by two experimental antigen+antibody systems: (i) lipopolysaccharide-protein complex (LPPC) of Azospirillum brasilense Sp245+rabbit anti-LPPC; and (ii) human IgG (hIgG)+sheep anti-hIgG. Our measurements by the two methods for both types of systems gave, on the average, the same result: the volume fraction of the IICP biopolymers is about 30%; accordingly, the volume fraction of buffer solvent is 70%.

Structural Basis of Substrate Specificity and Regiochemistry in the MycF/TylF Family of Sugar O -Methyltransferases.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bernard, Steffen M.; Akey, David L.; Tripathi, Ashootosh

Sugar moieties in natural products are frequently modified by O-methylation. In the biosynthesis of the macrolide antibiotic mycinamicin, methylation of a 6'-deoxyallose substituent occurs in a stepwise manner first at the 2'- and then the 3'-hydroxyl groups to produce the mycinose moiety in the final product. The timing and placement of the O-methylations impact final stage C-H functionalization reactions mediated by the P450 monooxygenase MycG. The structural basis of pathway ordering and substrate specificity is unknown. A series of crystal structures of MycF, the 3'-O-methyltransferase, including the free enzyme and complexes with S-adenosyl homocysteine (SAH), substrate, product, and unnatural substrates,more » show that SAM binding induces substantial ordering that creates the binding site for the natural substrate, and a bound metal ion positions the substrate for catalysis. A single amino acid substitution relaxed the 2'-methoxy specificity but retained regiospecificity. The engineered variant produced a new mycinamicin analog, demonstrating the utility of structural information to facilitate bioengineering approaches for the chemoenzymatic synthesis of complex small molecules containing modified sugars. Using the MycF substrate complex and the modeled substrate complex of a 4'-specific homolog, active site residues were identified that correlate with the 3'- or 4'- specificity of MycF family members and define the protein and substrate features that direct the regiochemistry of methyltransfer. Lastly, this classification scheme will be useful in the annotation of new secondary metabolite pathways that utilize this family of enzymes.« less

Structural Basis of Substrate Specificity and Regiochemistry in the MycF/TylF Family of Sugar O -Methyltransferases.

DOE PAGES

Bernard, Steffen M.; Akey, David L.; Tripathi, Ashootosh; ...

2015-02-18

Sugar moieties in natural products are frequently modified by O-methylation. In the biosynthesis of the macrolide antibiotic mycinamicin, methylation of a 6'-deoxyallose substituent occurs in a stepwise manner first at the 2'- and then the 3'-hydroxyl groups to produce the mycinose moiety in the final product. The timing and placement of the O-methylations impact final stage C-H functionalization reactions mediated by the P450 monooxygenase MycG. The structural basis of pathway ordering and substrate specificity is unknown. A series of crystal structures of MycF, the 3'-O-methyltransferase, including the free enzyme and complexes with S-adenosyl homocysteine (SAH), substrate, product, and unnatural substrates,more » show that SAM binding induces substantial ordering that creates the binding site for the natural substrate, and a bound metal ion positions the substrate for catalysis. A single amino acid substitution relaxed the 2'-methoxy specificity but retained regiospecificity. The engineered variant produced a new mycinamicin analog, demonstrating the utility of structural information to facilitate bioengineering approaches for the chemoenzymatic synthesis of complex small molecules containing modified sugars. Using the MycF substrate complex and the modeled substrate complex of a 4'-specific homolog, active site residues were identified that correlate with the 3'- or 4'- specificity of MycF family members and define the protein and substrate features that direct the regiochemistry of methyltransfer. Lastly, this classification scheme will be useful in the annotation of new secondary metabolite pathways that utilize this family of enzymes.« less

Characterization of Conserved Tandem Donor Sites and Intronic Motifs Required for Alternative Splicing in Corticosteroid Receptor Genes

PubMed Central

Qian, Xiaoxiao; Matthews, Laura; Lightman, Stafford; Ray, David; Norman, Michael

2015-01-01

Alternative splicing events from tandem donor sites result in mRNA variants coding for additional amino acids in the DNA binding domain of both the glucocorticoid (GR) and mineralocorticoid (MR) receptors. We now show that expression of both splice variants is extensively conserved in mammalian species, providing strong evidence for their functional significance. An exception to the conservation of the MR tandem splice site (an A at position +5 of the MR+12 donor site in the mouse) was predicted to decrease U1 small nuclear RNA binding. In accord with this prediction, we were unable to detect the MR+12 variant in this species. The one exception to the conservation of the GR tandem splice site, an A at position +3 of the platypus GRγ donor site that was predicted to enhance binding of U1 snRNA, was unexpectedly associated with decreased expression of the variant from the endogenous gene as well as a minigene. An intronic pyrimidine motif present in both GR and MR genes was found to be critical for usage of the downstream donor site, and overexpression of TIA1/TIAL1 RNA binding proteins, which are known to bind such motifs, led to a marked increase in the proportion of GRγ and MR+12. These results provide striking evidence for conservation of a complex splicing mechanism that involves processes other than stochastic spliceosome binding and identify a mechanism that would allow regulation of variant expression. PMID:19819975

Comparison of self-processing of foot-and-mouth disease virus leader proteinase and porcine reproductive and respiratory syndrome virus leader proteinase nsp1α

DOE Office of Scientific and Technical Information (OSTI.GOV)

Steinberger, Jutta; Kontaxis, Georg; Rancan, Chiara

The foot-and-mouth disease virus leader proteinase (Lb{sup pro}) cleaves itself off the nascent viral polyprotein. NMR studies on the monomeric variant Lb{sup pro} L200F provide structural evidence for intramolecular self-processing. {sup 15}N-HSQC measurements of Lb{sup pro} L200F showed specifically shifted backbone signals in the active and substrate binding sites compared to the monomeric variant sLb{sup pro}, lacking six C-terminal residues. This indicates transient intramolecular interactions between the C-terminal extension (CTE) of one molecule and its own active site. Contrastingly, the porcine reproductive and respiratory syndrome virus (PRRSV) leader proteinase nsp1α, with a papain-like fold like Lb{sup pro}, stably binds itsmore » own CTE. Parts of the β-sheet domains but none of the α-helical domains of Lb{sup pro} and nsp1α superimpose; consequently, the α-helical domain of nsp1α is oriented differently relative to its β-sheet domain. This provides a large interaction surface for the CTE with the globular domain, stabilising the intramolecular complex. Consequently, self-processing inactivates nsp1α but not Lb{sup pro}. - Highlights: • We examine self-processing of the leader protease of foot-and-mouth disease virus. • NMR analysis strongly supports intramolecular self-processing. • Self-processing is a dynamic process with no stable complex. • Structural comparison with nsp1α of PRRSV which forms stable intramolecular complex. • Subdomain orientation explains differences in stability of intramolecular complexes.« less

Analysis of RNA-Seq datasets reveals enrichment of tissue-specific splice variants for nuclear envelope proteins.

PubMed

Capitanchik, Charlotte; Dixon, Charles; Swanson, Selene K; Florens, Laurence; Kerr, Alastair R W; Schirmer, Eric C

2018-06-18

Nuclear envelopathies/laminopathies yield tissue-specific pathologies, yet arise from mutation of ubiquitously-expressed genes. One possible explanation of this tissue specificity is that tissue-specific partners become disrupted from larger complexes, but a little investigated alternate hypothesis is that the mutated proteins themselves have tissue-specific splice variants. Here, we analyze RNA-Seq datasets to identify muscle-specific splice variants of nuclear envelope genes that could be relevant to the study of laminopathies, particularly muscular dystrophies, that are not currently annotated in sequence databases. Notably, we found novel isoforms or tissue-specificity of isoforms for: Lap2, linked to cardiomyopathy; Nesprin 2, linked to Emery-Dreifuss muscular dystrophy and Lmo7, a regulator of the emerin gene that is linked to Emery-Dreifuss muscular dystrophy. Interestingly, the muscle-specific exon in Lmo7 is rich in serine phosphorylation motifs, suggesting an important regulatory function. Evidence for muscle-specific splice variants in non-nuclear envelope proteins linked to other muscular dystrophies was also found. Tissue-specific variants were also indicated for several nucleoporins including Nup54, Nup133, Nup153 and Nup358/RanBP2. We confirmed expression of novel Lmo7 and RanBP2 variants with RT-PCR and found that specific knockdown of the Lmo7 variant caused a reduction in myogenic index during mouse C2C12 myogenesis. Global analysis revealed an enrichment of tissue-specific splice variants for nuclear envelope proteins in general compared to the rest of the genome, suggesting that splice variants contribute to regulating its tissue-specific functions.

Evaluating whole genome sequence data from the Genetic Absence Epilepsy Rat from Strasbourg and its related non-epileptic strain

PubMed Central

Powell, Kim L.; Zhu, Mingfu; Campbell, C. Ryan; Maia, Jessica M.; Ren, Zhong; Jones, Nigel C.; O’Brien, Terence J.; Petrovski, Slavé

2017-01-01

Objective The Genetic Absence Epilepsy Rats from Strasbourg (GAERS) are an inbreed Wistar rat strain widely used as a model of genetic generalised epilepsy with absence seizures. As in humans, the genetic architecture that results in genetic generalized epilepsy in GAERS is poorly understood. Here we present the strain-specific variants found among the epileptic GAERS and their related Non-Epileptic Control (NEC) strain. The GAERS and NEC represent a powerful opportunity to identify neurobiological factors that are associated with the genetic generalised epilepsy phenotype. Methods We performed whole genome sequencing on adult epileptic GAERS and adult NEC rats, a strain derived from the same original Wistar colony. We also generated whole genome sequencing on four double-crossed (GAERS with NEC) F2 selected for high-seizing (n = 2) and non-seizing (n = 2) phenotypes. Results Specific to the GAERS genome, we identified 1.12 million single nucleotide variants, 296.5K short insertion-deletions, and 354 putative copy number variants that result in complete or partial loss/duplication of 41 genes. Of the GAERS-specific variants that met high quality criteria, 25 are annotated as stop codon gain/loss, 56 as putative essential splice sites, and 56 indels are predicted to result in a frameshift. Subsequent screening against the two F2 progeny sequenced for having the highest and two F2 progeny for having the lowest seizure burden identified only the selected Cacna1h GAERS-private protein-coding variant as exclusively co-segregating with the two high-seizing F2 rats. Significance This study highlights an approach for using whole genome sequencing to narrow down to a manageable candidate list of genetic variants in a complex genetic epilepsy animal model, and suggests utility of this sequencing design to investigate other spontaneously occurring animal models of human disease. PMID:28708842

Selective destruction of cells infected with human immunodeficiency virus

DOEpatents

Keener, William K.; Ward, Thomas E.

2003-09-30

Compositions and methods for selectively killing a cell containing a viral protease are disclosed. The composition is a variant of a protein synthesis inactivating toxin wherein a viral protease cleavage site is interposed between the A and B chains. The variant of the type II ribosome-inactivating protein is activated by digestion of the viral protease cleavage site by the specific viral protease. The activated ribosome-inactivating protein then kills the cell by inactivating cellular ribosomes. A preferred embodiment of the invention is specific for human immunodeficiency virus (HIV) and uses ricin as the ribosome-inactivating protein. In another preferred embodiment of the invention, the variant of the ribosome-inactivating protein is modified by attachment of one or more hydrophobic agents. The hydrophobic agent facilitates entry of the variant of the ribosome-inactivating protein into cells and can lead to incorporation of the ribosome-inactivating protein into viral particles. Still another preferred embodiment of the invention includes a targeting moiety attached to the variants of the ribosome-inactivating protein to target the agent to HIV infectable cells.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xia, Shuangluo; Vashishtha, Ashwani; Bulkley, David

During DNA synthesis, base stacking and Watson-Crick (WC) hydrogen bonding increase the stability of nascent base pairs when they are in a ternary complex. To evaluate the contribution of base stacking to the incorporation efficiency of dNTPs when a DNA polymerase encounters an abasic site, we varied the penultimate base pairs (PBs) adjacent to the abasic site using all 16 possible combinations. We then determined pre-steady-state kinetic parameters with an RB69 DNA polymerase variant and solved nine structures of the corresponding ternary complexes. The efficiency of incorporation for incoming dNTPs opposite an abasic site varied between 2- and 210-fold dependingmore » on the identity of the PB. We propose that the A rule can be extended to encompass the fact that DNA polymerase can bypass dA/abasic sites more efficiently than other dN/abasic sites. Crystal structures of the ternary complexes show that the surface of the incoming base was stacked against the PB's interface and that the kinetic parameters for dNMP incorporation were consistent with specific features of base stacking, such as surface area and partial charge-charge interactions between the incoming base and the PB. Without a templating nucleotide residue, an incoming dNTP has no base with which it can hydrogen bond and cannot be desolvated, so that these surrounding water molecules become ordered and remain on the PB's surface in the ternary complex. When these water molecules are on top of a hydrophobic patch on the PB, they destabilize the ternary complex, and the incorporation efficiency of incoming dNTPs is reduced.« less

A CT-rich haplotype in intron 4 of SNCA confers risk for Lewy body pathology in Alzheimer’s disease and affects SNCA expression

PubMed Central

Lutz, Michael W.; Saul, Robert; Linnertz, Colton; Glenn, Omolara-Chinue; Roses, Allen D.; Chiba-Falek, Ornit

2015-01-01

INTRODUCTION We recently showed that tagging-SNPs across the SNCA locus were significantly associated with increased risk for LB pathology in AD cases. However, the actual genetic variant(s) that underlie the observed associations remain elusive. METHODS We used a bioinformatics algorithm to catalogue Structural-Variants in a region of SNCA-intron4, followed by phased-sequencing. We performed a genetic-association analysis in autopsy series of LBV/AD cases compared with AD-only controls. We investigated the biological functions by expression analysis using temporal-cortex samples. RESULTS We identified four distinct haplotypes within a highly-polymorphic-low-complexity CT-rich region. We showed that a specific haplotype conferred risk to develop LBV/AD. We demonstrated that the CT-rich site acts as an enhancer element, where the risk haplotype was significantly associated with elevated levels of SNCA-mRNA. DISCUSSION We have discovered a novel haplotype in a CT-rich region in SNCA that contributes to LB pathology in AD patients, possibly via cis-regulation of the gene expression. PMID:26079410

[Microsurgical anatomy importance of A1-anterior communicating artery complex].

PubMed

Monroy-Sosa, Alejandro; Pérez-Cruz, Julio César; Reyes-Soto, Gervith; Delgado-Hernández, Carlos; Macías-Duvignau, Mario Alberto; Delgado-Reyes, Luis

2013-01-01

The anterior cerebral artery originates from the bifurcation of the internal carotid artery lateral to the optic chiasm, then joins with its contralateral counterpart via the anterior communicating artery. A1-anterior communicating artery complex is the most frequent anatomical variants and is the major site of aneurysms between 30 to 37%. Know the anatomy microsurgical, variants anatomical and importance of complex precommunicating segment-artery anterior communicating in surgery neurological of the pathology vascular, mainly aneurysms, in Mexican population. The study was performed in 30 brains injected. Microanatomy was studied (length and diameter) of A1-anterior communicating artery complex and its variants. 60 segments A1, the average length of left side was 11.35 mm and 11.84 mm was right. The average diameter of left was 1.67 mm and the right was 1.64 mm. The average number of perforators on the left side was 7.9 and the right side was 7.5. Anterior communicating artery was found in 29 brains of the optic chiasm, its course depended on the length of the A1 segment. The average length of the segment was 2.84 mm, the average diameter was 1.41 mm and the average number of perforators was 3.27. A1-anterior communicating artery complex variants were found in 18 (60%) and the presence of two blister-like aneurysms. It is necessary to understand the A1-anterior communicating artery complex microanatomy of its variants to have a three-dimensional vision during aneurysm surgery.

Incorporation of native antibodies and Fc-fusion proteins on DNA nanostructures via a modular conjugation strategy† †Electronic supplementary information (ESI) available: Experimental methods, DNA origami design, DNA sequences, and additional experimental data. See DOI: 10.1039/c7cc04178k

PubMed Central

Rosier, Bas J. H. M.; Cremers, Glenn A. O.; Engelen, Wouter; Merkx, Maarten; Brunsveld, Luc

2017-01-01

A photocrosslinkable protein G variant was used as an adapter protein to covalently and site-specifically conjugate an antibody and an Fc-fusion protein to an oligonucleotide. This modular approach enables straightforward decoration of DNA nanostructures with complex native proteins while retaining their innate binding affinity, allowing precise control over the nanoscale spatial organization of such proteins for in vitro and in vivo biomedical applications. PMID:28617516

Optimization of Photoactive Protein Z for Fast and Efficient Site-Specific Conjugation of Native IgG

PubMed Central

2015-01-01

Antibody conjugates have been used in a variety of applications from immunoassays to drug conjugates. However, it is becoming increasingly clear that in order to maximize an antibody’s antigen binding ability and to produce homogeneous antibody-conjugates, the conjugated molecule should be attached onto IgG site-specifically. We previously developed a facile method for the site-specific modification of full length, native IgGs by engineering a recombinant Protein Z that forms a covalent link to the Fc domain of IgG upon exposure to long wavelength UV light. To further improve the efficiency of Protein Z production and IgG conjugation, we constructed a panel of 13 different Protein Z variants with the UV-active amino acid benzoylphenylalanine (BPA) in different locations. By using this panel of Protein Z to cross-link a range of IgGs from different hosts, including human, mouse, and rat, we discovered two previously unknown Protein Z variants, L17BPA and K35BPA, that are capable of cross-linking many commonly used IgG isotypes with efficiencies ranging from 60% to 95% after only 1 h of UV exposure. When compared to existing site-specific methods, which often require cloning or enzymatic reactions, the Protein Z-based method described here, utilizing the L17BPA, K35BPA, and the previously described Q32BPA variants, represents a vastly more accessible and efficient approach that is compatible with nearly all native IgGs, thus making site-specific conjugation more accessible to the general research community. PMID:25121619

Optimization of photoactive protein Z for fast and efficient site-specific conjugation of native IgG.

PubMed

Hui, James Z; Tsourkas, Andrew

2014-09-17

Antibody conjugates have been used in a variety of applications from immunoassays to drug conjugates. However, it is becoming increasingly clear that in order to maximize an antibody's antigen binding ability and to produce homogeneous antibody-conjugates, the conjugated molecule should be attached onto IgG site-specifically. We previously developed a facile method for the site-specific modification of full length, native IgGs by engineering a recombinant Protein Z that forms a covalent link to the Fc domain of IgG upon exposure to long wavelength UV light. To further improve the efficiency of Protein Z production and IgG conjugation, we constructed a panel of 13 different Protein Z variants with the UV-active amino acid benzoylphenylalanine (BPA) in different locations. By using this panel of Protein Z to cross-link a range of IgGs from different hosts, including human, mouse, and rat, we discovered two previously unknown Protein Z variants, L17BPA and K35BPA, that are capable of cross-linking many commonly used IgG isotypes with efficiencies ranging from 60% to 95% after only 1 h of UV exposure. When compared to existing site-specific methods, which often require cloning or enzymatic reactions, the Protein Z-based method described here, utilizing the L17BPA, K35BPA, and the previously described Q32BPA variants, represents a vastly more accessible and efficient approach that is compatible with nearly all native IgGs, thus making site-specific conjugation more accessible to the general research community.

Preferential Allele Expression Analysis Identifies Shared Germline and Somatic Driver Genes in Advanced Ovarian Cancer

PubMed Central

Halabi, Najeeb M.; Martinez, Alejandra; Al-Farsi, Halema; Mery, Eliane; Puydenus, Laurence; Pujol, Pascal; Khalak, Hanif G.; McLurcan, Cameron; Ferron, Gwenael; Querleu, Denis; Al-Azwani, Iman; Al-Dous, Eman; Mohamoud, Yasmin A.; Malek, Joel A.; Rafii, Arash

2016-01-01

Identifying genes where a variant allele is preferentially expressed in tumors could lead to a better understanding of cancer biology and optimization of targeted therapy. However, tumor sample heterogeneity complicates standard approaches for detecting preferential allele expression. We therefore developed a novel approach combining genome and transcriptome sequencing data from the same sample that corrects for sample heterogeneity and identifies significant preferentially expressed alleles. We applied this analysis to epithelial ovarian cancer samples consisting of matched primary ovary and peritoneum and lymph node metastasis. We find that preferentially expressed variant alleles include germline and somatic variants, are shared at a relatively high frequency between patients, and are in gene networks known to be involved in cancer processes. Analysis at a patient level identifies patient-specific preferentially expressed alleles in genes that are targets for known drugs. Analysis at a site level identifies patterns of site specific preferential allele expression with similar pathways being impacted in the primary and metastasis sites. We conclude that genes with preferentially expressed variant alleles can act as cancer drivers and that targeting those genes could lead to new therapeutic strategies. PMID:26735499

BAC Modification through Serial or Simultaneous Use of CRE/Lox Technology

PubMed Central

Parrish, Mark; Unruh, Jay; Krumlauf, Robb

2011-01-01

Bacterial Artificial Chromosomes (BACs) are vital tools in mouse genomic analyses because of their ability to propagate large inserts. The size of these constructs, however, prevents the use of conventional molecular biology techniques for modification and manipulation. Techniques such as recombineering and Cre/Lox methodologies have thus become heavily relied upon for such purposes. In this work, we investigate the applicability of Lox variant sites for serial and/or simultaneous manipulations of BACs. We show that Lox spacer mutants are very specific, and inverted repeat variants reduce Lox reaction rates through reducing the affinity of Cre for the site, while retaining some functionality. Employing these methods, we produced serial modifications encompassing four independent changes which generated a mouse HoxB BAC with fluorescent reporter proteins inserted into four adjacent Hox genes. We also generated specific, simultaneous deletions using combinations of spacer variants and inverted repeat variants. These techniques will facilitate BAC manipulations and open a new repertoire of methods for BAC and genome manipulation. PMID:21197414

Fine-mapping inflammatory bowel disease loci to single variant resolution

PubMed Central

Huang, Hailiang; Fang, Ming; Jostins, Luke; Mirkov, Maša Umićević; Boucher, Gabrielle; Anderson, Carl A; Andersen, Vibeke; Cleynen, Isabelle; Cortes, Adrian; Crins, François; D'Amato, Mauro; Deffontaine, Valérie; Dimitrieva, Julia; Docampo, Elisa; Elansary, Mahmoud; Farh, Kyle Kai-How; Franke, Andre; Gori, Ann-Stephan; Goyette, Philippe; Halfvarson, Jonas; Haritunians, Talin; Knight, Jo; Lawrance, Ian C; Lees, Charlie W; Louis, Edouard; Mariman, Rob; Meuwissen, Theo; Mni, Myriam; Momozawa, Yukihide; Parkes, Miles; Spain, Sarah L; Théâtre, Emilie; Trynka, Gosia; Satsangi, Jack; van Sommeren, Suzanne; Vermeire, Severine; Xavier, Ramnik J; Weersma, Rinse K; Duerr, Richard H; Mathew, Christopher G; Rioux, John D; McGovern, Dermot PB; Cho, Judy H; Georges, Michel; Daly, Mark J; Barrett, Jeffrey C

2017-01-01

Summary The inflammatory bowel diseases (IBD) are chronic gastrointestinal inflammatory disorders that affect millions worldwide. Genome-wide association studies have identified 200 IBD-associated loci, but few have been conclusively resolved to specific functional variants. Here we report fine-mapping of 94 IBD loci using high-density genotyping in 67,852 individuals. We pinpointed 18 associations to a single causal variant with >95% certainty, and an additional 27 associations to a single variant with >50% certainty. These 45 variants are significantly enriched for protein-coding changes (n=13), direct disruption of transcription factor binding sites (n=3) and tissue specific epigenetic marks (n=10), with the latter category showing enrichment in specific immune cells among associations stronger in CD and in gut mucosa among associations stronger in UC. The results of this study suggest that high-resolution fine-mapping in large samples can convert many GWAS discoveries into statistically convincing causal variants, providing a powerful substrate for experimental elucidation of disease mechanisms. PMID:28658209

Forced Ambiguity of the Leucine Codons for Multiple-Site-Specific Incorporation of a Noncanonical Amino Acid

PubMed Central

Kwon, Inchan; Choi, Eun Sil

2016-01-01

Multiple-site-specific incorporation of a noncanonical amino acid into a recombinant protein would be a very useful technique to generate multiple chemical handles for bioconjugation and multivalent binding sites for the enhanced interaction. Previously combination of a mutant yeast phenylalanyl-tRNA synthetase variant and the yeast phenylalanyl-tRNA containing the AAA anticodon was used to incorporate a noncanonical amino acid into multiple UUU phenylalanine (Phe) codons in a site-specific manner. However, due to the less selective codon recognition of the AAA anticodon, there was significant misincorporation of a noncanonical amino acid into unwanted UUC Phe codons. To enhance codon selectivity, we explored degenerate leucine (Leu) codons instead of Phe degenerate codons. Combined use of the mutant yeast phenylalanyl-tRNA containing the CAA anticodon and the yPheRS_naph variant allowed incorporation of a phenylalanine analog, 2-naphthylalanine, into murine dihydrofolate reductase in response to multiple UUG Leu codons, but not to other Leu codon sites. Despite the moderate UUG codon occupancy by 2-naphthylalaine, these results successfully demonstrated that the concept of forced ambiguity of the genetic code can be achieved for the Leu codons, available for multiple-site-specific incorporation. PMID:27028506

Forced Ambiguity of the Leucine Codons for Multiple-Site-Specific Incorporation of a Noncanonical Amino Acid.

PubMed

Kwon, Inchan; Choi, Eun Sil

2016-01-01

Multiple-site-specific incorporation of a noncanonical amino acid into a recombinant protein would be a very useful technique to generate multiple chemical handles for bioconjugation and multivalent binding sites for the enhanced interaction. Previously combination of a mutant yeast phenylalanyl-tRNA synthetase variant and the yeast phenylalanyl-tRNA containing the AAA anticodon was used to incorporate a noncanonical amino acid into multiple UUU phenylalanine (Phe) codons in a site-specific manner. However, due to the less selective codon recognition of the AAA anticodon, there was significant misincorporation of a noncanonical amino acid into unwanted UUC Phe codons. To enhance codon selectivity, we explored degenerate leucine (Leu) codons instead of Phe degenerate codons. Combined use of the mutant yeast phenylalanyl-tRNA containing the CAA anticodon and the yPheRS_naph variant allowed incorporation of a phenylalanine analog, 2-naphthylalanine, into murine dihydrofolate reductase in response to multiple UUG Leu codons, but not to other Leu codon sites. Despite the moderate UUG codon occupancy by 2-naphthylalaine, these results successfully demonstrated that the concept of forced ambiguity of the genetic code can be achieved for the Leu codons, available for multiple-site-specific incorporation.

Inhibition of SCF ubiquitin ligases by engineered ubiquitin variants that target the Cul1 binding site on the Skp1–F-box interface

DOE PAGES

Gorelik, Maryna; Orlicky, Stephen; Sartori, Maria A.; ...

2016-03-14

Skp1–Cul1–F-box (SCF) E3 ligases play key roles in multiple cellular processes through ubiquitination and subsequent degradation of substrate proteins. Although Skp1 and Cul1 are invariant components of all SCF complexes, the 69 different human F-box proteins are variable substrate binding modules that determine specificity. SCF E3 ligases are activated in many cancers and inhibitors could have therapeutic potential. Here, we used phage display to develop specific ubiquitin-based inhibitors against two F-box proteins, Fbw7 and Fbw11. Unexpectedly, the ubiquitin variants bind at the interface of Skp1 and F-box proteins and inhibit ligase activity by preventing Cul1 binding to the same surface.more » Using structure-based design and phage display, we modified the initial inhibitors to generate broad-spectrum inhibitors that targeted many SCF ligases, or conversely, a highly specific inhibitor that discriminated between even the close homologs Fbw11 and Fbw1. We propose that most F-box proteins can be targeted by this approach for basic research and for potential cancer therapies.« less

Refining the accuracy of validated target identification through coding variant fine-mapping in type 2 diabetes.

PubMed

Mahajan, Anubha; Wessel, Jennifer; Willems, Sara M; Zhao, Wei; Robertson, Neil R; Chu, Audrey Y; Gan, Wei; Kitajima, Hidetoshi; Taliun, Daniel; Rayner, N William; Guo, Xiuqing; Lu, Yingchang; Li, Man; Jensen, Richard A; Hu, Yao; Huo, Shaofeng; Lohman, Kurt K; Zhang, Weihua; Cook, James P; Prins, Bram Peter; Flannick, Jason; Grarup, Niels; Trubetskoy, Vassily Vladimirovich; Kravic, Jasmina; Kim, Young Jin; Rybin, Denis V; Yaghootkar, Hanieh; Müller-Nurasyid, Martina; Meidtner, Karina; Li-Gao, Ruifang; Varga, Tibor V; Marten, Jonathan; Li, Jin; Smith, Albert Vernon; An, Ping; Ligthart, Symen; Gustafsson, Stefan; Malerba, Giovanni; Demirkan, Ayse; Tajes, Juan Fernandez; Steinthorsdottir, Valgerdur; Wuttke, Matthias; Lecoeur, Cécile; Preuss, Michael; Bielak, Lawrence F; Graff, Marielisa; Highland, Heather M; Justice, Anne E; Liu, Dajiang J; Marouli, Eirini; Peloso, Gina Marie; Warren, Helen R; Afaq, Saima; Afzal, Shoaib; Ahlqvist, Emma; Almgren, Peter; Amin, Najaf; Bang, Lia B; Bertoni, Alain G; Bombieri, Cristina; Bork-Jensen, Jette; Brandslund, Ivan; Brody, Jennifer A; Burtt, Noël P; Canouil, Mickaël; Chen, Yii-Der Ida; Cho, Yoon Shin; Christensen, Cramer; Eastwood, Sophie V; Eckardt, Kai-Uwe; Fischer, Krista; Gambaro, Giovanni; Giedraitis, Vilmantas; Grove, Megan L; de Haan, Hugoline G; Hackinger, Sophie; Hai, Yang; Han, Sohee; Tybjærg-Hansen, Anne; Hivert, Marie-France; Isomaa, Bo; Jäger, Susanne; Jørgensen, Marit E; Jørgensen, Torben; Käräjämäki, Annemari; Kim, Bong-Jo; Kim, Sung Soo; Koistinen, Heikki A; Kovacs, Peter; Kriebel, Jennifer; Kronenberg, Florian; Läll, Kristi; Lange, Leslie A; Lee, Jung-Jin; Lehne, Benjamin; Li, Huaixing; Lin, Keng-Hung; Linneberg, Allan; Liu, Ching-Ti; Liu, Jun; Loh, Marie; Mägi, Reedik; Mamakou, Vasiliki; McKean-Cowdin, Roberta; Nadkarni, Girish; Neville, Matt; Nielsen, Sune F; Ntalla, Ioanna; Peyser, Patricia A; Rathmann, Wolfgang; Rice, Kenneth; Rich, Stephen S; Rode, Line; Rolandsson, Olov; Schönherr, Sebastian; Selvin, Elizabeth; Small, Kerrin S; Stančáková, Alena; Surendran, Praveen; Taylor, Kent D; Teslovich, Tanya M; Thorand, Barbara; Thorleifsson, Gudmar; Tin, Adrienne; Tönjes, Anke; Varbo, Anette; Witte, Daniel R; Wood, Andrew R; Yajnik, Pranav; Yao, Jie; Yengo, Loïc; Young, Robin; Amouyel, Philippe; Boeing, Heiner; Boerwinkle, Eric; Bottinger, Erwin P; Chowdhury, Rajiv; Collins, Francis S; Dedoussis, George; Dehghan, Abbas; Deloukas, Panos; Ferrario, Marco M; Ferrières, Jean; Florez, Jose C; Frossard, Philippe; Gudnason, Vilmundur; Harris, Tamara B; Heckbert, Susan R; Howson, Joanna M M; Ingelsson, Martin; Kathiresan, Sekar; Kee, Frank; Kuusisto, Johanna; Langenberg, Claudia; Launer, Lenore J; Lindgren, Cecilia M; Männistö, Satu; Meitinger, Thomas; Melander, Olle; Mohlke, Karen L; Moitry, Marie; Morris, Andrew D; Murray, Alison D; de Mutsert, Renée; Orho-Melander, Marju; Owen, Katharine R; Perola, Markus; Peters, Annette; Province, Michael A; Rasheed, Asif; Ridker, Paul M; Rivadineira, Fernando; Rosendaal, Frits R; Rosengren, Anders H; Salomaa, Veikko; Sheu, Wayne H-H; Sladek, Rob; Smith, Blair H; Strauch, Konstantin; Uitterlinden, André G; Varma, Rohit; Willer, Cristen J; Blüher, Matthias; Butterworth, Adam S; Chambers, John Campbell; Chasman, Daniel I; Danesh, John; van Duijn, Cornelia; Dupuis, Josée; Franco, Oscar H; Franks, Paul W; Froguel, Philippe; Grallert, Harald; Groop, Leif; Han, Bok-Ghee; Hansen, Torben; Hattersley, Andrew T; Hayward, Caroline; Ingelsson, Erik; Kardia, Sharon L R; Karpe, Fredrik; Kooner, Jaspal Singh; Köttgen, Anna; Kuulasmaa, Kari; Laakso, Markku; Lin, Xu; Lind, Lars; Liu, Yongmei; Loos, Ruth J F; Marchini, Jonathan; Metspalu, Andres; Mook-Kanamori, Dennis; Nordestgaard, Børge G; Palmer, Colin N A; Pankow, James S; Pedersen, Oluf; Psaty, Bruce M; Rauramaa, Rainer; Sattar, Naveed; Schulze, Matthias B; Soranzo, Nicole; Spector, Timothy D; Stefansson, Kari; Stumvoll, Michael; Thorsteinsdottir, Unnur; Tuomi, Tiinamaija; Tuomilehto, Jaakko; Wareham, Nicholas J; Wilson, James G; Zeggini, Eleftheria; Scott, Robert A; Barroso, Inês; Frayling, Timothy M; Goodarzi, Mark O; Meigs, James B; Boehnke, Michael; Saleheen, Danish; Morris, Andrew P; Rotter, Jerome I; McCarthy, Mark I

2018-04-01

We aggregated coding variant data for 81,412 type 2 diabetes cases and 370,832 controls of diverse ancestry, identifying 40 coding variant association signals (P < 2.2 × 10 -7 ); of these, 16 map outside known risk-associated loci. We make two important observations. First, only five of these signals are driven by low-frequency variants: even for these, effect sizes are modest (odds ratio ≤1.29). Second, when we used large-scale genome-wide association data to fine-map the associated variants in their regional context, accounting for the global enrichment of complex trait associations in coding sequence, compelling evidence for coding variant causality was obtained for only 16 signals. At 13 others, the associated coding variants clearly represent 'false leads' with potential to generate erroneous mechanistic inference. Coding variant associations offer a direct route to biological insight for complex diseases and identification of validated therapeutic targets; however, appropriate mechanistic inference requires careful specification of their causal contribution to disease predisposition.

Evolutionary transitions to new DNA methyltransferases through target site expansion and shrinkage.

PubMed

Rockah-Shmuel, Liat; Tawfik, Dan S

2012-12-01

DNA-binding and modifying proteins show high specificity but also exhibit a certain level of promiscuity. Such latent promiscuous activities comprise the starting points for new protein functions, but this hypothesis presents a paradox: a new activity can only evolve if it already exists. How then, do novel activities evolve? DNA methyltransferases, for example, are highly divergent in their target sites, but how transitions toward novel sites occur remains unknown. We performed laboratory evolution of the DNA methyltransferase M.HaeIII. We found that new target sites emerged primarily through expansion of the original site, GGCC, and the subsequent shrinkage of evolved expanded sites. Variants evolved for sites that are promiscuously methylated by M.HaeIII [GG((A)/(T))CC and GGCGCC] carried mutations in 'gate-keeper' residues. They could thereby methylate novel target sites such as GCGC and GGATCC that were neither selected for nor present in M.HaeIII. These 'generalist' intermediates were further evolved to obtain variants with novel target specificities. Our results demonstrate the ease by which new DNA-binding and modifying specificities evolve and the mechanism by which they occur at both the protein and DNA levels.

The Principal Genetic Determinants for Nasopharyngeal Carcinoma in China Involve the HLA Class I Antigen Recognition Groove

PubMed Central

Tang, Minzhong; Lautenberger, James A.; Gao, Xiaojiang; Sezgin, Efe; Hendrickson, Sher L.; Troyer, Jennifer L.; David, Victor A.; Guan, Li; Mcintosh, Carl E.; Guo, Xiuchan; Zheng, Yuming; Liao, Jian; Deng, Hong; Malasky, Michael; Kessing, Bailey; Winkler, Cheryl A.; Carrington, Mary; dé The, Guy; Zeng, Yi; O'Brien, Stephen J.

2012-01-01

Nasopharyngeal carcinoma (NPC) is an epithelial malignancy facilitated by Epstein-Barr Virus infection. Here we resolve the major genetic influences for NPC incidence using a genome-wide association study (GWAS), independent cohort replication, and high-resolution molecular HLA class I gene typing including 4,055 study participants from the Guangxi Zhuang Autonomous Region and Guangdong province of southern China. We detect and replicate strong association signals involving SNPs, HLA alleles, and amino acid (aa) variants across the major histocompatibility complex-HLA-A, HLA –B, and HLA -C class I genes (PHLA-A-aa-site-62 = 7.4×10−29; P HLA-B-aa-site-116 = 6.5×10−19; P HLA-C-aa-site-156 = 6.8×10−8 respectively). Over 250 NPC-HLA associated variants within HLA were analyzed in concert to resolve separate and largely independent HLA-A, -B, and -C gene influences. Multivariate logistical regression analysis collapsed significant associations in adjacent genes spanning 500 kb (OR2H1, GABBR1, HLA-F, and HCG9) as proxies for peptide binding motifs carried by HLA- A*11:01. A similar analysis resolved an independent association signal driven by HLA-B*13:01, B*38:02, and B*55:02 alleles together. NPC resistance alleles carrying the strongly associated amino acid variants implicate specific class I peptide recognition motifs in HLA-A and -B peptide binding groove as conferring strong genetic influence on the development of NPC in China. PMID:23209447

Regression and Data Mining Methods for Analyses of Multiple Rare Variants in the Genetic Analysis Workshop 17 Mini-Exome Data

PubMed Central

Bailey-Wilson, Joan E.; Brennan, Jennifer S.; Bull, Shelley B; Culverhouse, Robert; Kim, Yoonhee; Jiang, Yuan; Jung, Jeesun; Li, Qing; Lamina, Claudia; Liu, Ying; Mägi, Reedik; Niu, Yue S.; Simpson, Claire L.; Wang, Libo; Yilmaz, Yildiz E.; Zhang, Heping; Zhang, Zhaogong

2012-01-01

Group 14 of Genetic Analysis Workshop 17 examined several issues related to analysis of complex traits using DNA sequence data. These issues included novel methods for analyzing rare genetic variants in an aggregated manner (often termed collapsing rare variants), evaluation of various study designs to increase power to detect effects of rare variants, and the use of machine learning approaches to model highly complex heterogeneous traits. Various published and novel methods for analyzing traits with extreme locus and allelic heterogeneity were applied to the simulated quantitative and disease phenotypes. Overall, we conclude that power is (as expected) dependent on locus-specific heritability or contribution to disease risk, large samples will be required to detect rare causal variants with small effect sizes, extreme phenotype sampling designs may increase power for smaller laboratory costs, methods that allow joint analysis of multiple variants per gene or pathway are more powerful in general than analyses of individual rare variants, population-specific analyses can be optimal when different subpopulations harbor private causal mutations, and machine learning methods may be useful for selecting subsets of predictors for follow-up in the presence of extreme locus heterogeneity and large numbers of potential predictors. PMID:22128066

Altered Substrate Specificity of Drug-Resistant Human Immunodeficiency Virus Type 1 Protease

PubMed Central

Dauber, Deborah S.; Ziermann, Rainer; Parkin, Neil; Maly, Dustin J.; Mahrus, Sami; Harris, Jennifer L.; Ellman, Jon A.; Petropoulos, Christos; Craik, Charles S.

2002-01-01

Resistance to human immunodeficiency virus type 1 protease (HIV PR) inhibitors results primarily from the selection of multiple mutations in the protease region. Because many of these mutations are selected for the ability to decrease inhibitor binding in the active site, they also affect substrate binding and potentially substrate specificity. This work investigates the substrate specificity of a panel of clinically derived protease inhibitor-resistant HIV PR variants. To compare protease specificity, we have used positional-scanning, synthetic combinatorial peptide libraries as well as a select number of individual substrates. The subsite preferences of wild-type HIV PR determined by using the substrate libraries are consistent with prior reports, validating the use of these libraries to compare specificity among a panel of HIV PR variants. Five out of seven protease variants demonstrated subtle differences in specificity that may have significant impacts on their abilities to function in viral maturation. Of these, four variants demonstrated up to fourfold changes in the preference for valine relative to alanine at position P2 when tested on individual peptide substrates. This change correlated with a common mutation in the viral NC/p1 cleavage site. These mutations may represent a mechanism by which severely compromised, drug-resistant viral strains can increase fitness levels. Understanding the altered substrate specificity of drug-resistant HIV PR should be valuable in the design of future generations of protease inhibitors as well as in elucidating the molecular basis of regulation of proteolysis in HIV. PMID:11773410

Mass Spectrometric Determination of ILPR G-quadruplex Binding Sites in Insulin and IGF-2

PubMed Central

Xiao, JunFeng

2009-01-01

The insulin-linked polymorphic region (ILPR) of the human insulin gene promoter region forms G-quadruplex structures in vitro. Previous studies show that insulin and insulin-like growth factor-2 (IGF-2) exhibit high affinity binding in vitro to 2-repeat sequences of ILPR variants a and h, but negligible binding to variant i. Two-repeat sequences of variants a and h form intramolecular G-quadruplex structures that are not evidenced for variant i. Here we report on the use of protein digestion combined with affinity capture and MALDI-MS detection to pinpoint ILPR binding sites in insulin and IGF-2. Peptides captured by ILPR variants a and h were sequenced by MALDI-MS/MS, LC-MS and in silico digestion. On-bead digestion of insulin-ILPR variant a complexes supported the conclusions. The results indicate that the sequence VCG(N)RGF is generally present in the captured peptides and is likely involved in the affinity binding interactions of the proteins with the ILPR G-quadruplexes. The significance of arginine in the interactions was studied by comparing the affinities of synthesized peptides VCGERGF and VCGEAGF with ILPR variant a. Peptides from other regions of the proteins that are connected through disulfide linkages were also detected in some capture experiments. Identification of binding sites could facilitate design of DNA binding ligands for capture and detection of insulin and IGF-2. The interactions may have biological significance as well. PMID:19747845

Exome Sequencing Identified a Splice Site Mutation in FHL1 that Causes Uruguay Syndrome, an X-Linked Disorder With Skeletal Muscle Hypertrophy and Premature Cardiac Death.

PubMed

Xue, Yuan; Schoser, Benedikt; Rao, Aliz R; Quadrelli, Roberto; Vaglio, Alicia; Rupp, Verena; Beichler, Christine; Nelson, Stanley F; Schapacher-Tilp, Gudrun; Windpassinger, Christian; Wilcox, William R

2016-04-01

Previously, we reported a rare X-linked disorder, Uruguay syndrome in a single family. The main features are pugilistic facies, skeletal deformities, and muscular hypertrophy despite a lack of exercise and cardiac ventricular hypertrophy leading to premature death. An ≈19 Mb critical region on X chromosome was identified through identity-by-descent analysis of 3 affected males. Exome sequencing was conducted on one affected male to identify the disease-causing gene and variant. A splice site variant (c.502-2A>G) in the FHL1 gene was highly suspicious among other candidate genes and variants. FHL1A is the predominant isoform of FHL1 in cardiac and skeletal muscle. Sequencing cDNA showed the splice site variant led to skipping of exons 6 of the FHL1A isoform, equivalent to the FHL1C isoform. Targeted analysis showed that this splice site variant cosegregated with disease in the family. Western blot and immunohistochemical analysis of muscle from the proband showed a significant decrease in protein expression of FHL1A. Real-time polymerase chain reaction analysis of different isoforms of FHL1 demonstrated that the FHL1C is markedly increased. Mutations in the FHL1 gene have been reported in disorders with skeletal and cardiac myopathy but none has the skeletal or facial phenotype seen in patients with Uruguay syndrome. Our data suggest that a novel FHL1 splice site variant results in the absence of FHL1A and the abundance of FHL1C, which may contribute to the complex and severe phenotype. Mutation screening of the FHL1 gene should be considered for patients with uncharacterized myopathies and cardiomyopathies. © 2016 American Heart Association, Inc.

Coronary heart disease-associated variation in TCF21 disrupts a miR-224 binding site and miRNA-mediated regulation.

PubMed

Miller, Clint L; Haas, Ulrike; Diaz, Roxanne; Leeper, Nicholas J; Kundu, Ramendra K; Patlolla, Bhagat; Assimes, Themistocles L; Kaiser, Frank J; Perisic, Ljubica; Hedin, Ulf; Maegdefessel, Lars; Schunkert, Heribert; Erdmann, Jeanette; Quertermous, Thomas; Sczakiel, Georg

2014-03-01

Genome-wide association studies (GWAS) have identified chromosomal loci that affect risk of coronary heart disease (CHD) independent of classical risk factors. One such association signal has been identified at 6q23.2 in both Caucasians and East Asians. The lead CHD-associated polymorphism in this region, rs12190287, resides in the 3' untranslated region (3'-UTR) of TCF21, a basic-helix-loop-helix transcription factor, and is predicted to alter the seed binding sequence for miR-224. Allelic imbalance studies in circulating leukocytes and human coronary artery smooth muscle cells (HCASMC) showed significant imbalance of the TCF21 transcript that correlated with genotype at rs12190287, consistent with this variant contributing to allele-specific expression differences. 3' UTR reporter gene transfection studies in HCASMC showed that the disease-associated C allele has reduced expression compared to the protective G allele. Kinetic analyses in vitro revealed faster RNA-RNA complex formation and greater binding of miR-224 with the TCF21 C allelic transcript. In addition, in vitro probing with Pb2+ and RNase T1 revealed structural differences between the TCF21 variants in proximity of the rs12190287 variant, which are predicted to provide greater access to the C allele for miR-224 binding. miR-224 and TCF21 expression levels were anti-correlated in HCASMC, and miR-224 modulates the transcriptional response of TCF21 to transforming growth factor-β (TGF-β) and platelet derived growth factor (PDGF) signaling in an allele-specific manner. Lastly, miR-224 and TCF21 were localized in human coronary artery lesions and anti-correlated during atherosclerosis. Together, these data suggest that miR-224 interaction with the TCF21 transcript contributes to allelic imbalance of this gene, thus partly explaining the genetic risk for coronary heart disease associated at 6q23.2. These studies implicating rs12190287 in the miRNA-dependent regulation of TCF21, in conjunction with previous studies showing that this variant modulates transcriptional regulation through activator protein 1 (AP-1), suggests a unique bimodal level of complexity previously unreported for disease-associated variants.

Crystal structure of a cytochrome P450 2B6 genetic variant in complex with the inhibitor 4-(4-chlorophenyl)imidazole at 2.0-A resolution.

PubMed

Gay, Sean C; Shah, Manish B; Talakad, Jyothi C; Maekawa, Keiko; Roberts, Arthur G; Wilderman, P Ross; Sun, Ling; Yang, Jane Y; Huelga, Stephanie C; Hong, Wen-Xu; Zhang, Qinghai; Stout, C David; Halpert, James R

2010-04-01

The structure of the K262R genetic variant of human cytochrome P450 2B6 in complex with the inhibitor 4-(4-chlorophenyl)imidazole (4-CPI) has been determined using X-ray crystallography to 2.0-A resolution. Production of diffraction quality crystals was enabled through a combination of protein engineering, chaperone coexpression, modifications to the purification protocol, and the use of unique facial amphiphiles during crystallization. The 2B6-4-CPI complex is virtually identical to the rabbit 2B4 structure bound to the same inhibitor with respect to the arrangement of secondary structural elements and the placement of active site residues. The structure supports prior P450 2B6 homology models based on other mammalian cytochromes P450 and is consistent with the limited site-directed mutagenesis studies on 2B6 and extensive studies on P450 2B4 and 2B1. Although the K262R genetic variant shows unaltered binding of 4-CPI, altered binding affinity, kinetics, and/or product profiles have been previously shown with several other ligands. On the basis of new P450 2B6 crystal structure and previous 2B4 structures, substitutions at residue 262 affect a hydrogen-bonding network connecting the G and H helices, where subtle differences could be transduced to the active site. Docking experiments indicate that the closed protein conformation allows smaller ligands such as ticlopidine to bind to the 2B6 active site in the expected orientation. However, it is unknown whether 2B6 undergoes structural reorganization to accommodate bulkier molecules, as previously inferred from multiple P450 2B4 crystal structures.

Functional genetic selection of Helix 66 in Escherichia coli 23S rRNA identified the eukaryotic-binding sequence for ribosomal protein L2

PubMed Central

Kitahara, Kei; Kajiura, Akimasa; Sato, Neuza Satomi; Suzuki, Tsutomu

2007-01-01

Ribosomal protein L2 is a highly conserved primary 23S rRNA-binding protein. L2 specifically recognizes the internal bulge sequence in Helix 66 (H66) of 23S rRNA and is localized to the intersubunit space through formation of bridge B7b with 16S rRNA. The L2-binding site in H66 is highly conserved in prokaryotic ribosomes, whereas the corresponding site in eukaryotic ribosomes has evolved into distinct classes of sequences. We performed a systematic genetic selection of randomized rRNA sequences in Escherichia coli, and isolated 20 functional variants of the L2-binding site. The isolated variants consisted of eukaryotic sequences, in addition to prokaryotic sequences. These results suggest that L2/L8e does not recognize a specific base sequence of H66, but rather a characteristic architecture of H66. The growth phenotype of the isolated variants correlated well with their ability of subunit association. Upon continuous cultivation of a deleterious variant, we isolated two spontaneous mutations within domain IV of 23S rRNA that compensated for its weak subunit association, and alleviated its growth defect, implying that functional interactions between intersubunit bridges compensate ribosomal function. PMID:17553838

Monitoring Ras Interactions with the Nucleotide Exchange Factor Son of Sevenless (Sos) Using Site-specific NMR Reporter Signals and Intrinsic Fluorescence*

PubMed Central

Vo, Uybach; Vajpai, Navratna; Flavell, Liz; Bobby, Romel; Breeze, Alexander L.; Embrey, Kevin J.; Golovanov, Alexander P.

2016-01-01

The activity of Ras is controlled by the interconversion between GTP- and GDP-bound forms partly regulated by the binding of the guanine nucleotide exchange factor Son of Sevenless (Sos). The details of Sos binding, leading to nucleotide exchange and subsequent dissociation of the complex, are not completely understood. Here, we used uniformly 15N-labeled Ras as well as [13C]methyl-Met,Ile-labeled Sos for observing site-specific details of Ras-Sos interactions in solution. Binding of various forms of Ras (loaded with GDP and mimics of GTP or nucleotide-free) at the allosteric and catalytic sites of Sos was comprehensively characterized by monitoring signal perturbations in the NMR spectra. The overall affinity of binding between these protein variants as well as their selected functional mutants was also investigated using intrinsic fluorescence. The data support a positive feedback activation of Sos by Ras·GTP with Ras·GTP binding as a substrate for the catalytic site of activated Sos more weakly than Ras·GDP, suggesting that Sos should actively promote unidirectional GDP → GTP exchange on Ras in preference of passive homonucleotide exchange. Ras·GDP weakly binds to the catalytic but not to the allosteric site of Sos. This confirms that Ras·GDP cannot properly activate Sos at the allosteric site. The novel site-specific assay described may be useful for design of drugs aimed at perturbing Ras-Sos interactions. PMID:26565026

Fine-mapping inflammatory bowel disease loci to single-variant resolution.

PubMed

Huang, Hailiang; Fang, Ming; Jostins, Luke; Umićević Mirkov, Maša; Boucher, Gabrielle; Anderson, Carl A; Andersen, Vibeke; Cleynen, Isabelle; Cortes, Adrian; Crins, François; D'Amato, Mauro; Deffontaine, Valérie; Dmitrieva, Julia; Docampo, Elisa; Elansary, Mahmoud; Farh, Kyle Kai-How; Franke, Andre; Gori, Ann-Stephan; Goyette, Philippe; Halfvarson, Jonas; Haritunians, Talin; Knight, Jo; Lawrance, Ian C; Lees, Charlie W; Louis, Edouard; Mariman, Rob; Meuwissen, Theo; Mni, Myriam; Momozawa, Yukihide; Parkes, Miles; Spain, Sarah L; Théâtre, Emilie; Trynka, Gosia; Satsangi, Jack; van Sommeren, Suzanne; Vermeire, Severine; Xavier, Ramnik J; Weersma, Rinse K; Duerr, Richard H; Mathew, Christopher G; Rioux, John D; McGovern, Dermot P B; Cho, Judy H; Georges, Michel; Daly, Mark J; Barrett, Jeffrey C

2017-07-13

Inflammatory bowel diseases are chronic gastrointestinal inflammatory disorders that affect millions of people worldwide. Genome-wide association studies have identified 200 inflammatory bowel disease-associated loci, but few have been conclusively resolved to specific functional variants. Here we report fine-mapping of 94 inflammatory bowel disease loci using high-density genotyping in 67,852 individuals. We pinpoint 18 associations to a single causal variant with greater than 95% certainty, and an additional 27 associations to a single variant with greater than 50% certainty. These 45 variants are significantly enriched for protein-coding changes (n = 13), direct disruption of transcription-factor binding sites (n = 3), and tissue-specific epigenetic marks (n = 10), with the last category showing enrichment in specific immune cells among associations stronger in Crohn's disease and in gut mucosa among associations stronger in ulcerative colitis. The results of this study suggest that high-resolution fine-mapping in large samples can convert many discoveries from genome-wide association studies into statistically convincing causal variants, providing a powerful substrate for experimental elucidation of disease mechanisms.

Selective Destruction Of Cells Infected With The Human Immunodeficiency Virus

DOEpatents

Keener, William K.; Ward, Thomas E.

2006-03-28

Compositions and methods for selectively killing a cell containing a viral protease are disclosed. The composition is a varient of a protein synthesis inactivating toxin wherein a viral protease cleavage site is interposed between the A and B chains. The variant of the type II ribosome-inactivating protein is activated by digestion of the viral protease cleavage site by the specific viral protease. The activated ribosome-inactivating protein then kills the cell by inactivating cellular ribosomes. A preferred embodiment of the invention is specific for human immunodeficiency virus (HIV) and uses ricin as the ribosome-inactivating protein. In another preferred embodiment of the invention, the variant of the ribosome-inactivating protein is modified by attachment of one or more hydrophobic agents. The hydrophobic agent facilitates entry of the variant of the ribosome-inactivating protein into cells and can lead to incorporation of the ribosome-inactivating protein into viral particles. Still another preferred embodiment of the invention includes a targeting moiety attached to the variants of the ribosome-inactivating protein to target the agent to HIV infectable cells.

Staphylococcus aureus innate immune evasion is lineage-specific: a bioinfomatics study.

PubMed

McCarthy, Alex J; Lindsay, Jodi A

2013-10-01

Staphylococcus aureus is a major human pathogen, and is targeted by the host innate immune system. In response, S. aureus genomes encode dozens of secreted proteins that inhibit complement, chemotaxis and neutrophil activation resulting in successful evasion of innate immune responses. These proteins include immune evasion cluster proteins (IEC; Chp, Sak, Scn), staphylococcal superantigen-like proteins (SSLs), phenol soluble modulins (PSMs) and several leukocidins. Biochemical studies have indicated that genetic variants of these proteins can have unique functions. To ascertain the scale of genetic variation in secreted immune evasion proteins, whole genome sequences of 88 S. aureus isolates, representing 25 clonal complex (CC) lineages, in the public domain were analysed across 43 genes encoding 38 secreted innate immune evasion protein complexes. Twenty-three genes were variable, with between 2 and 15 variants, and the variants had lineage-specific distributions. They include genes encoding Eap, Ecb, Efb, Flipr/Flipr-like, Hla, Hld, Hlg, Sbi, Scin-B/C and 13 SSLs. Most of these protein complexes inhibit complement, chemotaxis and neutrophil activation suggesting that isolates from each S. aureus lineage respond to the innate immune system differently. In contrast, protein complexes that lyse neutrophils (LukSF-PVL, LukMF, LukED and PSMs) were highly conserved, but can be carried on mobile genetic elements (MGEs). MGEs also encode proteins with narrow host-specificities arguing that their acquisition has important roles in host/environmental adaptation. In conclusion, this data suggests that each lineage of S. aureus evades host immune responses differently, and that isolates can adapt to new host environments by acquiring MGEs and the immune evasion protein complexes that they encode. Cocktail therapeutics that targets multiple variant proteins may be the most appropriate strategy for controlling S. aureus infections. Copyright © 2013 Elsevier B.V. All rights reserved.

Deep Sequencing-guided Design of a High Affinity Dual Specificity Antibody to Target Two Angiogenic Factors in Neovascular Age-related Macular Degeneration* ♦

PubMed Central

Koenig, Patrick; Lee, Chingwei V.; Sanowar, Sarah; Wu, Ping; Stinson, Jeremy; Harris, Seth F.; Fuh, Germaine

2015-01-01

The development of dual targeting antibodies promises therapies with improved efficacy over mono-specific antibodies. Here, we engineered a Two-in-One VEGF/angiopoietin 2 antibody with dual action Fab (DAF) as a potential therapeutic for neovascular age-related macular degeneration. Crystal structures of the VEGF/angiopoietin 2 DAF in complex with its two antigens showed highly overlapping binding sites. To achieve sufficient affinity of the DAF to block both angiogenic factors, we turned to deep mutational scanning in the complementarity determining regions (CDRs). By mutating all three CDRs of each antibody chain simultaneously, we were able not only to identify affinity improving single mutations but also mutation pairs from different CDRs that synergistically improve both binding functions. Furthermore, insights into the cooperativity between mutations allowed us to identify fold-stabilizing mutations in the CDRs. The data obtained from deep mutational scanning reveal that the majority of the 52 CDR residues are utilized differently for the two antigen binding function and permit, for the first time, the engineering of several DAF variants with sub-nanomolar affinity against two structurally unrelated antigens. The improved variants show similar blocking activity of receptor binding as the high affinity mono-specific antibodies against these two proteins, demonstrating the feasibility of generating a dual specificity binding surface with comparable properties to individual high affinity mono-specific antibodies. PMID:26088137

Stereotypic and complex phrase types provide structural evidence for a multi-message display in humpback whales (Megaptera novaeangliae).

PubMed

Murray, Anita; Dunlop, Rebecca A; Noad, Michael J; Goldizen, Anne W

2018-02-01

Male humpback whales produce a mating display called "song." Behavioral studies indicate song has inter- and/or intra-sexual functionality, suggesting song may be a multi-message display. Multi-message displays often include stereotypic components that convey group membership for mate attraction and/or male-male interactions, and complex components that convey individual quality for courtship. Humpback whale song contains sounds ("units") arranged into sequences ("phrases"). Repetitions of a specific phrase create a "theme." Within a theme, imperfect phrase repetitions ("phrase variants") create variability among phrases of the same type ("phrase type"). The hypothesis that song contains stereotypic and complex phrase types, structural characteristics consistent with a multi-message display, is investigated using recordings of 17 east Australian males (8:2004, 9:2011). Phrase types are categorized as stereotypic or complex using number of unit types, number of phrase variants, and the proportion of phrases that is unique to an individual versus shared amongst males. Unit types are determined using self-organizing maps. Phrase variants are determined by Levenshtein distances between phrases. Stereotypic phrase types have smaller numbers of unit types and shared phrase variants. Complex phrase types have larger numbers of unit types and unique phrase variants. This study supports the hypothesis that song could be a multi-message display.

Stepwise Loop Insertion Strategy for Active Site Remodeling to Generate Novel Enzyme Functions.

PubMed

Hoque, Md Anarul; Zhang, Yong; Chen, Liuqing; Yang, Guangyu; Khatun, Mst Afroza; Chen, Haifeng; Hao, Liu; Feng, Yan

2017-05-19

The remodeling of active sites to generate novel biocatalysts is an attractive and challenging task. We developed a stepwise loop insertion strategy (StLois), in which randomized residue pairs are inserted into active site loops. The phosphotriesterase-like lactonase from Geobacillus kaustophilus (GkaP-PLL) was used to investigate StLois's potential for changing enzyme function. By inserting six residues into active site loop 7, the best variant ML7-B6 demonstrated a 16-fold further increase in catalytic efficiency toward ethyl-paraoxon compared with its initial template, that is a 609-fold higher, >10 7 fold substrate specificity shift relative to that of wild-type lactonase. The remodeled variants displayed 760-fold greater organophosphate hydrolysis activity toward the organophosphates parathion, diazinon, and chlorpyrifos. Structure and docking computations support the source of notably inverted enzyme specificity. Considering the fundamental importance of active site loops, the strategy has potential for the rapid generation of novel enzyme functions by loop remodeling.

Two variants of the major serine protease inhibitor from the sea anemone Stichodactyla helianthus, expressed in Pichia pastoris.

PubMed

García-Fernández, Rossana; Ziegelmüller, Patrick; González, Lidice; Mansur, Manuel; Machado, Yoan; Redecke, Lars; Hahn, Ulrich; Betzel, Christian; Chávez, María de Los Ángeles

2016-07-01

The major protease inhibitor from the sea anemone Stichodactyla helianthus (ShPI-1) is a non-specific inhibitor that binds trypsin and other trypsin-like enzymes, as well as chymotrypsin, and human neutrophil elastase. We performed site-directed mutagenesis of ShPI-1 to produce two variants (rShPI-1/K13L and rShPI/Y15S) that were expressed in Pichia pastoris, purified, and characterized. After a single purification step, 65 mg and 15 mg of protein per liter of culture supernatant were obtained for rShPI-1/K13L and rShPI/Y15S, respectively. Functional studies demonstrated a 100-fold decreased trypsin inhibitory activity as result of the K13L substitution at the reactive (P1) site. This protein variant has a novel tight-binding inhibitor activity of pancreatic elastase and increased activity toward neutrophil elastase in comparison to rShPI-1A. In contrast, the substitution Y15S at P2' site did not affect the Ki value against trypsin, but did reduce activity 10-fold against chymotrypsin and neutrophil elastase. Our results provide two new ShPI-1 variants with modified inhibitory activities, one of them with increased biomedical potential. This study also offers new insight into the functional impact of the P1 and P2' sites on ShPI-1 specificity. Copyright © 2016 Elsevier Inc. All rights reserved.

Single substitutions to closely related amino acids contribute to the functional diversification of an insect-inducible, positively selected plant cystatin.

PubMed

Rasoolizadeh, Asieh; Goulet, Marie-Claire; Sainsbury, Frank; Cloutier, Conrad; Michaud, Dominique

2016-04-01

A causal link has been reported between positively selected amino acids in plant cystatins and the inhibitory range of these proteins against insect digestive cysteine (Cys) proteases. Here we assessed the impact of single substitutions to closely related amino acids on the contribution of positive selection to cystatin diversification. Cystatin sequence alignments, while confirming hypervariability, indicated a preference for related amino acids at positively selected sites. For example, the non-polar residues leucine (Leu), isoleucine (Ile) and valine (Val) were shown to predominate at positively selected site 2 in the N-terminal region, unlike selected sites 6 and 10, where polar residues are preferred. The model cystatin SlCYS8 and single variants with Leu, Ile or Val at position 2 were compared with regard to their ability to bind digestive proteases of the coleopteran pest Leptinotarsa decemlineata and to induce compensatory responses in this insect. A functional proteomics procedure to capture target Cys proteases in midgut extracts allowed confirmation of distinct binding profiles for the cystatin variants. A shotgun proteomics procedure to monitor whole Cys protease complements revealed protease family specific compensatory responses in the insect, dependent on the variant ingested. Our data confirm the contribution of closely related amino acids to the functional diversity of positively selected plant cystatins in a broader structure/function context imposing physicochemical constraints to primary structure alterations. They also underline the complexity of protease/inhibitor interactions in plant-insect systems, and the challenges still to be met in order to harness the full potential of ectopically expressed protease inhibitors in crop protection. © 2016 Federation of European Biochemical Societies.

Directed evolution of glutathione transferases towards a selective glutathione-binding site and improved oxidative stability.

PubMed

Axarli, Irine; Muleta, Abdi W; Chronopoulou, Evangelia G; Papageorgiou, Anastassios C; Labrou, Nikolaos E

2017-01-01

Glutathione transferases (GSTs) are a family of detoxification enzymes that catalyze the conjugation of glutathione (GSH) to electrophilic compounds. A library of alpha class GSTs was constructed by DNA shuffling using the DNA encoding the human glutathione transferase A1-1 (hGSTA1-1) and the rat glutathione transferase A1-1 (rGSTA1-1). Activity screening of the library allowed the selection of a chimeric enzyme variant (GSTD4) that displayed high affinity towards GSH and GSH-Sepharose affinity adsorbent, higher k cat /K m and improved thermal stability, compared to the parent enzymes. The crystal structures of the GSTD4 enzyme in free form and in complex with GSH were determined to 1.6Šand 2.3Šresolution, respectively. Analysis of the GSTD4 structure showed subtle conformational changes in the GSH-binding site and in electron-sharing network that may contribute to the increased GSH affinity. The shuffled variant GSTD4 was further optimized for improved oxidative stability employing site-saturation mutagenesis. The Cys112Ser mutation confers optimal oxidative stability and kinetic properties in the GSTD4 enzyme. DNA shuffling allowed the creation of a chimeric enzyme variant with improved properties, compared to the parent enzymes. X-ray crystallography shed light on how recombination of a specific segment from homologous GSTA1-1 together with point mutations gives rise to a new functionally competent enzyme with improved binding, catalytic properties and stability. Such an engineered GST would be useful in biotechnology as affinity tool in affinity chromatography as well as a biocatalytic matrix for the construction of biochips or enzyme biosensors. Copyright © 2016 Elsevier B.V. All rights reserved.

Constitutive turnover of histone H2A.Z at yeast promoters requires the preinitiation complex

PubMed Central

Tramantano, Michael; Sun, Lu; Au, Christy; Labuz, Daniel; Liu, Zhimin; Chou, Mindy; Shen, Chen; Luk, Ed

2016-01-01

The assembly of the preinitiation complex (PIC) occurs upstream of the +1 nucleosome which, in yeast, obstructs the transcription start site and is frequently assembled with the histone variant H2A.Z. To understand the contribution of the transcription machinery in the disassembly of the +1 H2A.Z nucleosome, conditional mutants were used to block PIC assembly. A quantitative ChIP-seq approach, which allows detection of global occupancy change, was employed to measure H2A.Z occupancy. Blocking PIC assembly resulted in promoter-specific H2A.Z accumulation, indicating that the PIC is required to evict H2A.Z. By contrast, H2A.Z eviction was unaffected upon depletion of INO80, a remodeler previously reported to displace nucleosomal H2A.Z. Robust PIC-dependent H2A.Z eviction was observed at active and infrequently transcribed genes, indicating that constitutive H2A.Z turnover is a general phenomenon. Finally, sites with strong H2A.Z turnover precisely mark transcript starts, providing a new metric for identifying cryptic and alternative sites of initiation. DOI: http://dx.doi.org/10.7554/eLife.14243.001 PMID:27438412

GETPrime: a gene- or transcript-specific primer database for quantitative real-time PCR.

PubMed

Gubelmann, Carine; Gattiker, Alexandre; Massouras, Andreas; Hens, Korneel; David, Fabrice; Decouttere, Frederik; Rougemont, Jacques; Deplancke, Bart

2011-01-01

The vast majority of genes in humans and other organisms undergo alternative splicing, yet the biological function of splice variants is still very poorly understood in large part because of the lack of simple tools that can map the expression profiles and patterns of these variants with high sensitivity. High-throughput quantitative real-time polymerase chain reaction (qPCR) is an ideal technique to accurately quantify nucleic acid sequences including splice variants. However, currently available primer design programs do not distinguish between splice variants and also differ substantially in overall quality, functionality or throughput mode. Here, we present GETPrime, a primer database supported by a novel platform that uniquely combines and automates several features critical for optimal qPCR primer design. These include the consideration of all gene splice variants to enable either gene-specific (covering the majority of splice variants) or transcript-specific (covering one splice variant) expression profiling, primer specificity validation, automated best primer pair selection according to strict criteria and graphical visualization of the latter primer pairs within their genomic context. GETPrime primers have been extensively validated experimentally, demonstrating high transcript specificity in complex samples. Thus, the free-access, user-friendly GETPrime database allows fast primer retrieval and visualization for genes or groups of genes of most common model organisms, and is available at http://updepla1srv1.epfl.ch/getprime/. Database URL: http://deplanckelab.epfl.ch.

GETPrime: a gene- or transcript-specific primer database for quantitative real-time PCR

PubMed Central

Gubelmann, Carine; Gattiker, Alexandre; Massouras, Andreas; Hens, Korneel; David, Fabrice; Decouttere, Frederik; Rougemont, Jacques; Deplancke, Bart

2011-01-01

The vast majority of genes in humans and other organisms undergo alternative splicing, yet the biological function of splice variants is still very poorly understood in large part because of the lack of simple tools that can map the expression profiles and patterns of these variants with high sensitivity. High-throughput quantitative real-time polymerase chain reaction (qPCR) is an ideal technique to accurately quantify nucleic acid sequences including splice variants. However, currently available primer design programs do not distinguish between splice variants and also differ substantially in overall quality, functionality or throughput mode. Here, we present GETPrime, a primer database supported by a novel platform that uniquely combines and automates several features critical for optimal qPCR primer design. These include the consideration of all gene splice variants to enable either gene-specific (covering the majority of splice variants) or transcript-specific (covering one splice variant) expression profiling, primer specificity validation, automated best primer pair selection according to strict criteria and graphical visualization of the latter primer pairs within their genomic context. GETPrime primers have been extensively validated experimentally, demonstrating high transcript specificity in complex samples. Thus, the free-access, user-friendly GETPrime database allows fast primer retrieval and visualization for genes or groups of genes of most common model organisms, and is available at http://updepla1srv1.epfl.ch/getprime/. Database URL: http://deplanckelab.epfl.ch. PMID:21917859

FUN-LDA: A Latent Dirichlet Allocation Model for Predicting Tissue-Specific Functional Effects of Noncoding Variation: Methods and Applications.

PubMed

Backenroth, Daniel; He, Zihuai; Kiryluk, Krzysztof; Boeva, Valentina; Pethukova, Lynn; Khurana, Ekta; Christiano, Angela; Buxbaum, Joseph D; Ionita-Laza, Iuliana

2018-05-03

We describe a method based on a latent Dirichlet allocation model for predicting functional effects of noncoding genetic variants in a cell-type- and/or tissue-specific way (FUN-LDA). Using this unsupervised approach, we predict tissue-specific functional effects for every position in the human genome in 127 different tissues and cell types. We demonstrate the usefulness of our predictions by using several validation experiments. Using eQTL data from several sources, including the GTEx project, Geuvadis project, and TwinsUK cohort, we show that eQTLs in specific tissues tend to be most enriched among the predicted functional variants in relevant tissues in Roadmap. We further show how these integrated functional scores can be used for (1) deriving the most likely cell or tissue type causally implicated for a complex trait by using summary statistics from genome-wide association studies and (2) estimating a tissue-based correlation matrix of various complex traits. We found large enrichment of heritability in functional components of relevant tissues for various complex traits, and FUN-LDA yielded higher enrichment estimates than existing methods. Finally, using experimentally validated functional variants from the literature and variants possibly implicated in disease by previous studies, we rigorously compare FUN-LDA with state-of-the-art functional annotation methods and show that FUN-LDA has better prediction accuracy and higher resolution than these methods. In particular, our results suggest that tissue- and cell-type-specific functional prediction methods tend to have substantially better prediction accuracy than organism-level prediction methods. Scores for each position in the human genome and for each ENCODE and Roadmap tissue are available online (see Web Resources). Copyright © 2018 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

Family studies to find rare high risk variants in migraine.

PubMed

Hansen, Rikke Dyhr; Christensen, Anne Francke; Olesen, Jes

2017-12-01

Migraine has long been known as a common complex disease caused by genetic and environmental factors. The pathophysiology and the specific genetic susceptibility are poorly understood. Common variants only explain a small part of the heritability of migraine. It is thought that rare genetic variants with bigger effect size may be involved in the disease. Since migraine has a tendency to cluster in families, a family approach might be the way to find these variants. This is also indicated by identification of migraine-associated loci in classical linkage-analyses in migraine families. A single migraine study using a candidate-gene approach was performed in 2010 identifying a rare mutation in the TRESK potassium channel segregating in a large family with migraine with aura, but this finding has later become questioned. The technologies of next-generation sequencing (NGS) now provides an affordable tool to investigate the genetic variation in the entire exome or genome. The family-based study design using NGS is described in this paper. We also review family studies using NGS that have been successful in finding rare variants in other common complex diseases in order to argue the promising application of a family approach to migraine. PubMed was searched to find studies that looked for rare genetic variants in common complex diseases through a family-based design using NGS, excluding studies looking for de-novo mutations, or using a candidate-gene approach and studies on cancer. All issues from Nature Genetics and PLOS genetics 2014, 2015 and 2016 (UTAI June) were screened for relevant papers. Reference lists from included and other relevant papers were also searched. For the description of the family-based study design using NGS an in-house protocol was used. Thirty-two successful studies, which covered 16 different common complex diseases, were included in this paper. We also found a single migraine study. Twenty-three studies found one or a few family specific variants (less than five), while other studies found several possible variants. Not all of them were genome wide significant. Four studies performed follow-up analyses in unrelated cases and controls and calculated odds ratios that supported an association between detected variants and risk of disease. Studies of 11 diseases identified rare variants that segregated fully or to a large degree with the disease in the pedigrees. It is possible to find rare high risk variants for common complex diseases through a family-based approach. One study using a family approach and NGS to find rare variants in migraine has already been published but with strong limitations. More studies are under way.

Phenotypic dissection of bone mineral density reveals skeletal site specificity and facilitates the identification of novel loci in the genetic regulation of bone mass attainment.

PubMed

Kemp, John P; Medina-Gomez, Carolina; Estrada, Karol; St Pourcain, Beate; Heppe, Denise H M; Warrington, Nicole M; Oei, Ling; Ring, Susan M; Kruithof, Claudia J; Timpson, Nicholas J; Wolber, Lisa E; Reppe, Sjur; Gautvik, Kaare; Grundberg, Elin; Ge, Bing; van der Eerden, Bram; van de Peppel, Jeroen; Hibbs, Matthew A; Ackert-Bicknell, Cheryl L; Choi, Kwangbom; Koller, Daniel L; Econs, Michael J; Williams, Frances M K; Foroud, Tatiana; Zillikens, M Carola; Ohlsson, Claes; Hofman, Albert; Uitterlinden, André G; Davey Smith, George; Jaddoe, Vincent W V; Tobias, Jonathan H; Rivadeneira, Fernando; Evans, David M

2014-06-01

Heritability of bone mineral density (BMD) varies across skeletal sites, reflecting different relative contributions of genetic and environmental influences. To quantify the degree to which common genetic variants tag and environmental factors influence BMD, at different sites, we estimated the genetic (rg) and residual (re) correlations between BMD measured at the upper limbs (UL-BMD), lower limbs (LL-BMD) and skull (SK-BMD), using total-body DXA scans of ∼ 4,890 participants recruited by the Avon Longitudinal Study of Parents and their Children (ALSPAC). Point estimates of rg indicated that appendicular sites have a greater proportion of shared genetic architecture (LL-/UL-BMD rg = 0.78) between them, than with the skull (UL-/SK-BMD rg = 0.58 and LL-/SK-BMD rg = 0.43). Likewise, the residual correlation between BMD at appendicular sites (r(e) = 0.55) was higher than the residual correlation between SK-BMD and BMD at appendicular sites (r(e) = 0.20-0.24). To explore the basis for the observed differences in rg and re, genome-wide association meta-analyses were performed (n ∼ 9,395), combining data from ALSPAC and the Generation R Study identifying 15 independent signals from 13 loci associated at genome-wide significant level across different skeletal regions. Results suggested that previously identified BMD-associated variants may exert site-specific effects (i.e. differ in the strength of their association and magnitude of effect across different skeletal sites). In particular, variants at CPED1 exerted a larger influence on SK-BMD and UL-BMD when compared to LL-BMD (P = 2.01 × 10(-37)), whilst variants at WNT16 influenced UL-BMD to a greater degree when compared to SK- and LL-BMD (P = 2.31 × 10(-14)). In addition, we report a novel association between RIN3 (previously associated with Paget's disease) and LL-BMD (rs754388: β = 0.13, SE = 0.02, P = 1.4 × 10(-10)). Our results suggest that BMD at different skeletal sites is under a mixture of shared and specific genetic and environmental influences. Allowing for these differences by performing genome-wide association at different skeletal sites may help uncover new genetic influences on BMD.

Phenotypic Dissection of Bone Mineral Density Reveals Skeletal Site Specificity and Facilitates the Identification of Novel Loci in the Genetic Regulation of Bone Mass Attainment

PubMed Central

Estrada, Karol; St Pourcain, Beate; Heppe, Denise H. M.; Warrington, Nicole M.; Oei, Ling; Ring, Susan M.; Kruithof, Claudia J.; Timpson, Nicholas J.; Wolber, Lisa E.; Reppe, Sjur; Gautvik, Kaare; Grundberg, Elin; Ge, Bing; van der Eerden, Bram; van de Peppel, Jeroen; Hibbs, Matthew A.; Ackert-Bicknell, Cheryl L.; Choi, Kwangbom; Koller, Daniel L.; Econs, Michael J.; Williams, Frances M. K.; Foroud, Tatiana; Carola Zillikens, M.; Ohlsson, Claes; Hofman, Albert; Uitterlinden, André G.; Davey Smith, George; Jaddoe, Vincent W. V.; Tobias, Jonathan H.; Rivadeneira, Fernando; Evans, David M.

2014-01-01

Heritability of bone mineral density (BMD) varies across skeletal sites, reflecting different relative contributions of genetic and environmental influences. To quantify the degree to which common genetic variants tag and environmental factors influence BMD, at different sites, we estimated the genetic (rg) and residual (re) correlations between BMD measured at the upper limbs (UL-BMD), lower limbs (LL-BMD) and skull (SK-BMD), using total-body DXA scans of ∼4,890 participants recruited by the Avon Longitudinal Study of Parents and their Children (ALSPAC). Point estimates of rg indicated that appendicular sites have a greater proportion of shared genetic architecture (LL-/UL-BMD rg = 0.78) between them, than with the skull (UL-/SK-BMD rg = 0.58 and LL-/SK-BMD rg = 0.43). Likewise, the residual correlation between BMD at appendicular sites (re = 0.55) was higher than the residual correlation between SK-BMD and BMD at appendicular sites (re = 0.20–0.24). To explore the basis for the observed differences in rg and re, genome-wide association meta-analyses were performed (n∼9,395), combining data from ALSPAC and the Generation R Study identifying 15 independent signals from 13 loci associated at genome-wide significant level across different skeletal regions. Results suggested that previously identified BMD-associated variants may exert site-specific effects (i.e. differ in the strength of their association and magnitude of effect across different skeletal sites). In particular, variants at CPED1 exerted a larger influence on SK-BMD and UL-BMD when compared to LL-BMD (P = 2.01×10−37), whilst variants at WNT16 influenced UL-BMD to a greater degree when compared to SK- and LL-BMD (P = 2.31×10−14). In addition, we report a novel association between RIN3 (previously associated with Paget's disease) and LL-BMD (rs754388: β = 0.13, SE = 0.02, P = 1.4×10−10). Our results suggest that BMD at different skeletal sites is under a mixture of shared and specific genetic and environmental influences. Allowing for these differences by performing genome-wide association at different skeletal sites may help uncover new genetic influences on BMD. PMID:24945404

Molecular Darwinism: The Contingency of Spontaneous Genetic Variation

PubMed Central

Arber, Werner

2011-01-01

The availability of spontaneously occurring genetic variants is an important driving force of biological evolution. Largely thanks to experimental investigations by microbial geneticists, we know today that several different molecular mechanisms contribute to the overall genetic variations. These mechanisms can be assigned to three natural strategies to generate genetic variants: 1) local sequence changes, 2) intragenomic reshuffling of DNA segments, and 3) acquisition of a segment of foreign DNA. In these processes, specific gene products are involved in cooperation with different nongenetic elements. Some genetic variations occur fully at random along the DNA filaments, others rather with a statistical reproducibility, although at many possible sites. We have to be aware that evolution in natural ecosystems is of higher complexity than under most laboratory conditions, not at least in view of symbiotic associations and the occurrence of horizontal gene transfer. The encountered contingency of genetic variation can possibly best ensure a long-term persistence of life under steadily changing living conditions. PMID:21979160

Molecular Darwinism: the contingency of spontaneous genetic variation.

PubMed

Arber, Werner

2011-01-01

The availability of spontaneously occurring genetic variants is an important driving force of biological evolution. Largely thanks to experimental investigations by microbial geneticists, we know today that several different molecular mechanisms contribute to the overall genetic variations. These mechanisms can be assigned to three natural strategies to generate genetic variants: 1) local sequence changes, 2) intragenomic reshuffling of DNA segments, and 3) acquisition of a segment of foreign DNA. In these processes, specific gene products are involved in cooperation with different nongenetic elements. Some genetic variations occur fully at random along the DNA filaments, others rather with a statistical reproducibility, although at many possible sites. We have to be aware that evolution in natural ecosystems is of higher complexity than under most laboratory conditions, not at least in view of symbiotic associations and the occurrence of horizontal gene transfer. The encountered contingency of genetic variation can possibly best ensure a long-term persistence of life under steadily changing living conditions.

Histone H1 phosphorylation is associated with transcription by RNA polymerases I and II

PubMed Central

Zheng, Yupeng; John, Sam; Pesavento, James J.; Schultz-Norton, Jennifer R.; Schiltz, R. Louis; Baek, Sonjoon; Nardulli, Ann M.; Hager, Gordon L.; Kelleher, Neil L.

2010-01-01

Histone H1 phosphorylation affects chromatin condensation and function, but little is known about how specific phosphorylations impact the function of H1 variants in higher eukaryotes. In this study, we show that specific sites in H1.2 and H1.4 of human cells are phosphorylated only during mitosis or during both mitosis and interphase. Antisera generated to individual H1.2/H1.4 interphase phosphorylations reveal that they are distributed throughout nuclei and enriched in nucleoli. Moreover, interphase phosphorylated H1.4 is enriched at active 45S preribosomal RNA gene promoters and is rapidly induced at steroid hormone response elements by hormone treatment. Our results imply that site-specific interphase H1 phosphorylation facilitates transcription by RNA polymerases I and II and has an unanticipated function in ribosome biogenesis and control of cell growth. Differences in the numbers, structure, and locations of interphase phosphorylation sites may contribute to the functional diversity of H1 variants. PMID:20439994

Genetic selection for a highly functional cysteine-less membrane protein using site-saturation mutagenesis

PubMed Central

Arendt, Cassandra S.; Ri, Keirei; Yates, Phillip A.; Ullman, Buddy

2007-01-01

We describe an efficient method for generating highly functional membrane proteins with variant amino acids at defined positions that couples a modified site-saturation strategy with functional genetic selection. We applied this method to the production of a cysteine-less variant of the Crithidia fasciculata inosine-guanosine permease CfNT2, in order to facilitate biochemical studies using thiol-specific modifying reagents. Of ten endogenous cysteine residues in CfNT2, two cannot be replaced with serine or alanine without loss of function. High-quality single- and double-mutant libraries were produced by combining a previously reported site-saturation mutagenesis scheme based on the Quikchange method with a novel gel purification step that effectively eliminated template DNA from the products. Following selection for functional complementation in S. cerevisiae cells auxotrophic for purines, several highly functional non-cysteine substitutions were efficiently identified at each desired position, allowing the construction of cysteine-less variants of CfNT2 that retained wild-type affinity for inosine. This combination of an improved site-saturation mutagenesis technique and positive genetic selection provides a simple and efficient means to identify functional and perhaps unexpected amino acid variants at a desired position. PMID:17481563

Cold-dependent alternative splicing of a Jumonji C domain-containing gene MtJMJC5 in Medicago truncatula.

PubMed

Shen, Yingfang; Wu, Xiaopei; Liu, Demei; Song, Shengjing; Liu, Dengcai; Wang, Haiqing

2016-05-27

Histone methylation is an epigenetic modification mechanism that regulates gene expression in eukaryotic cells. Jumonji C domain-containing demethylases are involved in removal of methyl groups at lysine or arginine residues. The JmjC domain-only member, JMJ30/JMJD5 of Arabidopsis, is a component of the plant circadian clock. Although some plant circadian clock genes undergo alternative splicing in response to external cues, there is no evidence that JMJ30/JMJD5 is regulated by alternative splicing. In this study, the expression of an Arabidopsis JMJ30/JMJD5 ortholog in Medicago truncatula, MtJMJC5, in response to circadian clock and abiotic stresses were characterized. The results showed that MtJMJC5 oscillates with a circadian rhythm, and undergoes cold specifically induced alternative splicing. The cold-induced alternative splicing could be reversed after ambient temperature returning to the normal. Sequencing results revealed four alternative splicing RNA isoforms including a full-length authentic protein encoding variant, and three premature termination condon-containing variants due to alternative 3' splice sites at the first and second intron. Under cold treatment, the variants that share a common 3' alternative splicing site at the second intron were intensively up-regulated while the authentic protein encoding variant and the premature termination condon-containing variant only undergoing a 3' alternative splicing at the first intron were down regulated. Although all the premature termination condon-harboring alternative splicing variants were sensitive to nonsense-mediated decay, the premature termination codon-harboring alternative splicing variants sharing the 3' alternative splicing site at the second intron showed less sensitivity than the one only containing the 3' alternative slicing site at the first intron under cold treatment. These results suggest that the cold-dependent alternative splicing of MtJMJC5 is likely a species or genus-specific mechanism of gene expression regulation on RNA levels, and might play a role in epigenetic regulation of the link between the circadian clock and ambient temperature fluctuation in Medicago. Copyright © 2016 Elsevier Inc. All rights reserved.

VarioML framework for comprehensive variation data representation and exchange.

PubMed

Byrne, Myles; Fokkema, Ivo Fac; Lancaster, Owen; Adamusiak, Tomasz; Ahonen-Bishopp, Anni; Atlan, David; Béroud, Christophe; Cornell, Michael; Dalgleish, Raymond; Devereau, Andrew; Patrinos, George P; Swertz, Morris A; Taschner, Peter Em; Thorisson, Gudmundur A; Vihinen, Mauno; Brookes, Anthony J; Muilu, Juha

2012-10-03

Sharing of data about variation and the associated phenotypes is a critical need, yet variant information can be arbitrarily complex, making a single standard vocabulary elusive and re-formatting difficult. Complex standards have proven too time-consuming to implement. The GEN2PHEN project addressed these difficulties by developing a comprehensive data model for capturing biomedical observations, Observ-OM, and building the VarioML format around it. VarioML pairs a simplified open specification for describing variants, with a toolkit for adapting the specification into one's own research workflow. Straightforward variant data can be captured, federated, and exchanged with no overhead; more complex data can be described, without loss of compatibility. The open specification enables push-button submission to gene variant databases (LSDBs) e.g., the Leiden Open Variation Database, using the Cafe Variome data publishing service, while VarioML bidirectionally transforms data between XML and web-application code formats, opening up new possibilities for open source web applications building on shared data. A Java implementation toolkit makes VarioML easily integrated into biomedical applications. VarioML is designed primarily for LSDB data submission and transfer scenarios, but can also be used as a standard variation data format for JSON and XML document databases and user interface components. VarioML is a set of tools and practices improving the availability, quality, and comprehensibility of human variation information. It enables researchers, diagnostic laboratories, and clinics to share that information with ease, clarity, and without ambiguity.

VarioML framework for comprehensive variation data representation and exchange

PubMed Central

2012-01-01

Background Sharing of data about variation and the associated phenotypes is a critical need, yet variant information can be arbitrarily complex, making a single standard vocabulary elusive and re-formatting difficult. Complex standards have proven too time-consuming to implement. Results The GEN2PHEN project addressed these difficulties by developing a comprehensive data model for capturing biomedical observations, Observ-OM, and building the VarioML format around it. VarioML pairs a simplified open specification for describing variants, with a toolkit for adapting the specification into one's own research workflow. Straightforward variant data can be captured, federated, and exchanged with no overhead; more complex data can be described, without loss of compatibility. The open specification enables push-button submission to gene variant databases (LSDBs) e.g., the Leiden Open Variation Database, using the Cafe Variome data publishing service, while VarioML bidirectionally transforms data between XML and web-application code formats, opening up new possibilities for open source web applications building on shared data. A Java implementation toolkit makes VarioML easily integrated into biomedical applications. VarioML is designed primarily for LSDB data submission and transfer scenarios, but can also be used as a standard variation data format for JSON and XML document databases and user interface components. Conclusions VarioML is a set of tools and practices improving the availability, quality, and comprehensibility of human variation information. It enables researchers, diagnostic laboratories, and clinics to share that information with ease, clarity, and without ambiguity. PMID:23031277

Innate immune activity conditions the effect of regulatory variants upon monocyte gene expression.

PubMed

Fairfax, Benjamin P; Humburg, Peter; Makino, Seiko; Naranbhai, Vivek; Wong, Daniel; Lau, Evelyn; Jostins, Luke; Plant, Katharine; Andrews, Robert; McGee, Chris; Knight, Julian C

2014-03-07

To systematically investigate the impact of immune stimulation upon regulatory variant activity, we exposed primary monocytes from 432 healthy Europeans to interferon-γ (IFN-γ) or differing durations of lipopolysaccharide and mapped expression quantitative trait loci (eQTLs). More than half of cis-eQTLs identified, involving hundreds of genes and associated pathways, are detected specifically in stimulated monocytes. Induced innate immune activity reveals multiple master regulatory trans-eQTLs including the major histocompatibility complex (MHC), coding variants altering enzyme and receptor function, an IFN-β cytokine network showing temporal specificity, and an interferon regulatory factor 2 (IRF2) transcription factor-modulated network. Induced eQTL are significantly enriched for genome-wide association study loci, identifying context-specific associations to putative causal genes including CARD9, ATM, and IRF8. Thus, applying pathophysiologically relevant immune stimuli assists resolution of functional genetic variants.

Read count-based method for high-throughput allelic genotyping of transposable elements and structural variants.

PubMed

Kuhn, Alexandre; Ong, Yao Min; Quake, Stephen R; Burkholder, William F

2015-07-08

Like other structural variants, transposable element insertions can be highly polymorphic across individuals. Their functional impact, however, remains poorly understood. Current genome-wide approaches for genotyping insertion-site polymorphisms based on targeted or whole-genome sequencing remain very expensive and can lack accuracy, hence new large-scale genotyping methods are needed. We describe a high-throughput method for genotyping transposable element insertions and other types of structural variants that can be assayed by breakpoint PCR. The method relies on next-generation sequencing of multiplex, site-specific PCR amplification products and read count-based genotype calls. We show that this method is flexible, efficient (it does not require rounds of optimization), cost-effective and highly accurate. This method can benefit a wide range of applications from the routine genotyping of animal and plant populations to the functional study of structural variants in humans.

Discovery and validation of information theory-based transcription factor and cofactor binding site motifs.

PubMed

Lu, Ruipeng; Mucaki, Eliseos J; Rogan, Peter K

2017-03-17

Data from ChIP-seq experiments can derive the genome-wide binding specificities of transcription factors (TFs) and other regulatory proteins. We analyzed 765 ENCODE ChIP-seq peak datasets of 207 human TFs with a novel motif discovery pipeline based on recursive, thresholded entropy minimization. This approach, while obviating the need to compensate for skewed nucleotide composition, distinguishes true binding motifs from noise, quantifies the strengths of individual binding sites based on computed affinity and detects adjacent cofactor binding sites that coordinate with the targets of primary, immunoprecipitated TFs. We obtained contiguous and bipartite information theory-based position weight matrices (iPWMs) for 93 sequence-specific TFs, discovered 23 cofactor motifs for 127 TFs and revealed six high-confidence novel motifs. The reliability and accuracy of these iPWMs were determined via four independent validation methods, including the detection of experimentally proven binding sites, explanation of effects of characterized SNPs, comparison with previously published motifs and statistical analyses. We also predict previously unreported TF coregulatory interactions (e.g. TF complexes). These iPWMs constitute a powerful tool for predicting the effects of sequence variants in known binding sites, performing mutation analysis on regulatory SNPs and predicting previously unrecognized binding sites and target genes. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Monitoring Ras Interactions with the Nucleotide Exchange Factor Son of Sevenless (Sos) Using Site-specific NMR Reporter Signals and Intrinsic Fluorescence.

PubMed

Vo, Uybach; Vajpai, Navratna; Flavell, Liz; Bobby, Romel; Breeze, Alexander L; Embrey, Kevin J; Golovanov, Alexander P

2016-01-22

The activity of Ras is controlled by the interconversion between GTP- and GDP-bound forms partly regulated by the binding of the guanine nucleotide exchange factor Son of Sevenless (Sos). The details of Sos binding, leading to nucleotide exchange and subsequent dissociation of the complex, are not completely understood. Here, we used uniformly (15)N-labeled Ras as well as [(13)C]methyl-Met,Ile-labeled Sos for observing site-specific details of Ras-Sos interactions in solution. Binding of various forms of Ras (loaded with GDP and mimics of GTP or nucleotide-free) at the allosteric and catalytic sites of Sos was comprehensively characterized by monitoring signal perturbations in the NMR spectra. The overall affinity of binding between these protein variants as well as their selected functional mutants was also investigated using intrinsic fluorescence. The data support a positive feedback activation of Sos by Ras·GTP with Ras·GTP binding as a substrate for the catalytic site of activated Sos more weakly than Ras·GDP, suggesting that Sos should actively promote unidirectional GDP → GTP exchange on Ras in preference of passive homonucleotide exchange. Ras·GDP weakly binds to the catalytic but not to the allosteric site of Sos. This confirms that Ras·GDP cannot properly activate Sos at the allosteric site. The novel site-specific assay described may be useful for design of drugs aimed at perturbing Ras-Sos interactions. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

DNA methylation signatures of chronic low-grade inflammation are associated with complex diseases.

PubMed

Ligthart, Symen; Marzi, Carola; Aslibekyan, Stella; Mendelson, Michael M; Conneely, Karen N; Tanaka, Toshiko; Colicino, Elena; Waite, Lindsay L; Joehanes, Roby; Guan, Weihua; Brody, Jennifer A; Elks, Cathy; Marioni, Riccardo; Jhun, Min A; Agha, Golareh; Bressler, Jan; Ward-Caviness, Cavin K; Chen, Brian H; Huan, Tianxiao; Bakulski, Kelly; Salfati, Elias L; Fiorito, Giovanni; Wahl, Simone; Schramm, Katharina; Sha, Jin; Hernandez, Dena G; Just, Allan C; Smith, Jennifer A; Sotoodehnia, Nona; Pilling, Luke C; Pankow, James S; Tsao, Phil S; Liu, Chunyu; Zhao, Wei; Guarrera, Simonetta; Michopoulos, Vasiliki J; Smith, Alicia K; Peters, Marjolein J; Melzer, David; Vokonas, Pantel; Fornage, Myriam; Prokisch, Holger; Bis, Joshua C; Chu, Audrey Y; Herder, Christian; Grallert, Harald; Yao, Chen; Shah, Sonia; McRae, Allan F; Lin, Honghuang; Horvath, Steve; Fallin, Daniele; Hofman, Albert; Wareham, Nicholas J; Wiggins, Kerri L; Feinberg, Andrew P; Starr, John M; Visscher, Peter M; Murabito, Joanne M; Kardia, Sharon L R; Absher, Devin M; Binder, Elisabeth B; Singleton, Andrew B; Bandinelli, Stefania; Peters, Annette; Waldenberger, Melanie; Matullo, Giuseppe; Schwartz, Joel D; Demerath, Ellen W; Uitterlinden, André G; van Meurs, Joyce B J; Franco, Oscar H; Chen, Yii-Der Ida; Levy, Daniel; Turner, Stephen T; Deary, Ian J; Ressler, Kerry J; Dupuis, Josée; Ferrucci, Luigi; Ong, Ken K; Assimes, Themistocles L; Boerwinkle, Eric; Koenig, Wolfgang; Arnett, Donna K; Baccarelli, Andrea A; Benjamin, Emelia J; Dehghan, Abbas

2016-12-12

Chronic low-grade inflammation reflects a subclinical immune response implicated in the pathogenesis of complex diseases. Identifying genetic loci where DNA methylation is associated with chronic low-grade inflammation may reveal novel pathways or therapeutic targets for inflammation. We performed a meta-analysis of epigenome-wide association studies (EWAS) of serum C-reactive protein (CRP), which is a sensitive marker of low-grade inflammation, in a large European population (n = 8863) and trans-ethnic replication in African Americans (n = 4111). We found differential methylation at 218 CpG sites to be associated with CRP (P < 1.15 × 10 -7 ) in the discovery panel of European ancestry and replicated (P < 2.29 × 10 -4 ) 58 CpG sites (45 unique loci) among African Americans. To further characterize the molecular and clinical relevance of the findings, we examined the association with gene expression, genetic sequence variants, and clinical outcomes. DNA methylation at nine (16%) CpG sites was associated with whole blood gene expression in cis (P < 8.47 × 10 -5 ), ten (17%) CpG sites were associated with a nearby genetic variant (P < 2.50 × 10 -3 ), and 51 (88%) were also associated with at least one related cardiometabolic entity (P < 9.58 × 10 -5 ). An additive weighted score of replicated CpG sites accounted for up to 6% inter-individual variation (R2) of age-adjusted and sex-adjusted CRP, independent of known CRP-related genetic variants. We have completed an EWAS of chronic low-grade inflammation and identified many novel genetic loci underlying inflammation that may serve as targets for the development of novel therapeutic interventions for inflammation.

Genome-wide co-localization of Polycomb orthologs and their effects on gene expression in human fibroblasts

PubMed Central

2014-01-01

Background Polycomb group proteins form multicomponent complexes that are important for establishing lineage-specific patterns of gene expression. Mammalian cells encode multiple permutations of the prototypic Polycomb repressive complex 1 (PRC1) with little evidence for functional specialization. An aim of this study is to determine whether the multiple orthologs that are co-expressed in human fibroblasts act on different target genes and whether their genomic location changes during cellular senescence. Results Deep sequencing of chromatin immunoprecipitated with antibodies against CBX6, CBX7, CBX8, RING1 and RING2 reveals that the orthologs co-localize at multiple sites. PCR-based validation at representative loci suggests that a further six PRC1 proteins have similar binding patterns. Importantly, sequential chromatin immunoprecipitation with antibodies against different orthologs implies that multiple variants of PRC1 associate with the same DNA. At many loci, the binding profiles have a distinctive architecture that is preserved in two different types of fibroblast. Conversely, there are several hundred loci at which PRC1 binding is cell type-specific and, contrary to expectations, the presence of PRC1 does not necessarily equate with transcriptional silencing. Interestingly, the PRC1 binding profiles are preserved in senescent cells despite changes in gene expression. Conclusions The multiple permutations of PRC1 in human fibroblasts congregate at common rather than specific sites in the genome and with overlapping but distinctive binding profiles in different fibroblasts. The data imply that the effects of PRC1 complexes on gene expression are more subtle than simply repressing the loci at which they bind. PMID:24485159

Functional Incompatibility between the Generic NF-κB Motif and a Subtype-Specific Sp1III Element Drives the Formation of the HIV-1 Subtype C Viral Promoter

PubMed Central

Verma, Anjali; Rajagopalan, Pavithra; Lotke, Rishikesh; Varghese, Rebu; Selvam, Deepak; Kundu, Tapas K.

2016-01-01

ABSTRACT Of the various genetic subtypes of human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2) and simian immunodeficiency virus (SIV), only in subtype C of HIV-1 is a genetically variant NF-κB binding site found at the core of the viral promoter in association with a subtype-specific Sp1III motif. How the subtype-associated variations in the core transcription factor binding sites (TFBS) influence gene expression from the viral promoter has not been examined previously. Using panels of infectious viral molecular clones, we demonstrate that subtype-specific NF-κB and Sp1III motifs have evolved for optimal gene expression, and neither of the motifs can be replaced by a corresponding TFBS variant. The variant NF-κB motif binds NF-κB with an affinity 2-fold higher than that of the generic NF-κB site. Importantly, in the context of an infectious virus, the subtype-specific Sp1III motif demonstrates a profound loss of function in association with the generic NF-κB motif. An additional substitution of the Sp1III motif fully restores viral replication, suggesting that the subtype C-specific Sp1III has evolved to function with the variant, but not generic, NF-κB motif. A change of only two base pairs in the central NF-κB motif completely suppresses viral transcription from the provirus and converts the promoter into heterochromatin refractory to tumor necrosis factor alpha (TNF-α) induction. The present work represents the first demonstration of functional incompatibility between an otherwise functional NF-κB motif and a unique Sp1 site in the context of an HIV-1 promoter. Our work provides important leads as to the evolution of the HIV-1 subtype C viral promoter with relevance for gene expression regulation and viral latency. IMPORTANCE Subtype-specific genetic variations provide a powerful tool to examine how these variations offer a replication advantage to specific viral subtypes, if any. Only in subtype C of HIV-1 are two genetically distinct transcription factor binding sites positioned at the most critical location of the viral promoter. Since a single promoter regulates viral gene expression, the promoter variations can play a critical role in determining the replication fitness of the viral strains. Our work for the first time provides a scientific explanation for the presence of a unique NF-κB binding motif in subtype C, a major HIV-1 genetic family responsible for half of the global HIV-1 infections. The results offer compelling evidence that the subtype C viral promoter not only is stronger but also is endowed with a qualitative gain-of-function advantage. The genetically variant NF-κB and the Sp1III motifs may be respond differently to specific cell signal pathways, and these mechanisms must be examined. PMID:27194770

The E2A splice variant E47 regulates the differentiation of projection neurons via p57(KIP2) during cortical development.

PubMed

Pfurr, Sabrina; Chu, Yu-Hsuan; Bohrer, Christian; Greulich, Franziska; Beattie, Robert; Mammadzada, Könül; Hils, Miriam; Arnold, Sebastian J; Taylor, Verdon; Schachtrup, Kristina; Uhlenhaut, N Henriette; Schachtrup, Christian

2017-11-01

During corticogenesis, distinct classes of neurons are born from progenitor cells located in the ventricular and subventricular zones, from where they migrate towards the pial surface to assemble into highly organized layer-specific circuits. However, the precise and coordinated transcriptional network activity defining neuronal identity is still not understood. Here, we show that genetic depletion of the basic helix-loop-helix (bHLH) transcription factor E2A splice variant E47 increased the number of Tbr1-positive deep layer and Satb2-positive upper layer neurons at E14.5, while depletion of the alternatively spliced E12 variant did not affect layer-specific neurogenesis. While ChIP-Seq identified a big overlap for E12- and E47-specific binding sites in embryonic NSCs, including sites at the cyclin-dependent kinase inhibitor (CDKI) Cdkn1c gene locus, RNA-Seq revealed a unique transcriptional regulation by each splice variant. E47 activated the expression of the CDKI Cdkn1c through binding to a distal enhancer. Finally, overexpression of E47 in embryonic NSCs in vitro impaired neurite outgrowth, and overexpression of E47 in vivo by in utero electroporation disturbed proper layer-specific neurogenesis and upregulated p57(KIP2) expression. Overall, this study identifies E2A target genes in embryonic NSCs and demonstrates that E47 regulates neuronal differentiation via p57(KIP2). © 2017. Published by The Company of Biologists Ltd.

Structure-Based Alteration of Substrate Specificity and Catalytic Activity of Sulfite Oxidase from Sulfite Oxidation to Nitrate Reduction

DOE Office of Scientific and Technical Information (OSTI.GOV)

Qiu, James A.; Wilson, Heather L.; Rajagopalan, K.V.

Eukaryotic sulfite oxidase is a dimeric protein that contains the molybdenum cofactor and catalyzes the metabolically essential conversion of sulfite to sulfate as the terminal step in the metabolism of cysteine and methionine. Nitrate reductase is an evolutionarily related molybdoprotein in lower organisms that is essential for growth on nitrate. In this study, we describe human and chicken sulfite oxidase variants in which the active site has been modified to alter substrate specificity and activity from sulfite oxidation to nitrate reduction. On the basis of sequence alignments and the known crystal structure of chicken sulfite oxidase, two residues are conservedmore » in nitrate reductases that align with residues in the active site of sulfite oxidase. On the basis of the crystal structure of yeast nitrate reductase, both positions were mutated in human sulfite oxidase and chicken sulfite oxidase. The resulting double-mutant variants demonstrated a marked decrease in sulfite oxidase activity but gained nitrate reductase activity. An additional methionine residue in the active site was proposed to be important in nitrate catalysis, and therefore, the triple variant was also produced. The nitrate reducing ability of the human sulfite oxidase triple mutant was nearly 3-fold greater than that of the double mutant. To obtain detailed structural data for the active site of these variants, we introduced the analogous mutations into chicken sulfite oxidase to perform crystallographic analysis. The crystal structures of the Mo domains of the double and triple mutants were determined to 2.4 and 2.1 {angstrom} resolution, respectively.« less

Mutations in mitochondrial complex I assembly factor NDUFAF3 cause Leigh syndrome.

PubMed

Baertling, Fabian; Sánchez-Caballero, Laura; Timal, Sharita; van den Brand, Mariël Am; Ngu, Lock Hock; Distelmaier, Felix; Rodenburg, Richard Jt; Nijtmans, Leo Gj

2017-03-01

NDUFAF3 is an assembly factor of mitochondrial respiratory chain complex I. Variants in NDUFAF3 have been identified as a cause of severe multisystem mitochondrial disease. In a patient presenting with Leigh syndrome, which has hitherto not been described as a clinical feature of NDUFAF3 deficiency, we identified a novel homozygous variant and confirmed its pathogenicity in patient fibroblasts studies. Furthermore, we present an analysis of complex I assembly routes representative of each functional module and, thereby, link NDUFAF3 to a specific step in complex I assembly. Therefore, our report expands the phenotype of NDUFAF3 deficiency and further characterizes the role of NDUFAF3 in complex I biogenesis. Copyright © 2016 Elsevier Inc. All rights reserved.

Conformational analysis of HAMLET, the folding variant of human alpha-lactalbumin associated with apoptosis.

PubMed

Casbarra, Annarita; Birolo, Leila; Infusini, Giuseppe; Dal Piaz, Fabrizio; Svensson, Malin; Pucci, Piero; Svanborg, Catharina; Marino, Gennaro

2004-05-01

A combination of hydrogen/deuterium (H/D) exchange and limited proteolysis experiments coupled to mass spectrometry analysis was used to depict the conformation in solution of HAMLET, the folding variant of human alpha-lactalbumin, complexed to oleic acid, that induces apoptosis in tumor and immature cells. Although near- and far-UV CD and fluorescence spectroscopy were not able to discriminate between HAMLET and apo-alpha-lactalbumin, H/D exchange experiments clearly showed that they correspond to two distinct conformational states, with HAMLET incorporating a greater number of deuterium atoms than the apo and holo forms. Complementary proteolysis experiments revealed that HAMLET and apo are both accessible to proteases in the beta-domain but showed substantial differences in accessibility to proteases at specific sites. The overall results indicated that the conformational changes associated with the release of Ca2+ are not sufficient to induce the HAMLET conformation. Metal depletion might represent the first event to produce a partial unfolding in the beta-domain of alpha-lactalbumin, but some more unfolding is needed to generate the active conformation HAMLET, very likely allowing the protein to bind the C18:1 fatty acid moiety. On the basis of these data, a putative binding site of the oleic acid, which stabilizes the HAMLET conformation, is proposed.

Sanger Confirmation Is Required to Achieve Optimal Sensitivity and Specificity in Next-Generation Sequencing Panel Testing.

PubMed

Mu, Wenbo; Lu, Hsiao-Mei; Chen, Jefferey; Li, Shuwei; Elliott, Aaron M

2016-11-01

Next-generation sequencing (NGS) has rapidly replaced Sanger sequencing as the method of choice for diagnostic gene-panel testing. For hereditary-cancer testing, the technical sensitivity and specificity of the assay are paramount as clinicians use results to make important clinical management and treatment decisions. There is significant debate within the diagnostics community regarding the necessity of confirming NGS variant calls by Sanger sequencing, considering that numerous laboratories report having 100% specificity from the NGS data alone. Here we report our results from 20,000 hereditary-cancer NGS panels spanning 47 genes, in which all 7845 nonpolymorphic variants were Sanger- sequenced. Of these, 98.7% were concordant between NGS and Sanger sequencing and 1.3% were identified as NGS false-positives, located mainly in complex genomic regions (A/T-rich regions, G/C-rich regions, homopolymer stretches, and pseudogene regions). Simulating a false-positive rate of zero by adjusting the variant-calling quality-score thresholds decreased the sensitivity of the assay from 100% to 97.8%, resulting in the missed detection of 176 Sanger-confirmed variants, the majority in complex genomic regions (n = 114) and mosaic mutations (n = 7). The data illustrate the importance of setting quality thresholds for panel testing only after thousands of samples have been processed and the necessity of Sanger confirmation of NGS variants to maintain the highest possible sensitivity. Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

Deep Sequencing-guided Design of a High Affinity Dual Specificity Antibody to Target Two Angiogenic Factors in Neovascular Age-related Macular Degeneration.

PubMed

Koenig, Patrick; Lee, Chingwei V; Sanowar, Sarah; Wu, Ping; Stinson, Jeremy; Harris, Seth F; Fuh, Germaine

2015-09-04

The development of dual targeting antibodies promises therapies with improved efficacy over mono-specific antibodies. Here, we engineered a Two-in-One VEGF/angiopoietin 2 antibody with dual action Fab (DAF) as a potential therapeutic for neovascular age-related macular degeneration. Crystal structures of the VEGF/angiopoietin 2 DAF in complex with its two antigens showed highly overlapping binding sites. To achieve sufficient affinity of the DAF to block both angiogenic factors, we turned to deep mutational scanning in the complementarity determining regions (CDRs). By mutating all three CDRs of each antibody chain simultaneously, we were able not only to identify affinity improving single mutations but also mutation pairs from different CDRs that synergistically improve both binding functions. Furthermore, insights into the cooperativity between mutations allowed us to identify fold-stabilizing mutations in the CDRs. The data obtained from deep mutational scanning reveal that the majority of the 52 CDR residues are utilized differently for the two antigen binding function and permit, for the first time, the engineering of several DAF variants with sub-nanomolar affinity against two structurally unrelated antigens. The improved variants show similar blocking activity of receptor binding as the high affinity mono-specific antibodies against these two proteins, demonstrating the feasibility of generating a dual specificity binding surface with comparable properties to individual high affinity mono-specific antibodies. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Bioengineered nisin derivatives with enhanced activity in complex matrices

PubMed Central

Rouse, Susan; Field, Des; Daly, Karen M.; O'Connor, Paula M.; Cotter, Paul D.; Hill, Colin; Ross, R. Paul

2012-01-01

Summary Nisin A is the best known and most extensively characterized lantibiotic. As it is ribosomally synthesized, bioengineering‐based strategies can be used to generate variants. We have previously demonstrated that bioengineering of the hinge region of nisin A can result in the generation of variants with enhanced anti‐microbial activity against Gram‐positive pathogens. Here we created a larger bank of hinge variant producers and screened for producers that exhibit enhanced bioactivity as assessed by agar‐based assays against a selection of target strains. Further analysis of 12 ‘lead’ variants reveals that in many cases enhanced bioactivity is not attributable to enhanced specific activity but is instead as a consequence of an enhanced ability to diffuse through complex polymers. In the case of two variants, which contain the residues SVA and NAK, respectively, within the hinge region, we demonstrate that this enhanced trait enables the peptides to dramatically outperform nisin A with respect to controlling Listeria monocytogenes in commercially produced chocolate milk that contains carrageenan as a stabilizer. PMID:22260415

Global impact of RNA splicing on transcriptome remodeling in the heart.

PubMed

Gao, Chen; Wang, Yibin

2012-08-01

In the eukaryotic transcriptome, both the numbers of genes and different RNA species produced by each gene contribute to the overall complexity. These RNA species are generated by the utilization of different transcriptional initiation or termination sites, or more commonly, from different messenger RNA (mRNA) splicing events. Among the 30,000+ genes in human genome, it is estimated that more than 95% of them can generate more than one gene product via alternative RNA splicing. The protein products generated from different RNA splicing variants can have different intracellular localization, activity, or tissue-distribution. Therefore, alternative RNA splicing is an important molecular process that contributes to the overall complexity of the genome and the functional specificity and diversity among different cell types. In this review, we will discuss current efforts to unravel the full complexity of the cardiac transcriptome using a deep-sequencing approach, and highlight the potential of this technology to uncover the global impact of RNA splicing on the transcriptome during development and diseases of the heart.

Characterization of a spliced variant of human IRF-3 promoter and its regulation by the transcription factor Sp1.

PubMed

Ren, Wei; Zhu, Liang-Hua; Xu, Hua-Guo; Jin, Rui; Zhou, Guo-Ping

2012-06-01

Interferon regulatory factor 3 (IRF-3), an essential transcriptional regulator of the interferon genes, plays an important role in host defense against viral and microbial infection as well as in cell growth regulation. Promoter plays a crucial role in gene transcription. We have reported the characterization of the wide type of human IRF-3 promoter, but the characterization of the spliced variant of human IRF-3 Int2V1 promoter has not been systematically analyzed. To observe the spliced variant of human IRF-3 promoter, we have cloned the human IRF-3 gene promoter region containing 300 nucleotides upstream the transcription start site (TSS). Transient transfection of 5' deleted promoter-reporter constructs and luciferase assay illustrated the region -159/-100 relative to the TSS is sufficient for full promoter activity. This region contains GATA1 and specific protein-1 (Sp1) transcription factor binding sites. Interestingly, mutation of this Sp1 site reduced the promoter activity by 50%. However, overexpression of Sp1 increased the transcription activity by 2.4-fold. These results indicated that the spliced variant of human IRF-3 gene core promoter was located within the region -159/-100 relative to the TSS. Sp1 transcription factor upregulates the spliced variant of human IRF-3 gene promoter.

Extreme Entropy-Enthalpy Compensation in a Drug Resistant Variant of HIV-1 Protease

PubMed Central

King, Nancy M.; Prabu-Jeyabalan, Moses; Bandaranayake, Rajintha M.; Nalam, Madhavi N. L.; Nalivaika, Ellen A.; Özen, Ayşegül; Haliloglu, Türkan; Yılmaz, Neşe Kurt; Schiffer, Celia A.

2012-01-01

The development of HIV-1 protease inhibitors has been the historic paradigm of rational structure-based drug design, where structural and thermodynamic analyses have assisted in the discovery of novel inhibitors. While the total enthalpy and entropy change upon binding determine the affinity, often the thermodynamics are considered in terms of inhibitor properties only. In the current study, profound changes are observed in the binding thermodynamics of a drug resistant variant compared to wild-type HIV-1 protease, irrespective of the inhibitor bound. This variant (Flap+) has a combination of flap and active site mutations and exhibits extremely large entropy-enthalpy compensation compared to wild-type protease, 5–15 kcal/mol, while losing only 1–3 kcal/mol in total binding free energy for any of six FDA approved inhibitors. Although entropy-enthalpy compensation has been previously observed for a variety of systems, never have changes of this magnitude been reported. The co-crystal structures of Flap+ protease with four of the inhibitors were determined and compared with complexes of both the wildtype protease and another drug resistant variant that does not exhibit this energetic compensation. Structural changes conserved across the Flap+ complexes, which are more pronounced for the flaps covering the active site, likely contribute to the thermodynamic compensation. The finding that drug resistant mutations can profoundly modulate the relative thermodynamic properties of a therapeutic target independent of the inhibitor presents a new challenge for rational drug design. PMID:22712830

Investigation of protein selectivity in multimodal chromatography using in silico designed Fab fragment variants.

PubMed

Karkov, Hanne Sophie; Krogh, Berit Olsen; Woo, James; Parimal, Siddharth; Ahmadian, Haleh; Cramer, Steven M

2015-11-01

In this study, a unique set of antibody Fab fragments was designed in silico and produced to examine the relationship between protein surface properties and selectivity in multimodal chromatographic systems. We hypothesized that multimodal ligands containing both hydrophobic and charged moieties would interact strongly with protein surface regions where charged groups and hydrophobic patches were in close spatial proximity. Protein surface property characterization tools were employed to identify the potential multimodal ligand binding regions on the Fab fragment of a humanized antibody and to evaluate the impact of mutations on surface charge and hydrophobicity. Twenty Fab variants were generated by site-directed mutagenesis, recombinant expression, and affinity purification. Column gradient experiments were carried out with the Fab variants in multimodal, cation-exchange, and hydrophobic interaction chromatographic systems. The results clearly indicated that selectivity in the multimodal system was different from the other chromatographic modes examined. Column retention data for the reduced charge Fab variants identified a binding site comprising light chain CDR1 as the main electrostatic interaction site for the multimodal and cation-exchange ligands. Furthermore, the multimodal ligand binding was enhanced by additional hydrophobic contributions as evident from the results obtained with hydrophobic Fab variants. The use of in silico protein surface property analyses combined with molecular biology techniques, protein expression, and chromatographic evaluations represents a previously undescribed and powerful approach for investigating multimodal selectivity with complex biomolecules. © 2015 Wiley Periodicals, Inc.

Centromere reference models for human chromosomes X and Y satellite arrays

PubMed Central

Miga, Karen H.; Newton, Yulia; Jain, Miten; Altemose, Nicolas; Willard, Huntington F.; Kent, W. James

2014-01-01

The human genome sequence remains incomplete, with multimegabase-sized gaps representing the endogenous centromeres and other heterochromatic regions. Available sequence-based studies within these sites in the genome have demonstrated a role in centromere function and chromosome pairing, necessary to ensure proper chromosome segregation during cell division. A common genomic feature of these regions is the enrichment of long arrays of near-identical tandem repeats, known as satellite DNAs, which offer a limited number of variant sites to differentiate individual repeat copies across millions of bases. This substantial sequence homogeneity challenges available assembly strategies and, as a result, centromeric regions are omitted from ongoing genomic studies. To address this problem, we utilize monomer sequence and ordering information obtained from whole-genome shotgun reads to model two haploid human satellite arrays on chromosomes X and Y, resulting in an initial characterization of 3.83 Mb of centromeric DNA within an individual genome. To further expand the utility of each centromeric reference sequence model, we evaluate sites within the arrays for short-read mappability and chromosome specificity. Because satellite DNAs evolve in a concerted manner, we use these centromeric assemblies to assess the extent of sequence variation among 366 individuals from distinct human populations. We thus identify two satellite array variants in both X and Y centromeres, as determined by array length and sequence composition. This study provides an initial sequence characterization of a regional centromere and establishes a foundation to extend genomic characterization to these sites as well as to other repeat-rich regions within complex genomes. PMID:24501022

STATIC AND KINETIC SITE-SPECIFIC PROTEIN-DNA PHOTOCROSSLINKING: ANALYSIS OF BACTERIAL TRANSCRIPTION INITIATION COMPLEXES

PubMed Central

Naryshkin, Nikolai; Druzhinin, Sergei; Revyakin, Andrei; Kim, Younggyu; Mekler, Vladimir; Ebright, Richard H.

2009-01-01

Static site-specific protein-DNA photocrosslinking permits identification of protein-DNA interactions within multiprotein-DNA complexes. Kinetic site-specific protein-DNA photocrosslinking--involving rapid-quench-flow mixing and pulsed-laser irradiation--permits elucidation of pathways and kinetics of formation of protein-DNA interactions within multiprotein-DNA complexes. We present detailed protocols for application of static and kinetic site-specific protein-DNA photocrosslinking to bacterial transcription initiation complexes. PMID:19378179

Specific insertions of zinc finger domains into Gag-Pol yield engineered retroviral vectors with selective integration properties

PubMed Central

Lim, Kwang-il; Klimczak, Ryan; Yu, Julie H.; Schaffer, David V.

2010-01-01

Retroviral vectors offer benefits of efficient delivery and stable gene expression; however, their clinical use raises the concerns of insertional mutagenesis and potential oncogenesis due to genomic integration preferences in transcriptional start sites (TSS). We have shifted the integration preferences of retroviral vectors by generating a library of viral variants with a DNA-binding domain inserted at random positions throughout murine leukemia virus Gag-Pol, then selecting for variants that are viable and exhibit altered integration properties. We found seven permissive zinc finger domain (ZFD) insertion sites throughout Gag-Pol, including within p12, reverse transcriptase, and integrase. Comprehensive genome integration analysis showed that several ZFD insertions yielded retroviral vector variants with shifted integration patterns that did not favor TSS. Furthermore, integration site analysis revealed selective integration for numerous mutants. For example, two retroviral variants with a given ZFD at appropriate positions in Gag-Pol strikingly integrated primarily into four common sites out of 3.1 × 109 possible human genome locations (P = 4.6 × 10-29). Our findings demonstrate that insertion of DNA-binding motifs into multiple locations in Gag-Pol can make considerable progress toward engineering safer retroviral vectors that integrate into a significantly narrowed pool of sites on human genome and overcome the preference for TSS. PMID:20616052

Integrative analysis of omics summary data reveals putative mechanisms underlying complex traits.

PubMed

Wu, Yang; Zeng, Jian; Zhang, Futao; Zhu, Zhihong; Qi, Ting; Zheng, Zhili; Lloyd-Jones, Luke R; Marioni, Riccardo E; Martin, Nicholas G; Montgomery, Grant W; Deary, Ian J; Wray, Naomi R; Visscher, Peter M; McRae, Allan F; Yang, Jian

2018-03-02

The identification of genes and regulatory elements underlying the associations discovered by GWAS is essential to understanding the aetiology of complex traits (including diseases). Here, we demonstrate an analytical paradigm of prioritizing genes and regulatory elements at GWAS loci for follow-up functional studies. We perform an integrative analysis that uses summary-level SNP data from multi-omics studies to detect DNA methylation (DNAm) sites associated with gene expression and phenotype through shared genetic effects (i.e., pleiotropy). We identify pleiotropic associations between 7858 DNAm sites and 2733 genes. These DNAm sites are enriched in enhancers and promoters, and >40% of them are mapped to distal genes. Further pleiotropic association analyses, which link both the methylome and transcriptome to 12 complex traits, identify 149 DNAm sites and 66 genes, indicating a plausible mechanism whereby the effect of a genetic variant on phenotype is mediated by genetic regulation of transcription through DNAm.

Genetics and Genomics of Single-Gene Cardiovascular Diseases: Common Hereditary Cardiomyopathies as Prototypes of Single-Gene Disorders

PubMed Central

Marian, Ali J.; van Rooij, Eva; Roberts, Robert

2016-01-01

This is the first of 2 review papers on genetics and genomics appearing as part of the series on “omics.” Genomics pertains to all components of an organism’s genes, whereas genetics involves analysis of a specific gene(s) in the context of heredity. The paper provides introductory comments, describes the basis of human genetic diversity, and addresses the phenotypic consequences of genetic variants. Rare variants with large effect sizes are responsible for single-gene disorders, whereas complex polygenic diseases are typically due to multiple genetic variants, each exerting a modest effect size. To illustrate the clinical implications of genetic variants with large effect sizes, 3 common forms of hereditary cardiomyopathies are discussed as prototypic examples of single-gene disorders, including their genetics, clinical manifestations, pathogenesis, and treatment. The genetic basis of complex traits is discussed in a separate paper. PMID:28007145

Heterozygous KIDINS220/ARMS nonsense variants cause spastic paraplegia, intellectual disability, nystagmus, and obesity.

PubMed

Josifova, Dragana J; Monroe, Glen R; Tessadori, Federico; de Graaff, Esther; van der Zwaag, Bert; Mehta, Sarju G; Harakalova, Magdalena; Duran, Karen J; Savelberg, Sanne M C; Nijman, Isaäc J; Jungbluth, Heinz; Hoogenraad, Casper C; Bakkers, Jeroen; Knoers, Nine V; Firth, Helen V; Beales, Philip L; van Haaften, Gijs; van Haelst, Mieke M

2016-06-01

We identified de novo nonsense variants in KIDINS220/ARMS in three unrelated patients with spastic paraplegia, intellectual disability, nystagmus, and obesity (SINO). KIDINS220 is an essential scaffold protein coordinating neurotrophin signal pathways in neurites and is spatially and temporally regulated in the brain. Molecular analysis of patients' variants confirmed expression and translation of truncated transcripts similar to recently characterized alternative terminal exon splice isoforms of KIDINS220 KIDINS220 undergoes extensive alternative splicing in specific neuronal populations and developmental time points, reflecting its complex role in neuronal maturation. In mice and humans, KIDINS220 is alternative spliced in the middle region as well as in the last exon. These full-length and KIDINS220 splice variants occur at precise moments in cortical, hippocampal, and motor neuron development, with splice variants similar to the variants seen in our patients and lacking the last exon of KIDINS220 occurring in adult rather than in embryonic brain. We conducted tissue-specific expression studies in zebrafish that resulted in spasms, confirming a functional link with disruption of the KIDINS220 levels in developing neurites. This work reveals a crucial physiological role of KIDINS220 in development and provides insight into how perturbation of the complex interplay of KIDINS220 isoforms and their relative expression can affect neuron control and human metabolism. Altogether, we here show that de novo protein-truncating KIDINS220 variants cause a new syndrome, SINO. This is the first report of KIDINS220 variants causing a human disease. © The Author 2016. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Expression, purification and characterisation of two variant cysteine peptidases from Trypanosoma congolense with active site substitutions.

PubMed

Pillay, Davita; Boulangé, Alain F; Coetzer, Theresa H T

2010-12-01

Congopain, the major cysteine peptidase of Trypanosoma congolense is an attractive candidate for an anti-disease vaccine and target for the design of specific inhibitors. A complicating factor for the inclusion of congopain in a vaccine is that multiple variants of congopain are present in the genome of the parasite. In order to determine whether the variant congopain-like genes code for peptidases with enzymatic activities different to those of congopain, two variants were cloned and expressed. Two truncated catalytic domain variants were recombinantly expressed in Pichia pastoris. The two expressed catalytic domain variants differed slightly from one another in substrate preferences and also from that of C2 (the recombinant truncated form of congopain). Surprisingly, a variant with the catalytic triad Ser(25), His(159) and Asn(175) was shown to be active against classical cysteine peptidase substrates and inhibited by E-64, a class-specific cysteine protease inhibitor. Both catalytic domain clones and C2 had pH optima of either 6.0 or 6.5 implying that these congopain-like proteases are likely to be expressed and active in the bloodstream of the host animal. Copyright © 2010 Elsevier Inc. All rights reserved.

Structural basis for recognition of centromere histone variant CenH3 by the chaperone Scm3

PubMed Central

Zhou, Zheng; Feng, Hanqiao; Zhou, Bing-Rui; Ghirlando, Rodolfo; Hu, Kaifeng; Zwolak, Adam; Miller Jenkins, Lisa M.; Xiao, Hua; Tjandra, Nico; Wu, Carl; Bai, Yawen

2011-01-01

The centromere is a unique chromosomal locus that ensures accurate segregation of chromosomes during cell division by directing the assembly of a multiprotein complex, the kinetochore1. The centromere is marked by a conserved variant of conventional histone H3 termed CenH3 or CENP-A2. A conserved motif of CenH3, the CATD, defined by loop 1 and helix 2 of the histone fold, is necessary and sufficient for specifying centromere functions of CenH33, 4. The structural basis of this specification is of outstanding interest. Yeast Scm3 and human HJURP are conserved nonhistone proteins that interact physically with the (CenH3-H4)2 heterotetramer and are required for the deposition of CenH3 at centromeres in vivo5, 6, 7, 8, 9, 10, 11, 12, 13. Here we have elucidated the structural basis for recognition of budding yeast CenH3 (Cse4) by Scm3. We solved the structure of the Cse4-binding domain (CBD) of Scm3 complexed with Cse4 and H4 in a single chain model. An α-helix and an irregular loop at the conserved N-terminus and a shorter α-helix at the C-terminus of Scm3-CBD wraps around the Cse4-H4 dimer. Four Cse4-specific residues in the N-terminal region of helix 2 are sufficient for specific recognition by conserved and functionally important residues in the N-terminal helix of Scm3 through formation of a hydrophobic cluster. Scm3-CBD induces major conformational changes and sterically occludes DNA binding sites in the structure of Cse4 and H4. These findings have implications for the assembly and architecture of the centromeric nucleosome. PMID:21412236

SNPs located at CpG sites modulate genome-epigenome interaction

USDA-ARS?s Scientific Manuscript database

DNA methylation is an important molecular-level phenotype that links genotypes and complex disease traits. Previous studies have found local correlation between genetic variants and DNA methylation levels (cis-meQTLs). However, general mechanisms underlying cis-meQTLs are unclear. We conducted a cis...

Differences in N-linked glycosylation between human surfactant protein-B variants of the C or T allele at the single-nucleotide polymorphism at position 1580: implications for disease.

PubMed Central

Wang, Guirong; Christensen, Neil D; Wigdahl, Brian; Guttentag, Susan H; Floros, Joanna

2003-01-01

Human surfactant protein-B (SP-B), a hydrophobic protein, is essential for normal lung function. SP-B is expressed and secreted by specific lung cell types, i.e. alveolar type II and Clara cells, of the respiratory epithelium. The SP-B precursor (42 kDa) undergoes post-translational processing to generate an 8 kDa mature SP-B. A single-nucleotide polymorphism (SNP) at nucleotide 1580 (C/T) in exon 4 of SP-B that changes amino acid 131 from threonine to isoleucine (Thr131-->Ile) is associated with several pulmonary diseases. The Thr131-->Ile substitution can eliminate a potential N-linked glycosylation site, Asn129-Gln-Thr131, which is present in the SP-B variant of the C allele (ACT/Thr) but not in that of the T allele (ATT/Ile). To determine whether the C allele SP-B variant is indeed glycosylated at Asn(129)-Gln-Thr131, we first generated stably transfected Chinese hamster ovary cell lines that expressed each version of SP-B, and developed specific SP-B polyclonal anti-peptide antibodies. Using both the stably transfected cell lines and fetal lung explants, we observed that the C allele variant is indeed glycosylated at the Asn129-Gln-Thr131 site, whereas the T allele variant, which served as a control, is not. In addition, we also confirmed that both SP-B variants contain another N-linked glycosylation site, Asn311-Ser-Ser313. Given its association with several pulmonary diseases, this finding provides useful information for future studies in disease systems associated with this SNP. Further, we speculate that, given the fact that this SNP is found frequently in the general population, N-linked glycosylation at residue Asn129 interferes with SP-B processing, secretion and folding under certain disease conditions. PMID:12356334

Candidate Sequence Variants and Fetal Hemoglobin in Children with Sickle Cell Disease Treated with Hydroxyurea

PubMed Central

Green, Nancy S.; Ender, Katherine L.; Pashankar, Farzana; Driscoll, Catherine; Giardina, Patricia J.; Mullen, Craig A.; Clark, Lorraine N.; Manwani, Deepa; Crotty, Jennifer; Kisselev, Sergey; Neville, Kathleen A.; Hoppe, Carolyn; Barral, Sandra

2013-01-01

Background Fetal hemoglobin level is a heritable complex trait that strongly correlates swith the clinical severity of sickle cell disease. Only few genetic loci have been identified as robustly associated with fetal hemoglobin in patients with sickle cell disease, primarily adults. The sole approved pharmacologic therapy for this disease is hydroxyurea, with effects largely attributable to induction of fetal hemoglobin. Methodology/Principal Findings In a multi-site observational analysis of children with sickle cell disease, candidate single nucleotide polymorphisms associated with baseline fetal hemoglobin levels in adult sickle cell disease were examined in children at baseline and induced by hydroxyurea therapy. For baseline levels, single marker analysis demonstrated significant association with BCL11A and the beta and epsilon globin loci (HBB and HBE, respectively), with an additive attributable variance from these loci of 23%. Among a subset of children on hydroxyurea, baseline fetal hemoglobin levels explained 33% of the variance in induced levels. The variant in HBE accounted for an additional 13% of the variance in induced levels, while variants in the HBB and BCL11A loci did not contribute beyond baseline levels. Conclusions/Significance These findings clarify the overlap between baseline and hydroxyurea-induced fetal hemoglobin levels in pediatric disease. Studies assessing influences of specific sequence variants in these and other genetic loci in larger populations and in unusual hydroxyurea responders are needed to further understand the maintenance and therapeutic induction of fetal hemoglobin in pediatric sickle cell disease. PMID:23409025

Investigating the Structural Impacts of I64T and P311S Mutations in APE1-DNA Complex: A Molecular Dynamics Approach

PubMed Central

Doss, C. George Priya; NagaSundaram, N.

2012-01-01

Background Elucidating the molecular dynamic behavior of Protein-DNA complex upon mutation is crucial in current genomics. Molecular dynamics approach reveals the changes on incorporation of variants that dictate the structure and function of Protein-DNA complexes. Deleterious mutations in APE1 protein modify the physicochemical property of amino acids that affect the protein stability and dynamic behavior. Further, these mutations disrupt the binding sites and prohibit the protein to form complexes with its interacting DNA. Principal Findings In this study, we developed a rapid and cost-effective method to analyze variants in APE1 gene that are associated with disease susceptibility and evaluated their impacts on APE1-DNA complex dynamic behavior. Initially, two different in silico approaches were used to identify deleterious variants in APE1 gene. Deleterious scores that overlap in these approaches were taken in concern and based on it, two nsSNPs with IDs rs61730854 (I64T) and rs1803120 (P311S) were taken further for structural analysis. Significance Different parameters such as RMSD, RMSF, salt bridge, H-bonds and SASA applied in Molecular dynamic study reveals that predicted deleterious variants I64T and P311S alters the structure as well as affect the stability of APE1-DNA interacting functions. This study addresses such new methods for validating functional polymorphisms of human APE1 which is critically involved in causing deficit in repair capacity, which in turn leads to genetic instability and carcinogenesis. PMID:22384055

Directed evolution to re-adapt a co-evolved network within an enzyme.

PubMed

Strafford, John; Payongsri, Panwajee; Hibbert, Edward G; Morris, Phattaraporn; Batth, Sukhjeet S; Steadman, David; Smith, Mark E B; Ward, John M; Hailes, Helen C; Dalby, Paul A

2012-01-01

We have previously used targeted active-site saturation mutagenesis to identify a number of transketolase single mutants that improved activity towards either glycolaldehyde (GA), or the non-natural substrate propionaldehyde (PA). Here, all attempts to recombine the singles into double mutants led to unexpected losses of specific activity towards both substrates. A typical trade-off occurred between soluble expression levels and specific activity for all single mutants, but many double mutants decreased both properties more severely suggesting a critical loss of protein stability or native folding. Statistical coupling analysis (SCA) of a large multiple sequence alignment revealed a network of nine co-evolved residues that affected all but one double mutant. Such networks maintain important functional properties such as activity, specificity, folding, stability, and solubility and may be rapidly disrupted by introducing one or more non-naturally occurring mutations. To identify variants of this network that would accept and improve upon our best D469 mutants for activity towards PA, we created a library of random single, double and triple mutants across seven of the co-evolved residues, combining our D469 variants with only naturally occurring mutations at the remaining sites. A triple mutant cluster at D469, E498 and R520 was found to behave synergistically for the specific activity towards PA. Protein expression was severely reduced by E498D and improved by R520Q, yet variants containing both mutations led to improved specific activity and enzyme expression, but with loss of solubility and the formation of inclusion bodies. D469S and R520Q combined synergistically to improve k(cat) 20-fold for PA, more than for any previous transketolase mutant. R520Q also doubled the specific activity of the previously identified D469T to create our most active transketolase mutant to date. Our results show that recombining active-site mutants obtained by saturation mutagenesis can rapidly destabilise critical networks of co-evolved residues, whereas beneficial single mutants can be retained and improved upon by randomly recombining them with natural variants at other positions in the network. Copyright © 2011 Elsevier B.V. All rights reserved.

The role of water molecules in stereoselectivity of glucose/galactose-binding protein

NASA Astrophysics Data System (ADS)

Kim, Minsup; Cho, Art E.

2016-11-01

Using molecular dynamics (MD) simulation methods, we attempted to explain the experimental results on ligand specificity of glucose/galactose-binding protein (GGBP) to β-D-glucose and β-D-galactose. For the simulation, a three-dimensional structure of GGBP was prepared, and homology modeling was performed to generate variant structures of GGBP with mutations at Asp14. Then, docking was carried out to find a reasonable β-D-glucose and β-D-galactose binding conformations with GGBP. Subsequent molecular dynamics simulations of β-D-glucose-GGBP and β-D-galactose-GGBP complexes and estimation of the orientation and stability of water molecules at the binding site revealed how water molecules influence ligand specificity. In our simulation, water molecules mediated interactions of β-D-glucose or β-D-galactose with residue 14 of GGBP. In this mechanism, the Phe16Ala mutant leaves both sugar molecules free to move, and the specific role of water molecules were eliminated, while the wild type, Asp14Asn mutant, and Asp14Glu mutant make hydrogen bond interactions with β-D-glucose more favorable. Our results demonstrate that bound water molecules at the binding site of GGBP are related to localized conformational change, contributing to ligand specificity of GGBP for β-D-glucose over β-D-galactose.

Structural and functional insight into the carbohydrate receptor binding of F4 fimbriae-producing enterotoxigenic Escherichia coli.

PubMed

Moonens, Kristof; Van den Broeck, Imke; De Kerpel, Maia; Deboeck, Francine; Raymaekers, Hanne; Remaut, Han; De Greve, Henri

2015-03-27

Enterotoxigenic Escherichia coli (ETEC) strains are important causes of intestinal disease in humans and lead to severe production losses in animal farming. A range of fimbrial adhesins in ETEC strains determines host and tissue tropism. ETEC strains expressing F4 fimbriae are associated with neonatal and post-weaning diarrhea in piglets. Three naturally occurring variants of F4 fimbriae (F4ab, F4ac, and F4ad) exist that differ in the primary sequence of their major adhesive subunit FaeG, and each features a related yet distinct receptor binding profile. Here the x-ray structure of FaeGad bound to lactose provides the first structural insight into the receptor specificity and mode of binding by the poly-adhesive F4 fimbriae. A small D'-D″-α1-α2 subdomain grafted on the immunoglobulin-like core of FaeG hosts the carbohydrate binding site. Two short amino acid stretches Phe(150)-Glu(152) and Val(166)-Glu(170) of FaeGad bind the terminal galactose in the lactosyl unit and provide affinity and specificity to the interaction. A hemagglutination-based assay with E. coli expressing mutant F4ad fimbriae confirmed the elucidated co-complex structure. Interestingly, the crucial D'-α1 loop that borders the FaeGad binding site adopts a different conformation in the two other FaeG variants and hints at a heterogeneous binding pocket among the FaeG serotypes. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

DBATE: database of alternative transcripts expression.

PubMed

Bianchi, Valerio; Colantoni, Alessio; Calderone, Alberto; Ausiello, Gabriele; Ferrè, Fabrizio; Helmer-Citterich, Manuela

2013-01-01

The use of high-throughput RNA sequencing technology (RNA-seq) allows whole transcriptome analysis, providing an unbiased and unabridged view of alternative transcript expression. Coupling splicing variant-specific expression with its functional inference is still an open and difficult issue for which we created the DataBase of Alternative Transcripts Expression (DBATE), a web-based repository storing expression values and functional annotation of alternative splicing variants. We processed 13 large RNA-seq panels from human healthy tissues and in disease conditions, reporting expression levels and functional annotations gathered and integrated from different sources for each splicing variant, using a variant-specific annotation transfer pipeline. The possibility to perform complex queries by cross-referencing different functional annotations permits the retrieval of desired subsets of splicing variant expression values that can be visualized in several ways, from simple to more informative. DBATE is intended as a novel tool to help appreciate how, and possibly why, the transcriptome expression is shaped. DATABASE URL: http://bioinformatica.uniroma2.it/DBATE/.

Crystallographic analysis of 1,2,3-trichloropropane biodegradation by the haloalkane dehalogenase DhaA31.

PubMed

Lahoda, Maryna; Mesters, Jeroen R; Stsiapanava, Alena; Chaloupkova, Radka; Kuty, Michal; Damborsky, Jiri; Kuta Smatanova, Ivana

2014-02-01

Haloalkane dehalogenases catalyze the hydrolytic cleavage of carbon-halogen bonds, which is a key step in the aerobic mineralization of many environmental pollutants. One important pollutant is the toxic and anthropogenic compound 1,2,3-trichloropropane (TCP). Rational design was combined with saturation mutagenesis to obtain the haloalkane dehalogenase variant DhaA31, which displays an increased catalytic activity towards TCP. Here, the 1.31 Å resolution crystal structure of substrate-free DhaA31, the 1.26 Å resolution structure of DhaA31 in complex with TCP and the 1.95 Å resolution structure of wild-type DhaA are reported. Crystals of the enzyme-substrate complex were successfully obtained by adding volatile TCP to the reservoir after crystallization at pH 6.5 and room temperature. Comparison of the substrate-free structure with that of the DhaA31 enzyme-substrate complex reveals that the nucleophilic Asp106 changes its conformation from an inactive to an active state during the catalytic cycle. The positions of three chloride ions found inside the active site of the enzyme indicate a possible pathway for halide release from the active site through the main tunnel. Comparison of the DhaA31 variant with wild-type DhaA revealed that the introduced substitutions reduce the volume and the solvent-accessibility of the active-site pocket.

Removing the Active-Site Flap in Lipase A from Candida antarctica Produces a Functional Enzyme without Interfacial Activation.

PubMed

Wikmark, Ylva; Engelmark Cassimjee, Karim; Lihammar, Richard; Bäckvall, Jan-E

2016-01-01

A mobile region is proposed to be a flap that covers the active site of Candida antarctica lipase A. Removal of the mobile region retains the functional properties of the enzyme. Interestingly interfacial activation, required for the wild-type enzyme, was not observed for the truncated variant, although stability, activity, and stereoselectivity were very similar for the wild-type and variant enzymes. The variant followed classical Michaelis-Menten kinetics, unlike the wild type. Both gave the same relative specificity in the transacylation of a primary and a secondary alcohol in organic solvent. Furthermore, both showed the same enantioselectivity in transacylation of alcohols and the hydrolysis of alcohol esters, as well as in the hydrolysis of esters chiral at the acid part. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Structure and dynamics of mesophilic variants from the homing endonuclease I-DmoI

NASA Astrophysics Data System (ADS)

Alba, Josephine; Marcaida, Maria Jose; Prieto, Jesus; Montoya, Guillermo; Molina, Rafael; D'Abramo, Marco

2017-12-01

I-DmoI, from the hyperthermophilic archaeon Desulfurococcus mobilis, belongs to the LAGLIDADG homing endonuclease protein family. Its members are highly specific enzymes capable of recognizing long DNA target sequences, thus providing potential tools for genome manipulation. Working towards this particular application, many efforts have been made to generate mesophilic variants of I-DmoI that function at lower temperatures than the wild-type. Here, we report a structural and computational analysis of two I-DmoI mesophilic mutants. Despite very limited structural variations between the crystal structures of these variants and the wild-type, a different dynamical behaviour near the cleavage sites is observed. In particular, both the dynamics of the water molecules and the protein perturbation effect on the cleavage site correlate well with the changes observed in the experimental enzymatic activity.

Regulatory polymorphisms modulate the expression of HLA class II molecules and promote autoimmunity

PubMed Central

Raj, Prithvi; Rai, Ekta; Song, Ran; Khan, Shaheen; Wakeland, Benjamin E; Viswanathan, Kasthuribai; Arana, Carlos; Liang, Chaoying; Zhang, Bo; Dozmorov, Igor; Carr-Johnson, Ferdicia; Mitrovic, Mitja; Wiley, Graham B; Kelly, Jennifer A; Lauwerys, Bernard R; Olsen, Nancy J; Cotsapas, Chris; Garcia, Christine K; Wise, Carol A; Harley, John B; Nath, Swapan K; James, Judith A; Jacob, Chaim O; Tsao, Betty P; Pasare, Chandrashekhar; Karp, David R; Li, Quan Zhen; Gaffney, Patrick M; Wakeland, Edward K

2016-01-01

Targeted sequencing of sixteen SLE risk loci among 1349 Caucasian cases and controls produced a comprehensive dataset of the variations causing susceptibility to systemic lupus erythematosus (SLE). Two independent disease association signals in the HLA-D region identified two regulatory regions containing 3562 polymorphisms that modified thirty-seven transcription factor binding sites. These extensive functional variations are a new and potent facet of HLA polymorphism. Variations modifying the consensus binding motifs of IRF4 and CTCF in the XL9 regulatory complex modified the transcription of HLA-DRB1, HLA-DQA1 and HLA-DQB1 in a chromosome-specific manner, resulting in a 2.5-fold increase in the surface expression of HLA-DR and DQ molecules on dendritic cells with SLE risk genotypes, which increases to over 4-fold after stimulation. Similar analyses of fifteen other SLE risk loci identified 1206 functional variants tightly linked with disease-associated SNPs and demonstrated that common disease alleles contain multiple causal variants modulating multiple immune system genes. DOI: http://dx.doi.org/10.7554/eLife.12089.001 PMID:26880555

Mutation load in melanoma is affected by MC1R genotype.

PubMed

Johansson, Peter A; Pritchard, Antonia L; Patch, Ann-Marie; Wilmott, James S; Pearson, John V; Waddell, Nicola; Scolyer, Richard A; Mann, Graham J; Hayward, Nicholas K

2017-03-01

Whole-genome sequencing of matched germline and tumour pairs in a well-characterized cohort of melanoma patients allowed investigation of associations between melanoma body site, age at melanoma onset and MC1R variant status with overall mutation burden and specific base pair changes observed in the corresponding melanoma. We observed statistically significant associations between mutation burden in melanoma and body site, age at onset and MC1R genotype, for both ultraviolet radiation (UVR) signature changes (C>T and CC>TT) and non-UVR base pair substitutions, as well as with overall variant load. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Cavβ2 transcription start site variants modulate calcium handling in newborn rat cardiomyocytes.

PubMed

Moreno, Cristian; Hermosilla, Tamara; Morales, Danna; Encina, Matías; Torres-Díaz, Leandro; Díaz, Pablo; Sarmiento, Daniela; Simon, Felipe; Varela, Diego

2015-12-01

In the heart, the main pathway for calcium influx is mediated by L-type calcium channels, a multi-subunit complex composed of the pore-forming subunit CaV1.2 and the auxiliary subunits CaVα2δ1 and CaVβ2. To date, five distinct CaVβ2 transcriptional start site (TSS) variants (CaVβ2a-e) varying only in the composition and length of the N-terminal domain have been described, each of them granting distinct biophysical properties to the L-type current. However, the physiological role of these variants in Ca(2+) handling in the native tissue has not been explored. Our results show that four of these variants are present in neonatal rat cardiomyocytes. The contribution of those CaVβ2 TSS variants on endogenous L-type current and Ca(2+) handling was explored by adenoviral-mediated overexpression of each CaVβ2 variant in cultured newborn rat cardiomyocytes. As expected, all CaVβ2 TSS variants increased L-type current density and produced distinctive changes on L-type calcium channel (LTCC) current activation and inactivation kinetics. The characteristics of the induced calcium transients were dependent on the TSS variant overexpressed. Moreover, the amplitude of the calcium transients varied depending on the subunit involved, being higher in cardiomyocytes transduced with CaVβ2a and smaller in CaVβ2d. Interestingly, the contribution of Ca(2+) influx and Ca(2+) release on total calcium transients, as well as the sarcoplasmic calcium content, was found to be TSS-variant-dependent. Remarkably, determination of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) messenger RNA (mRNA) abundance and cell size change indicates that CaVβ2 TSS variants modulate the cardiomyocyte hypertrophic state. In summary, we demonstrate that expression of individual CaVβ2 TSS variants regulates calcium handling in cardiomyocytes and, consequently, has significant repercussion in the development of hypertrophy.

New CD20 alternative splice variants: molecular identification and differential expression within hematological B cell malignancies.

PubMed

Gamonet, Clémentine; Bole-Richard, Elodie; Delherme, Aurélia; Aubin, François; Toussirot, Eric; Garnache-Ottou, Francine; Godet, Yann; Ysebaert, Loïc; Tournilhac, Olivier; Caroline, Dartigeas; Larosa, Fabrice; Deconinck, Eric; Saas, Philippe; Borg, Christophe; Deschamps, Marina; Ferrand, Christophe

2015-01-01

CD20 is a B cell lineage-specific marker expressed by normal and leukemic B cells and targeted by several antibody immunotherapies. We have previously shown that the protein from a CD20 mRNA splice variant (D393-CD20) is expressed at various levels in leukemic B cells or lymphoma B cells but not in resting, sorted B cells from the peripheral blood of healthy donors. Western blot (WB) analysis of B malignancy primary samples showed additional CD20 signals. Deep molecular PCR analysis revealed four new sequences corresponding to in-frame CD20 splice variants (D657-CD20, D618-CD20, D480-CD20, and D177-CD20) matching the length of WB signals. We demonstrated that the cell spliceosome machinery can process ex vivo D480-, D657-, and D618-CD20 transcript variants by involving canonical sites associated with cryptic splice sites. Results of specific and quantitative RT-PCR assays showed that these CD20 splice variants are differentially expressed in B malignancies. Moreover, Epstein-Barr virus (EBV) transformation modified the CD20 splicing profile and mainly increased the D393-CD20 variant transcripts. Finally, investigation of three cohorts of chronic lymphocytic leukemia (CLL) patients showed that the total CD20 splice variant expression was higher in a stage B and C sample collection compared to routinely collected CLL samples or relapsed refractory stage A, B, or C CLL. The involvement of these newly discovered alternative CD20 transcript variants in EBV transformation makes them interesting molecular indicators, as does their association with oncogenesis rather than non-oncogenic B cell diseases, differential expression in B cell malignancies, and correlation with CLL stage and some predictive CLL markers. This potential should be investigated in further studies.

Genetic variants in RNA-induced silencing complex genes and prostate cancer.

PubMed

Nikolić, Z; Savić Pavićević, D; Vučić, N; Cerović, S; Vukotić, V; Brajušković, G

2017-04-01

The purpose of this study is to evaluate the potential association between genetic variants in genes encoding the components of RNA-induced silencing complex and prostate cancer (PCa) risk. Genetic variants chosen for this study are rs3742330 in DICER1, rs4961280 in AGO2, rs784567 in TARBP2, rs7813 in GEMIN4 and rs197414 in GEMIN3. The study involved 355 PCa patients, 360 patients with benign prostatic hyperplasia and 318 healthy controls. For individuals diagnosed with PCa, clinicopathological characteristics including serum prostate-specific antigen level at diagnosis, Gleason score (GS) and clinical stage were determined. Genotyping was performed using high-resolution melting analysis, PCR-RFLP, TaqMan SNP Genotyping Assay and real-time PCR-based genotyping assay using specific probes. Allelic and genotypic associations were evaluated by unconditional linear and logistic regression methods. The study provided no evidence of association between the analyzed genetic variants and PCa risk. Nevertheless, allele A of rs784567 was found to confer the reduced risk of higher serum PSA level at diagnosis (P = 0.046; Difference = -66.64, 95 % CI -131.93 to 1.35, for log-additive model). Furthermore, rs4961280, as well as rs3742330, were shown to be associated with GS. These variants, together with rs7813, were found to be associated with the lower clinical stage of PCa. Also, rs3742330 minor allele G was found to be associated with lower PCa aggressiveness (P = 0.036; OR 0.14, 95 % CI 0.023-1.22, for recessive model). According to our data, rs3742330, rs4961280 and rs7813 qualify for potentially protective genetic variants against PCa progression. These variants were not shown to be associated with PCa risk.

NMR Studies of Peroxidases.

NASA Astrophysics Data System (ADS)

Veitch, Nigel Charles

Available from UMI in association with The British Library. Requires signed TDF. Peroxidases are a haem-containing group of enzymes with a wide diversity of function within biological systems. While a common characteristic is the ability to catalyse the conversion of hydrogen peroxide to water, it is the accompanying processes of hormone synthesis and degradation which have generated such a high level of interest. However, information at the molecular level is limited to a single well-resolved crystal structure, that of yeast cytochrome c peroxidase. This thesis presents a strategy for the investigation of peroxidase structure and function based on proton nuclear magnetic resonance spectroscopy, a technique which has the ability to address aspects of both protein structure and protein dynamics in solution. The application of one- and two-dimensional NMR techniques has been developed in the context of plant peroxidases, notably the isoenzyme HRP-C derived from the horseradish root. Characterisation of the proton NMR spectra of HRP -C in resting and ligated states provided new information enabling the structure of the binding site for aromatic donor molecules, such as indole-3-propionic, ferulic and benzhydroxamic acids, to be resolved. In order to overcome difficulties encountered with a protein of the complexity of peroxidase, additional information was obtained from chemical shift parameters and the use of peroxidase variants produced by site-directed mutagenesis. A comparative study using NMR spectroscopy was undertaken for wild-type recombinant HRP-C expressed in Escherichia coli, and two protein variants with substitutions made to residues located on the distal side of the haem pocket, Phe41 to Val and Arg38 to Lys. NMR analyses of a plant peroxidase from barley grains and the fungal peroxidase from Coprinus cinereus were also successful using methods conceived with HRP-C. Examination of three specifically constructed recombinant protein variants of C. cinereus peroxidase was essential in confirming the identity of residues participating in the aromatic donor molecule binding site of peroxidases.

Homo sapiens-Specific Binding Site Variants within Brain Exclusive Enhancers Are Subject to Accelerated Divergence across Human Population.

PubMed

Zehra, Rabail; Abbasi, Amir Ali

2018-03-01

Empirical assessments of human accelerated noncoding DNA frgaments have delineated presence of many cis-regulatory elements. Enhancers make up an important category of such accelerated cis-regulatory elements that efficiently control the spatiotemporal expression of many developmental genes. Establishing plausible reasons for accelerated enhancer sequence divergence in Homo sapiens has been termed significant in various previously published studies. This acceleration by including closely related primates and archaic human data has the potential to open up evolutionary avenues for deducing present-day brain structure. This study relied on empirically confirmed brain exclusive enhancers to avoid any misjudgments about their regulatory status and categorized among them a subset of enhancers with an exceptionally accelerated rate of lineage specific divergence in humans. In this assorted set, 13 distinct transcription factor binding sites were located that possessed unique existence in humans. Three of 13 such sites belonging to transcription factors SOX2, RUNX1/3, and FOS/JUND possessed single nucleotide variants that made them unique to H. sapiens upon comparisons with Neandertal and Denisovan orthologous sequences. These variants modifying the binding sites in modern human lineage were further substantiated as single nucleotide polymorphisms via exploiting 1000 Genomes Project Phase3 data. Long range haplotype based tests laid out evidence of positive selection to be governing in African population on two of the modern human motif modifying alleles with strongest results for SOX2 binding site. In sum, our study acknowledges acceleration in noncoding regulatory landscape of the genome and highlights functional parts within it to have undergone accelerated divergence in present-day human population.

Distinct patterns of mitochondrial genome diversity in bonobos (Pan paniscus) and humans.

PubMed

Zsurka, Gábor; Kudina, Tatiana; Peeva, Viktoriya; Hallmann, Kerstin; Elger, Christian E; Khrapko, Konstantin; Kunz, Wolfram S

2010-09-02

We have analyzed the complete mitochondrial genomes of 22 Pan paniscus (bonobo, pygmy chimpanzee) individuals to assess the detailed mitochondrial DNA (mtDNA) phylogeny of this close relative of Homo sapiens. We identified three major clades among bonobos that separated approximately 540,000 years ago, as suggested by Bayesian analysis. Incidentally, we discovered that the current reference sequence for bonobo likely is a hybrid of the mitochondrial genomes of two distant individuals. When comparing spectra of polymorphic mtDNA sites in bonobos and humans, we observed two major differences: (i) Of all 31 bonobo mtDNA homoplasies, i.e. nucleotide changes that occurred independently on separate branches of the phylogenetic tree, 13 were not homoplasic in humans. This indicates that at least a part of the unstable sites of the mitochondrial genome is species-specific and difficult to be explained on the basis of a mutational hotspot concept. (ii) A comparison of the ratios of non-synonymous to synonymous changes (dN/dS) among polymorphic positions in bonobos and in 4902 Homo sapiens mitochondrial genomes revealed a remarkable difference in the strength of purifying selection in the mitochondrial genes of the F0F1-ATPase complex. While in bonobos this complex showed a similar low value as complexes I and IV, human haplogroups displayed 2.2 to 7.6 times increased dN/dS ratios when compared to bonobos. Some variants of mitochondrially encoded subunits of the ATPase complex in humans very likely decrease the efficiency of energy conversion leading to production of extra heat. Thus, we hypothesize that the species-specific release of evolutionary constraints for the mitochondrial genes of the proton-translocating ATPase is a consequence of altered heat homeostasis in modern humans.

Distinct patterns of mitochondrial genome diversity in bonobos (Pan paniscus) and humans

PubMed Central

2010-01-01

Background We have analyzed the complete mitochondrial genomes of 22 Pan paniscus (bonobo, pygmy chimpanzee) individuals to assess the detailed mitochondrial DNA (mtDNA) phylogeny of this close relative of Homo sapiens. Results We identified three major clades among bonobos that separated approximately 540,000 years ago, as suggested by Bayesian analysis. Incidentally, we discovered that the current reference sequence for bonobo likely is a hybrid of the mitochondrial genomes of two distant individuals. When comparing spectra of polymorphic mtDNA sites in bonobos and humans, we observed two major differences: (i) Of all 31 bonobo mtDNA homoplasies, i.e. nucleotide changes that occurred independently on separate branches of the phylogenetic tree, 13 were not homoplasic in humans. This indicates that at least a part of the unstable sites of the mitochondrial genome is species-specific and difficult to be explained on the basis of a mutational hotspot concept. (ii) A comparison of the ratios of non-synonymous to synonymous changes (dN/dS) among polymorphic positions in bonobos and in 4902 Homo sapiens mitochondrial genomes revealed a remarkable difference in the strength of purifying selection in the mitochondrial genes of the F0F1-ATPase complex. While in bonobos this complex showed a similar low value as complexes I and IV, human haplogroups displayed 2.2 to 7.6 times increased dN/dS ratios when compared to bonobos. Conclusions Some variants of mitochondrially encoded subunits of the ATPase complex in humans very likely decrease the efficiency of energy conversion leading to production of extra heat. Thus, we hypothesize that the species-specific release of evolutionary constraints for the mitochondrial genes of the proton-translocating ATPase is a consequence of altered heat homeostasis in modern humans. PMID:20813043

Synthesis of Trypsin-Resistant Variants of the Listeria-Active Bacteriocin Salivaricin P▿

PubMed Central

O'Shea, Eileen F.; O'Connor, Paula M.; Cotter, Paul D.; Ross, R. Paul; Hill, Colin

2010-01-01

Two-component salivaricin P-like bacteriocins have demonstrated potential as antimicrobials capable of controlling infections in the gastrointestinal tract (GIT). The anti-Listeria activity of salivaricin P is optimal when the individual peptides Sln1 and Sln2 are added in succession at a 1:1 ratio. However, as degradation by digestive proteases may compromise the functionality of these peptides within the GIT, we investigated the potential to create salivaricin variants with enhanced resistance to the intestinal protease trypsin. A total of 11 variants of the salivaricin P components, in which conservative modifications at the trypsin-specific cleavage sites were explored in order to protect the peptides from trypsin degradation while maintaining their potent antimicrobial activity, were generated. Analysis of these variants revealed that eight were resistant to trypsin digestion while retaining antimicrobial activity. Combining the complementary trypsin-resistant variants Sln1-5 and Sln2-3 resulted in a MIC50 of 300 nM against Listeria monocytogenes, a 3.75-fold reduction in activity compared to the level for wild-type salivaricin P. This study demonstrates the potential of engineering bacteriocin variants which are resistant to specific protease action but which retain significant antimicrobial activity. PMID:20581174

Site-Directed Immobilization of BMP-2: Two Approaches for the Production of Innovative Osteoinductive Scaffolds.

PubMed

Tabisz, Barbara; Schmitz, Werner; Schmitz, Michael; Luehmann, Tessa; Heusler, Eva; Rybak, Jens-Christoph; Meinel, Lorenz; Fiebig, Juliane E; Mueller, Thomas D; Nickel, Joachim

2017-03-13

The regenerative potential of bone is strongly impaired in pathological conditions, such as nonunion fractures. To support bone regeneration various scaffolds have been developed in the past, which have been functionalized with osteogenic growth factors such as bone morphogenetic proteins (BMPs). However, most of them required supra-physiological levels of these proteins leading to burst releases, thereby causing severe side effects. Site-specific, covalent coupling of BMP2 to implant materials might be an optimal strategy in order to overcome these problems. Therefore, we created a BMP-2 variant (BMP2-K3Plk) containing a noncanonical amino acid (propargyl-l-lysine) substitution introduced by genetic code expansion that allows for site-specific and covalent immobilization onto polymeric scaffold materials. To directly compare different coupling strategies, we also produced a BMP2 variant containing an additional cysteine residue (BMP2-A2C) allowing covalent coupling by thioether formation. The BMP2-K3Plk mutant was coupled to functionalized beads by a copper-catalyzed azide-alkyne cycloaddition (CuAAC) either directly or via a short biotin-PEG linker both with high specificity. After exposing the BMP-coated beads to C2C12 cells, ALP expression appeared locally restricted in close proximity to these beads, showing that both coupled BMP2 variants trigger cell differentiation. The advantage of our approach over non-site-directed immobilization techniques is the ability to produce fully defined osteogenic surfaces, allowing for lower BMP2 loads and concomitant higher bioactivities, for example, due to controlled orientation toward BMP2 receptors. Such products might provide superior bone healing capabilities with potential safety advantages as of homogeneous product outcome.

Semisynthetic Enzymes by Protein-Peptide Site-Directed Covalent Conjugation: Methods and Applications.

PubMed

Palomo, Jose M

2017-01-01

This chapter describes the rational design and synthesis of semisynthetic lipases by site-directed incorporation of tailor-made peptides on the lipase-lid site to improve its activity, specificity, and enantioselectivity in specific biotransformations. Cysteine was genetically introduced at a particular point of the oligopeptide lid of the enzyme, and cysteine-containing peptides, complementary to the amino acid sequence on the lid site of Geobacillus thermocatenulatus lipase (BTL), were covalently attached on the lid of two different cysteine-BTL variants based on a fast thiol-disulfide exchange ligation followed by desulfurization. The BTL variants were initially immobilized on solid support to introduce the advantages of solid-state chemistry, such as quantitative transformations, easy purification, and recyclability. In the two different immobilized variants BTL-A193C and BTL-L230C, the cysteine was then activated with 2-dipyridyldisulfide to help the disulfide exchange with the peptide, generating the semisynthetic enzyme in high yield. Excellent results of improvement of activity and selectivity were obtained. For example, the peptide-BTL conjugate (at position 193) was 40-fold more active than the corresponding unmodified enzyme for the hydrolysis of per-acetylated thymidine at pH 5, or fourfold in the desymmetrization of dimethyl-3-phenylglutarate at pH 7. The new enzyme also exhibited excellent enantioselectivity in the desymmetrization reaction with enantiomeric excess (ee) of >99% when compared to that of the unmodified enzyme (ee=78%). © 2017 Elsevier Inc. All rights reserved.

Amino acid changes in disease-associated variants differ radically from variants observed in the 1000 genomes project dataset.

PubMed

de Beer, Tjaart A P; Laskowski, Roman A; Parks, Sarah L; Sipos, Botond; Goldman, Nick; Thornton, Janet M

2013-01-01

The 1000 Genomes Project data provides a natural background dataset for amino acid germline mutations in humans. Since the direction of mutation is known, the amino acid exchange matrix generated from the observed nucleotide variants is asymmetric and the mutabilities of the different amino acids are very different. These differences predominantly reflect preferences for nucleotide mutations in the DNA (especially the high mutation rate of the CpG dinucleotide, which makes arginine mutability very much higher than other amino acids) rather than selection imposed by protein structure constraints, although there is evidence for the latter as well. The variants occur predominantly on the surface of proteins (82%), with a slight preference for sites which are more exposed and less well conserved than random. Mutations to functional residues occur about half as often as expected by chance. The disease-associated amino acid variant distributions in OMIM are radically different from those expected on the basis of the 1000 Genomes dataset. The disease-associated variants preferentially occur in more conserved sites, compared to 1000 Genomes mutations. Many of the amino acid exchange profiles appear to exhibit an anti-correlation, with common exchanges in one dataset being rare in the other. Disease-associated variants exhibit more extreme differences in amino acid size and hydrophobicity. More modelling of the mutational processes at the nucleotide level is needed, but these observations should contribute to an improved prediction of the effects of specific variants in humans.

Evaluation of Bioinformatic Programmes for the Analysis of Variants within Splice Site Consensus Regions

PubMed Central

Tang, Rongying; Prosser, Debra O.; Love, Donald R.

2016-01-01

The increasing diagnostic use of gene sequencing has led to an expanding dataset of novel variants that lie within consensus splice junctions. The challenge for diagnostic laboratories is the evaluation of these variants in order to determine if they affect splicing or are merely benign. A common evaluation strategy is to use in silico analysis, and it is here that a number of programmes are available online; however, currently, there are no consensus guidelines on the selection of programmes or protocols to interpret the prediction results. Using a collection of 222 pathogenic mutations and 50 benign polymorphisms, we evaluated the sensitivity and specificity of four in silico programmes in predicting the effect of each variant on splicing. The programmes comprised Human Splice Finder (HSF), Max Entropy Scan (MES), NNSplice, and ASSP. The MES and ASSP programmes gave the highest performance based on Receiver Operator Curve analysis, with an optimal cut-off of score reduction of 10%. The study also showed that the sensitivity of prediction is affected by the level of conservation of individual positions, with in silico predictions for variants at positions −4 and +7 within consensus splice sites being largely uninformative. PMID:27313609

Novel insights regarding the operational characteristics and teleological purpose of the renal Na+-K+-Cl2 cotransporter (NKCC2s) splice variants.

PubMed

Brunet, Geneviève M; Gagnon, Edith; Simard, Charles F; Daigle, Nikolas D; Caron, Luc; Noël, Micheline; Lefoll, Marie-Hélène; Bergeron, Marc J; Isenring, Paul

2005-10-01

The absorptive Na(+)-K(+)-Cl(-) cotransporter (NKCC2) is a polytopic protein that forms homooligomeric complexes in the apical membrane of the thick ascending loop of Henle (TAL). It occurs in at least four splice variants (called B, A, F, and AF) that are identical to one another except for a short region in the membrane-associated domain. Although each of these variants exhibits unique functional properties and distributions along the TAL, their teleological purpose and structural organization remain poorly defined. In the current work, we provide additional insight in these regards by showing in mouse that the administration of either furosemide or an H(2)O-rich diet, which are predicted to alter NKCC2 expression in the TAL, exerts differential effects on mRNA levels for the variants, increasing those of A (furosemide) but decreasing those of F and AF (furosemide or H(2)O). Based on a yeast two-hybrid mapping analysis, we also show that the formation of homooligomeric complexes is mediated by two self-interacting domains in the COOH terminus (residues 671 to 816 and 910 to 1098), and that these complexes could probably include more than one type of variant. Taken together, the data reported here suggest that A, F, and AF each play unique roles that are adapted to specific physiological needs, and that the accomplishment of such roles is coordinated through the splicing machinery as well as complex NKCC2-NKCC2 interactions.

Karyotype diversity and 2C DNA content in species of the Caesalpinia group.

PubMed

Rodrigues, Polliana Silva; Souza, Margarete Magalhães; Melo, Cláusio Antônio Ferreira; Pereira, Telma Nair Santana; Corrêa, Ronan Xavier

2018-04-11

The Leguminosae family is the third-largest family of angiosperms, and Caesalpinioideae is its second-largest subfamily. A great number of species (approximately 205) are found in the Caesalpinia group within this subfamily; together with these species' phenotypic plasticity and the similarities in their morphological descriptors, make this a complex group for taxonomic and phylogenetic studies. The objective of the present work was to evaluate the karyotypic diversity and the 2C DNA content variation in 10 species of the Caesalpinia group, representing six genera: Paubrasilia, Caesalpinia, Cenostigma, Poincianella, Erythrostemon and Libidibia. The GC-rich heterochromatin and 45S rDNA sites (which are used as chromosome markers) were located to evaluate the karyotype diversity in the clade. The variation in the 2C DNA content was determined through flow cytometry. The fluorochrome banding indicated that the chromomycin A 3 + /4',6-diamidino-2-phenylindole - blocks were exclusively in the terminal regions of the chromosomes, coinciding with 45S rDNA sites in all analyzed species. Physical mapping of the species (through fluorescence in situ hybridization) revealed variation in the size of the hybridization signals and in the number and distribution of the 45S rDNA sites. All hybridization sites were in the terminal regions of the chromosomes. In addition, all species had a hybridization site in the fourth chromosome pair. The 2C DNA content ranged from 1.54 pg in Erythrostemon calycina to 2.82 pg in the Paubrasilia echinata large-leaf variant. The Pa. echinata small-leaf variant was isolated from the other leaf variants through Scoot-Knott clustering. The chromosome diversity and the variation in the 2C DNA content reinforce that the actual taxonomy and clustering of the analyzed taxa requires more genera that were previously proposed. This fact indicates that taxonomy, phylogeny and cytoevolutionary inference related to the complex Caesalpinia group have to be done through integrative evaluation.

Site-specific photoconjugation of antibodies using chemically synthesized IgG-binding domains.

PubMed

Perols, Anna; Karlström, Amelie Eriksson

2014-03-19

Site-specific labeling of antibodies can be performed using the immunoglobulin-binding Z domain, derived from staphylococcal protein A (SpA), which has a well-characterized binding site in the Fc region of antibodies. By introducing a photoactivable probe in the Z domain, a covalent bond can be formed between the Z domain and the antibody by irradiation with UV light. The aim of this study was to improve the conjugation yield for labeling of different subclasses of IgG having different sequence composition, using a photoactivated Z domain variant. Four different variants of the Z domain (Z5BPA, Z5BBA, Z32BPA, and Z32BBA) were synthesized to investigate the influence of the position of the photoactivable probe and the presence of a flexible linker between the probe and the protein. For two of the variants, the photoreactive benzophenone group was introduced as part of an amino acid side chain by incorporation of the unnatural amino acid benzoylphenylalanine (BPA) during peptide synthesis. For the other two variants, the photoreactive benzophenone group was attached via a flexible linker by coupling of benzoylbenzoic acid (BBA) to the ε-amino group of a selectively deprotected lysine residue. Photoconjugation experiments using human IgG1, mouse IgG1, and mouse IgG2A demonstrated efficient conjugation for all antibodies. It was shown that differences in linker length had a large impact on the conjugation efficiency for labeling of mouse IgG1, whereas the positioning of the photoactivable probe in the sequence of the protein had a larger effect for mouse IgG2A. Conjugation to human IgG1 was only to a minor extent affected by position or linker length. For each subclass of antibody, the best variant tested using a standard conjugation protocol resulted in conjugation efficiencies of 41-66%, which corresponds to on average approximately one Z domain attached to each antibody. As a combination of the two best performing variants, Z5BBA and Z32BPA, a Z domain variant with two photoactivable probes (Z5BBA32BPA) was also synthesized with the aim of targeting a wider panel of antibody subclasses and species. This new reagent could efficiently couple to all antibody subclasses that were targeted by the single benzophenone-labeled Z domain variants, with conjugation efficiencies of 26-41%.

Molecular modeling and structural analysis of nAChR variants uncovers the mechanism of resistance to snake toxins.

PubMed

Gunasekaran, D; Sridhar, J; Suryanarayanan, V; Manimaran, N C; Singh, Sanjeev Kumar

2017-06-01

Nicotinic acetylcholine receptors (nAChRs) are neuromuscular proteins responsible for muscle contraction upon binding with chemical stimulant acetylcholine (ACh). The α-neurotoxins of snake mimic the structure of ACh and attacks nAChRs, which block the flow of ACh and leads to numbness and paralysis. The toxin-binding site of alpha subunit in the nAChRs is highly conserved throughout chordate lineages with few exceptions in resistance organisms. In this study, we have analyzed the sequence and structures of toxin-binding/resistant nAChRs and their interaction stability with toxins through molecular docking and molecular dynamics simulation (MDS). We have reported the potential glycosylation residues within the toxin-binding cleft adding sugar moieties through N-linked glycosylation in resistant organisms. Residue variations at key positions alter the secondary structure of binding cleft, which might interfere with toxin binding and it could be one of the possible explanations for the resistance to snake venoms. Analysis of nAChR-α-neurotoxin complexes has confirmed the key interacting residues. In addition, drastic variation in the binding stability of Mongoose nAChR-α-Bungarotoxin (α-BTX) and human nAChR-α-BTX complexes were found at specific phase of MDS. Our findings suggest that specific mutations in the binding site of toxin are potentially preventing the formation of stable complex of receptor-toxin, which might lead to mechanism of resistance. This in silico study on the binding cleft of nAChR and the findings of interacting residues will assist in designing potential inhibitors as therapeutic targets.

VariantBam: filtering and profiling of next-generational sequencing data using region-specific rules.

PubMed

Wala, Jeremiah; Zhang, Cheng-Zhong; Meyerson, Matthew; Beroukhim, Rameen

2016-07-01

We developed VariantBam, a C ++ read filtering and profiling tool for use with BAM, CRAM and SAM sequencing files. VariantBam provides a flexible framework for extracting sequencing reads or read-pairs that satisfy combinations of rules, defined by any number of genomic intervals or variant sites. We have implemented filters based on alignment data, sequence motifs, regional coverage and base quality. For example, VariantBam achieved a median size reduction ratio of 3.1:1 when applied to 10 lung cancer whole genome BAMs by removing large tags and selecting for only high-quality variant-supporting reads and reads matching a large dictionary of sequence motifs. Thus VariantBam enables efficient storage of sequencing data while preserving the most relevant information for downstream analysis. VariantBam and full documentation are available at github.com/jwalabroad/VariantBam rameen@broadinstitute.org Supplementary data are available at Bioinformatics online. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Molecular and structural determinants of adamantyl susceptibility to HLA-DRs allelic variants: an in silico approach to understand the mechanism of MLEs

NASA Astrophysics Data System (ADS)

Zaheer-ul-Haq; Khan, Waqasuddin

2011-01-01

Class II major histocompatibility complex (MHC II) molecules as expressed by antigen-presenting cells are heterodimeric cell-surface glycoprotein receptors that are fundamental in initiating and propagating an immune response by presenting tumor-associated antigenic peptides to CD4+/TH cells. The loading efficiency of such peptides can be improved by small organic compounds (MHC Loading Enhancers—MLEs), that convert the non-receptive peptide conformation of MHC II to a peptide-receptive conformation. In a reversible reaction, these compounds open up the binding site of MHC II molecules by specific interactions with a yet undefined pocket. Here, we performed molecular docking and molecular dynamics simulation studies of adamantyl compounds on the predicted cavity around the P1 pocket of 2 allelic variants of HLA-DRs. The purpose was to investigate the suitability of adamantyl compounds as MLEs at the dimorphic β86 position. Docking studies revealed that besides numerous molecular interactions formed by the adamantyl compounds, Asnβ82, Tyrβ83, and Thrβ90 are the crucial amino acid residues that are characterized as the "sensors" of peptide loading. Molecular dynamics simulation studies exposed the dynamical structural changes that HLA-DRs adopted as a response to binding of 3-(1-adamantyl)-5-hydrazidocarbonyl-1H-pyrazole (AdCaPy). The conformations of AdCaPy complexed with the Glyβ86 HLA-DR allelic variant are well correlated with the stabilized form of peptide-loaded HLA-DRs, further confirming the role of AdCaPy as a MLE. Hydrogen bonding interaction analysis clearly demonstrated that after making suitable contacts with AdCaPy, HLA-DR changes its local conformation. However, AdCaPy complexed with HLA-DR having Valβ86 at the dimorphic position did not accommodate AdCaPy as MLE due to steric hindrance caused by the valine.

Structural and functional characterization of phosphomimetic mutants of cytochrome c at threonine 28 and serine 47.

PubMed

Guerra-Castellano, Alejandra; Díaz-Moreno, Irene; Velázquez-Campoy, Adrián; De la Rosa, Miguel A; Díaz-Quintana, Antonio

2016-04-01

Protein function is frequently modulated by post-translational modifications of specific residues. Cytochrome c, in particular, is phosphorylated in vivo at threonine 28 and serine 47. However, the effect of such modifications on the physiological functions of cytochrome c - namely, the transfer of electrons in the respiratory electron transport chain and the triggering of programmed cell death - is still unknown. Here we replace each of these two residues by aspartate, in order to mimic phosphorylation, and report the structural and functional changes in the resulting cytochrome c variants. We find that the T28D mutant causes a 30-mV decrease on the midpoint redox potential and lowers the affinity for the distal site of Arabidopsis thaliana cytochrome c1 in complex III. Both the T28D and S47D variants display a higher efficiency as electron donors for the cytochrome c oxidase activity of complex IV. In both protein mutants, the peroxidase activity is significantly higher, which is related to the ability of cytochrome c to leave the mitochondria and reach the cytoplasm. We also find that both mutations at serine 47 (S47D and S47A) impair the ability of cytoplasmic cytochrome c to activate the caspases cascade, which is essential for triggering programmed cell death. Copyright © 2016 Elsevier B.V. All rights reserved.

Structure of Arabidopsis thaliana FUT1 Reveals a Variant of the GT-B Class Fold and Provides Insight into Xyloglucan Fucosylation

PubMed Central

Chazalet, Valérie

2016-01-01

The plant cell wall is a complex and dynamic network made mostly of cellulose, hemicelluloses, and pectins. Xyloglucan, the major hemicellulosic component in Arabidopsis thaliana, is biosynthesized in the Golgi apparatus by a series of glycan synthases and glycosyltransferases before export to the wall. A better understanding of the xyloglucan biosynthetic machinery will give clues toward engineering plants with improved wall properties or designing novel xyloglucan-based biomaterials. The xyloglucan-specific α2-fucosyltransferase FUT1 catalyzes the transfer of fucose from GDP-fucose to terminal galactosyl residues on xyloglucan side chains. Here, we present crystal structures of Arabidopsis FUT1 in its apoform and in a ternary complex with GDP and a xylo-oligosaccharide acceptor (named XLLG). Although FUT1 is clearly a member of the large GT-B fold family, like other fucosyltransferases of known structures, it contains a variant of the GT-B fold. In particular, it includes an extra C-terminal region that is part of the acceptor binding site. Our crystal structures support previous findings that FUT1 behaves as a functional dimer. Mutational studies and structure comparison with other fucosyltransferases suggest that FUT1 uses a SN2-like reaction mechanism similar to that of protein-O-fucosyltransferase 2. Thus, our results provide new insights into the mechanism of xyloglucan fucosylation in the Golgi. PMID:27637560

Antioxidant Defense Enzyme Genes and Asthma Susceptibility: Gender-Specific Effects and Heterogeneity in Gene-Gene Interactions between Pathogenetic Variants of the Disease

PubMed Central

Polonikov, Alexey V.; Ivanov, Vladimir P.; Bogomazov, Alexey D.; Freidin, Maxim B.; Illig, Thomas; Solodilova, Maria A.

2014-01-01

Oxidative stress resulting from an increased amount of reactive oxygen species and an imbalance between oxidants and antioxidants plays an important role in the pathogenesis of asthma. The present study tested the hypothesis that genetic susceptibility to allergic and nonallergic variants of asthma is determined by complex interactions between genes encoding antioxidant defense enzymes (ADE). We carried out a comprehensive analysis of the associations between adult asthma and 46 single nucleotide polymorphisms of 34 ADE genes and 12 other candidate genes of asthma in Russian population using set association analysis and multifactor dimensionality reduction approaches. We found for the first time epistatic interactions between ADE genes underlying asthma susceptibility and the genetic heterogeneity between allergic and nonallergic variants of the disease. We identified GSR (glutathione reductase) and PON2 (paraoxonase 2) as novel candidate genes for asthma susceptibility. We observed gender-specific effects of ADE genes on the risk of asthma. The results of the study demonstrate complexity and diversity of interactions between genes involved in oxidative stress underlying susceptibility to allergic and nonallergic asthma. PMID:24895604

Direct and indirect effectiveness of influenza vaccination delivered to children at school preceding an epidemic caused by 3 new influenza virus variants.

PubMed

Glezen, W Paul; Gaglani, Manjusha J; Kozinetz, Claudia A; Piedra, Pedro A

2010-12-01

Influenza is an uncontrolled epidemic disease that is vaccine preventable. New recommendations for universal immunization present a challenge to the implementation of vaccine delivery. This field trial examines the effectiveness of school-based clinics for vaccine delivery before an epidemic caused by 3 new influenza virus variants not contained in the vaccine. Live attenuated influenza vaccine (LAIV) was offered to eligible children in elementary schools of eastern Bell County, Texas. Age-specific rates of medically attended acute respiratory illness for health plan members at the intervention site were compared with those for members at comparison sites during the epidemic, defined by viral surveillance at all sites. Almost 48% of children in elementary schools were vaccinated. Significant herd protection attributed to LAIV was detected for all age groups except 12-17-year-old students, who were not offered free vaccine. Approximately 2500 medical encounters were prevented at the intervention site. Inactivated vaccine provided marginal protection against the epidemic viruses. LAIV delivered to elementary-school children before an epidemic caused by 3 new variant influenza viruses generated significant cross-protection for the recipients and indirect (herd) protection for the community.

Communication between Thiamin Cofactors in the Escherichia coli Pyruvate Dehydrogenase Complex E1 Component Active Centers EVIDENCE FOR A DIRECT PATHWAY BETWEEN THE 4′-AMINOPYRIMIDINE N1′ ATOMS

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nemeria, Natalia S; Arjunan, Palaniappa; Chandrasekhar, Krishnamoorthy

2010-11-03

Kinetic, spectroscopic, and structural analysis tested the hypothesis that a chain of residues connecting the 4{prime}-aminopyrimidine N1{prime} atoms of thiamin diphosphates (ThDPs) in the two active centers of the Escherichia coli pyruvate dehydrogenase complex E1 component provides a signal transduction pathway. Substitution of the three acidic residues (Glu{sup 571}, Glu{sup 235}, and Glu{sup 237}) and Arg{sup 606} resulted in impaired binding of the second ThDP, once the first active center was filled, suggesting a pathway for communication between the two ThDPs. (1) Steady-state kinetic and fluorescence quenching studies revealed that upon E571A, E235A, E237A, and R606A substitutions, ThDP binding inmore » the second active center was affected. (2) Analysis of the kinetics of thiazolium C2 hydrogen/deuterium exchange of enzyme-bound ThDP suggests half-of-the-sites reactivity for the E1 component, with fast (activated site) and slow exchanging sites (dormant site). The E235A and E571A variants gave no evidence for the slow exchanging site, indicating that only one of two active sites is filled with ThDP. (3) Titration of the E235A and E237A variants with methyl acetylphosphonate monitored by circular dichroism suggested that only half of the active sites were filled with a covalent predecarboxylation intermediate analog. (4) Crystal structures of E235A and E571A in complex with ThDP revealed the structural basis for the spectroscopic and kinetic observations and showed that either substitution affects cofactor binding, despite the fact that Glu{sup 235} makes no direct contact with the cofactor. The role of the conserved Glu{sup 571} residue in both catalysis and cofactor orientation is revealed by the combined results for the first time.« less

Structural characterization of O- and C-glycosylating variants of the landomycin glycosyltransferase LanGT2.

PubMed

Tam, Heng Keat; Härle, Johannes; Gerhardt, Stefan; Rohr, Jürgen; Wang, Guojun; Thorson, Jon S; Bigot, Aurélien; Lutterbeck, Monika; Seiche, Wolfgang; Breit, Bernhard; Bechthold, Andreas; Einsle, Oliver

2015-02-23

The structures of the O-glycosyltransferase LanGT2 and the engineered, C-C bond-forming variant LanGT2S8Ac show how the replacement of a single loop can change the functionality of the enzyme. Crystal structures of the enzymes in complex with a nonhydrolyzable nucleotide-sugar analogue revealed that there is a conformational transition to create the binding sites for the aglycon substrate. This induced-fit transition was explored by molecular docking experiments with various aglycon substrates. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Removal of either N-glycan site from the envelope receptor binding domain of Moloney and Friend but not AKV mouse ecotropic gammaretroviruses alters receptor usage

DOE Office of Scientific and Technical Information (OSTI.GOV)

Knoper, Ryan C.; Ferrarone, John; Yan Yuhe

2009-09-01

Three N-linked glycosylation sites were removed from the envelope glycoproteins of Friend, Moloney, and AKV mouse ecotropic gammaretroviruses: gs1 and gs2, in the receptor binding domain; and gs8, in a region implicated in post-binding cell fusion. Mutants were tested for their ability to infect rodent cells expressing 4 CAT-1 receptor variants. Three mutants (Mo-gs1, Mo-gs2, and Fr-gs1) infect NIH 3T3 and rat XC cells, but are severely restricted in Mus dunni cells and Lec8, a Chinese hamster cell line susceptible to ecotropic virus. This restriction is reproduced in ferret cells expressing M. dunni dCAT-1, but not in cells expressing NIHmore » 3T3 mCAT-1. Virus binding assays, pseudotype assays, and the use of glycosylation inhibitors further suggest that restriction is primarily due to receptor polymorphism and, in M. dunni cells, to glycosylation of cellular proteins. Virus envelope glycan size or type does not affect infectivity. Thus, host range variation due to N-glycan deletion is receptor variant-specific, cell-specific, virus type-specific, and glycan site-specific.« less

SPECIATION OF ORGANICS IN WATER

EPA Science Inventory

We describe herein a method for determining constants for simultaneously occurring, site-specific "microequilibria" (as with tautomers) for organics in water. The method is based in part on modeling temperature-variant Raman spectra according to the van't Hoff equation....

Exome sequencing in an admixed isolated population indicates NFXL1 variants confer a risk for specific language impairment.

PubMed

Villanueva, Pía; Nudel, Ron; Hoischen, Alexander; Fernández, María Angélica; Simpson, Nuala H; Gilissen, Christian; Reader, Rose H; Jara, Lillian; Echeverry, María Magdalena; Echeverry, Maria Magdalena; Francks, Clyde; Baird, Gillian; Conti-Ramsden, Gina; O'Hare, Anne; Bolton, Patrick F; Hennessy, Elizabeth R; Palomino, Hernán; Carvajal-Carmona, Luis; Veltman, Joris A; Cazier, Jean-Baptiste; De Barbieri, Zulema; Fisher, Simon E; Newbury, Dianne F

2015-03-01

Children affected by Specific Language Impairment (SLI) fail to acquire age appropriate language skills despite adequate intelligence and opportunity. SLI is highly heritable, but the understanding of underlying genetic mechanisms has proved challenging. In this study, we use molecular genetic techniques to investigate an admixed isolated founder population from the Robinson Crusoe Island (Chile), who are affected by a high incidence of SLI, increasing the power to discover contributory genetic factors. We utilize exome sequencing in selected individuals from this population to identify eight coding variants that are of putative significance. We then apply association analyses across the wider population to highlight a single rare coding variant (rs144169475, Minor Allele Frequency of 4.1% in admixed South American populations) in the NFXL1 gene that confers a nonsynonymous change (N150K) and is significantly associated with language impairment in the Robinson Crusoe population (p = 2.04 × 10-4, 8 variants tested). Subsequent sequencing of NFXL1 in 117 UK SLI cases identified four individuals with heterozygous variants predicted to be of functional consequence. We conclude that coding variants within NFXL1 confer an increased risk of SLI within a complex genetic model.

A SNP in the HTT promoter alters NF-κB binding and is a bidirectional genetic modifier of Huntington disease.

PubMed

Bečanović, Kristina; Nørremølle, Anne; Neal, Scott J; Kay, Chris; Collins, Jennifer A; Arenillas, David; Lilja, Tobias; Gaudenzi, Giulia; Manoharan, Shiana; Doty, Crystal N; Beck, Jessalyn; Lahiri, Nayana; Portales-Casamar, Elodie; Warby, Simon C; Connolly, Colúm; De Souza, Rebecca A G; Tabrizi, Sarah J; Hermanson, Ola; Langbehn, Douglas R; Hayden, Michael R; Wasserman, Wyeth W; Leavitt, Blair R

2015-06-01

Cis-regulatory variants that alter gene expression can modify disease expressivity, but none have previously been identified in Huntington disease (HD). Here we provide in vivo evidence in HD patients that cis-regulatory variants in the HTT promoter are bidirectional modifiers of HD age of onset. HTT promoter analysis identified a NF-κB binding site that regulates HTT promoter transcriptional activity. A non-coding SNP, rs13102260:G > A, in this binding site impaired NF-κB binding and reduced HTT transcriptional activity and HTT protein expression. The presence of the rs13102260 minor (A) variant on the HD disease allele was associated with delayed age of onset in familial cases, whereas the presence of the rs13102260 (A) variant on the wild-type HTT allele was associated with earlier age of onset in HD patients in an extreme case-based cohort. Our findings suggest a previously unknown mechanism linking allele-specific effects of rs13102260 on HTT expression to HD age of onset and have implications for HTT silencing treatments that are currently in development.

Modes of Interaction of KMT2 Histone H3 Lysine 4 Methyltransferase/COMPASS Complexes with Chromatin

PubMed Central

Bochyńska, Agnieszka; Lüscher-Firzlaff, Juliane

2018-01-01

Regulation of gene expression is achieved by sequence-specific transcriptional regulators, which convey the information that is contained in the sequence of DNA into RNA polymerase activity. This is achieved by the recruitment of transcriptional co-factors. One of the consequences of co-factor recruitment is the control of specific properties of nucleosomes, the basic units of chromatin, and their protein components, the core histones. The main principles are to regulate the position and the characteristics of nucleosomes. The latter includes modulating the composition of core histones and their variants that are integrated into nucleosomes, and the post-translational modification of these histones referred to as histone marks. One of these marks is the methylation of lysine 4 of the core histone H3 (H3K4). While mono-methylation of H3K4 (H3K4me1) is located preferentially at active enhancers, tri-methylation (H3K4me3) is a mark found at open and potentially active promoters. Thus, H3K4 methylation is typically associated with gene transcription. The class 2 lysine methyltransferases (KMTs) are the main enzymes that methylate H3K4. KMT2 enzymes function in complexes that contain a necessary core complex composed of WDR5, RBBP5, ASH2L, and DPY30, the so-called WRAD complex. Here we discuss recent findings that try to elucidate the important question of how KMT2 complexes are recruited to specific sites on chromatin. This is embedded into short overviews of the biological functions of KMT2 complexes and the consequences of H3K4 methylation. PMID:29498679

Redesigning the specificity of protein-DNA interactions with Rosetta.

PubMed

Thyme, Summer; Baker, David

2014-01-01

Building protein tools that can selectively bind or cleave specific DNA sequences requires efficient technologies for modifying protein-DNA interactions. Computational design is one method for accomplishing this goal. In this chapter, we present the current state of protein-DNA interface design with the Rosetta macromolecular modeling program. The LAGLIDADG endonuclease family of DNA-cleaving enzymes, under study as potential gene therapy reagents, has been the main testing ground for these in silico protocols. At this time, the computational methods are most useful for designing endonuclease variants that can accommodate small numbers of target site substitutions. Attempts to engineer for more extensive interface changes will likely benefit from an approach that uses the computational design results in conjunction with a high-throughput directed evolution or screening procedure. The family of enzymes presents an engineering challenge because their interfaces are highly integrated and there is significant coordination between the binding and catalysis events. Future developments in the computational algorithms depend on experimental feedback to improve understanding and modeling of these complex enzymatic features. This chapter presents both the basic method of design that has been successfully used to modulate specificity and more advanced procedures that incorporate DNA flexibility and other properties that are likely necessary for reliable modeling of more extensive target site changes.

A survey of tools for variant analysis of next-generation genome sequencing data

PubMed Central

Pabinger, Stephan; Dander, Andreas; Fischer, Maria; Snajder, Rene; Sperk, Michael; Efremova, Mirjana; Krabichler, Birgit; Speicher, Michael R.; Zschocke, Johannes

2014-01-01

Recent advances in genome sequencing technologies provide unprecedented opportunities to characterize individual genomic landscapes and identify mutations relevant for diagnosis and therapy. Specifically, whole-exome sequencing using next-generation sequencing (NGS) technologies is gaining popularity in the human genetics community due to the moderate costs, manageable data amounts and straightforward interpretation of analysis results. While whole-exome and, in the near future, whole-genome sequencing are becoming commodities, data analysis still poses significant challenges and led to the development of a plethora of tools supporting specific parts of the analysis workflow or providing a complete solution. Here, we surveyed 205 tools for whole-genome/whole-exome sequencing data analysis supporting five distinct analytical steps: quality assessment, alignment, variant identification, variant annotation and visualization. We report an overview of the functionality, features and specific requirements of the individual tools. We then selected 32 programs for variant identification, variant annotation and visualization, which were subjected to hands-on evaluation using four data sets: one set of exome data from two patients with a rare disease for testing identification of germline mutations, two cancer data sets for testing variant callers for somatic mutations, copy number variations and structural variations, and one semi-synthetic data set for testing identification of copy number variations. Our comprehensive survey and evaluation of NGS tools provides a valuable guideline for human geneticists working on Mendelian disorders, complex diseases and cancers. PMID:23341494

Deep comparative genomics among Chlamydia trachomatis lymphogranuloma venereum isolates highlights genes potentially involved in pathoadaptation.

PubMed

Borges, Vítor; Gomes, João Paulo

2015-06-01

Lymphogranuloma venereum (LGV) is a human sexually transmitted disease caused by the obligate intracellular bacterium Chlamydia trachomatis (serovars L1-L3). LGV clinical manifestations range from severe ulcerative proctitis (anorectal syndrome), primarily caused by the epidemic L2b strains, to painful inguinal lymphadenopathy (the typical LGV bubonic form). Besides potential host-related factors, the differential disease severity and tissue tropism among LGV strains is likely a function of the genetic backbone of the strains. We aimed to characterize the genetic variability among LGV strains as strain- or serovar-specific mutations may underlie phenotypic signatures, and to investigate the mutational events that occurred throughout the pathoadaptation of the epidemic L2b lineage. By analyzing 20 previously published genomes from L1, L2, L2b and L3 strains and two new genomes from L2b strains, we detected 1497 variant sites and about 100 indels, affecting 453 genes and 144 intergenic regions, with 34 genes displaying a clear overrepresentation of nonsynonymous mutations. Effectors and/or type III secretion substrates (almost all of those described in the literature) and inclusion membrane proteins showed amino acid changes that were about fivefold more frequent than silent changes. More than 120 variant sites occurred in plasmid-regulated virulence genes, and 66% yielded amino acid changes. The identified serovar-specific variant sites revealed that the L2b-specific mutations are likely associated with higher fitness and pointed out potential targets for future highly discriminatory diagnostic/typing tests. By evaluating the evolutionary pathway beyond the L2b clonal radiation, we observed that 90.2% of the intra-L2b variant sites occurring in coding regions involve nonsynonymous mutations, where CT456/tarp has been the main target. Considering the progress on C. trachomatis genetic manipulation, this study may constitute an important contribution for prioritizing study targets for functional genomics aiming to dissect the impact of the identified intra-LGV polymorphisms on virulence or tropism dissimilarities among LGV strains. Copyright © 2015 Elsevier B.V. All rights reserved.

Two Oxidation Sites for Low Redox Potential Substrates

PubMed Central

Morales, María; Mate, María J.; Romero, Antonio; Martínez, María Jesús; Martínez, Ángel T.; Ruiz-Dueñas, Francisco J.

2012-01-01

Versatile peroxidase shares with manganese peroxidase and lignin peroxidase the ability to oxidize Mn2+ and high redox potential aromatic compounds, respectively. Moreover, it is also able to oxidize phenols (and low redox potential dyes) at two catalytic sites, as shown by biphasic kinetics. A high efficiency site (with 2,6-dimethoxyphenol and p-hydroquinone catalytic efficiencies of ∼70 and ∼700 s−1 mm−1, respectively) was localized at the same exposed Trp-164 responsible for high redox potential substrate oxidation (as shown by activity loss in the W164S variant). The second site, characterized by low catalytic efficiency (∼3 and ∼50 s−1 mm−1 for 2,6-dimethoxyphenol and p-hydroquinone, respectively) was localized at the main heme access channel. Steady-state and transient-state kinetics for oxidation of phenols and dyes at the latter site were improved when side chains of residues forming the heme channel edge were removed in single and multiple variants. Among them, the E140G/K176G, E140G/P141G/K176G, and E140G/W164S/K176G variants attained catalytic efficiencies for oxidation of 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate) at the heme channel similar to those of the exposed tryptophan site. The heme channel enlargement shown by x-ray diffraction of the E140G, P141G, K176G, and E140G/K176G variants would allow a better substrate accommodation near the heme, as revealed by the up to 26-fold lower Km values (compared with native VP). The resulting interactions were shown by the x-ray structure of the E140G-guaiacol complex, which includes two H-bonds of the substrate with Arg-43 and Pro-139 in the distal heme pocket (at the end of the heme channel) and several hydrophobic interactions with other residues and the heme cofactor. PMID:23071108

Crystal structure of the S187F variant of human liver alanine: Aminotransferase associated with primary hyperoxaluria type I and its functional implications

PubMed Central

Oppici, Elisa; Fodor, Krisztian; Paiardini, Alessandro; Williams, Chris; Voltattorni, Carla Borri; Wilmanns, Matthias; Cellini, Barbara

2013-01-01

The substitution of Ser187, a residue located far from the active site of human liver peroxisomal alanine:glyoxylate aminotransferase (AGT), by Phe gives rise to a variant associated with primary hyperoxaluria type I. Unexpectedly, previous studies revealed that the recombinant form of S187F exhibits a remarkable loss of catalytic activity, an increased pyridoxal 5′-phosphate (PLP) binding affinity and a different coenzyme binding mode compared with normal AGT. To shed light on the structural elements responsible for these defects, we solved the crystal structure of the variant to a resolution of 2.9 Å. Although the overall conformation of the variant is similar to that of normal AGT, we noticed: (i) a displacement of the PLP-binding Lys209 and Val185, located on the re and si side of PLP, respectively, and (ii) slight conformational changes of other active site residues, in particular Trp108, the base stacking residue with the pyridine cofactor moiety. This active site perturbation results in a mispositioning of the AGT-pyridoxamine 5′-phosphate (PMP) complex and of the external aldimine, as predicted by molecular modeling studies. Taken together, both predicted and observed movements caused by the S187F mutation are consistent with the following functional properties of the variant: (i) a 300- to 500-fold decrease in both the rate constant of L-alanine half-transamination and the kcat of the overall transamination, (ii) a different PMP binding mode and affinity, and (iii) a different microenvironment of the external aldimine. Proposals for the treatment of patients bearing S187F mutation are discussed on the basis of these results. Proteins 2013; 81:1457–1465. © 2013 Wiley Periodicals, Inc. PMID:23589421

Leveraging cross-species transcription factor binding site patterns: from diabetes risk loci to disease mechanisms.

PubMed

Claussnitzer, Melina; Dankel, Simon N; Klocke, Bernward; Grallert, Harald; Glunk, Viktoria; Berulava, Tea; Lee, Heekyoung; Oskolkov, Nikolay; Fadista, Joao; Ehlers, Kerstin; Wahl, Simone; Hoffmann, Christoph; Qian, Kun; Rönn, Tina; Riess, Helene; Müller-Nurasyid, Martina; Bretschneider, Nancy; Schroeder, Timm; Skurk, Thomas; Horsthemke, Bernhard; Spieler, Derek; Klingenspor, Martin; Seifert, Martin; Kern, Michael J; Mejhert, Niklas; Dahlman, Ingrid; Hansson, Ola; Hauck, Stefanie M; Blüher, Matthias; Arner, Peter; Groop, Leif; Illig, Thomas; Suhre, Karsten; Hsu, Yi-Hsiang; Mellgren, Gunnar; Hauner, Hans; Laumen, Helmut

2014-01-16

Genome-wide association studies have revealed numerous risk loci associated with diverse diseases. However, identification of disease-causing variants within association loci remains a major challenge. Divergence in gene expression due to cis-regulatory variants in noncoding regions is central to disease susceptibility. We show that integrative computational analysis of phylogenetic conservation with a complexity assessment of co-occurring transcription factor binding sites (TFBS) can identify cis-regulatory variants and elucidate their mechanistic role in disease. Analysis of established type 2 diabetes risk loci revealed a striking clustering of distinct homeobox TFBS. We identified the PRRX1 homeobox factor as a repressor of PPARG2 expression in adipose cells and demonstrate its adverse effect on lipid metabolism and systemic insulin sensitivity, dependent on the rs4684847 risk allele that triggers PRRX1 binding. Thus, cross-species conservation analysis at the level of co-occurring TFBS provides a valuable contribution to the translation of genetic association signals to disease-related molecular mechanisms. Copyright © 2014 Elsevier Inc. All rights reserved.

Reference genotype and exome data from an Australian Aboriginal population for health-based research

PubMed Central

Tang, Dave; Anderson, Denise; Francis, Richard W.; Syn, Genevieve; Jamieson, Sarra E.; Lassmann, Timo; Blackwell, Jenefer M.

2016-01-01

Genetic analyses, including genome-wide association studies and whole exome sequencing (WES), provide powerful tools for the analysis of complex and rare genetic diseases. To date there are no reference data for Aboriginal Australians to underpin the translation of health-based genomic research. Here we provide a catalogue of variants called after sequencing the exomes of 72 Aboriginal individuals to a depth of 20X coverage in ∼80% of the sequenced nucleotides. We determined 320,976 single nucleotide variants (SNVs) and 47,313 insertions/deletions using the Genome Analysis Toolkit. We had previously genotyped a subset of the Aboriginal individuals (70/72) using the Illumina Omni2.5 BeadChip platform and found ~99% concordance at overlapping sites, which suggests high quality genotyping. Finally, we compared our SNVs to six publicly available variant databases, such as dbSNP and the Exome Sequencing Project, and 70,115 of our SNVs did not overlap any of the single nucleotide polymorphic sites in all the databases. Our data set provides a useful reference point for genomic studies on Aboriginal Australians. PMID:27070114

Reference genotype and exome data from an Australian Aboriginal population for health-based research.

PubMed

Tang, Dave; Anderson, Denise; Francis, Richard W; Syn, Genevieve; Jamieson, Sarra E; Lassmann, Timo; Blackwell, Jenefer M

2016-04-12

Genetic analyses, including genome-wide association studies and whole exome sequencing (WES), provide powerful tools for the analysis of complex and rare genetic diseases. To date there are no reference data for Aboriginal Australians to underpin the translation of health-based genomic research. Here we provide a catalogue of variants called after sequencing the exomes of 72 Aboriginal individuals to a depth of 20X coverage in ∼80% of the sequenced nucleotides. We determined 320,976 single nucleotide variants (SNVs) and 47,313 insertions/deletions using the Genome Analysis Toolkit. We had previously genotyped a subset of the Aboriginal individuals (70/72) using the Illumina Omni2.5 BeadChip platform and found ~99% concordance at overlapping sites, which suggests high quality genotyping. Finally, we compared our SNVs to six publicly available variant databases, such as dbSNP and the Exome Sequencing Project, and 70,115 of our SNVs did not overlap any of the single nucleotide polymorphic sites in all the databases. Our data set provides a useful reference point for genomic studies on Aboriginal Australians.

Friedreich's Ataxia Variants I154F and W155R Diminish Frataxin-Based Activation of the Iron-Sulfur Cluster Assembly Complex

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tsai, Chi-Lin; Bridwell-Rabb, Jennifer; Barondeau, David P

2011-11-07

Friedreich's ataxia (FRDA) is a progressive neurodegenerative disease that has been linked to defects in the protein frataxin (Fxn). Most FRDA patients have a GAA expansion in the first intron of their Fxn gene that decreases protein expression. Some FRDA patients have a GAA expansion on one allele and a missense mutation on the other allele. Few functional details are known for the ~15 different missense mutations identified in FRDA patients. Here in vitro evidence is presented that indicates the FRDA I154F and W155R variants bind more weakly to the complex of Nfs1, Isd11, and Isu2 and thereby are defectivemore » in forming the four-component SDUF complex that constitutes the core of the Fe-S cluster assembly machine. The binding affinities follow the trend Fxn ~ I154F > W155F > W155A ~ W155R. The Fxn variants also have diminished ability to function as part of the SDUF complex to stimulate the cysteine desulfurase reaction and facilitate Fe-S cluster assembly. Four crystal structures, including the first for a FRDA variant, reveal specific rearrangements associated with the loss of function and lead to a model for Fxn-based activation of the Fe-S cluster assembly complex. Importantly, the weaker binding and lower activity for FRDA variants correlate with the severity of disease progression. Together, these results suggest that Fxn facilitates sulfur transfer from Nfs1 to Isu2 and that these in vitro assays are sensitive and appropriate for deciphering functional defects and mechanistic details for human Fe-S cluster biosynthesis.« less

Genetic variations in the DNA replication origins of human papillomavirus family correlate with their oncogenic potential.

PubMed

Yilmaz, Gulden; Biswas-Fiss, Esther E; Biswas, Subhasis B

2018-04-01

Human papillomaviruses (HPVs) encompass a large family of viruses that range from benign to highly carcinogenic. The crucial differences between benign and carcinogenic types of HPV remain unknown, except that the two HPV types differ in the frequency of DNA replication. We have systematically analyzed the mechanism of HPV DNA replication initiation in low-risk and high-risk HPVs. Our results demonstrate that HPV-encoded E2 initiator protein and its four binding sites in the replication origin play pivotal roles in determining the destiny of the HPV-infected cell. We have identified strain-specific single nucleotide variations in E2 binding sites found only in the high-risk HPVs. We have demonstrated that these variations result in attenuated formation of the E2-DNA complex. E2 binding to these sites is linked to the activation of the DNA replication origin as well as initiation of DNA replication. Both electrophoretic mobility shift assay and atomic force microscopy studies demonstrated that binding of E2 from either low- or high-risk HPVs with variant binding sequences lacked multimeric E2-DNA complex formation in vitro. These results provided a molecular basis of differential DNA replication in the two types of HPVs and pointed to a correlation with the development of cancer. Copyright © 2017. Published by Elsevier B.V.

Extending Thymidine Kinase Activity to the Catalytic Repertoire of Human Deoxycytidine Kinase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hazra, Saugata; Sabini, Eliszbetta; Ort, Stephan

Salvage of nucleosides in the cytosol of human cells is carried out by deoxycytidine kinase (dCK) and thymidine kinase 1 (TK1). Whereas TK1 is only responsible for thymidine phosphorylation, dCK is capable of converting dC, dA, and dG into their monophosphate forms. Using structural data on dCK, we predicted that select mutations at the active site would, in addition to making the enzyme faster, expand the catalytic repertoire of dCK to include thymidine. Specifically, we hypothesized that steric repulsion between the methyl group of the thymine base and Arg104 is the main factor preventing the phosphorylation of thymidine by wild-typemore » dCK. Here we present kinetic data on several dCK variants where Arg104 has been replaced by select residues, all performed in combination with the mutation of Asp133 to an alanine. We show that several hydrophobic residues at position 104 endow dCK with thymidine kinase activity. Depending on the exact nature of the mutations, the enzyme's substrate preference is modified. The R104M-D133A double mutant is a pyrimidine-specific enzyme due to large K{sub m} values with purines. The crystal structure of the double mutant R104M-D133A in complex with the L-form of thymidine supplies a structural explanation for the ability of this variant to phosphorylate thymidine and thymidine analogs. The replacement of Arg104 by a smaller residue allows L-dT to bind deeper into the active site, making space for the C5-methyl group of the thymine base. The unique catalytic properties of several of the mutants make them good candidates for suicide-gene/protein-therapy applications.« less

Kinetics of Mismatch Formation opposite Lesions by the Replicative DNA Polymerase from Bacteriophage RB69

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hogg, Matthew; Rudnicki, Jean; Midkiff, John

2010-04-12

The fidelity of DNA replication is under constant threat from the formation of lesions within the genome. Oxidation of DNA bases leads to the formation of altered DNA bases such as 8-oxo-7,8-dihydroguanine, commonly called 8-oxoG, and 2-hydroxyadenenine, or 2-OHA. In this work we have examined the incorporation kinetics opposite these two oxidatively derived lesions as well as an abasic site analogue by the replicative DNA polymerase from bacteriophage RB69. We compared the kinetic parameters for both wild type and the low fidelity L561A variant. While nucleotide incorporation rates (k{sub pol}) were generally higher for the variant, the presence of amore » lesion in the templating position reduced the ability of both the wild-type and variant DNA polymerases to form ternary enzyme-DNA-dNTP complexes. Thus, the L561A substitution does not significantly affect the ability of the RB69 DNA polymerase to recognize damaged DNA; instead, the mutation increases the probability that nucleotide incorporation will occur. We have also solved the crystal structure of the L561A variant forming an 8-oxoG {center_dot} dATP mispair and show that the propensity for forming this mispair depends on an enlarged polymerase active site.« less

Comprehensive analysis of the mutation spectrum in 301 German ALS families.

PubMed

Müller, Kathrin; Brenner, David; Weydt, Patrick; Meyer, Thomas; Grehl, Torsten; Petri, Susanne; Grosskreutz, Julian; Schuster, Joachim; Volk, Alexander E; Borck, Guntram; Kubisch, Christian; Klopstock, Thomas; Zeller, Daniel; Jablonka, Sibylle; Sendtner, Michael; Klebe, Stephan; Knehr, Antje; Günther, Kornelia; Weis, Joachim; Claeys, Kristl G; Schrank, Berthold; Sperfeld, Anne-Dorte; Hübers, Annemarie; Otto, Markus; Dorst, Johannes; Meitinger, Thomas; Strom, Tim M; Andersen, Peter M; Ludolph, Albert C; Weishaupt, Jochen H

2018-04-12

Recent advances in amyotrophic lateral sclerosis (ALS) genetics have revealed that mutations in any of more than 25 genes can cause ALS, mostly as an autosomal-dominant Mendelian trait. Detailed knowledge about the genetic architecture of ALS in a specific population will be important for genetic counselling but also for genotype-specific therapeutic interventions. Here we combined fragment length analysis, repeat-primed PCR, Southern blotting, Sanger sequencing and whole exome sequencing to obtain a comprehensive profile of genetic variants in ALS disease genes in 301 German pedigrees with familial ALS. We report C9orf72 mutations as well as variants in consensus splice sites and non-synonymous variants in protein-coding regions of ALS genes. We furthermore estimate their pathogenicity by taking into account type and frequency of the respective variant as well as segregation within the families. 49% of our German ALS families carried a likely pathogenic variant in at least one of the earlier identified ALS genes. In 45% of the ALS families, likely pathogenic variants were detected in C9orf72, SOD1, FUS, TARDBP or TBK1 , whereas the relative contribution of the other ALS genes in this familial ALS cohort was 4%. We identified several previously unreported rare variants and demonstrated the absence of likely pathogenic variants in some of the recently described ALS disease genes. We here present a comprehensive genetic characterisation of German familial ALS. The present findings are of importance for genetic counselling in clinical practice, for molecular research and for the design of diagnostic gene panels or genotype-specific therapeutic interventions in Europe. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

Stabilization of bacterially expressed erythropoietin by single site-specific introduction of short branched PEG chains at naturally occurring glycosylation sites.

PubMed

Hoffmann, E; Streichert, K; Nischan, N; Seitz, C; Brunner, T; Schwagerus, S; Hackenberger, C P R; Rubini, M

2016-05-24

The covalent attachment of polyethylene glycol (PEG) to therapeutic proteins can improve their physicochemical properties. In this work we utilized the non-natural amino acid p-azidophenylalanine (pAzF) in combination with the chemoselective Staudinger-phosphite reaction to install branched PEG chains to recombinant unglycosylated erythropoietin (EPO) at each single naturally occurring glycosylation site. PEGylation with two short 750 or 2000 Da PEG units at positions 24, 38, or 83 significantly decreased unspecific aggregation and proteolytic degradation while biological activity in vitro was preserved or even increased in comparison to full-glycosylated EPO. This site-specific bioconjugation approach permits to analyse the impact of PEGylation at single positions. These results represent an important step towards the engineering of site-specifically modified EPO variants from bacterial expression with increased therapeutic efficacy.

Biocatalytic Conversion of Avermectin to 4″-Oxo-Avermectin: Improvement of Cytochrome P450 Monooxygenase Specificity by Directed Evolution▿ †

PubMed Central

Trefzer, Axel; Jungmann, Volker; Molnár, István; Botejue, Ajit; Buckel, Dagmar; Frey, Gerhard; Hill, D. Steven; Jörg, Mario; Ligon, James M.; Mason, Dylan; Moore, David; Pachlatko, J. Paul; Richardson, Toby H.; Spangenberg, Petra; Wall, Mark A.; Zirkle, Ross; Stege, Justin T.

2007-01-01

Discovery of the CYP107Z subfamily of cytochrome P450 oxidases (CYPs) led to an alternative biocatalytic synthesis of 4″-oxo-avermectin, a key intermediate for the commercial production of the semisynthetic insecticide emamectin. However, under industrial process conditions, these wild-type CYPs showed lower yields due to side product formation. Molecular evolution employing GeneReassembly was used to improve the regiospecificity of these enzymes by a combination of random mutagenesis, protein structure-guided site-directed mutagenesis, and recombination of multiple natural and synthetic CYP107Z gene fragments. To assess the specificity of CYP mutants, a miniaturized, whole-cell biocatalytic reaction system that allowed high-throughput screening of large numbers of variants was developed. In an iterative process consisting of four successive rounds of GeneReassembly evolution, enzyme variants with significantly improved specificity for the production of 4″-oxo-avermectin were identified; these variants could be employed for a more economical industrial biocatalytic process to manufacture emamectin. PMID:17483257

Genomic variants in the FTO gene are associated with sporadic amyotrophic lateral sclerosis in Greek patients.

PubMed

Mitropoulos, Konstantinos; Merkouri Papadima, Eleni; Xiromerisiou, Georgia; Balasopoulou, Angeliki; Charalampidou, Kyriaki; Galani, Vasiliki; Zafeiri, Krystallia-Vassiliki; Dardiotis, Efthymios; Ralli, Styliani; Deretzi, Georgia; John, Anne; Kydonopoulou, Kyriaki; Papadopoulou, Elpida; di Pardo, Alba; Akcimen, Fulya; Loizedda, Annalisa; Dobričić, Valerija; Novaković, Ivana; Kostić, Vladimir S; Mizzi, Clint; Peters, Brock A; Basak, Nazli; Orrù, Sandro; Kiskinis, Evangelos; Cooper, David N; Gerou, Spyridon; Drmanac, Radoje; Bartsakoulia, Marina; Tsermpini, Evangelia-Eirini; Hadjigeorgiou, Georgios M; Ali, Bassam R; Katsila, Theodora; Patrinos, George P

2017-12-08

Amyotrophic lateral sclerosis (ALS) is a devastating disease whose complex pathology has been associated with a strong genetic component in the context of both familial and sporadic disease. Herein, we adopted a next-generation sequencing approach to Greek patients suffering from sporadic ALS (together with their healthy counterparts) in order to explore further the genetic basis of sporadic ALS (sALS). Whole-genome sequencing analysis of Greek sALS patients revealed a positive association between FTO and TBC1D1 gene variants and sALS. Further, linkage disequilibrium analyses were suggestive of a specific disease-associated haplotype for FTO gene variants. Genotyping for these variants was performed in Greek, Sardinian, and Turkish sALS patients. A lack of association between FTO and TBC1D1 variants and sALS in patients of Sardinian and Turkish descent may suggest a founder effect in the Greek population. FTO was found to be highly expressed in motor neurons, while in silico analyses predicted an impact on FTO and TBC1D1 mRNA splicing for the genomic variants in question. To our knowledge, this is the first study to present a possible association between FTO gene variants and the genetic etiology of sALS. In addition, the next-generation sequencing-based genomics approach coupled with the two-step validation strategy described herein has the potential to be applied to other types of human complex genetic disorders in order to identify variants of clinical significance.

Whole genome sequencing and imputation in isolated populations identify genetic associations with medically-relevant complex traits

PubMed Central

Southam, Lorraine; Gilly, Arthur; Süveges, Dániel; Farmaki, Aliki-Eleni; Schwartzentruber, Jeremy; Tachmazidou, Ioanna; Matchan, Angela; Rayner, Nigel W.; Tsafantakis, Emmanouil; Karaleftheri, Maria; Xue, Yali; Dedoussis, George; Zeggini, Eleftheria

2017-01-01

Next-generation association studies can be empowered by sequence-based imputation and by studying founder populations. Here we report ∼9.5 million variants from whole-genome sequencing (WGS) of a Cretan-isolated population, and show enrichment of rare and low-frequency variants with predicted functional consequences. We use a WGS-based imputation approach utilizing 10,422 reference haplotypes to perform genome-wide association analyses and observe 17 genome-wide significant, independent signals, including replicating evidence for association at eight novel low-frequency variant signals. Two novel cardiometabolic associations are at lead variants unique to the founder population sequences: chr16:70790626 (high-density lipoprotein levels beta −1.71 (SE 0.25), P=1.57 × 10−11, effect allele frequency (EAF) 0.006); and rs145556679 (triglycerides levels beta −1.13 (SE 0.17), P=2.53 × 10−11, EAF 0.013). Our findings add empirical support to the contribution of low-frequency variants in complex traits, demonstrate the advantage of including population-specific sequences in imputation panels and exemplify the power gains afforded by population isolates. PMID:28548082

NMR analysis of cleaved Escherichia coli thioredoxin (1-73/74-108) and its P76A variant: cis/trans peptide isomerization.

PubMed Central

Yu, W. F.; Tung, C. S.; Wang, H.; Tasayco, M. L.

2000-01-01

Inspection of high resolution three-dimensional (3D) structures from the protein database reveals an increasing number of cis-Xaa-Pro and cis-Xaa-Yaa peptide bonds. However, we are still far from being able to predict whether these bonds will remain cis upon single-site substitution of Pro or Yaa and/or cleavage of a peptide bond close to it in the sequence. We have chosen oxidized Escherichia coli thioredoxin (Trx), a member of the Trx superfamily with a single alpha/beta domain and cis P76 to determine the effect of single-site substitution and/or cleavage on this isomer. Standard two-dimensional (2D) NMR analysis were performed on cleaved Trx (1-73/74-108) and its P76A variant. Analysis of the NOE connectivities indicates remarkable similarity between the secondary and supersecondary structure of the noncovalent complexes and Trx. Analysis of the 2D version of the HCCH-TOCSY and HMQC-NOESY-HMQC and 13C-filtered HMQC-NOESY spectra of cleaved Trx with uniformly 13C-labeled 175 and P76 shows surprising conservation of both cis P76 and packing of 175 against W31. A similar NMR analysis of its P76A variant provides no evidence for cis A76 and shows only subtle local changes in both the packing of 175 and the interstrand connectivities between its most protected hydrophobic strands (beta2 and beta4). Indeed, a molecular simulation model for the trans P76A variant of Trx shows only subtle local changes around the substitution site. In conclusion, cleavage of R73 is insufficient to provoke cis/trans isomerization of P76, but cleavage and single-site substitution (P76A) favors the trans isomer. PMID:10739243

Engineered Cpf1 variants with altered PAM specificities.

PubMed

Gao, Linyi; Cox, David B T; Yan, Winston X; Manteiga, John C; Schneider, Martin W; Yamano, Takashi; Nishimasu, Hiroshi; Nureki, Osamu; Crosetto, Nicola; Zhang, Feng

2017-08-01

The RNA-guided endonuclease Cpf1 is a promising tool for genome editing in eukaryotic cells. However, the utility of the commonly used Acidaminococcus sp. BV3L6 Cpf1 (AsCpf1) and Lachnospiraceae bacterium ND2006 Cpf1 (LbCpf1) is limited by their requirement of a TTTV protospacer adjacent motif (PAM) in the DNA substrate. To address this limitation, we performed a structure-guided mutagenesis screen to increase the targeting range of Cpf1. We engineered two AsCpf1 variants carrying the mutations S542R/K607R and S542R/K548V/N552R, which recognize TYCV and TATV PAMs, respectively, with enhanced activities in vitro and in human cells. Genome-wide assessment of off-target activity using BLISS indicated that these variants retain high DNA-targeting specificity, which we further improved by introducing an additional non-PAM-interacting mutation. Introducing the identified PAM-interacting mutations at their corresponding positions in LbCpf1 similarly altered its PAM specificity. Together, these variants increase the targeting range of Cpf1 by approximately threefold in human coding sequences to one cleavage site per ∼11 bp.

Engineered Cpf1 variants with altered PAM specificities increase genome targeting range

PubMed Central

Gao, Linyi; Cox, David B.T.; Yan, Winston X.; Manteiga, John C.; Schneider, Martin W.; Yamano, Takashi; Nishimasu, Hiroshi; Nureki, Osamu; Crosetto, Nicola; Zhang, Feng

2017-01-01

The RNA-guided endonuclease Cpf1 is a promising tool for genome editing in eukaryotic cells1–7. However, the utility of the commonly used Acidaminococcus sp. BV3L6 Cpf1 (AsCpf1) and Lachnospiraceae bacterium ND2006 Cpf1 (LbCpf1) is limited by their requirement of a TTTV protospacer adjacent motif (PAM) in the DNA substrate. To address this limitation, we performed a structure-guided mutagenesis screen to increase the targeting range of Cpf1. We engineered two AsCpf1 variants carrying the mutations S542R/K607R and S542R/K548V/N552R, which recognize TYCV and TATV PAMs, respectively, with enhanced activities in vitro and in human cells. Genome-wide assessment of off-target activity using BLISS7 assay indicated that these variants retain high DNA targeting specificity, which we further improved by introducing an additional non-PAM-interacting mutation. Introducing the identified mutations at their corresponding positions in LbCpf1 similarly altered its PAM specificity. Together, these variants increase the targeting range of Cpf1 by approximately three-fold in human coding sequences to one cleavage site per ~11 bp. PMID:28581492

Identification of hemoglobin variants by top-down mass spectrometry using selected diagnostic product ions.

PubMed

Coelho Graça, Didia; Hartmer, Ralf; Jabs, Wolfgang; Beris, Photis; Clerici, Lorella; Stoermer, Carsten; Samii, Kaveh; Hochstrasser, Denis; Tsybin, Yury O; Scherl, Alexander; Lescuyer, Pierre

2015-04-01

Hemoglobin disorder diagnosis is a complex procedure combining several analytical steps. Due to the lack of specificity of the currently used protein analysis methods, the identification of uncommon hemoglobin variants (proteoforms) can become a hard task to accomplish. The aim of this work was to develop a mass spectrometry-based approach to quickly identify mutated protein sequences within globin chain variants. To reach this goal, a top-down electron transfer dissociation mass spectrometry method was developed for hemoglobin β chain analysis. A diagnostic product ion list was established with a color code strategy allowing to quickly and specifically localize a mutation in the hemoglobin β chain sequence. The method was applied to the analysis of rare hemoglobin β chain variants and an (A)γ-β fusion protein. The results showed that the developed data analysis process allows fast and reliable interpretation of top-down electron transfer dissociation mass spectra by nonexpert users in the clinical area.

Characterization of Squalene Epoxidase of Saccharomyces cerevisiae by Applying Terbinafine-Sensitive Variants▿

PubMed Central

Ruckenstuhl, Christoph; Lang, Silvia; Poschenel, Andrea; Eidenberger, Armin; Baral, Pravas Kumar; Kohút, Peter; Hapala, Ivan; Gruber, Karl; Turnowsky, Friederike

2007-01-01

Squalene epoxidase (SE) is the target of terbinafine, which specifically inhibits the fungal enzyme in a noncompetitive manner. On the basis of functional homologies to p-hydroxybenzoate hydroxylase (PHBH) from Pseudomonas fluorescens, the Erg1 protein contains two flavin adenine dinucleotide (FAD) domains and one nucleotide binding (NB) site. By in vitro mutagenesis of the ERG1 gene, which codes for the Saccharomyces cerevisiae SE, we isolated erg1 alleles that conferred increased terbinafine sensitivity or that showed a lethal phenotype when they were expressed in erg1-knockout strain KLN1. All but one of the amino acid substitutions affected conserved FAD/nucleotide binding sites. The G25S, D335X (W, F, P), and G210A substitutions in the FADI, FADII, and NB sites, respectively, rendered the SE variants nonfunctional. The G30S and L37P variants exhibited decreased enzymatic activity, accompanied by a sevenfold increase in erg1 mRNA levels and an altered sterol composition, and rendered KLN1 more sensitive not only to allylamines (10 to 25 times) but also to other ergosterol biosynthesis inhibitors. The R269G variant exhibited moderately reduced SE activity and a 5- to 10-fold increase in allylamine sensitivity but no cross-sensitivity to the other ergosterol biosynthesis inhibitors. To further elucidate the roles of specific amino acids in SE function and inhibitor interaction, a homology model of Erg1p was built on the basis of the crystal structure of PHBH. All experimental data obtained with the sensitive Erg1 variants support this model. In addition, the amino acids responsible for terbinafine resistance, although they are distributed along the sequence of Erg1p, cluster on the surface of the Erg1p model, giving rise to a putative binding site for allylamines. PMID:17043127

The genomic landscape of rapid repeated evolutionary ...

EPA Pesticide Factsheets

Atlantic killifish populations have rapidly adapted to normally lethal levels of pollution in four urban estuaries. Through analysis of 384 whole killifish genome sequences and comparative transcriptomics in four pairs of sensitive and tolerant populations, we identify the aryl hydrocarbon receptor–based signaling pathway as a shared target of selection. This suggests evolutionary constraint on adaptive solutions to complex toxicant mixtures at each site. However, distinct molecular variants apparently contribute to adaptive pathway modification among tolerant populations. Selection also targets other toxicity-mediatinggenes and genes of connected signaling pathways; this indicates complex tolerance phenotypes and potentially compensatory adaptations. Molecular changes are consistent with selection on standing genetic variation. In killifish, high nucleotide diversityhas likely been a crucial substrate for selective sweeps to propel rapid adaptation. This manuscript describes genomic evaluations that contribute to our understanding of the ecological and evolutionary risks associated with chronic contaminant exposures to wildlife populations. Here, we assessed genetic patterns associated with long-term response to an important class of highly toxic environmental pollutants. Specifically, chemical-specific tolerance has rapidly and repeatedly evolved in an estuarine fish species resident to estuaries of the Atlantic U.S. coast. We used laboratory studies to ch

A population-specific uncommon variant in GRIN3A associated with schizophrenia.

PubMed

Takata, Atsushi; Iwayama, Yoshimi; Fukuo, Yasuhisa; Ikeda, Masashi; Okochi, Tomo; Maekawa, Motoko; Toyota, Tomoko; Yamada, Kazuo; Hattori, Eiji; Ohnishi, Tetsuo; Toyoshima, Manabu; Ujike, Hiroshi; Inada, Toshiya; Kunugi, Hiroshi; Ozaki, Norio; Nanko, Shinichiro; Nakamura, Kazuhiko; Mori, Norio; Kanba, Shigenobu; Iwata, Nakao; Kato, Tadafumi; Yoshikawa, Takeo

2013-03-15

Genome-wide association studies have successfully identified several common variants showing robust association with schizophrenia. However, individually, these variants only produce a weak effect. To identify genetic variants with larger effect sizes, increasing attention is now being paid to uncommon and rare variants. From the 1000 Genomes Project data, we selected 47 candidate single nucleotide variants (SNVs), which were: 1) uncommon (minor allele frequency < 5%); 2) Asian-specific; 3) missense, nonsense, or splice site variants predicted to be damaging; and 4) located in candidate genes for schizophrenia and bipolar disorder. We examined their association with schizophrenia, using a Japanese case-control cohort (2012 cases and 2781 control subjects). Additional meta-analysis was performed using genotyping data from independent Han-Chinese case-control (333 cases and 369 control subjects) and family samples (9 trios and 284 quads). We identified disease association of a missense variant in GRIN3A (p.R480G, rs149729514, p = .00042, odds ratio [OR] = 1.58), encoding a subunit of the N-methyl-D-aspartate type glutamate receptor, with study-wide significance (threshold p = .0012). This association was supported by meta-analysis (combined p = 3.3 × 10(-5), OR = 1.61). Nominally significant association was observed in missense variants from FAAH, DNMT1, MYO18B, and CFB, with ORs of risk alleles ranging from 1.41 to 2.35. The identified SNVs, particularly the GRIN3A R480G variant, are good candidates for further replication studies and functional evaluation. The results of this study indicate that association analyses focusing on uncommon and rare SNVs are a promising way to discover risk variants with larger effects. Copyright © 2013 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.

Natural selection on marine carnivores elaborated a diverse family of classical MHC class I genes exhibiting haplotypic gene content variation and allelic polymorphism

PubMed Central

Norman, Paul J.; Parham, Peter

2012-01-01

Pinnipeds, marine carnivores, diverged from terrestrial carnivores ~45 million years ago, before their adaptation to marine environments. This lifestyle change exposed pinnipeds to different microbiota and pathogens, with probable impact on their MHC class I genes. Investigating this question, genomic sequences were determined for 71 MHC class I variants: 27 from harbor seal and 44 from gray seal. These variants form three MHC class I gene lineages, one comprising a pseudogene. The second, a candidate nonclassical MHC class I gene, comprises a nonpolymorphic transcribed gene related to dog DLA-79 and giant panda Aime-1906. The third is the diversity lineage, which includes 62 of the 71 seal MHC class I variants. All are transcribed, and they minimally represent six harbor and 12 gray seal MHC class I genes. Besides species-specific differences in gene number, seal MHC class I haplotypes exhibit gene content variation and allelic polymorphism. Patterns of sequence variation, and of positions for positively selected sites, indicate the diversity lineage genes are the seals’ classical MHC class I genes. Evidence that expansion of diversity lineage genes began before gray and harbor seals diverged is the presence in both species of two distinctive sublineages of diversity lineage genes. Pointing to further expansion following the divergence are the presence of species-specific genes and greater MHC class I diversity in gray seals than harbor seals. The elaboration of a complex variable family of classical MHC class I genes in pinnipeds contrasts with the single, highly polymorphic classical MHC class I gene of dog and giant panda, terrestrial carnivores. PMID:23001684

Antigenic variants of yellow fever virus with an altered neurovirulence phenotype in mice.

PubMed

Ryman, K D; Xie, H; Ledger, T N; Campbell, G A; Barrett, A D

1997-04-14

The live-attenuated yellow fever (YF) vaccine virus, strain 17D-204, has long been known to consist of a heterologous population of virions. Gould et al. (J. Gen. Virol. 70, 1889-1894 (1989)) previously demonstrated that variant viruses exhibiting a YF wild-type-specific envelope (E) protein epitope are present at low frequency in the vaccine pool and were able to isolate representative virus variants with and without this epitope, designated 17D(+wt) and 17D(-wt), respectively. These variants were employed here in an investigation of YF virus pathogenesis in the mouse model. Both the 17D-204 parent and the 17D(+wt) variant viruses were lethal for adult outbred mice by the intracerebral route of inoculation. However, the 17D(-wt) variant was significantly attenuated (18% mortality rate) and replicated to much lower titer in the brains of infected mice. A single amino acid substitution in the envelope (E) protein at E-240 (Ala-->Val) was identified as responsible for the restricted replication of the 17D(-wt) variant in vivo. The 17D(+wt) variant has an additional second-site mutation, believed to encode a reversion to the neurovirulence phenotype of the 17D-204 parent virus. The amino acid substitution in the E protein at E-173 (Thr-->Ile) of the 17D(+wt) variant which results in the appearance of the wild-type-specific epitope or nucleotide changes in the 5' and 3' noncoding regions of the virus are proposed as a candidates.

C1q-targeted inhibition of the classical complement pathway prevents injury in a novel mouse model of acute motor axonal neuropathy.

PubMed

McGonigal, Rhona; Cunningham, Madeleine E; Yao, Denggao; Barrie, Jennifer A; Sankaranarayanan, Sethu; Fewou, Simon N; Furukawa, Koichi; Yednock, Ted A; Willison, Hugh J

2016-03-02

Guillain-Barré syndrome (GBS) is an autoimmune disease that results in acute paralysis through inflammatory attack on peripheral nerves, and currently has limited, non-specific treatment options. The pathogenesis of the acute motor axonal neuropathy (AMAN) variant is mediated by complement-fixing anti-ganglioside antibodies that directly bind and injure the axon at sites of vulnerability such as nodes of Ranvier and nerve terminals. Consequently, the complement cascade is an attractive target to reduce disease severity. Recently, C5 complement component inhibitors that block the formation of the membrane attack complex and subsequent downstream injury have been shown to be efficacious in an in vivo anti-GQ1b antibody-mediated mouse model of the GBS variant Miller Fisher syndrome (MFS). However, since gangliosides are widely expressed in neurons and glial cells, injury in this model was not targeted exclusively to the axon and there are currently no pure mouse models for AMAN. Additionally, C5 inhibition does not prevent the production of early complement fragments such as C3a and C3b that can be deleterious via their known role in immune cell and macrophage recruitment to sites of neuronal damage. In this study, we first developed a new in vivo transgenic mouse model of AMAN using mice that express complex gangliosides exclusively in neurons, thereby enabling specific targeting of axons with anti-ganglioside antibodies. Secondly, we have evaluated the efficacy of a novel anti-C1q antibody (M1) that blocks initiation of the classical complement cascade, in both the newly developed anti-GM1 antibody-mediated AMAN model and our established MFS model in vivo. Anti-C1q monoclonal antibody treatment attenuated complement cascade activation and deposition, reduced immune cell recruitment and axonal injury, in both mouse models of GBS, along with improvement in respiratory function. These results demonstrate that neutralising C1q function attenuates injury with a consequent neuroprotective effect in acute GBS models and promises to be a useful new target for human therapy.

Genotype-specific signal generation based on digestion of 3-way DNA junctions: application to KRAS variation detection.

PubMed

Amicarelli, Giulia; Adlerstein, Daniel; Shehi, Erlet; Wang, Fengfei; Makrigiorgos, G Mike

2006-10-01

Genotyping methods that reveal single-nucleotide differences are useful for a wide range of applications. We used digestion of 3-way DNA junctions in a novel technology, OneCutEventAmplificatioN (OCEAN) that allows sequence-specific signal generation and amplification. We combined OCEAN with peptide-nucleic-acid (PNA)-based variant enrichment to detect and simultaneously genotype v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) codon 12 sequence variants in human tissue specimens. We analyzed KRAS codon 12 sequence variants in 106 lung cancer surgical specimens. We conducted a PNA-PCR reaction that suppresses wild-type KRAS amplification and genotyped the product with a set of OCEAN reactions carried out in fluorescence microplate format. The isothermal OCEAN assay enabled a 3-way DNA junction to form between the specific target nucleic acid, a fluorescently labeled "amplifier", and an "anchor". The amplifier-anchor contact contains the recognition site for a restriction enzyme. Digestion produces a cleaved amplifier and generation of a fluorescent signal. The cleaved amplifier dissociates from the 3-way DNA junction, allowing a new amplifier to bind and propagate the reaction. The system detected and genotyped KRAS sequence variants down to approximately 0.3% variant-to-wild-type alleles. PNA-PCR/OCEAN had a concordance rate with PNA-PCR/sequencing of 93% to 98%, depending on the exact implementation. Concordance rate with restriction endonuclease-mediated selective-PCR/sequencing was 89%. OCEAN is a practical and low-cost novel technology for sequence-specific signal generation. Reliable analysis of KRAS sequence alterations in human specimens circumvents the requirement for sequencing. Application is expected in genotyping KRAS codon 12 sequence variants in surgical specimens or in bodily fluids, as well as single-base variations and sequence alterations in other genes.

Insights into mechanisms of bacterial antigenic variation derived from the complete genome sequence of Anaplasma marginale.

PubMed

Palmer, Guy H; Futse, James E; Knowles, Donald P; Brayton, Kelly A

2006-10-01

Persistence of Anaplasma spp. in the animal reservoir host is required for efficient tick-borne transmission of these pathogens to animals and humans. Using A. marginale infection of its natural reservoir host as a model, persistent infection has been shown to reflect sequential cycles in which antigenic variants emerge, replicate, and are controlled by the immune system. Variation in the immunodominant outer-membrane protein MSP2 is generated by a process of gene conversion, in which unique hypervariable region sequences (HVRs) located in pseudogenes are recombined into a single operon-linked msp2 expression site. Although organisms expressing whole HVRs derived from pseudogenes emerge early in infection, long-term persistent infection is dependent on the generation of complex mosaics in which segments from different HVRs recombine into the expression site. The resulting combinatorial diversity generates the number of variants both predicted and shown to emerge during persistence.

Klebsiella aerogenes UreF: Identification of the UreG Binding Site and Role in Enhancing the Fidelity of Urease Activation†

PubMed Central

Boer, Jodi L.; Hausinger, Robert P.

2012-01-01

The Ni-containing active site of Klebsiella aerogenes urease is assembled through the concerted action of the UreD, UreE, UreF, and UreG accessory proteins. UreE functions as a metallochaperone that delivers Ni to a complex of UreD—UreF—UreG bound to urease apoprotein, with UreG serving as a GTPase during enzyme activation. The present study focuses on the role of UreF, previously proposed to act as a GTPase activating protein (GAP) of UreG. Sixteen conserved UreF surface residues that may play roles in protein:protein interactions were independently changed to Ala. When produced in the context of the entire urease gene cluster, cell-free extracts of nine site-directed mutants had less than 10% of the wild-type urease activity. Enrichment of the variant forms of UreF, as the UreE-F fusion proteins, uniformly resulted in co-purification of UreD and urease apoprotein; whereas UreG bound to only a subset of the species. Notably, reduced interaction with UreG correlated with the low activity mutants. The affected residues in UreF map to a distinct surface on the crystal structure, defining the UreG binding site. In contrast to the hypothesis that UreF is a GAP, the UreD—UreF—UreG—urease apoprotein complex containing K165A UreF exhibited significantly greater levels of GTPase activity than that containing the wild-type protein. Additional studies demonstrated the UreG GTPase activity was largely uncoupled from urease activation for the complex containing this UreF variant. Further experiments with these complexes provided evidence that UreF gates the GTPase activity of UreG to enhance the fidelity of urease metallocenter assembly, especially in the presence of the non-cognate metal Zn. PMID:22369361

BMP15 c.-9C>G promoter sequence variant may contribute to the cause of non-syndromic premature ovarian failure.

PubMed

Fonseca, Dora Janeth; Ortega-Recalde, Oscar; Esteban-Perez, Clara; Moreno-Ortiz, Harold; Patiño, Liliana Catherine; Bermúdez, Olga María; Ortiz, Angela María; Restrepo, Carlos M; Lucena, Elkin; Laissue, Paul

2014-11-01

BMP15 has drawn particular attention in the pathophysiology of reproduction, as its mutations in mammalian species have been related to different reproductive phenotypes. In humans, BMP15 coding regions have been sequenced in large panels of women with premature ovarian failure (POF), but only some mutations have been definitely validated as causing the phenotype. A functional association between the BMP15 c.-9C>G promoter polymorphism and cause of POF have been reported. The aim of this study was to determine the potential functional effect of this sequence variant on specific BMP15 promoter transactivation disturbances. Bioinformatics was used to identify transcription factor binding sites located on the promoter region of BMP15. Reverse transcription polymerase chain reaction was used to study specific gene expression in ovarian tissue. Luciferase reporter assays were used to establish transactivation disturbances caused by the BMP15 c.-9C>G variant. The c.-9C>G variant was found to modify the PITX1 transcription factor binding site. PITX1 and BMP15 co-expressed in human and mouse ovarian tissue, and PITX1 transactivated both BMP15 promoter versions (-9C and -9G). It was found that the BMP15 c.-9G allele was related to BMP15 increased transcription, supporting c.-9C>G as a causal agent of POF. Copyright © 2014 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

Kinetic stabilization of Bacillus licheniformis alpha-amylase through introduction of hydrophobic residues at the surface.

PubMed

Machius, Mischa; Declerck, Nathalie; Huber, Robert; Wiegand, Georg

2003-03-28

It is generally assumed that in proteins hydrophobic residues are not favorable at solvent-exposed sites, and that amino acid substitutions on the surface have little effect on protein thermostability. Contrary to these assumptions, we have identified hyperthermostable variants of Bacillus licheniformis alpha-amylase (BLA) that result from the incorporation of hydrophobic residues at the surface. Under highly destabilizing conditions, a variant combining five stabilizing mutations unfolds 32 times more slowly and at a temperature 13 degrees C higher than the wild-type. Crystal structure analysis at 1.7 A resolution suggests that stabilization is achieved through (a) extension of the concept of increased hydrophobic packing, usually applied to cavities, to surface indentations, (b) introduction of favorable aromatic-aromatic interactions on the surface, (c) specific stabilization of intrinsic metal binding sites, and (d) stabilization of a beta-sheet by introducing a residue with high beta-sheet forming propensity. All mutated residues are involved in forming complex, cooperative interaction networks that extend from the interior of the protein to its surface and which may therefore constitute "weak points" where BLA unfolding is initiated. This might explain the unexpectedly large effect induced by some of the substitutions on the kinetic stability of BLA. Our study shows that substantial protein stabilization can be achieved by stabilizing surface positions that participate in underlying cooperatively formed substructures. At such positions, even the apparently thermodynamically unfavorable introduction of hydrophobic residues should be explored.

Characterization of the two intra-individual sequence variants in the 18S rRNA gene in the plant parasitic nematode, Rotylenchulus reniformis.

PubMed

Nyaku, Seloame T; Sripathi, Venkateswara R; Kantety, Ramesh V; Gu, Yong Q; Lawrence, Kathy; Sharma, Govind C

2013-01-01

The 18S rRNA gene is fundamental to cellular and organismal protein synthesis and because of its stable persistence through generations it is also used in phylogenetic analysis among taxa. Sequence variation in this gene within a single species is rare, but it has been observed in few metazoan organisms. More frequently it has mostly been reported in the non-transcribed spacer region. Here, we have identified two sequence variants within the near full coding region of 18S rRNA gene from a single reniform nematode (RN) Rotylenchulus reniformis labeled as reniform nematode variant 1 (RN_VAR1) and variant 2 (RN_VAR2). All sequences from three of the four isolates had both RN variants in their sequences; however, isolate 13B had only RN variant 2 sequence. Specific variable base sites (96 or 5.5%) were found within the 18S rRNA gene that can clearly distinguish the two 18S rDNA variants of RN, in 11 (25.0%) and 33 (75.0%) of the 44 RN clones, for RN_VAR1 and RN_VAR2, respectively. Neighbor-joining trees show that the RN_VAR1 is very similar to the previously existing R. reniformis sequence in GenBank, while the RN_VAR2 sequence is more divergent. This is the first report of the identification of two major variants of the 18S rRNA gene in the same single RN, and documents the specific base variation between the two variants, and hypothesizes on simultaneous co-existence of these two variants for this gene.

Characterization of the Two Intra-Individual Sequence Variants in the 18S rRNA Gene in the Plant Parasitic Nematode, Rotylenchulus reniformis

PubMed Central

Nyaku, Seloame T.; Sripathi, Venkateswara R.; Kantety, Ramesh V.; Gu, Yong Q.; Lawrence, Kathy; Sharma, Govind C.

2013-01-01

The 18S rRNA gene is fundamental to cellular and organismal protein synthesis and because of its stable persistence through generations it is also used in phylogenetic analysis among taxa. Sequence variation in this gene within a single species is rare, but it has been observed in few metazoan organisms. More frequently it has mostly been reported in the non-transcribed spacer region. Here, we have identified two sequence variants within the near full coding region of 18S rRNA gene from a single reniform nematode (RN) Rotylenchulus reniformis labeled as reniform nematode variant 1 (RN_VAR1) and variant 2 (RN_VAR2). All sequences from three of the four isolates had both RN variants in their sequences; however, isolate 13B had only RN variant 2 sequence. Specific variable base sites (96 or 5.5%) were found within the 18S rRNA gene that can clearly distinguish the two 18S rDNA variants of RN, in 11 (25.0%) and 33 (75.0%) of the 44 RN clones, for RN_VAR1 and RN_VAR2, respectively. Neighbor-joining trees show that the RN_VAR1 is very similar to the previously existing R. reniformis sequence in GenBank, while the RN_VAR2 sequence is more divergent. This is the first report of the identification of two major variants of the 18S rRNA gene in the same single RN, and documents the specific base variation between the two variants, and hypothesizes on simultaneous co-existence of these two variants for this gene. PMID:23593343

Exome Sequencing in an Admixed Isolated Population Indicates NFXL1 Variants Confer a Risk for Specific Language Impairment

PubMed Central

Villanueva, Pía; Nudel, Ron; Hoischen, Alexander; Fernández, María Angélica; Simpson, Nuala H.; Gilissen, Christian; Reader, Rose H.; Jara, Lillian; Echeverry, Maria Magdalena; Francks, Clyde; Baird, Gillian; Conti-Ramsden, Gina; O’Hare, Anne; Bolton, Patrick F.; Hennessy, Elizabeth R.; Palomino, Hernán; Carvajal-Carmona, Luis; Veltman, Joris A.; Cazier, Jean-Baptiste; De Barbieri, Zulema

2015-01-01

Children affected by Specific Language Impairment (SLI) fail to acquire age appropriate language skills despite adequate intelligence and opportunity. SLI is highly heritable, but the understanding of underlying genetic mechanisms has proved challenging. In this study, we use molecular genetic techniques to investigate an admixed isolated founder population from the Robinson Crusoe Island (Chile), who are affected by a high incidence of SLI, increasing the power to discover contributory genetic factors. We utilize exome sequencing in selected individuals from this population to identify eight coding variants that are of putative significance. We then apply association analyses across the wider population to highlight a single rare coding variant (rs144169475, Minor Allele Frequency of 4.1% in admixed South American populations) in the NFXL1 gene that confers a nonsynonymous change (N150K) and is significantly associated with language impairment in the Robinson Crusoe population (p = 2.04 × 10–4, 8 variants tested). Subsequent sequencing of NFXL1 in 117 UK SLI cases identified four individuals with heterozygous variants predicted to be of functional consequence. We conclude that coding variants within NFXL1 confer an increased risk of SLI within a complex genetic model. PMID:25781923

A unique H2A histone variant occupies the transcriptional start site of active genes.

PubMed

Soboleva, Tatiana A; Nekrasov, Maxim; Pahwa, Anuj; Williams, Rohan; Huttley, Gavin A; Tremethick, David J

2011-12-04

Transcriptional activation is controlled by chromatin, which needs to be unfolded and remodeled to ensure access to the transcription start site (TSS). However, the mechanisms that yield such an 'open' chromatin structure, and how these processes are coordinately regulated during differentiation, are poorly understood. We identify the mouse (Mus musculus) H2A histone variant H2A.Lap1 as a previously undescribed component of the TSS of active genes expressed during specific stages of spermatogenesis. This unique chromatin landscape also includes a second histone variant, H2A.Z. In the later stages of round spermatid development, H2A.Lap1 dynamically loads onto the inactive X chromosome, enabling the transcriptional activation of previously repressed genes. Mechanistically, we show that H2A.Lap1 imparts unique unfolding properties to chromatin. We therefore propose that H2A.Lap1 coordinately regulates gene expression by directly opening the chromatin structure of the TSS at genes regulated during spermatogenesis.

Characterization of recombinant monoclonal antibody variants detected by hydrophobic interaction chromatography and imaged capillary isoelectric focusing electrophoresis.

PubMed

King, Cory; Patel, Rekha; Ponniah, Gomathinayagam; Nowak, Christine; Neill, Alyssa; Gu, Zhenyu; Liu, Hongcheng

2018-05-15

In-depth characterization of the commonly observed variants is critical to the successful development of recombinant monoclonal antibody therapeutics. Multiple peaks of a recombinant monoclonal antibody were observed when analyzed by hydrophobic interaction chromatography and imaged capillary isoelectric focusing. The potential modification causing the heterogeneity was localized to F(ab')2 region by analyzing the antibody after IdeS digestion using hydrophobic interaction chromatography. LC-MS analysis identified asparagine deamidation as the root cause of the observed multiple variants. While the isoelectric focusing method is expected to separate deamidated species, the similar profile observed in hydrophobic interaction chromatography indicates that the single site deamidation caused differences in hydrophobicity. Forced degradation demonstrated that the susceptible asparagine residue is highly exposed, which is expected as it is located in the light chain complementarity determining region. Deamidation of this single site decreased the mAb binding affinity to its specific antigen. Copyright © 2018 Elsevier B.V. All rights reserved.

Electrochemistry suggests proton access from the exit site to the binuclear center in Paracoccus denitrificans cytochrome c oxidase pathway variants.

PubMed

Meyer, Thomas; Melin, Frédéric; Richter, Oliver-M H; Ludwig, Bernd; Kannt, Aimo; Müller, Hanne; Michel, Hartmut; Hellwig, Petra

2015-02-27

Two different pathways through which protons access cytochrome c oxidase operate during oxygen reduction from the mitochondrial matrix, or the bacterial cytoplasm. Here, we use electrocatalytic current measurements to follow oxygen reduction coupled to proton uptake in cytochrome c oxidase isolated from Paracoccus denitrificans. Wild type enzyme and site-specific variants with defects in both proton uptake pathways (K354M, D124N and K354M/D124N) were immobilized on gold nanoparticles, and oxygen reduction was probed electrochemically in the presence of varying concentrations of Zn(2+) ions, which are known to inhibit both the entry and the exit proton pathways in the enzyme. Our data suggest that under these conditions substrate protons gain access to the oxygen reduction site via the exit pathway. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Population- and individual-specific regulatory variation in Sardinia.

PubMed

Pala, Mauro; Zappala, Zachary; Marongiu, Mara; Li, Xin; Davis, Joe R; Cusano, Roberto; Crobu, Francesca; Kukurba, Kimberly R; Gloudemans, Michael J; Reinier, Frederic; Berutti, Riccardo; Piras, Maria G; Mulas, Antonella; Zoledziewska, Magdalena; Marongiu, Michele; Sorokin, Elena P; Hess, Gaelen T; Smith, Kevin S; Busonero, Fabio; Maschio, Andrea; Steri, Maristella; Sidore, Carlo; Sanna, Serena; Fiorillo, Edoardo; Bassik, Michael C; Sawcer, Stephen J; Battle, Alexis; Novembre, John; Jones, Chris; Angius, Andrea; Abecasis, Gonçalo R; Schlessinger, David; Cucca, Francesco; Montgomery, Stephen B

2017-05-01

Genetic studies of complex traits have mainly identified associations with noncoding variants. To further determine the contribution of regulatory variation, we combined whole-genome and transcriptome data for 624 individuals from Sardinia to identify common and rare variants that influence gene expression and splicing. We identified 21,183 expression quantitative trait loci (eQTLs) and 6,768 splicing quantitative trait loci (sQTLs), including 619 new QTLs. We identified high-frequency QTLs and found evidence of selection near genes involved in malarial resistance and increased multiple sclerosis risk, reflecting the epidemiological history of Sardinia. Using family relationships, we identified 809 segregating expression outliers (median z score of 2.97), averaging 13.3 genes per individual. Outlier genes were enriched for proximal rare variants, providing a new approach to study large-effect regulatory variants and their relevance to traits. Our results provide insight into the effects of regulatory variants and their relationship to population history and individual genetic risk.

Genetic variants of ghrelin in metabolic disorders.

PubMed

Ukkola, Olavi

2011-11-01

An increasing understanding of the role of genes in the development of obesity may reveal genetic variants that, in combination with conventional risk factors, may help to predict an individual's risk for developing metabolic disorders. Accumulating evidence indicates that ghrelin plays a role in regulating food intake and energy homeostasis and it is a reasonable candidate gene for obesity-related co-morbidities. In cross-sectional studies low total ghrelin concentrations and some genetic polymorphisms of ghrelin have been associated with obesity-associated diseases. The present review highlights many of the important problems in association studies of genetic variants and complex diseases. It is known that population-specific differences in reported associations exist. We therefore conclude that more studies on variants of ghrelin gene are needed to perform in different populations to get deeper understanding on the relationship of ghrelin gene and its variants to obesity. Copyright © 2011 Elsevier Inc. All rights reserved.

Multiple conformational states of the hammerhead ribozyme, broad time range of relaxation and topology of dynamics

PubMed Central

Menger, Marcus; Eckstein, Fritz; Porschke, Dietmar

2000-01-01

The dynamics of a hammerhead ribozyme was analyzed by measurements of fluorescence-detected temperature jump relaxation. The ribozyme was substituted at different positions by 2-aminopurine (2-AP) as fluorescence indicator; these substitutions do not inhibit catalysis. The general shape of relaxation curves reported from different positions of the ribozyme is very similar: a fast decrease of fluorescence, mainly due to physical quenching, is followed by a slower increase of fluorescence due to conformational relaxation. In most cases at least three relaxation time constants in the time range from a few microseconds to ~200 ms are required for fitting. Although the relaxation at different positions of the ribozyme is similar in general, suggesting a global type of ribozyme dynamics, a close examination reveals differences, indicating an individual local response. For example, 2-AP in a tetraloop reports mainly the local loop dynamics known from isolated loops, whereas 2-AP located at the core, e.g. at the cleavage site or its vicinity, also reports relatively large amplitudes of slower components of the ribozyme dynamics. A variant with an A→G substitution in domain II, resulting in an inactive form, leads to the appearance of a particularly slow relaxation process (τ ≈200 ms). Addition of Mg2+ ions induces a reduction of amplitudes and in most cases a general increase of time constants. Differences between the hammerhead variants are clearly demonstrated by subtraction of relaxation curves recorded under corresponding conditions. The changes induced in the relaxation response by Mg2+ are very similar to those induced by Ca2+. The relaxation data do not provide any evidence for formation of Mg2+-inner sphere complexes in hammerhead ribozymes, because a Mg2+-specific relaxation effect was not visible. However, a Mg2+-specific effect was found for a dodeca-riboadenylate substituted with 2-AP, showing that the fluorescence of 2-AP is able to indicate inner sphere complexation. Amplitudes and time constants show that the equilibrium constant of inner sphere complexation is 1.2, corresponding to 55% inner sphere state of the Mg2+ complexes; the rate constant 6.6 × 103 s–1 for inner sphere complexation is relatively low and shows the existence of some barrier(s) on the way to inner sphere complexes. PMID:11071929

Variations in paranasal sinus anatomy: implications for the pathophysiology of chronic rhinosinusitis and safety of endoscopic sinus surgery.

PubMed

Nouraei, S A R; Elisay, A R; Dimarco, A; Abdi, R; Majidi, H; Madani, S A; Andrews, P J

2009-02-01

To study the radiologic anatomy of the paranasal sinuses in patients with and without chronic rhinosinusitis to assess whether anatomic variations are associated with disease pathology, and to identify those variants that may impact operative safety. Tertiary referral otolaryngology unit. Incidence and nature of anatomic variants with potential impact on operative safety, and the presence or absence of sinus mucosal disease and its correlation with anatomic variants with a potential impact on mucociliary clearance. We reviewed 278 computed tomographic scans from patients with rhinosinusitis symptoms to investigate anatomic variations that may predispose to sinusitis or impact on operative safety. The incidence of variants with potential impact on sinus drainage was compared between patients with and without sinus mucosal disease with logistic regression. A closed osteomeatal complex was identified in 148 patients (53%), followed by concha bullosa in 98 patients (35%). Closed osteomeatal complex and nasal polyposis were independent risk factors for sinus mucosal disease. Anatomic variants with a potential impact on operative safety included anterior clinoid process pneumatization (18%), infraorbital ethmoid cell (12%), sphenomaxillary plate (11%), and supraorbital recess (6%). In 92% of patients, the level difference between the roof of the ethmoid cavity and the cribriform plate was Keros I. Bony anatomic variants do not increase the risk of sinus mucosal disease. However, anatomic variants with a potential impact on operative safety occur frequently and need to be specifically sought as part of preoperative evaluation.

Structural, kinetic, and thermodynamic studies of specificity designed HIV-1 protease.

PubMed

Alvizo, Oscar; Mittal, Seema; Mayo, Stephen L; Schiffer, Celia A

2012-07-01

HIV-1 protease recognizes and cleaves more than 12 different substrates leading to viral maturation. While these substrates share no conserved motif, they are specifically selected for and cleaved by protease during viral life cycle. Drug resistant mutations evolve within the protease that compromise inhibitor binding but allow the continued recognition of all these substrates. While the substrate envelope defines a general shape for substrate recognition, successfully predicting the determinants of substrate binding specificity would provide additional insights into the mechanism of altered molecular recognition in resistant proteases. We designed a variant of HIV protease with altered specificity using positive computational design methods and validated the design using X-ray crystallography and enzyme biochemistry. The engineered variant, Pr3 (A28S/D30F/G48R), was designed to preferentially bind to one out of three of HIV protease's natural substrates; RT-RH over p2-NC and CA-p2. In kinetic assays, RT-RH binding specificity for Pr3 increased threefold compared to the wild-type (WT), which was further confirmed by isothermal titration calorimetry. Crystal structures of WT protease and the designed variant in complex with RT-RH, CA-p2, and p2-NC were determined. Structural analysis of the designed complexes revealed that one of the engineered substitutions (G48R) potentially stabilized heterogeneous flap conformations, thereby facilitating alternate modes of substrate binding. Our results demonstrate that while substrate specificity could be engineered in HIV protease, the structural pliability of protease restricted the propagation of interactions as predicted. These results offer new insights into the plasticity and structural determinants of substrate binding specificity of the HIV-1 protease. Copyright © 2012 The Protein Society.

Regulation of MVM NS1 by protein kinase C: impact of mutagenesis at consensus phosphorylation sites on replicative functions and cytopathic effects.

PubMed

Corbau, R; Duverger, V; Rommelaere, J; Nüesch, J P

2000-12-05

Minute virus of mice NS1, an 83-kDa mainly nuclear phosphoprotein, is the only viral nonstructural protein required in all cell types and it is involved in multiple processes necessary for virus propagation. The diversity of functions assigned to NS1, together with the variation of its complex phosphorylation pattern during infection, suggested that the various activities of NS1 could be regulated by distinct phosphorylation events. So far, it has been demonstrated that NS1 replicative functions, in particular, DNA-unwinding activities, are regulated by protein kinase C (PKC), as exemplified by the modulation of NS1 helicase activity by PKClambda phosphorylation. In order to determine further impact of phosphorylation on NS1 functions, including the induction of cytopathic effects, a mutational approach was pursued in order to produce NS1 variants harboring amino acid substitutions at candidate PKC target residues. Besides the determination of two additional in vivo phosphorylation sites in NS1, this mutagenesis allowed the segregation of distinct NS1 functions from one another, generating NS1 variants with a distinct activity profile. Thus, we obtained NS1 mutants that were fully proficient for trans activation of the viral P38 promoter, while being impaired in their replicative functions. Moreover, the alterations of specific PKC phosphorylation sites gave rise to NS1 polypeptides that exerted reduced cytotoxicity, leading to sustained gene expression, while keeping functions necessary for progeny virus production, i.e., viral DNA replication and activation of the capsid gene promoter. These data suggested that in the course of a viral infection, NS1 may undergo a shift from productive to cytotoxic functions as a result of a phosphorylation-dependent regulation. Copyright 2000 Academic Press.

Structure-guided evolution of antigenically distinct adeno-associated virus variants for immune evasion.

PubMed

Tse, Longping Victor; Klinc, Kelli A; Madigan, Victoria J; Castellanos Rivera, Ruth M; Wells, Lindsey F; Havlik, L Patrick; Smith, J Kennon; Agbandje-McKenna, Mavis; Asokan, Aravind

2017-06-13

Preexisting neutralizing antibodies (NAbs) against adeno-associated viruses (AAVs) pose a major, unresolved challenge that restricts patient enrollment in gene therapy clinical trials using recombinant AAV vectors. Structural studies suggest that despite a high degree of sequence variability, antibody recognition sites or antigenic hotspots on AAVs and other related parvoviruses might be evolutionarily conserved. To test this hypothesis, we developed a structure-guided evolution approach that does not require selective pressure exerted by NAbs. This strategy yielded highly divergent antigenic footprints that do not exist in natural AAV isolates. Specifically, synthetic variants obtained by evolving murine antigenic epitopes on an AAV serotype 1 capsid template can evade NAbs without compromising titer, transduction efficiency, or tissue tropism. One lead AAV variant generated by combining multiple evolved antigenic sites effectively evades polyclonal anti-AAV1 neutralizing sera from immunized mice and rhesus macaques. Furthermore, this variant displays robust immune evasion in nonhuman primate and human serum samples at dilution factors as high as 1:5, currently mandated by several clinical trials. Our results provide evidence that antibody recognition of AAV capsids is conserved across species. This approach can be applied to any AAV strain to evade NAbs in prospective patients for human gene therapy.

A 5000-Fold Increase in the Specificity of a Bacterial Phosphotriesterase for Malathion through Combinatorial Active Site Mutagenesis

PubMed Central

Naqvi, Tatheer; Warden, Andrew C.; French, Nigel; Sugrue, Elena; Carr, Paul D.; Jackson, Colin J.; Scott, Colin

2014-01-01

Phosphotriesterases (PTEs) have been isolated from a range of bacterial species, including Agrobcaterium radiobacter (PTEAr), and are efficient enzymes with broad substrate ranges. The turnover rate of PTEAr for the common organophosphorous insecticide malathion is lower than expected based on its physical properties; principally the pka of its leaving group. In this study, we rationalise the turnover rate of PTEAr for malathion using computational docking of the substrate into a high resolution crystal structure of the enzyme, suggesting that malathion is too large for the PTEAr binding pocket. Protein engineering through combinatorial active site saturation testing (CASTing) was then used to increase the rate of malathion turnover. Variants from a CASTing library in which Ser308 and Tyr309 were mutated yielded variants with increased activity towards malathion. The most active PTEAr variant carried Ser308Leu and Tyr309Ala substitutions, which resulted in a ca. 5000-fold increase in k cat/K M for malathion. X-ray crystal structures for the PTEAr Ser308Leu\\Tyr309Ala variant demonstrate that the access to the binding pocket was enhanced by the replacement of the bulky Tyr309 residue with the smaller alanine residue. PMID:24721933

Disruption of Chemoreceptor Signaling Arrays by High Levels of CheW, the Receptor-Kinase Coupling Protein

PubMed Central

Cardozo, Marcos J.; Massazza, Diego A.; Parkinson, John S.; Studdert, Claudia A.

2017-01-01

Summary During chemotactic signaling by Escherichia coli, the small cytoplasmic CheW protein couples the histidine kinase CheA to chemoreceptor control. Although essential for assembly and operation of receptor signaling complexes, CheW in stoichiometric excess disrupts chemotactic behavior. To explore the mechanism of the CheW excess effect, we measured the physiological consequences of high cellular levels of wild-type CheW and of several CheW variants with reduced or enhanced binding affinities for receptor molecules. We found that high levels of CheW interfered with trimer assembly, prevented CheA activation, blocked cluster formation, disrupted chemotactic ability, and elevated receptor methylation levels. The severity of these effects paralleled the receptor binding affinities of the CheW variants. Because trimer formation may be an obligate step in the assembly of ternary signaling complexes and higher-order receptor arrays, we suggest that all CheW excess effects stem from disruption of trimer assembly. We propose that the CheW-binding sites in receptor dimers overlap their trimer contact sites and that high levels of CheW saturate the receptor binding sites, preventing trimer assembly. The CheW-trapped receptor dimers seem to be improved substrates for methyltransferase reactions, but cannot activate CheA or assemble into clusters, processes that are essential for chemotactic signaling. PMID:20487303

Dual allosteric activation mechanisms in monomeric human glucokinase

PubMed Central

Whittington, A. Carl; Larion, Mioara; Bowler, Joseph M.; Ramsey, Kristen M.; Brüschweiler, Rafael; Miller, Brian G.

2015-01-01

Cooperativity in human glucokinase (GCK), the body’s primary glucose sensor and a major determinant of glucose homeostatic diseases, is fundamentally different from textbook models of allostery because GCK is monomeric and contains only one glucose-binding site. Prior work has demonstrated that millisecond timescale order-disorder transitions within the enzyme’s small domain govern cooperativity. Here, using limited proteolysis, we map the site of disorder in unliganded GCK to a 30-residue active-site loop that closes upon glucose binding. Positional randomization of the loop, coupled with genetic selection in a glucokinase-deficient bacterium, uncovers a hyperactive GCK variant with substantially reduced cooperativity. Biochemical and structural analysis of this loop variant and GCK variants associated with hyperinsulinemic hypoglycemia reveal two distinct mechanisms of enzyme activation. In α-type activation, glucose affinity is increased, the proteolytic susceptibility of the active site loop is suppressed and the 1H-13C heteronuclear multiple quantum coherence (HMQC) spectrum of 13C-Ile–labeled enzyme resembles the glucose-bound state. In β-type activation, glucose affinity is largely unchanged, proteolytic susceptibility of the loop is enhanced, and the 1H-13C HMQC spectrum reveals no perturbation in ensemble structure. Leveraging both activation mechanisms, we engineer a fully noncooperative GCK variant, whose functional properties are indistinguishable from other hexokinase isozymes, and which displays a 100-fold increase in catalytic efficiency over wild-type GCK. This work elucidates specific structural features responsible for generating allostery in a monomeric enzyme and suggests a general strategy for engineering cooperativity into proteins that lack the structural framework typical of traditional allosteric systems. PMID:26283387

Dual allosteric activation mechanisms in monomeric human glucokinase.

PubMed

Whittington, A Carl; Larion, Mioara; Bowler, Joseph M; Ramsey, Kristen M; Brüschweiler, Rafael; Miller, Brian G

2015-09-15

Cooperativity in human glucokinase (GCK), the body's primary glucose sensor and a major determinant of glucose homeostatic diseases, is fundamentally different from textbook models of allostery because GCK is monomeric and contains only one glucose-binding site. Prior work has demonstrated that millisecond timescale order-disorder transitions within the enzyme's small domain govern cooperativity. Here, using limited proteolysis, we map the site of disorder in unliganded GCK to a 30-residue active-site loop that closes upon glucose binding. Positional randomization of the loop, coupled with genetic selection in a glucokinase-deficient bacterium, uncovers a hyperactive GCK variant with substantially reduced cooperativity. Biochemical and structural analysis of this loop variant and GCK variants associated with hyperinsulinemic hypoglycemia reveal two distinct mechanisms of enzyme activation. In α-type activation, glucose affinity is increased, the proteolytic susceptibility of the active site loop is suppressed and the (1)H-(13)C heteronuclear multiple quantum coherence (HMQC) spectrum of (13)C-Ile-labeled enzyme resembles the glucose-bound state. In β-type activation, glucose affinity is largely unchanged, proteolytic susceptibility of the loop is enhanced, and the (1)H-(13)C HMQC spectrum reveals no perturbation in ensemble structure. Leveraging both activation mechanisms, we engineer a fully noncooperative GCK variant, whose functional properties are indistinguishable from other hexokinase isozymes, and which displays a 100-fold increase in catalytic efficiency over wild-type GCK. This work elucidates specific structural features responsible for generating allostery in a monomeric enzyme and suggests a general strategy for engineering cooperativity into proteins that lack the structural framework typical of traditional allosteric systems.

OPTIMIZATION OF RAMAN SPECTROSCOPY FOR SPECIATION OF ORGANICS IN WATER

EPA Science Inventory

We describe herein a method for determining constants for simultaneously occurring, site-specific "microequilibria" (as with tautomers) for organics in water. The method is based in part on modeling temperature-variant Raman spectra according to the van't Hoff equation. Spectra a...

Vaccine-elicited antibody that neutralizes H5N1 influenza and variants binds the receptor site and polymorphic sites

DOE PAGES

Winarski, Katie L.; Thornburg, Natalie J.; Yu, Yingchun; ...

2015-07-13

Antigenic drift of circulating seasonal influenza viruses necessitates an international vaccine effort to reduce the impact on human health. A critical feature of the seasonal vaccine is that it stimulates an already primed immune system to diversify memory B cells to recognize closely related, but antigenically distinct, influenza glycoproteins (hemagglutinins). Influenza pandemics arise when hemagglutinins to which no preexisting adaptive immunity exists acquire the capacity to infect humans. Hemagglutinin 5 is one subtype to which little preexisting immunity exists and is only a few acquired mutations away from the ability to transmit efficiently between ferrets, and possibly humans. In thismore » paper, we describe the structure and molecular mechanism of neutralization by H5.3, a vaccine-elicited antibody that neutralizes hemagglutinin 5 viruses and variants with expanded host range. H5.3 binds in the receptor-binding site, forming contacts that recapitulate many of the sialic acid interactions, as well as multiple peripheral interactions, yet is not sensitive to mutations that alter sialic acid binding. H5.3 is highly specific for a subset of H5 strains, and this specificity arises from interactions to the periphery of the receptor-binding site. Finally, H5.3 is also extremely potent, despite retaining germ line-like conformational flexibility.« less

Vaccine-elicited antibody that neutralizes H5N1 influenza and variants binds the receptor site and polymorphic sites

DOE Office of Scientific and Technical Information (OSTI.GOV)

Winarski, Katie L.; Thornburg, Natalie J.; Yu, Yingchun

Antigenic drift of circulating seasonal influenza viruses necessitates an international vaccine effort to reduce the impact on human health. A critical feature of the seasonal vaccine is that it stimulates an already primed immune system to diversify memory B cells to recognize closely related, but antigenically distinct, influenza glycoproteins (hemagglutinins). Influenza pandemics arise when hemagglutinins to which no preexisting adaptive immunity exists acquire the capacity to infect humans. Hemagglutinin 5 is one subtype to which little preexisting immunity exists and is only a few acquired mutations away from the ability to transmit efficiently between ferrets, and possibly humans. In thismore » paper, we describe the structure and molecular mechanism of neutralization by H5.3, a vaccine-elicited antibody that neutralizes hemagglutinin 5 viruses and variants with expanded host range. H5.3 binds in the receptor-binding site, forming contacts that recapitulate many of the sialic acid interactions, as well as multiple peripheral interactions, yet is not sensitive to mutations that alter sialic acid binding. H5.3 is highly specific for a subset of H5 strains, and this specificity arises from interactions to the periphery of the receptor-binding site. Finally, H5.3 is also extremely potent, despite retaining germ line-like conformational flexibility.« less

Genome-Wide Profiling of RNA–Protein Interactions Using CLIP-Seq

PubMed Central

Stork, Cheryl; Zheng, Sika

2017-01-01

UV crosslinking immunoprecipitation (CLIP) is an increasingly popular technique to study protein–RNA interactions in tissues and cells. Whole cells or tissues are ultraviolet irradiated to generate a covalent bond between RNA and proteins that are in close contact. After partial RNase digestion, antibodies specific to an RNA binding protein (RBP) or a protein–epitope tag is then used to immunoprecipitate the protein–RNA complexes. After stringent washing and gel separation the RBP–RNA complex is excised. The RBP is protease digested to allow purification of the bound RNA. Reverse transcription of the RNA followed by high-throughput sequencing of the cDNA library is now often used to identify protein bound RNA on a genome-wide scale. UV irradiation can result in cDNA truncations and/or mutations at the crosslink sites, which complicates the alignment of the sequencing library to the reference genome and the identification of the crosslinking sites. Meanwhile, one or more amino acids of a crosslinked RBP can remain attached to its bound RNA due to incomplete digestion of the protein. As a result, reverse transcriptase may not read through the crosslink sites, and produce cDNA ending at the crosslinked nucleotide. This is harnessed by one variant of CLIP methods to identify crosslinking sites at a nucleotide resolution. This method, individual nucleotide resolution CLIP (iCLIP) circularizes cDNA to capture the truncated cDNA and also increases the efficiency of ligating sequencing adapters to the library. Here, we describe the detailed procedure of iCLIP. PMID:26965263

Activation of a cryptic splice site in the mitochondrial elongation factor GFM1 causes combined OXPHOS deficiency☆

PubMed Central

Simon, Mariella T.; Ng, Bobby G.; Friederich, Marisa W.; Wang, Raymond Y.; Boyer, Monica; Kircher, Martin; Collard, Renata; Buckingham, Kati J.; Chang, Richard; Shendure, Jay; Nickerson, Deborah A.; Bamshad, Michael J.; Van Hove, Johan L.K.; Freeze, Hudson H.; Abdenur, Jose E.

2017-01-01

We report the clinical, biochemical, and molecular findings in two brothers with encephalopathy and multi-systemic disease. Abnormal transferrin glycoforms were suggestive of a type I congenital disorder of glycosylation (CDG). While exome sequencing was negative for CDG related candidate genes, the testing revealed compound heterozygous mutations in the mitochondrial elongation factor G gene (GFM1). One of the mutations had been reported previously while the second, novel variant was found deep in intron 6, activating a cryptic splice site. Functional studies demonstrated decreased GFM1 protein levels, suggested disrupted assembly of mitochondrial complexes III and V and decreased activities of mitochondrial complexes I and IV, all indicating combined OXPHOS deficiency. PMID:28216230

DPP10 splice variants are localized in distinct neuronal populations and act to differentially regulate the inactivation properties of Kv4-based ion channels.

PubMed

Jerng, Henry H; Lauver, Aaron D; Pfaffinger, Paul J

2007-08-01

Dipeptidyl peptidase-like proteins (DPLs) and Kv-channel-interacting proteins (KChIPs) join Kv4 pore-forming subunits to form multi-protein complexes that underlie subthreshold A-type currents (I(SA)) in neuronal somatodendritic compartments. Here, we characterize the functional effects and brain distributions of N-terminal variants belonging to the DPL dipeptidyl peptidase 10 (DPP10). In the Kv4.2+KChIP3+DPP10 channel complex, all DPP10 variants accelerate channel gating kinetics; however, the splice variant DPP10a produces uniquely fast inactivation kinetics that accelerates with increasing depolarization. This DPP10a-specific inactivation dominates in co-expression studies with KChIP4a and other DPP10 isoforms. Real-time qRT-PCR and in situ hybridization analyses reveal differential expression of DPP10 variants in rat brain. DPP10a transcripts are prominently expressed in the cortex, whereas DPP10c and DPP10d mRNAs exhibit more diffuse distributions. Our results suggest that DPP10a underlies rapid inactivation of cortical I(SA), and the regulation of isoform expression may contribute to the variable inactivation properties of I(SA) across different brain regions.

REGULATED VESICULAR TRAFFICKING OF SPECIFIC PCDH15 AND VLGR1 VARIANTS IN AUDITORY HAIR CELLS

PubMed Central

Zallocchi, Marisa; Delimont, Duane; Meehan, Daniel T.; Cosgrove, Dominic

2012-01-01

Usher syndrome is a genetically heterogeneous disorder characterized by hearing and balance dysfunction and progressive retinitis pigmentosa. Mouse models carrying mutations for the nine Usher-associated genes have splayed stereocilia and some show delayed maturation of ribbon synapses suggesting these proteins may play different roles in terminal differentiation of auditory hair cells. The presence of the Usher proteins at the basal and apical aspects of the neurosensory epithelia suggests the existence of regulated trafficking through specific transport proteins and routes. Immature mouse cochleae and UB/OC-1 cells were used in this work to address whether specific variants of PCDH15 and VLGR1 are being selectively transported to opposite poles of the hair cells. Confocal co-localization studies between apical and basal vesicular markers and the different PCDH15 and VLGR1 variants along with sucrose density gradients and the use of vesicle trafficking inhibitors show the existence of Usher protein complexes in at least two vesicular sub-pools. The apically trafficked pool co-localized with the early endosomal vesicle marker, rab5, while the basally trafficked pool associates with membrane microdomains and SNAP25. Moreover, co-immunoprecipitation experiments between SNAP25 and VLGR1 show a physical interaction of these two proteins in organ of Corti and brain. Collectively, these findings establish the existence of a differential vesicular trafficking mechanism for specific Usher protein variants in mouse cochlear hair cells, with the apical variants playing a potential role in endosomal recycling and stereocilia development/maintenance and the basolateral variants involved in vesicle docking and/or fusion through SNAP25-mediated interactions. PMID:23035094

TATES: Efficient Multivariate Genotype-Phenotype Analysis for Genome-Wide Association Studies

PubMed Central

van der Sluis, Sophie; Posthuma, Danielle; Dolan, Conor V.

2013-01-01

To date, the genome-wide association study (GWAS) is the primary tool to identify genetic variants that cause phenotypic variation. As GWAS analyses are generally univariate in nature, multivariate phenotypic information is usually reduced to a single composite score. This practice often results in loss of statistical power to detect causal variants. Multivariate genotype–phenotype methods do exist but attain maximal power only in special circumstances. Here, we present a new multivariate method that we refer to as TATES (Trait-based Association Test that uses Extended Simes procedure), inspired by the GATES procedure proposed by Li et al (2011). For each component of a multivariate trait, TATES combines p-values obtained in standard univariate GWAS to acquire one trait-based p-value, while correcting for correlations between components. Extensive simulations, probing a wide variety of genotype–phenotype models, show that TATES's false positive rate is correct, and that TATES's statistical power to detect causal variants explaining 0.5% of the variance can be 2.5–9 times higher than the power of univariate tests based on composite scores and 1.5–2 times higher than the power of the standard MANOVA. Unlike other multivariate methods, TATES detects both genetic variants that are common to multiple phenotypes and genetic variants that are specific to a single phenotype, i.e. TATES provides a more complete view of the genetic architecture of complex traits. As the actual causal genotype–phenotype model is usually unknown and probably phenotypically and genetically complex, TATES, available as an open source program, constitutes a powerful new multivariate strategy that allows researchers to identify novel causal variants, while the complexity of traits is no longer a limiting factor. PMID:23359524

Design of an allosterically modulated doxycycline and doxorubicin drug-binding protein.

PubMed

Schmidt, Karin; Gardill, Bernd R; Kern, Alina; Kirchweger, Peter; Börsch, Michael; Muller, Yves A

2018-05-14

The allosteric interplay between distant functional sites present in a single protein provides for one of the most important regulatory mechanisms in biological systems. While the design of ligand-binding sites into proteins remains challenging, this holds even truer for the coupling of a newly engineered binding site to an allosteric mechanism that regulates the ligand affinity. Here it is shown how computational design algorithms enabled the introduction of doxycycline- and doxorubicin-binding sites into the serine proteinase inhibitor (serpin) family member α1-antichymotrypsin. Further engineering allowed exploitation of the proteinase-triggered serpin-typical S-to-R transition to modulate the ligand affinities. These design variants follow strategies observed in naturally occurring plasma globulins that allow for the targeted delivery of hormones in the blood. By analogy, we propose that the variants described in the present study could be further developed to allow for the delivery of the antibiotic doxycycline and the anticancer compound doxorubicin to tissues/locations that express specific proteinases, such as bacterial infection sites or tumor cells secreting matrix metalloproteinases.

Both Epistasis and Diversifying Selection Drive the Structural Evolution of the Ebola Virus Glycoprotein Mucin-Like Domain.

PubMed

Ibeh, Neke; Nshogozabahizi, Jean Claude; Aris-Brosou, Stéphane

2016-06-01

Throughout the last 3 decades, Ebola virus (EBOV) outbreaks have been confined to isolated areas within Central Africa; however, the 2014 variant reached unprecedented transmission and mortality rates. While the outbreak was still under way, it was reported that the variant leading up to this outbreak evolved faster than previous EBOV variants, but evidence for diversifying selection was undetermined. Here, we test this selection hypothesis and show that while previous EBOV outbreaks were preceded by bursts of diversification, evidence for site-specific diversifying selection during the emergence of the 2014 EBOV clade is weak. However, we show strong evidence supporting an interplay between selection and correlated evolution (epistasis), particularly in the mucin-like domain (MLD) of the EBOV glycoprotein. By reconstructing ancestral structures of the MLD, we further propose a structural mechanism explaining how the substitutions that accumulated between 1918 and 1969 distorted the MLD, while more recent epistatic substitutions restored part of the structure, with the most recent substitution being adaptive. We suggest that it is this complex interplay between weak selection, epistasis, and structural constraints that has shaped the evolution of the 2014 EBOV variant. The role that selection plays in the emergence of viral epidemics remains debated, particularly in the context of the 2014 EBOV outbreak. Most critically, should such evidence exist, it is generally unclear how this relates to function and increased virulence. Here, we show that the viral lineage leading up to the 2014 outbreak underwent a complex interplay between selection and correlated evolution (epistasis) in a protein region that is critical for immune evasion. We then reconstructed the three-dimensional structure of this domain and showed that the initial mutations in this lineage deformed the structure, while subsequent mutations restored part of the structure. Along this mutational path, the first and last mutations were adaptive, while the intervening ones were epistatic. Altogether, we provide a mechanistic model that explains how selection and epistasis acted on the structural constraints that materialized during the 2014 EBOV outbreak. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Multimer Formation Explains Allelic Suppression of PRDM9 Recombination Hotspots.

PubMed

Baker, Christopher L; Petkova, Pavlina; Walker, Michael; Flachs, Petr; Mihola, Ondrej; Trachtulec, Zdenek; Petkov, Petko M; Paigen, Kenneth

2015-09-01

Genetic recombination during meiosis functions to increase genetic diversity, promotes elimination of deleterious alleles, and helps assure proper segregation of chromatids. Mammalian recombination events are concentrated at specialized sites, termed hotspots, whose locations are determined by PRDM9, a zinc finger DNA-binding histone methyltransferase. Prdm9 is highly polymorphic with most alleles activating their own set of hotspots. In populations exhibiting high frequencies of heterozygosity, questions remain about the influences different alleles have in heterozygous individuals where the two variant forms of PRDM9 typically do not activate equivalent populations of hotspots. We now find that, in addition to activating its own hotspots, the presence of one Prdm9 allele can modify the activity of hotspots activated by the other allele. PRDM9 function is also dosage sensitive; Prdm9+/- heterozygous null mice have reduced numbers and less active hotspots and increased numbers of aberrant germ cells. In mice carrying two Prdm9 alleles, there is allelic competition; the stronger Prdm9 allele can partially or entirely suppress chromatin modification and recombination at hotspots of the weaker allele. In cell cultures, PRDM9 protein variants form functional heteromeric complexes which can bind hotspots sequences. When a heteromeric complex binds at a hotspot of one PRDM9 variant, the other PRDM9 variant, which would otherwise not bind, can still methylate hotspot nucleosomes. We propose that in heterozygous individuals the underlying molecular mechanism of allelic suppression results from formation of PRDM9 heteromers, where the DNA binding activity of one protein variant dominantly directs recombination initiation towards its own hotspots, effectively titrating down recombination by the other protein variant. In natural populations with many heterozygous individuals, allelic competition will influence the recombination landscape.

Multimer Formation Explains Allelic Suppression of PRDM9 Recombination Hotspots

PubMed Central

Baker, Christopher L.; Petkova, Pavlina; Walker, Michael; Flachs, Petr; Mihola, Ondrej; Trachtulec, Zdenek; Petkov, Petko M.; Paigen, Kenneth

2015-01-01

Genetic recombination during meiosis functions to increase genetic diversity, promotes elimination of deleterious alleles, and helps assure proper segregation of chromatids. Mammalian recombination events are concentrated at specialized sites, termed hotspots, whose locations are determined by PRDM9, a zinc finger DNA-binding histone methyltransferase. Prdm9 is highly polymorphic with most alleles activating their own set of hotspots. In populations exhibiting high frequencies of heterozygosity, questions remain about the influences different alleles have in heterozygous individuals where the two variant forms of PRDM9 typically do not activate equivalent populations of hotspots. We now find that, in addition to activating its own hotspots, the presence of one Prdm9 allele can modify the activity of hotspots activated by the other allele. PRDM9 function is also dosage sensitive; Prdm9 +/- heterozygous null mice have reduced numbers and less active hotspots and increased numbers of aberrant germ cells. In mice carrying two Prdm9 alleles, there is allelic competition; the stronger Prdm9 allele can partially or entirely suppress chromatin modification and recombination at hotspots of the weaker allele. In cell cultures, PRDM9 protein variants form functional heteromeric complexes which can bind hotspots sequences. When a heteromeric complex binds at a hotspot of one PRDM9 variant, the other PRDM9 variant, which would otherwise not bind, can still methylate hotspot nucleosomes. We propose that in heterozygous individuals the underlying molecular mechanism of allelic suppression results from formation of PRDM9 heteromers, where the DNA binding activity of one protein variant dominantly directs recombination initiation towards its own hotspots, effectively titrating down recombination by the other protein variant. In natural populations with many heterozygous individuals, allelic competition will influence the recombination landscape. PMID:26368021

Complex phenotype of dyskeratosis congenita and mood dysregulation with novel homozygous RTEL1 and TPH1 variants.

PubMed

Ungar, Rachel A; Giri, Neelam; Pao, Maryland; Khincha, Payal P; Zhou, Weiyin; Alter, Blanche P; Savage, Sharon A

2018-06-01

Dyskeratosis congenita (DC) is an inherited bone marrow failure syndrome caused by germline mutations in telomere biology genes. Patients have extremely short telomeres for their age and a complex phenotype including oral leukoplakia, abnormal skin pigmentation, and dysplastic nails in addition to bone marrow failure, pulmonary fibrosis, stenosis of the esophagus, lacrimal ducts and urethra, developmental anomalies, and high risk of cancer. We evaluated a patient with features of DC, mood dysregulation, diabetes, and lack of pubertal development. Family history was not available but genome-wide genotyping was consistent with consanguinity. Whole exome sequencing identified 82 variants of interest in 80 genes based on the following criteria: homozygous, <0.1% minor allele frequency in public and in-house databases, nonsynonymous, and predicted deleterious by multiple in silico prediction programs. Six genes were identified likely contributory to the clinical presentation. The cause of DC is likely due to homozygous splice site variants in regulator of telomere elongation helicase 1, a known DC and telomere biology gene. A homozygous, missense variant in tryptophan hydroxylase 1 may be clinically important as this gene encodes the rate limiting step in serotonin biosynthesis, a biologic pathway connected with mood disorders. Four additional genes (SCN4A, LRP4, GDAP1L1, and SPTBN5) had rare, missense homozygous variants that we speculate may contribute to portions of the clinical phenotype. This case illustrates the value of conducting detailed clinical and genomic evaluations on rare patients in order to identify new areas of research into the functional consequences of rare variants and their contribution to human disease. © 2018 Wiley Periodicals, Inc.

Artificial Metalloproteins Containing Co 4O 4Cubane Active Sites

DOE Office of Scientific and Technical Information (OSTI.GOV)

Olshansky, Lisa; Huerta-Lavorie, Raul; Nguyen, Andy I.

Artificial metalloproteins (ArMs) containing Co 4O 4 cubane active sites were constructed via biotin-streptavidin technology. Stabilized by hydrogen bonds (H-bonds), terminal and cofacial Co III-OH 2 moieties are observed crystallographically in a series of immobilized cubane sites. Solution electrochemistry provided correlations of oxidation potential and pH. For variants containing Ser and Phe adjacent to the metallocofactor, 1e -/1H + chemistry predominates until pH 8, above which the oxidation becomes pH-independent. Installation of Tyr proximal to the Co 4O 4 active site provided a single H-bond to one of a set of cofacial Co III-OH 2 groups. With this variant, multi-emore » - /multi-H + chemistry is observed, along with a change in mechanism at pH 9.5 that is consistent with Tyr deprotonation. Finally, with structural similarities to both the oxygen-evolving complex of photosystem II (H-bonded Tyr) and to thin film water oxidation catalysts (Co 4O 4 core), these findings bridge synthetic and biological systems for water oxidation, highlighting the importance of secondary sphere interactions in mediating multi-e - /multi-H + reactivity.« less

Artificial Metalloproteins Containing Co 4O 4Cubane Active Sites

DOE PAGES

Olshansky, Lisa; Huerta-Lavorie, Raul; Nguyen, Andy I.; ...

2018-02-05

Artificial metalloproteins (ArMs) containing Co 4O 4 cubane active sites were constructed via biotin-streptavidin technology. Stabilized by hydrogen bonds (H-bonds), terminal and cofacial Co III-OH 2 moieties are observed crystallographically in a series of immobilized cubane sites. Solution electrochemistry provided correlations of oxidation potential and pH. For variants containing Ser and Phe adjacent to the metallocofactor, 1e -/1H + chemistry predominates until pH 8, above which the oxidation becomes pH-independent. Installation of Tyr proximal to the Co 4O 4 active site provided a single H-bond to one of a set of cofacial Co III-OH 2 groups. With this variant, multi-emore » - /multi-H + chemistry is observed, along with a change in mechanism at pH 9.5 that is consistent with Tyr deprotonation. Finally, with structural similarities to both the oxygen-evolving complex of photosystem II (H-bonded Tyr) and to thin film water oxidation catalysts (Co 4O 4 core), these findings bridge synthetic and biological systems for water oxidation, highlighting the importance of secondary sphere interactions in mediating multi-e - /multi-H + reactivity.« less

Two strategies to engineer flexible loops for improved enzyme thermostability

PubMed Central

Yu, Haoran; Yan, Yihan; Zhang, Cheng; Dalby, Paul A.

2017-01-01

Flexible sites are potential targets for engineering the stability of enzymes. Nevertheless, the success rate of the rigidifying flexible sites (RFS) strategy is still low due to a limited understanding of how to determine the best mutation candidates. In this study, two parallel strategies were applied to identify mutation candidates within the flexible loops of Escherichia coli transketolase (TK). The first was a “back to consensus mutations” approach, and the second was computational design based on ΔΔG calculations in Rosetta. Forty-nine single variants were generated and characterised experimentally. From these, three single-variants I189H, A282P, D143K were found to be more thermostable than wild-type TK. The combination of A282P with H192P, a variant constructed previously, resulted in the best all-round variant with a 3-fold improved half-life at 60 °C, 5-fold increased specific activity at 65 °C, 1.3-fold improved kcat and a Tm increased by 5 °C above that of wild type. Based on a statistical analysis of the stability changes for all variants, the qualitative prediction accuracy of the Rosetta program reached 65.3%. Both of the two strategies investigated were useful in guiding mutation candidates to flexible loops, and had the potential to be used for other enzymes. PMID:28145457

Studying Gene and Gene-Environment Effects of Uncommon and Common Variants on Continuous Traits: A Marker-Set Approach Using Gene-Trait Similarity Regression

PubMed Central

Tzeng, Jung-Ying; Zhang, Daowen; Pongpanich, Monnat; Smith, Chris; McCarthy, Mark I.; Sale, Michèle M.; Worrall, Bradford B.; Hsu, Fang-Chi; Thomas, Duncan C.; Sullivan, Patrick F.

2011-01-01

Genomic association analyses of complex traits demand statistical tools that are capable of detecting small effects of common and rare variants and modeling complex interaction effects and yet are computationally feasible. In this work, we introduce a similarity-based regression method for assessing the main genetic and interaction effects of a group of markers on quantitative traits. The method uses genetic similarity to aggregate information from multiple polymorphic sites and integrates adaptive weights that depend on allele frequencies to accomodate common and uncommon variants. Collapsing information at the similarity level instead of the genotype level avoids canceling signals that have the opposite etiological effects and is applicable to any class of genetic variants without the need for dichotomizing the allele types. To assess gene-trait associations, we regress trait similarities for pairs of unrelated individuals on their genetic similarities and assess association by using a score test whose limiting distribution is derived in this work. The proposed regression framework allows for covariates, has the capacity to model both main and interaction effects, can be applied to a mixture of different polymorphism types, and is computationally efficient. These features make it an ideal tool for evaluating associations between phenotype and marker sets defined by linkage disequilibrium (LD) blocks, genes, or pathways in whole-genome analysis. PMID:21835306

Synonymous mutations in RNASEH2A create cryptic splice sites impairing RNase H2 enzyme function in Aicardi-Goutières syndrome

PubMed Central

Rice, Gillian I.; Reijns, Martin A.M.; Coffin, Stephanie R.; Forte, Gabriella M.A.; Anderson, Beverley H.; Szynkiewicz, Marcin; Gornall, Hannah; Gent, David; Leitch, Andrea; Botella, Maria P.; Fazzi, Elisa; Gener, Blanca; Lagae, Lieven; Olivieri, Ivana; Orcesi, Simona; Swoboda, Kathryn J.; Perrino, Fred W.; Jackson, Andrew P.; Crow, Yanick J.

2013-01-01

Aicardi-Goutières syndrome (AGS) is an inflammatory disorder resulting from mutations in TREX1, RNASEH2A/2B/2C, SAMHD1 or ADAR1. Here we provide molecular, biochemical and cellular evidence for the pathogenicity of two synonymous variants in RNASEH2A. Firstly, the c.69G>A (p.Val23Val) mutation causes the formation of a splice donor site within exon 1, resulting in an out of frame deletion at the end of exon 1, leading to reduced RNase H2 protein levels. The second mutation, c.75C>T (p.Arg25Arg), also introduces a splice donor site within exon 1, and the internal deletion of 18 amino acids. The truncated protein still forms a heterotrimeric RNase H2 complex, but lacks catalytic activity. However, as a likely result of leaky splicing, a small amount of full-length active protein is apparently produced in an individual homozygous for this mutation. Recognition of the disease causing status of these variants allows for diagnostic testing in relevant families. PMID:23592335

Informatic search strategies to discover analogues and variants of natural product archetypes.

PubMed

Johnston, Chad W; Connaty, Alex D; Skinnider, Michael A; Li, Yong; Grunwald, Alyssa; Wyatt, Morgan A; Kerr, Russell G; Magarvey, Nathan A

2016-03-01

Natural products are a crucial source of antimicrobial agents, but reliance on low-resolution bioactivity-guided approaches has led to diminishing interest in discovery programmes. Here, we demonstrate that two in-house automated informatic platforms can be used to target classes of biologically active natural products, specifically, peptaibols. We demonstrate that mass spectrometry-based informatic approaches can be used to detect natural products with high sensitivity, identifying desired agents present in complex microbial extracts. Using our specialised software packages, we could elaborate specific branches of chemical space, uncovering new variants of trichopolyn and demonstrating a way forward in mining natural products as a valuable source of potential pharmaceutical agents.

Sent packing: protein engineering generates a new crystal form of Pseudomonas aeruginosa DsbA1 with increased catalytic surface accessibility

DOE Office of Scientific and Technical Information (OSTI.GOV)

McMahon, Roisin M., E-mail: r.mcmahon1@uq.edu.au; Coinçon, Mathieu; Tay, Stephanie

The crystal structure of a P. aeruginosa DsbA1 variant is more suitable for fragment-based lead discovery efforts to identify inhibitors of this antimicrobial drug target. In the reported structures the active site of the protein can simultaneously bind multiple ligands introduced in the crystallization solution or via soaking. Pseudomonas aeruginosa is an opportunistic human pathogen for which new antimicrobial drug options are urgently sought. P. aeruginosa disulfide-bond protein A1 (PaDsbA1) plays a pivotal role in catalyzing the oxidative folding of multiple virulence proteins and as such holds great promise as a drug target. As part of a fragment-based lead discoverymore » approach to PaDsbA1 inhibitor development, the identification of a crystal form of PaDsbA1 that was more suitable for fragment-soaking experiments was sought. A previously identified crystallization condition for this protein was unsuitable, as in this crystal form of PaDsbA1 the active-site surface loops are engaged in the crystal packing, occluding access to the target site. A single residue involved in crystal-packing interactions was substituted with an amino acid commonly found at this position in closely related enzymes, and this variant was successfully used to generate a new crystal form of PaDsbA1 in which the active-site surface is more accessible for soaking experiments. The PaDsbA1 variant displays identical redox character and in vitro activity to wild-type PaDsbA1 and is structurally highly similar. Two crystal structures of the PaDsbA1 variant were determined in complex with small molecules bound to the protein active site. These small molecules (MES, glycerol and ethylene glycol) were derived from the crystallization or cryoprotectant solutions and provide a proof of principle that the reported crystal form will be amenable to co-crystallization and soaking with small molecules designed to target the protein active-site surface.« less

Neutralization Escape Variants of Human Immunodeficiency Virus Type 1 Are Transmitted from Mother to Infant

PubMed Central

Wu, Xueling; Parast, Adam B.; Richardson, Barbra A.; Nduati, Ruth; John-Stewart, Grace; Mbori-Ngacha, Dorothy; Rainwater, Stephanie M. J.; Overbaugh, Julie

2006-01-01

Maternal passive immunity typically plays a critical role in protecting infants from new infections; however, the specific contribution of neutralizing antibodies in limiting mother-to-child transmission of human immunodeficiency virus type 1 is unclear. By examining cloned envelope variants from 12 transmission pairs, we found that vertically transmitted variants were more resistant to neutralization by maternal plasma than were maternal viral variants near the time of transmission. The vertically transmitted envelope variants were poorly neutralized by monoclonal antibodies biz, 2G12, 2F5, and 4E10 individually or in combination. Despite the fact that the infant viruses were among the most neutralization resistant in the mother, they had relatively few glycosylation sites. Moreover, the transmitted variants elicited de novo neutralizing antibodies in the infants, indicating that they were not inherently difficult to neutralize. The neutralization resistance of vertically transmitted viruses is in contrast to the relative neutralization sensitivity of viruses sexually transmitted within discordant couples, suggesting that the antigenic properties of viruses that are favored for transmission may differ depending upon mode of transmission. PMID:16378985

A Bioinformatics Approach to the Identification of Variants Associated with Type 1 and Type 2 Diabetes Mellitus that Reside in Functionally Validated miRNAs Binding Sites.

PubMed

Ghaedi, Hamid; Bastami, Milad; Jahani, Mohammad Mehdi; Alipoor, Behnam; Tabasinezhad, Maryam; Ghaderi, Omar; Nariman-Saleh-Fam, Ziba; Mirfakhraie, Reza; Movafagh, Abolfazl; Omrani, Mir Davood; Masotti, Andrea

2016-06-01

The present work is aimed at finding variants associated with Type 1 and Type 2 diabetes mellitus (DM) that reside in functionally validated miRNAs binding sites and that can have a functional role in determining diabetes and related pathologies. Using bioinformatics analyses we obtained a database of validated polymorphic miRNA binding sites which has been intersected with genes related to DM or to variants associated and/or in linkage disequilibrium (LD) with it and is reported in genome-wide association studies (GWAS). The workflow we followed allowed us to find variants associated with DM that also reside in functional miRNA binding sites. These data have been demonstrated to have a functional role by impairing the functions of genes implicated in biological processes linked to DM. In conclusion, our work emphasized the importance of SNPs located in miRNA binding sites. The results discussed in this work may constitute the basis of further works aimed at finding functional candidates and variants affecting protein structure and function, transcription factor binding sites, and non-coding epigenetic variants, contributing to widen the knowledge about the pathogenesis of this important disease.

What can we learn about lipoprotein metabolism and coronary heart disease from studying rare variants?

PubMed

Jeff, Janina M; Peloso, Gina M; Do, Ron

2016-04-01

Rare variant association studies (RVAS) target the class of genetic variation with frequencies less than 1%. Recently, investigators have used exome sequencing in RVAS to identify rare alleles responsible for Mendelian diseases but have experienced greater difficulty discovering such alleles for complex diseases. In this review, we describe what we have learned about lipoprotein metabolism and coronary heart disease through the conduct of RVAS. Rare protein-altering genetic variation can provide important insights that are not as easily attainable from common variant association studies. First, RVAS can facilitate gene discovery by identifying novel rare protein-altering variants in specific genes that are associated with disease. Second, rare variant associations can provide supportive evidence for putative drug targets for novel therapies. Finally, rare variants can uncover new pathways and reveal new biologic mechanisms. The field of human genetics has already made tremendous progress in understanding lipoprotein metabolism and the causes of coronary heart disease in the context of rare variants. As next generation sequencing becomes more cost-effective, RVAS with larger sample sizes will be conducted. This will lead to more novel rare variant discoveries and the translation of genomic data into biological knowledge and clinical insights for cardiovascular disease.

Fc-specific biotinylation of antibody using an engineered photoactivatable Z-Biotin and its biosensing application.

PubMed

Yang, Hong-Ming; Bao, Ru-Meng; Yu, Chang-Mei; Lv, Yan-Na; Zhang, Wei-Fen; Tang, Jin-Bao

2017-01-01

The development of a site-specific and covalent attachment methodology is crucial for antibody-biotin conjugates to preserve the antigen-binding ability of antibodies and yield homogeneous products. In this study, an engineered photoactivatable Z-domain variant [an UV-active amino acid benzoylphenylalanine (Bpa) was genetically incorporated into the Z-domain] carrying one biotin molecule (Z Bpa -Biotin) was prepared by employing aminoacyl-tRNA synthetase/suppressor tRNA and Avitag/BirA techniques. The site-specific and covalent attachment of IgG-biotin conjugates, viz. photo-biotinylated IgG, was successfully achieved after UV exposure by combining the inherent Fc-binding capability of the Z-domain with the formation of covalent bond by the photo-crosslinker. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis assay showed that more than 90% of IgGs conjugated with Z Bpa -Biotin molecules suffered 3 h UV irradiation. Further pepsin digestion analysis confirmed that the Z Bpa -Biotin was conjugated to the Fc fragment of IgG without interference. We took the tumor biomarker carcinoembryoic antigen (CEA) as model to evaluate the detection efficiency of the site-specific photo-biotinylated IgG in biosensing application using surface plasmon resonance (SPR) technology. The photo-biotinylated IgG coated surface gave a limit of detection (LOD) of 2 ng mL -1 , is 5-fold lower than that of the randomly NHS-biotinylated IgG (10 ng mL -1 ). Given that the (strept)avidin-biotin complex is extensively used in immunoassays, the proposed method for biotinylated IgG provides a powerful approach to further expand related applications. Copyright © 2016 Elsevier B.V. All rights reserved.

Fast, scalable prediction of deleterious noncoding variants from functional and population genomic data.

PubMed

Huang, Yi-Fei; Gulko, Brad; Siepel, Adam

2017-04-01

Many genetic variants that influence phenotypes of interest are located outside of protein-coding genes, yet existing methods for identifying such variants have poor predictive power. Here we introduce a new computational method, called LINSIGHT, that substantially improves the prediction of noncoding nucleotide sites at which mutations are likely to have deleterious fitness consequences, and which, therefore, are likely to be phenotypically important. LINSIGHT combines a generalized linear model for functional genomic data with a probabilistic model of molecular evolution. The method is fast and highly scalable, enabling it to exploit the 'big data' available in modern genomics. We show that LINSIGHT outperforms the best available methods in identifying human noncoding variants associated with inherited diseases. In addition, we apply LINSIGHT to an atlas of human enhancers and show that the fitness consequences at enhancers depend on cell type, tissue specificity, and constraints at associated promoters.

Nanopore Force Spectroscopy of Aptamer–Ligand Complexes

PubMed Central

Arnaut, Vera; Langecker, Martin; Simmel, Friedrich C.

2013-01-01

The stability of aptamer–ligand complexes is probed in nanopore-based dynamic force spectroscopy experiments. Specifically, the ATP-binding aptamer is investigated using a backward translocation technique, in which the molecules are initially pulled through an α-hemolysin nanopore from the cis to the trans side of a lipid bilayer membrane, allowed to refold and interact with their target, and then translocated back in the trans–cis direction. From these experiments, the distribution of bound and unbound complexes is determined, which in turn allows determination of the dissociation constant Kd ≈ 0.1 mM of the aptamer and of voltage-dependent unfolding rates. The experiments also reveal differences in binding of the aptamer to AMP, ADP, or ATP ligands. Investigation of an aptamer variant with a stabilized ATP-binding site indicates fast conformational switching of the original aptamer before ATP binding. Nanopore force spectroscopy is also used to study binding of the thrombin-binding aptamer to its target. To detect aptamer–target interactions in this case, the stability of the ligand-free aptamer—containing G-quadruplexes—is tuned via the potassium content of the buffer. Although the presence of thrombin was detected, limitations of the method for aptamers with strong secondary structures and complexes with nanomolar Kd were identified. PMID:24010663

Point mutation impairs centromeric CENH3 loading and induces haploid plants.

PubMed

Karimi-Ashtiyani, Raheleh; Ishii, Takayoshi; Niessen, Markus; Stein, Nils; Heckmann, Stefan; Gurushidze, Maia; Banaei-Moghaddam, Ali Mohammad; Fuchs, Jörg; Schubert, Veit; Koch, Kerstin; Weiss, Oda; Demidov, Dmitri; Schmidt, Klaus; Kumlehn, Jochen; Houben, Andreas

2015-09-08

The chromosomal position of the centromere-specific histone H3 variant CENH3 (also called "CENP-A") is the assembly site for the kinetochore complex of active centromeres. Any error in transcription, translation, modification, or incorporation can affect the ability to assemble intact CENH3 chromatin and can cause centromere inactivation [Allshire RC, Karpen GH (2008) Nat Rev Genet 9 (12):923-937]. Here we show that a single-point amino acid exchange in the centromere-targeting domain of CENH3 leads to reduced centromere loading of CENH3 in barley, sugar beet, and Arabidopsis thaliana. Haploids were obtained after cenh3 L130F-complemented cenh3-null mutant plants were crossed with wild-type A. thaliana. In contrast, in a noncompeting situation (i.e., centromeres possessing only mutated or only wild-type CENH3), no uniparental chromosome elimination occurs during early embryogenesis. The high degree of evolutionary conservation of the identified mutation site offers promising opportunities for application in a wide range of crop species in which haploid technology is of interest.

Point mutation impairs centromeric CENH3 loading and induces haploid plants

PubMed Central

Karimi-Ashtiyani, Raheleh; Ishii, Takayoshi; Niessen, Markus; Stein, Nils; Heckmann, Stefan; Gurushidze, Maia; Banaei-Moghaddam, Ali Mohammad; Fuchs, Jörg; Schubert, Veit; Koch, Kerstin; Weiss, Oda; Demidov, Dmitri; Schmidt, Klaus; Kumlehn, Jochen; Houben, Andreas

2015-01-01

The chromosomal position of the centromere-specific histone H3 variant CENH3 (also called “CENP-A”) is the assembly site for the kinetochore complex of active centromeres. Any error in transcription, translation, modification, or incorporation can affect the ability to assemble intact CENH3 chromatin and can cause centromere inactivation [Allshire RC, Karpen GH (2008) Nat Rev Genet 9 (12):923–937]. Here we show that a single-point amino acid exchange in the centromere-targeting domain of CENH3 leads to reduced centromere loading of CENH3 in barley, sugar beet, and Arabidopsis thaliana. Haploids were obtained after cenh3 L130F-complemented cenh3-null mutant plants were crossed with wild-type A. thaliana. In contrast, in a noncompeting situation (i.e., centromeres possessing only mutated or only wild-type CENH3), no uniparental chromosome elimination occurs during early embryogenesis. The high degree of evolutionary conservation of the identified mutation site offers promising opportunities for application in a wide range of crop species in which haploid technology is of interest. PMID:26294252

TelAP1 links telomere complexes with developmental expression site silencing in African trypanosomes

PubMed Central

Reis, Helena; Schwebs, Marie; Dietz, Sabrina; Janzen, Christian J; Butter, Falk

2018-01-01

Abstract During its life cycle, Trypanosoma brucei shuttles between a mammalian host and the tsetse fly vector. In the mammalian host, immune evasion of T. brucei bloodstream form (BSF) cells relies on antigenic variation, which includes monoallelic expression and periodic switching of variant surface glycoprotein (VSG) genes. The active VSG is transcribed from only 1 of the 15 subtelomeric expression sites (ESs). During differentiation from BSF to the insect-resident procyclic form (PCF), the active ES is transcriptionally silenced. We used mass spectrometry-based interactomics to determine the composition of telomere protein complexes in T. brucei BSF and PCF stages to learn more about the structure and functions of telomeres in trypanosomes. Our data suggest a different telomere complex composition in the two forms of the parasite. One of the novel telomere-associated proteins, TelAP1, forms a complex with telomeric proteins TbTRF, TbRAP1 and TbTIF2 and influences ES silencing kinetics during developmental differentiation. PMID:29385523

Mitochondrial generation of superoxide and hydrogen peroxide as the source of mitochondrial redox signaling.

PubMed

Brand, Martin D

2016-11-01

This review examines the generation of reactive oxygen species by mammalian mitochondria, and the status of different sites of production in redox signaling and pathology. Eleven distinct mitochondrial sites associated with substrate oxidation and oxidative phosphorylation leak electrons to oxygen to produce superoxide or hydrogen peroxide: oxoacid dehydrogenase complexes that feed electrons to NAD + ; respiratory complexes I and III, and dehydrogenases, including complex II, that use ubiquinone as acceptor. The topologies, capacities, and substrate dependences of each site have recently clarified. Complex III and mitochondrial glycerol 3-phosphate dehydrogenase generate superoxide to the external side of the mitochondrial inner membrane as well as the matrix, the other sites generate superoxide and/or hydrogen peroxide exclusively in the matrix. These different site-specific topologies are important for redox signaling. The net rate of superoxide or hydrogen peroxide generation depends on the substrates present and the antioxidant systems active in the matrix and cytosol. The rate at each site can now be measured in complex substrate mixtures. In skeletal muscle mitochondria in media mimicking muscle cytosol at rest, four sites dominate, two in complex I and one each in complexes II and III. Specific suppressors of two sites have been identified, the outer ubiquinone-binding site in complex III (site III Qo ) and the site in complex I active during reverse electron transport (site I Q ). These suppressors prevent superoxide/hydrogen peroxide production from a specific site without affecting oxidative phosphorylation, making them excellent tools to investigate the status of the sites in redox signaling, and to suppress the sites to prevent pathologies. They allow the cellular roles of mitochondrial superoxide/hydrogen peroxide production to be investigated without catastrophic confounding bioenergetic effects. They show that sites III Qo and I Q are active in cells and have important roles in redox signaling (e.g. hypoxic signaling and ER-stress) and in causing oxidative damage in a variety of biological contexts. Copyright © 2016 Elsevier Inc. All rights reserved.

Potential high-frequency off-target mutagenesis induced by CRISPR/Cas9 in Arabidopsis and its prevention.

PubMed

Zhang, Qiang; Xing, Hui-Li; Wang, Zhi-Ping; Zhang, Hai-Yan; Yang, Fang; Wang, Xue-Chen; Chen, Qi-Jun

2018-03-01

We present novel observations of high-specificity SpCas9 variants, sgRNA expression strategies based on mutant sgRNA scaffold and tRNA processing system, and CRISPR/Cas9-mediated T-DNA integrations. Specificity of CRISPR/Cas9 tools has been a major concern along with the reports of their successful applications. We report unexpected observations of high frequency off-target mutagenesis induced by CRISPR/Cas9 in T1 Arabidopsis mutants although the sgRNA was predicted to have a high specificity score. We also present evidence that the off-target effects were further exacerbated in the T2 progeny. To prevent the off-target effects, we tested and optimized two strategies in Arabidopsis, including introduction of a mCherry cassette for a simple and reliable isolation of Cas9-free mutants and the use of highly specific mutant SpCas9 variants. Optimization of the mCherry vectors and subsequent validation found that fusion of tRNA with the mutant rather than the original sgRNA scaffold significantly improves editing efficiency. We then examined the editing efficiency of eight high-specificity SpCas9 variants in combination with the improved tRNA-sgRNA fusion strategy. Our results suggest that highly specific SpCas9 variants require a higher level of expression than their wild-type counterpart to maintain high editing efficiency. Additionally, we demonstrate that T-DNA can be inserted into the cleavage sites of CRISPR/Cas9 targets with high frequency. Altogether, our results suggest that in plants, continuous attention should be paid to off-target effects induced by CRISPR/Cas9 in current and subsequent generations, and that the tools optimized in this report will be useful in improving genome editing efficiency and specificity in plants and other organisms.

A rhodium(III) complex for high-affinity DNA base-pair mismatch recognition

PubMed Central

Junicke, Henrik; Hart, Jonathan R.; Kisko, Jennifer; Glebov, Oleg; Kirsch, Ilan R.; Barton, Jacqueline K.

2003-01-01

A rhodium(III) complex, rac-[Rh(bpy)2phzi]3+ (bpy, 2,2′-bipyridine; phzi, benzo[a]phenazine-5,6-quinone diimine) has been designed as a sterically demanding intercalator targeted to destabilized mismatched sites in double-helical DNA. The complex is readily synthesized by condensation of the phenazine quinone with the corresponding diammine complex. Upon photoactivation, the complex promotes direct strand scission at single-base mismatch sites within the DNA duplex. As with the parent mismatch-specific reagent, [Rh(bpy)2(chrysi)]3+ [chrysene-5,6-quinone diimine (chrysi)], mismatch selectivity depends on the helix destabilization associated with mispairing. Unlike the parent chrysi complex, the phzi analogue binds and cleaves with high affinity and efficiency. The specific binding constants for CA, CC, and CT mismatches within a 31-mer oligonucleotide duplex are 0.3, 1, and 6 × 107 M−1, respectively; site-specific photocleavage is evident at nanomolar concentrations. Moreover, the specificity, defined as the ratio in binding affinities for mispaired vs. well paired sites, is maintained. The increase in affinity is attributed to greater stability in the mismatched site associated with stacking by the heterocyclic aromatic ligand. The high-affinity complex is also applied in the differential cleavage of DNA obtained from cell lines deficient in mismatch repair vs. those proficient in mismatch repair. Agreement is found between photocleavage by the mismatch-specific probes and deficiency in mismatch repair. This mismatch-specific targeting, therefore, offers a potential strategy for new chemotherapeutic design. PMID:12610209

Unraveling Entropic Rate Acceleration Induced by Solvent Dynamics in Membrane Enzymes.

PubMed

Kürten, Charlotte; Syrén, Per-Olof

2016-01-16

Enzyme catalysis evolved in an aqueous environment. The influence of solvent dynamics on catalysis is, however, currently poorly understood and usually neglected. The study of water dynamics in enzymes and the associated thermodynamical consequences is highly complex and has involved computer simulations, nuclear magnetic resonance (NMR) experiments, and calorimetry. Water tunnels that connect the active site with the surrounding solvent are key to solvent displacement and dynamics. The protocol herein allows for the engineering of these motifs for water transport, which affects specificity, activity and thermodynamics. By providing a biophysical framework founded on theory and experiments, the method presented herein can be used by researchers without previous expertise in computer modeling or biophysical chemistry. The method will advance our understanding of enzyme catalysis on the molecular level by measuring the enthalpic and entropic changes associated with catalysis by enzyme variants with obstructed water tunnels. The protocol can be used for the study of membrane-bound enzymes and other complex systems. This will enhance our understanding of the importance of solvent reorganization in catalysis as well as provide new catalytic strategies in protein design and engineering.

Amino Acid Variation in HLA Class II Proteins Is a Major Determinant of Humoral Response to Common Viruses

PubMed Central

Hammer, Christian; Begemann, Martin; McLaren, Paul J.; Bartha, István; Michel, Angelika; Klose, Beate; Schmitt, Corinna; Waterboer, Tim; Pawlita, Michael; Schulz, Thomas F.; Ehrenreich, Hannelore; Fellay, Jacques

2015-01-01

The magnitude of the human antibody response to viral antigens is highly variable. To explore the human genetic contribution to this variability, we performed genome-wide association studies of the immunoglobulin G response to 14 pathogenic viruses in 2,363 immunocompetent adults. Significant associations were observed in the major histocompatibility complex region on chromosome 6 for influenza A virus, Epstein-Barr virus, JC polyomavirus, and Merkel cell polyomavirus. Using local imputation and fine mapping, we identified specific amino acid residues in human leucocyte antigen (HLA) class II proteins as the most probable causal variants underlying these association signals. Common HLA-DRβ1 haplotypes showed virus-specific patterns of humoral-response regulation. We observed an overlap between variants affecting the humoral response to influenza A and EBV and variants previously associated with autoimmune diseases related to these viruses. The results of this study emphasize the central and pathogen-specific role of HLA class II variation in the modulation of humoral immune response to viral antigens in humans. PMID:26456283

Determination of a Screening Metric for High Diversity DNA Libraries.

PubMed

Guido, Nicholas J; Handerson, Steven; Joseph, Elaine M; Leake, Devin; Kung, Li A

2016-01-01

The fields of antibody engineering, enzyme optimization and pathway construction rely increasingly on screening complex variant DNA libraries. These highly diverse libraries allow researchers to sample a maximized sequence space; and therefore, more rapidly identify proteins with significantly improved activity. The current state of the art in synthetic biology allows for libraries with billions of variants, pushing the limits of researchers' ability to qualify libraries for screening by measuring the traditional quality metrics of fidelity and diversity of variants. Instead, when screening variant libraries, researchers typically use a generic, and often insufficient, oversampling rate based on a common rule-of-thumb. We have developed methods to calculate a library-specific oversampling metric, based on fidelity, diversity, and representation of variants, which informs researchers, prior to screening the library, of the amount of oversampling required to ensure that the desired fraction of variant molecules will be sampled. To derive this oversampling metric, we developed a novel alignment tool to efficiently measure frequency counts of individual nucleotide variant positions using next-generation sequencing data. Next, we apply a method based on the "coupon collector" probability theory to construct a curve of upper bound estimates of the sampling size required for any desired variant coverage. The calculated oversampling metric will guide researchers to maximize their efficiency in using highly variant libraries.

Adeno-Associated Virus Type 2 Wild-Type and Vector-Mediated Genomic Integration Profiles of Human Diploid Fibroblasts Analyzed by Third-Generation PacBio DNA Sequencing

PubMed Central

Hüser, Daniela; Gogol-Döring, Andreas; Chen, Wei

2014-01-01

ABSTRACT Genome-wide analysis of adeno-associated virus (AAV) type 2 integration in HeLa cells has shown that wild-type AAV integrates at numerous genomic sites, including AAVS1 on chromosome 19q13.42. Multiple GAGY/C repeats, resembling consensus AAV Rep-binding sites are preferred, whereas rep-deficient AAV vectors (rAAV) regularly show a random integration profile. This study is the first study to analyze wild-type AAV integration in diploid human fibroblasts. Applying high-throughput third-generation PacBio-based DNA sequencing, integration profiles of wild-type AAV and rAAV are compared side by side. Bioinformatic analysis reveals that both wild-type AAV and rAAV prefer open chromatin regions. Although genomic features of AAV integration largely reproduce previous findings, the pattern of integration hot spots differs from that described in HeLa cells before. DNase-Seq data for human fibroblasts and for HeLa cells reveal variant chromatin accessibility at preferred AAV integration hot spots that correlates with variant hot spot preferences. DNase-Seq patterns of these sites in human tissues, including liver, muscle, heart, brain, skin, and embryonic stem cells further underline variant chromatin accessibility. In summary, AAV integration is dependent on cell-type-specific, variant chromatin accessibility leading to random integration profiles for rAAV, whereas wild-type AAV integration sites cluster near GAGY/C repeats. IMPORTANCE Adeno-associated virus type 2 (AAV) is assumed to establish latency by chromosomal integration of its DNA. This is the first genome-wide analysis of wild-type AAV2 integration in diploid human cells and the first to compare wild-type to recombinant AAV vector integration side by side under identical experimental conditions. Major determinants of wild-type AAV integration represent open chromatin regions with accessible consensus AAV Rep-binding sites. The variant chromatin accessibility of different human tissues or cell types will have impact on vector targeting to be considered during gene therapy. PMID:25031342

A comprehensive collection of annotations to interpret sequence variation in human mitochondrial transfer RNAs.

PubMed

Diroma, Maria Angela; Lubisco, Paolo; Attimonelli, Marcella

2016-11-08

The abundance of biological data characterizing the genomics era is contributing to a comprehensive understanding of human mitochondrial genetics. Nevertheless, many aspects are still unclear, specifically about the variability of the 22 human mitochondrial transfer RNA (tRNA) genes and their involvement in diseases. The complex enrichment and isolation of tRNAs in vitro leads to an incomplete knowledge of their post-transcriptional modifications and three-dimensional folding, essential for correct tRNA functioning. An accurate annotation of mitochondrial tRNA variants would be definitely useful and appreciated by mitochondrial researchers and clinicians since the most of bioinformatics tools for variant annotation and prioritization available so far cannot shed light on the functional role of tRNA variations. To this aim, we updated our MToolBox pipeline for mitochondrial DNA analysis of high throughput and Sanger sequencing data by integrating tRNA variant annotations in order to identify and characterize relevant variants not only in protein coding regions, but also in tRNA genes. The annotation step in the pipeline now provides detailed information for variants mapping onto the 22 mitochondrial tRNAs. For each mt-tRNA position along the entire genome, the relative tRNA numbering, tRNA type, cloverleaf secondary domains (loops and stems), mature nucleotide and interactions in the three-dimensional folding were reported. Moreover, pathogenicity predictions for tRNA and rRNA variants were retrieved from the literature and integrated within the annotations provided by MToolBox, both in the stand-alone version and web-based tool at the Mitochondrial Disease Sequence Data Resource (MSeqDR) website. All the information available in the annotation step of MToolBox were exploited to generate custom tracks which can be displayed in the GBrowse instance at MSeqDR website. To the best of our knowledge, specific data regarding mitochondrial variants in tRNA genes were introduced for the first time in a tool for mitochondrial genome analysis, supporting the interpretation of genetic variants in specific genomic contexts.

Allele frequencies of variants in ultra conserved elements identify selective pressure on transcription factor binding.

PubMed

Silla, Toomas; Kepp, Katrin; Tai, E Shyong; Goh, Liang; Davila, Sonia; Catela Ivkovic, Tina; Calin, George A; Voorhoeve, P Mathijs

2014-01-01

Ultra-conserved genes or elements (UCGs/UCEs) in the human genome are extreme examples of conservation. We characterized natural variations in 2884 UCEs and UCGs in two distinct populations; Singaporean Chinese (n = 280) and Italian (n = 501) by using a pooled sample, targeted capture, sequencing approach. We identify, with high confidence, in these regions the abundance of rare SNVs (MAF<0.5%) of which 75% is not present in dbSNP137. UCEs association studies for complex human traits can use this information to model expected background variation and thus necessary power for association studies. By combining our data with 1000 Genome Project data, we show in three independent datasets that prevalent UCE variants (MAF>5%) are more often found in relatively less-conserved nucleotides within UCEs, compared to rare variants. Moreover, prevalent variants are less likely to overlap transcription factor binding site. Using SNPfold we found no significant influence of RNA secondary structure on UCE conservation. All together, these results suggest UCEs are not under selective pressure as a stretch of DNA but are under differential evolutionary pressure on the single nucleotide level.

Mutation spectrum of homogentisic acid oxidase (HGD) in alkaptonuria.

PubMed

Vilboux, Thierry; Kayser, Michael; Introne, Wendy; Suwannarat, Pim; Bernardini, Isa; Fischer, Roxanne; O'Brien, Kevin; Kleta, Robert; Huizing, Marjan; Gahl, William A

2009-12-01

Alkaptonuria (AKU) is a rare autosomal recessive metabolic disorder, characterized by accumulation of homogentisic acid, leading to darkened urine, pigmentation of connective tissue (ochronosis), joint and spine arthritis, and destruction of cardiac valves. AKU is due to mutations in the homogentisate dioxygenase gene (HGD) that converts homogentisic acid to maleylacetoacetic acid in the tyrosine catabolic pathway. Here we report a comprehensive mutation analysis of 93 patients enrolled in our study, as well as an extensive update of all previously published HGD mutations associated with AKU. Within our patient cohort, we identified 52 HGD variants, of which 22 were novel. This yields a total of 91 identified HGD variations associated with AKU to date, including 62 missense, 13 splice site, 10 frameshift, 5 nonsense, and 1 no-stop mutation. Most HGD variants reside in exons 3, 6, 8, and 13. We assessed the potential effect of all missense variations on protein function, using five bioinformatic tools specifically designed for interpretation of missense variants (SIFT, POLYPHEN, PANTHER, PMUT, and SNAP). We also analyzed the potential effect of splice-site variants using two different tools (BDGP and NetGene2). This study provides valuable resources for molecular analysis of alkaptonuria and expands our knowledge of the molecular basis of this disease.

Mutation spectrum of homogentisic acid oxidase (HGD) in alkaptonuria

PubMed Central

Vilboux, Thierry; Kayser, Michael; Introne, Wendy; Suwannarat, Pim; Bernardini, Isa; Fischer, Roxanne; O’Brien, Kevin; Kleta, Robert; Huizing, Marjan; Gahl, William A.

2009-01-01

Alkaptonuria (AKU) is a rare autosomal recessive metabolic disorder, characterized by accumulation of homogentisic acid, leading to darkened urine, pigmentation of connective tissue (ochronosis), joint and spine arthritis, and destruction of cardiac valves. AKU is due to mutations in the homogentisate dioxygenase gene, HGD, that converts homogentisic acid to maleylacetoacetic acid in the tyrosine catabolic pathway. Here we report a comprehensive mutation analysis of 93 patients enrolled in our study, as well as an extensive update of all previously published HGD mutations associated with AKU. Within our patient cohort, we identified 52 HGD variants, of which 22 were novel. This yields a total of 91 identified HGD variations associated with AKU to date, including 62 missense, 13 splice site, 10 frameshift, 5 nonsense and 1 no-stop mutation. Most HGD variants reside in exons 3, 6, 8 and 13. We assessed the potential effect of all missense variations on protein function, using 5 bioinformatic tools specifically designed for interpretation of missense variants (SIFT, POLYPHEN, PANTHER, PMUT and SNAP). We also analyzed the potential effect of splice site variants using two different tools (BDGP and NetGene2). This study provides valuable resources for molecular analysis of alkaptonuria and expands our knowledge of the molecular basis of this disease. PMID:19862842

Allelic Variants of Complement Genes Associated with Dense Deposit Disease

PubMed Central

Abrera-Abeleda, Maria Asuncion; Nishimura, Carla; Frees, Kathy; Jones, Michael; Maga, Tara; Katz, Louis M.; Zhang, Yuzhou

2011-01-01

The alternative pathway of the complement cascade plays a role in the pathogenesis of dense deposit disease (DDD). Deficiency of complement factor H and mutations in CFH associate with the development of DDD, but it is unknown whether allelic variants in other complement genes also associate with this disease. We studied patients with DDD and identified previously unreported sequence alterations in several genes in addition to allelic variants and haplotypes common to patients with DDD. We found that the likelihood of developing DDD increases with the presence of two or more risk alleles in CFH and C3. To determine the functional consequence of this finding, we measured the activity of the alternative pathway in serum samples from phenotypically normal controls genotyped for variants in CFH and C3. Alternative pathway activity was higher in the presence of variants associated with DDD. Taken together, these data confirm that DDD is a complex genetic disease and may provide targets for the development of disease-specific therapies. PMID:21784901

A pooling-based approach to mapping genetic variants associated with DNA methylation

PubMed Central

Kaplow, Irene M.; MacIsaac, Julia L.; Mah, Sarah M.; McEwen, Lisa M.; Kobor, Michael S.; Fraser, Hunter B.

2015-01-01

DNA methylation is an epigenetic modification that plays a key role in gene regulation. Previous studies have investigated its genetic basis by mapping genetic variants that are associated with DNA methylation at specific sites, but these have been limited to microarrays that cover <2% of the genome and cannot account for allele-specific methylation (ASM). Other studies have performed whole-genome bisulfite sequencing on a few individuals, but these lack statistical power to identify variants associated with DNA methylation. We present a novel approach in which bisulfite-treated DNA from many individuals is sequenced together in a single pool, resulting in a truly genome-wide map of DNA methylation. Compared to methods that do not account for ASM, our approach increases statistical power to detect associations while sharply reducing cost, effort, and experimental variability. As a proof of concept, we generated deep sequencing data from a pool of 60 human cell lines; we evaluated almost twice as many CpGs as the largest microarray studies and identified more than 2000 genetic variants associated with DNA methylation. We found that these variants are highly enriched for associations with chromatin accessibility and CTCF binding but are less likely to be associated with traits indirectly linked to DNA, such as gene expression and disease phenotypes. In summary, our approach allows genome-wide mapping of genetic variants associated with DNA methylation in any tissue of any species, without the need for individual-level genotype or methylation data. PMID:25910490

A pooling-based approach to mapping genetic variants associated with DNA methylation

DOE PAGES

Kaplow, Irene M.; MacIsaac, Julia L.; Mah, Sarah M.; ...

2015-04-24

DNA methylation is an epigenetic modification that plays a key role in gene regulation. Previous studies have investigated its genetic basis by mapping genetic variants that are associated with DNA methylation at specific sites, but these have been limited to microarrays that cover <2% of the genome and cannot account for allele-specific methylation (ASM). Other studies have performed whole-genome bisulfite sequencing on a few individuals, but these lack statistical power to identify variants associated with DNA methylation. We present a novel approach in which bisulfite-treated DNA from many individuals is sequenced together in a single pool, resulting in a trulymore » genome-wide map of DNA methylation. Compared to methods that do not account for ASM, our approach increases statistical power to detect associations while sharply reducing cost, effort, and experimental variability. As a proof of concept, we generated deep sequencing data from a pool of 60 human cell lines; we evaluated almost twice as many CpGs as the largest microarray studies and identified more than 2000 genetic variants associated with DNA methylation. Here we found that these variants are highly enriched for associations with chromatin accessibility and CTCF binding but are less likely to be associated with traits indirectly linked to DNA, such as gene expression and disease phenotypes. In summary, our approach allows genome-wide mapping of genetic variants associated with DNA methylation in any tissue of any species, without the need for individual-level genotype or methylation data.« less

Assessing the Spectrum of Germline Variation in Fanconi Anemia Genes among Patients with Head and Neck Carcinoma before Age 50

PubMed Central

Chandrasekharappa, Settara C.; Chinn, Steven B.; Donovan, Frank X.; Chowdhury, Naweed I.; Kamat, Aparna; Adeyemo, Adebowale A.; Thomas, James W.; Vemulapalli, Meghana; Hussey, Caroline S.; Reid, Holly H.; Mullikin, James C.; Wei, Qingyi; Sturgis, Erich M.

2018-01-01

Patients with Fanconi anemia (FA) have increased risk for head and neck squamous cell carcinoma (HNSCC). We sought to determine the prevalence of undiagnosed FA and FA carriers in patients with HNSCC and an age cutoff for FA genetic screening. Screening germline DNA from 417 HNSCC patients under age 50 revealed 194 FA gene variants in 185 patients (44%). The variant spectrum was comprised of 183 nonsynonymous point mutations, nine indels, one large deletion, and one synonymous variant predicted to effect splicing. 108 patients (26%) had at least one rare variant predicted to be damaging, and 57 (14%) had at least one rare variant predicted to be damaging and previously reported. Fifteen patients carried two rare variants, or an X-linked variant, in an FA gene. Overall, we did not identify an age cutoff for FA screening among young HNSCC patients, as there were no significant differences in mutation rates when patients were stratified by age, tumor site, ethnicity, smoking status, or human papillomavirus status. However, we observed an increased burden, or mutation load, of FA gene variants in FANCD2, FANCE, and FANCL in our HNSCC patient cohort relative to the 1000 Genomes population. FANCE and FANCL, components of the core complex, are known to be responsible for the recruitment and ubiquitination, respectively, of FANCD2, a critical step in the FA DNA repair pathway. FA germline functional variants offer a novel area of study in HNSCC tumorigenesis, and the increased mutation burden of critical genes indicates the importance of the FA pathway in HNSCC. PMID:28678401

Bifunctional chelating agent for the design and development of site specific radiopharmaceuticals and biomolecule conjugation strategy

DOEpatents

Katti, Kattesh V.; Prabhu, Kandikere R.; Gali, Hariprasad; Pillarsetty, Nagavara Kishore; Volkert, Wynn A.

2003-10-21

There is provided a method of labeling a biomolecule with a transition metal or radiometal in a site specific manner to produce a diagnostic or therapeutic pharmaceutical compound by synthesizing a P.sub.2 N.sub.2 -bifunctional chelating agent intermediate, complexing the intermediate with a radio metal or a transition metal, and covalently linking the resulting metal-complexed bifunctional chelating agent with a biomolecule in a site specific manner. Also provided is a method of synthesizing the --PR.sub.2 containing biomolecules by synthesizing a P.sub.2 N.sub.2 -bifunctional chelating agent intermediate, complexing the intermediate with a radiometal or a transition metal, and covalently linking the resulting radio metal-complexed bifunctional chelating agent with a biomolecule in a site specific manner. There is provided a therapeutic or diagnostic agent comprising a --PR.sub.2 containing biomolecule.

Study designs for identification of rare disease variants in complex diseases: the utility of family-based designs.

PubMed

Ionita-Laza, Iuliana; Ottman, Ruth

2011-11-01

The recent progress in sequencing technologies makes possible large-scale medical sequencing efforts to assess the importance of rare variants in complex diseases. The results of such efforts depend heavily on the use of efficient study designs and analytical methods. We introduce here a unified framework for association testing of rare variants in family-based designs or designs based on unselected affected individuals. This framework allows us to quantify the enrichment in rare disease variants in families containing multiple affected individuals and to investigate the optimal design of studies aiming to identify rare disease variants in complex traits. We show that for many complex diseases with small values for the overall sibling recurrence risk ratio, such as Alzheimer's disease and most cancers, sequencing affected individuals with a positive family history of the disease can be extremely advantageous for identifying rare disease variants. In contrast, for complex diseases with large values of the sibling recurrence risk ratio, sequencing unselected affected individuals may be preferable.

Molecular dynamics simulations revealed structural differences among WRKY domain-DNA interaction in barley (Hordeum vulgare).

PubMed

Pandey, Bharati; Grover, Abhinav; Sharma, Pradeep

2018-02-12

The WRKY transcription factors are a class of DNA-binding proteins involved in diverse plant processes play critical roles in response to abiotic and biotic stresses. Genome-wide divergence analysis of WRKY gene family in Hordeum vulgare provided a framework for molecular evolution and functional roles. So far, the crystal structure of WRKY from barley has not been resolved; moreover, knowledge of the three-dimensional structure of WRKY domain is pre-requisites for exploring the protein-DNA recognition mechanisms. Homology modelling based approach was used to generate structures for WRKY DNA binding domain (DBD) and its variants using AtWRKY1 as a template. Finally, the stability and conformational changes of the generated model in unbound and bound form was examined through atomistic molecular dynamics (MD) simulations for 100 ns time period. In this study, we investigated the comparative binding pattern of WRKY domain and its variants with W-box cis-regulatory element using molecular docking and dynamics (MD) simulations assays. The atomic insight into WRKY domain exhibited significant variation in the intermolecular hydrogen bonding pattern, leading to the structural anomalies in the variant type and differences in the DNA-binding specificities. Based on the MD analysis, residual contribution and interaction contour, wild-type WRKY (HvWRKY46) were found to interact with DNA through highly conserved heptapeptide in the pre- and post-MD simulated complexes, whereas heptapeptide interaction with DNA was missing in variants (I and II) in post-MD complexes. Consequently, through principal component analysis, wild-type WRKY was also found to be more stable by obscuring a reduced conformational space than the variant I (HvWRKY34). Lastly, high binding free energy for wild-type and variant II allowed us to conclude that wild-type WRKY-DNA complex was more stable relative to variants I. The results of our study revealed complete dynamic and structural information about WRKY domain-DNA interactions. However, no structure base information reported to date for WRKY variants and their mechanism of interaction with DNA. Our findings highlighted the importance of selecting a sequence to generate newer transgenic plants that would be increasingly tolerance to stress conditions.

Global Displacement of Canine Parvovirus by a Host-Adapted Variant: Structural Comparison between Pandemic Viruses with Distinct Host Ranges

PubMed Central

Organtini, Lindsey J.; Allison, Andrew B.; Lukk, Tiit; Parrish, Colin R.

2014-01-01

Canine parvovirus type 2 (CPV-2) emerged in 1978 and spread worldwide within 2 years. Subsequently, CPV-2 was completely replaced by the variant CPV-2a, which is characterized by four specific capsid (VP2) mutations. The X-ray crystal structure of the CPV-2a capsid shows that each mutation confers small local changes. The loss of a hydrogen bond and introduction of a glycine residue likely introduce flexibility to sites that control interactions with the host receptor, antibodies, and sialic acids. PMID:25410876

Association of transforming growth-factor alpha gene polymorphisms with nonsyndromic cleft palate only (CPO)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shiang, R.; Lidral, A.C.; Ardinger, H.H.

1993-10-01

Genetic analysis and tissue-specific expression studies support a role for transforming growth-factor alpha (TGFA) in craniofacial development. Previous studies have confirmed an association of alleles for TGFA with nonsyndromic cleft lip with or without cleft palate (CL/P) in humans. The authors carried out a retrospective association study to determine whether specific allelic variants of the TGFA gene are also associated with cleft palate only (CPO). The PCR products from 12 overlapping sets of primers to the TGFA cDNA were examined by using single-strand conformational polymorphism analysis. Four DNA polymorphic sites for TGFA were identified in the 3[prime] untranslated region ofmore » the TGFA gene. These variants, as well as previously identified RFLPs for TGFA, were characterized in case and control populations for CPO by using X[sup 2] analysis. A significant association between alleles of TGFA and CPO was identified which further supports a role for this gene as one of the genetic determinants of craniofacial development. Sequence analysis of the variants disclosed a cluster of three variable sites within 30 bp of each other in the 3[prime] untranslated region previously associated with an antisense transcript. These studies extend the role for TGFA in craniofacial morphogenesis and support an interrelated mechanism underlying nonsyndromic forms of CL/P. 46 refs., 3 figs., 3 tabs.« less

The tissue inhibitors of metalloproteinases (TIMPs): An ancient family with structural and functional diversity

PubMed Central

Brew, Keith; Nagase, Hideaki

2010-01-01

Tissue inhibitors of metalloproteinases (TIMPs) are widely distributed in the animal kingdom and the human genome contains four paralogous genes encoding TIMPs 1 to 4. TIMPs were originally characterized as inhibitors of matrix metalloproteinases (MMPs), but their range of activities has now been found to be broader as it includes the inhibition of several of the disintegrin-metalloproteinases, ADAMs and ADAMTSs. TIMPs are therefore key regulators of the metalloproteinases that degrade the extracellular matrix and shed cell surface molecules. Structural studies of TIMP–MMP complexes have elucidated the inhibition mechanism of TIMPs and the multiple sites through which they interact with target enzymes, allowing the generation of TIMP variants that selectively inhibit different groups of metalloproteinases. Engineering such variants is complicated by the fact that TIMPs can undergo changes in molecular dynamics induced by their interactions with proteases. TIMPs also have biological activities that are independent of metalloproteinases; these include effects on cell growth and differentiation, cell migration, anti-angiogenesis, anti- and pro-apoptosis, and synaptic plasticity. Receptors responsible for some of these activities have been identified and their signaling pathways have been investigated. A series of studies using mice with specific TIMP gene deletions has illuminated the importance of these molecules in biology and pathology. PMID:20080133

Anatomy and histology of the sacroiliac joints.

PubMed

Egund, Niels; Jurik, Anne Grethe

2014-07-01

The anatomy of joints provides an important basis for understanding the nature and imaging of pathologic lesions and their imaging appearance. This applies especially to the sacroiliac (SI) joints, which play a major role in the diagnosis of spondyloarthritis. They are composed of two different joint portions, a cartilage-covered portion ventrally and a ligamentous portion dorsally, and thus rather complex anatomically. Knowledge of anatomy and the corresponding normal imaging findings are important in the imaging diagnosis of sacroiliitis, especially by MR imaging. A certain distinction between the two joint portions by MR imaging is only obtainable by axial slice orientation. Together with a perpendicular coronal slice orientation, it provides adequate anatomical information and thereby a possibility for detecting the anatomical site of disease-specific characteristics and normal variants simulating disease. This overview describes current knowledge about the normal macroscopic and microscopic anatomy of the SI joints. Thieme Medical Publishers 333 Seventh Avenue, New York, NY 10001, USA.

Biochemical and structural characterization of Klebsiella pneumoniae oxamate amidohydrolase in the uric acid degradation pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hicks, Katherine A.; Ealick, Steven E.

HpxW from the ubiquitous pathogenKlebsiella pneumoniaeis involved in a novel uric acid degradation pathway downstream from the formation of oxalurate. Specifically, HpxW is an oxamate amidohydrolase which catalyzes the conversion of oxamate to oxalate and is a member of the Ntn-hydrolase superfamily. HpxW is autoprocessed from an inactive precursor to form a heterodimer, resulting in a 35.5 kDa α subunit and a 20 kDa β subunit. Here, the structure of HpxW is presented and the substrate complex is modeled. In addition, the steady-state kinetics of this enzyme and two active-site variants were characterized. These structural and biochemical studies provide furthermore » insight into this class of enzymes and allow a mechanism for catalysis consistent with other members of the Ntn-hydrolase superfamily to be proposed.« less

Atmospheric studies in complex terrain: a planning guide for future studies

DOE Office of Scientific and Technical Information (OSTI.GOV)

Orgill, M.M.

The objective of this study is to assist the US Department of Energy in Conducting its atmospheric studies in complex terrain (ASCOT0 by defining various complex terrain research systems and relating these options to specific landforms sites. This includes: (1) reviewing past meteorological and diffusion research on complex terrain; (2) relating specific terrain-induced airflow phenomena to specific landforms and time and space scales; (3) evaluating the technical difficulty of modeling and measuring terrain-induced airflow phenomena; and (4) avolving severdal research options and proposing candidate sites for continuing and expanding field and modeling work. To evolve research options using variable candidatemore » sites, four areas were considered: site selection, terrain uniqueness and quantification, definition of research problems and research plans. 36 references, 111 figures, 20 tables.« less

Plant centromeres.

PubMed

Lamb, J C; Yu, W; Han, F; Birchler, J A

2008-01-01

Plant centromeres are generally composed of tandem arrays of simple repeats that are typical of a particular species, but that evolve rapidly. Centromere specific retroelements are also present. These arrays associate with a centromere specific variant of histone H3 that anchors the site of the kinetochore. Although such DNA arrays are typical of the centromere, the specification of centromere activity has an epigenetic component as shown by the fact that centromeres are formed in the absence of such repeats and that centromeres in dicentric chromosomes regularly undergo inactivation.

Extrinsic Tryptophans as NMR Probes of Allosteric Coupling in Membrane Proteins: Application to the A2A Adenosine Receptor.

PubMed

Eddy, Matthew T; Gao, Zhan-Guo; Mannes, Philip; Patel, Nilkanth; Jacobson, Kenneth A; Katritch, Vsevolod; Stevens, Raymond C; Wüthrich, Kurt

2018-06-20

Tryptophan indole 15 N- 1 H signals are well separated in nuclear magnetic resonance (NMR) spectra of proteins. Assignment of the indole 15 N- 1 H signals therefore enables one to obtain site-specific information on complex proteins in supramacromolecular systems, even when extensive assignment of backbone 15 N- 1 H resonances is challenging. Here we exploit the unique indole 15 N- 1 H chemical shift by introducing extrinsic tryptophan reporter residues at judiciously chosen locations in a membrane protein for increased coverage of structure and function by NMR. We demonstrate this approach with three variants of the human A 2A adenosine receptor (A 2A AR), a class A G protein-coupled receptor, each containing a single extrinsic tryptophan near the receptor intracellular surface, in helix V, VI, or VII, respectively. We show that the native A 2A AR global protein fold and ligand binding activity are preserved in these A 2A AR variants. The indole 15 N- 1 H signals from the extrinsic tryptophan reporter residues show different responses to variable efficacy of drugs bound to the receptor orthosteric cavity, and the indole 15 N- 1 H chemical shift of the tryptophan introduced at the intracellular end of helix VI is sensitive to conformational changes resulting from interactions with a polypeptide from the carboxy terminus of the Gα S intracellular partner protein. Introducing extrinsic tryptophans into proteins in complex supramolecular systems thus opens new avenues for NMR investigations in solution.

GARLIC: a bioinformatic toolkit for aetiologically connecting diseases and cell type-specific regulatory maps

PubMed Central

Nikolić, Miloš; Papantonis, Argyris

2017-01-01

Abstract Genome-wide association studies (GWAS) have emerged as a powerful tool to uncover the genetic basis of human common diseases, which often show a complex, polygenic and multi-factorial aetiology. These studies have revealed that 70–90% of all single nucleotide polymorphisms (SNPs) associated with common complex diseases do not occur within genes (i.e. they are non-coding), making the discovery of disease-causative genetic variants and the elucidation of the underlying pathological mechanisms far from straightforward. Based on emerging evidences suggesting that disease-associated SNPs are frequently found within cell type-specific regulatory sequences, here we present GARLIC (GWAS-based Prediction Toolkit for Connecting Diseases and Cell Types), a user-friendly, multi-purpose software with an associated database and online viewer that, using global maps of cis-regulatory elements, can aetiologically connect human diseases with relevant cell types. Additionally, GARLIC can be used to retrieve potential disease-causative genetic variants overlapping regulatory sequences of interest. Overall, GARLIC can satisfy several important needs within the field of medical genetics, thus potentially assisting in the ultimate goal of uncovering the elusive and complex genetic basis of common human disorders. PMID:28007912

The C-terminal domain of CblD interacts with CblC and influences intracellular cobalamin partitioning.

PubMed

Gherasim, Carmen; Hannibal, Luciana; Rajagopalan, Deepa; Jacobsen, Donald W; Banerjee, Ruma

2013-05-01

Mutations in cobalamin or B12 trafficking genes needed for cofactor assimilation and targeting lead to inborn errors of cobalamin metabolism. The gene corresponding to one of these loci, cblD, affects both the mitochondrial and cytoplasmic pathways for B12 processing. We have demonstrated that fibroblast cell lines from patients with mutations in CblD, can dealkylate exogenously supplied methylcobalamin (MeCbl), an activity catalyzed by the CblC protein, but show imbalanced intracellular partitioning of the cofactor into the MeCbl and 5'-deoxyadenosylcobalamin (AdoCbl) pools. These results confirm that CblD functions downstream of CblC in the cofactor assimilation pathway and that it plays an important role in controlling the traffic of the cofactor between the competing cytoplasmic and mitochondrial routes for MeCbl and AdoCbl synthesis, respectively. In this study, we report the interaction of CblC with four CblD protein variants with variable N-terminal start sites. We demonstrate that a complex between CblC and CblD can be isolated particularly under conditions that permit dealkylation of alkylcobalamin by CblC or in the presence of the corresponding dealkylated and oxidized product, hydroxocobalamin (HOCbl). A weak CblC·CblD complex is also seen in the presence of cyanocobalamin. Formation of the CblC·CblD complex is observed with all four CblD variants tested suggesting that the N-terminal 115 residues missing in the shortest variant are not essential for this interaction. Furthermore, limited proteolysis of the CblD variants indicates the presence of a stable C-terminal domain spanning residues ∼116-296. Our results are consistent with an adapter function for CblD, which in complex with CblC·HOCbl, or possibly the less oxidized CblC·cob(II)alamin, partitions the cofactor between AdoCbl and MeCbl assimilation pathways. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

The C-terminal domain of CblD interacts with CblC and influences intracellular cobalamin partitioning☆

PubMed Central

Gherasim, Carmen; Hannibal, Luciana; Rajagopalan, Deepa; Jacobsen, Donald W.; Banerjee, Ruma

2013-01-01

Mutations in cobalamin or B12 trafficking genes needed for cofactor assimilation and targeting lead to inborn errors of cobalamin metabolism. The gene corresponding to one of these loci, cblD, affects both the mitochondrial and cytoplasmic pathways for B12 processing. We have demonstrated that fibroblast cell lines from patients with mutations in CblD, can dealkylate exogenously supplied methylcobalamin (MeCbl), an activity catalyzed by the CblC protein, but show imbalanced intracellular partitioning of the cofactor into the MeCbl and 5′-deoxyadenosylcobalamin (AdoCbl) pools. These results confirm that CblD functions downstream of CblC in the cofactor assimilation pathway and that it plays an important role in controlling the traffic of the cofactor between the competing cytoplasmic and mitochondrial routes for MeCbl and AdoCbl synthesis, respectively. In this study, we report the interaction of CblC with four CblD protein variants with variable N-terminal start sites. We demonstrate that a complex between CblC and CblD can be isolated particularly under conditions that permit dealkylation of alkylcobalamin by CblC or in the presence of the corresponding dealkylated and oxidized product, hydroxocobalamin (HOCbl). A weak CblC·CblD complex is also seen in the presence of cyanocobalamin. Formation of the CblC·CblD complex is observed with all four CblD variants tested suggesting that the N-terminal 115 residues missing in the shortest variant are not essential for this interaction. Furthermore, limited proteolysis of the CblD variants indicates the presence of a stable C-terminal domain spanning residues ~116–296. Our results are consistent with an adapter function for CblD, which in complex with CblC·HOCbl, or possibly the less oxidized CblC·cob(II)alamin, partitions the cofactor between AdoCbl and MeCbl assimilation pathways. PMID:23415655

Characterization of a carbon-carbon hydrolase from Mycobacterium tuberculosis involved in cholesterol metabolism.

PubMed

Lack, Nathan A; Yam, Katherine C; Lowe, Edward D; Horsman, Geoff P; Owen, Robin L; Sim, Edith; Eltis, Lindsay D

2010-01-01

In the recently identified cholesterol catabolic pathway of Mycobacterium tuberculosis, 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate hydrolase (HsaD) is proposed to catalyze the hydrolysis of a carbon-carbon bond in 4,5-9,10-diseco-3-hydroxy-5,9,17-tri-oxoandrosta-1(10),2-diene-4-oic acid (DSHA), the cholesterol meta-cleavage product (MCP) and has been implicated in the intracellular survival of the pathogen. Herein, purified HsaD demonstrated 4-33 times higher specificity for DSHA (k(cat)/K(m) = 3.3 +/- 0.3 x 10(4) m(-1) s(-1)) than for the biphenyl MCP 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA) and the synthetic analogue 8-(2-chlorophenyl)-2-hydroxy-5-methyl-6-oxoocta-2,4-dienoic acid (HOPODA), respectively. The S114A variant of HsaD, in which the active site serine was substituted with alanine, was catalytically impaired and bound DSHA with a K(d) of 51 +/- 2 mum. The S114A.DSHA species absorbed maximally at 456 nm, 60 nm red-shifted versus the DSHA enolate. Crystal structures of the variant in complex with HOPDA, HOPODA, or DSHA to 1.8-1.9 Aindicate that this shift is due to the enzyme-induced strain of the enolate. These data indicate that the catalytic serine catalyzes tautomerization. A second role for this residue is suggested by a solvent molecule whose position in all structures is consistent with its activation by the serine for the nucleophilic attack of the substrate. Finally, the alpha-helical lid covering the active site displayed a ligand-dependent conformational change involving differences in side chain carbon positions of up to 6.7 A, supporting a two-conformation enzymatic mechanism. Overall, these results provide novel insights into the determinants of specificity in a mycobacterial cholesterol-degrading enzyme as well as into the mechanism of MCP hydrolases.

Characterization of a Carbon-Carbon Hydrolase from Mycobacterium tuberculosis Involved in Cholesterol Metabolism*

PubMed Central

Lack, Nathan A.; Yam, Katherine C.; Lowe, Edward D.; Horsman, Geoff P.; Owen, Robin L.; Sim, Edith; Eltis, Lindsay D.

2010-01-01

In the recently identified cholesterol catabolic pathway of Mycobacterium tuberculosis, 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate hydrolase (HsaD) is proposed to catalyze the hydrolysis of a carbon-carbon bond in 4,5–9,10-diseco-3-hydroxy-5,9,17-tri-oxoandrosta-1(10),2-diene-4-oic acid (DSHA), the cholesterol meta-cleavage product (MCP) and has been implicated in the intracellular survival of the pathogen. Herein, purified HsaD demonstrated 4–33 times higher specificity for DSHA (kcat/Km = 3.3 ± 0.3 × 104 m−1 s−1) than for the biphenyl MCP 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA) and the synthetic analogue 8-(2-chlorophenyl)-2-hydroxy-5-methyl-6-oxoocta-2,4-dienoic acid (HOPODA), respectively. The S114A variant of HsaD, in which the active site serine was substituted with alanine, was catalytically impaired and bound DSHA with a Kd of 51 ± 2 μm. The S114A·DSHA species absorbed maximally at 456 nm, 60 nm red-shifted versus the DSHA enolate. Crystal structures of the variant in complex with HOPDA, HOPODA, or DSHA to 1.8–1.9 Åindicate that this shift is due to the enzyme-induced strain of the enolate. These data indicate that the catalytic serine catalyzes tautomerization. A second role for this residue is suggested by a solvent molecule whose position in all structures is consistent with its activation by the serine for the nucleophilic attack of the substrate. Finally, the α-helical lid covering the active site displayed a ligand-dependent conformational change involving differences in side chain carbon positions of up to 6.7 Å, supporting a two-conformation enzymatic mechanism. Overall, these results provide novel insights into the determinants of specificity in a mycobacterial cholesterol-degrading enzyme as well as into the mechanism of MCP hydrolases. PMID:19875455

First evidence for an association of a functional variant in the microRNA-510 target site of the serotonin receptor-type 3E gene with diarrhea predominant irritable bowel syndrome.

PubMed

Kapeller, Johannes; Houghton, Lesley A; Mönnikes, Hubert; Walstab, Jutta; Möller, Dorothee; Bönisch, Heinz; Burwinkel, Barbara; Autschbach, Frank; Funke, Benjamin; Lasitschka, Felix; Gassler, Nikolaus; Fischer, Christine; Whorwell, Peter J; Atkinson, Wendy; Fell, Catherine; Büchner, Karl J; Schmidtmann, Marco; van der Voort, Ivo; Wisser, Anna-Sophia; Berg, Thomas; Rappold, Gudrun; Niesler, Beate

2008-10-01

Diarrhea predominant irritable bowel syndrome (IBS-D) is a complex disorder related to dysfunctions in the serotonergic system. As cis-regulatory variants can play a role in the etiology of complex conditions, we investigated the untranslated regions (UTRs) of the serotonin receptor type 3 subunit genes HTR3A and HTR3E. Mutation analysis was carried out in a pilot sample of 200 IBS patients and 100 healthy controls from the UK. The novel HTR3E 3'-UTR variant c.*76G>A (rs62625044) was associated with female IBS-D (P = 0.033, OR = 8.53). This association was confirmed in a replication study, including 119 IBS-D patients and 195 controls from Germany (P = 0.0046, OR = 4.92). Pooled analysis resulted in a highly significant association of c.*76G>A with female IBS-D (P = 0.0002, OR = 5.39). In a reporter assay, c.*76G>A affected binding of miR-510 to the HTR3E 3'-UTR and caused elevated luciferase expression. HTR3E and miR-510 co-localize in enterocytes of the gut epithelium as shown by in situ hybridization and RT-PCR. This is the first example indicating micro RNA-related expression regulation of a serotonin receptor gene with a cis-regulatory variant affecting this regulation and appearing to be associated with female IBS-D.

Characterization of C-terminally engineered laccases.

PubMed

Liu, Yingli; Cusano, Angela Maria; Wallace, Erin C; Mekmouche, Yasmina; Ullah, Sana; Robert, Viviane; Tron, Thierry

2014-08-01

Extremities of proteins are potent sites for functionalization. Carboxy terminus variants of the Trametes sp. strain C30 LAC3 laccase were generated and produced in Saccharomyces cerevisiae. A variant deleted of the last 13 residues (CΔ) and its 6 His tagged counterpart (CΔ6H) were found active enzymes. The production of CΔ6H resulted in the synthesis of a unusually high proportion of highly glycosylated forms of the enzyme therefore allowing the additional purification of a hyper-glycosylated form of CΔ6H noted CΔ6Hh. Properties of CΔ, CΔ6H and CΔ6Hh were compared. Globally, LAC3 catalytic efficiency was moderately affected by terminal modifications except in CΔ for which the kcat/KM ratio decreased 4 fold (with syringaldazine as substrate) and 10 fold (with ABTS as substrate) respectively. The catalytic parameters kcat and KM of CΔ6H and CΔ6Hh were found to be strictly comparable revealing that over glycosylation does not affect the enzyme catalytic efficiency. To the contrary, in vitro deglycosylation of laccase drastically reduced its activity. So, despite a complex glycosylated pattern observed for some of the variant enzymes, terminal sequences of laccases appear to be appropriate sites for the functionalization/immobilization of laccase. Copyright © 2014 Elsevier B.V. All rights reserved.

Complex nature of SNP genotype effects on gene expression in primary human leucocytes.

PubMed

Heap, Graham A; Trynka, Gosia; Jansen, Ritsert C; Bruinenberg, Marcel; Swertz, Morris A; Dinesen, Lotte C; Hunt, Karen A; Wijmenga, Cisca; Vanheel, David A; Franke, Lude

2009-01-07

Genome wide association studies have been hugely successful in identifying disease risk variants, yet most variants do not lead to coding changes and how variants influence biological function is usually unknown. We correlated gene expression and genetic variation in untouched primary leucocytes (n = 110) from individuals with celiac disease - a common condition with multiple risk variants identified. We compared our observations with an EBV-transformed HapMap B cell line dataset (n = 90), and performed a meta-analysis to increase power to detect non-tissue specific effects. In celiac peripheral blood, 2,315 SNP variants influenced gene expression at 765 different transcripts (< 250 kb from SNP, at FDR = 0.05, cis expression quantitative trait loci, eQTLs). 135 of the detected SNP-probe effects (reflecting 51 unique probes) were also detected in a HapMap B cell line published dataset, all with effects in the same allelic direction. Overall gene expression differences within the two datasets predominantly explain the limited overlap in observed cis-eQTLs. Celiac associated risk variants from two regions, containing genes IL18RAP and CCR3, showed significant cis genotype-expression correlations in the peripheral blood but not in the B cell line datasets. We identified 14 genes where a SNP affected the expression of different probes within the same gene, but in opposite allelic directions. By incorporating genetic variation in co-expression analyses, functional relationships between genes can be more significantly detected. In conclusion, the complex nature of genotypic effects in human populations makes the use of a relevant tissue, large datasets, and analysis of different exons essential to enable the identification of the function for many genetic risk variants in common diseases.

Pleiotropic associations of risk variants identified for other cancers with lung cancer risk: the PAGE and TRICL consortia.

PubMed

Park, S Lani; Fesinmeyer, Megan D; Timofeeva, Maria; Caberto, Christian P; Kocarnik, Jonathan M; Han, Younghun; Love, Shelly-Ann; Young, Alicia; Dumitrescu, Logan; Lin, Yi; Goodloe, Robert; Wilkens, Lynne R; Hindorff, Lucia; Fowke, Jay H; Carty, Cara; Buyske, Steven; Schumacher, Frederick R; Butler, Anne; Dilks, Holli; Deelman, Ewa; Cote, Michele L; Chen, Wei; Pande, Mala; Christiani, David C; Field, John K; Bickebller, Heike; Risch, Angela; Heinrich, Joachim; Brennan, Paul; Wang, Yufei; Eisen, Timothy; Houlston, Richard S; Thun, Michael; Albanes, Demetrius; Caporaso, Neil; Peters, Ulrike; North, Kari E; Heiss, Gerardo; Crawford, Dana C; Bush, William S; Haiman, Christopher A; Landi, Maria Teresa; Hung, Rayjean J; Kooperberg, Charles; Amos, Christopher I; Le Marchand, Loïc; Cheng, Iona

2014-04-01

Genome-wide association studies have identified hundreds of genetic variants associated with specific cancers. A few of these risk regions have been associated with more than one cancer site; however, a systematic evaluation of the associations between risk variants for other cancers and lung cancer risk has yet to be performed. We included 18023 patients with lung cancer and 60543 control subjects from two consortia, Population Architecture using Genomics and Epidemiology (PAGE) and Transdisciplinary Research in Cancer of the Lung (TRICL). We examined 165 single-nucleotide polymorphisms (SNPs) that were previously associated with at least one of 16 non-lung cancer sites. Study-specific logistic regression results underwent meta-analysis, and associations were also examined by race/ethnicity, histological cell type, sex, and smoking status. A Bonferroni-corrected P value of 2.5×10(-5) was used to assign statistical significance. The breast cancer SNP LSP1 rs3817198 was associated with an increased risk of lung cancer (odds ratio [OR] = 1.10; 95% confidence interval [CI] = 1.05 to 1.14; P = 2.8×10(-6)). This association was strongest for women with adenocarcinoma (P = 1.2×10(-4)) and not statistically significant in men (P = .14) with this cell type (P het by sex = .10). Two glioma risk variants, TERT rs2853676 and CDKN2BAS1 rs4977756, which are located in regions previously associated with lung cancer, were associated with increased risk of adenocarcinoma (OR = 1.16; 95% CI = 1.10 to 1.22; P = 1.1×10(-8)) and squamous cell carcinoma (OR = 1.13; CI = 1.07 to 1.19; P = 2.5×10(-5)), respectively. Our findings demonstrate a novel pleiotropic association between the breast cancer LSP1 risk region marked by variant rs3817198 and lung cancer risk.

Mapping rare and common causal alleles for complex human diseases

PubMed Central

Raychaudhuri, Soumya

2011-01-01

Advances in genotyping and sequencing technologies have revolutionized the genetics of complex disease by locating rare and common variants that influence an individual’s risk for diseases, such as diabetes, cancers, and psychiatric disorders. However, to capitalize on this data for prevention and therapies requires the identification of causal alleles and a mechanistic understanding for how these variants contribute to the disease. After discussing the strategies currently used to map variants for complex diseases, this Primer explores how variants may be prioritized for follow-up functional studies and the challenges and approaches for assessing the contributions of rare and common variants to disease phenotypes. PMID:21962507

Immune Escape Variants of H9N2 Influenza Viruses Containing Deletions at the Hemagglutinin Receptor Binding Site Retain Fitness In Vivo and Display Enhanced Zoonotic Characteristics.

PubMed

Peacock, Thomas P; Benton, Donald J; James, Joe; Sadeyen, Jean-Remy; Chang, Pengxiang; Sealy, Joshua E; Bryant, Juliet E; Martin, Stephen R; Shelton, Holly; Barclay, Wendy S; Iqbal, Munir

2017-07-15

H9N2 avian influenza viruses are enzootic in poultry across Asia and North Africa, where they pose a threat to human health as both zoonotic agents and potential pandemic candidates. Poultry vaccination against H9N2 viruses has been employed in many regions; however, vaccine effectiveness is frequently compromised due to antigenic drift arising from amino acid substitutions in the major influenza virus antigen hemagglutinin (HA). Using selection with HA-specific monoclonal antibodies, we previously identified H9N2 antibody escape mutants that contained deletions of amino acids in the 220 loop of the HA receptor binding sites (RBSs). Here we analyzed the impact of these deletions on virus zoonotic infection characteristics and fitness. We demonstrated that mutant viruses with RBS deletions are able to escape polyclonal antiserum binding and are able to infect and be transmitted between chickens. We showed that the deletion mutants have increased binding to human-like receptors and greater replication in primary human airway cells; however, the mutant HAs also displayed reduced pH and thermal stability. In summary, we infer that variant influenza viruses with deletions in the 220 loop could arise in the field due to immune selection pressure; however, due to reduced HA stability, we conclude that these viruses are unlikely to be transmitted from human to human by the airborne route, a prerequisite for pandemic emergence. Our findings underscore the complex interplay between antigenic drift and viral fitness for avian influenza viruses as well as the challenges of predicting which viral variants may pose the greatest threats for zoonotic and pandemic emergence. IMPORTANCE Avian influenza viruses, such as H9N2, cause disease in poultry as well as occasionally infecting humans and are therefore considered viruses with pandemic potential. Many countries have introduced vaccination of poultry to try to control the disease burden; however, influenza viruses are able to rapidly evolve to escape immune pressure in a process known as "antigenic drift." Previously, we experimentally generated antigenic-drift variants in the laboratory, and here, we test our "drifted" viruses to assess their zoonotic infection characteristics and transmissibility in chickens. We found that the drifted viruses were able to infect and be transmitted between chickens and showed increased binding to human-like receptors. However, the drift mutant viruses displayed reduced stability, and we predict that they are unlikely to be transmitted from human to human and cause an influenza pandemic. These results demonstrate the complex relationship between antigenic drift and the potential of avian influenza viruses to infect humans. Copyright © 2017 Peacock et al.

Rare variant testing across methods and thresholds using the multi-kernel sequence kernel association test (MK-SKAT).

PubMed

Urrutia, Eugene; Lee, Seunggeun; Maity, Arnab; Zhao, Ni; Shen, Judong; Li, Yun; Wu, Michael C

Analysis of rare genetic variants has focused on region-based analysis wherein a subset of the variants within a genomic region is tested for association with a complex trait. Two important practical challenges have emerged. First, it is difficult to choose which test to use. Second, it is unclear which group of variants within a region should be tested. Both depend on the unknown true state of nature. Therefore, we develop the Multi-Kernel SKAT (MK-SKAT) which tests across a range of rare variant tests and groupings. Specifically, we demonstrate that several popular rare variant tests are special cases of the sequence kernel association test which compares pair-wise similarity in trait value to similarity in the rare variant genotypes between subjects as measured through a kernel function. Choosing a particular test is equivalent to choosing a kernel. Similarly, choosing which group of variants to test also reduces to choosing a kernel. Thus, MK-SKAT uses perturbation to test across a range of kernels. Simulations and real data analyses show that our framework controls type I error while maintaining high power across settings: MK-SKAT loses power when compared to the kernel for a particular scenario but has much greater power than poor choices.

Operating Room Telephone Microbial Flora

DTIC Science & Technology

2005-05-24

times of the bacteria most frequently implicated in surgical site infections on hands and inanimate surfaces . These bacteria are: S . aureus, CNS...variants of S . aureus and Enterococcus spp. Due to the lack of studies specific to telephone surfaces , we also used plastic surfaces as a substitute search...comparison with tube Coagulase test on S . aureus isolates has been found to have a relative sensitivity of 99.4%, a relative specificity of 95.5% and

Folate-genetics and colorectal neoplasia: What we know and need to know next

USDA-ARS?s Scientific Manuscript database

The metabolism of folate involves a complex network of polymorphic enzymes that may explain a proportion of the risk associated with colorectal neoplasia. Over 60 observational studies primarily in non-Hispanic White populations have been conducted on selected genetic variants in specific genes, MTH...

Teaching Information Systems Development via Process Variants

ERIC Educational Resources Information Center

Tan, Wee-Kek; Tan, Chuan-Hoo

2010-01-01

Acquiring the knowledge to assemble an integrated Information System (IS) development process that is tailored to the specific needs of a project has become increasingly important. It is therefore necessary for educators to impart to students this crucial skill. However, Situational Method Engineering (SME) is an inherently complex process that…

Assessing the genetic overlap between BMI and cognitive function

PubMed Central

Marioni, R E; Yang, J; Dykiert, D; Mõttus, R; Campbell, A; Ibrahim-Verbaas, Carla A; Bressler, Jan; Debette, Stephanie; Schuur, Maaike; Smith, Albert V; Davies, Gail; Bennett, David A; Deary, Ian J; Ikram, M Arfan; Launer, Lenore J; Fitzpatrick, Annette L; Seshadri, Sudha; van Duijn, Cornelia M; Mosely Jr, Thomas H; Davies, G; Hayward, C; Porteous, D J; Visscher, P M; Deary, I J

2016-01-01

Obesity and low cognitive function are associated with multiple adverse health outcomes across the life course. They have a small phenotypic correlation (r=−0.11; high body mass index (BMI)−low cognitive function), but whether they have a shared genetic aetiology is unknown. We investigated the phenotypic and genetic correlations between the traits using data from 6815 unrelated, genotyped members of Generation Scotland, an ethnically homogeneous cohort from five sites across Scotland. Genetic correlations were estimated using the following: same-sample bivariate genome-wide complex trait analysis (GCTA)–GREML; independent samples bivariate GCTA–GREML using Generation Scotland for cognitive data and four other samples (n=20 806) for BMI; and bivariate LDSC analysis using the largest genome-wide association study (GWAS) summary data on cognitive function (n=48 462) and BMI (n=339 224) to date. The GWAS summary data were also used to create polygenic scores for the two traits, with within- and cross-trait prediction taking place in the independent Generation Scotland cohort. A large genetic correlation of −0.51 (s.e. 0.15) was observed using the same-sample GCTA–GREML approach compared with −0.10 (s.e. 0.08) from the independent-samples GCTA–GREML approach and −0.22 (s.e. 0.03) from the bivariate LDSC analysis. A genetic profile score using cognition-specific genetic variants accounts for 0.08% (P=0.020) of the variance in BMI and a genetic profile score using BMI-specific variants accounts for 0.42% (P=1.9 × 10−7) of the variance in cognitive function. Seven common genetic variants are significantly associated with both traits at P<5 × 10−5, which is significantly more than expected by chance (P=0.007). All these results suggest there are shared genetic contributions to BMI and cognitive function. PMID:26857597

Characterization of Centromeric Histone H3 (CENH3) Variants in Cultivated and Wild Carrots (Daucus sp.)

PubMed Central

Dunemann, Frank; Schrader, Otto; Budahn, Holger; Houben, Andreas

2014-01-01

In eukaryotes, centromeres are the assembly sites for the kinetochore, a multi-protein complex to which spindle microtubules are attached at mitosis and meiosis, thereby ensuring segregation of chromosomes during cell division. They are specified by incorporation of CENH3, a centromere specific histone H3 variant which replaces canonical histone H3 in the nucleosomes of functional centromeres. To lay a first foundation of a putative alternative haploidization strategy based on centromere-mediated genome elimination in cultivated carrots, in the presented research we aimed at the identification and cloning of functional CENH3 genes in Daucus carota and three distantly related wild species of genus Daucus varying in basic chromosome numbers. Based on mining the carrot transcriptome followed by a subsequent PCR-based cloning, homologous coding sequences for CENH3s of the four Daucus species were identified. The ORFs of the CENH3 variants were very similar, and an amino acid sequence length of 146 aa was found in three out of the four species. Comparison of Daucus CENH3 amino acid sequences with those of other plant CENH3s as well as their phylogenetic arrangement among other dicot CENH3s suggest that the identified genes are authentic CENH3 homologs. To verify the location of the CENH3 protein in the kinetochore regions of the Daucus chromosomes, a polyclonal antibody based on a peptide corresponding to the N-terminus of DcCENH3 was developed and used for anti-CENH3 immunostaining of mitotic root cells. The chromosomal location of CENH3 proteins in the centromere regions of the chromosomes could be confirmed. For genetic localization of the CENH3 gene in the carrot genome, a previously constructed linkage map for carrot was used for mapping a CENH3-specific simple sequence repeat (SSR) marker, and the CENH3 locus was mapped on the carrot chromosome 9. PMID:24887084

Shared activity patterns arising at genetic susceptibility loci reveal underlying genomic and cellular architecture of human disease.

PubMed

Baillie, J Kenneth; Bretherick, Andrew; Haley, Christopher S; Clohisey, Sara; Gray, Alan; Neyton, Lucile P A; Barrett, Jeffrey; Stahl, Eli A; Tenesa, Albert; Andersson, Robin; Brown, J Ben; Faulkner, Geoffrey J; Lizio, Marina; Schaefer, Ulf; Daub, Carsten; Itoh, Masayoshi; Kondo, Naoto; Lassmann, Timo; Kawai, Jun; Mole, Damian; Bajic, Vladimir B; Heutink, Peter; Rehli, Michael; Kawaji, Hideya; Sandelin, Albin; Suzuki, Harukazu; Satsangi, Jack; Wells, Christine A; Hacohen, Nir; Freeman, Thomas C; Hayashizaki, Yoshihide; Carninci, Piero; Forrest, Alistair R R; Hume, David A

2018-03-01

Genetic variants underlying complex traits, including disease susceptibility, are enriched within the transcriptional regulatory elements, promoters and enhancers. There is emerging evidence that regulatory elements associated with particular traits or diseases share similar patterns of transcriptional activity. Accordingly, shared transcriptional activity (coexpression) may help prioritise loci associated with a given trait, and help to identify underlying biological processes. Using cap analysis of gene expression (CAGE) profiles of promoter- and enhancer-derived RNAs across 1824 human samples, we have analysed coexpression of RNAs originating from trait-associated regulatory regions using a novel quantitative method (network density analysis; NDA). For most traits studied, phenotype-associated variants in regulatory regions were linked to tightly-coexpressed networks that are likely to share important functional characteristics. Coexpression provides a new signal, independent of phenotype association, to enable fine mapping of causative variants. The NDA coexpression approach identifies new genetic variants associated with specific traits, including an association between the regulation of the OCT1 cation transporter and genetic variants underlying circulating cholesterol levels. NDA strongly implicates particular cell types and tissues in disease pathogenesis. For example, distinct groupings of disease-associated regulatory regions implicate two distinct biological processes in the pathogenesis of ulcerative colitis; a further two separate processes are implicated in Crohn's disease. Thus, our functional analysis of genetic predisposition to disease defines new distinct disease endotypes. We predict that patients with a preponderance of susceptibility variants in each group are likely to respond differently to pharmacological therapy. Together, these findings enable a deeper biological understanding of the causal basis of complex traits.

Shared activity patterns arising at genetic susceptibility loci reveal underlying genomic and cellular architecture of human disease

PubMed Central

Gray, Alan; Neyton, Lucile P. A.; Barrett, Jeffrey; Stahl, Eli A.; Tenesa, Albert; Andersson, Robin; Brown, J. Ben; Faulkner, Geoffrey J.; Lizio, Marina; Schaefer, Ulf; Daub, Carsten; Kondo, Naoto; Lassmann, Timo; Kawai, Jun; Kawaji, Hideya; Suzuki, Harukazu; Satsangi, Jack; Wells, Christine A.; Hacohen, Nir; Freeman, Thomas C.; Hayashizaki, Yoshihide; Forrest, Alistair R. R.; Hume, David A.

2018-01-01

Genetic variants underlying complex traits, including disease susceptibility, are enriched within the transcriptional regulatory elements, promoters and enhancers. There is emerging evidence that regulatory elements associated with particular traits or diseases share similar patterns of transcriptional activity. Accordingly, shared transcriptional activity (coexpression) may help prioritise loci associated with a given trait, and help to identify underlying biological processes. Using cap analysis of gene expression (CAGE) profiles of promoter- and enhancer-derived RNAs across 1824 human samples, we have analysed coexpression of RNAs originating from trait-associated regulatory regions using a novel quantitative method (network density analysis; NDA). For most traits studied, phenotype-associated variants in regulatory regions were linked to tightly-coexpressed networks that are likely to share important functional characteristics. Coexpression provides a new signal, independent of phenotype association, to enable fine mapping of causative variants. The NDA coexpression approach identifies new genetic variants associated with specific traits, including an association between the regulation of the OCT1 cation transporter and genetic variants underlying circulating cholesterol levels. NDA strongly implicates particular cell types and tissues in disease pathogenesis. For example, distinct groupings of disease-associated regulatory regions implicate two distinct biological processes in the pathogenesis of ulcerative colitis; a further two separate processes are implicated in Crohn’s disease. Thus, our functional analysis of genetic predisposition to disease defines new distinct disease endotypes. We predict that patients with a preponderance of susceptibility variants in each group are likely to respond differently to pharmacological therapy. Together, these findings enable a deeper biological understanding of the causal basis of complex traits. PMID:29494619

Synonymous mutations in RNASEH2A create cryptic splice sites impairing RNase H2 enzyme function in Aicardi-Goutières syndrome.

PubMed

Rice, Gillian I; Reijns, Martin A M; Coffin, Stephanie R; Forte, Gabriella M A; Anderson, Beverley H; Szynkiewicz, Marcin; Gornall, Hannah; Gent, David; Leitch, Andrea; Botella, Maria P; Fazzi, Elisa; Gener, Blanca; Lagae, Lieven; Olivieri, Ivana; Orcesi, Simona; Swoboda, Kathryn J; Perrino, Fred W; Jackson, Andrew P; Crow, Yanick J

2013-08-01

Aicardi-Goutières syndrome is an inflammatory disorder resulting from mutations in TREX1, RNASEH2A/2B/2C, SAMHD1, or ADAR1. Here, we provide molecular, biochemical, and cellular evidence for the pathogenicity of two synonymous variants in RNASEH2A. Firstly, the c.69G>A (p.Val23Val) mutation causes the formation of a splice donor site within exon 1, resulting in an out of frame deletion at the end of exon 1, leading to reduced RNase H2 protein levels. The second mutation, c.75C>T (p.Arg25Arg), also introduces a splice donor site within exon 1, and the internal deletion of 18 amino acids. The truncated protein still forms a heterotrimeric RNase H2 complex, but lacks catalytic activity. However, as a likely result of leaky splicing, a small amount of full-length active protein is apparently produced in an individual homozygous for this mutation. Recognition of the disease causing status of these variants allows for diagnostic testing in relevant families. © 2013 WILEY PERIODICALS, INC.

Reengineered glucose oxidase for amperometric glucose determination in diabetes analytics.

PubMed

Arango Gutierrez, Erik; Mundhada, Hemanshu; Meier, Thomas; Duefel, Hartmut; Bocola, Marco; Schwaneberg, Ulrich

2013-12-15

Glucose oxidase is an oxidoreductase exhibiting a high β-D-glucose specificity and high stability which renders glucose oxidase well-suited for applications in diabetes care. Nevertheless, GOx activity is highly oxygen dependent which can lead to inaccuracies in amperometric β-D-glucose determinations. Therefore a directed evolution campaign with two rounds of random mutagenesis (SeSaM followed by epPCR), site saturation mutagenesis studies on individual positions, and one simultaneous site saturation library (OmniChange; 4 positions) was performed. A diabetes care well suited mediator (quinone diimine) was selected and the GOx variant (T30V I94V) served as starting point. For directed GOx evolution a microtiter plate detection system based on the quinone diimine mediator was developed and the well-known ABTS-assay was applied in microtiter plate format to validate oxygen independency of improved GOx variants. Two iterative rounds of random diversity generation and screening yielded to two subsets of amino acid positions which mainly improved activity (A173, A332) and oxygen independency (F414, V560). Simultaneous site saturation of all four positions with a reduced subset of amino acids using the OmniChange method yielded finally variant V7 with a 37-fold decreased oxygen dependency (mediator activity: 7.4 U/mg WT, 47.5 U/mg V7; oxygen activity: 172.3 U/mg WT, 30.1 U/mg V7). V7 is still highly β-D-glucose specific, highly active with the quinone diimine mediator and thermal resistance is retained (prerequisite for GOx coating of diabetes test stripes). The latter properties and V7's oxygen insensitivity make V7 a very promising candidate to replace standard GOx in diabetes care applications. Copyright © 2013 Elsevier B.V. All rights reserved.

Common and specific liability to addiction: approaches to association studies of opioid addiction.

PubMed

Nielsen, David A; Kreek, Mary Jeanne

2012-06-01

Opioid addiction, whether to opiates such as heroin and morphine, and/or to non-medical use of opioids, is a major problem worldwide. Although drug-induced and environmental factors are essential for the liability to develop opioid addiction, the genetic background of an individual is now known also to play a substantial role. The overall goal of this article is to address the common and specific liabilities to addiction in the context of approaches to studies of one addiction, opioid addiction. Literature on identifying genetic variants that may play a role in the development of opioid addiction was reviewed. A substantial number of genetic variants have been reported to be associated with opioid addiction. No single variant has been found in any of the reported GWAS studies with a substantial effect size on the liability to develop heroin addiction. It appears that there is a complex interaction of a large number of variants, some rare, some common, which interact with the environment and in response to specific drugs of abuse to increase the liability of developing opioid addiction. In spite of the inherent difficulties in obtaining large well-phenotyped cohorts for genetic studies, new findings have been reported that are being used to develop testable hypotheses into the biological basis of opioid addiction. Copyright © 2012. Published by Elsevier Ireland Ltd.

Potent and Specific Inhibition of Glycosidases by Small Artificial Binding Proteins (Affitins)

PubMed Central

Mechaly, Ariel E.; Obal, Gonzalo; Béhar, Ghislaine; Mouratou, Barbara; Oppezzo, Pablo; Alzari, Pedro M.; Pecorari, Frédéric

2014-01-01

Glycosidases are associated with various human diseases. The development of efficient and specific inhibitors may provide powerful tools to modulate their activity. However, achieving high selectivity is a major challenge given that glycosidases with different functions can have similar enzymatic mechanisms and active-site architectures. As an alternative approach to small-chemical compounds, proteinaceous inhibitors might provide a better specificity by involving a larger surface area of interaction. We report here the design and characterization of proteinaceous inhibitors that specifically target endoglycosidases representative of the two major mechanistic classes; retaining and inverting glycosidases. These inhibitors consist of artificial affinity proteins, Affitins, selected against the thermophilic CelD from Clostridium thermocellum and lysozyme from hen egg. They were obtained from libraries of Sac7d variants, which involve either the randomization of a surface or the randomization of a surface and an artificially-extended loop. Glycosidase binders exhibited affinities in the nanomolar range with no cross-recognition, with efficient inhibition of lysozyme (Ki = 45 nM) and CelD (Ki = 95 and 111 nM), high expression yields in Escherichia coli, solubility, and thermal stabilities up to 81.1°C. The crystal structures of glycosidase-Affitin complexes validate our library designs. We observed that Affitins prevented substrate access by two modes of binding; covering or penetrating the catalytic site via the extended loop. In addition, Affitins formed salt-bridges with residues essential for enzymatic activity. These results lead us to propose the use of Affitins as versatile selective glycosidase inhibitors and, potentially, as enzymatic inhibitors in general. PMID:24823716

Potent and specific inhibition of glycosidases by small artificial binding proteins (affitins).

PubMed

Correa, Agustín; Pacheco, Sabino; Mechaly, Ariel E; Obal, Gonzalo; Béhar, Ghislaine; Mouratou, Barbara; Oppezzo, Pablo; Alzari, Pedro M; Pecorari, Frédéric

2014-01-01

Glycosidases are associated with various human diseases. The development of efficient and specific inhibitors may provide powerful tools to modulate their activity. However, achieving high selectivity is a major challenge given that glycosidases with different functions can have similar enzymatic mechanisms and active-site architectures. As an alternative approach to small-chemical compounds, proteinaceous inhibitors might provide a better specificity by involving a larger surface area of interaction. We report here the design and characterization of proteinaceous inhibitors that specifically target endoglycosidases representative of the two major mechanistic classes; retaining and inverting glycosidases. These inhibitors consist of artificial affinity proteins, Affitins, selected against the thermophilic CelD from Clostridium thermocellum and lysozyme from hen egg. They were obtained from libraries of Sac7d variants, which involve either the randomization of a surface or the randomization of a surface and an artificially-extended loop. Glycosidase binders exhibited affinities in the nanomolar range with no cross-recognition, with efficient inhibition of lysozyme (Ki = 45 nM) and CelD (Ki = 95 and 111 nM), high expression yields in Escherichia coli, solubility, and thermal stabilities up to 81.1°C. The crystal structures of glycosidase-Affitin complexes validate our library designs. We observed that Affitins prevented substrate access by two modes of binding; covering or penetrating the catalytic site via the extended loop. In addition, Affitins formed salt-bridges with residues essential for enzymatic activity. These results lead us to propose the use of Affitins as versatile selective glycosidase inhibitors and, potentially, as enzymatic inhibitors in general.

Caenorhabditis elegans Evolves a New Architecture for the Multi-aminoacyl-tRNA Synthetase Complex*

PubMed Central

Havrylenko, Svitlana; Legouis, Renaud; Negrutskii, Boris; Mirande, Marc

2011-01-01

MARS is an evolutionary conserved supramolecular assembly of aminoacyl-tRNA synthetases found in eukaryotes. This complex was thought to be ubiquitous in the deuterostome and protostome clades of bilaterians because similar complexes were isolated from arthropods and vertebrates. However, several features of the component enzymes suggested that in the nematode Caenorhabditis elegans, a species grouped with arthropods in modern phylogeny, this complex might not exist, or should display a significantly different structural organization. C. elegans was also taken as a model system to study in a multicellular organism amenable to experimental approaches, the reason for existence of these supramolecular entities. Here, using a proteomic approach, we have characterized the components of MARS in C. elegans. We show that this organism evolved a specific structural organization of this complex, which contains several bona fide components of the MARS complexes known so far, but also displays significant variations. These data highlight molecular evolution events that took place after radiation of bilaterians. Remarkably, it shows that expansion of MARS assembly in metazoans is not linear, but is the result of additions but also of subtractions along evolution. We then undertook an experimental approach, using inactivation of the endogenous copy of methionyl-tRNA synthetase by RNAi and expression of transgenic variants, to understand the role in complex assembly and the in vivo functionality, of the eukaryotic-specific domains appended to aminoacyl-tRNA synthetases. We show that rescue of the worms and assembly of transgenic variants into MARS rest on the presence of these appended domains. PMID:21685384

Caenorhabditis elegans evolves a new architecture for the multi-aminoacyl-tRNA synthetase complex.

PubMed

Havrylenko, Svitlana; Legouis, Renaud; Negrutskii, Boris; Mirande, Marc

2011-08-12

MARS is an evolutionary conserved supramolecular assembly of aminoacyl-tRNA synthetases found in eukaryotes. This complex was thought to be ubiquitous in the deuterostome and protostome clades of bilaterians because similar complexes were isolated from arthropods and vertebrates. However, several features of the component enzymes suggested that in the nematode Caenorhabditis elegans, a species grouped with arthropods in modern phylogeny, this complex might not exist, or should display a significantly different structural organization. C. elegans was also taken as a model system to study in a multicellular organism amenable to experimental approaches, the reason for existence of these supramolecular entities. Here, using a proteomic approach, we have characterized the components of MARS in C. elegans. We show that this organism evolved a specific structural organization of this complex, which contains several bona fide components of the MARS complexes known so far, but also displays significant variations. These data highlight molecular evolution events that took place after radiation of bilaterians. Remarkably, it shows that expansion of MARS assembly in metazoans is not linear, but is the result of additions but also of subtractions along evolution. We then undertook an experimental approach, using inactivation of the endogenous copy of methionyl-tRNA synthetase by RNAi and expression of transgenic variants, to understand the role in complex assembly and the in vivo functionality, of the eukaryotic-specific domains appended to aminoacyl-tRNA synthetases. We show that rescue of the worms and assembly of transgenic variants into MARS rest on the presence of these appended domains.

Complex Tissue-Specific Patterns and Distribution of Multiple RAGE Splice Variants in Different Mammals

PubMed Central

López-Díez, Raquel; Rastrojo, Alberto; Villate, Olatz; Aguado, Begoña

2013-01-01

The receptor for advanced glycosylation end products (RAGE) is a multiligand receptor involved in diverse cell signaling pathways. Previous studies show that this gene expresses several splice variants in human, mouse, and dog. Alternative splicing (AS) plays an important role in expanding transcriptomic and proteomic diversity, and it has been related to disease. AS is also one of the main evolutionary mechanisms in mammalian genomes. However, limited information is available regarding the AS of RAGE in a wide context of mammalian tissues. In this study, we examined in detail the different RAGE mRNAs generated by AS from six mammals, including two primates (human and monkey), two artiodactyla (cow and pig), and two rodentia (mouse and rat) in 6–18 different tissues including fetal, adult, and tumor. By nested reverse transcription-polymerase chain reaction (RT-PCR) we identified a high number of splice variants including noncoding transcripts and predicted coding ones with different potential protein modifications affecting mainly the transmembrane and ligand-binding domains that could influence their biological function. However, analysis of RNA-seq data enabled detecting only the most abundant splice variants. More than 80% of the detected RT-PCR variants (87 of 101 transcripts) are novel (different exon/intron structure to the previously described ones), and interestingly, 20–60% of the total transcripts (depending on the species) are noncoding ones that present tissue specificity. Our results suggest that RAGE undergoes extensive AS in mammals, with different expression patterns among adult, fetal, and tumor tissues. Moreover, most splice variants seem to be species specific, especially the noncoding variants, with only two (canonical human Tv1-RAGE, and human N-truncated or Tv10-RAGE) conserved among the six different species. This could indicate a special evolution pattern of this gene at mRNA level. PMID:24273313

Site-Specific Protein Labeling via Sortase-Mediated Transpeptidation

PubMed Central

Antos, John M.; Ingram, Jessica; Fang, Tao; Pishesha, Novalia; Truttmann, Matthias C.; Ploegh, Hidde L.

2017-01-01

Strategies for site-specific protein modification are highly desirable for the construction of conjugates containing non-genetically encoded functional groups. Ideally, these strategies should proceed under mild conditions, and be compatible with a wide range of protein targets and non-natural moieties. The transpeptidation reaction catalyzed by bacterial sortases is a prominent strategy for protein derivatization that possesses these features. Naturally occurring or engineered variants of sortase A from Staphylococcus aureus catalyze a ligation reaction between a five amino acid substrate motif (LPXTG) and oligoglycine nucleophiles. By pairing proteins and synthetic peptides that possess these ligation handles, it is possible to install modifications onto the protein N- or C-terminus in site-specific fashion. As described in this unit, the successful implementation of sortase-mediated labeling involves straightforward solid-phase synthesis and molecular biology techniques, and this method is compatible with proteins in solution or on the surface of live cells. PMID:19365788

Plant-expressed cocaine hydrolase variants of butyrylcholinesterase exhibit altered allosteric effects of cholinesterase activity and increased inhibitor sensitivity.

PubMed

Larrimore, Katherine E; Kazan, I Can; Kannan, Latha; Kendle, R Player; Jamal, Tameem; Barcus, Matthew; Bolia, Ashini; Brimijoin, Stephen; Zhan, Chang-Guo; Ozkan, S Banu; Mor, Tsafrir S

2017-09-05

Butyrylcholinesterase (BChE) is an enzyme with broad substrate and ligand specificities and may function as a generalized bioscavenger by binding and/or hydrolyzing various xenobiotic agents and toxicants, many of which target the central and peripheral nervous systems. Variants of BChE were rationally designed to increase the enzyme's ability to hydrolyze the psychoactive enantiomer of cocaine. These variants were cloned, and then expressed using the magnICON transient expression system in plants and their enzymatic properties were investigated. In particular, we explored the effects that these site-directed mutations have over the enzyme kinetics with various substrates of BChE. We further compared the affinity of various anticholinesterases including organophosphorous nerve agents and pesticides toward these BChE variants relative to the wild type enzyme. In addition to serving as a therapy for cocaine addiction-related diseases, enhanced bioscavenging against other harmful agents could add to the practicality and versatility of the plant-derived recombinant enzyme as a multivalent therapeutic.

Plasmonic bio-sensing for the Fenna-Matthews-Olson complex

NASA Astrophysics Data System (ADS)

Chen, Guang-Yin; Lambert, Neill; Shih, Yen-An; Liu, Meng-Han; Chen, Yueh-Nan; Nori, Franco

2017-01-01

We study theoretically the bio-sensing capabilities of metal nanowire surface plasmons. As a specific example, we couple the nanowire to specific sites (bacteriochlorophyll) of the Fenna-Matthews-Olson (FMO) photosynthetic pigment protein complex. In this hybrid system, we find that when certain sites of the FMO complex are subject to either the suppression of inter-site transitions or are entirely disconnected from the complex, the resulting variations in the excitation transfer rates through the complex can be monitored through the corresponding changes in the scattering spectra of the incident nanowire surface plasmons. We also find that these changes can be further enhanced by changing the ratio of plasmon-site couplings. The change of the Fano lineshape in the scattering spectra further reveals that “site 5” in the FMO complex plays a distinct role from other sites. Our results provide a feasible way, using single photons, to detect mutation-induced, or bleaching-induced, local defects or modifications of the FMO complex, and allows access to both the local and global properties of the excitation transfer in such systems.

The Role of Distant Mutations and Allosteric Regulation on LovD Active Site Dynamics

PubMed Central

Jiménez-Osés, Gonzalo; Osuna, Sílvia; Gao, Xue; Sawaya, Michael R.; Gilson, Lynne; Collier, Steven J.; Huisman, Gjalt W.; Yeates, Todd O.; Tang, Yi; Houk, K. N.

2014-01-01

Natural enzymes have evolved to perform their cellular functions under complex selective pressures, which often require their catalytic activities to be regulated by other proteins. We contrasted a natural enzyme, LovD, which acts on a protein-bound (LovF) acyl substrate, with a laboratory-generated variant that was transformed by directed evolution to accept instead a small free acyl thioester, and no longer requires the acyl carrier protein. The resulting 29-mutant variant is 1000-fold more efficient in the synthesis of the drug simvastatin than the wild-type LovD. This is the first non-patent report of the enzyme currently used for the manufacture of simvastatin, as well as the intermediate evolved variants. Crystal structures and microsecond molecular dynamics simulations revealed the mechanism by which the laboratory-generated mutations free LovD from dependence on protein-protein interactions. Mutations dramatically altered conformational dynamics of the catalytic residues, obviating the need for allosteric modulation by the acyl carrier LovF. PMID:24727900

PUF60 variants cause a syndrome of ID, short stature, microcephaly, coloboma, craniofacial, cardiac, renal and spinal features

PubMed Central

Low, Karen J; Ansari, Morad; Abou Jamra, Rami; Clarke, Angus; El Chehadeh, Salima; FitzPatrick, David R; Greenslade, Mark; Henderson, Alex; Hurst, Jane; Keller, Kory; Kuentz, Paul; Prescott, Trine; Roessler, Franziska; Selmer, Kaja K; Schneider, Michael C; Stewart, Fiona; Tatton-Brown, Katrina; Thevenon, Julien; Vigeland, Magnus D; Vogt, Julie; Willems, Marjolaine; Zonana, Jonathan; Study, D D D; Smithson, Sarah F

2017-01-01

PUF60 encodes a nucleic acid-binding protein, a component of multimeric complexes regulating RNA splicing and transcription. In 2013, patients with microdeletions of chromosome 8q24.3 including PUF60 were found to have developmental delay, microcephaly, craniofacial, renal and cardiac defects. Very similar phenotypes have been described in six patients with variants in PUF60, suggesting that it underlies the syndrome. We report 12 additional patients with PUF60 variants who were ascertained using exome sequencing: six through the Deciphering Developmental Disorders Study and six through similar projects. Detailed phenotypic analysis of all patients was undertaken. All 12 patients had de novo heterozygous PUF60 variants on exome analysis, each confirmed by Sanger sequencing: four frameshift variants resulting in premature stop codons, three missense variants that clustered within the RNA recognition motif of PUF60 and five essential splice-site (ESS) variant. Analysis of cDNA from a fibroblast cell line derived from one of the patients with an ESS variants revealed aberrant splicing. The consistent feature was developmental delay and most patients had short stature. The phenotypic variability was striking; however, we observed similarities including spinal segmentation anomalies, congenital heart disease, ocular colobomata, hand anomalies and (in two patients) unilateral renal agenesis/horseshoe kidney. Characteristic facial features included micrognathia, a thin upper lip and long philtrum, narrow almond-shaped palpebral fissures, synophrys, flared eyebrows and facial hypertrichosis. Heterozygote loss-of-function variants in PUF60 cause a phenotype comprising growth/developmental delay and craniofacial, cardiac, renal, ocular and spinal anomalies, adding to disorders of human development resulting from aberrant RNA processing/spliceosomal function. PMID:28327570

Active Site Mutations Change the Cleavage Specificity of Neprilysin

PubMed Central

Sexton, Travis; Hitchcook, Lisa J.; Rodgers, David W.; Bradley, Luke H.; Hersh, Louis B.

2012-01-01

Neprilysin (NEP), a member of the M13 subgroup of the zinc-dependent endopeptidase family is a membrane bound peptidase capable of cleaving a variety of physiological peptides. We have generated a series of neprilysin variants containing mutations at either one of two active site residues, Phe563 and Ser546. Among the mutants studied in detail we observed changes in their activity towards leucine5-enkephalin, insulin B chain, and amyloid β1–40. For example, NEPF563I displayed an increase in preference towards cleaving leucine5-enkephalin relative to insulin B chain, while mutant NEPS546E was less discriminating than neprilysin. Mutants NEPF563L and NEPS546E exhibit different cleavage site preferences than neprilysin with insulin B chain and amyloid ß1–40 as substrates. These data indicate that it is possible to alter the cleavage site specificity of neprilysin opening the way for the development of substrate specific or substrate exclusive forms of the enzyme with enhanced therapeutic potential. PMID:22384224

Effects of organizational complexity and resources on construction site risk.

PubMed

Forteza, Francisco J; Carretero-Gómez, Jose M; Sesé, Albert

2017-09-01

Our research is aimed at studying the relationship between risk level and organizational complexity and resources on constructions sites. Our general hypothesis is that site complexity increases risk, whereas more resources of the structure decrease risk. A Structural Equation Model (SEM) approach was adopted to validate our theoretical model. To develop our study, 957 building sites in Spain were visited and assessed in 2003-2009. All needed data were obtained using a specific tool developed by the authors to assess site risk, structure and resources (Construction Sites Risk Assessment Tool, or CONSRAT). This tool operationalizes the variables to fit our model, specifically, via a site risk index (SRI) and 10 organizational variables. Our random sample is composed largely of small building sites with general high levels of risk, moderate complexity, and low resources on site. The model obtained adequate fit, and results showed empirical evidence that the factors of complexity and resources can be considered predictors of site risk level. Consequently, these results can help companies, managers of construction and regulators to identify which organizational aspects should be improved to prevent risks on sites and consequently accidents. Copyright © 2017 National Safety Council and Elsevier Ltd. All rights reserved.

Incorporation of Amino Acids with Long-Chain Terminal Olefins into Proteins.

PubMed

Exner, Matthias P; Köhling, Sebastian; Rivollier, Julie; Gosling, Sandrine; Srivastava, Puneet; Palyancheva, Zheni I; Herdewijn, Piet; Heck, Marie-Pierre; Rademann, Jörg; Budisa, Nediljko

2016-02-29

The increasing need for site-specific protein decorations that mimic natural posttranslational modifications requires access to a variety of noncanonical amino acids with moieties enabling bioorthogonal conjugation chemistry. Here we present the incorporation of long-chain olefinic amino acids into model proteins with rational variants of pyrrolysyl-tRNA synthetase (PylRS). Nε-heptenoyl lysine was incorporated for the first time using the known promiscuous variant PylRS(Y306A/Y384F), and Nε-pentenoyl lysine was incorporated in significant yields with the novel variant PylRS(C348A/Y384F). This is the only example of rational modification at position C348 to enlarge the enzyme's binding pocket. Furthermore, we demonstrate the feasibility of our chosen amino acids in the thiol-ene conjugation reaction with a thiolated polysaccharide.

BRCA1 and BRCA2 mutation analysis of early-onset and familial breast cancer cases in Mexico.

PubMed

Ruiz-Flores, Pablo; Sinilnikova, Olga M; Badzioch, Michael; Calderon-Garcidueñas, A L; Chopin, Sandrine; Fabrice, Odefrey; González-Guerrero, J F; Szabo, Csilla; Lenoir, Gilbert; Goldgar, David E; Barrera-Saldaña, Hugo A

2002-12-01

The entire coding regions of BRCA1 and BRCA2 were screened for mutations by heteroduplex analysis in 51 Mexican breast cancer patients. One BRCA1 and one BRCA2 truncating mutation each was identified in the group of 32 (6%) early-onset breast cancer patients (< or =35 years). Besides these two likely deleterious mutations, eight rare variants of unknown significance, mostly in the BRCA2 gene, were detected in six of 32 (19%) early-onset breast cancer cases and in three of 17 (18%) site-specific breast cancer families, one containing a male breast cancer case. No mutations or rare sequence variants have been identified in two additional families including each an early-onset breast cancer case and an ovarian cancer patient. The two truncating mutations (BRCA1 3857delT; BRCA2 2663-2664insA) and six of the rare variants have never been reported before and may be of country-specific origin. The majority of the alterations appeared to be distinct, with only one of them being observed in more than one family. Copyright 2002 Wiley-Liss, Inc.

Second generation engineering of transketolase for polar aromatic aldehyde substrates.

PubMed

Payongsri, Panwajee; Steadman, David; Hailes, Helen C; Dalby, Paul A

2015-04-01

Transketolase has significant industrial potential for the asymmetric synthesis of carboncarbon bonds with new chiral centres. Variants evolved on propanal were found previously with nascent activity on polar aromatic aldehydes 3-formylbenzoic acid (3-FBA), 4-formylbenzoic acid (4-FBA), and 3-hydroxybenzaldehyde (3-HBA), suggesting a potential novel route to analogues of chloramphenicol. Here we evolved improved transketolase activities towards aromatic aldehydes, by saturation mutagenesis of two active-site residues (R358 and S385), predicted to interact with the aromatic substituents. S385 variants selectively controlled the aromatic substrate preference, with up to 13-fold enhanced activities, and KM values comparable to those of natural substrates with wild-type transketolase. S385E even completely removed the substrate inhibition for 3-FBA, observed in all previous variants. The mechanisms of catalytic improvement were both mutation type and substrate dependent. S385E improved 3-FBA activity via kcat, but reduced 4-FBA activity via KM. Conversely, S385Y/T improved 3-FBA activity via KM and 4-FBA activity via kcat. This suggested that both substrate proximity and active-site orientation are very sensitive to mutation. Comparison of all variant activities on each substrate indicated different binding modes for the three aromatic substrates, supported by computational docking. This highlights a potential divergence in the evolution of different substrate specificities, with implications for enzyme engineering. Copyright © 2015 Elsevier Inc. All rights reserved.

Convergent roles of de novo mutations and common variants in schizophrenia in tissue-specific and spatiotemporal co-expression network.

PubMed

Jia, Peilin; Chen, Xiangning; Fanous, Ayman H; Zhao, Zhongming

2018-05-24

Genetic components susceptible to complex disease such as schizophrenia include a wide spectrum of variants, including common variants (CVs) and de novo mutations (DNMs). Although CVs and DNMs differ by origin, it remains elusive whether and how they interact at the gene, pathway, and network levels that leads to the disease. In this work, we characterized the genes harboring schizophrenia-associated CVs (CVgenes) and the genes harboring DNMs (DNMgenes) using measures from network, tissue-specific expression profile, and spatiotemporal brain expression profile. We developed an algorithm to link the DNMgenes and CVgenes in spatiotemporal brain co-expression networks. DNMgenes tended to have central roles in the human protein-protein interaction (PPI) network, evidenced in their high degree and high betweenness values. DNMgenes and CVgenes connected with each other significantly more often than with other genes in the networks. However, only CVgenes remained significantly connected after adjusting for their degree. In our gene co-expression PPI network, we found DNMgenes and CVgenes connected in a tissue-specific fashion, and such a pattern was similar to that in GTEx brain but not in other GTEx tissues. Importantly, DNMgene-CVgene subnetworks were enriched with pathways of chromatin remodeling, MHC protein complex binding, and neurotransmitter activities. In summary, our results unveiled that both DNMgenes and CVgenes contributed to a core set of biologically important pathways and networks, and their interactions may attribute to the risk for schizophrenia. Our results also suggested a stronger biological effect of DNMgenes than CVgenes in schizophrenia.

Experimental Assessment of Splicing Variants Using Expression Minigenes and Comparison with In Silico Predictions

PubMed Central

Sharma, Neeraj; Sosnay, Patrick R.; Ramalho, Anabela S.; Douville, Christopher; Franca, Arianna; Gottschalk, Laura B.; Park, Jeenah; Lee, Melissa; Vecchio-Pagan, Briana; Raraigh, Karen S.; Amaral, Margarida D.; Karchin, Rachel; Cutting, Garry R.

2015-01-01

Assessment of the functional consequences of variants near splice sites is a major challenge in the diagnostic laboratory. To address this issue, we created expression minigenes (EMGs) to determine the RNA and protein products generated by splice site variants (n = 10) implicated in cystic fibrosis (CF). Experimental results were compared with the splicing predictions of eight in silico tools. EMGs containing the full-length Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) coding sequence and flanking intron sequences generated wild-type transcript and fully processed protein in Human Embryonic Kidney (HEK293) and CF bronchial epithelial (CFBE41o-) cells. Quantification of variant induced aberrant mRNA isoforms was concordant using fragment analysis and pyrosequencing. The splicing patterns of c.1585−1G>A and c.2657+5G>A were comparable to those reported in primary cells from individuals bearing these variants. Bioinformatics predictions were consistent with experimental results for 9/10 variants (MES), 8/10 variants (NNSplice), and 7/10 variants (SSAT and Sroogle). Programs that estimate the consequences of mis-splicing predicted 11/16 (HSF and ASSEDA) and 10/16 (Fsplice and SplicePort) experimentally observed mRNA isoforms. EMGs provide a robust experimental approach for clinical interpretation of splice site variants and refinement of in silico tools. PMID:25066652

Racial Differences in DNA-Methylation of CpG Sites Within Preterm-Promoting Genes and Gene Variants.

PubMed

Salihu, H M; Das, R; Morton, L; Huang, H; Paothong, A; Wilson, R E; Aliyu, M H; Salemi, J L; Marty, P J

2016-08-01

Objective To evaluate the role DNA methylation may play in genes associated with preterm birth for higher rates of preterm births in African-American women. Methods Fetal cord blood samples from births collected at delivery and maternal demographic and medical information were used in a cross-sectional study to examine fetal DNA methylation of genes implicated in preterm birth among black and non-black infants. Allele-specific DNA methylation analysis was performed using a methylation bead array. Targeted maximum likelihood estimation was applied to examine the relationship between race and fetal DNA methylation of candidate preterm birth genes. Receiver-operating characteristic analyses were then conducted to validate the CpG site methylation marker within the two racial groups. Bootstrapping, a method of validation and replication, was employed. Results 42 CpG sites were screened within 20 candidate gene variants reported consistently in the literature as being associated with preterm birth. Of these, three CpG sites on TNFAIP8 and PON1 genes (corresponding to: cg23917399; cg07086380; and cg07404485, respectively) were significantly differentially methylated between black and non-black individuals. The three CpG sites showed lower methylation status among infants of black women. Bootstrapping validated and replicated results. Conclusion for Practice Our study identified significant differences in levels of methylation on specific genes between black and non-black individuals. Understanding the genetic/epigenetic mechanisms that lead to preterm birth may lead to enhanced prevention strategies to reduce morbidity and mortality by eventually providing a means to identify individuals with a genetic predisposition to preterm labor.

Multiplexed enrichment of rare DNA variants via sequence-selective and temperature-robust amplification

PubMed Central

Wu, Lucia R.; Chen, Sherry X.; Wu, Yalei; Patel, Abhijit A.; Zhang, David Yu

2018-01-01

Rare DNA-sequence variants hold important clinical and biological information, but existing detection techniques are expensive, complex, allele-specific, or don’t allow for significant multiplexing. Here, we report a temperature-robust polymerase-chain-reaction method, which we term blocker displacement amplification (BDA), that selectively amplifies all sequence variants, including single-nucleotide variants (SNVs), within a roughly 20-nucleotide window by 1,000-fold over wild-type sequences. This allows for easy detection and quantitation of hundreds of potential variants originally at ≤0.1% in allele frequency. BDA is compatible with inexpensive thermocycler instrumentation and employs a rationally designed competitive hybridization reaction to achieve comparable enrichment performance across annealing temperatures ranging from 56 °C to 64 °C. To show the sequence generality of BDA, we demonstrate enrichment of 156 SNVs and the reliable detection of single-digit copies. We also show that the BDA detection of rare driver mutations in cell-free DNA samples extracted from the blood plasma of lung-cancer patients is highly consistent with deep sequencing using molecular lineage tags, with a receiver operator characteristic accuracy of 95%. PMID:29805844

m6ASNP: a tool for annotating genetic variants by m6A function.

PubMed

Jiang, Shuai; Xie, Yubin; He, Zhihao; Zhang, Ya; Zhao, Yuli; Chen, Li; Zheng, Yueyuan; Miao, Yanyan; Zuo, Zhixiang; Ren, Jian

2018-05-01

Large-scale genome sequencing projects have identified many genetic variants for diverse diseases. A major goal of these projects is to characterize these genetic variants to provide insight into their function and roles in diseases. N6-methyladenosine (m6A) is one of the most abundant RNA modifications in eukaryotes. Recent studies have revealed that aberrant m6A modifications are involved in many diseases. In this study, we present a user-friendly web server called "m6ASNP" that is dedicated to the identification of genetic variants that target m6A modification sites. A random forest model was implemented in m6ASNP to predict whether the methylation status of an m6A site is altered by the variants that surround the site. In m6ASNP, genetic variants in a standard variant call format (VCF) are accepted as the input data, and the output includes an interactive table that contains the genetic variants annotated by m6A function. In addition, statistical diagrams and a genome browser are provided to visualize the characteristics and to annotate the genetic variants. We believe that m6ASNP is a very convenient tool that can be used to boost further functional studies investigating genetic variants. The web server "m6ASNP" is implemented in JAVA and PHP and is freely available at [60].

Alteration of the C-terminal ligand specificity of the erbin PDZ domain by allosteric mutational effects.

PubMed

Murciano-Calles, Javier; McLaughlin, Megan E; Erijman, Ariel; Hooda, Yogesh; Chakravorty, Nishant; Martinez, Jose C; Shifman, Julia M; Sidhu, Sachdev S

2014-10-23

Modulation of protein binding specificity is important for basic biology and for applied science. Here we explore how binding specificity is conveyed in PDZ (postsynaptic density protein-95/discs large/zonula occludens-1) domains, small interaction modules that recognize various proteins by binding to an extended C terminus. Our goal was to engineer variants of the Erbin PDZ domain with altered specificity for the most C-terminal position (position 0) where a Val is strongly preferred by the wild-type domain. We constructed a library of PDZ domains by randomizing residues in direct contact with position 0 and in a loop that is close to but does not contact position 0. We used phage display to select for PDZ variants that bind to 19 peptide ligands differing only at position 0. To verify that each obtained PDZ domain exhibited the correct binding specificity, we selected peptide ligands for each domain. Despite intensive efforts, we were only able to evolve Erbin PDZ domain variants with selectivity for the aliphatic C-terminal side chains Val, Ile and Leu. Interestingly, many PDZ domains with these three distinct specificities contained identical amino acids at positions that directly contact position 0 but differed in the loop that does not contact position 0. Computational modeling of the selected PDZ domains shows how slight conformational changes in the loop region propagate to the binding site and result in different binding specificities. Our results demonstrate that second-sphere residues could be crucial in determining protein binding specificity. Copyright © 2014 Elsevier Ltd. All rights reserved.

VarMod: modelling the functional effects of non-synonymous variants

PubMed Central

Pappalardo, Morena; Wass, Mark N.

2014-01-01

Unravelling the genotype–phenotype relationship in humans remains a challenging task in genomics studies. Recent advances in sequencing technologies mean there are now thousands of sequenced human genomes, revealing millions of single nucleotide variants (SNVs). For non-synonymous SNVs present in proteins the difficulties of the problem lie in first identifying those nsSNVs that result in a functional change in the protein among the many non-functional variants and in turn linking this functional change to phenotype. Here we present VarMod (Variant Modeller) a method that utilises both protein sequence and structural features to predict nsSNVs that alter protein function. VarMod develops recent observations that functional nsSNVs are enriched at protein–protein interfaces and protein–ligand binding sites and uses these characteristics to make predictions. In benchmarking on a set of nearly 3000 nsSNVs VarMod performance is comparable to an existing state of the art method. The VarMod web server provides extensive resources to investigate the sequence and structural features associated with the predictions including visualisation of protein models and complexes via an interactive JSmol molecular viewer. VarMod is available for use at http://www.wasslab.org/varmod. PMID:24906884

SNCA 3'UTR genetic variants in patients with Parkinson's disease and REM sleep behavior disorder.

PubMed

Toffoli, M; Dreussi, E; Cecchin, E; Valente, M; Sanvilli, N; Montico, M; Gagno, S; Garziera, M; Polano, M; Savarese, M; Calandra-Buonaura, G; Placidi, F; Terzaghi, M; Toffoli, G; Gigli, G L

2017-07-01

REM sleep behavior disorder (RBD) is an early marker of Parkinson's disease (PD); however, it is still unclear which patients with RBD will eventually develop PD. Single nucleotide polymorphisms (SNPs) in the 3'untranslated region (3'UTR) of alpha-synuclein (SNCA) have been associated with PD, but at present, no data is available about RBD. The 3'UTR hosts regulatory regions involved in gene expression control, such as microRNA binding sites. The aim of this study was to determine RBD specific genetic features associated to an increased risk of progression to PD, by sequencing of the SNCA-3'UTR in patients with "idiopathic" RBD (iRBD) and in patients with PD. We recruited 113 consecutive patients with a diagnosis of iRBD (56 patients) or PD (with or without RBD, 57 patients). Sequencing of SNCA-3'UTR was performed on genomic DNA extracted from peripheral blood samples. Bioinformatic analyses were carried out to predict the potential effect of the identified genetic variants on microRNA binding. We found three SNCA-3'UTR SNPs (rs356165, rs3857053, rs1045722) to be more frequent in PD patients than in iRBD patients (p = 0.014, 0.008, and 0.008, respectively). Four new or previously reported but not annotated specific genetic variants (KP876057, KP876056, NM_000345.3:c*860T>A, NM_000345.3:c*2320A>T) have been observed in the RBD population. The in silico approach highlighted that these variants could affect microRNA-mediated gene expression control. Our data show specific SNPs in the SNCA-3'UTR that may bear a risk for RBD to be associated with PD. Moreover, new genetic variants were identified in patients with iRBD.

Identification of a functional enhancer variant within the chronic pancreatitis-associated SPINK1 c.101A>G (p.Asn34Ser)-containing haplotype.

PubMed

Boulling, Arnaud; Masson, Emmanuelle; Zou, Wen-Bin; Paliwal, Sumit; Wu, Hao; Issarapu, Prachand; Bhaskar, Seema; Génin, Emmanuelle; Cooper, David N; Li, Zhao-Shen; Chandak, Giriraj R; Liao, Zhuan; Chen, Jian-Min; Férec, Claude

2017-08-01

The haplotype harboring the SPINK1 c.101A>G (p.Asn34Ser) variant (also known as rs17107315:T>C) represents the most important heritable risk factor for idiopathic chronic pancreatitis identified to date. The causal variant contained within this risk haplotype has however remained stubbornly elusive. Herein, we set out to resolve this enigma by employing a hypothesis-driven approach. First, we searched for variants in strong linkage disequilibrium (LD) with rs17107315:T>C using HaploReg v4.1. Second, we identified two candidate SNPs by visual inspection of sequences spanning all 25 SNPs found to be in LD with rs17107315:T>C, guided by prior knowledge of pancreas-specific transcription factors and their cognate binding sites. Third, employing a novel cis-regulatory module (CRM)-guided approach to further filter the two candidate SNPs yielded a solitary candidate causal variant. Finally, combining data from phylogenetic conservation and chromatin accessibility, cotransfection transactivation experiments, and population genetic studies, we suggest that rs142703147:C>A, which disrupts a PTF1L-binding site within an evolutionarily conserved HNF1A-PTF1L CRM located ∼4 kb upstream of the SPINK1 promoter, contributes to the aforementioned chronic pancreatitis risk haplotype. Further studies are required not only to improve the characterization of this functional SNP but also to identify other functional components that might contribute to this high-risk haplotype. © 2017 Wiley Periodicals, Inc.

An anthrax toxin variant with an improved activity in tumor targeting

PubMed Central

Wein, Alexander N.; Peters, Diane E.; Valivullah, Zaheer; Hoover, Benjamin J.; Tatineni, Aparna; Ma, Qian; Fattah, Rasem; Bugge, Thomas H.; Leppla, Stephen H.; Liu, Shihui

2015-01-01

Anthrax lethal toxin (LT) is an A-B type toxin secreted by Bacillus anthracis, consisting of the cellular binding moiety, protective antigen (PA), and the catalytic moiety, lethal factor (LF). To target cells, PA binds to cell-surface receptors and is then proteolytically processed forming a LF-binding competent PA oligomer where each LF binding site is comprised of three subsites on two adjacent PA monomers. We previously generated PA-U2-R200A, a urokinase-activated PA variant with LF-binding subsite II residue Arg200 mutated to Ala, and PA-L1-I210A, a matrix metalloproteinase-activated PA variant with subsite III residue Ile210 mutated to Ala. PA-U2-R200A and PA-L1-I210A displayed reduced cytotoxicity when used singly. However, when combined, they formed LF-binding competent heterogeneous oligomers by intermolecular complementation, and achieved high specificity in tumor targeting. Nevertheless, each of these proteins, in particular PA-L1-I210A, retained residual LF-binding ability. In this work, we screened a library containing all possible amino acid substitutions for LF-binding site to find variants with activity strictly dependent upon intermolecular complementation. PA-I207R was identified as an excellent replacement for the original clockwise-side variant, PA-I210A. Consequently, the new combination of PA-L1-I207R and PA-U2-R200A showed potent anti-tumor activity and low toxicity, exceeding the performance of the original combination, and warranting further investigation. PMID:26584669

Replacement of oxidizable residues predicted by QM-MM simulation of a fungal laccase generates variants with higher operational stability.

PubMed

Avelar, Mayra; Pastor, Nina; Ramirez-Ramirez, Joaquin; Ayala, Marcela

2018-01-01

In this work, we sought to obtain a more stable laccase with higher operational stability for the oxidation of phenols. During this reaction, phenoxy free radicals are produced that gradually inactivate the enzyme; the inactivation rate depends on the phenol chemical nature. In order to predict residues prone to oxidize within the active site, we simulated activated states of the catalytic region of a fungal laccase using QM-MM tools (Quantum Mechanics-Molecular Mechanics). After simulating the electron distribution in both the basal and activated state (plus or minus one electron) of several conformations of Coriolopsis gallica laccase, residues that could be susceptible to oxidation were identified, according to the values of spin density obtained from calculations. Three targets were selected (F357, F413, and F475) to be replaced by site-directed mutagenesis with less oxidizable residues such as leucine, alanine, and isoleucine. The resulting variants displayed a higher specific activity (from 1.5-to 4-fold) than the parental enzyme. Catalyst depletion during phenol oxidation was 2.5-fold lower for the variants, reflecting a higher operational stability. Copyright © 2017 Elsevier Inc. All rights reserved.

Further mapping of 10q26 supports strong association of HTRA1 polymorphisms with age-related macular degeneration.

PubMed

Gibbs, Daniel; Yang, Zhenglin; Constantine, Ryan; Ma, Xiang; Camp, Nicola J; Yang, Xian; Chen, Hayou; Jorgenson, Adam; Hau, Vincent; Dewan, Andrew; Zeng, Jiexi; Harmon, Jennifer; Buehler, Jeanette; Brand, John M; Hoh, Josephine; Cameron, D Joshua; Dixit, Manjusha; Tong, Zongzhong; Zhang, Kang

2008-02-01

Age-related macular degeneration (AMD) is a complex disorder with genetic and environmental influences. The genetic influences affecting AMD are not well understood and few genes have been consistently implicated and replicated for this disease. A polymorphism (rs11200638) in a transcription factor binding site of the HTRA1 gene has been described, in previous reports, as being most significantly associated with AMD. In this paper, we investigate haplotype association and individual polymorphic association by genotyping additional variants in the AMD risk-associated region of chromosome 10q26. We demonstrate that rs11200638 in the promoter region and rs2293870 in exon 1 of HTRA1, are among the most significantly associated variants for advanced forms of AMD.

Combined effects of ankylosing spondylitis-associated ERAP1 polymorphisms outside the catalytic and peptide-binding sites on the processing of natural HLA-B27 ligands.

PubMed

Martín-Esteban, Adrian; Gómez-Molina, Patricia; Sanz-Bravo, Alejandro; López de Castro, José A

2014-02-14

ERAP1 polymorphism involving residues 528 and 575/725 is associated with ankylosing spondylitis among HLA-B27-positive individuals. We used four recombinant variants to address the combined effects of the K528R and D575N polymorphism on the processing of HLA-B27 ligands. The hydrolysis of a fluorogenic substrate, Arg-528/Asp-575 < Lys-528/Asp-575 < Arg-528/Asn-575 < Lys-528/Asn-575, indicated that the relative activity of variants carrying Arg-528 or Lys-528 depends on residue 575. Asp-575 conferred lower activity than Asn-575, but the difference depended on residue 528. The same hierarchy was observed with synthetic precursors of HLA-B27 ligands, but the effects were peptide-dependent. Sometimes the epitope yields were variant-specific at all times. For other peptides, concomitant generation and destruction led to similar epitope amounts with all the variants at long, but not at short, digestion times. The generation/destruction balance of two related HLA-B27 ligands was analyzed in vitro and in live cells. Their relative yields at long digestion times were comparable with those from HLA-B27-positive cells, suggesting that ERAP1 was a major determinant of the abundance of these peptides in vivo. The hydrolysis of fluorogenic and peptide substrates by an HLA-B27 ligand or a shorter peptide, respectively, was increasingly inhibited as a function of ERAP1 activity, indicating that residues 528 and 575 affect substrate inhibition of ERAP1 trimming. The significant and complex effects of co-occurring ERAP1 polymorphisms on multiple HLA-B27 ligands, and their potential to alter the immunological and pathogenetic features of HLA-B27 as a function of the ERAP1 context, explain the epistatic association of both molecules in ankylosing spondylitis.

Combined Effects of Ankylosing Spondylitis-associated ERAP1 Polymorphisms Outside the Catalytic and Peptide-binding Sites on the Processing of Natural HLA-B27 Ligands*

PubMed Central

Martín-Esteban, Adrian; Gómez-Molina, Patricia; Sanz-Bravo, Alejandro; López de Castro, José A.

2014-01-01

ERAP1 polymorphism involving residues 528 and 575/725 is associated with ankylosing spondylitis among HLA-B27-positive individuals. We used four recombinant variants to address the combined effects of the K528R and D575N polymorphism on the processing of HLA-B27 ligands. The hydrolysis of a fluorogenic substrate, Arg-528/Asp-575 < Lys-528/Asp-575 < Arg-528/Asn-575 < Lys-528/Asn-575, indicated that the relative activity of variants carrying Arg-528 or Lys-528 depends on residue 575. Asp-575 conferred lower activity than Asn-575, but the difference depended on residue 528. The same hierarchy was observed with synthetic precursors of HLA-B27 ligands, but the effects were peptide-dependent. Sometimes the epitope yields were variant-specific at all times. For other peptides, concomitant generation and destruction led to similar epitope amounts with all the variants at long, but not at short, digestion times. The generation/destruction balance of two related HLA-B27 ligands was analyzed in vitro and in live cells. Their relative yields at long digestion times were comparable with those from HLA-B27-positive cells, suggesting that ERAP1 was a major determinant of the abundance of these peptides in vivo. The hydrolysis of fluorogenic and peptide substrates by an HLA-B27 ligand or a shorter peptide, respectively, was increasingly inhibited as a function of ERAP1 activity, indicating that residues 528 and 575 affect substrate inhibition of ERAP1 trimming. The significant and complex effects of co-occurring ERAP1 polymorphisms on multiple HLA-B27 ligands, and their potential to alter the immunological and pathogenetic features of HLA-B27 as a function of the ERAP1 context, explain the epistatic association of both molecules in ankylosing spondylitis. PMID:24352655

How important are rare variants in common disease?

PubMed

Saint Pierre, Aude; Génin, Emmanuelle

2014-09-01

Genome-wide association studies have uncovered hundreds of common genetic variants involved in complex diseases. However, for most complex diseases, these common genetic variants only marginally contribute to disease susceptibility. It is now argued that rare variants located in different genes could in fact play a more important role in disease susceptibility than common variants. These rare genetic variants were not captured by genome-wide association studies using single nucleotide polymorphism-chips but with the advent of next-generation sequencing technologies, they have become detectable. It is now possible to study their contribution to common disease by resequencing samples of cases and controls or by using new genotyping exome arrays that cover rare alleles. In this review, we address the question of the contribution of rare variants in common disease by taking the examples of different diseases for which some resequencing studies have already been performed, and by summarizing the results of simulation studies conducted so far to investigate the genetic architecture of complex traits in human. So far, empirical data have not allowed the exclusion of many models except the most extreme ones involving only a small number of rare variants with large effects contributing to complex disease. To unravel the genetic architecture of complex disease, case-control data will not be sufficient, and alternative study designs need to be proposed together with methodological developments. © The Author 2014. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Analysis of prostate-specific antigen transcripts in chimpanzees, cynomolgus monkeys, baboons, and African green monkeys.

PubMed

Mubiru, James N; Yang, Alice S; Olsen, Christian; Nayak, Sudhir; Livi, Carolina B; Dick, Edward J; Owston, Michael; Garcia-Forey, Magdalena; Shade, Robert E; Rogers, Jeffrey

2014-01-01

The function of prostate-specific antigen (PSA) is to liquefy the semen coagulum so that the released sperm can fuse with the ovum. Fifteen spliced variants of the PSA gene have been reported in humans, but little is known about alternative splicing in nonhuman primates. Positive selection has been reported in sex- and reproductive-related genes from sea urchins to Drosophila to humans; however, there are few studies of adaptive evolution of the PSA gene. Here, using polymerase chain reaction (PCR) product cloning and sequencing, we study PSA transcript variant heterogeneity in the prostates of chimpanzees (Pan troglodytes), cynomolgus monkeys (Macaca fascicularis), baboons (Papio hamadryas anubis), and African green monkeys (Chlorocebus aethiops). Six PSA variants were identified in the chimpanzee prostate, but only two variants were found in cynomolgus monkeys, baboons, and African green monkeys. In the chimpanzee the full-length transcript is expressed at the same magnitude as the transcripts that retain intron 3. We have found previously unidentified splice variants of the PSA gene, some of which might be linked to disease conditions. Selection on the PSA gene was studied in 11 primate species by computational methods using the sequences reported here for African green monkey, cynomolgus monkey, baboon, and chimpanzee and other sequences available in public databases. A codon-based analysis (dN/dS) of the PSA gene identified potential adaptive evolution at five residue sites (Arg45, Lys70, Gln144, Pro189, and Thr203).

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kaplow, Irene M.; MacIsaac, Julia L.; Mah, Sarah M.

DNA methylation is an epigenetic modification that plays a key role in gene regulation. Previous studies have investigated its genetic basis by mapping genetic variants that are associated with DNA methylation at specific sites, but these have been limited to microarrays that cover <2% of the genome and cannot account for allele-specific methylation (ASM). Other studies have performed whole-genome bisulfite sequencing on a few individuals, but these lack statistical power to identify variants associated with DNA methylation. We present a novel approach in which bisulfite-treated DNA from many individuals is sequenced together in a single pool, resulting in a trulymore » genome-wide map of DNA methylation. Compared to methods that do not account for ASM, our approach increases statistical power to detect associations while sharply reducing cost, effort, and experimental variability. As a proof of concept, we generated deep sequencing data from a pool of 60 human cell lines; we evaluated almost twice as many CpGs as the largest microarray studies and identified more than 2000 genetic variants associated with DNA methylation. Here we found that these variants are highly enriched for associations with chromatin accessibility and CTCF binding but are less likely to be associated with traits indirectly linked to DNA, such as gene expression and disease phenotypes. In summary, our approach allows genome-wide mapping of genetic variants associated with DNA methylation in any tissue of any species, without the need for individual-level genotype or methylation data.« less

Variants in members of the cadherin-catenin complex, CDH1 and CTNND1, cause blepharocheilodontic syndrome.

PubMed

Kievit, Anneke; Tessadori, Federico; Douben, Hannie; Jordens, Ingrid; Maurice, Madelon; Hoogeboom, Jeannette; Hennekam, Raoul; Nampoothiri, Sheela; Kayserili, Hülya; Castori, Marco; Whiteford, Margo; Motter, Connie; Melver, Catherine; Cunningham, Michael; Hing, Anne; Kokitsu-Nakata, Nancy M; Vendramini-Pittoli, Siulan; Richieri-Costa, Antonio; Baas, Annette F; Breugem, Corstiaan C; Duran, Karen; Massink, Maarten; Derksen, Patrick W B; van IJcken, Wilfred F J; van Unen, Leontine; Santos-Simarro, Fernando; Lapunzina, Pablo; Gil-da Silva Lopes, Vera L; Lustosa-Mendes, Elaine; Krall, Max; Slavotinek, Anne; Martinez-Glez, Victor; Bakkers, Jeroen; van Gassen, Koen L I; de Klein, Annelies; van den Boogaard, Marie-José H; van Haaften, Gijs

2018-02-01

Blepharocheilodontic syndrome (BCDS) consists of lagophthalmia, ectropion of the lower eyelids, distichiasis, euryblepharon, cleft lip/palate and dental anomalies and has autosomal dominant inheritance with variable expression. We identified heterozygous variants in two genes of the cadherin-catenin complex, CDH1, encoding E-cadherin, and CTNND1, encoding p120 catenin delta1 in 15 of 17 BCDS index patients, as was recently described in a different publication. CDH1 plays an essential role in epithelial cell adherence; CTNND1 binds to CDH1 and controls the stability of the complex. Functional experiments in zebrafish and human cells showed that the CDH1 variants impair the cell adhesion function of the cadherin-catenin complex in a dominant-negative manner. Variants in CDH1 have been linked to familial hereditary diffuse gastric cancer and invasive lobular breast cancer; however, no cases of gastric or breast cancer have been reported in our BCDS cases. Functional experiments reported here indicated the BCDS variants comprise a distinct class of CDH1 variants. Altogether, we identified the genetic cause of BCDS enabling DNA diagnostics and counseling, in addition we describe a novel class of dominant negative CDH1 variants.

TERT promoter mutations in bladder cancer affect patient survival and disease recurrence through modification by a common polymorphism.

PubMed

Rachakonda, P Sivaramakrishna; Hosen, Ismail; de Verdier, Petra J; Fallah, Mahdi; Heidenreich, Barbara; Ryk, Charlotta; Wiklund, N Peter; Steineck, Gunnar; Schadendorf, Dirk; Hemminki, Kari; Kumar, Rajiv

2013-10-22

The telomerase reverse transcriptase (TERT) promoter, an important element of telomerase expression, has emerged as a target of cancer-specific mutations. Originally described in melanoma, the mutations in TERT promoter have been shown to be common in certain other tumor types that include glioblastoma, hepatocellular carcinoma, and bladder cancer. To fully define the occurrence and effect of the TERT promoter mutations, we investigated tumors from a well-characterized series of 327 patients with urothelial cell carcinoma of bladder. The somatic mutations, mainly at positions -124 and -146 bp from ATG start site that create binding motifs for E-twenty six/ternary complex factors (Ets/TCF), affected 65.4% of the tumors, with even distribution across different stages and grades. Our data showed that a common polymorphism rs2853669, within a preexisting Ets2 binding site in the TERT promoter, acts as a modifier of the effect of the mutations on survival and tumor recurrence. The patients with the mutations showed poor survival in the absence [hazard ratio (HR) 2.19, 95% confidence interval (CI) 1.02-4.70] but not in the presence (HR 0.42, 95% CI 0.18-1.01) of the variant allele of the polymorphism. The mutations in the absence of the variant allele were highly associated with the disease recurrence in patients with Tis, Ta, and T1 tumors (HR 1.85, 95% CI 1.11-3.08). The TERT promoter mutations are the most common somatic lesions in bladder cancer with clinical implications. The association of the mutations with patient survival and disease recurrence, subject to modification by a common polymorphism, can be a unique putative marker with individualized prognostic potential.

The generation of a 1-hydroxy-2-naphthoate 1,2-dioxygenase by single point mutations of salicylate 1,2-dioxygenase--rational design of mutants and the crystal structures of the A85H and W104Y variants.

PubMed

Ferraroni, Marta; Steimer, Lenz; Matera, Irene; Bürger, Sibylle; Scozzafava, Andrea; Stolz, Andreas; Briganti, Fabrizio

2012-12-01

Key amino acid residues of the salicylate 1,2-dioxygenase (SDO), an iron (II) class III ring cleaving dioxygenase from Pseudaminobacter salicylatoxidans BN12, were selected, based on amino acid sequence alignments and structural analysis of the enzyme, and modified by site-directed mutagenesis to obtain variant forms with altered catalytic properties. SDO shares with 1-hydroxy-2-naphthoate dioxygenase (1H2NDO) its unique ability to oxidatively cleave monohydroxylated aromatic compounds. Nevertheless SDO is more versatile with respect to 1H2NDO and other known gentisate dioxygenases (GDOs) because it cleaves not only gentisate and 1-hydroxy-2-naphthoate (1H2NC) but also salicylate and substituted salicylates. Several enzyme variants of SDO were rationally designed to simulate 1H2NDO. The basic kinetic parameters for the SDO mutants L38Q, M46V, A85H and W104Y were determined. The enzyme variants L38Q, M46V, A85H demonstrated higher catalytic efficiencies toward 1-hydroxy-2-naphthoate (1H2NC) compared to gentisate. Remarkably, the enzyme variant A85H effectively cleaved 1H2NC but did not oxidize gentisate at all. The W104Y SDO mutant exhibited reduced reaction rates for all substrates tested. The crystal structures of the A85H and W104Y variants were solved and analyzed. The substitution of Ala85 with a histidine residue caused significant changes in the orientation of the loop containing this residue which is involved in the active site closing upon substrate binding. In SDO A85H this specific loop shifts away from the active site and thus opens the cavity favoring the binding of bulkier substrates. Since this loop also interacts with the N-terminal residues of the vicinal subunit, the structure and packing of the holoenzyme might be also affected. Copyright © 2012 Elsevier Inc. All rights reserved.

Rapid functional analysis of computationally complex rare human IRF6 gene variants using a novel zebrafish model.

PubMed

Li, Edward B; Truong, Dawn; Hallett, Shawn A; Mukherjee, Kusumika; Schutte, Brian C; Liao, Eric C

2017-09-01

Large-scale sequencing efforts have captured a rapidly growing catalogue of genetic variations. However, the accurate establishment of gene variant pathogenicity remains a central challenge in translating personal genomics information to clinical decisions. Interferon Regulatory Factor 6 (IRF6) gene variants are significant genetic contributors to orofacial clefts. Although approximately three hundred IRF6 gene variants have been documented, their effects on protein functions remain difficult to interpret. Here, we demonstrate the protein functions of human IRF6 missense gene variants could be rapidly assessed in detail by their abilities to rescue the irf6 -/- phenotype in zebrafish through variant mRNA microinjections at the one-cell stage. The results revealed many missense variants previously predicted by traditional statistical and computational tools to be loss-of-function and pathogenic retained partial or full protein function and rescued the zebrafish irf6 -/- periderm rupture phenotype. Through mRNA dosage titration and analysis of the Exome Aggregation Consortium (ExAC) database, IRF6 missense variants were grouped by their abilities to rescue at various dosages into three functional categories: wild type function, reduced function, and complete loss-of-function. This sensitive and specific biological assay was able to address the nuanced functional significances of IRF6 missense gene variants and overcome many limitations faced by current statistical and computational tools in assigning variant protein function and pathogenicity. Furthermore, it unlocked the possibility for characterizing yet undiscovered human IRF6 missense gene variants from orofacial cleft patients, and illustrated a generalizable functional genomics paradigm in personalized medicine.

Quantitative imaging of single mRNA splice variants in living cells

NASA Astrophysics Data System (ADS)

Lee, Kyuwan; Cui, Yi; Lee, Luke P.; Irudayaraj, Joseph

2014-06-01

Alternative messenger RNA (mRNA) splicing is a fundamental process of gene regulation, and errors in RNA splicing are known to be associated with a variety of different diseases. However, there is currently a lack of quantitative technologies for monitoring mRNA splice variants in cells. Here, we show that a combination of plasmonic dimer probes and hyperspectral imaging can be used to detect and quantify mRNA splice variants in living cells. The probes are made from gold nanoparticles functionalized with oligonucleotides and can hybridize to specific mRNA sequences, forming nanoparticle dimers that exhibit distinct spectral shifts due to plasmonic coupling. With this approach, we show that the spatial and temporal distribution of three selected splice variants of the breast cancer susceptibility gene, BRCA1, can be monitored at single-copy resolution by measuring the hybridization dynamics of the nanoplasmonic dimers. Our study provides insights into RNA and its transport in living cells, which could improve our understanding of cellular protein complexes, pharmacogenomics, genetic diagnosis and gene therapies.

Phonetic Spelling Filter for Keyword Selection in Drug Mention Mining from Social Media

PubMed Central

Pimpalkhute, Pranoti; Patki, Apurv; Nikfarjam, Azadeh; Gonzalez, Graciela

2014-01-01

Social media postings are rich in information that often remain hidden and inaccessible for automatic extraction due to inherent limitations of the site’s APIs, which mostly limit access via specific keyword-based searches (and limit both the number of keywords and the number of postings that are returned). When mining social media for drug mentions, one of the first problems to solve is how to derive a list of variants of the drug name (common misspellings) that can capture a sufficient number of postings. We present here an approach that filters the potential variants based on the intuition that, faced with the task of writing an unfamiliar, complex word (the drug name), users will tend to revert to phonetic spelling, and we thus give preference to variants that reflect the phonemes of the correct spelling. The algorithm allowed us to capture 50.4 – 56.0 % of the user comments using only about 18% of the variants. PMID:25717407

Low-Pass Genome-Wide Sequencing and Variant Inference Using Identity-by-Descent in an Isolated Human Population

PubMed Central

Gusev, A.; Shah, M. J.; Kenny, E. E.; Ramachandran, A.; Lowe, J. K.; Salit, J.; Lee, C. C.; Levandowsky, E. C.; Weaver, T. N.; Doan, Q. C.; Peckham, H. E.; McLaughlin, S. F.; Lyons, M. R.; Sheth, V. N.; Stoffel, M.; De La Vega, F. M.; Friedman, J. M.; Breslow, J. L.

2012-01-01

Whole-genome sequencing in an isolated population with few founders directly ascertains variants from the population bottleneck that may be rare elsewhere. In such populations, shared haplotypes allow imputation of variants in unsequenced samples without resorting to complex statistical methods as in studies of outbred cohorts. We focus on an isolated population cohort from the Pacific Island of Kosrae, Micronesia, where we previously collected SNP array and rich phenotype data for the majority of the population. We report identification of long regions with haplotypes co-inherited between pairs of individuals and methodology to leverage such shared genetic content for imputation. Our estimates show that sequencing as few as 40 personal genomes allows for inference in up to 60% of the 3000-person cohort at the average locus. We ascertained a pilot data set of whole-genome sequences from seven Kosraean individuals, with average 5× coverage. This assay identified 5,735,306 unique sites of which 1,212,831 were previously unknown. Additionally, these variants are unusually enriched for alleles that are rare in other populations when compared to geographic neighbors (published Korean genome SJK). We used the presence of shared haplotypes between the seven Kosraen individuals to estimate expected imputation accuracy of known and novel homozygous variants at 99.6% and 97.3%, respectively. This study presents whole-genome analysis of a homogenous isolate population with emphasis on optimal rare variant inference. PMID:22135348

Common variants in Mendelian kidney disease genes and their association with renal function.

PubMed

Parsa, Afshin; Fuchsberger, Christian; Köttgen, Anna; O'Seaghdha, Conall M; Pattaro, Cristian; de Andrade, Mariza; Chasman, Daniel I; Teumer, Alexander; Endlich, Karlhans; Olden, Matthias; Chen, Ming-Huei; Tin, Adrienne; Kim, Young J; Taliun, Daniel; Li, Man; Feitosa, Mary; Gorski, Mathias; Yang, Qiong; Hundertmark, Claudia; Foster, Meredith C; Glazer, Nicole; Isaacs, Aaron; Rao, Madhumathi; Smith, Albert V; O'Connell, Jeffrey R; Struchalin, Maksim; Tanaka, Toshiko; Li, Guo; Hwang, Shih-Jen; Atkinson, Elizabeth J; Lohman, Kurt; Cornelis, Marilyn C; Johansson, Asa; Tönjes, Anke; Dehghan, Abbas; Couraki, Vincent; Holliday, Elizabeth G; Sorice, Rossella; Kutalik, Zoltan; Lehtimäki, Terho; Esko, Tõnu; Deshmukh, Harshal; Ulivi, Sheila; Chu, Audrey Y; Murgia, Federico; Trompet, Stella; Imboden, Medea; Kollerits, Barbara; Pistis, Giorgio; Harris, Tamara B; Launer, Lenore J; Aspelund, Thor; Eiriksdottir, Gudny; Mitchell, Braxton D; Boerwinkle, Eric; Schmidt, Helena; Hofer, Edith; Hu, Frank; Demirkan, Ayse; Oostra, Ben A; Turner, Stephen T; Ding, Jingzhong; Andrews, Jeanette S; Freedman, Barry I; Giulianini, Franco; Koenig, Wolfgang; Illig, Thomas; Döring, Angela; Wichmann, H-Erich; Zgaga, Lina; Zemunik, Tatijana; Boban, Mladen; Minelli, Cosetta; Wheeler, Heather E; Igl, Wilmar; Zaboli, Ghazal; Wild, Sarah H; Wright, Alan F; Campbell, Harry; Ellinghaus, David; Nöthlings, Ute; Jacobs, Gunnar; Biffar, Reiner; Ernst, Florian; Homuth, Georg; Kroemer, Heyo K; Nauck, Matthias; Stracke, Sylvia; Völker, Uwe; Völzke, Henry; Kovacs, Peter; Stumvoll, Michael; Mägi, Reedik; Hofman, Albert; Uitterlinden, Andre G; Rivadeneira, Fernando; Aulchenko, Yurii S; Polasek, Ozren; Hastie, Nick; Vitart, Veronique; Helmer, Catherine; Wang, Jie Jin; Stengel, Bénédicte; Ruggiero, Daniela; Bergmann, Sven; Kähönen, Mika; Viikari, Jorma; Nikopensius, Tiit; Province, Michael; Colhoun, Helen; Doney, Alex; Robino, Antonietta; Krämer, Bernhard K; Portas, Laura; Ford, Ian; Buckley, Brendan M; Adam, Martin; Thun, Gian-Andri; Paulweber, Bernhard; Haun, Margot; Sala, Cinzia; Mitchell, Paul; Ciullo, Marina; Vollenweider, Peter; Raitakari, Olli; Metspalu, Andres; Palmer, Colin; Gasparini, Paolo; Pirastu, Mario; Jukema, J Wouter; Probst-Hensch, Nicole M; Kronenberg, Florian; Toniolo, Daniela; Gudnason, Vilmundur; Shuldiner, Alan R; Coresh, Josef; Schmidt, Reinhold; Ferrucci, Luigi; van Duijn, Cornelia M; Borecki, Ingrid; Kardia, Sharon L R; Liu, Yongmei; Curhan, Gary C; Rudan, Igor; Gyllensten, Ulf; Wilson, James F; Franke, Andre; Pramstaller, Peter P; Rettig, Rainer; Prokopenko, Inga; Witteman, Jacqueline; Hayward, Caroline; Ridker, Paul M; Bochud, Murielle; Heid, Iris M; Siscovick, David S; Fox, Caroline S; Kao, W Linda; Böger, Carsten A

2013-12-01

Many common genetic variants identified by genome-wide association studies for complex traits map to genes previously linked to rare inherited Mendelian disorders. A systematic analysis of common single-nucleotide polymorphisms (SNPs) in genes responsible for Mendelian diseases with kidney phenotypes has not been performed. We thus developed a comprehensive database of genes for Mendelian kidney conditions and evaluated the association between common genetic variants within these genes and kidney function in the general population. Using the Online Mendelian Inheritance in Man database, we identified 731 unique disease entries related to specific renal search terms and confirmed a kidney phenotype in 218 of these entries, corresponding to mutations in 258 genes. We interrogated common SNPs (minor allele frequency >5%) within these genes for association with the estimated GFR in 74,354 European-ancestry participants from the CKDGen Consortium. However, the top four candidate SNPs (rs6433115 at LRP2, rs1050700 at TSC1, rs249942 at PALB2, and rs9827843 at ROBO2) did not achieve significance in a stage 2 meta-analysis performed in 56,246 additional independent individuals, indicating that these common SNPs are not associated with estimated GFR. The effect of less common or rare variants in these genes on kidney function in the general population and disease-specific cohorts requires further research.

Heterozygous variants in ACTL6A, encoding a component of the BAF complex, are associated with intellectual disability.

PubMed

Marom, Ronit; Jain, Mahim; Burrage, Lindsay C; Song, I-Wen; Graham, Brett H; Brown, Chester W; Stevens, Servi J C; Stegmann, Alexander P A; Gunter, Andrew T; Kaplan, Julie D; Gavrilova, Ralitza H; Shinawi, Marwan; Rosenfeld, Jill A; Bae, Yangjin; Tran, Alyssa A; Chen, Yuqing; Lu, James T; Gibbs, Richard A; Eng, Christine; Yang, Yaping; Rousseau, Justine; de Vries, Bert B A; Campeau, Philippe M; Lee, Brendan

2017-10-01

Pathogenic variants in genes encoding components of the BRG1-associated factor (BAF) chromatin remodeling complex have been associated with intellectual disability syndromes. We identified heterozygous, novel variants in ACTL6A, a gene encoding a component of the BAF complex, in three subjects with varying degrees of intellectual disability. Two subjects have missense variants affecting highly conserved amino acid residues within the actin-like domain. Missense mutations in the homologous region in yeast actin were previously reported to be dominant lethal and were associated with impaired binding of the human ACTL6A to β-actin and BRG1. A third subject has a splicing variant that creates an in-frame deletion. Our findings suggest that the variants identified in our subjects may have a deleterious effect on the function of the protein by disturbing the integrity of the BAF complex. Thus, ACTL6A gene mutation analysis should be considered in patients with intellectual disability, learning disabilities, or developmental language disorder. © 2017 Wiley Periodicals, Inc.

Structures of MART-1 26/27-35Peptide/HLA-A2 Complexes Reveal a Remarkable Disconnect between Antigen Structural Homology and T Cell Recognition

DOE Office of Scientific and Technical Information (OSTI.GOV)

Borbulevych, Oleg Y; Insaidoo, Francis K; Baxter, Tiffany K

2008-09-17

Small structural changes in peptides presented by major histocompatibility complex (MHC) molecules often result in large changes in immunogenicity, supporting the notion that T cell receptors are exquisitely sensitive to antigen structure. Yet there are striking examples of TCR recognition of structurally dissimilar ligands. The resulting unpredictability of how T cells will respond to different or modified antigens impacts both our understanding of the physical bases for TCR specificity as well as efforts to engineer peptides for immunomodulation. In cancer immunotherapy, epitopes and variants derived from the MART-1/Melan-A protein are widely used as clinical vaccines. Two overlapping epitopes spanning aminomore » acid residues 26 through 35 are of particular interest: numerous clinical studies have been performed using variants of the MART-1 26-35 decamer, although only the 27-35 nonamer has been found on the surface of targeted melanoma cells. Here, we show that the 26-35 and 27-35 peptides adopt strikingly different conformations when bound to HLA-A2. Nevertheless, clonally distinct MART-1{sub 26/27-35}-reactive T cells show broad cross-reactivity towards these ligands. Simultaneously, however, many of the cross-reactive T cells remain unable to recognize anchor-modified variants with very subtle structural differences. These dichotomous observations challenge our thinking about how structural information on unligated peptide/MHC complexes should be best used when addressing questions of TCR specificity. Our findings also indicate that caution is warranted in the design of immunotherapeutics based on the MART-1 26/27-35 epitopes, as neither cross-reactivity nor selectivity is predictable based on the analysis of the structures alone.« less

Structures of MART-126/27–35 Peptide/HLA-A2 Complexes Reveal a Remarkable Disconnect between Antigen Structural Homology and T Cell Recognition

PubMed Central

Borbulevych, Oleg Y.; Insaidoo, Francis K.; Baxter, Tiffany K.; Powell, Daniel J.; Johnson, Laura A.; Restifo, Nicholas P.; Baker, Brian M.

2007-01-01

Small structural changes in peptides presented by major histocompatibility complex (MHC) molecules often result in large changes in immunogenicity, supporting the notion that T cell receptors are exquisitely sensitive to antigen structure. Yet there are striking examples of TCR recognition of structurally dissimilar ligands. The resulting unpredictability of how T cells will respond to different or modified antigens impacts both our understanding of the physical bases for TCR specificity as well as efforts to engineer peptides for immunomodulation. In cancer immunotherapy, epitopes and variants derived from the MART-1/Melan-A protein are widely used as clinical vaccines. Two overlapping epitopes spanning amino acid residues 26 through 35 are of particular interest: numerous clinical studies have been performed using variants of the MART-1 26–35 decamer, although only the 27–35 nonamer has been found on the surface of targeted melanoma cells. Here, we show that the 26–35 and 27–35 peptides adopt strikingly different conformations when bound to HLA-A2. Nevertheless, clonally distinct MART-126/27–35-reactive T cells show broad cross-reactivity towards these ligands. Simultaneously, however, many of the cross-reactive T cells remain unable to recognize anchor-modified variants with very subtle structural differences. These dichotomous observations challenge our thinking about how structural information on unligated peptide/MHC complexes should be best used when addressing questions of TCR specificity. Our findings also indicate that caution is warranted in the design of immunotherapeutics based on the MART-1 26/27–35 epitopes, as neither cross-reactivity nor selectivity is predictable based on the analysis of the structures alone. PMID:17719062

Mesotrypsin Signature Mutation in a Chymotrypsin C (CTRC) Variant Associated with Chronic Pancreatitis.

PubMed

Szabó, András; Ludwig, Maren; Hegyi, Eszter; Szépeová, Renata; Witt, Heiko; Sahin-Tóth, Miklós

2015-07-10

Human chymotrypsin C (CTRC) protects against pancreatitis by degrading trypsinogen and thereby curtailing harmful intra-pancreatic trypsinogen activation. Loss-of-function mutations in CTRC increase the risk for chronic pancreatitis. Here we describe functional analysis of eight previously uncharacterized natural CTRC variants tested for potential defects in secretion, proteolytic stability, and catalytic activity. We found that all variants were secreted from transfected cells normally, and none suffered proteolytic degradation by trypsin. Five variants had normal enzymatic activity, whereas variant p.R29Q was catalytically inactive due to loss of activation by trypsin and variant p.S239C exhibited impaired activity possibly caused by disulfide mispairing. Surprisingly, variant p.G214R had increased activity on a small chromogenic peptide substrate but was markedly defective in cleaving bovine β-casein or the natural CTRC substrates human cationic trypsinogen and procarboxypeptidase A1. Mutation p.G214R is analogous to the evolutionary mutation in human mesotrypsin, which rendered this trypsin isoform resistant to proteinaceous inhibitors and conferred its ability to cleave these inhibitors. Similarly to the mesotrypsin phenotype, CTRC variant p.G214R was inhibited poorly by eglin C, ecotin, or a CTRC-specific variant of SGPI-2, and it readily cleaved the reactive-site peptide bonds in eglin C and ecotin. We conclude that CTRC variants p.R29Q, p.G214R, and p.S239C are risk factors for chronic pancreatitis. Furthermore, the mesotrypsin-like CTRC variant highlights how the same natural mutation in homologous pancreatic serine proteases can evolve a new physiological role or lead to pathology, determined by the biological context of protease function. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Role of DNA conformation & energetic insights in Msx-1-DNA recognition as revealed by molecular dynamics studies on specific and nonspecific complexes.

PubMed

Kachhap, Sangita; Singh, Balvinder

2015-01-01

In most of homeodomain-DNA complexes, glutamine or lysine is present at 50th position and interacts with 5th and 6th nucleotide of core recognition region. Molecular dynamics simulations of Msx-1-DNA complex (Q50-TG) and its variant complexes, that is specific (Q50K-CC), nonspecific (Q50-CC) having mutation in DNA and (Q50K-TG) in protein, have been carried out. Analysis of protein-DNA interactions and structure of DNA in specific and nonspecific complexes show that amino acid residues use sequence-dependent shape of DNA to interact. The binding free energies of all four complexes were analysed to define role of amino acid residue at 50th position in terms of binding strength considering the variation in DNA on stability of protein-DNA complexes. The order of stability of protein-DNA complexes shows that specific complexes are more stable than nonspecific ones. Decomposition analysis shows that N-terminal amino acid residues have been found to contribute maximally in binding free energy of protein-DNA complexes. Among specific protein-DNA complexes, K50 contributes more as compared to Q50 towards binding free energy in respective complexes. The sequence dependence of local conformation of DNA enables Q50/Q50K to make hydrogen bond with nucleotide(s) of DNA. The changes in amino acid sequence of protein are accommodated and stabilized around TAAT core region of DNA having variation in nucleotides.

Defective mitochondrial RNA processing due to PNPT1 variants causes Leigh syndrome.

PubMed

Matilainen, Sanna; Carroll, Christopher J; Richter, Uwe; Euro, Liliya; Pohjanpelto, Max; Paetau, Anders; Isohanni, Pirjo; Suomalainen, Anu

2017-09-01

Leigh syndrome is a severe infantile encephalopathy with an exceptionally variable genetic background. We studied the exome of a child manifesting with Leigh syndrome at one month of age and progressing to death by the age of 2.4 years, and identified novel compound heterozygous variants in PNPT1, encoding the polynucleotide phosphorylase (PNPase). Expression of the wild type PNPT1 in the subject's myoblasts functionally complemented the defects, and the pathogenicity was further supported by structural predictions and protein and RNA analyses. PNPase is a key enzyme in mitochondrial RNA metabolism, with suggested roles in mitochondrial RNA import and degradation. The variants were predicted to locate in the PNPase active site and disturb the RNA processing activity of the enzyme. The PNPase trimer formation was not affected, but specific RNA processing intermediates derived from mitochondrial transcripts of the ND6 subunit of Complex I, as well as small mRNA fragments, accumulated in the subject's myoblasts. Mitochondrial RNA processing mediated by the degradosome consisting of hSUV3 and PNPase is poorly characterized, and controversy on the role and location of PNPase within human mitochondria exists. Our evidence indicates that PNPase activity is essential for the correct maturation of the ND6 transcripts, and likely for the efficient removal of degradation intermediates. Loss of its activity will result in combined respiratory chain deficiency, and a classic respiratory chain-deficiency-associated disease, Leigh syndrome, indicating an essential role for the enzyme for normal function of the mitochondrial respiratory chain. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Missense dopamine transporter mutations associate with adult parkinsonism and ADHD

PubMed Central

Hansen, Freja H.; Skjørringe, Tina; Yasmeen, Saiqa; Arends, Natascha V.; Sahai, Michelle A.; Erreger, Kevin; Andreassen, Thorvald F.; Holy, Marion; Hamilton, Peter J.; Neergheen, Viruna; Karlsborg, Merete; Newman, Amy H.; Pope, Simon; Heales, Simon J.R.; Friberg, Lars; Law, Ian; Pinborg, Lars H.; Sitte, Harald H.; Loland, Claus; Shi, Lei; Weinstein, Harel; Galli, Aurelio; Hjermind, Lena E.; Møller, Lisbeth B.; Gether, Ulrik

2014-01-01

Parkinsonism and attention deficit hyperactivity disorder (ADHD) are widespread brain disorders that involve disturbances of dopaminergic signaling. The sodium-coupled dopamine transporter (DAT) controls dopamine homeostasis, but its contribution to disease remains poorly understood. Here, we analyzed a cohort of patients with atypical movement disorder and identified 2 DAT coding variants, DAT-Ile312Phe and a presumed de novo mutant DAT-Asp421Asn, in an adult male with early-onset parkinsonism and ADHD. According to DAT single-photon emission computed tomography (DAT-SPECT) scans and a fluoro-deoxy-glucose-PET/MRI (FDG-PET/MRI) scan, the patient suffered from progressive dopaminergic neurodegeneration. In heterologous cells, both DAT variants exhibited markedly reduced dopamine uptake capacity but preserved membrane targeting, consistent with impaired catalytic activity. Computational simulations and uptake experiments suggested that the disrupted function of the DAT-Asp421Asn mutant is the result of compromised sodium binding, in agreement with Asp421 coordinating sodium at the second sodium site. For DAT-Asp421Asn, substrate efflux experiments revealed a constitutive, anomalous efflux of dopamine, and electrophysiological analyses identified a large cation leak that might further perturb dopaminergic neurotransmission. Our results link specific DAT missense mutations to neurodegenerative early-onset parkinsonism. Moreover, the neuropsychiatric comorbidity provides additional support for the idea that DAT missense mutations are an ADHD risk factor and suggests that complex DAT genotype and phenotype correlations contribute to different dopaminergic pathologies. PMID:24911152

Biphasic responses in multi-site phosphorylation systems.

PubMed

Suwanmajo, Thapanar; Krishnan, J

2013-12-06

Multi-site phosphorylation systems are repeatedly encountered in cellular biology and multi-site modification is a basic building block of post-translational modification. In this paper, we demonstrate how distributive multi-site modification mechanisms by a single kinase/phosphatase pair can lead to biphasic/partial biphasic dose-response characteristics for the maximally phosphorylated substrate at steady state. We use simulations and analysis to uncover a hidden competing effect which is responsible for this and analyse how it may be accentuated. We build on this to analyse different variants of multi-site phosphorylation mechanisms showing that some mechanisms are intrinsically not capable of displaying this behaviour. This provides both a consolidated understanding of how and under what conditions biphasic responses are obtained in multi-site phosphorylation and a basis for discriminating between different mechanisms based on this. We also demonstrate how this behaviour may be combined with other behaviour such as threshold and bistable responses, demonstrating the capacity of multi-site phosphorylation systems to act as complex molecular signal processors.

H3.3 demarcates GC-rich coding and subtelomeric regions and serves as potential memory mark for virulence gene expression in Plasmodium falciparum

PubMed Central

Fraschka, Sabine Anne-Kristin; Henderson, Rob Wilhelmus Maria; Bártfai, Richárd

2016-01-01

Histones, by packaging and organizing the DNA into chromatin, serve as essential building blocks for eukaryotic life. The basic structure of the chromatin is established by four canonical histones (H2A, H2B, H3 and H4), while histone variants are more commonly utilized to alter the properties of specific chromatin domains. H3.3, a variant of histone H3, was found to have diverse localization patterns and functions across species but has been rather poorly studied in protists. Here we present the first genome-wide analysis of H3.3 in the malaria-causing, apicomplexan parasite, P. falciparum, which revealed a complex occupancy profile consisting of conserved and parasite-specific features. In contrast to other histone variants, PfH3.3 primarily demarcates euchromatic coding and subtelomeric repetitive sequences. Stable occupancy of PfH3.3 in these regions is largely uncoupled from the transcriptional activity and appears to be primarily dependent on the GC-content of the underlying DNA. Importantly, PfH3.3 specifically marks the promoter region of an active and poised, but not inactive antigenic variation (var) gene, thereby potentially contributing to immune evasion. Collectively, our data suggest that PfH3.3, together with other histone variants, indexes the P. falciparum genome to functionally distinct domains and contribute to a key survival strategy of this deadly pathogen. PMID:27555062

High Throughput Engineering to Revitalize a Vestigial Electron Transfer Pathway in Bacterial Photosynthetic Reaction Centers*

PubMed Central

Faries, Kaitlyn M.; Kressel, Lucas L.; Wander, Marc J.; Holten, Dewey; Laible, Philip D.; Kirmaier, Christine; Hanson, Deborah K.

2012-01-01

Photosynthetic reaction centers convert light energy into chemical energy in a series of transmembrane electron transfer reactions, each with near 100% yield. The structures of reaction centers reveal two symmetry-related branches of cofactors (denoted A and B) that are functionally asymmetric; purple bacterial reaction centers use the A pathway exclusively. Previously, site-specific mutagenesis has yielded reaction centers capable of transmembrane charge separation solely via the B branch cofactors, but the best overall electron transfer yields are still low. In an attempt to better realize the architectural and energetic factors that underlie the directionality and yields of electron transfer, sites within the protein-cofactor complex were targeted in a directed molecular evolution strategy that implements streamlined mutagenesis and high throughput spectroscopic screening. The polycistronic approach enables efficient construction and expression of a large number of variants of a heteroligomeric complex that has two intimately regulated subunits with high sequence similarity, common features of many prokaryotic and eukaryotic transmembrane protein assemblies. The strategy has succeeded in the discovery of several mutant reaction centers with increased efficiency of the B pathway; they carry multiple substitutions that have not been explored or linked using traditional approaches. This work expands our understanding of the structure-function relationships that dictate the efficiency of biological energy-conversion reactions, concepts that will aid the design of bio-inspired assemblies capable of both efficient charge separation and charge stabilization. PMID:22247556

Crystallization of recombinant cyclo-oxygenase-2

NASA Astrophysics Data System (ADS)

Stevens, Anna M.; Pawlitz, Jennifer L.; Kurumbail, Ravi G.; Gierse, James K.; Moreland, Kirby T.; Stegeman, Roderick A.; Loduca, Jina Y.; Stallings, William C.

1999-01-01

The integral membrane protein, prostaglandin H 2 synthase, or cyclo-oxygenase (COX), catalyses the first step in the conversion of arachidonic acid to prostaglandins (PGs) and is the target of nonsteroidal anti-inflammatory drugs (NSAIDs). Two isoforms are known. The constitutive enzyme, COX-1, is present in most tissues and is responsible for the physiological production of PGs. The isoform responsible for the elevated production of PGs during inflammation is COX-2 which is induced specifically at inflammatory sites. Three-dimensional structures of inhibitor complexes of COX-2, and of site variants of COX-2 which mimic COX-1, provide insight into the structural basis for selective inhibition of COX-2. Additionally, structures of COX-2 mutants and complexes with the substrate can provide a clearer understanding of the catalytic mechanism of the reaction. A crystallization protocol has been developed for COX-2 which reproducibly yields diffraction quality crystals. Polyethyleneglycol 550 monomethylether (MMP550) and MgCl 2 were systematically varied and used in conjunction with the detergent β- D-octylglucopyranoside ( β-OG). As a result of many crystallization trials, we determined that the initial β-OG concentration should be held constant, allowing the salt concentration to modulate the critical micelle concentration (CMC) of the detergent. Over 25 crystal structures have been solved using crystals generated from this system. Most crystals belong to the space group P2 12 12, with lattice constants of a=180, b=134, c=120 Å in a pseudo body-centered lattice.

Whole-genome sequencing identifies EN1 as a determinant of bone density and fracture

PubMed Central

Zheng, Hou-Feng; Forgetta, Vincenzo; Hsu, Yi-Hsiang; Estrada, Karol; Rosello-Diez, Alberto; Leo, Paul J; Dahia, Chitra L; Park-Min, Kyung Hyun; Tobias, Jonathan H; Kooperberg, Charles; Kleinman, Aaron; Styrkarsdottir, Unnur; Liu, Ching-Ti; Uggla, Charlotta; Evans, Daniel S; Nielson, Carrie M; Walter, Klaudia; Pettersson-Kymmer, Ulrika; McCarthy, Shane; Eriksson, Joel; Kwan, Tony; Jhamai, Mila; Trajanoska, Katerina; Memari, Yasin; Min, Josine; Huang, Jie; Danecek, Petr; Wilmot, Beth; Li, Rui; Chou, Wen-Chi; Mokry, Lauren E; Moayyeri, Alireza; Claussnitzer, Melina; Cheng, Chia-Ho; Cheung, Warren; Medina-Gómez, Carolina; Ge, Bing; Chen, Shu-Huang; Choi, Kwangbom; Oei, Ling; Fraser, James; Kraaij, Robert; Hibbs, Matthew A; Gregson, Celia L; Paquette, Denis; Hofman, Albert; Wibom, Carl; Tranah, Gregory J; Marshall, Mhairi; Gardiner, Brooke B; Cremin, Katie; Auer, Paul; Hsu, Li; Ring, Sue; Tung, Joyce Y; Thorleifsson, Gudmar; Enneman, Anke W; van Schoor, Natasja M; de Groot, Lisette C.P.G.M.; van der Velde, Nathalie; Melin, Beatrice; Kemp, John P; Christiansen, Claus; Sayers, Adrian; Zhou, Yanhua; Calderari, Sophie; van Rooij, Jeroen; Carlson, Chris; Peters, Ulrike; Berlivet, Soizik; Dostie, Josée; Uitterlinden, Andre G; Williams, Stephen R.; Farber, Charles; Grinberg, Daniel; LaCroix, Andrea Z; Haessler, Jeff; Chasman, Daniel I; Giulianini, Franco; Rose, Lynda M; Ridker, Paul M; Eisman, John A; Nguyen, Tuan V; Center, Jacqueline R; Nogues, Xavier; Garcia-Giralt, Natalia; Launer, Lenore L; Gudnason, Vilmunder; Mellström, Dan; Vandenput, Liesbeth; Karlsson, Magnus K; Ljunggren, Östen; Svensson, Olle; Hallmans, Göran; Rousseau, François; Giroux, Sylvie; Bussière, Johanne; Arp, Pascal P; Koromani, Fjorda; Prince, Richard L; Lewis, Joshua R; Langdahl, Bente L; Hermann, A Pernille; Jensen, Jens-Erik B; Kaptoge, Stephen; Khaw, Kay-Tee; Reeve, Jonathan; Formosa, Melissa M; Xuereb-Anastasi, Angela; Åkesson, Kristina; McGuigan, Fiona E; Garg, Gaurav; Olmos, Jose M; Zarrabeitia, Maria T; Riancho, Jose A; Ralston, Stuart H; Alonso, Nerea; Jiang, Xi; Goltzman, David; Pastinen, Tomi; Grundberg, Elin; Gauguier, Dominique; Orwoll, Eric S; Karasik, David; Davey-Smith, George; Smith, Albert V; Siggeirsdottir, Kristin; Harris, Tamara B; Zillikens, M Carola; van Meurs, Joyce BJ; Thorsteinsdottir, Unnur; Maurano, Matthew T; Timpson, Nicholas J; Soranzo, Nicole; Durbin, Richard; Wilson, Scott G; Ntzani, Evangelia E; Brown, Matthew A; Stefansson, Kari; Hinds, David A; Spector, Tim; Cupples, L Adrienne; Ohlsson, Claes; Greenwood, Celia MT; Jackson, Rebecca D; Rowe, David W; Loomis, Cynthia A; Evans, David M; Ackert-Bicknell, Cheryl L; Joyner, Alexandra L; Duncan, Emma L; Kiel, Douglas P; Rivadeneira, Fernando; Richards, J Brent

2016-01-01

SUMMARY The extent to which low-frequency (minor allele frequency [MAF] between 1–5%) and rare (MAF ≤ 1%) variants contribute to complex traits and disease in the general population is largely unknown. Bone mineral density (BMD) is highly heritable, is a major predictor of osteoporotic fractures and has been previously associated with common genetic variants1–8, and rare, population-specific, coding variants9. Here we identify novel non-coding genetic variants with large effects on BMD (ntotal = 53,236) and fracture (ntotal = 508,253) in individuals of European ancestry from the general population. Associations for BMD were derived from whole-genome sequencing (n=2,882 from UK10K), whole-exome sequencing (n= 3,549), deep imputation of genotyped samples using a combined UK10K/1000Genomes reference panel (n=26,534), and de-novo replication genotyping (n= 20,271). We identified a low-frequency non-coding variant near a novel locus, EN1, with an effect size 4-fold larger than the mean of previously reported common variants for lumbar spine BMD8 (rs11692564[T], MAF = 1.7%, replication effect size = +0.20 standard deviations [SD], Pmeta = 2×10−14), which was also associated with a decreased risk of fracture (OR = 0.85; P = 2×10−11; ncases = 98,742 and ncontrols = 409,511). Using an En1Cre/flox mouse model, we observed that conditional loss of En1 results in low bone mass, likely as a consequence of high bone turn-over. We also identified a novel low-frequency non-coding variant with large effects on BMD near WNT16 (rs148771817[T], MAF = 1.1%, replication effect size = +0.39 SD, Pmeta = 1×10−11). In general, there was an excess of association signals arising from deleterious coding and conserved non-coding variants. These findings provide evidence that low-frequency non-coding variants have large effects on BMD and fracture, thereby providing rationale for whole-genome sequencing and improved imputation reference panels to study the genetic architecture of complex traits and disease in the general population. PMID:26367794

Twelve novel HGD gene variants identified in 99 alkaptonuria patients: focus on 'black bone disease' in Italy.

PubMed

Nemethova, Martina; Radvanszky, Jan; Kadasi, Ludevit; Ascher, David B; Pires, Douglas E V; Blundell, Tom L; Porfirio, Berardino; Mannoni, Alessandro; Santucci, Annalisa; Milucci, Lia; Sestini, Silvia; Biolcati, Gianfranco; Sorge, Fiammetta; Aurizi, Caterina; Aquaron, Robert; Alsbou, Mohammed; Lourenço, Charles Marques; Ramadevi, Kanakasabapathi; Ranganath, Lakshminarayan R; Gallagher, James A; van Kan, Christa; Hall, Anthony K; Olsson, Birgitta; Sireau, Nicolas; Ayoob, Hana; Timmis, Oliver G; Sang, Kim-Hanh Le Quan; Genovese, Federica; Imrich, Richard; Rovensky, Jozef; Srinivasaraghavan, Rangan; Bharadwaj, Shruthi K; Spiegel, Ronen; Zatkova, Andrea

2016-01-01

Alkaptonuria (AKU) is an autosomal recessive disorder caused by mutations in homogentisate-1,2-dioxygenase (HGD) gene leading to the deficiency of HGD enzyme activity. The DevelopAKUre project is underway to test nitisinone as a specific treatment to counteract this derangement of the phenylalanine-tyrosine catabolic pathway. We analysed DNA of 40 AKU patients enrolled for SONIA1, the first study in DevelopAKUre, and of 59 other AKU patients sent to our laboratory for molecular diagnostics. We identified 12 novel DNA variants: one was identified in patients from Brazil (c.557T>A), Slovakia (c.500C>T) and France (c.440T>C), three in patients from India (c.469+6T>C, c.650-85A>G, c.158G>A), and six in patients from Italy (c.742A>G, c.614G>A, c.1057A>C, c.752G>A, c.119A>C, c.926G>T). Thus, the total number of potential AKU-causing variants found in 380 patients reported in the HGD mutation database is now 129. Using mCSM and DUET, computational approaches based on the protein 3D structure, the novel missense variants are predicted to affect the activity of the enzyme by three mechanisms: decrease of stability of individual protomers, disruption of protomer-protomer interactions or modification of residues in the region of the active site. We also present an overview of AKU in Italy, where so far about 60 AKU cases are known and DNA analysis has been reported for 34 of them. In this rather small group, 26 different HGD variants affecting function were described, indicating rather high heterogeneity. Twelve of these variants seem to be specific for Italy.

Twelve novel HGD gene variants identified in 99 alkaptonuria patients: focus on ‘black bone disease' in Italy

PubMed Central

Nemethova, Martina; Radvanszky, Jan; Kadasi, Ludevit; Ascher, David B; Pires, Douglas E V; Blundell, Tom L; Porfirio, Berardino; Mannoni, Alessandro; Santucci, Annalisa; Milucci, Lia; Sestini, Silvia; Biolcati, Gianfranco; Sorge, Fiammetta; Aurizi, Caterina; Aquaron, Robert; Alsbou, Mohammed; Marques Lourenço, Charles; Ramadevi, Kanakasabapathi; Ranganath, Lakshminarayan R; Gallagher, James A; van Kan, Christa; Hall, Anthony K; Olsson, Birgitta; Sireau, Nicolas; Ayoob, Hana; Timmis, Oliver G; Le Quan Sang, Kim-Hanh; Genovese, Federica; Imrich, Richard; Rovensky, Jozef; Srinivasaraghavan, Rangan; Bharadwaj, Shruthi K; Spiegel, Ronen; Zatkova, Andrea

2016-01-01

Alkaptonuria (AKU) is an autosomal recessive disorder caused by mutations in homogentisate-1,2-dioxygenase (HGD) gene leading to the deficiency of HGD enzyme activity. The DevelopAKUre project is underway to test nitisinone as a specific treatment to counteract this derangement of the phenylalanine-tyrosine catabolic pathway. We analysed DNA of 40 AKU patients enrolled for SONIA1, the first study in DevelopAKUre, and of 59 other AKU patients sent to our laboratory for molecular diagnostics. We identified 12 novel DNA variants: one was identified in patients from Brazil (c.557T>A), Slovakia (c.500C>T) and France (c.440T>C), three in patients from India (c.469+6T>C, c.650–85A>G, c.158G>A), and six in patients from Italy (c.742A>G, c.614G>A, c.1057A>C, c.752G>A, c.119A>C, c.926G>T). Thus, the total number of potential AKU-causing variants found in 380 patients reported in the HGD mutation database is now 129. Using mCSM and DUET, computational approaches based on the protein 3D structure, the novel missense variants are predicted to affect the activity of the enzyme by three mechanisms: decrease of stability of individual protomers, disruption of protomer-protomer interactions or modification of residues in the region of the active site. We also present an overview of AKU in Italy, where so far about 60 AKU cases are known and DNA analysis has been reported for 34 of them. In this rather small group, 26 different HGD variants affecting function were described, indicating rather high heterogeneity. Twelve of these variants seem to be specific for Italy. PMID:25804398

Genomic analysis identifies masqueraders of full-term cerebral palsy.

PubMed

Takezawa, Yusuke; Kikuchi, Atsuo; Haginoya, Kazuhiro; Niihori, Tetsuya; Numata-Uematsu, Yurika; Inui, Takehiko; Yamamura-Suzuki, Saeko; Miyabayashi, Takuya; Anzai, Mai; Suzuki-Muromoto, Sato; Okubo, Yukimune; Endo, Wakaba; Togashi, Noriko; Kobayashi, Yasuko; Onuma, Akira; Funayama, Ryo; Shirota, Matsuyuki; Nakayama, Keiko; Aoki, Yoko; Kure, Shigeo

2018-05-01

Cerebral palsy is a common, heterogeneous neurodevelopmental disorder that causes movement and postural disabilities. Recent studies have suggested genetic diseases can be misdiagnosed as cerebral palsy. We hypothesized that two simple criteria, that is, full-term births and nonspecific brain MRI findings, are keys to extracting masqueraders among cerebral palsy cases due to the following: (1) preterm infants are susceptible to multiple environmental factors and therefore demonstrate an increased risk of cerebral palsy and (2) brain MRI assessment is essential for excluding environmental causes and other particular disorders. A total of 107 patients-all full-term births-without specific findings on brain MRI were identified among 897 patients diagnosed with cerebral palsy who were followed at our center. DNA samples were available for 17 of the 107 cases for trio whole-exome sequencing and array comparative genomic hybridization. We prioritized variants in genes known to be relevant in neurodevelopmental diseases and evaluated their pathogenicity according to the American College of Medical Genetics guidelines. Pathogenic/likely pathogenic candidate variants were identified in 9 of 17 cases (52.9%) within eight genes: CTNNB1 , CYP2U1 , SPAST , GNAO1 , CACNA1A , AMPD2 , STXBP1 , and SCN2A . Five identified variants had previously been reported. No pathogenic copy number variations were identified. The AMPD2 missense variant and the splice-site variants in CTNNB1 and AMPD2 were validated by in vitro functional experiments. The high rate of detecting causative genetic variants (52.9%) suggests that patients diagnosed with cerebral palsy in full-term births without specific MRI findings may include genetic diseases masquerading as cerebral palsy.

Multiple splicing events involved in regulation of human aromatase expression by a novel promoter, I.6.

PubMed

Shozu, M; Zhao, Y; Bulun, S E; Simpson, E R

1998-04-01

The expression of aromatase is regulated in a tissue-specific fashion through alternative use of multiple promoter-specific first exons. To date, eight different first exons have been reported in human aromatase, namely I.1., I.2, I.3. I.4, I.5, PII, 2a, and 1f. Recently, we have found a new putative exon I in a RACE-generated library of THP-1 cells and have conducted studies to characterize this new exon I. We confirmed that the constructs containing -1552/+17 or less flanking sequence of this exon function as a promoter in THP-1 cells, JEG-3 cells and osteoblast-like cells obtained from a human fetus. Results of transfection assays using a series of deletion constructs and mutation constructs indicate that a 1-bp mismatch of the consensus TATA-like box (TTTAAT) and the consensus sequence of the initiator site, which is located 45 bp downstream of the putative TATA box, were functioning cooperatively as a core promoter. The putative transcription site was confirmed by the results of RT-PCR southern blot analysis. We examined the regulation and the expression of this exon, I.6, in several human cells and tissues by RT-PCR Southern blot analysis. THP-1 cells (mononuclear leukemic origin) and JEG-3 cells (choriocarcinoma origin) expressed exon I.6 in serum-free media. The level of expression was increased by serum and phorbol myristyl acetate (PMA) in both cell lines. Adipose stromal cells also expressed exon I.6 in the presence of PMA. In fetal osteoblasts, the expression of exon I.6 was increased most effectively by serum and less so by dexamethasone (DEX) + IL-1beta and DEX + IL-11, whereas induction by serum was suppressed by the addition of DEX. The level of expression was low in granulosa cells in culture and did not change with forskolin. On the other hand, dibutyryl cAMP suppressed PMA-stimulated expression of exon I.6 in THP-1 cells and adipose stromal cells. This result supports the hypothesis that the expression of exon I.6 is regulated mainly via an AP-1 binding site that is found upstream of the initiator site of the promoter region. Expression of exon I.6-specific transcripts was examined in several human tissues. Testis and bone obtained from normal adults expressed exon I.6. Testicular tumor and hepatic carcinoma expressed high levels of exon I.6, whereas granulosa cell tumor did not. Fetal liver and bone also showed a significant level of exon I.6 expression, but not so much as testicular tumor and hepatic tumor. Several splicing variants of exon I.6 were detected especially in THP-1 and JEG-3 cells, and to a lesser extent in primary cultures and tissue samples. These variants were identified as an unspliced form, a form spliced at the end of exon I.4, a form spliced at the end of exon I.3 (truncated) and a form spliced 220 bp downstream of the 3' end of exon I.6. The last variant revealed a new splicing site. Because most of the splicing variants contain the sequence specific for exon I.3, RT-PCR specific for exon I.3 can coamplify these splicing variants of exon I.6 transcripts. These results suggests that it is necessary to examine the expression of I.6 in tissues that are known to express exon I.3 such as breast adipose tissue, in which promoter usage of exon I of the aromatase gene switches from exon I.4 to I.3 in the course of malignant transformation.

HaploReg v4: systematic mining of putative causal variants, cell types, regulators and target genes for human complex traits and disease.

PubMed

Ward, Lucas D; Kellis, Manolis

2016-01-04

More than 90% of common variants associated with complex traits do not affect proteins directly, but instead the circuits that control gene expression. This has increased the urgency of understanding the regulatory genome as a key component for translating genetic results into mechanistic insights and ultimately therapeutics. To address this challenge, we developed HaploReg (http://compbio.mit.edu/HaploReg) to aid the functional dissection of genome-wide association study (GWAS) results, the prediction of putative causal variants in haplotype blocks, the prediction of likely cell types of action, and the prediction of candidate target genes by systematic mining of comparative, epigenomic and regulatory annotations. Since first launching the website in 2011, we have greatly expanded HaploReg, increasing the number of chromatin state maps to 127 reference epigenomes from ENCODE 2012 and Roadmap Epigenomics, incorporating regulator binding data, expanding regulatory motif disruption annotations, and integrating expression quantitative trait locus (eQTL) variants and their tissue-specific target genes from GTEx, Geuvadis, and other recent studies. We present these updates as HaploReg v4, and illustrate a use case of HaploReg for attention deficit hyperactivity disorder (ADHD)-associated SNPs with putative brain regulatory mechanisms. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Molecular Basis for the Catalytic Specificity of the CTX-M Extended-Spectrum β-Lactamases

DOE PAGES

Adamski, Carolyn J.; Cardenas, Ana Maria; Brown, Nicholas G.; ...

2014-12-09

We report that extended-spectrum β-lactamases (ESBLs) pose a threat to public health because of their ability to confer resistance to extended-spectrum cephalosporins such as cefotaxime. The CTX-M β-lactamases are the most widespread ESBL enzymes among antibiotic resistant bacteria. Many of the active site residues are conserved between the CTX-M family and non-ESBL β-lactamases such as TEM-1, but the residues Ser237 and Arg276 are specific to the CTX-M family, suggesting that they may help to define the increased specificity for cefotaxime hydrolysis. To test this hypothesis, site-directed mutagenesis of these positions was performed in the CTX-M-14 β-lactamase. Substitutions of Ser237 andmore » Arg276 with their TEM-1 counterparts, Ala237 and Asn276, had a modest effect on cefotaxime hydrolysis, as did removal of the Arg276 side chain in an R276A mutant. The S237A:R276N and S237A:R276A double mutants, however, exhibited 29- and 14-fold losses in catalytic efficiency for cefotaxime hydrolysis, respectively, while the catalytic efficiency for benzylpenicillin hydrolysis was unchanged. Therefore, together, the Ser237 and Arg276 residues are important contributors to the cefotaximase substrate profile of the enzyme. High-resolution crystal structures of the CTX-M-14 S70G, S70G:S237A, and S70G:S237A:R276A variants alone and in complex with cefotaxime show that residues Ser237 and Arg276 in the wild-type enzyme promote the expansion of the active site to accommodate cefotaxime and favor a conformation of cefotaxime that allows optimal contacts between the enzyme and substrate. In conclusion, the conservation of these residues, linked to their effects on structure and catalysis, imply that their coevolution is an important specificity determinant in the CTX-M family.« less

Structure-based non-canonical amino acid design to covalently crosslink an antibody–antigen complex

PubMed Central

Xu, Jianqing; Tack, Drew; Hughes, Randall A.; Ellington, Andrew D.; Gray, Jeffrey J.

2014-01-01

Engineering antibodies to utilize non-canonical amino acids (NCAA) should greatly expand the utility of an already important biological reagent. In particular, introducing crosslinking reagents into antibody complementarity determining regions (CDRs) should provide a means to covalently crosslink residues at the antibody–antigen interface. Unfortunately, finding the optimum position for crosslinking two proteins is often a matter of iterative guessing, even when the interface is known in atomic detail. Computer-aided antibody design can potentially greatly restrict the number of variants that must be explored in order to identify successful crosslinking sites. We have therefore used Rosetta to guide the introduction of an oxidizable crosslinking NCAA, l-3,4-dihydroxyphenylalanine (l-DOPA), into the CDRs of the anti-protective antigen scFv antibody M18, and have measured crosslinking to its cognate antigen, domain 4 of the anthrax protective antigen. Computed crosslinking distance, solvent accessibility, and interface energetics were three factors considered that could impact the efficiency of l-DOPA-mediated crosslinking. In the end, 10 variants were synthesized, and crosslinking efficiencies were generally 10% or higher, with the best variant crosslinking to 52% of the available antigen. The results suggest that computational analysis can be used in a pipeline for engineering crosslinking antibodies. The rules learned from l-DOPA crosslinking of antibodies may also be generalizable to the formation of other crosslinked interfaces and complexes. PMID:23680795

Modulatory mechanisms and multiple functions of somatodendritic A-type K+ channel auxiliary subunits

PubMed Central

Jerng, Henry H.; Pfaffinger, Paul J.

2014-01-01

Auxiliary subunits are non-conducting, modulatory components of the multi-protein ion channel complexes that underlie normal neuronal signaling. They interact with the pore-forming α-subunits to modulate surface distribution, ion conductance, and channel gating properties. For the somatodendritic subthreshold A-type potassium (ISA) channel based on Kv4 α-subunits, two types of auxiliary subunits have been extensively studied: Kv channel-interacting proteins (KChIPs) and dipeptidyl peptidase-like proteins (DPLPs). KChIPs are cytoplasmic calcium-binding proteins that interact with intracellular portions of the Kv4 subunits, whereas DPLPs are type II transmembrane proteins that associate with the Kv4 channel core. Both KChIPs and DPLPs genes contain multiple start sites that are used by various neuronal populations to drive the differential expression of functionally distinct N-terminal variants. In turn, these N-terminal variants generate tremendous functional diversity across the nervous system. Here, we focus our review on (1) the molecular mechanism underlying the unique properties of different N-terminal variants, (2) the shaping of native ISA properties by the concerted actions of KChIPs and DPLP variants, and (3) the surprising ways that KChIPs and DPLPs coordinate the activity of multiple channels to fine-tune neuronal excitability. Unlocking the unique contributions of different auxiliary subunit N-terminal variants may provide an important opportunity to develop novel targeted therapeutics to treat numerous neurological disorders. PMID:24723849

Modulatory mechanisms and multiple functions of somatodendritic A-type K (+) channel auxiliary subunits.

PubMed

Jerng, Henry H; Pfaffinger, Paul J

2014-01-01

Auxiliary subunits are non-conducting, modulatory components of the multi-protein ion channel complexes that underlie normal neuronal signaling. They interact with the pore-forming α-subunits to modulate surface distribution, ion conductance, and channel gating properties. For the somatodendritic subthreshold A-type potassium (ISA) channel based on Kv4 α-subunits, two types of auxiliary subunits have been extensively studied: Kv channel-interacting proteins (KChIPs) and dipeptidyl peptidase-like proteins (DPLPs). KChIPs are cytoplasmic calcium-binding proteins that interact with intracellular portions of the Kv4 subunits, whereas DPLPs are type II transmembrane proteins that associate with the Kv4 channel core. Both KChIPs and DPLPs genes contain multiple start sites that are used by various neuronal populations to drive the differential expression of functionally distinct N-terminal variants. In turn, these N-terminal variants generate tremendous functional diversity across the nervous system. Here, we focus our review on (1) the molecular mechanism underlying the unique properties of different N-terminal variants, (2) the shaping of native ISA properties by the concerted actions of KChIPs and DPLP variants, and (3) the surprising ways that KChIPs and DPLPs coordinate the activity of multiple channels to fine-tune neuronal excitability. Unlocking the unique contributions of different auxiliary subunit N-terminal variants may provide an important opportunity to develop novel targeted therapeutics to treat numerous neurological disorders.

Molecular evolution of HIV-1 integrase during the 20 years prior to the first approval of integrase inhibitors.

PubMed

Meixenberger, Karolin; Yousef, Kaveh Pouran; Smith, Maureen Rebecca; Somogyi, Sybille; Fiedler, Stefan; Bartmeyer, Barbara; Hamouda, Osamah; Bannert, Norbert; von Kleist, Max; Kücherer, Claudia

2017-11-14

Detailed knowledge of the evolutionary potential of polymorphic sites in a viral protein is important for understanding the development of drug resistance in the presence of an inhibitor. We therefore set out to analyse the molecular evolution of the HIV-1 subtype B integrase at the inter-patient level in Germany during a 20-year period prior to the first introduction of integrase strand inhibitors (INSTIs). We determined 337 HIV-1 integrase subtype B sequences (amino acids 1-278) from stored plasma samples of antiretroviral treatment-naïve individuals newly diagnosed with HIV-1 between 1986 and 2006. Shannon entropy was calculated to determine the variability at each amino acid position. Time trends in the frequency of amino acid variants were identified by linear regression. Direct coupling analysis was applied to detect covarying sites. Twenty-two time trends in the frequency of amino acid variants demonstrated either single amino acid exchanges or variation in the degree of polymorphy. Covariation was observed for 17 amino acid variants with a temporal trend. Some minor INSTI resistance mutations (T124A, V151I, K156 N, T206S, S230 N) and some INSTI-selected mutations (M50I, L101I, T122I, T124 N, T125A, M154I, G193E, V201I) were identified at overall frequencies >5%. Among these, the frequencies of L101I, T122I, and V201I increased over time, whereas the frequency of M154I decreased. Moreover, L101I, T122I, T124A, T125A, M154I, and V201I covaried with non-resistance-associated variants. Time-trending, covarying polymorphisms indicate that long-term evolutionary changes of the HIV-1 integrase involve defined clusters of possibly structurally or functionally associated sites independent of selective pressure through INSTIs at the inter-patient level. Linkage between polymorphic resistance- and non-resistance-associated sites can impact the selection of INSTI resistance mutations in complex ways. Identification of these sites can help in improving genotypic resistance assays, resistance prediction algorithms, and the development of new integrase inhibitors.

A variational Bayes discrete mixture test for rare variant association

PubMed Central

Logsdon, Benjamin A.; Dai, James Y.; Auer, Paul L.; Johnsen, Jill M.; Ganesh, Santhi K.; Smith, Nicholas L.; Wilson, James G.; Tracy, Russell P.; Lange, Leslie A.; Jiao, Shuo; Rich, Stephen S.; Lettre, Guillaume; Carlson, Christopher S.; Jackson, Rebecca D.; O’Donnell, Christopher J.; Wurfel, Mark M.; Nickerson, Deborah A.; Tang, Hua; Reiner, Alexander P.; Kooperberg, Charles

2014-01-01

Recently, many statistical methods have been proposed to test for associations between rare genetic variants and complex traits. Most of these methods test for association by aggregating genetic variations within a predefined region, such as a gene. Although there is evidence that “aggregate” tests are more powerful than the single marker test, these tests generally ignore neutral variants and therefore are unable to identify specific variants driving the association with phenotype. We propose a novel aggregate rare-variant test that explicitly models a fraction of variants as neutral, tests associations at the gene-level, and infers the rare-variants driving the association. Simulations show that in the practical scenario where there are many variants within a given region of the genome with only a fraction causal our approach has greater power compared to other popular tests such as the Sequence Kernel Association Test (SKAT), the Weighted Sum Statistic (WSS), and the collapsing method of Morris and Zeggini (MZ). Our algorithm leverages a fast variational Bayes approximate inference methodology to scale to exome-wide analyses, a significant computational advantage over exact inference model selection methodologies. To demonstrate the efficacy of our methodology we test for associations between von Willebrand Factor (VWF) levels and VWF missense rare-variants imputed from the National Heart, Lung, and Blood Institute’s Exome Sequencing project into 2,487 African Americans within the VWF gene. Our method suggests that a relatively small fraction (~10%) of the imputed rare missense variants within VWF are strongly associated with lower VWF levels in African Americans. PMID:24482836

Establishment of Sf9 Transformants Constitutively Expressing PBAN Receptor Variants: Application to Functional Evaluation

PubMed Central

Lee, Jae Min; Hull, J. Joe; Kawai, Takeshi; Tsuneizumi, Kazuhide; Kurihara, Masaaki; Tanokura, Masaru; Nagata, Koji; Nagasawa, Hiromichi; Matsumoto, Shogo

2012-01-01

To facilitate further evaluation of pheromone biosynthesis activating neuropeptide receptor (PBANR) functionality and regulation, we generated cultured insect cell lines constitutively expressing green fluorescent protein chimeras of the recently identified Bombyx mori PBANR (BommoPBANR) and Pseudaletia separata PBANR (PsesePBANR) variants. Fluorescent chimeras included the BommoPBANR-A, -B, and -C variants and the PsesePBANR-B and -C variants. Cell lines expressing non-chimeric BommoPBANR-B and -C variants were also generated. Functional evaluation of these transformed cell lines using confocal laser microscopy revealed that a Rhodamine Red-labeled PBAN derivative (RR-C10PBANR2K) specifically co-localized with all of the respective PBANR variants at the plasma membrane. Near complete internalization of the fluorescent RR-C10PBANR2K ligand 30 min after binding was observed in all cell lines except those expressing the BommoPBANR-A variant, in which the ligand/receptor complex remained at the plasma membrane. Fluorescent Ca2+ imaging further showed that the BommoPBANR-A cell line exhibited drastically different Ca2+ mobilization kinetics at a number of RR-C10PBANR2K concentrations including 10 μM. These observations demonstrate a clear functional difference between the BommoPBANR-A variant and the BommoPBANR-B and -C variants in terms of receptor regulation and activation of downstream effector molecules. We also found that, contrary to previous reports, ligand-induced internalization of BommoPBANR-B and BommoPBANR-C in cell lines stably expressing these variants occurred in the absence of extracellular Ca2+. PMID:22654874

A variational Bayes discrete mixture test for rare variant association.

PubMed

Logsdon, Benjamin A; Dai, James Y; Auer, Paul L; Johnsen, Jill M; Ganesh, Santhi K; Smith, Nicholas L; Wilson, James G; Tracy, Russell P; Lange, Leslie A; Jiao, Shuo; Rich, Stephen S; Lettre, Guillaume; Carlson, Christopher S; Jackson, Rebecca D; O'Donnell, Christopher J; Wurfel, Mark M; Nickerson, Deborah A; Tang, Hua; Reiner, Alexander P; Kooperberg, Charles

2014-01-01

Recently, many statistical methods have been proposed to test for associations between rare genetic variants and complex traits. Most of these methods test for association by aggregating genetic variations within a predefined region, such as a gene. Although there is evidence that "aggregate" tests are more powerful than the single marker test, these tests generally ignore neutral variants and therefore are unable to identify specific variants driving the association with phenotype. We propose a novel aggregate rare-variant test that explicitly models a fraction of variants as neutral, tests associations at the gene-level, and infers the rare-variants driving the association. Simulations show that in the practical scenario where there are many variants within a given region of the genome with only a fraction causal our approach has greater power compared to other popular tests such as the Sequence Kernel Association Test (SKAT), the Weighted Sum Statistic (WSS), and the collapsing method of Morris and Zeggini (MZ). Our algorithm leverages a fast variational Bayes approximate inference methodology to scale to exome-wide analyses, a significant computational advantage over exact inference model selection methodologies. To demonstrate the efficacy of our methodology we test for associations between von Willebrand Factor (VWF) levels and VWF missense rare-variants imputed from the National Heart, Lung, and Blood Institute's Exome Sequencing project into 2,487 African Americans within the VWF gene. Our method suggests that a relatively small fraction (~10%) of the imputed rare missense variants within VWF are strongly associated with lower VWF levels in African Americans.

Structure of Mandelate Racemase with Bound Intermediate Analogues Benzohydroxamate and Cupferron†

PubMed Central

Lietzan, Adam D.; Nagar, Mitesh; Pellmann, Elise A.; Bourque, Jennifer R.; Bearne, Stephen L.; St Maurice, Martin

2012-01-01

Mandelate racemase (MR, EC 5.1.2.2) from Pseudomonas putida catalyzes the Mg2+-dependent interconversion of the enantiomers of mandelate, stabilizing the altered substrate in the transition state by 26 kcal/mol relative to the substrate in the ground state. To understand the origins of this binding discrimination, we solved the X-ray crystal structures of wild-type MR complexed with two analogues of the putative aci-carboxylate intermediate, benzohydroxamate and cupferron, to 2.2-Å resolution. Benzohydroxamate is shown to be a reasonable mimic of the transition state/intermediate since its binding affinity to 21 MR variants correlates well with changes in the free energy of transition state stabilization afforded by these variants. Both benzohydroxamate and cupferron chelate the active site divalent metal ion and are bound in a conformation with the phenyl ring coplanar with the hydroxamate and diazeniumdiolate moieties, respectively. Structural overlays of MR complexed with benzohydroxamate, cupferron, and the ground state analogue (S)-atrolacatate reveal that the para-carbon of the substrate phenyl ring moves by 0.8–1.2 Å between the ground state and intermediate state, consistent with the proposal that the phenyl ring moves during MR catalysis while the polar groups remain relatively fixed. Although the overall protein structure of MR with bound intermediate analogues is very similar to MR with bound (S)-atrolactate, the intermediate-Mg2+ distance shortens, suggesting a tighter complex with the catalytic Mg2+. In addition, Tyr 54 moves nearer to the phenyl ring of the bound intermediate analogues, contributing to an overall constriction of the active site cavity. However, site-directed mutagenesis experiments revealed that the role of Tyr 54 in MR catalysis is relatively minor, suggesting that alterations in enzyme structure that contribute to discrimination between the altered substrate in the transition state and the ground state by this proficient enzyme are extremely subtle. PMID:22264153

Thermophilic Enzyme or Mesophilic Enzyme with Enhanced Thermostability: Can We Draw a Line?

PubMed

Jing, Xiaomin; Evangelista Falcon, Wilfredo; Baudry, Jerome; Serpersu, Engin H

2017-07-27

Aminoglycoside nucleotidyltransferase 4' (ANT) is a homodimeric enzyme that modifies the C4'-OH site of aminoglycoside antibiotics by nucleotidylation. A few single- and double-residue mutants of this enzyme (T130K, D80Y, and D80Y/T130K) from Bacillus stearothermophilus show increased thermostability. This article investigates how such residue replacements, which are distant from the active site and monomer-monomer interface, result in various changes of the thermostability of the enzyme. In this work, we show that the thermodynamic properties of enzyme-ligand complexes and protein dynamics may be indicators of a thermophilic behavior. Our data suggests that one of the single-site mutants of ANT, D80Y, may be a thermophilic protein and the other thermostable mutant, T130K, is actually a more heat-stable variant of the mesophilic wild type (WT) with a higher T m . Our data also suggest that T130K and D80Y adopt different global dynamics strategies to achieve different levels of thermostability enhancement and that the differences between the properties of the species can be described in terms of global dynamics rather than in terms of specific structural features. Thermophilicity of the D80Y comes at the cost of less favorable thermodynamic parameters for ligand binding relative to WT. On the other hand, the T130K species exhibits the same affinity to ligands and the same thermodynamic parameters of complex formation as the WT enzyme. These observations suggest that a quantitative characterization of ligand binding and protein dynamics can be used to differentiate thermophilic proteins from their simply more heat-stable mesophilic counterparts.

Unraveling the Molecular Complexity of O-Glycosylated Endogenous (N-Terminal) pro-B-Type Natriuretic Peptide Forms in Blood Plasma of Patients with Severe Heart Failure.

PubMed

Halfinger, Bernhard; Hammerer-Lercher, Angelika; Amplatz, Benno; Sarg, Bettina; Kremser, Leopold; Lindner, Herbert H

2017-01-01

Currently, N-terminal pro-B-type natriuretic peptide (NT-proBNP) and its physiologically active counterpart, BNP, are most frequently used as biomarkers for diagnosis, prognosis, and disease monitoring of heart failure (HF). Commercial NT-proBNP and BNP immunoassays cross-react to varying degrees with unprocessed proBNP, which is also found in the circulation. ProBNP processing and immunoassay response are related to O-linked glycosylation of NT-proBNP and proBNP. There is a clear and urgent need to identify the glycosylation sites in the endogenously circulating peptides requested by the community to gain further insights into the different naturally occurring forms. The glycosylation sites of (NT-) proBNP (NT-proBNP and/or proBNP) were characterized in leftovers of heparinized plasma samples of severe HF patients (NT-proBNP: >10000 ng/L) by using tandem immunoaffinity purification, sequential exoglycosidase treatment for glycan trimming, β-elimination and Michael addition chemistry, as well as high-resolution nano-flow liquid chromatography electrospray multistage mass spectrometry. We describe 9 distinct glycosylation sites on circulating (NT-) proBNP in HF patients. Differentially glycosylated variants were detected based on highly accurate mass determination and multistage mass spectrometry. Remarkably, for each of the identified proteolytic glycopeptides, a nonglycosylated form also was detectable. Our results directly demonstrate for the first time a rather complex distribution of the endogenously circulating glycoforms by mass spectrometric analysis in HF patients, and show 9 glycosites in human (NT-) proBNP. This information may also have an impact on commercial immunoassays applying antibodies specific for the central region of (NT-) proBNP, which detect mostly nonglycosylated forms. © 2016 American Association for Clinical Chemistry.

Disease resistance through impairment of α-SNAP–NSF interaction and vesicular trafficking by soybean Rhg1

PubMed Central

Bayless, Adam M.; Smith, John M.; Song, Junqi; McMinn, Patrick H.; Teillet, Alice; August, Benjamin K.

2016-01-01

α-SNAP [soluble NSF (N-ethylmaleimide–sensitive factor) attachment protein] and NSF proteins are conserved across eukaryotes and sustain cellular vesicle trafficking by mediating disassembly and reuse of SNARE protein complexes, which facilitate fusion of vesicles to target membranes. However, certain haplotypes of the Rhg1 (resistance to Heterodera glycines 1) locus of soybean possess multiple repeat copies of an α-SNAP gene (Glyma.18G022500) that encodes atypical amino acids at a highly conserved functional site. These Rhg1 loci mediate resistance to soybean cyst nematode (SCN; H. glycines), the most economically damaging pathogen of soybeans worldwide. Rhg1 is widely used in agriculture, but the mechanisms of Rhg1 disease resistance have remained unclear. In the present study, we found that the resistance-type Rhg1 α-SNAP is defective in interaction with NSF. Elevated in planta expression of resistance-type Rhg1 α-SNAPs depleted the abundance of SNARE-recycling 20S complexes, disrupted vesicle trafficking, induced elevated abundance of NSF, and caused cytotoxicity. Soybean, due to ancient genome duplication events, carries other loci that encode canonical (wild-type) α-SNAPs. Expression of these α-SNAPs counteracted the cytotoxicity of resistance-type Rhg1 α-SNAPs. For successful growth and reproduction, SCN dramatically reprograms a set of plant root cells and must sustain this sedentary feeding site for 2–4 weeks. Immunoblots and electron microscopy immunolocalization revealed that resistance-type α-SNAPs specifically hyperaccumulate relative to wild-type α-SNAPs at the nematode feeding site, promoting the demise of this biotrophic interface. The paradigm of disease resistance through a dysfunctional variant of an essential gene may be applicable to other plant–pathogen interactions. PMID:27821740

Integration of multiethnic fine-mapping and genomic annotation to prioritize candidate functional SNPs at prostate cancer susceptibility regions

PubMed Central

Han, Ying; Hazelett, Dennis J.; Wiklund, Fredrik; Schumacher, Fredrick R.; Stram, Daniel O.; Berndt, Sonja I.; Wang, Zhaoming; Rand, Kristin A.; Hoover, Robert N.; Machiela, Mitchell J.; Yeager, Merideth; Burdette, Laurie; Chung, Charles C.; Hutchinson, Amy; Yu, Kai; Xu, Jianfeng; Travis, Ruth C.; Key, Timothy J.; Siddiq, Afshan; Canzian, Federico; Takahashi, Atsushi; Kubo, Michiaki; Stanford, Janet L.; Kolb, Suzanne; Gapstur, Susan M.; Diver, W. Ryan; Stevens, Victoria L.; Strom, Sara S.; Pettaway, Curtis A.; Al Olama, Ali Amin; Kote-Jarai, Zsofia; Eeles, Rosalind A.; Yeboah, Edward D.; Tettey, Yao; Biritwum, Richard B.; Adjei, Andrew A.; Tay, Evelyn; Truelove, Ann; Niwa, Shelley; Chokkalingam, Anand P.; Isaacs, William B.; Chen, Constance; Lindstrom, Sara; Le Marchand, Loic; Giovannucci, Edward L.; Pomerantz, Mark; Long, Henry; Li, Fugen; Ma, Jing; Stampfer, Meir; John, Esther M.; Ingles, Sue A.; Kittles, Rick A.; Murphy, Adam B.; Blot, William J.; Signorello, Lisa B.; Zheng, Wei; Albanes, Demetrius; Virtamo, Jarmo; Weinstein, Stephanie; Nemesure, Barbara; Carpten, John; Leske, M. Cristina; Wu, Suh-Yuh; Hennis, Anselm J. M.; Rybicki, Benjamin A.; Neslund-Dudas, Christine; Hsing, Ann W.; Chu, Lisa; Goodman, Phyllis J.; Klein, Eric A.; Zheng, S. Lilly; Witte, John S.; Casey, Graham; Riboli, Elio; Li, Qiyuan; Freedman, Matthew L.; Hunter, David J.; Gronberg, Henrik; Cook, Michael B.; Nakagawa, Hidewaki; Kraft, Peter; Chanock, Stephen J.; Easton, Douglas F.; Henderson, Brian E.; Coetzee, Gerhard A.; Conti, David V.; Haiman, Christopher A.

2015-01-01

Interpretation of biological mechanisms underlying genetic risk associations for prostate cancer is complicated by the relatively large number of risk variants (n = 100) and the thousands of surrogate SNPs in linkage disequilibrium. Here, we combined three distinct approaches: multiethnic fine-mapping, putative functional annotation (based upon epigenetic data and genome-encoded features), and expression quantitative trait loci (eQTL) analyses, in an attempt to reduce this complexity. We examined 67 risk regions using genotyping and imputation-based fine-mapping in populations of European (cases/controls: 8600/6946), African (cases/controls: 5327/5136), Japanese (cases/controls: 2563/4391) and Latino (cases/controls: 1034/1046) ancestry. Markers at 55 regions passed a region-specific significance threshold (P-value cutoff range: 3.9 × 10−4–5.6 × 10−3) and in 30 regions we identified markers that were more significantly associated with risk than the previously reported variants in the multiethnic sample. Novel secondary signals (P < 5.0 × 10−6) were also detected in two regions (rs13062436/3q21 and rs17181170/3p12). Among 666 variants in the 55 regions with P-values within one order of magnitude of the most-associated marker, 193 variants (29%) in 48 regions overlapped with epigenetic or other putative functional marks. In 11 of the 55 regions, cis-eQTLs were detected with nearby genes. For 12 of the 55 regions (22%), the most significant region-specific, prostate-cancer associated variant represented the strongest candidate functional variant based on our annotations; the number of regions increased to 20 (36%) and 27 (49%) when examining the 2 and 3 most significantly associated variants in each region, respectively. These results have prioritized subsets of candidate variants for downstream functional evaluation. PMID:26162851

Integration of multiethnic fine-mapping and genomic annotation to prioritize candidate functional SNPs at prostate cancer susceptibility regions.

PubMed

Han, Ying; Hazelett, Dennis J; Wiklund, Fredrik; Schumacher, Fredrick R; Stram, Daniel O; Berndt, Sonja I; Wang, Zhaoming; Rand, Kristin A; Hoover, Robert N; Machiela, Mitchell J; Yeager, Merideth; Burdette, Laurie; Chung, Charles C; Hutchinson, Amy; Yu, Kai; Xu, Jianfeng; Travis, Ruth C; Key, Timothy J; Siddiq, Afshan; Canzian, Federico; Takahashi, Atsushi; Kubo, Michiaki; Stanford, Janet L; Kolb, Suzanne; Gapstur, Susan M; Diver, W Ryan; Stevens, Victoria L; Strom, Sara S; Pettaway, Curtis A; Al Olama, Ali Amin; Kote-Jarai, Zsofia; Eeles, Rosalind A; Yeboah, Edward D; Tettey, Yao; Biritwum, Richard B; Adjei, Andrew A; Tay, Evelyn; Truelove, Ann; Niwa, Shelley; Chokkalingam, Anand P; Isaacs, William B; Chen, Constance; Lindstrom, Sara; Le Marchand, Loic; Giovannucci, Edward L; Pomerantz, Mark; Long, Henry; Li, Fugen; Ma, Jing; Stampfer, Meir; John, Esther M; Ingles, Sue A; Kittles, Rick A; Murphy, Adam B; Blot, William J; Signorello, Lisa B; Zheng, Wei; Albanes, Demetrius; Virtamo, Jarmo; Weinstein, Stephanie; Nemesure, Barbara; Carpten, John; Leske, M Cristina; Wu, Suh-Yuh; Hennis, Anselm J M; Rybicki, Benjamin A; Neslund-Dudas, Christine; Hsing, Ann W; Chu, Lisa; Goodman, Phyllis J; Klein, Eric A; Zheng, S Lilly; Witte, John S; Casey, Graham; Riboli, Elio; Li, Qiyuan; Freedman, Matthew L; Hunter, David J; Gronberg, Henrik; Cook, Michael B; Nakagawa, Hidewaki; Kraft, Peter; Chanock, Stephen J; Easton, Douglas F; Henderson, Brian E; Coetzee, Gerhard A; Conti, David V; Haiman, Christopher A

2015-10-01

Interpretation of biological mechanisms underlying genetic risk associations for prostate cancer is complicated by the relatively large number of risk variants (n = 100) and the thousands of surrogate SNPs in linkage disequilibrium. Here, we combined three distinct approaches: multiethnic fine-mapping, putative functional annotation (based upon epigenetic data and genome-encoded features), and expression quantitative trait loci (eQTL) analyses, in an attempt to reduce this complexity. We examined 67 risk regions using genotyping and imputation-based fine-mapping in populations of European (cases/controls: 8600/6946), African (cases/controls: 5327/5136), Japanese (cases/controls: 2563/4391) and Latino (cases/controls: 1034/1046) ancestry. Markers at 55 regions passed a region-specific significance threshold (P-value cutoff range: 3.9 × 10(-4)-5.6 × 10(-3)) and in 30 regions we identified markers that were more significantly associated with risk than the previously reported variants in the multiethnic sample. Novel secondary signals (P < 5.0 × 10(-6)) were also detected in two regions (rs13062436/3q21 and rs17181170/3p12). Among 666 variants in the 55 regions with P-values within one order of magnitude of the most-associated marker, 193 variants (29%) in 48 regions overlapped with epigenetic or other putative functional marks. In 11 of the 55 regions, cis-eQTLs were detected with nearby genes. For 12 of the 55 regions (22%), the most significant region-specific, prostate-cancer associated variant represented the strongest candidate functional variant based on our annotations; the number of regions increased to 20 (36%) and 27 (49%) when examining the 2 and 3 most significantly associated variants in each region, respectively. These results have prioritized subsets of candidate variants for downstream functional evaluation. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

The role of protein dynamics in the evolution of new enzyme function.

PubMed

Campbell, Eleanor; Kaltenbach, Miriam; Correy, Galen J; Carr, Paul D; Porebski, Benjamin T; Livingstone, Emma K; Afriat-Jurnou, Livnat; Buckle, Ashley M; Weik, Martin; Hollfelder, Florian; Tokuriki, Nobuhiko; Jackson, Colin J

2016-11-01

Enzymes must be ordered to allow the stabilization of transition states by their active sites, yet dynamic enough to adopt alternative conformations suited to other steps in their catalytic cycles. The biophysical principles that determine how specific protein dynamics evolve and how remote mutations affect catalytic activity are poorly understood. Here we examine a 'molecular fossil record' that was recently obtained during the laboratory evolution of a phosphotriesterase from Pseudomonas diminuta to an arylesterase. Analysis of the structures and dynamics of nine protein variants along this trajectory, and three rationally designed variants, reveals cycles of structural destabilization and repair, evolutionary pressure to 'freeze out' unproductive motions and sampling of distinct conformations with specific catalytic properties in bi-functional intermediates. This work establishes that changes to the conformational landscapes of proteins are an essential aspect of molecular evolution and that change in function can be achieved through enrichment of preexisting conformational sub-states.

An organelle-specific protein landscape identifies novel diseases and molecular mechanisms

PubMed Central

Boldt, Karsten; van Reeuwijk, Jeroen; Lu, Qianhao; Koutroumpas, Konstantinos; Nguyen, Thanh-Minh T.; Texier, Yves; van Beersum, Sylvia E. C.; Horn, Nicola; Willer, Jason R.; Mans, Dorus A.; Dougherty, Gerard; Lamers, Ideke J. C.; Coene, Karlien L. M.; Arts, Heleen H.; Betts, Matthew J.; Beyer, Tina; Bolat, Emine; Gloeckner, Christian Johannes; Haidari, Khatera; Hetterschijt, Lisette; Iaconis, Daniela; Jenkins, Dagan; Klose, Franziska; Knapp, Barbara; Latour, Brooke; Letteboer, Stef J. F.; Marcelis, Carlo L.; Mitic, Dragana; Morleo, Manuela; Oud, Machteld M.; Riemersma, Moniek; Rix, Susan; Terhal, Paulien A.; Toedt, Grischa; van Dam, Teunis J. P.; de Vrieze, Erik; Wissinger, Yasmin; Wu, Ka Man; Apic, Gordana; Beales, Philip L.; Blacque, Oliver E.; Gibson, Toby J.; Huynen, Martijn A.; Katsanis, Nicholas; Kremer, Hannie; Omran, Heymut; van Wijk, Erwin; Wolfrum, Uwe; Kepes, François; Davis, Erica E.; Franco, Brunella; Giles, Rachel H.; Ueffing, Marius; Russell, Robert B.; Roepman, Ronald; Al-Turki, Saeed; Anderson, Carl; Antony, Dinu; Barroso, Inês; Bentham, Jamie; Bhattacharya, Shoumo; Carss, Keren; Chatterjee, Krishna; Cirak, Sebahattin; Cosgrove, Catherine; Danecek, Petr; Durbin, Richard; Fitzpatrick, David; Floyd, Jamie; Reghan Foley, A.; Franklin, Chris; Futema, Marta; Humphries, Steve E.; Hurles, Matt; Joyce, Chris; McCarthy, Shane; Mitchison, Hannah M.; Muddyman, Dawn; Muntoni, Francesco; O'Rahilly, Stephen; Onoufriadis, Alexandros; Payne, Felicity; Plagnol, Vincent; Raymond, Lucy; Savage, David B.; Scambler, Peter; Schmidts, Miriam; Schoenmakers, Nadia; Semple, Robert; Serra, Eva; Stalker, Jim; van Kogelenberg, Margriet; Vijayarangakannan, Parthiban; Walter, Klaudia; Whittall, Ros; Williamson, Kathy

2016-01-01

Cellular organelles provide opportunities to relate biological mechanisms to disease. Here we use affinity proteomics, genetics and cell biology to interrogate cilia: poorly understood organelles, where defects cause genetic diseases. Two hundred and seventeen tagged human ciliary proteins create a final landscape of 1,319 proteins, 4,905 interactions and 52 complexes. Reverse tagging, repetition of purifications and statistical analyses, produce a high-resolution network that reveals organelle-specific interactions and complexes not apparent in larger studies, and links vesicle transport, the cytoskeleton, signalling and ubiquitination to ciliary signalling and proteostasis. We observe sub-complexes in exocyst and intraflagellar transport complexes, which we validate biochemically, and by probing structurally predicted, disruptive, genetic variants from ciliary disease patients. The landscape suggests other genetic diseases could be ciliary including 3M syndrome. We show that 3M genes are involved in ciliogenesis, and that patient fibroblasts lack cilia. Overall, this organelle-specific targeting strategy shows considerable promise for Systems Medicine. PMID:27173435

Variants in EXOSC9 Disrupt the RNA Exosome and Result in Cerebellar Atrophy with Spinal Motor Neuronopathy.

PubMed

Burns, David T; Donkervoort, Sandra; Müller, Juliane S; Knierim, Ellen; Bharucha-Goebel, Diana; Faqeih, Eissa Ali; Bell, Stephanie K; AlFaifi, Abdullah Y; Monies, Dorota; Millan, Francisca; Retterer, Kyle; Dyack, Sarah; MacKay, Sara; Morales-Gonzalez, Susanne; Giunta, Michele; Munro, Benjamin; Hudson, Gavin; Scavina, Mena; Baker, Laura; Massini, Tara C; Lek, Monkol; Hu, Ying; Ezzo, Daniel; AlKuraya, Fowzan S; Kang, Peter B; Griffin, Helen; Foley, A Reghan; Schuelke, Markus; Horvath, Rita; Bönnemann, Carsten G

2018-05-03

The exosome is a conserved multi-protein complex that is essential for correct RNA processing. Recessive variants in exosome components EXOSC3, EXOSC8, and RBM7 cause various constellations of pontocerebellar hypoplasia (PCH), spinal muscular atrophy (SMA), and central nervous system demyelination. Here, we report on four unrelated affected individuals with recessive variants in EXOSC9 and the effect of the variants on the function of the RNA exosome in vitro in affected individuals' fibroblasts and skeletal muscle and in vivo in zebrafish. The clinical presentation was severe, early-onset, progressive SMA-like motor neuronopathy, cerebellar atrophy, and in one affected individual, congenital fractures of the long bones. Three affected individuals of different ethnicity carried the homozygous c.41T>C (p.Leu14Pro) variant, whereas one affected individual was compound heterozygous for c.41T>C (p.Leu14Pro) and c.481C>T (p.Arg161 ∗ ). We detected reduced EXOSC9 in fibroblasts and skeletal muscle and observed a reduction of the whole multi-subunit exosome complex on blue-native polyacrylamide gel electrophoresis. RNA sequencing of fibroblasts and skeletal muscle detected significant >2-fold changes in genes involved in neuronal development and cerebellar and motor neuron degeneration, demonstrating the widespread effect of the variants. Morpholino oligonucleotide knockdown and CRISPR/Cas9-mediated mutagenesis of exosc9 in zebrafish recapitulated aspects of the human phenotype, as they have in other zebrafish models of exosomal disease. Specifically, portions of the cerebellum and hindbrain were absent, and motor neurons failed to develop and migrate properly. In summary, we show that variants in EXOSC9 result in a neurological syndrome combining cerebellar atrophy and spinal motoneuronopathy, thus expanding the list of human exosomopathies. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

De novo variants in RHOBTB2, an atypical Rho GTPase gene, cause epileptic encephalopathy.

PubMed

Belal, Hazrat; Nakashima, Mitsuko; Matsumoto, Hiroshi; Yokochi, Kenji; Taniguchi-Ikeda, Mariko; Aoto, Kazushi; Amin, Mohammed Badrul; Maruyama, Azusa; Nagase, Hiroaki; Mizuguchi, Takeshi; Miyatake, Satoko; Miyake, Noriko; Iijima, Kazumoto; Nonoyama, Shigeaki; Matsumoto, Naomichi; Saitsu, Hirotomo

2018-05-16

By whole exome sequencing, we identified three de novo RHOBTB2 variants in three patients with epileptic encephalopathies (EEs). Interestingly, all three patients showed acute encephalopathy (febrile status epilepticus), with magnetic resonance imaging revealing hemisphere swelling or reduced diffusion in various brain regions. RHOBTB2 encodes Rho-related BTB domain-containing protein 2, an atypical Rho GTPase that is a substrate-specific adaptor or itself is a substrate for the Cullin-3 (CUL3)-based ubiquitin/proteasome complex. Transient expression experiments in Neuro-2a cells revealed that mutant RHOBTB2 was more abundant than wild-type RHOBTB2. Co-expression of CUL3 with RHOBTB2 decreased the level of wild-type RHOBTB2 but not the level of any of the three mutants, indicating impaired CUL3 complex-dependent degradation of the three mutants. These data indicate that RHOBTB2 variants are a rare genetic cause of EEs, in which acute encephalopathy might be a characteristic feature, and that precise regulation of RHOBTB2 levels is essential for normal brain function. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Furaldehyde substrate specificity and kinetics of Saccharomyces cerevisiae alcohol dehydrogenase 1 variants.

PubMed

Laadan, Boaz; Wallace-Salinas, Valeria; Carlsson, Åsa Janfalk; Almeida, João Rm; Rådström, Peter; Gorwa-Grauslund, Marie F

2014-08-09

A previously discovered mutant of Saccharomyces cerevisiae alcohol dehydrogenase 1 (Adh1p) was shown to enable a unique NADH-dependent reduction of 5-hydroxymethylfurfural (HMF), a well-known inhibitor of yeast fermentation. In the present study, site-directed mutagenesis of both native and mutated ADH1 genes was performed in order to identify the key amino acids involved in this substrate shift, resulting in Adh1p-variants with different substrate specificities. In vitro activities of the Adh1p-variants using two furaldehydes, HMF and furfural, revealed that HMF reduction ability could be acquired after a single amino acid substitution (Y295C). The highest activity, however, was reached with the double mutation S110P Y295C. Kinetic characterization with both aldehydes and the in vivo primary substrate acetaldehyde also enabled to correlate the alterations in substrate affinity with the different amino acid substitutions. We demonstrated the key role of Y295C mutation in HMF reduction by Adh1p. We generated and kinetically characterized a group of protein variants using two furaldehyde compounds of industrial relevance. Also, we showed that there is a threshold after which higher in vitro HMF reduction activities do not correlate any more with faster in vivo rates of HMF conversion, indicating other cell limitations in the conversion of HMF.

Preferential association of a functional variant in complement receptor 2 with antibodies to double-stranded DNA

PubMed Central

Zhao, Jian; Giles, Brendan M; Taylor, Rhonda L; Yette, Gabriel A; Lough, Kara M; Ng, Han Leng; Abraham, Lawrence J; Wu, Hui; Kelly, Jennifer A; Glenn, Stuart B; Adler, Adam J; Williams, Adrienne H; Comeau, Mary E; Ziegler, Julie T; Marion, Miranda; Alarcón-Riquelme, Marta E; Alarcón, Graciela S; Anaya, Juan-Manuel; Bae, Sang-Cheol; Kim, Dam; Lee, Hye-Soon; Criswell, Lindsey A; Freedman, Barry I; Gilkeson, Gary S; Guthridge, Joel M; Jacob, Chaim O; James, Judith A; Kamen, Diane L; Merrill, Joan T; Sivils, Kathy Moser; Niewold, Timothy B; Petri, Michelle A; Ramsey-Goldman, Rosalind; Reveille, John D; Scofield, R Hal; Stevens, Anne M; Vilá, Luis M; Vyse, Timothy J; Kaufman, Kenneth M; Harley, John B; Langefeld, Carl D; Gaffney, Patrick M; Brown, Elizabeth E; Edberg, Jeffrey C; Kimberly, Robert P; Ulgiati, Daniela; Tsao, Betty P; Boackle, Susan A

2016-01-01

Objectives Systemic lupus erythematosus (SLE; OMIM 152700) is characterised by the production of antibodies to nuclear antigens. We previously identified variants in complement receptor 2 (CR2/CD21) that were associated with decreased risk of SLE. This study aimed to identify the causal variant for this association. Methods Genotyped and imputed genetic variants spanning CR2 were assessed for association with SLE in 15 750 case-control subjects from four ancestral groups. Allele-specific functional effects of associated variants were determined using quantitative real-time PCR, quantitative flow cytometry, electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP)-PCR. Results The strongest association signal was detected at rs1876453 in intron 1 of CR2 (pmeta=4.2×10−4, OR 0.85), specifically when subjects were stratified based on the presence of dsDNA autoantibodies (case-control pmeta=7.6×10−7, OR 0.71; case-only pmeta=1.9×10−4, OR 0.75). Although allele-specific effects on B cell CR2 mRNA or protein levels were not identified, levels of complement receptor 1 (CR1/CD35) mRNA and protein were significantly higher on B cells of subjects harbouring the minor allele (p=0.0248 and p=0.0006, respectively). The minor allele altered the formation of several DNA protein complexes by EMSA, including one containing CCCTC-binding factor (CTCF), an effect that was confirmed by ChIP-PCR. Conclusions These data suggest that rs1876453 in CR2 has long-range effects on gene regulation that decrease susceptibility to lupus. Since the minor allele at rs1876453 is preferentially associated with reduced risk of the highly specific dsDNA autoantibodies that are present in preclinical, active and severe lupus, understanding its mechanisms will have important therapeutic implications. PMID:25180293

Common genetic variants influence human subcortical brain structures.

PubMed

Hibar, Derrek P; Stein, Jason L; Renteria, Miguel E; Arias-Vasquez, Alejandro; Desrivières, Sylvane; Jahanshad, Neda; Toro, Roberto; Wittfeld, Katharina; Abramovic, Lucija; Andersson, Micael; Aribisala, Benjamin S; Armstrong, Nicola J; Bernard, Manon; Bohlken, Marc M; Boks, Marco P; Bralten, Janita; Brown, Andrew A; Chakravarty, M Mallar; Chen, Qiang; Ching, Christopher R K; Cuellar-Partida, Gabriel; den Braber, Anouk; Giddaluru, Sudheer; Goldman, Aaron L; Grimm, Oliver; Guadalupe, Tulio; Hass, Johanna; Woldehawariat, Girma; Holmes, Avram J; Hoogman, Martine; Janowitz, Deborah; Jia, Tianye; Kim, Sungeun; Klein, Marieke; Kraemer, Bernd; Lee, Phil H; Olde Loohuis, Loes M; Luciano, Michelle; Macare, Christine; Mather, Karen A; Mattheisen, Manuel; Milaneschi, Yuri; Nho, Kwangsik; Papmeyer, Martina; Ramasamy, Adaikalavan; Risacher, Shannon L; Roiz-Santiañez, Roberto; Rose, Emma J; Salami, Alireza; Sämann, Philipp G; Schmaal, Lianne; Schork, Andrew J; Shin, Jean; Strike, Lachlan T; Teumer, Alexander; van Donkelaar, Marjolein M J; van Eijk, Kristel R; Walters, Raymond K; Westlye, Lars T; Whelan, Christopher D; Winkler, Anderson M; Zwiers, Marcel P; Alhusaini, Saud; Athanasiu, Lavinia; Ehrlich, Stefan; Hakobjan, Marina M H; Hartberg, Cecilie B; Haukvik, Unn K; Heister, Angelien J G A M; Hoehn, David; Kasperaviciute, Dalia; Liewald, David C M; Lopez, Lorna M; Makkinje, Remco R R; Matarin, Mar; Naber, Marlies A M; McKay, D Reese; Needham, Margaret; Nugent, Allison C; Pütz, Benno; Royle, Natalie A; Shen, Li; Sprooten, Emma; Trabzuni, Daniah; van der Marel, Saskia S L; van Hulzen, Kimm J E; Walton, Esther; Wolf, Christiane; Almasy, Laura; Ames, David; Arepalli, Sampath; Assareh, Amelia A; Bastin, Mark E; Brodaty, Henry; Bulayeva, Kazima B; Carless, Melanie A; Cichon, Sven; Corvin, Aiden; Curran, Joanne E; Czisch, Michael; de Zubicaray, Greig I; Dillman, Allissa; Duggirala, Ravi; Dyer, Thomas D; Erk, Susanne; Fedko, Iryna O; Ferrucci, Luigi; Foroud, Tatiana M; Fox, Peter T; Fukunaga, Masaki; Gibbs, J Raphael; Göring, Harald H H; Green, Robert C; Guelfi, Sebastian; Hansell, Narelle K; Hartman, Catharina A; Hegenscheid, Katrin; Heinz, Andreas; Hernandez, Dena G; Heslenfeld, Dirk J; Hoekstra, Pieter J; Holsboer, Florian; Homuth, Georg; Hottenga, Jouke-Jan; Ikeda, Masashi; Jack, Clifford R; Jenkinson, Mark; Johnson, Robert; Kanai, Ryota; Keil, Maria; Kent, Jack W; Kochunov, Peter; Kwok, John B; Lawrie, Stephen M; Liu, Xinmin; Longo, Dan L; McMahon, Katie L; Meisenzahl, Eva; Melle, Ingrid; Mohnke, Sebastian; Montgomery, Grant W; Mostert, Jeanette C; Mühleisen, Thomas W; Nalls, Michael A; Nichols, Thomas E; Nilsson, Lars G; Nöthen, Markus M; Ohi, Kazutaka; Olvera, Rene L; Perez-Iglesias, Rocio; Pike, G Bruce; Potkin, Steven G; Reinvang, Ivar; Reppermund, Simone; Rietschel, Marcella; Romanczuk-Seiferth, Nina; Rosen, Glenn D; Rujescu, Dan; Schnell, Knut; Schofield, Peter R; Smith, Colin; Steen, Vidar M; Sussmann, Jessika E; Thalamuthu, Anbupalam; Toga, Arthur W; Traynor, Bryan J; Troncoso, Juan; Turner, Jessica A; Valdés Hernández, Maria C; van 't Ent, Dennis; van der Brug, Marcel; van der Wee, Nic J A; van Tol, Marie-Jose; Veltman, Dick J; Wassink, Thomas H; Westman, Eric; Zielke, Ronald H; Zonderman, Alan B; Ashbrook, David G; Hager, Reinmar; Lu, Lu; McMahon, Francis J; Morris, Derek W; Williams, Robert W; Brunner, Han G; Buckner, Randy L; Buitelaar, Jan K; Cahn, Wiepke; Calhoun, Vince D; Cavalleri, Gianpiero L; Crespo-Facorro, Benedicto; Dale, Anders M; Davies, Gareth E; Delanty, Norman; Depondt, Chantal; Djurovic, Srdjan; Drevets, Wayne C; Espeseth, Thomas; Gollub, Randy L; Ho, Beng-Choon; Hoffmann, Wolfgang; Hosten, Norbert; Kahn, René S; Le Hellard, Stephanie; Meyer-Lindenberg, Andreas; Müller-Myhsok, Bertram; Nauck, Matthias; Nyberg, Lars; Pandolfo, Massimo; Penninx, Brenda W J H; Roffman, Joshua L; Sisodiya, Sanjay M; Smoller, Jordan W; van Bokhoven, Hans; van Haren, Neeltje E M; Völzke, Henry; Walter, Henrik; Weiner, Michael W; Wen, Wei; White, Tonya; Agartz, Ingrid; Andreassen, Ole A; Blangero, John; Boomsma, Dorret I; Brouwer, Rachel M; Cannon, Dara M; Cookson, Mark R; de Geus, Eco J C; Deary, Ian J; Donohoe, Gary; Fernández, Guillén; Fisher, Simon E; Francks, Clyde; Glahn, David C; Grabe, Hans J; Gruber, Oliver; Hardy, John; Hashimoto, Ryota; Hulshoff Pol, Hilleke E; Jönsson, Erik G; Kloszewska, Iwona; Lovestone, Simon; Mattay, Venkata S; Mecocci, Patrizia; McDonald, Colm; McIntosh, Andrew M; Ophoff, Roel A; Paus, Tomas; Pausova, Zdenka; Ryten, Mina; Sachdev, Perminder S; Saykin, Andrew J; Simmons, Andy; Singleton, Andrew; Soininen, Hilkka; Wardlaw, Joanna M; Weale, Michael E; Weinberger, Daniel R; Adams, Hieab H H; Launer, Lenore J; Seiler, Stephan; Schmidt, Reinhold; Chauhan, Ganesh; Satizabal, Claudia L; Becker, James T; Yanek, Lisa; van der Lee, Sven J; Ebling, Maritza; Fischl, Bruce; Longstreth, W T; Greve, Douglas; Schmidt, Helena; Nyquist, Paul; Vinke, Louis N; van Duijn, Cornelia M; Xue, Luting; Mazoyer, Bernard; Bis, Joshua C; Gudnason, Vilmundur; Seshadri, Sudha; Ikram, M Arfan; Martin, Nicholas G; Wright, Margaret J; Schumann, Gunter; Franke, Barbara; Thompson, Paul M; Medland, Sarah E

2015-04-09

The highly complex structure of the human brain is strongly shaped by genetic influences. Subcortical brain regions form circuits with cortical areas to coordinate movement, learning, memory and motivation, and altered circuits can lead to abnormal behaviour and disease. To investigate how common genetic variants affect the structure of these brain regions, here we conduct genome-wide association studies of the volumes of seven subcortical regions and the intracranial volume derived from magnetic resonance images of 30,717 individuals from 50 cohorts. We identify five novel genetic variants influencing the volumes of the putamen and caudate nucleus. We also find stronger evidence for three loci with previously established influences on hippocampal volume and intracranial volume. These variants show specific volumetric effects on brain structures rather than global effects across structures. The strongest effects were found for the putamen, where a novel intergenic locus with replicable influence on volume (rs945270; P = 1.08 × 10(-33); 0.52% variance explained) showed evidence of altering the expression of the KTN1 gene in both brain and blood tissue. Variants influencing putamen volume clustered near developmental genes that regulate apoptosis, axon guidance and vesicle transport. Identification of these genetic variants provides insight into the causes of variability in human brain development, and may help to determine mechanisms of neuropsychiatric dysfunction.

Trans-Ethnic Meta-Analysis Identifies Common and Rare Variants Associated with Hepatocyte Growth Factor Levels in the Multi-Ethnic Study of Atherosclerosis (MESA)

PubMed Central

Larson, Nicholas B.; Berardi, Cecilia; Decker, Paul A.; Wassel, Christina L.; Kirsch, Phillip S.; Pankow, James S.; Sale, Michele M.; de Andrade, Mariza; Sicotte, Hugues; Tang, Weihong; Hanson, Naomi Q.; Tsai, Michael Y.; Taylor, Kent D.; Bielinski, Suzette J.

2015-01-01

Summary Hepatocyte growth factor (HGF) is a mesenchyme-derived pleiotropic factor that regulates cell growth, motility, mitogenesis, and morphogenesis in a variety of cells, and increased serum levels of HGF have been linked to a number of clinical and subclinical cardiovascular disease phenotypes. However, little is currently known regarding what genetic factors influence HGF levels, despite evidence of substantial genetic contributions to HGF variation. Based upon ethnicity-stratified single-variant association analysis and trans-ethnic meta-analysis of 6201 participants of the Multi-Ethnic Study of Atherosclerosis (MESA), we discovered five statistically significant common and low-frequency variants: HGF missense polymorphism rs5745687 (p.E299K) as well as four variants (rs16844364, rs4690098, rs114303452, rs3748034) within or in proximity to HGFAC. We also identified two significant ethnicity-specific gene-level associations (A1BG in African Americans; FASN in Chinese Americans) based upon low-frequency/rare variants, while meta-analysis of gene-level results identified a significant association for HGFAC. However, identified single-variant associations explained modest proportions of the total trait variation and were not significantly associated with coronary artery calcium or coronary heart disease. Our findings indicate genetic factors influencing circulating HGF levels may be complex and ethnically diverse. PMID:25998175

Allosteric alterations in the androgen receptor and activity in prostate cancer.

PubMed

Uo, Takuma; Plymate, Stephen R; Sprenger, Cynthia C

2017-09-01

Organisms have evolved to generate biological complexity in their proteome and transcriptome from a limited number of genes. This concept holds true for the androgen receptor, which displays a diversity of inclusion/exclusion events in its structural motifs as a mechanism of resistance to the most forefront anti-androgen therapies. More than 20 androgen receptor variants that lack various portions of ligand-binding domain have been identified in human prostate cancer (PCa) samples. Most of the variants are inactive on their own, with a few exceptions displaying constitutive activity. The full-length receptor and one or more variants can be co-expressed in the same cell under many circumstances, which raises the question of how these variants physically and functionally interact with the full-length receptor or one another in the course of PCa progression. To address this issue, in this review, we will characterize and discuss androgen receptor variants, including the novel variants discovered in the last couple of years (i) individually, (ii) with respect to their physical and functional interaction with one another and (iii) in clinical relevance. Here, we also introduce the very recent understanding of AR-Vs obtained through successful development of some AR-V-specific antibodies as well as identification of novel AR-Vs by data mining approaches. © 2017 Society for Endocrinology.

CD and MCD studies of the effects of component B variant binding on the biferrous active site of methane monooxygenase.

PubMed

Mitić, Natasa; Schwartz, Jennifer K; Brazeau, Brian J; Lipscomb, John D; Solomon, Edward I

2008-08-12

The multicomponent soluble form of methane monooxygenase (sMMO) catalyzes the oxidation of methane through the activation of O 2 at a nonheme biferrous center in the hydroxylase component, MMOH. Reactivity is limited without binding of the sMMO effector protein, MMOB. Past studies show that mutations of specific MMOB surface residues cause large changes in the rates of individual steps in the MMOH reaction cycle. To define the structural and mechanistic bases for these observations, CD, MCD, and VTVH MCD spectroscopies coupled with ligand-field (LF) calculations are used to elucidate changes occurring near and at the MMOH biferrous cluster upon binding of MMOB and the MMOB variants. Perturbations to both the CD and MCD are observed upon binding wild-type MMOB and the MMOB variant that similarly increases O 2 reactivity. MMOB variants that do not greatly increase O 2 reactivity fail to cause one or both of these changes. LF calculations indicate that reorientation of the terminal glutamate on Fe2 reproduces the spectral perturbations in MCD. Although this structural change allows O 2 to bridge the diiron site and shifts the redox active orbitals for good overlap, it is not sufficient for enhanced O 2 reactivity of the enzyme. Binding of the T111Y-MMOB variant to MMOH induces the MCD, but not CD changes, and causes only a small increase in reactivity. Thus, both the geometric rearrangement at Fe2 (observed in MCD) coupled with a more global conformational change that may control O 2 access (probed by CD), induced by MMOB binding, are critical factors in the reactivity of sMMO.

Site-specific identification of heparan and chondroitin sulfate glycosaminoglycans in hybrid proteoglycans.

PubMed

Noborn, Fredrik; Gomez Toledo, Alejandro; Green, Anders; Nasir, Waqas; Sihlbom, Carina; Nilsson, Jonas; Larson, Göran

2016-10-03

Heparan sulfate (HS) and chondroitin sulfate (CS) are complex polysaccharides that regulate important biological pathways in virtually all metazoan organisms. The polysaccharides often display opposite effects on cell functions with HS and CS structural motifs presenting unique binding sites for specific ligands. Still, the mechanisms by which glycan biosynthesis generates complex HS and CS polysaccharides required for the regulation of mammalian physiology remain elusive. Here we present a glycoproteomic approach that identifies and differentiates between HS and CS attachment sites and provides identity to the core proteins. Glycopeptides were prepared from perlecan, a complex proteoglycan known to be substituted with both HS and CS chains, further digested with heparinase or chondroitinase ABC to reduce the HS and CS chain lengths respectively, and thereafter analyzed by nLC-MS/MS. This protocol enabled the identification of three consensus HS sites and one hybrid site, carrying either a HS or a CS chain. Inspection of the amino acid sequence at the hybrid attachment locus indicates that certain peptide motifs may encode for the chain type selection process. This analytical approach will become useful when addressing fundamental questions in basic biology specifically in elucidating the functional roles of site-specific glycosylations of proteoglycans.

‘Nodophagy’

PubMed Central

Travassos, Leonardo H

2010-01-01

Autophagy is a homeostatic pathway that processes and recycles damaged organelles and other cytoplasmic contents. While studies have implicated autophagy in the immune response to infection, the understanding of how the autophagic machinery specifically targets intracellular pathogens has remained elusive. Two recent studies have uncovered an autophagy-mediated immune response to bacteria through their detection by Nod receptors. In particular, Nod1 and Nod2 recruit the autophagic protein ATG16L1 to the plasma membrane at the bacterial entry site to promote an autophagy-dependent elimination of bacteria. In addition, Nod2 and ATG16L1 synergize to initiate an adaptive immune response to bacterial invasion by enhancing major histocompatibility complex (MHC) class II antigen presentation. These findings link two Crohn disease-associated susceptibility genes and reveal that cells expressing the risk-associated variants of ATG16L1 are defective in autophagy-mediated bacterial handling and antigen presentation. This could lead to bacterial persistence and contribute to the pathogenesis of the disease. PMID:21327039

MicroCT-based phenomics in the zebrafish skeleton reveals virtues of deep phenotyping in a distributed organ system.

PubMed

Hur, Matthew; Gistelinck, Charlotte A; Huber, Philippe; Lee, Jane; Thompson, Marjorie H; Monstad-Rios, Adrian T; Watson, Claire J; McMenamin, Sarah K; Willaert, Andy; Parichy, David M; Coucke, Paul; Kwon, Ronald Y

2017-09-08

Phenomics, which ideally involves in-depth phenotyping at the whole-organism scale, may enhance our functional understanding of genetic variation. Here, we demonstrate methods to profile hundreds of phenotypic measures comprised of morphological and densitometric traits at a large number of sites within the axial skeleton of adult zebrafish. We show the potential for vertebral patterns to confer heightened sensitivity, with similar specificity, in discriminating mutant populations compared to analyzing individual vertebrae in isolation. We identify phenotypes associated with human brittle bone disease and thyroid stimulating hormone receptor hyperactivity. Finally, we develop allometric models and show their potential to aid in the discrimination of mutant phenotypes masked by alterations in growth. Our studies demonstrate virtues of deep phenotyping in a spatially distributed organ system. Analyzing phenotypic patterns may increase productivity in genetic screens, and facilitate the study of genetic variants associated with smaller effect sizes, such as those that underlie complex diseases.

Folding of a transcriptionally acting PreQ1 riboswitch

PubMed Central

Rieder, Ulrike; Kreutz, Christoph; Micura, Ronald

2010-01-01

7-Aminomethyl-7-deazaguanine (preQ1) sensitive mRNA domains belong to the smallest riboswitches known to date. Although recent efforts have revealed the three-dimensional architecture of the ligand–aptamer complex less is known about the molecular details of the ligand-induced response mechanism that modulates gene expression. We present an in vitro investigation on the ligand-induced folding process of the preQ1 responsive RNA element from Fusobacterium nucleatum using biophysical methods, including fluorescence and NMR spectroscopy of site-specifically labeled riboswitch variants. We provide evidence that the full-length riboswitch domain adopts two different coexisting stem-loop structures in the expression platform. Upon addition of preQ1, the equilibrium of the competing hairpins is significantly shifted. This system therefore, represents a finely tunable antiterminator/terminator interplay that impacts the in vivo cellular response mechanism. A model is presented how a riboswitch that provides no obvious overlap between aptamer and terminator stem-loop solves this communication problem by involving bistable sequence determinants. PMID:20534493

GenPlay Multi-Genome, a tool to compare and analyze multiple human genomes in a graphical interface.

PubMed

Lajugie, Julien; Fourel, Nicolas; Bouhassira, Eric E

2015-01-01

Parallel visualization of multiple individual human genomes is a complex endeavor that is rapidly gaining importance with the increasing number of personal, phased and cancer genomes that are being generated. It requires the display of variants such as SNPs, indels and structural variants that are unique to specific genomes and the introduction of multiple overlapping gaps in the reference sequence. Here, we describe GenPlay Multi-Genome, an application specifically written to visualize and analyze multiple human genomes in parallel. GenPlay Multi-Genome is ideally suited for the comparison of allele-specific expression and functional genomic data obtained from multiple phased genomes in a graphical interface with access to multiple-track operation. It also allows the analysis of data that have been aligned to custom genomes rather than to a standard reference and can be used as a variant calling format file browser and as a tool to compare different genome assembly, such as hg19 and hg38. GenPlay is available under the GNU public license (GPL-3) from http://genplay.einstein.yu.edu. The source code is available at https://github.com/JulienLajugie/GenPlay. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Diverse cross-reactive potential and Vbeta gene usage of an epitope-specific cytotoxic T-lymphocyte population in monkeys immunized with diverse human immunodeficiency virus type 1 Env immunogens.

PubMed

Hulot, Sandrine L; Seaman, Michael S; Sen, Pritha; Autissier, Patrick A; Manuel, Edwin R; Letvin, Norman L

2009-10-01

An ideal human immunodeficiency virus type 1 (HIV-1) vaccine would elicit potent cellular and humoral immune responses that recognize diverse strains of the virus. In the present study, combined methodologies (flow cytometry, Vbeta repertoire analysis, and complementarity-determining region 3 sequencing) were used to determine the clonality of CD8(+) T lymphocytes taking part in the recognition of variant epitope peptides elicited in Mamu-A*01-positive rhesus monkeys immunized with vaccines encoding diverse HIV-1 envelopes (Envs). Monkeys immunized with clade B Envs generated CD8(+) T lymphocytes that cross-recognized both clade B- and clade C-p41A epitope peptides using a large degree of diversity in Vbeta gene usage. However, with two monkeys immunized with clade C Env, one monkey exhibited p41A-specific cytotoxic T-lymphocytes (CTL) with the capacity for cross-recognition of variant epitopes, while the other monkey did not. These studies demonstrate that the cross-reactive potential of variant p41A epitope peptide-specific CTL populations can differ between monkeys that share the same restricting major histocompatibility complex class I molecule and receive the same vaccine immunogens.

The Molecular Basis of Drug Resistance against Hepatitis C Virus NS3/4A Protease Inhibitors

PubMed Central

Romano, Keith P.; Ali, Akbar; Aydin, Cihan; Soumana, Djade; Özen, Ayşegül; Deveau, Laura M.; Silver, Casey; Cao, Hong; Newton, Alicia; Petropoulos, Christos J.; Huang, Wei; Schiffer, Celia A.

2012-01-01

Hepatitis C virus (HCV) infects over 170 million people worldwide and is the leading cause of chronic liver diseases, including cirrhosis, liver failure, and liver cancer. Available antiviral therapies cause severe side effects and are effective only for a subset of patients, though treatment outcomes have recently been improved by the combination therapy now including boceprevir and telaprevir, which inhibit the viral NS3/4A protease. Despite extensive efforts to develop more potent next-generation protease inhibitors, however, the long-term efficacy of this drug class is challenged by the rapid emergence of resistance. Single-site mutations at protease residues R155, A156 and D168 confer resistance to nearly all inhibitors in clinical development. Thus, developing the next-generation of drugs that retain activity against a broader spectrum of resistant viral variants requires a comprehensive understanding of the molecular basis of drug resistance. In this study, 16 high-resolution crystal structures of four representative protease inhibitors – telaprevir, danoprevir, vaniprevir and MK-5172 – in complex with the wild-type protease and three major drug-resistant variants R155K, A156T and D168A, reveal unique molecular underpinnings of resistance to each drug. The drugs exhibit differential susceptibilities to these protease variants in both enzymatic and antiviral assays. Telaprevir, danoprevir and vaniprevir interact directly with sites that confer resistance upon mutation, while MK-5172 interacts in a unique conformation with the catalytic triad. This novel mode of MK-5172 binding explains its retained potency against two multi-drug-resistant variants, R155K and D168A. These findings define the molecular basis of HCV N3/4A protease inhibitor resistance and provide potential strategies for designing robust therapies against this rapidly evolving virus. PMID:22910833

Prediction, time variance, and classification of hydraulic response to recharge in two karst aquifers

USGS Publications Warehouse

Long, Andrew J.; Mahler, Barbara J.

2013-01-01

Many karst aquifers are rapidly filled and depleted and therefore are likely to be susceptible to changes in short-term climate variability. Here we explore methods that could be applied to model site-specific hydraulic responses, with the intent of simulating these responses to different climate scenarios from high-resolution climate models. We compare hydraulic responses (spring flow, groundwater level, stream base flow, and cave drip) at several sites in two karst aquifers: the Edwards aquifer (Texas, USA) and the Madison aquifer (South Dakota, USA). A lumped-parameter model simulates nonlinear soil moisture changes for estimation of recharge, and a time-variant convolution model simulates the aquifer response to this recharge. Model fit to data is 2.4% better for calibration periods than for validation periods according to the Nash–Sutcliffe coefficient of efficiency, which ranges from 0.53 to 0.94 for validation periods. We use metrics that describe the shapes of the impulse-response functions (IRFs) obtained from convolution modeling to make comparisons in the distribution of response times among sites and between aquifers. Time-variant IRFs were applied to 62% of the sites. Principal component analysis (PCA) of metrics describing the shapes of the IRFs indicates three principal components that together account for 84% of the variability in IRF shape: the first is related to IRF skewness and temporal spread and accounts for 51% of the variability; the second and third largely are related to time-variant properties and together account for 33% of the variability. Sites with IRFs that dominantly comprise exponential curves are separated geographically from those dominantly comprising lognormal curves in both aquifers as a result of spatial heterogeneity. The use of multiple IRF metrics in PCA is a novel method to characterize, compare, and classify the way in which different sites and aquifers respond to recharge. As convolution models are developed for additional aquifers, they could contribute to an IRF database and a general classification system for karst aquifers.

Recent Mitochondrial DNA Mutations Increase the Risk of Developing Common Late-Onset Human Diseases

PubMed Central

Hudson, Gavin; Gomez-Duran, Aurora; Wilson, Ian J.; Chinnery, Patrick F.

2014-01-01

Mitochondrial DNA (mtDNA) is highly polymorphic at the population level, and specific mtDNA variants affect mitochondrial function. With emerging evidence that mitochondrial mechanisms are central to common human diseases, it is plausible that mtDNA variants contribute to the “missing heritability” of several complex traits. Given the central role of mtDNA genes in oxidative phosphorylation, the same genetic variants would be expected to alter the risk of developing several different disorders, but this has not been shown to date. Here we studied 38,638 individuals with 11 major diseases, and 17,483 healthy controls. Imputing missing variants from 7,729 complete mitochondrial genomes, we captured 40.41% of European mtDNA variation. We show that mtDNA variants modifying the risk of developing one disease also modify the risk of developing other diseases, thus providing independent replication of a disease association in different case and control cohorts. High-risk alleles were more common than protective alleles, indicating that mtDNA is not at equilibrium in the human population, and that recent mutations interact with nuclear loci to modify the risk of developing multiple common diseases. PMID:24852434

Divergent biophysical properties, gating mechanisms, and possible functions of the two skeletal muscle Ca(V)1.1 calcium channel splice variants.

PubMed

Tuluc, Petronel; Flucher, Bernhard E

2011-12-01

Voltage-gated calcium channels are multi-subunit protein complexes that specifically allow calcium ions to enter the cell in response to membrane depolarization. But, for many years it seemed that the skeletal muscle calcium channel Ca(V)1.1 is the exception. The classical splice variant Ca(V)1.1a activates slowly, has a very small current amplitude and poor voltage sensitivity. In fact adult muscle fibers work perfectly well even in the absence of calcium influx. Recently a new splice variant of the skeletal muscle calcium channel Ca(V)1.1e has been characterized. The lack of the 19 amino acid exon 29 in this splice variant results in a rapidly activating calcium channel with high current amplitude and good voltage sensitivity. Ca(V)1.1e is the dominant channel in embryonic muscle, where the expression of this high calcium-conducting Ca(V)1.1 isoform readily explains developmental processes depending on L-type calcium currents. Moreover, the availability of these two structurally similar but functionally distinct channel variants facilitates the analysis of the molecular mechanisms underlying the unique current properties of the classical Ca(V)1.1a channel.

Identification of rare X-linked neuroligin variants by massively parallel sequencing in males with autism spectrum disorder.

PubMed

Steinberg, Karyn Meltz; Ramachandran, Dhanya; Patel, Viren C; Shetty, Amol C; Cutler, David J; Zwick, Michael E

2012-09-28

Autism spectrum disorder (ASD) is highly heritable, but the genetic risk factors for it remain largely unknown. Although structural variants with large effect sizes may explain up to 15% ASD, genome-wide association studies have failed to uncover common single nucleotide variants with large effects on phenotype. The focus within ASD genetics is now shifting to the examination of rare sequence variants of modest effect, which is most often achieved via exome selection and sequencing. This strategy has indeed identified some rare candidate variants; however, the approach does not capture the full spectrum of genetic variation that might contribute to the phenotype. We surveyed two loci with known rare variants that contribute to ASD, the X-linked neuroligin genes by performing massively parallel Illumina sequencing of the coding and noncoding regions from these genes in males from families with multiplex autism. We annotated all variant sites and functionally tested a subset to identify other rare mutations contributing to ASD susceptibility. We found seven rare variants at evolutionary conserved sites in our study population. Functional analyses of the three 3' UTR variants did not show statistically significant effects on the expression of NLGN3 and NLGN4X. In addition, we identified two NLGN3 intronic variants located within conserved transcription factor binding sites that could potentially affect gene regulation. These data demonstrate the power of massively parallel, targeted sequencing studies of affected individuals for identifying rare, potentially disease-contributing variation. However, they also point out the challenges and limitations of current methods of direct functional testing of rare variants and the difficulties of identifying alleles with modest effects.

Identification of rare X-linked neuroligin variants by massively parallel sequencing in males with autism spectrum disorder

PubMed Central

2012-01-01

Background Autism spectrum disorder (ASD) is highly heritable, but the genetic risk factors for it remain largely unknown. Although structural variants with large effect sizes may explain up to 15% ASD, genome-wide association studies have failed to uncover common single nucleotide variants with large effects on phenotype. The focus within ASD genetics is now shifting to the examination of rare sequence variants of modest effect, which is most often achieved via exome selection and sequencing. This strategy has indeed identified some rare candidate variants; however, the approach does not capture the full spectrum of genetic variation that might contribute to the phenotype. Methods We surveyed two loci with known rare variants that contribute to ASD, the X-linked neuroligin genes by performing massively parallel Illumina sequencing of the coding and noncoding regions from these genes in males from families with multiplex autism. We annotated all variant sites and functionally tested a subset to identify other rare mutations contributing to ASD susceptibility. Results We found seven rare variants at evolutionary conserved sites in our study population. Functional analyses of the three 3’ UTR variants did not show statistically significant effects on the expression of NLGN3 and NLGN4X. In addition, we identified two NLGN3 intronic variants located within conserved transcription factor binding sites that could potentially affect gene regulation. Conclusions These data demonstrate the power of massively parallel, targeted sequencing studies of affected individuals for identifying rare, potentially disease-contributing variation. However, they also point out the challenges and limitations of current methods of direct functional testing of rare variants and the difficulties of identifying alleles with modest effects. PMID:23020841

Incorporation of DPP6a and DPP6K variants in ternary Kv4 channel complex reconstitutes properties of A-type K current in rat cerebellar granule cells.

PubMed

Jerng, Henry H; Pfaffinger, Paul J

2012-01-01

Dipeptidyl peptidase-like protein 6 (DPP6) proteins co-assemble with Kv4 channel α-subunits and Kv channel-interacting proteins (KChIPs) to form channel protein complexes underlying neuronal somatodendritic A-type potassium current (I(SA)). DPP6 proteins are expressed as N-terminal variants (DPP6a, DPP6K, DPP6S, DPP6L) that result from alternative mRNA initiation and exhibit overlapping expression patterns. Here, we study the role DPP6 variants play in shaping the functional properties of I(SA) found in cerebellar granule (CG) cells using quantitative RT-PCR and voltage-clamp recordings of whole-cell currents from reconstituted channel complexes and native I(SA) channels. Differential expression of DPP6 variants was detected in rat CG cells, with DPP6K (41 ± 3%)>DPP6a (33 ± 3%)>DPP6S (18 ± 2%)>DPP6L (8 ± 3%). To better understand how DPP6 variants shape native neuronal I(SA), we focused on studying interactions between the two dominant variants, DPP6K and DPP6a. Although previous studies did not identify unique functional effects of DPP6K, we find that the unique N-terminus of DPP6K modulates the effects of KChIP proteins, slowing recovery and producing a negative shift in the steady-state inactivation curve. By contrast, DPP6a uses its distinct N-terminus to directly confer rapid N-type inactivation independently of KChIP3a. When DPP6a and DPP6K are co-expressed in ratios similar to those found in CG cells, their distinct effects compete in modulating channel function. The more rapid inactivation from DPP6a dominates during strong depolarization; however, DPP6K produces a negative shift in the steady-state inactivation curve and introduces a slow phase of recovery from inactivation. A direct comparison to the native CG cell I(SA) shows that these mixed effects are present in the native channels. Our results support the hypothesis that the precise expression and co-assembly of different auxiliary subunit variants are important factors in shaping the I(SA) functional properties in specific neuronal populations.

Structures of the Mycobacterium tuberculosis GlpX protein (class II fructose-1,6-bisphosphatase): implications for the active oligomeric state, catalytic mechanism and citrate inhibition

DOE PAGES

Wolf, Nina M.; Gutka, Hiten J.; Movahedzadeh, Farahnaz; ...

2018-04-03

The crystal structures of native class II fructose-1,6-bisphosphatase (FBPaseII) from Mycobacterium tuberculosis at 2.6 Å resolution and two active-site protein variants are presented. The variants were complexed with the reaction product fructose 6-phosphate (F6P). The Thr84Ala mutant is inactive, while the Thr84Ser mutant has a lower catalytic activity. The structures reveal the presence of a 222 tetramer, similar to those described for fructose-1,6/sedoheptulose-1,7-bisphosphatase from Synechocystis (strain 6803) as well as the equivalent enzyme from Thermosynechococcus elongatus . This homotetramer corresponds to a homologous oligomer that is present but not described in the crystal structure of FBPaseII from Escherichia coli and ismore » probably conserved in all FBPaseIIs. The constellation of amino-acid residues in the active site of FBPaseII from M. tuberculosis ( Mt FBPaseII) is conserved and is analogous to that described previously for the E. coli enzyme. Moreover, the structure of the active site of the partially active (Thr84Ser) variant and the analysis of the kinetics are consistent with the previously proposed catalytic mechanism. The presence of metabolites in the crystallization medium (for example citrate and malonate) and in the corresponding crystal structures of Mt FBPaseII, combined with their observed inhibitory effect, could suggest the existence of an uncharacterized inhibition of this class of enzymes besides the allosteric inhibition by adenosine monophosphate observed for the Synechocystis enzyme. The structural and functional insights derived from the structure of Mt FBPaseII will provide critical information for the design of lead inhibitors, which will be used to validate this target for future chemical intervention.« less

Novel mode of inhibition by D-tagatose 6-phosphate through a Heyns rearrangement in the active site of transaldolase B variants.

PubMed

Stellmacher, Lena; Sandalova, Tatyana; Schneider, Sarah; Schneider, Gunter; Sprenger, Georg A; Samland, Anne K

2016-04-01

Transaldolase B (TalB) and D-fructose-6-phosphate aldolase A (FSAA) from Escherichia coli are C-C bond-forming enzymes. Using kinetic inhibition studies and mass spectrometry, it is shown that enzyme variants of FSAA and TalB that exhibit D-fructose-6-phosphate aldolase activity are inhibited covalently and irreversibly by D-tagatose 6-phosphate (D-T6P), whereas no inhibition was observed for wild-type transaldolase B from E. coli. The crystal structure of the variant TalB(F178Y) with bound sugar phosphate was solved to a resolution of 1.46 Å and revealed a novel mode of covalent inhibition. The sugar is bound covalently via its C2 atom to the ℇ-NH2 group of the active-site residue Lys132. It is neither bound in the open-chain form nor as the closed-ring form of D-T6P, but has been converted to β-D-galactofuranose 6-phosphate (D-G6P), a five-membered ring structure. The furanose ring of the covalent adduct is formed via a Heyns rearrangement and subsequent hemiacetal formation. This reaction is facilitated by Tyr178, which is proposed to act as acid-base catalyst. The crystal structure of the inhibitor complex is compared with the structure of the Schiff-base intermediate of TalB(E96Q) formed with the substrate D-fructose 6-phosphate determined to a resolution of 2.20 Å. This comparison highlights the differences in stereochemistry at the C4 atom of the ligand as an essential determinant for the formation of the inhibitor adduct in the active site of the enzyme.

Structures of the Mycobacterium tuberculosis GlpX protein (class II fructose-1,6-bisphosphatase): implications for the active oligomeric state, catalytic mechanism and citrate inhibition

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wolf, Nina M.; Gutka, Hiten J.; Movahedzadeh, Farahnaz

The crystal structures of native class II fructose-1,6-bisphosphatase (FBPaseII) from Mycobacterium tuberculosis at 2.6 Å resolution and two active-site protein variants are presented. The variants were complexed with the reaction product fructose 6-phosphate (F6P). The Thr84Ala mutant is inactive, while the Thr84Ser mutant has a lower catalytic activity. The structures reveal the presence of a 222 tetramer, similar to those described for fructose-1,6/sedoheptulose-1,7-bisphosphatase from Synechocystis (strain 6803) as well as the equivalent enzyme from Thermosynechococcus elongatus . This homotetramer corresponds to a homologous oligomer that is present but not described in the crystal structure of FBPaseII from Escherichia coli and ismore » probably conserved in all FBPaseIIs. The constellation of amino-acid residues in the active site of FBPaseII from M. tuberculosis ( Mt FBPaseII) is conserved and is analogous to that described previously for the E. coli enzyme. Moreover, the structure of the active site of the partially active (Thr84Ser) variant and the analysis of the kinetics are consistent with the previously proposed catalytic mechanism. The presence of metabolites in the crystallization medium (for example citrate and malonate) and in the corresponding crystal structures of Mt FBPaseII, combined with their observed inhibitory effect, could suggest the existence of an uncharacterized inhibition of this class of enzymes besides the allosteric inhibition by adenosine monophosphate observed for the Synechocystis enzyme. The structural and functional insights derived from the structure of Mt FBPaseII will provide critical information for the design of lead inhibitors, which will be used to validate this target for future chemical intervention.« less

The Unique Morgue Ubiquitination Protein Is Conserved in a Diverse but Restricted Set of Invertebrates

PubMed Central

Zhou, Ying; Carpenter, Zachary W.; Brennan, Gregory

2009-01-01

Drosophila Morgue is a unique ubiquitination protein that facilitates programmed cell death and associates with DIAP1, a critical cell death inhibitor with E3 ubiquitin ligase activity. Morgue possesses a unique combination of functional domains typically associated with distinct types of ubiquitination enzymes. This includes an F box characteristic of the substrate-binding subunit in Skp, Cullin, and F box (SCF)-type ubiquitin E3 ligase complexes and a variant ubiquitin E2 conjugase domain where the active site cysteine is replaced by a glycine. Morgue also contains a single C4-type zinc finger motif. This architecture suggests potentially novel ubiquitination activities for Morgue. In this study, we address the evolutionary origins of this distinctive protein utilizing a combination of bioinformatics and molecular biology approaches. We find that Morgue exhibits widespread but restricted phylogenetic distribution among metazoans. Morgue proteins were identified in a wide range of Protostome phyla, including Arthropoda, Annelida, Mollusca, Nematoda, and Platyhelminthes. However, with one potential exception, Morgue was not detected in Deuterostomes, including Chordates, Hemichordates, or Echinoderms. Morgue was also not found in Ctenophora, Cnidaria, Placozoa, or Porifera. Characterization of Morgue sequences within specific animal lineages suggests that gene deletion or acquisition has occurred during divergence of nematodes and that at least one arachnid expresses an atypical form of Morgue consisting only of the variant E2 conjugase domain. Analysis of the organization of several morgue genes suggests that exon-shuffling events have contributed to the evolution of the Morgue protein. These results suggest that Morgue mediates conserved and distinctive ubiquitination functions in specific cell death pathways. PMID:19602541

[Fine mapping of complex disease susceptibility loci].

PubMed

Song, Qingfeng; Zhang, Hongxing; Ma, Yilong; Zhou, Gangqiao

2014-01-01

Genome-wide association studies (GWAS) using single nucleotide polymorphism (SNP) markers have identified more than 3800 susceptibility loci for more than 660 diseases or traits. However, the most significantly associated variants or causative variants in these loci and their biological functions have remained to be clarified. These causative variants can help to elucidate the pathogenesis and discover new biomarkers of complex diseases. One of the main goals in the post-GWAS era is to identify the causative variants and susceptibility genes, and clarify their functional aspects by fine mapping. For common variants, imputation or re-sequencing based strategies were implemented to increase the number of analyzed variants and help to identify the most significantly associated variants. In addition, functional element, expression quantitative trait locus (eQTL) and haplotype analyses were performed to identify functional common variants and susceptibility genes. For rare variants, fine mapping was carried out by re-sequencing, rare haplotype analysis, family-based analysis, burden test, etc.This review summarizes the strategies and problems for fine mapping.

Structural Basis for Inactivation of the Human Pyruvate Dehydrogenase Complex by Phosphorylation: Role of Disordered Phosphorylation Loops

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kato, Masato; Wynn, R. Max; Chuang, Jacinta L.

2009-09-11

We report the crystal structures of the phosporylated pyruvate dehydrogenase (E1p) component of the human pyruvate dehydrogenase complex (PDC). The complete phosphorylation at Ser264-{alpha} (site 1) of a variant E1p protein was achieved using robust pyruvate dehydrogenase kinase 4 free of the PDC core. We show that unlike its unmodified counterpart, the presence of a phosphoryl group at Ser264-{alpha} prevents the cofactor thiamine diphosphate-induced ordering of the two loops carrying the three phosphorylation sites. The disordering of these phosphorylation loops is caused by a previously unrecognized steric clash between the phosphoryl group at site 1 and a nearby Ser266-{alpha}, whichmore » nullifies a hydrogen-bonding network essential for maintaining the loop conformations. The disordered phosphorylation loops impede the binding of lipoyl domains of the PDC core to E1p, negating the reductive acetylation step. This results in the disruption of the substrate channeling in the PDC, leading to the inactivation of this catalytic machine.« less

Structural Insights into the Assembly of the Adeno-associated Virus Type 2 Rep68 Protein on the Integration Site AAVS1*

PubMed Central

Musayev, Faik N.; Zarate-Perez, Francisco; Bishop, Clayton; Burgner, John W.; Escalante, Carlos R.

2015-01-01

Adeno-associated virus (AAV) is the only eukaryotic virus with the property of establishing latency by integrating site-specifically into the human genome. The integration site known as AAVS1 is located in chromosome 19 and contains multiple GCTC repeats that are recognized by the AAV non-structural Rep proteins. These proteins are multifunctional, with an N-terminal origin-binding domain (OBD) and a helicase domain joined together by a short linker. As a first step to understand the process of site-specific integration, we proceeded to characterize the recognition and assembly of Rep68 onto the AAVS1 site. We first determined the x-ray structure of AAV-2 Rep68 OBD in complex with the AAVS1 DNA site. Specificity is achieved through the interaction of a glycine-rich loop that binds the major groove and an α-helix that interacts with a downstream minor groove on the same face of the DNA. Although the structure shows a complex with three OBD molecules bound to the AAVS1 site, we show by using analytical centrifugation and electron microscopy that the full-length Rep68 forms a heptameric complex. Moreover, we determined that a minimum of two direct repeats is required to form a stable complex and to melt DNA. Finally, we show that although the individual domains bind DNA poorly, complex assembly requires oligomerization and cooperation between its OBD, helicase, and the linker domains. PMID:26370092

VarMod: modelling the functional effects of non-synonymous variants.

PubMed

Pappalardo, Morena; Wass, Mark N

2014-07-01

Unravelling the genotype-phenotype relationship in humans remains a challenging task in genomics studies. Recent advances in sequencing technologies mean there are now thousands of sequenced human genomes, revealing millions of single nucleotide variants (SNVs). For non-synonymous SNVs present in proteins the difficulties of the problem lie in first identifying those nsSNVs that result in a functional change in the protein among the many non-functional variants and in turn linking this functional change to phenotype. Here we present VarMod (Variant Modeller) a method that utilises both protein sequence and structural features to predict nsSNVs that alter protein function. VarMod develops recent observations that functional nsSNVs are enriched at protein-protein interfaces and protein-ligand binding sites and uses these characteristics to make predictions. In benchmarking on a set of nearly 3000 nsSNVs VarMod performance is comparable to an existing state of the art method. The VarMod web server provides extensive resources to investigate the sequence and structural features associated with the predictions including visualisation of protein models and complexes via an interactive JSmol molecular viewer. VarMod is available for use at http://www.wasslab.org/varmod. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

Identification and Characterization of Alternative Promoters, Transcripts and Protein Isoforms of Zebrafish R2 Gene

PubMed Central

Shang, Hanqiao; Li, Qing; Feng, Guohui; Cui, Zongbin

2011-01-01

Ribonucleotide reductase (RNR) is the rate-limiting enzyme in the de novo synthesis of deoxyribonucleoside triphosphates. Expression of RNR subunits is closely associated with DNA replication and repair. Mammalian RNR M2 subunit (R2) functions exclusively in DNA replication of normal cells due to its S phase-specific expression and late mitotic degradation. Herein, we demonstrate the control of R2 expression through alternative promoters, splicing and polyadenylation sites in zebrafish. Three functional R2 promoters were identified to generate six transcript variants with distinct 5′ termini. The proximal promoter contains a conserved E2F binding site and two CCAAT boxes, which are crucial for the transcription of R2 gene during cell cycle. Activity of the distal promoter can be induced by DNA damage to generate four transcript variants through alternative splicing. In addition, two novel splice variants were found to encode distinct N-truncated R2 isoforms containing residues for enzymatic activity but no KEN box essential for its proteolysis. These two N-truncated R2 isoforms remained in the cytoplasm and were able to interact with RNR M1 subunit (R1). Thus, our results suggest that multilayered mechanisms control the differential expression and function of zebrafish R2 gene during cell cycle and under genotoxic stress. PMID:21887375

Identification and characterization of alternative promoters, transcripts and protein isoforms of zebrafish R2 gene.

PubMed

Shang, Hanqiao; Li, Qing; Feng, Guohui; Cui, Zongbin

2011-01-01

Ribonucleotide reductase (RNR) is the rate-limiting enzyme in the de novo synthesis of deoxyribonucleoside triphosphates. Expression of RNR subunits is closely associated with DNA replication and repair. Mammalian RNR M2 subunit (R2) functions exclusively in DNA replication of normal cells due to its S phase-specific expression and late mitotic degradation. Herein, we demonstrate the control of R2 expression through alternative promoters, splicing and polyadenylation sites in zebrafish. Three functional R2 promoters were identified to generate six transcript variants with distinct 5' termini. The proximal promoter contains a conserved E2F binding site and two CCAAT boxes, which are crucial for the transcription of R2 gene during cell cycle. Activity of the distal promoter can be induced by DNA damage to generate four transcript variants through alternative splicing. In addition, two novel splice variants were found to encode distinct N-truncated R2 isoforms containing residues for enzymatic activity but no KEN box essential for its proteolysis. These two N-truncated R2 isoforms remained in the cytoplasm and were able to interact with RNR M1 subunit (R1). Thus, our results suggest that multilayered mechanisms control the differential expression and function of zebrafish R2 gene during cell cycle and under genotoxic stress.

QuASAR-MPRA: accurate allele-specific analysis for massively parallel reporter assays.

PubMed

Kalita, Cynthia A; Moyerbrailean, Gregory A; Brown, Christopher; Wen, Xiaoquan; Luca, Francesca; Pique-Regi, Roger

2018-03-01

The majority of the human genome is composed of non-coding regions containing regulatory elements such as enhancers, which are crucial for controlling gene expression. Many variants associated with complex traits are in these regions, and may disrupt gene regulatory sequences. Consequently, it is important to not only identify true enhancers but also to test if a variant within an enhancer affects gene regulation. Recently, allele-specific analysis in high-throughput reporter assays, such as massively parallel reporter assays (MPRAs), have been used to functionally validate non-coding variants. However, we are still missing high-quality and robust data analysis tools for these datasets. We have further developed our method for allele-specific analysis QuASAR (quantitative allele-specific analysis of reads) to analyze allele-specific signals in barcoded read counts data from MPRA. Using this approach, we can take into account the uncertainty on the original plasmid proportions, over-dispersion, and sequencing errors. The provided allelic skew estimate and its standard error also simplifies meta-analysis of replicate experiments. Additionally, we show that a beta-binomial distribution better models the variability present in the allelic imbalance of these synthetic reporters and results in a test that is statistically well calibrated under the null. Applying this approach to the MPRA data, we found 602 SNPs with significant (false discovery rate 10%) allele-specific regulatory function in LCLs. We also show that we can combine MPRA with QuASAR estimates to validate existing experimental and computational annotations of regulatory variants. Our study shows that with appropriate data analysis tools, we can improve the power to detect allelic effects in high-throughput reporter assays. http://github.com/piquelab/QuASAR/tree/master/mpra. fluca@wayne.edu or rpique@wayne.edu. Supplementary data are available online at Bioinformatics. © The Author (2017). Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com

REDO: RNA Editing Detection in Plant Organelles Based on Variant Calling Results.

PubMed

Wu, Shuangyang; Liu, Wanfei; Aljohi, Hasan Awad; Alromaih, Sarah A; Alanazi, Ibrahim O; Lin, Qiang; Yu, Jun; Hu, Songnian

2018-05-01

RNA editing is a post-transcriptional or cotranscriptional process that changes the sequence of the precursor transcript by substitutions, insertions, or deletions. Almost all of the land plants undergo RNA editing in organelles (plastids and mitochondria). Although several software tools have been developed to identify RNA editing events, there has been a great challenge to distinguish true RNA editing events from genome variation, sequencing errors, and other factors. Here we introduce REDO, a comprehensive application tool for identifying RNA editing events in plant organelles based on variant call format files from RNA-sequencing data. REDO is a suite of Perl scripts that illustrate a bunch of attributes of RNA editing events in figures and tables. REDO can also detect RNA editing events in multiple samples simultaneously and identify the significant differential proportion of RNA editing loci. Comparing with similar tools, such as REDItools, REDO runs faster with higher accuracy, and more specificity at the cost of slightly lower sensitivity. Moreover, REDO annotates each RNA editing site in RNAs, whereas REDItools reports only possible RNA editing sites in genome, which need additional steps to obtain RNA editing profiles for RNAs. Overall, REDO can identify potential RNA editing sites easily and provide several functions such as detailed annotations, statistics, figures, and significantly differential proportion of RNA editing sites among different samples.

Correlations of carotenoid content and transcript abundances for fibrillin and carotenogenic enzymes in Capsicum annum fruit pericarp.

PubMed

Kilcrease, James; Rodriguez-Uribe, Laura; Richins, Richard D; Arcos, Juan Manuel Garcia; Victorino, Jesus; O'Connell, Mary A

2015-03-01

The fruits of Capsicum spp. are especially rich sites for carotenoid synthesis and accumulation, with cultivar-specific carotenoid accumulation profiles. Differences in chromoplast structure as well as carotenoid biosynthesis are correlated with distinct carotenoid accumulations and fruit color. In the present study, the inheritance of chromoplast shape, carotenoid accumulation profiles, and transcript levels of four genes were measured. Comparisons of these traits were conducted using fruit from contrasting variants, Costeño Amarillo versus Costeño Red, and from F1 hybrids; crosses between parental lines with novel versions of these traits. Intermediate chromoplast shapes were observed in the F1, but no association between specific carotenoid accumulation and chromoplast shape was detected. Increased total carotenoid content was associated with increased β-carotene and violaxanthin content. Transcript levels for phytoene synthase (Psy) and β-carotene hydroxylase (CrtZ-2) were positively correlated with increased levels of specific carotenoids. No correlation was detected between transcript levels of capsanthin/capsorubin synthase (Ccs) and carotenoid composition or chromoplast shape. Transcript levels of fibrillin, were differentially correlated with specific carotenoids, negatively correlated with accumulation of capsanthin, and positively correlated with violaxanthin. The regulation of carotenoid accumulation in chromoplasts in Capsicum fruit continues to be a complex process with multiple steps for control. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Simultaneous targeting of prostate stem cell antigen and prostate-specific membrane antigen improves the killing of prostate cancer cells using a novel modular T cell-retargeting system.

PubMed

Arndt, Claudia; Feldmann, Anja; Koristka, Stefanie; Cartellieri, Marc; Dimmel, Maria; Ehninger, Armin; Ehninger, Gerhard; Bachmann, Michael

2014-09-01

Recently, we described a novel modular platform technology in which T cell-recruitment and tumor-targeting domains of conventional bispecific antibodies are split to independent components, a universal effector module (EM) and replaceable monospecific/monovalent target modules (TMs) that form highly efficient T cell-retargeting complexes. Theoretically, our unique strategy should allow us to simultaneously retarget T cells to different tumor antigens by combining the EM with two or more different monovalent/monospecific TMs or even with bivalent/bispecific TMs, thereby overcoming limitations of a monospecific treatment such as the selection of target-negative tumor escape variants. In order to advance our recently introduced prostate stem cell antigen (PSCA)-specific modular system for a dual-targeting of prostate cancer cells, two additional TMs were constructed: a monovalent/monospecific TM directed against the prostate-specific membrane antigen (PSMA) and a bivalent/bispecific TM (bsTM) with specificity for PSMA and PSCA. The functionality of the novel dual-targeting strategies was analyzed by performing T cell activation and chromium release assays. Similar to the PSCA-specific modular system, the novel PSMA-specific modular system mediates an efficient target-dependent and -specific tumor cell lysis at low E:T ratios and picomolar Ab concentrations. Moreover, by combination of the EM with either the bispecific TM directed to PSMA and PSCA or both monospecifc TMs directed to either PSCA or PSMA, dual-specific targeting complexes were formed which allowed us to kill potential escape variants expressing only one or the other target antigen. Overall, the novel modular system represents a promising tool for multiple tumor targeting. © 2014 Wiley Periodicals, Inc.

Mutants of Cre recombinase with improved accuracy

PubMed Central

Eroshenko, Nikolai; Church, George M.

2013-01-01

Despite rapid advances in genome engineering technologies, inserting genes into precise locations in the human genome remains an outstanding problem. It has been suggested that site-specific recombinases can be adapted towards use as transgene delivery vectors. The specificity of recombinases can be altered either with directed evolution or via fusions to modular DNA-binding domains. Unfortunately, both wildtype and altered variants often have detectable activities at off-target sites. Here we use bacterial selections to identify mutations in the dimerization surface of Cre recombinase (R32V, R32M, and 303GVSdup) that improve the accuracy of recombination. The mutants are functional in bacteria, in human cells, and in vitro (except for 303GVSdup, which we did not purify), and have improved selectivity against both model off-target sites and the entire E. coli genome. We propose that destabilizing binding cooperativity may be a general strategy for improving the accuracy of dimeric DNA-binding proteins. PMID:24056590

Natural gene expression variation studies in yeast.

PubMed

Thompson, Dawn A; Cubillos, Francisco A

2017-01-01

The rise of sequence information across different yeast species and strains is driving an increasing number of studies in the emerging field of genomics to associate polymorphic variants, mRNA abundance and phenotypic differences between individuals. Here, we gathered evidence from recent studies covering several layers that define the genotype-phenotype gap, such as mRNA abundance, allele-specific expression and translation efficiency to demonstrate how genetic variants co-evolve and define an individual's genome. Moreover, we exposed several antecedents where inter- and intra-specific studies led to opposite conclusions, probably owing to genetic divergence. Future studies in this area will benefit from the access to a massive array of well-annotated genomes and new sequencing technologies, which will allow the fine breakdown of the complex layers that delineate the genotype-phenotype map. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Beyond genome-wide association studies: genetic heterogeneity and individual predisposition to cancer

PubMed Central

Galvan, Antonella; Ioannidis, John P.A.; Dragani, Tommaso A.

2010-01-01

Genome-wide association studies (GWAS) using population-based designs have identified many genetic loci associated with risk of a range of complex diseases including cancer; however, each locus exerts a very small effect and most heritability remains unexplained. Family-based pedigree studies have also suggested tentative loci linked to increased cancer risk, often characterized by pedigree-specificity. However, a comparison between the results of population-and those of family-based studies shows little concordance. Explanations for this unidentified genetic ‘dark matter’ of cancer include phenotype ascertainment issues, limited power, gene-gene and gene-environment interactions, population heterogeneity, parent-of-origin-specific effects, rare and unexplored variants. Many of these reasons converge towards the concept of genetic heterogeneity that might implicate hundreds of genetic variants in regulating cancer risk. Dissecting the dark matter is a challenging task. Further insights can be gained from both population association and pedigree studies. PMID:20106545

Genetic code expansion for multiprotein complex engineering.

PubMed

Koehler, Christine; Sauter, Paul F; Wawryszyn, Mirella; Girona, Gemma Estrada; Gupta, Kapil; Landry, Jonathan J M; Fritz, Markus Hsi-Yang; Radic, Ksenija; Hoffmann, Jan-Erik; Chen, Zhuo A; Zou, Juan; Tan, Piau Siong; Galik, Bence; Junttila, Sini; Stolt-Bergner, Peggy; Pruneri, Giancarlo; Gyenesei, Attila; Schultz, Carsten; Biskup, Moritz Bosse; Besir, Hueseyin; Benes, Vladimir; Rappsilber, Juri; Jechlinger, Martin; Korbel, Jan O; Berger, Imre; Braese, Stefan; Lemke, Edward A

2016-12-01

We present a baculovirus-based protein engineering method that enables site-specific introduction of unique functionalities in a eukaryotic protein complex recombinantly produced in insect cells. We demonstrate the versatility of this efficient and robust protein production platform, 'MultiBacTAG', (i) for the fluorescent labeling of target proteins and biologics using click chemistries, (ii) for glycoengineering of antibodies, and (iii) for structure-function studies of novel eukaryotic complexes using single-molecule Förster resonance energy transfer as well as site-specific crosslinking strategies.

Emerging applications of genome-editing technology to examine functionality of GWAS-associated variants for complex traits.

PubMed

Smith, Andrew J P; Deloukas, Panos; Munroe, Patricia B

2018-04-13

Over the last decade, genome-wide association studies (GWAS) have propelled the discovery of thousands of loci associated with complex diseases. The focus is now turning towards the function of these association signals, determining the causal variant(s) amongst those in strong linkage disequilibrium, and identifying their underlying mechanisms, such as long-range gene regulation. Genome-editing techniques utilising zinc-finger nucleases (ZFN), transcription activator-like effector nucleases (TALENs) and clustered regularly-interspaced short palindromic repeats with Cas9 nuclease (CRISPR-Cas9), are becoming the tools of choice to establish functionality for these variants, due to the ability to assess effects of single variants in vivo. This review will discuss examples of how these technologies have begun to aid functional analysis of GWAS loci for complex traits such as cardiovascular disease, type 2 diabetes, cancer, obesity and autoimmune disease. We focus on analysis of variants occurring within non-coding genomic regions, as these comprise the majority of GWAS variants, providing the greatest challenges to determining functionality, and compare editing strategies that provide different levels of evidence for variant functionality. The review describes molecular insights into some of these potentially causal variants, and how these may relate to the pathology of the trait, and look towards future directions for these technologies in post-GWAS analysis, such as base-editing.

Mapping replication dynamics in Trypanosoma brucei reveals a link with telomere transcription and antigenic variation

PubMed Central

Devlin, Rebecca; Marques, Catarina A; Paape, Daniel; Prorocic, Marko; Zurita-Leal, Andrea C; Campbell, Samantha J; Lapsley, Craig; Dickens, Nicholas; McCulloch, Richard

2016-01-01

Survival of Trypanosoma brucei depends upon switches in its protective Variant Surface Glycoprotein (VSG) coat by antigenic variation. VSG switching occurs by frequent homologous recombination, which is thought to require locus-specific initiation. Here, we show that a RecQ helicase, RECQ2, acts to repair DNA breaks, including in the telomeric site of VSG expression. Despite this, RECQ2 loss does not impair antigenic variation, but causes increased VSG switching by recombination, arguing against models for VSG switch initiation through direct generation of a DNA double strand break (DSB). Indeed, we show DSBs inefficiently direct recombination in the VSG expression site. By mapping genome replication dynamics, we reveal that the transcribed VSG expression site is the only telomeric site that is early replicating – a differential timing only seen in mammal-infective parasites. Specific association between VSG transcription and replication timing reveals a model for antigenic variation based on replication-derived DNA fragility. DOI: http://dx.doi.org/10.7554/eLife.12765.001 PMID:27228154

A thiamin-bound, pre-decarboxylation reaction intermediate analogue in the pyruvate dehydrogenase E1 subunit induces large scale disorder-to-order transformations in the enzyme and reveals novel structural features in the covalently bound adduct.

PubMed

Arjunan, Palaniappa; Sax, Martin; Brunskill, Andrew; Chandrasekhar, Krishnamoorthy; Nemeria, Natalia; Zhang, Sheng; Jordan, Frank; Furey, William

2006-06-02

The crystal structure of the E1 component from the Escherichia coli pyruvate dehydrogenase multienzyme complex (PDHc) has been determined with phosphonolactylthiamin diphosphate (PLThDP) in its active site. PLThDP serves as a structural and electrostatic analogue of the natural intermediate alpha-lactylthiamin diphosphate (LThDP), in which the carboxylate from the natural substrate pyruvate is replaced by a phosphonate group. This represents the first example of an experimentally determined, three-dimensional structure of a thiamin diphosphate (ThDP)-dependent enzyme containing a covalently bound, pre-decarboxylation reaction intermediate analogue and should serve as a model for the corresponding intermediates in other ThDP-dependent decarboxylases. Regarding the PDHc-specific reaction, the presence of PLThDP induces large scale conformational changes in the enzyme. In conjunction with the E1-PLThDP and E1-ThDP structures, analysis of a H407A E1-PLThDP variant structure shows that an interaction between His-407 and PLThDP is essential for stabilization of two loop regions in the active site that are otherwise disordered in the absence of intermediate analogue. This ordering completes formation of the active site and creates a new ordered surface likely involved in interactions with the lipoyl domains of E2s within the PDHc complex. The tetrahedral intermediate analogue is tightly held in the active site through direct hydrogen bonds to residues His-407, Tyr-599, and His-640 and reveals a new, enzyme-induced, strain-related feature that appears to aid in the decarboxylation process. This feature is almost certainly present in all ThDP-dependent decarboxylases; thus its inclusion in our understanding of general thiamin catalysis is important.

Common Variants in Mendelian Kidney Disease Genes and Their Association with Renal Function

PubMed Central

Fuchsberger, Christian; Köttgen, Anna; O’Seaghdha, Conall M.; Pattaro, Cristian; de Andrade, Mariza; Chasman, Daniel I.; Teumer, Alexander; Endlich, Karlhans; Olden, Matthias; Chen, Ming-Huei; Tin, Adrienne; Kim, Young J.; Taliun, Daniel; Li, Man; Feitosa, Mary; Gorski, Mathias; Yang, Qiong; Hundertmark, Claudia; Foster, Meredith C.; Glazer, Nicole; Isaacs, Aaron; Rao, Madhumathi; Smith, Albert V.; O’Connell, Jeffrey R.; Struchalin, Maksim; Tanaka, Toshiko; Li, Guo; Hwang, Shih-Jen; Atkinson, Elizabeth J.; Lohman, Kurt; Cornelis, Marilyn C.; Johansson, Åsa; Tönjes, Anke; Dehghan, Abbas; Couraki, Vincent; Holliday, Elizabeth G.; Sorice, Rossella; Kutalik, Zoltan; Lehtimäki, Terho; Esko, Tõnu; Deshmukh, Harshal; Ulivi, Sheila; Chu, Audrey Y.; Murgia, Federico; Trompet, Stella; Imboden, Medea; Kollerits, Barbara; Pistis, Giorgio; Harris, Tamara B.; Launer, Lenore J.; Aspelund, Thor; Eiriksdottir, Gudny; Mitchell, Braxton D.; Boerwinkle, Eric; Schmidt, Helena; Hofer, Edith; Hu, Frank; Demirkan, Ayse; Oostra, Ben A.; Turner, Stephen T.; Ding, Jingzhong; Andrews, Jeanette S.; Freedman, Barry I.; Giulianini, Franco; Koenig, Wolfgang; Illig, Thomas; Döring, Angela; Wichmann, H.-Erich; Zgaga, Lina; Zemunik, Tatijana; Boban, Mladen; Minelli, Cosetta; Wheeler, Heather E.; Igl, Wilmar; Zaboli, Ghazal; Wild, Sarah H.; Wright, Alan F.; Campbell, Harry; Ellinghaus, David; Nöthlings, Ute; Jacobs, Gunnar; Biffar, Reiner; Ernst, Florian; Homuth, Georg; Kroemer, Heyo K.; Nauck, Matthias; Stracke, Sylvia; Völker, Uwe; Völzke, Henry; Kovacs, Peter; Stumvoll, Michael; Mägi, Reedik; Hofman, Albert; Uitterlinden, Andre G.; Rivadeneira, Fernando; Aulchenko, Yurii S.; Polasek, Ozren; Hastie, Nick; Vitart, Veronique; Helmer, Catherine; Wang, Jie Jin; Stengel, Bénédicte; Ruggiero, Daniela; Bergmann, Sven; Kähönen, Mika; Viikari, Jorma; Nikopensius, Tiit; Province, Michael; Colhoun, Helen; Doney, Alex; Robino, Antonietta; Krämer, Bernhard K.; Portas, Laura; Ford, Ian; Buckley, Brendan M.; Adam, Martin; Thun, Gian-Andri; Paulweber, Bernhard; Haun, Margot; Sala, Cinzia; Mitchell, Paul; Ciullo, Marina; Vollenweider, Peter; Raitakari, Olli; Metspalu, Andres; Palmer, Colin; Gasparini, Paolo; Pirastu, Mario; Jukema, J. Wouter; Probst-Hensch, Nicole M.; Kronenberg, Florian; Toniolo, Daniela; Gudnason, Vilmundur; Shuldiner, Alan R.; Coresh, Josef; Schmidt, Reinhold; Ferrucci, Luigi; van Duijn, Cornelia M.; Borecki, Ingrid; Kardia, Sharon L.R.; Liu, Yongmei; Curhan, Gary C.; Rudan, Igor; Gyllensten, Ulf; Wilson, James F.; Franke, Andre; Pramstaller, Peter P.; Rettig, Rainer; Prokopenko, Inga; Witteman, Jacqueline; Hayward, Caroline; Ridker, Paul M.; Bochud, Murielle; Heid, Iris M.; Siscovick, David S.; Fox, Caroline S.; Kao, W. Linda; Böger, Carsten A.

2013-01-01

Many common genetic variants identified by genome-wide association studies for complex traits map to genes previously linked to rare inherited Mendelian disorders. A systematic analysis of common single-nucleotide polymorphisms (SNPs) in genes responsible for Mendelian diseases with kidney phenotypes has not been performed. We thus developed a comprehensive database of genes for Mendelian kidney conditions and evaluated the association between common genetic variants within these genes and kidney function in the general population. Using the Online Mendelian Inheritance in Man database, we identified 731 unique disease entries related to specific renal search terms and confirmed a kidney phenotype in 218 of these entries, corresponding to mutations in 258 genes. We interrogated common SNPs (minor allele frequency >5%) within these genes for association with the estimated GFR in 74,354 European-ancestry participants from the CKDGen Consortium. However, the top four candidate SNPs (rs6433115 at LRP2, rs1050700 at TSC1, rs249942 at PALB2, and rs9827843 at ROBO2) did not achieve significance in a stage 2 meta-analysis performed in 56,246 additional independent individuals, indicating that these common SNPs are not associated with estimated GFR. The effect of less common or rare variants in these genes on kidney function in the general population and disease-specific cohorts requires further research. PMID:24029420

The dual-function chaperone HycH improves assembly of the formate hydrogenlyase complex.

PubMed

Lindenstrauß, Ute; Skorupa, Philipp; McDowall, Jennifer S; Sargent, Frank; Pinske, Constanze

2017-08-11

The assembly of multi-protein complexes requires the concerted synthesis and maturation of its components and subsequently their co-ordinated interaction. The membrane-bound formate hydrogenlyase (FHL) complex is the primary hydrogen-producing enzyme in Escherichia coli and is composed of seven subunits mostly encoded within the hycA-I operon for [NiFe]-hydrogenase-3 (Hyd-3). The HycH protein is predicted to have an accessory function and is not part of the final structural FHL complex. In this work, a mutant strain devoid of HycH was characterised and found to have significantly reduced FHL activity due to the instability of the electron transfer subunits. HycH was shown to interact specifically with the unprocessed species of HycE, the catalytic hydrogenase subunit of the FHL complex, at different stages during the maturation and assembly of the complex. Variants of HycH were generated with the aim of identifying interacting residues and those that influence activity. The R70/71/K72, the Y79, the E81 and the Y128 variant exchanges interrupt the interaction with HycE without influencing the FHL activity. In contrast, FHL activity, but not the interaction with HycE, was negatively influenced by H37 exchanges with polar residues. Finally, a HycH Y30 variant was unstable. Surprisingly, an overlapping function between HycH with its homologous counterpart HyfJ from the operon encoding [NiFe]-hydrogenase-4 (Hyd-4) was identified and this is the first example of sharing maturation machinery components between Hyd-3 and Hyd-4 complexes. The data presented here show that HycH has a novel dual role as an assembly chaperone for a cytoplasmic [NiFe]-hydrogenase. © 2017 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

Discovery and characterization of antibody variants using mass spectrometry-based comparative analysis for biosimilar candidates of monoclonal antibody drugs.

PubMed

Li, Wenhua; Yang, Bin; Zhou, Dongmei; Xu, Jun; Ke, Zhi; Suen, Wen-Chen

2016-07-01

Liquid chromatography mass spectrometry (LC-MS) is the most commonly used technique for the characterization of antibody variants. MAb-X and mAb-Y are two approved IgG1 subtype monoclonal antibody drugs recombinantly produced in Chinese hamster ovary (CHO) cells. We report here that two unexpected and rare antibody variants have been discovered during cell culture process development of biosimilars for these two approved drugs through intact mass analysis. We then used comprehensive mass spectrometry-based comparative analysis including reduced light, heavy chains, and domain-specific mass as well as peptide mapping analysis to fully characterize the observed antibody variants. The "middle-up" mass comparative analysis demonstrated that the antibody variant from mAb-X biosimilar candidate was caused by mass variation of antibody crystalline fragment (Fc), whereas a different variant with mass variation in antibody antigen-binding fragment (Fab) from mAb-Y biosimilar candidate was identified. Endoproteinase Lys-C digested peptide mapping and tandem mass spectrometry analysis further revealed that a leucine to glutamine change in N-terminal 402 site of heavy chain was responsible for the generation of mAb-X antibody variant. Lys-C and trypsin coupled non-reduced and reduced peptide mapping comparative analysis showed that the formation of the light-heavy interchain trisulfide bond resulted in the mAb-Y antibody variant. These two cases confirmed that mass spectrometry-based comparative analysis plays a critical role for the characterization of monoclonal antibody variants, and biosimilar developers should start with a comprehensive structural assessment and comparative analysis to decrease the risk of the process development for biosimilars. Copyright © 2016 Elsevier B.V. All rights reserved.

Validation and optimization of the Ion Torrent S5 XL sequencer and Oncomine workflow for BRCA1 and BRCA2 genetic testing.

PubMed

Shin, Saeam; Kim, Yoonjung; Chul Oh, Seoung; Yu, Nae; Lee, Seung-Tae; Rak Choi, Jong; Lee, Kyung-A

2017-05-23

In this study, we validated the analytical performance of BRCA1/2 sequencing using Ion Torrent's new bench-top sequencer with amplicon panel with optimized bioinformatics pipelines. Using 43 samples that were previously validated by Illumina's MiSeq platform and/or by Sanger sequencing/multiplex ligation-dependent probe amplification, we amplified the target with the Oncomine™ BRCA Research Assay and sequenced on Ion Torrent S5 XL (Thermo Fisher Scientific, Waltham, MA, USA). We compared two bioinformatics pipelines for optimal processing of S5 XL sequence data: the Torrent Suite with a plug-in Torrent Variant Caller (Thermo Fisher Scientific), and commercial NextGENe software (Softgenetics, State College, PA, USA). All expected 681 single nucleotide variants, 15 small indels, and three copy number variants were correctly called, except one common variant adjacent to a rare variant on the primer-binding site. The sensitivity, specificity, false positive rate, and accuracy for detection of single nucleotide variant and small indels of S5 XL sequencing were 99.85%, 100%, 0%, and 99.99% for the Torrent Variant Caller and 99.85%, 99.99%, 0.14%, and 99.99% for NextGENe, respectively. The reproducibility of variant calling was 100%, and the precision of variant frequency also showed good performance with coefficients of variation between 0.32 and 5.29%. We obtained highly accurate data through uniform and sufficient coverage depth over all target regions and through optimization of the bioinformatics pipeline. We confirmed that our platform is accurate and practical for diagnostic BRCA1/2 testing in a clinical laboratory.

Nuclear Import of β-Dystroglycan Is Facilitated by Ezrin-Mediated Cytoskeleton Reorganization

PubMed Central

Vásquez-Limeta, Alejandra; Wagstaff, Kylie M.; Ortega, Arturo; Crouch, Dorothy H.; Jans, David A.; Cisneros, Bulmaro

2014-01-01

The β-dystroglycan (β-DG) protein has the ability to target to multiple sites in eukaryotic cells, being a member of diverse protein assemblies including the transmembranal dystrophin-associated complex, and a nuclear envelope-localised complex that contains emerin and lamins A/C and B1. We noted that the importin α2/β1-recognised nuclear localization signal (NLS) of β-DG is also a binding site for the cytoskeletal-interacting protein ezrin, and set out to determine whether ezrin binding might modulate β-DG nuclear translocation for the first time. Unexpectedly, we found that ezrin enhances rather than inhibits β-DG nuclear translocation in C2C12 myoblasts. Both overexpression of a phosphomimetic activated ezrin variant (Ez-T567D) and activation of endogenous ezrin through stimulation of the Rho pathway resulted in both formation of actin-rich surface protrusions and significantly increased nuclear translocation of β-DG as shown by quantitative microscopy and subcellular fractionation/Western analysis. In contrast, overexpression of a nonphosphorylatable inactive ezrin variant (Ez-T567A) or inhibition of Rho signaling, decreased nuclear translocation of β-DG concomitant with a lack of cell surface protrusions. Further, a role for the actin cytoskeleton in ezrin enhancement of β-DG nuclear translocation was implicated by the observation that an ezrin variant lacking its actin-binding domain failed to enhance nuclear translocation of β-DG, while disruption of the actin cytoskeleton led to a reduction in β-DG nuclear localization. Finally, we show that ezrin-mediated cytoskeletal reorganization enhances nuclear translocation of the cytoplasmic but not the transmembranal fraction of β-DG. This is the first study showing that cytoskeleton reorganization can modulate nuclear translocation of β-DG, with the implication that β-DG can respond to cytoskeleton-driven changes in cell morphology by translocating from the cytoplasm to the nucleus to orchestrate nuclear processes in response to the functional requirements of the cell. PMID:24599031

Clinical Applications of Molecular Genetic Discoveries

PubMed Central

Marian, A.J.

2015-01-01

Genome-wide association studies (GWAS) of complex traits have mapped more than 15,000 common single nucleotide variants (SNVs). Likewise, applications of massively parallel nucleic acid sequencing technologies often referred to as Next Generation Sequencing, to molecular genetic studies of complex traits have catalogued a large number of rare variants (population frequency of <0.01) in cases with complex traits. Moreover, high throughput nucleic acid sequencing, variant burden analysis, and linkage studies are illuminating the presence of large number of SNVs in cases and families with single gene disorders. The plethora of the genetic variants has exposed the formidable challenge of identifying the causal and pathogenic variants from the enormous number of innocuous common and rare variants that exist in the population as well as in an individual genome. The arduous task of identifying the causal and pathogenic variants is further compounded by the pleiotropic effects of the variants, complexity of cis and trans interactions in the genome, variability in phenotypic expression of the disease, as well as phenotypic plasticity, and the multifarious determinants of the phenotype. Population genetic studies offer the initial roadmaps and have the potential to elucidate novel pathways involved in the pathogenesis of the disease. However, the genome of an individual is unique, rendering unambiguous identification of the causal or pathogenic variant in a single individual exceedingly challenging. Yet, the focus of the practice of medicine is on the individual, as Sir William Osler elegantly expressed in his insightful quotation: “The good physician treats the disease; the great physician treats the patient who has the disease.” The daunting task facing physicians, patients, and researchers alike is to apply the modern genetic discoveries to care of the individual with or at risk of the disease. PMID:26548329

Assessing the spectrum of germline variation in Fanconi anemia genes among patients with head and neck carcinoma before age 50.

PubMed

Chandrasekharappa, Settara C; Chinn, Steven B; Donovan, Frank X; Chowdhury, Naweed I; Kamat, Aparna; Adeyemo, Adebowale A; Thomas, James W; Vemulapalli, Meghana; Hussey, Caroline S; Reid, Holly H; Mullikin, James C; Wei, Qingyi; Sturgis, Erich M

2017-10-15

Patients with Fanconi anemia (FA) have an increased risk for head and neck squamous cell carcinoma (HNSCC). The authors sought to determine the prevalence of undiagnosed FA and FA carriers among patients with HNSCC as well as an age cutoff for FA genetic screening. Germline DNA samples from 417 patients with HNSCC aged <50 years were screened for sequence variants by targeted next-generation sequencing of the entire length of 16 FA genes. The sequence revealed 194 FA gene variants in 185 patients (44%). The variant spectrum was comprised of 183 nonsynonymous point mutations, 9 indels, 1 large deletion, and 1 synonymous variant that was predicted to effect splicing. One hundred eight patients (26%) had at least 1 rare variant that was predicted to be damaging, and 57 (14%) had at least 1 rare variant that was predicted to be damaging and had been previously reported. Fifteen patients carried 2 rare variants or an X-linked variant in an FA gene. Overall, an age cutoff for FA screening was not identified among young patients with HNSCC, because there were no significant differences in mutation rates when patients were stratified by age, tumor site, ethnicity, smoking status, or human papillomavirus status. However, an increased burden, or mutation load, of FA gene variants was observed in carriers of the genes FA complementation group D2 (FANCD2), FANCE, and FANCL in the HNSCC patient cohort relative to the 1000 Genomes population. FA germline functional variants offer a novel area of study in HNSCC tumorigenesis. FANCE and FANCL, which are components of the core complex, are known to be responsible for the recruitment and ubiquitination, respectively, of FANCD2, a critical step in the FA DNA repair pathway. In the current cohort, the increased mutation load of FANCD2, FANCE, and FANCL variants among younger patients with HNSCC indicates the importance of the FA pathway in HNSCC. Cancer 2017;123:3943-54. © 2017 American Cancer Society. © 2017 American Cancer Society.

An In-Depth Characterization of the Major Psoriasis Susceptibility Locus Identifies Candidate Susceptibility Alleles within an HLA-C Enhancer Element

PubMed Central

Clop, Alex; Bertoni, Anna; Spain, Sarah L.; Simpson, Michael A.; Pullabhatla, Venu; Tonda, Raul; Hundhausen, Christian; Di Meglio, Paola; De Jong, Pieter; Hayday, Adrian C.; Nestle, Frank O.; Barker, Jonathan N.; Bell, Robert J. A.; Capon, Francesca; Trembath, Richard C.

2013-01-01

Psoriasis is an immune-mediated skin disorder that is inherited as a complex genetic trait. Although genome-wide association scans (GWAS) have identified 36 disease susceptibility regions, more than 50% of the genetic variance can be attributed to a single Major Histocompatibility Complex (MHC) locus, known as PSORS1. Genetic studies indicate that HLA-C is the strongest PSORS1 candidate gene, since markers tagging HLA-Cw*0602 consistently generate the most significant association signals in GWAS. However, it is unclear whether HLA-Cw*0602 is itself the causal PSORS1 allele, especially as the role of SNPs that may affect its expression has not been investigated. Here, we have undertaken an in-depth molecular characterization of the PSORS1 interval, with a view to identifying regulatory variants that may contribute to disease susceptibility. By analysing high-density SNP data, we refined PSORS1 to a 179 kb region encompassing HLA-C and the neighbouring HCG27 pseudogene. We compared multiple MHC sequences spanning this refined locus and identified 144 candidate susceptibility variants, which are unique to chromosomes bearing HLA-Cw*0602. In parallel, we investigated the epigenetic profile of the critical PSORS1 interval and uncovered three enhancer elements likely to be active in T lymphocytes. Finally we showed that nine candidate susceptibility SNPs map within a HLA-C enhancer and that three of these variants co-localise with binding sites for immune-related transcription factors. These data indicate that SNPs affecting HLA-Cw*0602 expression are likely to contribute to psoriasis susceptibility and highlight the importance of integrating multiple experimental approaches in the investigation of complex genomic regions such as the MHC. PMID:23990973

Analysis of Nidogen-1/Laminin γ1 Interaction by Cross-Linking, Mass Spectrometry, and Computational Modeling Reveals Multiple Binding Modes

PubMed Central

Lössl, Philip; Kölbel, Knut; Tänzler, Dirk; Nannemann, David; Ihling, Christian H.; Keller, Manuel V.; Schneider, Marian; Zaucke, Frank; Meiler, Jens; Sinz, Andrea

2014-01-01

We describe the detailed structural investigation of nidogen-1/laminin γ1 complexes using full-length nidogen-1 and a number of laminin γ1 variants. The interactions of nidogen-1 with laminin variants γ1 LEb2–4, γ1 LEb2–4 N836D, γ1 short arm, and γ1 short arm N836D were investigated by applying a combination of (photo-)chemical cross-linking, high-resolution mass spectrometry, and computational modeling. In addition, surface plasmon resonance and ELISA studies were used to determine kinetic constants of the nidogen-1/laminin γ1 interaction. Two complementary cross-linking strategies were pursued to analyze solution structures of laminin γ1 variants and nidogen-1. The majority of distance information was obtained with the homobifunctional amine-reactive cross-linker bis(sulfosuccinimidyl)glutarate. In a second approach, UV-induced cross-linking was performed after incorporation of the diazirine-containing unnatural amino acids photo-leucine and photo-methionine into laminin γ1 LEb2–4, laminin γ1 short arm, and nidogen-1. Our results indicate that Asn-836 within laminin γ1 LEb3 domain is not essential for complex formation. Cross-links between laminin γ1 short arm and nidogen-1 were found in all protein regions, evidencing several additional contact regions apart from the known interaction site. Computational modeling based on the cross-linking constraints indicates the existence of a conformational ensemble of both the individual proteins and the nidogen-1/laminin γ1 complex. This finding implies different modes of interaction resulting in several distinct protein-protein interfaces. PMID:25387007

Tunable allosteric library of caspase-3 identifies coupling between conserved water molecules and conformational selection

PubMed Central

Maciag, Joseph J.; Mackenzie, Sarah H.; Tucker, Matthew B.; Schipper, Joshua L.; Swartz, Paul; Clark, A. Clay

2016-01-01

The native ensemble of caspases is described globally by a complex energy landscape where the binding of substrate selects for the active conformation, whereas targeting an allosteric site in the dimer interface selects an inactive conformation that contains disordered active-site loops. Mutations and posttranslational modifications stabilize high-energy inactive conformations, with mostly formed, but distorted, active sites. To examine the interconversion of active and inactive states in the ensemble, we used detection of related solvent positions to analyze 4,995 waters in 15 high-resolution (<2.0 Å) structures of wild-type caspase-3, resulting in 450 clusters with the most highly conserved set containing 145 water molecules. The data show that regions of the protein that contact the conserved waters also correspond to sites of posttranslational modifications, suggesting that the conserved waters are an integral part of allosteric mechanisms. To test this hypothesis, we created a library of 19 caspase-3 variants through saturation mutagenesis in a single position of the allosteric site of the dimer interface, and we show that the enzyme activity varies by more than four orders of magnitude. Altogether, our database consists of 37 high-resolution structures of caspase-3 variants, and we demonstrate that the decrease in activity correlates with a loss of conserved water molecules. The data show that the activity of caspase-3 can be fine-tuned through globally desolvating the active conformation within the native ensemble, providing a mechanism for cells to repartition the ensemble and thus fine-tune activity through conformational selection. PMID:27681633

Tunable allosteric library of caspase-3 identifies coupling between conserved water molecules and conformational selection.

PubMed

Maciag, Joseph J; Mackenzie, Sarah H; Tucker, Matthew B; Schipper, Joshua L; Swartz, Paul; Clark, A Clay

2016-10-11

The native ensemble of caspases is described globally by a complex energy landscape where the binding of substrate selects for the active conformation, whereas targeting an allosteric site in the dimer interface selects an inactive conformation that contains disordered active-site loops. Mutations and posttranslational modifications stabilize high-energy inactive conformations, with mostly formed, but distorted, active sites. To examine the interconversion of active and inactive states in the ensemble, we used detection of related solvent positions to analyze 4,995 waters in 15 high-resolution (<2.0 Å) structures of wild-type caspase-3, resulting in 450 clusters with the most highly conserved set containing 145 water molecules. The data show that regions of the protein that contact the conserved waters also correspond to sites of posttranslational modifications, suggesting that the conserved waters are an integral part of allosteric mechanisms. To test this hypothesis, we created a library of 19 caspase-3 variants through saturation mutagenesis in a single position of the allosteric site of the dimer interface, and we show that the enzyme activity varies by more than four orders of magnitude. Altogether, our database consists of 37 high-resolution structures of caspase-3 variants, and we demonstrate that the decrease in activity correlates with a loss of conserved water molecules. The data show that the activity of caspase-3 can be fine-tuned through globally desolvating the active conformation within the native ensemble, providing a mechanism for cells to repartition the ensemble and thus fine-tune activity through conformational selection.

Structure and genetic variability of envelope glycoproteins of two antigenic variants of caprine arthritis-encephalitis lentivirus.

PubMed

Knowles, D P; Cheevers, W P; McGuire, T C; Brassfield, A L; Harwood, W G; Stem, T A

1991-11-01

To define the structure of the caprine arthritis-encephalitis virus (CAEV) env gene and characterize genetic changes which occur during antigenic variation, we sequenced the env genes of CAEV-63 and CAEV-Co, two antigenic variants of CAEV defined by serum neutralization. The deduced primary translation product of the CAEV env gene consists of a 60- to 80-amino-acid signal peptide followed by an amino-terminal surface protein (SU) and a carboxy-terminal transmembrane protein (TM) separated by an Arg-Lys-Lys-Arg cleavage site. The signal peptide cleavage site was verified by amino-terminal amino acid sequencing of native CAEV-63 SU. In addition, immunoprecipitation of [35S]methionine-labeled CAEV-63 proteins by sera from goats immunized with recombinant vaccinia virus expressing the CAEV-63 env gene confirmed that antibodies induced by env-encoded recombinant proteins react specifically with native virion SU and TM. The env genes of CAEV-63 and CAEV-Co encode 28 conserved cysteines and 25 conserved potential N-linked glycosylation sites. Nucleotide sequence variability results in 62 amino acid changes and one deletion within the SU and 34 amino acid changes within the TM.

Structure and genetic variability of envelope glycoproteins of two antigenic variants of caprine arthritis-encephalitis lentivirus.

PubMed Central

Knowles, D P; Cheevers, W P; McGuire, T C; Brassfield, A L; Harwood, W G; Stem, T A

1991-01-01

To define the structure of the caprine arthritis-encephalitis virus (CAEV) env gene and characterize genetic changes which occur during antigenic variation, we sequenced the env genes of CAEV-63 and CAEV-Co, two antigenic variants of CAEV defined by serum neutralization. The deduced primary translation product of the CAEV env gene consists of a 60- to 80-amino-acid signal peptide followed by an amino-terminal surface protein (SU) and a carboxy-terminal transmembrane protein (TM) separated by an Arg-Lys-Lys-Arg cleavage site. The signal peptide cleavage site was verified by amino-terminal amino acid sequencing of native CAEV-63 SU. In addition, immunoprecipitation of [35S]methionine-labeled CAEV-63 proteins by sera from goats immunized with recombinant vaccinia virus expressing the CAEV-63 env gene confirmed that antibodies induced by env-encoded recombinant proteins react specifically with native virion SU and TM. The env genes of CAEV-63 and CAEV-Co encode 28 conserved cysteines and 25 conserved potential N-linked glycosylation sites. Nucleotide sequence variability results in 62 amino acid changes and one deletion within the SU and 34 amino acid changes within the TM. Images PMID:1656067

Increased Transcript Complexity in Genes Associated with Chronic Obstructive Pulmonary Disease

PubMed Central

Lackey, Lela; McArthur, Evonne; Laederach, Alain

2015-01-01

Genome-wide association studies aim to correlate genotype with phenotype. Many common diseases including Type II diabetes, Alzheimer’s, Parkinson’s and Chronic Obstructive Pulmonary Disease (COPD) are complex genetic traits with hundreds of different loci that are associated with varied disease risk. Identifying common features in the genes associated with each disease remains a challenge. Furthermore, the role of post-transcriptional regulation, and in particular alternative splicing, is still poorly understood in most multigenic diseases. We therefore compiled comprehensive lists of genes associated with Type II diabetes, Alzheimer’s, Parkinson’s and COPD in an attempt to identify common features of their corresponding mRNA transcripts within each gene set. The SERPINA1 gene is a well-recognized genetic risk factor of COPD and it produces 11 transcript variants, which is exceptional for a human gene. This led us to hypothesize that other genes associated with COPD, and complex disorders in general, are highly transcriptionally diverse. We found that COPD-associated genes have a statistically significant enrichment in transcript complexity stemming from a disproportionately high level of alternative splicing, however, Type II Diabetes, Alzheimer’s and Parkinson’s disease genes were not significantly enriched. We also identified a subset of transcriptionally complex COPD-associated genes (~40%) that are differentially expressed between mild, moderate and severe COPD. Although the genes associated with other lung diseases are not extensively documented, we found preliminary data that idiopathic pulmonary disease genes, but not cystic fibrosis modulators, are also more transcriptionally complex. Interestingly, complex COPD transcripts are more often the product of alternative acceptor site usage. To verify the biological importance of these alternative transcripts, we used RNA-sequencing analyses to determine that COPD-associated genes are frequently expressed in lung and liver tissues and are regulated in a tissue-specific manner. Additionally, many complex COPD-associated genes are spliced differently between COPD and non-COPD patients. Our analysis therefore suggests that post-transcriptional regulation, particularly alternative splicing, is an important feature specific to COPD disease etiology that warrants further investigation. PMID:26480348

Missense-depleted regions in population exomes implicate ras superfamily nucleotide-binding protein alteration in patients with brain malformation

PubMed Central

Ge, Xiaoyan; Gong, Henry; Dumas, Kevin; Litwin, Jessica; Phillips, Joanna J; Waisfisz, Quinten; Weiss, Marjan M; Hendriks, Yvonne; Stuurman, Kyra E; Nelson, Stanley F; Grody, Wayne W; Lee, Hane; Kwok, Pui-Yan; Shieh, Joseph T C

2016-01-01

Genomic sequence interpretation can miss clinically relevant missense variants for several reasons. Rare missense variants are numerous in the exome and difficult to prioritise. Affected genes may also not have existing disease association. To improve variant prioritisation, we leverage population exome data to identify intragenic missense-depleted regions (MDRs) genome-wide that may be important in disease. We then use missense depletion analyses to help prioritise undiagnosed disease exome variants. We demonstrate application of this strategy to identify a novel gene association for human brain malformation. We identified de novo missense variants that affect the GDP/GTP-binding site of ARF1 in three unrelated patients. Corresponding functional analysis suggests ARF1 GDP/GTP-activation is affected by the specific missense mutations associated with heterotopia. These findings expand the genetic pathway underpinning neurologic disease that classically includes FLNA. ARF1 along with ARFGEF2 add further evidence implicating ARF/GEFs in the brain. Using functional ontology, top MDR-containing genes were highly enriched for nucleotide-binding function, suggesting these may be candidates for human disease. Routine consideration of MDR in the interpretation of exome data for rare diseases may help identify strong genetic factors for many severe conditions, infertility/reduction in reproductive capability, and embryonic conditions contributing to preterm loss. PMID:28868155

Phosphorylation and Methylation of Proteasomal Proteins of the Haloarcheon Haloferax volcanii

DOE PAGES

Humbard, Matthew A.; Reuter, Christopher J.; Zuobi-Hasona, Kheir; ...

2010-01-01

Promore » teasomes are composed of 20S core particles (CPs) of α - and β -type subunits that associate with regulatory particle AAA ATPases such as the proteasome-activating nucleotidase (PAN) complexes of archaea. In this study, the roles and additional sites of post-translational modification of proteasomes were investigated using the archaeonHaloferax volcaniias a model. Indicative of phosphorylation, phosphatase-sensitive isoforms of α 1 and α 2 were detected by 2-DE immunoblot. To map these and other potential sites of post-translational modification, proteasomes were purified and analyzed by tandem mass spectrometry (MS/MS). Using this approach, several phosphosites were mapped including α 1 Thr147, α 2 Thr13/Ser14 and PAN-A Ser340. Multiple methylation sites were also mapped to α 1 , thus, revealing a new type of proteasomal modification. bing the biological role of α 1 and PAN-A phosphorylation by site-directed mutagenesis revealed dominant negative phenotypes for cell viability and/or pigmentation for α 1 variants including Thr147Ala, Thr158Ala and Ser58Ala. AnH. volcaniiRio1p Ser/Thr kinase homolog was purified and shown to catalyze autophosphorylation and phosphotransfer to α 1 . The α 1 variants in Thr and Ser residues that displayed dominant negative phenotypes were significantly reduced in their ability to accept phosphoryl groups from Rio1p, thus, providing an important link between cell physiology and proteasomal phosphorylation.« less

Probing the mechanistic role of the long α-helix in subunit L of respiratory Complex I from Escherichia coli by site-directed mutagenesis

PubMed Central

Belevich, Galina; Knuuti, Juho; Verkhovsky, Michael I; Wikström, Mårten; Verkhovskaya, Marina

2011-01-01

The C-terminus of the NuoL subunit of Complex I includes a long amphipathic α-helix positioned parallel to the membrane, which has been considered to function as a piston in the proton pumping machinery. Here, we have introduced three types of mutations into the nuoL gene to test the piston-like function. First, NuoL was truncated at its C- and N-termini, which resulted in low production of a fragile Complex I with negligible activity. Second, we mutated three partially conserved residues of the amphipathic α-helix: Asp and Lys residues and a Pro were substituted for acidic, basic or neutral residues. All these variants exhibited almost a wild-type phenotype. Third, several substitutions and insertions were made to reduce rigidity of the amphipathic α-helix, and/or to change its geometry. Most insertions/substitutions resulted in a normal growth phenotype, albeit often with reduced stability of Complex I. In contrast, insertion of six to seven amino acids at a site of the long α-helix between NuoL and M resulted in substantial loss of proton pumping efficiency. The implications of these results for the proton pumping mechanism of Complex I are discussed. PMID:22060017

Stability and function of interdomain linker variants of glucoamylase 1 from Aspergillus niger.

PubMed

Sauer, J; Christensen, T; Frandsen, T P; Mirgorodskaya, E; McGuire, K A; Driguez, H; Roepstorff, P; Sigurskjold, B W; Svensson, B

2001-08-07

Several variants of glucoamylase 1 (GA1) from Aspergillus niger were created in which the highly O-glycosylated peptide (aa 468--508) connecting the (alpha/alpha)(6)-barrel catalytic domain and the starch binding domain was substituted at the gene level by equivalent segments of glucoamylases from Hormoconis resinae, Humicola grisea, and Rhizopus oryzae encoding 5, 19, and 36 amino acid residues. Variants were constructed in which the H. resinae linker was elongated by proline-rich sequences as this linker itself apparently was too short to allow formation of the corresponding protein variant. Size and isoelectric point of GA1 variants reflected differences in linker length, posttranslational modification, and net charge. While calculated polypeptide chain molecular masses for wild-type GA1, a nonnatural proline-rich linker variant, H. grisea, and R. oryzae linker variants were 65,784, 63,777, 63,912, and 65,614 Da, respectively, MALDI-TOF-MS gave values of 82,042, 73,800, 73,413, and 90,793 Da, respectively, where the latter value could partly be explained by an N-glycosylation site introduced near the linker C-terminus. The k(cat) and K(m) for hydrolysis of maltooligodextrins and soluble starch, and the rate of hydrolysis of barley starch granules were essentially the same for the variants as for wild-type GA1. beta-Cyclodextrin, acarbose, and two heterobidentate inhibitors were found by isothermal titration calorimetry to bind to the catalytic and starch binding domains of the linker variants, indicating that the function of the active site and the starch binding site was maintained. The stability of GA1 linker variants toward GdnHCl and heat, however, was reduced compared to wild-type.

Major Histocompatibility Complex (MHC) Class I and MHC Class II Proteins: Conformational Plasticity in Antigen Presentation

PubMed Central

Wieczorek, Marek; Abualrous, Esam T.; Sticht, Jana; Álvaro-Benito, Miguel; Stolzenberg, Sebastian; Noé, Frank; Freund, Christian

2017-01-01

Antigen presentation by major histocompatibility complex (MHC) proteins is essential for adaptive immunity. Prior to presentation, peptides need to be generated from proteins that are either produced by the cell’s own translational machinery or that are funneled into the endo-lysosomal vesicular system. The prolonged interaction between a T cell receptor and specific pMHC complexes, after an extensive search process in secondary lymphatic organs, eventually triggers T cells to proliferate and to mount a specific cellular immune response. Once processed, the peptide repertoire presented by MHC proteins largely depends on structural features of the binding groove of each particular MHC allelic variant. Additionally, two peptide editors—tapasin for class I and HLA-DM for class II—contribute to the shaping of the presented peptidome by favoring the binding of high-affinity antigens. Although there is a vast amount of biochemical and structural information, the mechanism of the catalyzed peptide exchange for MHC class I and class II proteins still remains controversial, and it is not well understood why certain MHC allelic variants are more susceptible to peptide editing than others. Recent studies predict a high impact of protein intermediate states on MHC allele-specific peptide presentation, which implies a profound influence of MHC dynamics on the phenomenon of immunodominance and the development of autoimmune diseases. Here, we review the recent literature that describe MHC class I and II dynamics from a theoretical and experimental point of view and we highlight the similarities between MHC class I and class II dynamics despite the distinct functions they fulfill in adaptive immunity. PMID:28367149

Two-step interrogation then recognition of DNA binding site by Integration Host Factor: an architectural DNA-bending protein.

PubMed

Velmurugu, Yogambigai; Vivas, Paula; Connolly, Mitchell; Kuznetsov, Serguei V; Rice, Phoebe A; Ansari, Anjum

2018-02-28

The dynamics and mechanism of how site-specific DNA-bending proteins initially interrogate potential binding sites prior to recognition have remained elusive for most systems. Here we present these dynamics for Integration Host factor (IHF), a nucleoid-associated architectural protein, using a μs-resolved T-jump approach. Our studies show two distinct DNA-bending steps during site recognition by IHF. While the faster (∼100 μs) step is unaffected by changes in DNA or protein sequence that alter affinity by >100-fold, the slower (1-10 ms) step is accelerated ∼5-fold when mismatches are introduced at DNA sites that are sharply kinked in the specific complex. The amplitudes of the fast phase increase when the specific complex is destabilized and decrease with increasing [salt], which increases specificity. Taken together, these results indicate that the fast phase is non-specific DNA bending while the slow phase, which responds only to changes in DNA flexibility at the kink sites, is specific DNA kinking during site recognition. Notably, the timescales for the fast phase overlap with one-dimensional diffusion times measured for several proteins on DNA, suggesting that these dynamics reflect partial DNA bending during interrogation of potential binding sites by IHF as it scans DNA.

MCM5: a new actor in the link between DNA replication and Meier-Gorlin syndrome.

PubMed

Vetro, Annalisa; Savasta, Salvatore; Russo Raucci, Annalisa; Cerqua, Cristina; Sartori, Geppo; Limongelli, Ivan; Forlino, Antonella; Maruelli, Silvia; Perucca, Paola; Vergani, Debora; Mazzini, Giuliano; Mattevi, Andrea; Stivala, Lucia Anna; Salviati, Leonardo; Zuffardi, Orsetta

2017-05-01

Meier-Gorlin syndrome (MGORS) is a rare disorder characterized by primordial dwarfism, microtia, and patellar aplasia/hypoplasia. Recessive mutations in ORC1, ORC4, ORC6, CDT1, CDC6, and CDC45, encoding members of the pre-replication (pre-RC) and pre-initiation (pre-IC) complexes, and heterozygous mutations in GMNN, a regulator of cell-cycle progression and DNA replication, have already been associated with this condition. We performed whole-exome sequencing (WES) in a patient with a clinical diagnosis of MGORS and identified biallelic variants in MCM5. This gene encodes a subunit of the replicative helicase complex, which represents a component of the pre-RC. Both variants, a missense substitution within a conserved domain critical for the helicase activity, and a single base deletion causing a frameshift and a premature stop codon, were predicted to be detrimental for the MCM5 function. Although variants of MCM5 have never been reported in specific human diseases, defect of this gene in zebrafish causes a phenotype of growth restriction overlapping the one associated with orc1 depletion. Complementation experiments in yeast showed that the plasmid carrying the missense variant was unable to rescue the lethal phenotype caused by mcm5 deletion. Moreover cell-cycle progression was delayed in patient's cells, as already shown for mutations in the ORC1 gene. Altogether our findings support the role of MCM5 as a novel gene involved in MGORS, further emphasizing that this condition is caused by impaired DNA replication.

Mitochondrial hepato-encephalopathy due to deficiency of QIL1/MIC13 (C19orf70), a MICOS complex subunit.

PubMed

Zeharia, Avraham; Friedman, Jonathan R; Tobar, Ana; Saada, Ann; Konen, Osnat; Fellig, Yacov; Shaag, Avraham; Nunnari, Jodi; Elpeleg, Orly

2016-12-01

The mitochondrial inner membrane possesses distinct subdomains including cristae, which are lamellar structures invaginated into the mitochondrial matrix and contain the respiratory complexes. Generation of inner membrane domains requires the complex interplay between the respiratory complexes, mitochondrial lipids and the recently identified mitochondrial contact site and cristae organizing system (MICOS) complex. Proper organization of the mitochondrial inner membrane has recently been shown to be important for respiratory function in yeast. Here we aimed at a molecular diagnosis in a brother and sister from a consanguineous family who presented with a neurodegenerative disorder accompanied by hyperlactatemia, 3-methylglutaconic aciduria, disturbed hepatocellular function with abnormal cristae morphology in liver and cerebellar and vermis atrophy, which suggest mitochondrial dysfunction. Using homozygosity mapping and exome sequencing the patients were found to be homozygous for the p.(Gly15Glufs*75) variant in the QIL1/MIC13 (C19orf70) gene. QIL1/MIC13 is a constituent of MICOS, a six subunit complex that helps to form and/or stabilize cristae junctions and determine the placement, distribution and number of cristae within mitochondria. In patient fibroblasts both MICOS subunits QIL1/MIC13 and MIC10 were absent whereas MIC60 was present in a comparable abundance to that of the control. We conclude that QIL1/MIC13 deficiency in human, is associated with disassembly of the MICOS complex, with the associated aberration of cristae morphology and mitochondrial respiratory dysfunction. 3-Methylglutaconic aciduria is associated with variants in genes encoding mitochondrial inner membrane organizing determinants, including TAZ, DNAJC19, SERAC1 and QIL1/MIC13.

GenProBiS: web server for mapping of sequence variants to protein binding sites.

PubMed

Konc, Janez; Skrlj, Blaz; Erzen, Nika; Kunej, Tanja; Janezic, Dusanka

2017-07-03

Discovery of potentially deleterious sequence variants is important and has wide implications for research and generation of new hypotheses in human and veterinary medicine, and drug discovery. The GenProBiS web server maps sequence variants to protein structures from the Protein Data Bank (PDB), and further to protein-protein, protein-nucleic acid, protein-compound, and protein-metal ion binding sites. The concept of a protein-compound binding site is understood in the broadest sense, which includes glycosylation and other post-translational modification sites. Binding sites were defined by local structural comparisons of whole protein structures using the Protein Binding Sites (ProBiS) algorithm and transposition of ligands from the similar binding sites found to the query protein using the ProBiS-ligands approach with new improvements introduced in GenProBiS. Binding site surfaces were generated as three-dimensional grids encompassing the space occupied by predicted ligands. The server allows intuitive visual exploration of comprehensively mapped variants, such as human somatic mis-sense mutations related to cancer and non-synonymous single nucleotide polymorphisms from 21 species, within the predicted binding sites regions for about 80 000 PDB protein structures using fast WebGL graphics. The GenProBiS web server is open and free to all users at http://genprobis.insilab.org. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Genome-Independent Identification of RNA Editing by Mutual Information (GIREMI) | Informatics Technology for Cancer Research (ITCR)

Cancer.gov

Identification of single-nucleotide variants in RNA-seq data. Current version focuses on detection of RNA editing sites without requiring genome sequence data. New version is under development to separately identify RNA editing sites and genetic variants using RNA-seq data alone.

Integrated rare variant-based risk gene prioritization in disease case-control sequencing studies.

PubMed

Lin, Jhih-Rong; Zhang, Quanwei; Cai, Ying; Morrow, Bernice E; Zhang, Zhengdong D

2017-12-01

Rare variants of major effect play an important role in human complex diseases and can be discovered by sequencing-based genome-wide association studies. Here, we introduce an integrated approach that combines the rare variant association test with gene network and phenotype information to identify risk genes implicated by rare variants for human complex diseases. Our data integration method follows a 'discovery-driven' strategy without relying on prior knowledge about the disease and thus maintains the unbiased character of genome-wide association studies. Simulations reveal that our method can outperform a widely-used rare variant association test method by 2 to 3 times. In a case study of a small disease cohort, we uncovered putative risk genes and the corresponding rare variants that may act as genetic modifiers of congenital heart disease in 22q11.2 deletion syndrome patients. These variants were missed by a conventional approach that relied on the rare variant association test alone.

An integrated map of structural variation in 2,504 human genomes.

PubMed

Sudmant, Peter H; Rausch, Tobias; Gardner, Eugene J; Handsaker, Robert E; Abyzov, Alexej; Huddleston, John; Zhang, Yan; Ye, Kai; Jun, Goo; Fritz, Markus Hsi-Yang; Konkel, Miriam K; Malhotra, Ankit; Stütz, Adrian M; Shi, Xinghua; Casale, Francesco Paolo; Chen, Jieming; Hormozdiari, Fereydoun; Dayama, Gargi; Chen, Ken; Malig, Maika; Chaisson, Mark J P; Walter, Klaudia; Meiers, Sascha; Kashin, Seva; Garrison, Erik; Auton, Adam; Lam, Hugo Y K; Mu, Xinmeng Jasmine; Alkan, Can; Antaki, Danny; Bae, Taejeong; Cerveira, Eliza; Chines, Peter; Chong, Zechen; Clarke, Laura; Dal, Elif; Ding, Li; Emery, Sarah; Fan, Xian; Gujral, Madhusudan; Kahveci, Fatma; Kidd, Jeffrey M; Kong, Yu; Lameijer, Eric-Wubbo; McCarthy, Shane; Flicek, Paul; Gibbs, Richard A; Marth, Gabor; Mason, Christopher E; Menelaou, Androniki; Muzny, Donna M; Nelson, Bradley J; Noor, Amina; Parrish, Nicholas F; Pendleton, Matthew; Quitadamo, Andrew; Raeder, Benjamin; Schadt, Eric E; Romanovitch, Mallory; Schlattl, Andreas; Sebra, Robert; Shabalin, Andrey A; Untergasser, Andreas; Walker, Jerilyn A; Wang, Min; Yu, Fuli; Zhang, Chengsheng; Zhang, Jing; Zheng-Bradley, Xiangqun; Zhou, Wanding; Zichner, Thomas; Sebat, Jonathan; Batzer, Mark A; McCarroll, Steven A; Mills, Ryan E; Gerstein, Mark B; Bashir, Ali; Stegle, Oliver; Devine, Scott E; Lee, Charles; Eichler, Evan E; Korbel, Jan O

2015-10-01

Structural variants are implicated in numerous diseases and make up the majority of varying nucleotides among human genomes. Here we describe an integrated set of eight structural variant classes comprising both balanced and unbalanced variants, which we constructed using short-read DNA sequencing data and statistically phased onto haplotype blocks in 26 human populations. Analysing this set, we identify numerous gene-intersecting structural variants exhibiting population stratification and describe naturally occurring homozygous gene knockouts that suggest the dispensability of a variety of human genes. We demonstrate that structural variants are enriched on haplotypes identified by genome-wide association studies and exhibit enrichment for expression quantitative trait loci. Additionally, we uncover appreciable levels of structural variant complexity at different scales, including genic loci subject to clusters of repeated rearrangement and complex structural variants with multiple breakpoints likely to have formed through individual mutational events. Our catalogue will enhance future studies into structural variant demography, functional impact and disease association.

On the use of haplotype phylogeny to detect disease susceptibility loci

PubMed Central

Bardel, Claire; Danjean, Vincent; Hugot, Jean-Pierre; Darlu, Pierre; Génin, Emmanuelle

2005-01-01

Background The cladistic approach proposed by Templeton has been presented as promising for the study of the genetic factors involved in common diseases. This approach allows the joint study of multiple markers within a gene by considering haplotypes and grouping them in nested clades. The idea is to search for clades with an excess of cases as compared to the whole sample and to identify the mutations defining these clades as potential candidate disease susceptibility sites. However, the performance of this approach for the study of the genetic factors involved in complex diseases has never been studied. Results In this paper, we propose a new method to perform such a cladistic analysis and we estimate its power through simulations. We show that under models where the susceptibility to the disease is caused by a single genetic variant, the cladistic test is neither really more powerful to detect an association nor really more efficient to localize the susceptibility site than an individual SNP testing. However, when two interacting sites are responsible for the disease, the cladistic analysis greatly improves the probability to find the two susceptibility sites. The impact of the linkage disequilibrium and of the tree characteristics on the efficiency of the cladistic analysis are also discussed. An application on a real data set concerning the CARD15 gene and Crohn disease shows that the method can successfully identify the three variant sites that are involved in the disease susceptibility. Conclusion The use of phylogenies to group haplotypes is especially interesting to pinpoint the sites that are likely to be involved in disease susceptibility among the different markers identified within a gene. PMID:15904492

Expanding the utility of 4-cyano-L-phenylalanine as a vibrational reporter of protein environments.

PubMed

Bazewicz, Christopher G; Lipkin, Jacob S; Smith, Emily E; Liskov, Melanie T; Brewer, Scott H

2012-09-06

The ability to genetically incorporate amino acids modified with spectroscopic reporters site-specifically into proteins with high efficiency and fidelity has greatly enhanced the ability to probe local protein structure and dynamics. Here, we have synthesized the unnatural amino acid (UAA), 4-cyano-L-phenylalanine (pCNPhe), containing the nitrile vibrational reporter and three isotopomers ((15)N, (13)C, (13)C(15)N) of this UAA to enhance the ability of pCNPhe to study local protein environments. Each pCNPhe isotopic variant was genetically incorporated in an efficient, site-specific manner into superfolder green fluorescent protein (sfGFP) in response to an amber codon with high fidelity utilizing an engineered, orthogonal aminoacyl-tRNA synthetase. The isotopomers of 4-cyano-L-phenylalanine permitted the nitrile symmetric stretch vibration of these UAAs to be unambiguously assigned utilizing the magnitude and direction of the isotopic shift of this vibration. The sensitivity of the nitrile symmetric stretching frequency of each isotopic variant to the local environment was measured by individually incorporating the probes into two distinct local environments of sfGFP. The UAAs were also utilized in concert to probe multiple local environments in sfGFP simultaneously to increase the utility of 4-cyano-L-phenylalanine.

Increasing the hydrolysis constant of the reactive site upon introduction of an engineered Cys¹⁴-Cys³⁹ bond into the ovomucoid third domain from silver pheasant.

PubMed

Hemmi, Hikaru; Kumazaki, Takashi; Kojima, Shuichi; Yoshida, Takuya; Ohkubo, Tadayasu; Yokosawa, Hideyoshi; Miura, Kin-Ichiro; Kobayashi, Yuji

2011-08-01

P14C/N39C is the disulfide variant of the ovomucoid third domain from silver pheasant (OMSVP3) introducing an engineered Cys¹⁴-Cys³⁹ bond near the reactive site on the basis of the sequence homology between OMSVP3 and ascidian trypsin inhibitor. This variant exhibits a narrower inhibitory specificity. We have examined the effects of introducing a Cys¹⁴-Cys³⁹ bond into the flexible N-terminal loop of OMSVP3 on the thermodynamics of the reactive site peptide bond hydrolysis, as well as the thermal stability of reactive site intact inhibitors. P14C/N39C can be selectively cleaved by Streptomyces griseus protease B at the reactive site of OMSVP3 to form a reactive site modified inhibitor. The conversion rate of intact to modified P14C/N39C is much faster than that for wild type under any pH condition. The pH-independent hydrolysis constant (K(hyd) °) is estimated to be approximately 5.5 for P14C/N39C, which is higher than the value of 1.6 for natural OMSVP3. The reactive site modified form of P14C/N39C is thermodynamically more stable than the intact one. Thermal denaturation experiments using intact inhibitors show that the temperature at the midpoint of unfolding at pH 2.0 is 59 °C for P14C/N39C and 58 °C for wild type. There have been no examples, except P14C/N39C, where introducing an engineered disulfide causes a significant increase in K(hyd) °, but has no effect on the thermal stability. The site-specific disulfide introduction into the flexible N-terminal loop of natural Kazal-type inhibitors would be useful to further characterize the thermodynamics of the reactive site peptide bond hydrolysis. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.

Limits of variation, specific infectivity, and genome packaging of massively recoded poliovirus genomes.

PubMed

Song, Yutong; Gorbatsevych, Oleksandr; Liu, Ying; Mugavero, JoAnn; Shen, Sam H; Ward, Charles B; Asare, Emmanuel; Jiang, Ping; Paul, Aniko V; Mueller, Steffen; Wimmer, Eckard

2017-10-10

Computer design and chemical synthesis generated viable variants of poliovirus type 1 (PV1), whose ORF (6,189 nucleotides) carried up to 1,297 "Max" mutations (excess of overrepresented synonymous codon pairs) or up to 2,104 "SD" mutations (randomly scrambled synonymous codons). "Min" variants (excess of underrepresented synonymous codon pairs) are nonviable except for P2 Min , a variant temperature-sensitive at 33 and 39.5 °C. Compared with WT PV1, P2 Min displayed a vastly reduced specific infectivity (si) (WT, 1 PFU/118 particles vs. P2 Min , 1 PFU/35,000 particles), a phenotype that will be discussed broadly. Si of haploid PV presents cellular infectivity of a single genotype. We performed a comprehensive analysis of sequence and structures of the PV genome to determine if evolutionary conserved cis-acting packaging signal(s) were preserved after recoding. We showed that conserved synonymous sites and/or local secondary structures that might play a role in determining packaging specificity do not survive codon pair recoding. This makes it unlikely that numerous "cryptic, sequence-degenerate, dispersed RNA packaging signals mapping along the entire viral genome" [Patel N, et al. (2017) Nat Microbiol 2:17098] play the critical role in poliovirus packaging specificity. Considering all available evidence, we propose a two-step assembly strategy for +ssRNA viruses: step I, acquisition of packaging specificity, either ( a ) by specific recognition between capsid protein(s) and replication proteins (poliovirus), or ( b ) by the high affinity interaction of a single RNA packaging signal (PS) with capsid protein(s) (most +ssRNA viruses so far studied); step II, cocondensation of genome/capsid precursors in which an array of hairpin structures plays a role in virion formation.

Antibody humanization by molecular dynamics simulations-in-silico guided selection of critical backmutations.

PubMed

Margreitter, Christian; Mayrhofer, Patrick; Kunert, Renate; Oostenbrink, Chris

2016-06-01

Monoclonal antibodies represent the fastest growing class of biotherapeutic proteins. However, as they are often initially derived from rodent organisms, there is a severe risk of immunogenic reactions, hampering their applicability. The humanization of these antibodies remains a challenging task in the context of rational drug design. "Superhumanization" describes the direct transfer of the complementarity determining regions to a human germline framework, but this humanization approach often results in loss of binding affinity. In this study, we present a new approach for predicting promising backmutation sites using molecular dynamics simulations of the model antibody Ab2/3H6. The simulation method was developed in close conjunction with novel specificity experiments. Binding properties of mAb variants were evaluated directly from crude supernatants and confirmed using established binding affinity assays for purified antibodies. Our approach provides access to the dynamical features of the actual binding sites of an antibody, based solely on the antibody sequence. Thus we do not need structural data on the antibody-antigen complex and circumvent cumbersome methods to assess binding affinities. © 2016 The Authors Journal of Molecular Recognition Published by John Wiley & Sons Ltd. © 2016 The Authors Journal of Molecular Recognition Published by John Wiley & Sons Ltd.

Detection of ATM germline variants by the p53 mitotic centrosomal localization test in BRCA1/2-negative patients with early-onset breast cancer.

PubMed

Prodosmo, Andrea; Buffone, Amelia; Mattioni, Manlio; Barnabei, Agnese; Persichetti, Agnese; De Leo, Aurora; Appetecchia, Marialuisa; Nicolussi, Arianna; Coppa, Anna; Sciacchitano, Salvatore; Giordano, Carolina; Pinnarò, Paola; Sanguineti, Giuseppe; Strigari, Lidia; Alessandrini, Gabriele; Facciolo, Francesco; Cosimelli, Maurizio; Grazi, Gian Luca; Corrado, Giacomo; Vizza, Enrico; Giannini, Giuseppe; Soddu, Silvia

2016-09-06

Variant ATM heterozygotes have an increased risk of developing cancer, cardiovascular diseases, and diabetes. Costs and time of sequencing and ATM variant complexity make large-scale, general population screenings not cost-effective yet. Recently, we developed a straightforward, rapid, and inexpensive test based on p53 mitotic centrosomal localization (p53-MCL) in peripheral blood mononuclear cells (PBMCs) that diagnoses mutant ATM zygosity and recognizes tumor-associated ATM polymorphisms. Fresh PBMCs from 496 cancer patients were analyzed by p53-MCL: 90 cases with familial BRCA1/2-positive and -negative breast and/or ovarian cancer, 337 with sporadic cancers (ovarian, lung, colon, and post-menopausal breast cancers), and 69 with breast/thyroid cancer. Variants were confirmed by ATM sequencing. A total of seven individuals with ATM variants were identified, 5/65 (7.7 %) in breast cancer cases of familial breast and/or ovarian cancer and 2/69 (2.9 %) in breast/thyroid cancer. No variant ATM carriers were found among the other cancer cases. Excluding a single case in which both BRCA1 and ATM were mutated, no p53-MCL alterations were observed in BRCA1/2-positive cases. These data validate p53-MCL as reliable and specific test for germline ATM variants, confirm ATM as breast cancer susceptibility gene, and highlight a possible association with breast/thyroid cancers.

Insights on activity and stability of subtilisin E towards guanidinium chloride and sodium dodecylsulfate.

PubMed

Li, Zhenwei; Roccatano, Danilo; Lorenz, Michael; Martinez, Ronny; Schwaneberg, Ulrich

2014-01-01

A subtilisin E variant (M4) showing high activity and resistance towards guanidinium chloride (GdmCl) and sodium dodecylsulfate (SDS) was previously identified after three rounds of directed evolution [Li et al., ChemBioChem 2012, 13(5), 691-699.]. In this report, 10 additional positions, identified during directed subtilisin E evolution, were saturated on the previously reported SeSaM1-5 variant (S62/A153/G166/I205). Screening confirmed that chaotolerant variants included amino acid substitutions either in the active site, or the substrate binding pocket. Two variants, M5 (S62I/A153V/G166S/T224A/T240S) and M6 (S62I/A153V/G166S/I205V/N218S/T224A) were finally generated to maximize activity and stability in the presence of GdmCl or SDS. The inactivation concentration (IC50) of M6 using Suc-AAPF-pNA as substrate was significantly increased compared to M4 in the presence of GdmCl (IC50 (M4): 2.7M; IC50 (M6): 4.6M) and SDS (IC50 (M4): 1.5%; IC50 (M6): 4.0%). The half-life in 5M GdmCl was also significantly improved for M6 compared to M4 (t 1/2 (M4): 2min; t 1/2 (M6): 15min). M5 retained resistance towards GdmCl or SDS as in M4. The activity of M5 towards a complex protein substrate (Azocasein) was increased by ∼1.5 fold compared to M4 and M6. Circular dichroism (CD) analysis for subtilisin E wild type (WT) and three variants (M4, M5 and M6) indicated that secondary structures of all variants including wild type at 1-2M GdmCl (except M4) were not significantly perturbed, with unfolding occurring for WT and all three variants above 3M GdmCl. In SDS, the secondary structures of WT and all three variants remained intact at concentrations of 0.5 to 2.0% (w/v) SDS. Results suggest that subtilisin E inactivation occurred most likely due to inhibitory effect, since a general unfolding of the enzyme was not observed through circular dichroism. Such inhibition could be avoided by limiting the access of GdmCl and SDS to the active site and/or to residues involved in substrate binding. Copyright © 2013 Elsevier B.V. All rights reserved.

A camel-derived MERS-CoV with a variant spike protein cleavage site and distinct fusion activation properties

PubMed Central

Millet, Jean Kaoru; Goldstein, Monty E; Labitt, Rachael N; Hsu, Hung-Lun; Daniel, Susan; Whittaker, Gary R

2016-01-01

Middle East respiratory syndrome coronavirus (MERS-CoV) continues to circulate in both humans and camels, and the origin and evolution of the virus remain unclear. Here we characterize the spike protein of a camel-derived MERS-CoV (NRCE-HKU205) identified in 2013, early in the MERS outbreak. NRCE-HKU205 spike protein has a variant cleavage motif with regard to the S2′ fusion activation site—notably, a novel substitution of isoleucine for the otherwise invariant serine at the critical P1′ cleavage site position. The substitutions resulted in a loss of furin-mediated cleavage, as shown by fluorogenic peptide cleavage and western blot assays. Cell–cell fusion and pseudotyped virus infectivity assays demonstrated that the S2′ substitutions decreased spike-mediated fusion and viral entry. However, cathepsin and trypsin-like protease activation were retained, albeit with much reduced efficiency compared with the prototypical EMC/2012 human strain. We show that NRCE-HKU205 has more limited fusion activation properties possibly resulting in more restricted viral tropism and may represent an intermediate in the complex pattern of MERS-CoV ecology and evolution. PMID:27999426

Novel sequence variants in the TMIE gene in families with autosomal recessive nonsyndromic hearing impairment

PubMed Central

Santos, Regie Lyn P.; El-Shanti, Hatem; Sikandar, Shaheen; Lee, Kwanghyuk; Bhatti, Attya; Yan, Kai; Chahrour, Maria H.; McArthur, Nathan; Pham, Thanh L.; Mahasneh, Amjad Abdullah; Ahmad, Wasim

2010-01-01

To date, 37 genes have been identified for nonsyndromic hearing impairment (NSHI). Identifying the functional sequence variants within these genes and knowing their population-specific frequencies is of public health value, in particular for genetic screening for NSHI. To determine putatively functional sequence variants in the transmembrane inner ear (TMIE) gene in Pakistani and Jordanian families with autosomal recessive (AR) NSHI, four Jordanian and 168 Pakistani families with ARNSHI that is not due to GJB2 (CX26) were submitted to a genome scan. Two-point and multipoint parametric linkage analyses were performed, and families with logarithmic odds (LOD) scores of 1.0 or greater within the TMIE region underwent further DNA sequencing. The evolutionary conservation and location in predicted protein domains of amino acid residues where sequence variants occurred were studied to elucidate the possible effects of these sequence variants on function. Of seven families that were screened for TMIE, putatively functional sequence variants were found to segregate with hearing impairment in four families but were not seen in not less than 110 ethnically matched control chromosomes. The previously reported c.241C>T (p.R81C) variant was observed in two Pakistani families. Two novel variants, c.92A>G (p.E31G) and the splice site mutation c.212–2A>C, were identified in one Pakistani and one Jordanian family, respectively. The c.92A>G (p.E31G) variant occurred at a residue that is conserved in the mouse and is predicted to be extracellular. Conservation and potential functionality of previously published mutations were also examined. The prevalence of functional TMIE variants in Pakistani families is 1.7% [95% confidence interval (CI) 0.3–4.8]. Further studies on the spectrum, prevalence rates, and functional effect of sequence variants in the TMIE gene in other populations should demonstrate the true importance of this gene as a cause of hearing impairment. PMID:16389551

Chimeric TALE recombinases with programmable DNA sequence specificity.

PubMed

Mercer, Andrew C; Gaj, Thomas; Fuller, Roberta P; Barbas, Carlos F

2012-11-01

Site-specific recombinases are powerful tools for genome engineering. Hyperactivated variants of the resolvase/invertase family of serine recombinases function without accessory factors, and thus can be re-targeted to sequences of interest by replacing native DNA-binding domains (DBDs) with engineered zinc-finger proteins (ZFPs). However, imperfect modularity with particular domains, lack of high-affinity binding to all DNA triplets, and difficulty in construction has hindered the widespread adoption of ZFPs in unspecialized laboratories. The discovery of a novel type of DBD in transcription activator-like effector (TALE) proteins from Xanthomonas provides an alternative to ZFPs. Here we describe chimeric TALE recombinases (TALERs): engineered fusions between a hyperactivated catalytic domain from the DNA invertase Gin and an optimized TALE architecture. We use a library of incrementally truncated TALE variants to identify TALER fusions that modify DNA with efficiency and specificity comparable to zinc-finger recombinases in bacterial cells. We also show that TALERs recombine DNA in mammalian cells. The TALER architecture described herein provides a platform for insertion of customized TALE domains, thus significantly expanding the targeting capacity of engineered recombinases and their potential applications in biotechnology and medicine.

Structural and functional characterization of a ubiquitin variant engineered for tight and specific binding to an alpha-helical ubiquitin interacting motif.

PubMed

Manczyk, Noah; Yates, Bradley P; Veggiani, Gianluca; Ernst, Andreas; Sicheri, Frank; Sidhu, Sachdev S

2017-05-01

Ubiquitin interacting motifs (UIMs) are short α-helices found in a number of eukaryotic proteins. UIMs interact weakly but specifically with ubiquitin conjugated to other proteins, and in so doing, mediate specific cellular signals. Here we used phage display to generate ubiquitin variants (UbVs) targeting the N-terminal UIM of the yeast Vps27 protein. Selections yielded UbV.v27.1, which recognized the cognate UIM with high specificity relative to other yeast UIMs and bound with an affinity more than two orders of magnitude higher than that of ubiquitin. Structural and mutational studies of the UbV.v27.1-UIM complex revealed the molecular details for the enhanced affinity and specificity of UbV.v27.1, and underscored the importance of changes at the binding interface as well as at positions that do not contact the UIM. Our study highlights the power of the phage display approach for selecting UbVs with unprecedented affinity and high selectivity for particular α-helical UIM domains within proteomes, and it establishes a general approach for the development of inhibitors targeting interactions of this type. © 2017 The Protein Society.

Novel factor VIII variants with a modified furin cleavage site improve the efficacy of gene therapy for hemophilia A.

PubMed

Nguyen, G N; George, L A; Siner, J I; Davidson, R J; Zander, C B; Zheng, X L; Arruda, V R; Camire, R M; Sabatino, D E

2017-01-01

Essentials Factor (F) VIII is an inefficiently expressed protein. Furin deletion FVIII variants were purified and characterized using in vitro and in vivo assays. These minimally modified novel FVIII variants have enhanced function. These variants provide a strategy for increasing FVIII expression in hemophilia A gene therapy. Background The major challenge for developing gene-based therapies for hemophilia A is that human factor VIII (hFVIII) has intrinsic properties that result in inefficient biosynthesis. During intracellular processing, hFVIII is predominantly cleaved at a paired basic amino acid cleaving enzyme (PACE) or furin cleavage site to yield a heterodimer that is the major form of secreted protein. Previous studies with B-domain-deleted (BDD) canine FVIII and hFVIII-R1645H, both differing from hFVIII by a single amino acid at this site, suggested that these proteins are secreted mainly in a single polypeptide chain (SC) form and exhibit enhanced function. Objective We hypothesized that deletion(s) of the furin site modulates FVIII biology and may enhance its function. Methods A series of recombinant hFVIII-furin deletion variants were introduced into hFVIII-BDD [Δ1645, 1645-46(Δ2), 1645-47(Δ3), 1645-48(Δ4), or Δ1648] and characterized. Results In vitro, recombinant purified Δ3 and Δ4 were primarily SC and, interestingly, had 2-fold higher procoagulant activity compared with FVIII-BDD. In vivo, the variants also have improved hemostatic function. After adeno-associated viral (AAV) vector delivery, the expression of these variants is 2-4-fold higher than hFVIII-BDD. Protein challenges of each variant in mice tolerant to hFVIII-BDD showed no anti-FVIII immune response. Conclusions These data suggest that the furin deletion hFVIII variants are superior to hFVIII-BDD without increased immunogenicity. In the setting of gene-based therapeutics, these novel variants provide a unique strategy to increase FVIII expression, thus lowering the vector dose, a critical factor for hemophilia A gene therapy. © 2016 The Authors. Journal of Thrombosis and Haemostasis published by Wiley Periodicals, Inc. on behalf of International Society on Thrombosis and Haemostasis.

Hypervariability of ribosomal DNA at multiple chromosomal sites in lake trout (Salvelinus namaycush).

PubMed

Zhuo, L; Reed, K M; Phillips, R B

1995-06-01

Variation in the intergenic spacer (IGS) of the ribosomal DNA (rDNA) of lake trout (Salvelinus namaycush) was examined. Digestion of genomic DNA with restriction enzymes showed that almost every individual had a unique combination of length variants with most of this variation occurring within rather than between populations. Sequence analysis of a 2.3 kilobase (kb) EcoRI-DraI fragment spanning the 3' end of the 28S coding region and approximately 1.8 kb of the IGS revealed two blocks of repetitive DNA. Putative transcriptional termination sites were found approximately 220 bases (b) downstream from the end of the 28S coding region. Comparison of the 2.3-kb fragments with two longer (3.1 kb) fragments showed that the major difference in length resulted from variation in the number of short (89 b) repeats located 3' to the putative terminator. Repeat units within a single nucleolus organizer region (NOR) appeared relatively homogeneous and genetic analysis found variants to be stably inherited. A comparison of the number of spacer-length variants with the number of NORs found that the number of length variants per individual was always less than the number of NORs. Examination of spacer variants in five populations showed that populations with more NORs had more spacer variants, indicating that variants are present at different rDNA sites on nonhomologous chromosomes.

Comprehensive analysis of the MLH1 promoter region in 480 patients with colorectal cancer and 1150 controls reveals new variants including one with a heritable constitutional MLH1 epimutation.

PubMed

Morak, Monika; Ibisler, Ayseguel; Keller, Gisela; Jessen, Ellen; Laner, Andreas; Gonzales-Fassrainer, Daniela; Locher, Melanie; Massdorf, Trisari; Nissen, Anke M; Benet-Pagès, Anna; Holinski-Feder, Elke

2018-04-01

Germline defects in MLH1 , MSH2 , MSH6 and PMS2 predisposing for Lynch syndrome (LS) are mainly based on sequence changes, whereas a constitutional epimutation of MLH1 (CEM) is exceptionally rare. This abnormal MLH1 promoter methylation is not hereditary when arising de novo, whereas a stably heritable and variant-induced CEM was described for one single allele. We searched for MLH1 promoter variants causing a germline or somatic methylation induction or transcriptional repression. We analysed the MLH1 promoter sequence in five different patient groups with colorectal cancer (CRC) (n=480) composed of patients with i) CEM (n=16), ii) unsolved loss of MLH1 expression in CRC (n=37), iii) CpG-island methylator-phenotype CRC (n=102), iv) patients with LS (n=83) and v) MLH1-proficient CRC (n=242) as controls. 1150 patients with non-LS tumours also served as controls to correctly judge the results. We detected 10 rare MLH1 promoter variants. One novel, complex MLH1 variant c.-63_-58delins18 is present in a patient with CRC with CEM and his sister, both showing a complete allele-specific promoter methylation and transcriptional silencing. The other nine promoter variants detected in 17 individuals were not associated with methylation. For four of these, a normal, biallelic MLH1 expression was found in the patients' cDNA. We report the second promoter variant stably inducing a hereditary CEM. Concerning the classification of promoter variants, we discuss contradictory results from the literature for two variants, describe classification discrepancies between existing rules for five variants, suggest the (re-)classification of five promoter variants to (likely) benign and regard four variants as functionally unclear. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

The specification and global reprogramming of histone epigenetic marks during gamete formation and early embryo development in C. elegans.

PubMed

Samson, Mark; Jow, Margaret M; Wong, Catherine C L; Fitzpatrick, Colin; Aslanian, Aaron; Saucedo, Israel; Estrada, Rodrigo; Ito, Takashi; Park, Sung-kyu Robin; Yates, John R; Chu, Diana S

2014-10-01

In addition to the DNA contributed by sperm and oocytes, embryos receive parent-specific epigenetic information that can include histone variants, histone post-translational modifications (PTMs), and DNA methylation. However, a global view of how such marks are erased or retained during gamete formation and reprogrammed after fertilization is lacking. To focus on features conveyed by histones, we conducted a large-scale proteomic identification of histone variants and PTMs in sperm and mixed-stage embryo chromatin from C. elegans, a species that lacks conserved DNA methylation pathways. The fate of these histone marks was then tracked using immunostaining. Proteomic analysis found that sperm harbor ∼2.4 fold lower levels of histone PTMs than embryos and revealed differences in classes of PTMs between sperm and embryos. Sperm chromatin repackaging involves the incorporation of the sperm-specific histone H2A variant HTAS-1, a widespread erasure of histone acetylation, and the retention of histone methylation at sites that mark the transcriptional history of chromatin domains during spermatogenesis. After fertilization, we show HTAS-1 and 6 histone PTM marks distinguish sperm and oocyte chromatin in the new embryo and characterize distinct paternal and maternal histone remodeling events during the oocyte-to-embryo transition. These include the exchange of histone H2A that is marked by ubiquitination, retention of HTAS-1, removal of the H2A variant HTZ-1, and differential reprogramming of histone PTMs. This work identifies novel and conserved features of paternal chromatin that are specified during spermatogenesis and processed in the embryo. Furthermore, our results show that different species, even those with diverged DNA packaging and imprinting strategies, use conserved histone modification and removal mechanisms to reprogram epigenetic information.

Common genetic variants influence human subcortical brain structures

PubMed Central

Hibar, Derrek P.; Stein, Jason L.; Renteria, Miguel E.; Arias-Vasquez, Alejandro; Desrivières, Sylvane; Jahanshad, Neda; Toro, Roberto; Wittfeld, Katharina; Abramovic, Lucija; Andersson, Micael; Aribisala, Benjamin S.; Armstrong, Nicola J.; Bernard, Manon; Bohlken, Marc M.; Boks, Marco P.; Bralten, Janita; Brown, Andrew A.; Chakravarty, M. Mallar; Chen, Qiang; Ching, Christopher R. K.; Cuellar-Partida, Gabriel; den Braber, Anouk; Giddaluru, Sudheer; Goldman, Aaron L.; Grimm, Oliver; Guadalupe, Tulio; Hass, Johanna; Woldehawariat, Girma; Holmes, Avram J.; Hoogman, Martine; Janowitz, Deborah; Jia, Tianye; Kim, Sungeun; Klein, Marieke; Kraemer, Bernd; Lee, Phil H.; Olde Loohuis, Loes M.; Luciano, Michelle; Macare, Christine; Mather, Karen A.; Mattheisen, Manuel; Milaneschi, Yuri; Nho, Kwangsik; Papmeyer, Martina; Ramasamy, Adaikalavan; Risacher, Shannon L.; Roiz-Santiañez, Roberto; Rose, Emma J.; Salami, Alireza; Sämann, Philipp G.; Schmaal, Lianne; Schork, Andrew J.; Shin, Jean; Strike, Lachlan T.; Teumer, Alexander; van Donkelaar, Marjolein M. J.; van Eijk, Kristel R.; Walters, Raymond K.; Westlye, Lars T.; Whelan, Christopher D.; Winkler, Anderson M.; Zwiers, Marcel P.; Alhusaini, Saud; Athanasiu, Lavinia; Ehrlich, Stefan; Hakobjan, Marina M. H.; Hartberg, Cecilie B.; Haukvik, Unn K.; Heister, Angelien J. G. A. M.; Hoehn, David; Kasperaviciute, Dalia; Liewald, David C. M.; Lopez, Lorna M.; Makkinje, Remco R. R.; Matarin, Mar; Naber, Marlies A. M.; McKay, D. Reese; Needham, Margaret; Nugent, Allison C.; Pütz, Benno; Royle, Natalie A.; Shen, Li; Sprooten, Emma; Trabzuni, Daniah; van der Marel, Saskia S. L.; van Hulzen, Kimm J. E.; Walton, Esther; Wolf, Christiane; Almasy, Laura; Ames, David; Arepalli, Sampath; Assareh, Amelia A.; Bastin, Mark E.; Brodaty, Henry; Bulayeva, Kazima B.; Carless, Melanie A.; Cichon, Sven; Corvin, Aiden; Curran, Joanne E.; Czisch, Michael; de Zubicaray, Greig I.; Dillman, Allissa; Duggirala, Ravi; Dyer, Thomas D.; Erk, Susanne; Fedko, Iryna O.; Ferrucci, Luigi; Foroud, Tatiana M.; Fox, Peter T.; Fukunaga, Masaki; Gibbs, J. Raphael; Göring, Harald H. H.; Green, Robert C.; Guelfi, Sebastian; Hansell, Narelle K.; Hartman, Catharina A.; Hegenscheid, Katrin; Heinz, Andreas; Hernandez, Dena G.; Heslenfeld, Dirk J.; Hoekstra, Pieter J.; Holsboer, Florian; Homuth, Georg; Hottenga, Jouke-Jan; Ikeda, Masashi; Jack, Clifford R.; Jenkinson, Mark; Johnson, Robert; Kanai, Ryota; Keil, Maria; Kent, Jack W.; Kochunov, Peter; Kwok, John B.; Lawrie, Stephen M.; Liu, Xinmin; Longo, Dan L.; McMahon, Katie L.; Meisenzahl, Eva; Melle, Ingrid; Mohnke, Sebastian; Montgomery, Grant W.; Mostert, Jeanette C.; Mühleisen, Thomas W.; Nalls, Michael A.; Nichols, Thomas E.; Nilsson, Lars G.; Nöthen, Markus M.; Ohi, Kazutaka; Olvera, Rene L.; Perez-Iglesias, Rocio; Pike, G. Bruce; Potkin, Steven G.; Reinvang, Ivar; Reppermund, Simone; Rietschel, Marcella; Romanczuk-Seiferth, Nina; Rosen, Glenn D.; Rujescu, Dan; Schnell, Knut; Schofield, Peter R.; Smith, Colin; Steen, Vidar M.; Sussmann, Jessika E.; Thalamuthu, Anbupalam; Toga, Arthur W.; Traynor, Bryan J.; Troncoso, Juan; Turner, Jessica A.; Valdés Hernández, Maria C.; van ’t Ent, Dennis; van der Brug, Marcel; van der Wee, Nic J. A.; van Tol, Marie-Jose; Veltman, Dick J.; Wassink, Thomas H.; Westman, Eric; Zielke, Ronald H.; Zonderman, Alan B.; Ashbrook, David G.; Hager, Reinmar; Lu, Lu; McMahon, Francis J.; Morris, Derek W.; Williams, Robert W.; Brunner, Han G.; Buckner, Randy L.; Buitelaar, Jan K.; Cahn, Wiepke; Calhoun, Vince D.; Cavalleri, Gianpiero L.; Crespo-Facorro, Benedicto; Dale, Anders M.; Davies, Gareth E.; Delanty, Norman; Depondt, Chantal; Djurovic, Srdjan; Drevets, Wayne C.; Espeseth, Thomas; Gollub, Randy L.; Ho, Beng-Choon; Hoffmann, Wolfgang; Hosten, Norbert; Kahn, René S.; Le Hellard, Stephanie; Meyer-Lindenberg, Andreas; Müller-Myhsok, Bertram; Nauck, Matthias; Nyberg, Lars; Pandolfo, Massimo; Penninx, Brenda W. J. H.; Roffman, Joshua L.; Sisodiya, Sanjay M.; Smoller, Jordan W.; van Bokhoven, Hans; van Haren, Neeltje E. M.; Völzke, Henry; Walter, Henrik; Weiner, Michael W.; Wen, Wei; White, Tonya; Agartz, Ingrid; Andreassen, Ole A.; Blangero, John; Boomsma, Dorret I.; Brouwer, Rachel M.; Cannon, Dara M.; Cookson, Mark R.; de Geus, Eco J. C.; Deary, Ian J.; Donohoe, Gary; Fernández, Guillén; Fisher, Simon E.; Francks, Clyde; Glahn, David C.; Grabe, Hans J.; Gruber, Oliver; Hardy, John; Hashimoto, Ryota; Hulshoff Pol, Hilleke E.; Jönsson, Erik G.; Kloszewska, Iwona; Lovestone, Simon; Mattay, Venkata S.; Mecocci, Patrizia; McDonald, Colm; McIntosh, Andrew M.; Ophoff, Roel A.; Paus, Tomas; Pausova, Zdenka; Ryten, Mina; Sachdev, Perminder S.; Saykin, Andrew J.; Simmons, Andy; Singleton, Andrew; Soininen, Hilkka; Wardlaw, Joanna M.; Weale, Michael E.; Weinberger, Daniel R.; Adams, Hieab H. H.; Launer, Lenore J.; Seiler, Stephan; Schmidt, Reinhold; Chauhan, Ganesh; Satizabal, Claudia L.; Becker, James T.; Yanek, Lisa; van der Lee, Sven J.; Ebling, Maritza; Fischl, Bruce; Longstreth, W. T.; Greve, Douglas; Schmidt, Helena; Nyquist, Paul; Vinke, Louis N.; van Duijn, Cornelia M.; Xue, Luting; Mazoyer, Bernard; Bis, Joshua C.; Gudnason, Vilmundur; Seshadri, Sudha; Ikram, M. Arfan; Martin, Nicholas G.; Wright, Margaret J.; Schumann, Gunter; Franke, Barbara; Thompson, Paul M.; Medland, Sarah E.

2015-01-01

The highly complex structure of the human brain is strongly shaped by genetic influences1. Subcortical brain regions form circuits with cortical areas to coordinate movement2, learning, memory3 and motivation4, and altered circuits can lead to abnormal behaviour and disease2. To investigate how common genetic variants affect the structure of these brain regions, here we conduct genome-wide association studies of the volumes of seven subcortical regions and the intracranial volume derived from magnetic resonance images of 30,717 individuals from 50 cohorts. We identify five novel genetic variants influencing the volumes of the putamen and caudate nucleus. We also find stronger evidence for three loci with previously established influences on hippocampal volume5 and intracranial volume6. These variants show specific volumetric effects on brain structures rather than global effects across structures. The strongest effects were found for the putamen, where a novel intergenic locus with replicable influence on volume (rs945270; P = 1.08 × 10−33; 0.52% variance explained) showed evidence of altering the expression of the KTN1 gene in both brain and blood tissue. Variants influencing putamen volume clustered near developmental genes that regulate apoptosis, axon guidance and vesicle transport. Identification of these genetic variants provides insight into the causes of variability inhuman brain development, and may help to determine mechanisms of neuropsychiatric dysfunction. PMID:25607358

Association analysis of calpain 10 gene variants/haplotypes with gestational diabetes mellitus among Mexican women.

PubMed

Castro-Martínez, Anna Gabriela; Sánchez-Corona, José; Vázquez-Vargas, Adriana Patricia; García-Zapién, Alejandra Guadalupe; López-Quintero, Andres; Villalpando-Velazco, Héctor Javier; Flores-Martínez, Silvia Esperanza

2018-02-28

Gestational diabetes mellitus (GDM) is a metabolically complex disease with major genetic determinants. GDM has been associated with insulin resistance and dysfunction of pancreatic beta cells, so the GDM candidate genes are those that encode proteins modulating the function and secretion of insulin, such as that for calpain 10 (CAPN10). This study aimed to assess whether single nucleotide polymorphism (SNP)-43, SNP-44, SNP-63, and the indel-19 variant, and specific haplotypes of the CAPN10 gene were associated with gestational diabetes mellitus. We studied 116 patients with gestational diabetes mellitus and 83 women with normal glucose tolerance. Measurements of anthropometric and biochemical parameters were performed. SNP-43, SNP-44, and SNP-63 were identified by polymerase chain reaction (PCR)-restriction fragment length polymorphisms, while the indel-19 variant was detected by TaqMan qPCR assays.  The allele, genotype, and haplotype frequencies of the four variants did not differ significantly between women with gestational diabetes mellitus and controls. However, in women with gestational diabetes mellitus, glucose levels were significantly higher bearing the 3R/3R genotype than in carriers of the 3R/2R genotype of the indel-19 variant (p = 0.006). In conclusion, the 3R/3R genotype of the indel-19 variant of the CAPN-10 gene influenced increased glucose levels in these Mexican women with gestational diabetes mellitus.

Significant contribution of subtype G to HIV-1 genetic complexity in Nigeria identified by a newly developed subtyping assay specific for subtype G and CRF02_AG

PubMed Central

Heipertz, Richard A.; Ayemoba, Ojor; Sanders-Buell, Eric; Poltavee, Kultida; Pham, Phuc; Kijak, Gustavo H.; Lei, Esther; Bose, Meera; Howell, Shana; O'Sullivan, Anne Marie; Bates, Adam; Cervenka, Taylor; Kuroiwa, Janelle; Akintunde, Akindiran; Ibezim, Onyekachukwu; Alabi, Abraham; Okoye, Obumneke; Manak, Mark; Malia, Jennifer; Peel, Sheila; Maisaka, Mohammed; Singer, Darrell; O’Connell, Robert J.; Robb, Merlin L.; Kim, Jerome H.; Michael, Nelson L.; Njoku, Ogbonnaya; Tovanabutra, Sodsai

2016-01-01

Abstract While abundant sequence information is available from human immunodeficiency virus type 1 (HIV-1) subtypes A, B, C and CRF01_AE for HIV-1 vaccine design, sequences from West Africa are less represented. We sought to augment our understanding of HIV-1 variants circulating in 6 Nigerian cities as a step to subsequent HIV-1 vaccine development. The G/CRF02_AG multi-region hybridization assay (MHA) was developed to differentiate subtype G, CRF02_AG and their recombinants from other subtypes based on 7 HIV-1 segments. Plasma from 224 HIV-1 infected volunteers enrolled in a cohort examining HIV-1 prevalence, risk factor, and subtype from Makurdi (30), Abuja (18), Enugu (11), Kaduna (12), Tafa (95), and Ojo/Lagos (58) was analyzed using MHA. HIV-1 genomes from 42 samples were sequenced to validate the MHA and fully explore the recombinant structure of G and CRF02_AG variants. The sensitivity and specificity of MHA varied between 73–100% and 90–100%, respectively. The subtype distribution as identified by MHA among 224 samples revealed 38% CRF02_AG, 28% G, and 26% G/CRF02_AG recombinants while 8% remained nontypeable strains. In envelope (env) gp120, 38.84% of the samples reacted to a G probe while 31.25% reacted to a CRF02 (subtype A) probe. Full genome characterization of 42 sequences revealed the complexity of Nigerian HIV-1 variants. CRF02_AG, subtype G, and their recombinants were the major circulating HIV-1 variants in 6 Nigerian cities. High proportions of samples reacted to a G probe in env gp120 confirms that subtype G infections are abundant and should be considered in strategies for global HIV-1 vaccine development. PMID:27512845

Common Variants within Oxidative Phosphorylation Genes Influence Risk of Ischemic Stroke and Intracerebral Hemorrhage

PubMed Central

Anderson, Christopher D.; Biffi, Alessandro; Nalls, Michael A.; Devan, William J.; Schwab, Kristin; Ayres, Alison M.; Valant, Valerie; Ross, Owen A.; Rost, Natalia S.; Saxena, Richa; Viswanathan, Anand; Worrall, Bradford B.; Brott, Thomas G.; Goldstein, Joshua N.; Brown, Devin; Broderick, Joseph P.; Norrving, Bo; Greenberg, Steven M.; Silliman, Scott L.; Hansen, Björn M.; Tirschwell, David L.; Lindgren, Arne; Slowik, Agnieszka; Schmidt, Reinhold; Selim, Magdy; Roquer, Jaume; Montaner, Joan; Singleton, Andrew B.; Kidwell, Chelsea S.; Woo, Daniel; Furie, Karen L.; Meschia, James F.; Rosand, Jonathan

2013-01-01

Background and Purpose Prior studies demonstrated association between mitochondrial DNA variants and ischemic stroke (IS). We investigated whether variants within a larger set of oxidative phosphorylation (OXPHOS) genes encoded by both autosomal and mitochondrial DNA were associated with risk of IS and, based on our results, extended our investigation to intracerebral hemorrhage (ICH). Methods This association study employed a discovery cohort of 1643 individuals, a validation cohort of 2432 individuals for IS, and an extension cohort of 1476 individuals for ICH. Gene-set enrichment analysis (GSEA) was performed on all structural OXPHOS genes, as well as genes contributing to individual respiratory complexes. Gene-sets passing GSEA were tested by constructing genetic scores using common variants residing within each gene. Associations between each variant and IS that emerged in the discovery cohort were examined in validation and extension cohorts. Results IS was associated with genetic risk scores in OXPHOS as a whole (odds ratio (OR)=1.17, p=0.008) and Complex I (OR=1.06, p=0.050). Among IS subtypes, small vessel (SV) stroke showed association with OXPHOS (OR=1.16, p=0.007), Complex I (OR=1.13, p=0.027) and Complex IV (OR 1.14, p=0.018). To further explore this SV association, we extended our analysis to ICH, revealing association between deep hemispheric ICH and Complex IV (OR=1.08, p=0.008). Conclusions This pathway analysis demonstrates association between common genetic variants within OXPHOS genes and stroke. The associations for SV stroke and deep ICH suggest that genetic variation in OXPHOS influences small vessel pathobiology. Further studies are needed to identify culprit genetic variants and assess their functional consequences. PMID:23362085

Toward rules relating zinc finger protein sequences and DNA binding site preferences.

PubMed

Desjarlais, J R; Berg, J M

1992-08-15

Zinc finger proteins of the Cys2-His2 type consist of tandem arrays of domains, where each domain appears to contact three adjacent base pairs of DNA through three key residues. We have designed and prepared a series of variants of the central zinc finger within the DNA binding domain of Sp1 by using information from an analysis of a large data base of zinc finger protein sequences. Through systematic variations at two of the three contact positions (underlined), relatively specific recognition of sequences of the form 5'-GGGGN(G or T)GGG-3' has been achieved. These results provide the basis for rules that may develop into a code that will allow the design of zinc finger proteins with preselected DNA site specificity.

Immunogenicity of DNA Vaccine against H5N1 Containing Extended Kappa B Site: In Vivo Study in Mice and Chickens

PubMed Central

Redkiewicz, Patrycja; Stachyra, Anna; Sawicka, Róz∙a; Bocian, Katarzyna; Góra-Sochacka, Anna; Kosson, Piotr; Sirko, Agnieszka

2017-01-01

Influenza is one of the most important illnesses in the modern world, causing great public health losses each year due to the lack of medication and broadly protective, long-lasting vaccines. The development of highly immunogenic and safe vaccines is currently one of the major problems encountered in efficient influenza prevention. DNA vaccines represent a novel and powerful alternative to the conventional vaccine approaches. To improve the efficacy of the DNA vaccine against influenza H5N1, we inserted three repeated kappa B (κB) motifs, separated by a 5-bp nucleotide spacer, upstream of the cytomegalovirus promoter and downstream of the SV40 late polyadenylation signal. The κB motif is a specific DNA element (10pb-long) recognized by one of the most important transcription factors NFκB. NFκB is present in almost all animal cell types and upon cell stimulation under a variety of pathogenic conditions. NFκB is released from IκB and translocates to the nucleus and binds to κB sites, thereby leading to enhanced transcription and expression of downstream genes. We tested the variants of DNA vaccine with κB sites flanking the antigen expression cassette and without such sites in two animal models: chickens (broilers and layers) and mice (BALB/c). In chickens, the variant with κB sites stimulated stronger humoral response against the target antigen. In mice, the differences in humoral response were less apparent. Instead, it was possible to spot several gene expression differences in the spleens isolated from mice immunized with both variants. The results of our study indicate that modification of the sequence outside of the sequence encoding the antigen might enhance the immune response to the target but understanding the mechanisms responsible for this process requires further analysis. PMID:28883819

Exosites in the substrate specificity of blood coagulation reactions.

PubMed

Bock, P E; Panizzi, P; Verhamme, I M A

2007-07-01

The specificity of blood coagulation proteinases for substrate, inhibitor, and effector recognition is mediated by exosites on the surfaces of the catalytic domains, physically separated from the catalytic site. Some thrombin ligands bind specifically to either exosite I or II, while others engage both exosites. The involvement of different, overlapping constellations of exosite residues enables binding of structurally diverse ligands. The flexibility of the thrombin structure is central to the mechanism of complex formation and the specificity of exosite interactions. Encounter complex formation is driven by electrostatic ligand-exosite interactions, followed by conformational rearrangement to a stable complex. Exosites on some zymogens are in low affinity proexosite states and are expressed concomitant with catalytic site activation. The requirement for exosite expression controls the specificity of assembly of catalytic complexes on the coagulation pathway, such as the membrane-bound factor Xa*factor Va (prothrombinase) complex, and prevents premature assembly. Substrate recognition by prothrombinase involves a two-step mechanism with initial docking of prothrombin to exosites, followed by a conformational change to engage the FXa catalytic site. Prothrombin and its activation intermediates bind prothrombinase in two alternative conformations determined by the zymogen to proteinase transition that are hypothesized to involve prothrombin (pro)exosite I interactions with FVa, which underpin the sequential activation pathway. The role of exosites as the major source of substrate specificity has stimulated development of exosite-targeted anticoagulants for treatment of thrombosis.

Crystal structure of casein kinase-1, a phosphate-directed protein kinase.

PubMed Central

Xu, R M; Carmel, G; Sweet, R M; Kuret, J; Cheng, X

1995-01-01

The structure of a truncated variant of casein kinase-1 from Schizosaccharomyces pombe, has been determined in complex with MgATP at 2.0 A resolution. The model resembles the 'closed', ATP-bound conformations of the cyclin-dependent kinase 2 and the cAMP-dependent protein kinase, with clear differences in the structure of surface loops that impart unique features to casein kinase-1. The structure is of unphosphorylated, active conformation of casein kinase-1 and the peptide-binding site is fully accessible to substrate. Images PMID:7889932

Brittle cornea syndrome ZNF469 mutation carrier phenotype and segregation analysis of rare ZNF469 variants in familial keratoconus.

PubMed

Davidson, Alice E; Borasio, Edmondo; Liskova, Petra; Khan, Arif O; Hassan, Hala; Cheetham, Michael E; Plagnol, Vincent; Alkuraya, Fowzan S; Tuft, Stephen J; Hardcastle, Alison J

2015-01-06

Brittle cornea syndrome 1 (BCS1) is a rare recessive condition characterized by extreme thinning of the cornea and sclera, caused by mutations in ZNF469. Keratoconus is a relatively common disease characterized by progressive thinning and ectasia of the cornea. The etiology of keratoconus is complex and not yet understood, but rare ZNF469 variants have recently been associated with disease. We investigated the phenotype of BCS1 carriers with known pathogenic ZNF469 mutations, and recruited families in which aggregation of keratoconus was observed to establish if rare variants in ZNF469 segregated with disease. Patients and family members were recruited and underwent comprehensive anterior segment examination, including corneal topography. Blood samples were donated and genomic DNA was extracted. The coding sequence and splice sites of ZNF469 were PCR amplified and Sanger sequenced. Four carriers of three BCS1-associated ZNF469 loss-of-function mutations (p.[Glu1392Ter], p.[Gln1930Argfs*6], p.[Gln1930fs*133]) were examined and none had keratoconus. One carrier had partially penetrant features of BCS1, including joint hypermobility. ZNF469 sequencing in 11 keratoconus families identified 9 rare (minor allele frequency [MAF] ≤ 0.025) variants predicted to be potentially damaging. However, in each instance the rare variant(s) identified, including two previously reported as potentially keratoconus-associated, did not segregate with the disease. The presence of heterozygous loss-of-function alleles in the ZNF469 gene did not cause keratoconus in the individuals examined. None of the rare nonsynonymous ZNF469 variants identified in the familial cohort conferred a high risk of keratoconus; therefore, genetic variants contributing to disease pathogenesis in these 11 families remain to be identified. Copyright 2015 The Association for Research in Vision and Ophthalmology, Inc.

Comparative analysis of activator-Eσ54 complexes formed with nucleotide-metal fluoride analogues

PubMed Central

Burrows, Patricia C.; Joly, Nicolas; Nixon, B. Tracy; Buck, Martin

2009-01-01

Bacterial RNA polymerase (RNAP) containing the major variant σ54 factor forms open promoter complexes in a reaction in which specialized activator proteins hydrolyse ATP. Here we probe binding interactions between σ54-RNAP (Eσ54) and the ATPases associated with various cellular activities (AAA+) domain of the Escherichia coli activator protein, PspF, using nucleotide-metal fluoride (BeF and AlF) analogues representing ground and transition states of ATP, which allow complexes (that are otherwise too transient with ATP) to be captured. We show that the organization and functionality of the ADP–BeF- and ADP–AlF-dependent complexes greatly overlap. Our data support an activation pathway in which the initial ATP-dependent binding of the activator to the Eσ54 closed complex results in the re-organization of Eσ54 with respect to the transcription start-site. However, the nucleotide-dependent binding interactions between the activator and the Eσ54 closed complex are in themselves insufficient for forming open promoter complexes when linear double-stranded DNA is present in the initial closed complex. PMID:19553192

Functional analysis of dengue virus (DENV) type 2 envelope protein domain 3 type-specific and DENV complex-reactive critical epitope residues.

PubMed

Pitcher, Trevor J; Sarathy, Vanessa V; Matsui, Kiyohiko; Gromowski, Gregory D; Huang, Claire Y-H; Barrett, Alan D T

2015-02-01

The dengue virus (DENV) envelope protein domain 3 (ED3) is the target of potent virus neutralizing antibodies. The DENV-2 ED3 contains adjacent type-specific and DENV complex-reactive antigenic sites that are composed of a small number of residues that were previously demonstrated to be critical for antibody binding. Site-directed mutagenesis of a DENV-2 16681 infectious clone was used to mutate critical residues in the DENV-2 type-specific (K305A and P384A) and DENV complex-reactive (K310A) antigenic sites. The K305A mutant virus multiplied like the parent virus in mosquito and mammalian cells, as did the P384A mutant virus, which required a compensatory mutation (G330D) for viability. However, the K310A mutant virus could not be recovered. The DENV-2 type-specific critical residue mutations K305A and P384A+G330D reduced the ability of DENV-2 type-specific, but not DENV complex-reactive, mAbs to neutralize virus infectivity and this was directly correlated with mAb binding affinity to the rED3 mutants. © 2015 The Authors.

Pooled-DNA Sequencing for Elucidating New Genomic Risk Factors, Rare Variants Underlying Alzheimer's Disease.

PubMed

Jin, Sheng Chih; Benitez, Bruno A; Deming, Yuetiva; Cruchaga, Carlos

2016-01-01

Analyses of genome-wide association studies (GWAS) for complex disorders usually identify common variants with a relatively small effect size that only explain a small proportion of phenotypic heritability. Several studies have suggested that a significant fraction of heritability may be explained by low-frequency (minor allele frequency (MAF) of 1-5 %) and rare-variants that are not contained in the commercial GWAS genotyping arrays (Schork et al., Curr Opin Genet Dev 19:212, 2009). Rare variants can also have relatively large effects on risk for developing human diseases or disease phenotype (Cruchaga et al., PLoS One 7:e31039, 2012). However, it is necessary to perform next-generation sequencing (NGS) studies in a large population (>4,000 samples) to detect a significant rare-variant association. Several NGS methods, such as custom capture sequencing and amplicon-based sequencing, are designed to screen a small proportion of the genome, but most of these methods are limited in the number of samples that can be multiplexed (i.e. most sequencing kits only provide 96 distinct index). Additionally, the sequencing library preparation for 4,000 samples remains expensive and thus conducting NGS studies with the aforementioned methods are not feasible for most research laboratories.The need for low-cost large scale rare-variant detection makes pooled-DNA sequencing an ideally efficient and cost-effective technique to identify rare variants in target regions by sequencing hundreds to thousands of samples. Our recent work has demonstrated that pooled-DNA sequencing can accurately detect rare variants in targeted regions in multiple DNA samples with high sensitivity and specificity (Jin et al., Alzheimers Res Ther 4:34, 2012). In these studies we used a well-established pooled-DNA sequencing approach and a computational package, SPLINTER (short indel prediction by large deviation inference and nonlinear true frequency estimation by recursion) (Vallania et al., Genome Res 20:1711, 2010), for accurate identification of rare variants in large DNA pools. Given an average sequencing coverage of 30× per haploid genome, SPLINTER can detect rare variants and short indels up to 4 base pairs (bp) with high sensitivity and specificity (up to 1 haploid allele in a pool as large as 500 individuals). Step-by-step instructions on how to conduct pooled-DNA sequencing experiments and data analyses are described in this chapter.

Demographic history and rare allele sharing among human populations.

PubMed

Gravel, Simon; Henn, Brenna M; Gutenkunst, Ryan N; Indap, Amit R; Marth, Gabor T; Clark, Andrew G; Yu, Fuli; Gibbs, Richard A; Bustamante, Carlos D

2011-07-19

High-throughput sequencing technology enables population-level surveys of human genomic variation. Here, we examine the joint allele frequency distributions across continental human populations and present an approach for combining complementary aspects of whole-genome, low-coverage data and targeted high-coverage data. We apply this approach to data generated by the pilot phase of the Thousand Genomes Project, including whole-genome 2-4× coverage data for 179 samples from HapMap European, Asian, and African panels as well as high-coverage target sequencing of the exons of 800 genes from 697 individuals in seven populations. We use the site frequency spectra obtained from these data to infer demographic parameters for an Out-of-Africa model for populations of African, European, and Asian descent and to predict, by a jackknife-based approach, the amount of genetic diversity that will be discovered as sample sizes are increased. We predict that the number of discovered nonsynonymous coding variants will reach 100,000 in each population after ∼1,000 sequenced chromosomes per population, whereas ∼2,500 chromosomes will be needed for the same number of synonymous variants. Beyond this point, the number of segregating sites in the European and Asian panel populations is expected to overcome that of the African panel because of faster recent population growth. Overall, we find that the majority of human genomic variable sites are rare and exhibit little sharing among diverged populations. Our results emphasize that replication of disease association for specific rare genetic variants across diverged populations must overcome both reduced statistical power because of rarity and higher population divergence.

Fine Mapping of a Clubroot Resistance Gene in Chinese Cabbage Using SNP Markers Identified from Bulked Segregant RNA Sequencing

PubMed Central

Huang, Zhen; Peng, Gary; Liu, Xunjia; Deora, Abhinandan; Falk, Kevin C.; Gossen, Bruce D.; McDonald, Mary R.; Yu, Fengqun

2017-01-01

Clubroot, caused by Plasmodiophora brassicae, is an important disease of canola (Brassica napus) in western Canada and worldwide. In this study, a clubroot resistance gene (Rcr2) was identified and fine mapped in Chinese cabbage cv. “Jazz” using single-nucleotide polymorphisms (SNP) markers identified from bulked segregant RNA sequencing (BSR-Seq) and molecular markers were developed for use in marker assisted selection. In total, 203.9 million raw reads were generated from one pooled resistant (R) and one pooled susceptible (S) sample, and >173,000 polymorphic SNP sites were identified between the R and S samples. One significant peak was observed between 22 and 26 Mb of chromosome A03, which had been predicted by BSR-Seq to contain the causal gene Rcr2. There were 490 polymorphic SNP sites identified in the region. A segregating population consisting of 675 plants was analyzed with 15 SNP sites in the region using the Kompetitive Allele Specific PCR method, and Rcr2 was fine mapped between two SNP markers, SNP_A03_32 and SNP_A03_67 with 0.1 and 0.3 cM from Rcr2, respectively. Five SNP markers co-segregated with Rcr2 in this region. Variants were identified in 14 of 36 genes annotated in the Rcr2 target region. The numbers of poly variants differed among the genes. Four genes encode TIR-NBS-LRR proteins and two of them Bra019410 and Bra019413, had high numbers of polymorphic variants and so are the most likely candidates of Rcr2. PMID:28894454

Demographic history and rare allele sharing among human populations

PubMed Central

Gravel, Simon; Henn, Brenna M.; Gutenkunst, Ryan N.; Indap, Amit R.; Marth, Gabor T.; Clark, Andrew G.; Yu, Fuli; Gibbs, Richard A.; Bustamante, Carlos D.; Altshuler, David L.; Durbin, Richard M.; Abecasis, Gonçalo R.; Bentley, David R.; Chakravarti, Aravinda; Clark, Andrew G.; Collins, Francis S.; De La Vega, Francisco M.; Donnelly, Peter; Egholm, Michael; Flicek, Paul; Gabriel, Stacey B.; Gibbs, Richard A.; Knoppers, Bartha M.; Lander, Eric S.; Lehrach, Hans; Mardis, Elaine R.; McVean, Gil A.; Nickerson, Debbie A.; Peltonen, Leena; Schafer, Alan J.; Sherry, Stephen T.; Wang, Jun; Wilson, Richard K.; Gibbs, Richard A.; Deiros, David; Metzker, Mike; Muzny, Donna; Reid, Jeff; Wheeler, David; Wang, Jun; Li, Jingxiang; Jian, Min; Li, Guoqing; Li, Ruiqiang; Liang, Huiqing; Tian, Geng; Wang, Bo; Wang, Jian; Wang, Wei; Yang, Huanming; Zhang, Xiuqing; Zheng, Huisong; Lander, Eric S.; Altshuler, David L.; Ambrogio, Lauren; Bloom, Toby; Cibulskis, Kristian; Fennell, Tim J.; Gabriel, Stacey B.; Jaffe, David B.; Shefler, Erica; Sougnez, Carrie L.; Bentley, David R.; Gormley, Niall; Humphray, Sean; Kingsbury, Zoya; Koko-Gonzales, Paula; Stone, Jennifer; McKernan, Kevin J.; Costa, Gina L.; Ichikawa, Jeffry K.; Lee, Clarence C.; Sudbrak, Ralf; Lehrach, Hans; Borodina, Tatiana A.; Dahl, Andreas; Davydov, Alexey N.; Marquardt, Peter; Mertes, Florian; Nietfeld, Wilfiried; Rosenstiel, Philip; Schreiber, Stefan; Soldatov, Aleksey V.; Timmermann, Bernd; Tolzmann, Marius; Egholm, Michael; Affourtit, Jason; Ashworth, Dana; Attiya, Said; Bachorski, Melissa; Buglione, Eli; Burke, Adam; Caprio, Amanda; Celone, Christopher; Clark, Shauna; Conners, David; Desany, Brian; Gu, Lisa; Guccione, Lorri; Kao, Kalvin; Kebbel, Andrew; Knowlton, Jennifer; Labrecque, Matthew; McDade, Louise; Mealmaker, Craig; Minderman, Melissa; Nawrocki, Anne; Niazi, Faheem; Pareja, Kristen; Ramenani, Ravi; Riches, David; Song, Wanmin; Turcotte, Cynthia; Wang, Shally; Mardis, Elaine R.; Wilson, Richard K.; Dooling, David; Fulton, Lucinda; Fulton, Robert; Weinstock, George; Durbin, Richard M.; Burton, John; Carter, David M.; Churcher, Carol; Coffey, Alison; Cox, Anthony; Palotie, Aarno; Quail, Michael; Skelly, Tom; Stalker, James; Swerdlow, Harold P.; Turner, Daniel; De Witte, Anniek; Giles, Shane; Gibbs, Richard A.; Wheeler, David; Bainbridge, Matthew; Challis, Danny; Sabo, Aniko; Yu, Fuli; Yu, Jin; Wang, Jun; Fang, Xiaodong; Guo, Xiaosen; Li, Ruiqiang; Li, Yingrui; Luo, Ruibang; Tai, Shuaishuai; Wu, Honglong; Zheng, Hancheng; Zheng, Xiaole; Zhou, Yan; Li, Guoqing; Wang, Jian; Yang, Huanming; Marth, Gabor T.; Garrison, Erik P.; Huang, Weichun; Indap, Amit; Kural, Deniz; Lee, Wan-Ping; Leong, Wen Fung; Quinlan, Aaron R.; Stewart, Chip; Stromberg, Michael P.; Ward, Alistair N.; Wu, Jiantao; Lee, Charles; Mills, Ryan E.; Shi, Xinghua; Daly, Mark J.; DePristo, Mark A.; Altshuler, David L.; Ball, Aaron D.; Banks, Eric; Bloom, Toby; Browning, Brian L.; Cibulskis, Kristian; Fennell, Tim J.; Garimella, Kiran V.; Grossman, Sharon R.; Handsaker, Robert E.; Hanna, Matt; Hartl, Chris; Jaffe, David B.; Kernytsky, Andrew M.; Korn, Joshua M.; Li, Heng; Maguire, Jared R.; McCarroll, Steven A.; McKenna, Aaron; Nemesh, James C.; Philippakis, Anthony A.; Poplin, Ryan E.; Price, Alkes; Rivas, Manuel A.; Sabeti, Pardis C.; Schaffner, Stephen F.; Shefler, Erica; Shlyakhter, Ilya A.; Cooper, David N.; Ball, Edward V.; Mort, Matthew; Phillips, Andrew D.; Stenson, Peter D.; Sebat, Jonathan; Makarov, Vladimir; Ye, Kenny; Yoon, Seungtai C.; Bustamante, Carlos D.; Clark, Andrew G.; Boyko, Adam; Degenhardt, Jeremiah; Gravel, Simon; Gutenkunst, Ryan N.; Kaganovich, Mark; Keinan, Alon; Lacroute, Phil; Ma, Xin; Reynolds, Andy; Clarke, Laura; Flicek, Paul; Cunningham, Fiona; Herrero, Javier; Keenen, Stephen; Kulesha, Eugene; Leinonen, Rasko; McLaren, William M.; Radhakrishnan, Rajesh; Smith, Richard E.; Zalunin, Vadim; Zheng-Bradley, Xiangqun; Korbel, Jan O.; Stütz, Adrian M.; Humphray, Sean; Bauer, Markus; Cheetham, R. Keira; Cox, Tony; Eberle, Michael; James, Terena; Kahn, Scott; Murray, Lisa; Chakravarti, Aravinda; Ye, Kai; De La Vega, Francisco M.; Fu, Yutao; Hyland, Fiona C. L.; Manning, Jonathan M.; McLaughlin, Stephen F.; Peckham, Heather E.; Sakarya, Onur; Sun, Yongming A.; Tsung, Eric F.; Batzer, Mark A.; Konkel, Miriam K.; Walker, Jerilyn A.; Sudbrak, Ralf; Albrecht, Marcus W.; Amstislavskiy, Vyacheslav S.; Herwig, Ralf; Parkhomchuk, Dimitri V.; Sherry, Stephen T.; Agarwala, Richa; Khouri, Hoda M.; Morgulis, Aleksandr O.; Paschall, Justin E.; Phan, Lon D.; Rotmistrovsky, Kirill E.; Sanders, Robert D.; Shumway, Martin F.; Xiao, Chunlin; McVean, Gil A.; Auton, Adam; Iqbal, Zamin; Lunter, Gerton; Marchini, Jonathan L.; Moutsianas, Loukas; Myers, Simon; Tumian, Afidalina; Desany, Brian; Knight, James; Winer, Roger; Craig, David W.; Beckstrom-Sternberg, Steve M.; Christoforides, Alexis; Kurdoglu, Ahmet A.; Pearson, John V.; Sinari, Shripad A.; Tembe, Waibhav D.; Haussler, David; Hinrichs, Angie S.; Katzman, Sol J.; Kern, Andrew; Kuhn, Robert M.; Przeworski, Molly; Hernandez, Ryan D.; Howie, Bryan; Kelley, Joanna L.; Melton, S. Cord; Abecasis, Gonçalo R.; Li, Yun; Anderson, Paul; Blackwell, Tom; Chen, Wei; Cookson, William O.; Ding, Jun; Kang, Hyun Min; Lathrop, Mark; Liang, Liming; Moffatt, Miriam F.; Scheet, Paul; Sidore, Carlo; Snyder, Matthew; Zhan, Xiaowei; Zöllner, Sebastian; Awadalla, Philip; Casals, Ferran; Idaghdour, Youssef; Keebler, John; Stone, Eric A.; Zilversmit, Martine; Jorde, Lynn; Xing, Jinchuan; Eichler, Evan E.; Aksay, Gozde; Alkan, Can; Hajirasouliha, Iman; Hormozdiari, Fereydoun; Kidd, Jeffrey M.; Sahinalp, S. Cenk; Sudmant, Peter H.; Mardis, Elaine R.; Chen, Ken; Chinwalla, Asif; Ding, Li; Koboldt, Daniel C.; McLellan, Mike D.; Dooling, David; Weinstock, George; Wallis, John W.; Wendl, Michael C.; Zhang, Qunyuan; Durbin, Richard M.; Albers, Cornelis A.; Ayub, Qasim; Balasubramaniam, Senduran; Barrett, Jeffrey C.; Carter, David M.; Chen, Yuan; Conrad, Donald F.; Danecek, Petr; Dermitzakis, Emmanouil T.; Hu, Min; Huang, Ni; Hurles, Matt E.; Jin, Hanjun; Jostins, Luke; Keane, Thomas M.; Le, Si Quang; Lindsay, Sarah; Long, Quan; MacArthur, Daniel G.; Montgomery, Stephen B.; Parts, Leopold; Stalker, James; Tyler-Smith, Chris; Walter, Klaudia; Zhang, Yujun; Gerstein, Mark B.; Snyder, Michael; Abyzov, Alexej; Balasubramanian, Suganthi; Bjornson, Robert; Du, Jiang; Grubert, Fabian; Habegger, Lukas; Haraksingh, Rajini; Jee, Justin; Khurana, Ekta; Lam, Hugo Y. K.; Leng, Jing; Mu, Xinmeng Jasmine; Urban, Alexander E.; Zhang, Zhengdong; Li, Yingrui; Luo, Ruibang; Marth, Gabor T.; Garrison, Erik P.; Kural, Deniz; Quinlan, Aaron R.; Stewart, Chip; Stromberg, Michael P.; Ward, Alistair N.; Wu, Jiantao; Lee, Charles; Mills, Ryan E.; Shi, Xinghua; McCarroll, Steven A.; Banks, Eric; DePristo, Mark A.; Handsaker, Robert E.; Hartl, Chris; Korn, Joshua M.; Li, Heng; Nemesh, James C.; Sebat, Jonathan; Makarov, Vladimir; Ye, Kenny; Yoon, Seungtai C.; Degenhardt, Jeremiah; Kaganovich, Mark; Clarke, Laura; Smith, Richard E.; Zheng-Bradley, Xiangqun; Korbel, Jan O.; Humphray, Sean; Cheetham, R. Keira; Eberle, Michael; Kahn, Scott; Murray, Lisa; Ye, Kai; De La Vega, Francisco M.; Fu, Yutao; Peckham, Heather E.; Sun, Yongming A.; Batzer, Mark A.; Konkel, Miriam K.; Walker, Jerilyn A.; Xiao, Chunlin; Iqbal, Zamin; Desany, Brian; Blackwell, Tom; Snyder, Matthew; Xing, Jinchuan; Eichler, Evan E.; Aksay, Gozde; Alkan, Can; Hajirasouliha, Iman; Hormozdiari, Fereydoun; Kidd, Jeffrey M.; Chen, Ken; Chinwalla, Asif; Ding, Li; McLellan, Mike D.; Wallis, John W.; Hurles, Matt E.; Conrad, Donald F.; Walter, Klaudia; Zhang, Yujun; Gerstein, Mark B.; Snyder, Michael; Abyzov, Alexej; Du, Jiang; Grubert, Fabian; Haraksingh, Rajini; Jee, Justin; Khurana, Ekta; Lam, Hugo Y. K.; Leng, Jing; Mu, Xinmeng Jasmine; Urban, Alexander E.; Zhang, Zhengdong; Gibbs, Richard A.; Bainbridge, Matthew; Challis, Danny; Coafra, Cristian; Dinh, Huyen; Kovar, Christie; Lee, Sandy; Muzny, Donna; Nazareth, Lynne; Reid, Jeff; Sabo, Aniko; Yu, Fuli; Yu, Jin; Marth, Gabor T.; Garrison, Erik P.; Indap, Amit; Leong, Wen Fung; Quinlan, Aaron R.; Stewart, Chip; Ward, Alistair N.; Wu, Jiantao; Cibulskis, Kristian; Fennell, Tim J.; Gabriel, Stacey B.; Garimella, Kiran V.; Hartl, Chris; Shefler, Erica; Sougnez, Carrie L.; Wilkinson, Jane; Clark, Andrew G.; Gravel, Simon; Grubert, Fabian; Clarke, Laura; Flicek, Paul; Smith, Richard E.; Zheng-Bradley, Xiangqun; Sherry, Stephen T.; Khouri, Hoda M.; Paschall, Justin E.; Shumway, Martin F.; Xiao, Chunlin; McVean, Gil A.; Katzman, Sol J.; Abecasis, Gonçalo R.; Blackwell, Tom; Mardis, Elaine R.; Dooling, David; Fulton, Lucinda; Fulton, Robert; Koboldt, Daniel C.; Durbin, Richard M.; Balasubramaniam, Senduran; Coffey, Allison; Keane, Thomas M.; MacArthur, Daniel G.; Palotie, Aarno; Scott, Carol; Stalker, James; Tyler-Smith, Chris; Gerstein, Mark B.; Balasubramanian, Suganthi; Chakravarti, Aravinda; Knoppers, Bartha M.; Abecasis, Gonçalo R.; Bustamante, Carlos D.; Gharani, Neda; Gibbs, Richard A.; Jorde, Lynn; Kaye, Jane S.; Kent, Alastair; Li, Taosha; McGuire, Amy L.; McVean, Gil A.; Ossorio, Pilar N.; Rotimi, Charles N.; Su, Yeyang; Toji, Lorraine H.; TylerSmith, Chris; Brooks, Lisa D.; Felsenfeld, Adam L.; McEwen, Jean E.; Abdallah, Assya; Juenger, Christopher R.; Clemm, Nicholas C.; Collins, Francis S.; Duncanson, Audrey; Green, Eric D.; Guyer, Mark S.; Peterson, Jane L.; Schafer, Alan J.; Abecasis, Gonçalo R.; Altshuler, David L.; Auton, Adam; Brooks, Lisa D.; Durbin, Richard M.; Gibbs, Richard A.; Hurles, Matt E.; McVean, Gil A.

2011-01-01

High-throughput sequencing technology enables population-level surveys of human genomic variation. Here, we examine the joint allele frequency distributions across continental human populations and present an approach for combining complementary aspects of whole-genome, low-coverage data and targeted high-coverage data. We apply this approach to data generated by the pilot phase of the Thousand Genomes Project, including whole-genome 2–4× coverage data for 179 samples from HapMap European, Asian, and African panels as well as high-coverage target sequencing of the exons of 800 genes from 697 individuals in seven populations. We use the site frequency spectra obtained from these data to infer demographic parameters for an Out-of-Africa model for populations of African, European, and Asian descent and to predict, by a jackknife-based approach, the amount of genetic diversity that will be discovered as sample sizes are increased. We predict that the number of discovered nonsynonymous coding variants will reach 100,000 in each population after ∼1,000 sequenced chromosomes per population, whereas ∼2,500 chromosomes will be needed for the same number of synonymous variants. Beyond this point, the number of segregating sites in the European and Asian panel populations is expected to overcome that of the African panel because of faster recent population growth. Overall, we find that the majority of human genomic variable sites are rare and exhibit little sharing among diverged populations. Our results emphasize that replication of disease association for specific rare genetic variants across diverged populations must overcome both reduced statistical power because of rarity and higher population divergence. PMID:21730125

Mutation databases and other online sites as a resource for transfusion medicine: history and attributes.

PubMed

Blumenfeld, Olga O

2002-04-01

Recent advances in molecular biology and technology have provided evidence, at a molecular level, for long-known observations that the human genome is not unique but is characterized by individual sequence variation. At the present time, documentation of genetic variation occurring in a large number of genes is increasing exponentially. The characterization of alleles that encode a variety of blood group antigens has been particularly fruitful for transfusion medicine. Phenotypic variation, as identified by the serologic study of blood group variants, is required to identify the presence of a variant allele. Many of the other alleles currently recorded have been selected and identified on the basis of inherited disease traits. New approaches document single nucleotide polymorphisms that occur throughout the genome and best show how the DNA sequence varies in the human population. The primary data dealing with variant alleles or more general genomic variation are scattered throughout the scientific literature and only within the last few years has information begun to be organized into databases. This article provides guidance on how to access those databases online as a source of information about genetic variation for purposes of molecular, clinical, and diagnostic medicine, research, and teaching. The attributes of the sites are described. A more detailed view of the database dealing specifically with alleles of genes encoding the blood group antigens includes a brief preliminary analysis of the molecular basis for observed polymorphisms. Other online sites that may be particularly useful to the transfusion medicine readership as well as a brief historical account are also presented. Copyright 2002, Elsevier Science (USA). All rights reserved.

Genome-wide association analysis of eosinophilic esophagitis provides insight into the tissue specificity of this allergic disease.

PubMed

Kottyan, Leah C; Davis, Benjamin P; Sherrill, Joseph D; Liu, Kan; Rochman, Mark; Kaufman, Kenneth; Weirauch, Matthew T; Vaughn, Samuel; Lazaro, Sara; Rupert, Andrew M; Kohram, Mojtaba; Stucke, Emily M; Kemme, Katherine A; Magnusen, Albert; He, Hua; Dexheimer, Phillip; Chehade, Mirna; Wood, Robert A; Pesek, Robbie D; Vickery, Brian P; Fleischer, David M; Lindbad, Robert; Sampson, Hugh A; Mukkada, Vincent A; Putnam, Phil E; Abonia, J Pablo; Martin, Lisa J; Harley, John B; Rothenberg, Marc E

2014-08-01

Eosinophilic esophagitis (EoE) is a chronic inflammatory disorder associated with allergic hypersensitivity to food. We interrogated >1.5 million genetic variants in EoE cases of European ancestry and subsequently in a multi-site cohort with local and out-of-study control subjects. In addition to replicating association of the 5q22 locus (meta-analysis P=1.9×10(-16)), we identified an association at 2p23 spanning CAPN14 (P=2.5×10(-10)). CAPN14 was specifically expressed in the esophagus, was dynamically upregulated as a function of disease activity and genetic haplotype and after exposure of epithelial cells to interleukin (IL)-13, and was located in an epigenetic hotspot modified by IL-13. Genes neighboring the top 208 EoE-associated sequence variants were enriched for esophageal expression, and multiple loci for allergic sensitization were associated with EoE susceptibility (4.8×10(-2)

Genome-wide association analysis of eosinophilic esophagitis provides insight into the tissue specificity of this allergic disease

PubMed Central

Kottyan, Leah C.; Davis, Benjamin P.; Sherrill, Joseph D.; Liu, Kan; Rochman, Mark; Kaufman, Kenneth; Weirauch, Matthew T.; Vaughn, Samuel; Lazaro, Sara; Rupert, Andrew M.; Kohram, Mojtaba; Stucke, Emily M.; Kemme, Katherine A.; Magnusen, Albert; He, Hua; Dexheimer, Phillip; Chehade, Mirna; Wood, Robert A.; Pesek, Robbie D.; Vickery, Brian P.; Fleischer, David M.; Lindbad, Robert; Sampson, Hugh A.; Mukkada, Vince; Putnam, Phil E.; Abonia, J. Pablo; Martin, Lisa J.; Harley, John B.; Rothenberg, Marc E.

2014-01-01

Eosinophilic esophagitis (EoE) is a chronic inflammatory disorder associated with allergic hypersensitivity to food. We interrogated >1.5 million genetic variants in European EoE cases and subsequently in a multi-site cohort with local and out-of-study control subjects. In addition to replication of the 5q22 locus (meta-analysis p = 1.9×10−16), we identified association at 2p23 (encoding CAPN14, p = 2.5×10−10). CAPN14 was specifically expressed in the esophagus, dynamically upregulated as a function of disease activity and genetic haplotype and after exposure of epithelial cells to IL-13, and located in an epigenetic hotspot modified by IL-13. There was enriched esophageal expression for the genes neighboring the top 208 EoE sequence variants. Multiple allergic sensitization loci were associated with EoE susceptibility (4.8×10−2 < p < 5.1×10−11). We propose a model that elucidates the tissue specific nature of EoE that involves the interplay of allergic sensitization with an EoE-specific, IL-13–inducible esophageal response involving CAPN14. PMID:25017104

Structures of human thymidylate synthase R163K with dUMP, FdUMP and glutathione show asymmetric ligand binding

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gibson, Lydia M.; Celeste, Lesa R.; Lovelace, Leslie L.

Thymidylate synthase (TS) is a well validated target in cancer chemotherapy. Here, a new crystal form of the R163K variant of human TS (hTS) with five subunits per asymmetric part of the unit cell, all with loop 181-197 in the active conformation, is reported. This form allows binding studies by soaking crystals in artificial mother liquors containing ligands that bind in the active site. Using this approach, crystal structures of hTS complexes with FdUMP and dUMP were obtained, indicating that this form should facilitate high-throughput analysis of hTS complexes with drug candidates. Crystal soaking experiments using oxidized glutathione revealed thatmore » hTS binds this ligand. Interestingly, the two types of binding observed are both asymmetric. In one subunit of the physiological dimer covalent modification of the catalytic nucleophile Cys195 takes place, while in another dimer a noncovalent adduct with reduced glutathione is formed in one of the active sites.« less

Whole-Exome Sequencing Identifies Novel Variants for Tooth Agenesis.

PubMed

Dinckan, N; Du, R; Petty, L E; Coban-Akdemir, Z; Jhangiani, S N; Paine, I; Baugh, E H; Erdem, A P; Kayserili, H; Doddapaneni, H; Hu, J; Muzny, D M; Boerwinkle, E; Gibbs, R A; Lupski, J R; Uyguner, Z O; Below, J E; Letra, A

2018-01-01

Tooth agenesis is a common craniofacial abnormality in humans and represents failure to develop 1 or more permanent teeth. Tooth agenesis is complex, and variations in about a dozen genes have been reported as contributing to the etiology. Here, we combined whole-exome sequencing, array-based genotyping, and linkage analysis to identify putative pathogenic variants in candidate disease genes for tooth agenesis in 10 multiplex Turkish families. Novel homozygous and heterozygous variants in LRP6, DKK1, LAMA3, and COL17A1 genes, as well as known variants in WNT10A, were identified as likely pathogenic in isolated tooth agenesis. Novel variants in KREMEN1 were identified as likely pathogenic in 2 families with suspected syndromic tooth agenesis. Variants in more than 1 gene were identified segregating with tooth agenesis in 2 families, suggesting oligogenic inheritance. Structural modeling of missense variants suggests deleterious effects to the encoded proteins. Functional analysis of an indel variant (c.3607+3_6del) in LRP6 suggested that the predicted resulting mRNA is subject to nonsense-mediated decay. Our results support a major role for WNT pathways genes in the etiology of tooth agenesis while revealing new candidate genes. Moreover, oligogenic cosegregation was suggestive for complex inheritance and potentially complex gene product interactions during development, contributing to improved understanding of the genetic etiology of familial tooth agenesis.

Paraxial diffractive elements for space-variant linear transforms

NASA Astrophysics Data System (ADS)

Teiwes, Stephan; Schwarzer, Heiko; Gu, Ben-Yuan

1998-06-01

Optical linear transform architectures bear good potential for future developments of very powerful hybrid vision systems and neural network classifiers. The optical modules of such systems could be used as pre-processors to solve complex linear operations at very high speed in order to simplify an electronic data post-processing. However, the applicability of linear optical architectures is strongly connected with the fundamental question of how to implement a specific linear transform by optical means and physical imitations. The large majority of publications on this topic focusses on the optical implementation of space-invariant transforms by the well-known 4f-setup. Only few papers deal with approaches to implement selected space-variant transforms. In this paper, we propose a simple algebraic method to design diffractive elements for an optical architecture in order to realize arbitrary space-variant transforms. The design procedure is based on a digital model of scalar, paraxial wave theory and leads to optimal element transmission functions within the model. Its computational and physical limitations are discussed in terms of complexity measures. Finally, the design procedure is demonstrated by some examples. Firstly, diffractive elements for the realization of different rotation operations are computed and, secondly, a Hough transform element is presented. The correct optical functions of the elements are proved in computer simulation experiments.

Arabidopsis MZT1 homologs GIP1 and GIP2 are essential for centromere architecture.

PubMed

Batzenschlager, Morgane; Lermontova, Inna; Schubert, Veit; Fuchs, Jörg; Berr, Alexandre; Koini, Maria A; Houlné, Guy; Herzog, Etienne; Rutten, Twan; Alioua, Abdelmalek; Fransz, Paul; Schmit, Anne-Catherine; Chabouté, Marie-Edith

2015-07-14

Centromeres play a pivotal role in maintaining genome integrity by facilitating the recruitment of kinetochore and sister-chromatid cohesion proteins, both required for correct chromosome segregation. Centromeres are epigenetically specified by the presence of the histone H3 variant (CENH3). In this study, we investigate the role of the highly conserved γ-tubulin complex protein 3-interacting proteins (GIPs) in Arabidopsis centromere regulation. We show that GIPs form a complex with CENH3 in cycling cells. GIP depletion in the gip1gip2 knockdown mutant leads to a decreased CENH3 level at centromeres, despite a higher level of Mis18BP1/KNL2 present at both centromeric and ectopic sites. We thus postulate that GIPs are required to ensure CENH3 deposition and/or maintenance at centromeres. In addition, the recruitment at the centromere of other proteins such as the CENP-C kinetochore component and the cohesin subunit SMC3 is impaired in gip1gip2. These defects in centromere architecture result in aneuploidy due to severely altered centromeric cohesion. Altogether, we ascribe a central function to GIPs for the proper recruitment and/or stabilization of centromeric proteins essential in the specification of the centromere identity, as well as for centromeric cohesion in somatic cells.

A Single Glycan at the 99-Loop of Human Kallikrein-related Peptidase 2 Regulates Activation and Enzymatic Activity*

PubMed Central

Guo, Shihui; Skala, Wolfgang; Magdolen, Viktor; Briza, Peter; Biniossek, Martin L.; Schilling, Oliver; Kellermann, Josef; Brandstetter, Hans; Goettig, Peter

2016-01-01

Human kallikrein-related peptidase 2 (KLK2) is a key serine protease in semen liquefaction and prostate cancer together with KLK3/prostate-specific antigen. In order to decipher the function of its potential N-glycosylation site, we produced pro-KLK2 in Leishmania tarentolae cells and compared it with its non-glycosylated counterpart from Escherichia coli expression. Mass spectrometry revealed that Asn-95 carries a core glycan, consisting of two GlcNAc and three hexoses. Autocatalytic activation was retarded in glyco-pro-KLK2, whereas the activated glyco-form exhibited an increased proteolytic resistance. The specificity patterns obtained by the PICS (proteomic identification of protease cleavage sites) method are similar for both KLK2 variants, with a major preference for P1-Arg. However, glycosylation changes the enzymatic activity of KLK2 in a drastically substrate-dependent manner. Although glyco-KLK2 has a considerably lower catalytic efficiency than glycan-free KLK2 toward peptidic substrates with P2-Phe, the situation was reverted toward protein substrates, such as glyco-pro-KLK2 itself. These findings can be rationalized by the glycan-carrying 99-loop that prefers to cover the active site like a lid. By contrast, the non-glycosylated 99-loop seems to favor a wide open conformation, which mostly increases the apparent affinity for the substrates (i.e. by a reduction of Km). Also, the cleavage pattern and kinetics in autolytic inactivation of both KLK2 variants can be explained by a shift of the target sites due to the presence of the glycan. These striking effects of glycosylation pave the way to a deeper understanding of kallikrein-related peptidase biology and pathology. PMID:26582203

Comprehensive evaluation of disease- and trait-specific enrichment for eight functional elements among GWAS-identified variants.

PubMed

Markunas, Christina A; Johnson, Eric O; Hancock, Dana B

2017-07-01

Genome-wide association study (GWAS)-identified variants are enriched for functional elements. However, we have limited knowledge of how functional enrichment may differ by disease/trait and tissue type. We tested a broad set of eight functional elements for enrichment among GWAS-identified SNPs (p < 5×10 -8 ) from the NHGRI-EBI Catalog across seven disease/trait categories: cancer, cardiovascular disease, diabetes, autoimmune disease, psychiatric disease, neurological disease, and anthropometric traits. SNPs were annotated using HaploReg for the eight functional elements across any tissue: DNase sites, expression quantitative trait loci (eQTL), sequence conservation, enhancers, promoters, missense variants, sequence motifs, and protein binding sites. In addition, tissue-specific annotations were considered for brain vs. blood. Disease/trait SNPs were compared to a control set of 4809 SNPs matched to the GWAS SNPs (N = 1639) on allele frequency, gene density, distance to nearest gene, and linkage disequilibrium at ~3:1 ratio. Enrichment analyses were conducted using logistic regression, with Bonferroni correction. Overall, a significant enrichment was observed for all functional elements, except sequence motifs. Missense SNPs showed the strongest magnitude of enrichment. eQTLs were the only functional element significantly enriched across all diseases/traits. Magnitudes of enrichment were generally similar across diseases/traits, where enrichment was statistically significant. Blood vs. brain tissue effects on enrichment were dependent on disease/trait and functional element (e.g., cardiovascular disease: eQTLs P TissueDifference  = 1.28 × 10 -6 vs. enhancers P TissueDifference  = 0.94). Identifying disease/trait-relevant functional elements and tissue types could provide new insight into the underlying biology, by guiding a priori GWAS analyses (e.g., brain enhancer elements for psychiatric disease) or facilitating post hoc interpretation.

[Association study between 834+7G/A and +1332C/T polymorphisms in the growth arrest specific 6 gene and risk of severe preeclampsia in Chinese population].

PubMed

Ye, Liyan; Guan, Linbo; Fan, Ping; Liu, Xinghui; Liu, Rui; Chen, Jinxin; Zhu, Yue; Wei, Xin; Liu, Yu; Bai, Huai

2017-02-10

To investigate the relationship between polymorphisms of the growth arrest specific 6 (GAS6) gene and severe preeclampsia in a South West Han Chinese population. Blood samples from 167 patients with severe preeclampsia and 312 normal pregnant women as controls from Han Chinese in Chengdu area were analyzed by polymerase chain reaction-restriction fragment length polymorphisms. C and T allele frequencies for +1332C/T site were 85.63% and 14.37% in the patient group, respectively, and 78.04% and 21.96% in control group, respectively. The TT genotype and variant T allelic frequencies of the +1332C/T polymorphism were significantly lower in patients with severe preeclampsia than in the control group (both P<0.05), and the odds ratio for the risk of severe preeclampsia was 0.602 (95%CI: 0.401-0.904) in carriers for the variant T allele (χ 2 =6.045, P=0.014). G and A allele frequencies for 834+7G/A site were 72.75% and 27.25% in case group, respectively, and 74.36% and 25.64% in control group, respectively. The genotype and allele frequencies of the 834+7G/A polymorphism in patients with severe preeclampsia and controls showed no significant differences (both P>0.05). In addition, there was no significant association between the polymorphisms and blood pressure levels in the patient or control groups. The variant GAS6+1332 T allele is associated with a decreased risk for severe preeclampsia in a South West Han Chinese population. On the other hand, the 834+7G/A polymorphism has no effect on the severe preeclampsia.

Genetic and molecular characterization of the maize rp3 rust resistance locus.

PubMed Central

Webb, Craig A; Richter, Todd E; Collins, Nicholas C; Nicolas, Marie; Trick, Harold N; Pryor, Tony; Hulbert, Scot H

2002-01-01

In maize, the Rp3 gene confers resistance to common rust caused by Puccinia sorghi. Flanking marker analysis of rust-susceptible rp3 variants suggested that most of them arose via unequal crossing over, indicating that rp3 is a complex locus like rp1. The PIC13 probe identifies a nucleotide binding site-leucine-rich repeat (NBS-LRR) gene family that maps to the complex. Rp3 variants show losses of PIC13 family members relative to the resistant parents when probed with PIC13, indicating that the Rp3 gene is a member of this family. Gel blots and sequence analysis suggest that at least 9 family members are at the locus in most Rp3-carrying lines and that at least 5 of these are transcribed in the Rp3-A haplotype. The coding regions of 14 family members, isolated from three different Rp3-carrying haplotypes, had DNA sequence identities from 93 to 99%. Partial sequencing of clones of a BAC contig spanning the rp3 locus in the maize inbred line B73 identified five different PIC13 paralogues in a region of approximately 140 kb. PMID:12242248

Genome sequencing reveals loci under artificial selection that underlie disease phenotypes in the laboratory rat.

PubMed

Atanur, Santosh S; Diaz, Ana Garcia; Maratou, Klio; Sarkis, Allison; Rotival, Maxime; Game, Laurence; Tschannen, Michael R; Kaisaki, Pamela J; Otto, Georg W; Ma, Man Chun John; Keane, Thomas M; Hummel, Oliver; Saar, Kathrin; Chen, Wei; Guryev, Victor; Gopalakrishnan, Kathirvel; Garrett, Michael R; Joe, Bina; Citterio, Lorena; Bianchi, Giuseppe; McBride, Martin; Dominiczak, Anna; Adams, David J; Serikawa, Tadao; Flicek, Paul; Cuppen, Edwin; Hubner, Norbert; Petretto, Enrico; Gauguier, Dominique; Kwitek, Anne; Jacob, Howard; Aitman, Timothy J

2013-08-01

Large numbers of inbred laboratory rat strains have been developed for a range of complex disease phenotypes. To gain insights into the evolutionary pressures underlying selection for these phenotypes, we sequenced the genomes of 27 rat strains, including 11 models of hypertension, diabetes, and insulin resistance, along with their respective control strains. Altogether, we identified more than 13 million single-nucleotide variants, indels, and structural variants across these rat strains. Analysis of strain-specific selective sweeps and gene clusters implicated genes and pathways involved in cation transport, angiotensin production, and regulators of oxidative stress in the development of cardiovascular disease phenotypes in rats. Many of the rat loci that we identified overlap with previously mapped loci for related traits in humans, indicating the presence of shared pathways underlying these phenotypes in rats and humans. These data represent a step change in resources available for evolutionary analysis of complex traits in disease models. Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

Corepressors: custom tailoring and alterations while you wait

PubMed Central

Goodson, Michael; Jonas, Brian A.; Privalsky, Martin A.

2005-01-01

A diverse cadre of metazoan transcription factors mediate repression by recruiting protein complexes containing the SMRT (silencing mediator of retinoid and thyroid hormone receptor) or N-CoR (nuclear receptor corepressor) corepressors. SMRT and N-CoR nucleate the assembly of still larger corepressor complexes that perform the specific molecular incantations necessary to confer transcriptional repression. Although SMRT and N-CoR are paralogs and possess similar molecular architectures and mechanistic strategies, they nonetheless exhibit distinct molecular and biological properties. It is now clear that the functions of both SMRT and N-CoR are further diversified through alternative mRNA splicing, yielding a series of corepressor protein variants that participate in distinctive transcription factor partnerships and display distinguishable repression properties. This review will discuss what is known about the structure and actions of SMRT, N-CoR, and their splicing variants, and how alternative splicing may allow the functions of these corepressors to be adapted and tailored to different cells and to different developmental stages. PMID:16604171

Genome Sequencing Reveals Loci under Artificial Selection that Underlie Disease Phenotypes in the Laboratory Rat

PubMed Central

Atanur, Santosh S.; Diaz, Ana Garcia; Maratou, Klio; Sarkis, Allison; Rotival, Maxime; Game, Laurence; Tschannen, Michael R.; Kaisaki, Pamela J.; Otto, Georg W.; Ma, Man Chun John; Keane, Thomas M.; Hummel, Oliver; Saar, Kathrin; Chen, Wei; Guryev, Victor; Gopalakrishnan, Kathirvel; Garrett, Michael R.; Joe, Bina; Citterio, Lorena; Bianchi, Giuseppe; McBride, Martin; Dominiczak, Anna; Adams, David J.; Serikawa, Tadao; Flicek, Paul; Cuppen, Edwin; Hubner, Norbert; Petretto, Enrico; Gauguier, Dominique; Kwitek, Anne; Jacob, Howard; Aitman, Timothy J.

2013-01-01

Summary Large numbers of inbred laboratory rat strains have been developed for a range of complex disease phenotypes. To gain insights into the evolutionary pressures underlying selection for these phenotypes, we sequenced the genomes of 27 rat strains, including 11 models of hypertension, diabetes, and insulin resistance, along with their respective control strains. Altogether, we identified more than 13 million single-nucleotide variants, indels, and structural variants across these rat strains. Analysis of strain-specific selective sweeps and gene clusters implicated genes and pathways involved in cation transport, angiotensin production, and regulators of oxidative stress in the development of cardiovascular disease phenotypes in rats. Many of the rat loci that we identified overlap with previously mapped loci for related traits in humans, indicating the presence of shared pathways underlying these phenotypes in rats and humans. These data represent a step change in resources available for evolutionary analysis of complex traits in disease models. PaperClip PMID:23890820

Selection and explosive growth alter genetic architecture and hamper the detection of causal rare variants

PubMed Central

Zaitlen, Noah A.; Ye, Chun Jimmie; Witte, John S.

2016-01-01

The role of rare alleles in complex phenotypes has been hotly debated, but most rare variant association tests (RVATs) do not account for the evolutionary forces that affect genetic architecture. Here, we use simulation and numerical algorithms to show that explosive population growth, as experienced by human populations, can dramatically increase the impact of very rare alleles on trait variance. We then assess the ability of RVATs to detect causal loci using simulations and human RNA-seq data. Surprisingly, we find that statistical performance is worst for phenotypes in which genetic variance is due mainly to rare alleles, and explosive population growth decreases power. Although many studies have attempted to identify causal rare variants, few have reported novel associations. This has sometimes been interpreted to mean that rare variants make negligible contributions to complex trait heritability. Our work shows that RVATs are not robust to realistic human evolutionary forces, so general conclusions about the impact of rare variants on complex traits may be premature. PMID:27197206

Evolutional dynamics of 45S and 5S ribosomal DNA in ancient allohexaploid Atropa belladonna.

PubMed

Volkov, Roman A; Panchuk, Irina I; Borisjuk, Nikolai V; Hosiawa-Baranska, Marta; Maluszynska, Jolanta; Hemleben, Vera

2017-01-23

Polyploid hybrids represent a rich natural resource to study molecular evolution of plant genes and genomes. Here, we applied a combination of karyological and molecular methods to investigate chromosomal structure, molecular organization and evolution of ribosomal DNA (rDNA) in nightshade, Atropa belladonna (fam. Solanaceae), one of the oldest known allohexaploids among flowering plants. Because of their abundance and specific molecular organization (evolutionarily conserved coding regions linked to variable intergenic spacers, IGS), 45S and 5S rDNA are widely used in plant taxonomic and evolutionary studies. Molecular cloning and nucleotide sequencing of A. belladonna 45S rDNA repeats revealed a general structure characteristic of other Solanaceae species, and a very high sequence similarity of two length variants, with the only difference in number of short IGS subrepeats. These results combined with the detection of three pairs of 45S rDNA loci on separate chromosomes, presumably inherited from both tetraploid and diploid ancestor species, example intensive sequence homogenization that led to substitution/elimination of rDNA repeats of one parent. Chromosome silver-staining revealed that only four out of six 45S rDNA sites are frequently transcriptionally active, demonstrating nucleolar dominance. For 5S rDNA, three size variants of repeats were detected, with the major class represented by repeats containing all functional IGS elements required for transcription, the intermediate size repeats containing partially deleted IGS sequences, and the short 5S repeats containing severe defects both in the IGS and coding sequences. While shorter variants demonstrate increased rate of based substitution, probably in their transition into pseudogenes, the functional 5S rDNA variants are nearly identical at the sequence level, pointing to their origin from a single parental species. Localization of the 5S rDNA genes on two chromosome pairs further supports uniparental inheritance from the tetraploid progenitor. The obtained molecular, cytogenetic and phylogenetic data demonstrate complex evolutionary dynamics of rDNA loci in allohexaploid species of Atropa belladonna. The high level of sequence unification revealed in 45S and 5S rDNA loci of this ancient hybrid species have been seemingly achieved by different molecular mechanisms.

Large transcription units unify copy number variants and common fragile sites arising under replication stress.

PubMed

Wilson, Thomas E; Arlt, Martin F; Park, So Hae; Rajendran, Sountharia; Paulsen, Michelle; Ljungman, Mats; Glover, Thomas W

2015-02-01

Copy number variants (CNVs) resulting from genomic deletions and duplications and common fragile sites (CFSs) seen as breaks on metaphase chromosomes are distinct forms of structural chromosome instability precipitated by replication inhibition. Although they share a common induction mechanism, it is not known how CNVs and CFSs are related or why some genomic loci are much more prone to their occurrence. Here we compare large sets of de novo CNVs and CFSs in several experimental cell systems to each other and to overlapping genomic features. We first show that CNV hotpots and CFSs occurred at the same human loci within a given cultured cell line. Bru-seq nascent RNA sequencing further demonstrated that although genomic regions with low CNV frequencies were enriched in transcribed genes, the CNV hotpots that matched CFSs specifically corresponded to the largest active transcription units in both human and mouse cells. Consistently, active transcription units >1 Mb were robust cell-type-specific predictors of induced CNV hotspots and CFS loci. Unlike most transcribed genes, these very large transcription units replicated late and organized deletion and duplication CNVs into their transcribed and flanking regions, respectively, supporting a role for transcription in replication-dependent lesion formation. These results indicate that active large transcription units drive extreme locus- and cell-type-specific genomic instability under replication stress, resulting in both CNVs and CFSs as different manifestations of perturbed replication dynamics. © 2015 Wilson et al.; Published by Cold Spring Harbor Laboratory Press.

Engineering a monolignol 4- O-methyltransferase with high selectivity for the condensed lignin precursor coniferyl alchohol

DOE PAGES

Cai, Yuanheng; Shanklin, John; Mohammad -Wadud Bhuiya; ...

2015-09-16

Lignin, a rigid biopolymer in plant cell walls, is derived from the oxidative polymerization of three monolignols. The composition of monolignol monomers dictates the degree of lignin condensation, reactivity, and thus the degradability of plant cell walls. Guaiacyl lignin is regarded as the condensed structural unit. Polymerization of lignin is initiated through the deprotonation of the para-hydroxyl group of monolignols. Therefore, preferentially modifying the para-hydroxyl of a specific monolignol to deprive its dehydrogenation propensity would disturb the formation of particular lignin subunits. Here, we test the hypothesis that specific remodeling the active site of a monolignol 4-O-methyltransferase would create anmore » enzyme that specifically methylates the condensed guaiacyl lignin precursor coniferyl alcohol. Combining crystal structural information with combinatorial active site saturation mutagenesis and starting with the engineered promiscuous enzyme, MOMT5 (T133L/E165I/F175I/F166W/H169F), we incrementally remodeled its substrate binding pocket by the addition of four substitutions, i.e. M26H, S30R, V33S, and T319M, yielding a mutant enzyme capable of discriminately etherifying the para-hydroxyl of coniferyl alcohol even in the presence of excess sinapyl alcohol. The engineered enzyme variant has a substantially reduced substrate binding pocket that imposes a clear steric hindrance thereby excluding bulkier lignin precursors. Lastly, the resulting enzyme variant represents an excellent candidate for modulating lignin composition and/or structure in planta.« less

Large transcription units unify copy number variants and common fragile sites arising under replication stress

PubMed Central

Park, So Hae; Rajendran, Sountharia; Paulsen, Michelle; Ljungman, Mats; Glover, Thomas W.

2015-01-01

Copy number variants (CNVs) resulting from genomic deletions and duplications and common fragile sites (CFSs) seen as breaks on metaphase chromosomes are distinct forms of structural chromosome instability precipitated by replication inhibition. Although they share a common induction mechanism, it is not known how CNVs and CFSs are related or why some genomic loci are much more prone to their occurrence. Here we compare large sets of de novo CNVs and CFSs in several experimental cell systems to each other and to overlapping genomic features. We first show that CNV hotpots and CFSs occurred at the same human loci within a given cultured cell line. Bru-seq nascent RNA sequencing further demonstrated that although genomic regions with low CNV frequencies were enriched in transcribed genes, the CNV hotpots that matched CFSs specifically corresponded to the largest active transcription units in both human and mouse cells. Consistently, active transcription units >1 Mb were robust cell-type-specific predictors of induced CNV hotspots and CFS loci. Unlike most transcribed genes, these very large transcription units replicated late and organized deletion and duplication CNVs into their transcribed and flanking regions, respectively, supporting a role for transcription in replication-dependent lesion formation. These results indicate that active large transcription units drive extreme locus- and cell-type-specific genomic instability under replication stress, resulting in both CNVs and CFSs as different manifestations of perturbed replication dynamics. PMID:25373142

Engineering a Monolignol 4-O-Methyltransferase with High Selectivity for the Condensed Lignin Precursor Coniferyl Alcohol*

PubMed Central

Cai, Yuanheng; Bhuiya, Mohammad-Wadud; Shanklin, John; Liu, Chang-Jun

2015-01-01

Lignin, a rigid biopolymer in plant cell walls, is derived from the oxidative polymerization of three monolignols. The composition of monolignol monomers dictates the degree of lignin condensation, reactivity, and thus the degradability of plant cell walls. Guaiacyl lignin is regarded as the condensed structural unit. Polymerization of lignin is initiated through the deprotonation of the para-hydroxyl group of monolignols. Therefore, preferentially modifying the para-hydroxyl of a specific monolignol to deprive its dehydrogenation propensity would disturb the formation of particular lignin subunits. Here, we test the hypothesis that specific remodeling the active site of a monolignol 4-O-methyltransferase would create an enzyme that specifically methylates the condensed guaiacyl lignin precursor coniferyl alcohol. Combining crystal structural information with combinatorial active site saturation mutagenesis and starting with the engineered promiscuous enzyme, MOMT5 (T133L/E165I/F175I/F166W/H169F), we incrementally remodeled its substrate binding pocket by the addition of four substitutions, i.e. M26H, S30R, V33S, and T319M, yielding a mutant enzyme capable of discriminately etherifying the para-hydroxyl of coniferyl alcohol even in the presence of excess sinapyl alcohol. The engineered enzyme variant has a substantially reduced substrate binding pocket that imposes a clear steric hindrance thereby excluding bulkier lignin precursors. The resulting enzyme variant represents an excellent candidate for modulating lignin composition and/or structure in planta. PMID:26378240

The multi-state energy landscape of the SAM-I riboswitch: A single-molecule Förster resonance energy transfer spectroscopy study

NASA Astrophysics Data System (ADS)

Manz, Christoph; Kobitski, Andrei Yu.; Samanta, Ayan; Jäschke, Andres; Nienhaus, G. Ulrich

2018-03-01

RNA (ribonucleic acid) molecules are highly flexible biopolymers fluctuating at physiological temperatures among many different conformations that are represented by minima in a hierarchical conformational free energy landscape. Here we have employed single-molecule FRET (smFRET) to explore the energy landscape of the B. subtilis yitJ SAM-I riboswitch (RS). In this small RNA molecule, specific binding of an S-adenosyl-L-methionine (SAM) ligand in the aptamer domain regulates gene expression by inducing structural changes in another domain, the expression platform, causing transcription termination by the RNA polymerase. We have measured smFRET histograms over wide ranges of Mg2+ concentration for three RS variants that were specifically labeled with fluorescent dyes on different sites. In the analysis, different conformations are associated with discrete Gaussian model distributions, which are typically fairly broad on the FRET efficiency scale and thus can be extremely challenging to unravel due to their mutual overlap. Our earlier work on two SAM-I RS variants revealed four major conformations. By introducing a global fitting procedure which models both the Mg2+ concentration dependencies of the fractional populations and the average FRET efficiencies of the individual FRET distributions according to Mg2+ binding isotherms, we were able to consistently describe the histogram data of both variants at all studied Mg2+ concentrations. With the third FRET-labeled variant, however, we found significant deviations when applying the four-state model to the data. This can arise because the different FRET labeling of the new variant allows two states to be distinguished that were previously not separable due to overlap. Indeed, the resulting five-state model presented here consistently describes the smFRET histograms of all three variants as well as their variations with Mg2+ concentration. We also performed a triangulation of the donor position for two of the constructs to explore how the expression platform is oriented with respect to the aptamer.

A Rb1 promoter variant with reduced activity contributes to osteosarcoma susceptibility in irradiated mice

PubMed Central

2014-01-01

Background Syndromic forms of osteosarcoma (OS) account for less than 10% of all recorded cases of this malignancy. An individual OS predisposition is also possible by the inheritance of low penetrance alleles of tumor susceptibility genes, usually without evidence of a syndromic condition. Genetic variants involved in such a non-syndromic form of tumor predisposition are difficult to identify, given the low incidence of osteosarcoma cases and the genetic heterogeneity of patients. We recently mapped a major OS susceptibility QTL to mouse chromosome 14 by comparing alpha-radiation induced osteosarcoma in mouse strains which differ in their tumor susceptibility. Methods Tumor-specific allelic losses in murine osteosacoma were mapped along chromosome 14 using microsatellite markers and SNP allelotyping. Candidate gene search in the mapped interval was refined using PosMed data mining and mRNA expression analysis in normal osteoblasts. A strain-specific promoter variant in Rb1 was tested for its influence on mRNA expression using reporter assay. Results A common Rb1 allele derived from the BALB/cHeNhg strain was identified as the major determinant of radiation-induced OS risk at this locus. Increased OS-risk is linked with a hexanucleotide deletion in the promoter region which is predicted to change WT1 and SP1 transcription factor-binding sites. Both in-vitro reporter and in-vivo expression assays confirmed an approx. 1.5 fold reduced gene expression by this promoter variant. Concordantly, the 50% reduction in Rb1 expression in mice bearing a conditional hemizygous Rb1 deletion causes a significant rise of OS incidence following alpha-irradiation. Conclusion This is the first experimental demonstration of a functional and genetic link between reduced Rb1 expression from a common promoter variant and increased tumor risk after radiation exposure. We propose that a reduced Rb1 expression by common variants in regulatory regions can modify the risk for a malignant transformation of bone cells after radiation exposure. PMID:25092376

Peptidomimetic Escape Mechanisms Arise via Genetic Diversity in the Ligand-Binding Site of the Hepatitis C Virus NS3/4A Serine Protease

PubMed Central

Welsch, Christoph; Shimakami, Tetsuro; Hartmann, Christoph; Yang, Yan; Domingues, Francisco S.; Lengauer, Thomas; Zeuzem, Stefan; Lemon, Stanley M.

2011-01-01

Background & Aims It is a challenge to develop direct-acting antiviral agents (DAAs) that target the NS3/4A protease of hepatitis C virus (HCV) because resistant variants develop. Ketoamide compounds, designed to mimic the natural protease substrate, have been developed as inhibitors. However, clinical trials have revealed rapid selection of resistant mutants, most of which are considered to be pre-existing variants. Methods We identified residues near the ketoamide-binding site in X-ray structures of the genotype 1a protease, co-crystallized with boceprevir or a telaprevir-like ligand, and then identified variants at these positions in 219 genotype 1 sequences from a public database. We used side-chain modeling to assess the potential effects of these variants on the interaction between ketoamide and the protease, and compared these results with the phenotypic effects on ketoamide resistance, RNA replication capacity, and infectious virus yields in a cell culture model of infection. Results Thirteen natural binding-site variants with potential for ketoamide resistance were identified at 10 residues in the protease, near the ketoamide binding site. Rotamer analysis of amino acid side-chain conformations indicated that 2 variants (R155K and D168G) could affect binding of telaprevir more than boceprevir. Measurements of antiviral susceptibility in cell culture studies were consistent with this observation. Four variants (Q41H, I132V, R155K, and D168G) caused low-to-moderate levels of ketoamide resistance; 3 of these were highly fit (Q41H, I132V, and R155K). Conclusions Using a comprehensive sequence and structure-based analysis, we showed how natural variation in the HCV protease NS3/4A sequences might affect susceptibility to first-generation DAAs. These findings increase our understanding of the molecular basis of ketoamide resistance among naturally existing viral variants. PMID:22155364

CardioClassifier: disease- and gene-specific computational decision support for clinical genome interpretation.

PubMed

Whiffin, Nicola; Walsh, Roddy; Govind, Risha; Edwards, Matthew; Ahmad, Mian; Zhang, Xiaolei; Tayal, Upasana; Buchan, Rachel; Midwinter, William; Wilk, Alicja E; Najgebauer, Hanna; Francis, Catherine; Wilkinson, Sam; Monk, Thomas; Brett, Laura; O'Regan, Declan P; Prasad, Sanjay K; Morris-Rosendahl, Deborah J; Barton, Paul J R; Edwards, Elizabeth; Ware, James S; Cook, Stuart A

2018-01-25

PurposeInternationally adopted variant interpretation guidelines from the American College of Medical Genetics and Genomics (ACMG) are generic and require disease-specific refinement. Here we developed CardioClassifier (http://www.cardioclassifier.org), a semiautomated decision-support tool for inherited cardiac conditions (ICCs).MethodsCardioClassifier integrates data retrieved from multiple sources with user-input case-specific information, through an interactive interface, to support variant interpretation. Combining disease- and gene-specific knowledge with variant observations in large cohorts of cases and controls, we refined 14 computational ACMG criteria and created three ICC-specific rules.ResultsWe benchmarked CardioClassifier on 57 expertly curated variants and show full retrieval of all computational data, concordantly activating 87.3% of rules. A generic annotation tool identified fewer than half as many clinically actionable variants (64/219 vs. 156/219, Fisher's P = 1.1  ×  10 -18 ), with important false positives, illustrating the critical importance of disease and gene-specific annotations. CardioClassifier identified putatively disease-causing variants in 33.7% of 327 cardiomyopathy cases, comparable with leading ICC laboratories. Through addition of manually curated data, variants found in over 40% of cardiomyopathy cases are fully annotated, without requiring additional user-input data.ConclusionCardioClassifier is an ICC-specific decision-support tool that integrates expertly curated computational annotations with case-specific data to generate fast, reproducible, and interactive variant pathogenicity reports, according to best practice guidelines.GENETICS in MEDICINE advance online publication, 25 January 2018; doi:10.1038/gim.2017.258.

Directed evolution of new and improved enzyme functions using an evolutionary intermediate and multidirectional search.

PubMed

Porter, Joanne L; Boon, Priscilla L S; Murray, Tracy P; Huber, Thomas; Collyer, Charles A; Ollis, David L

2015-02-20

The ease with which enzymes can be adapted from their native roles and engineered to function specifically for industrial or commercial applications is crucial to enabling enzyme technology to advance beyond its current state. Directed evolution is a powerful tool for engineering enzymes with improved physical and catalytic properties and can be used to evolve enzymes where lack of structural information may thwart the use of rational design. In this study, we take the versatile and diverse α/β hydrolase fold framework, in the form of dienelactone hydrolase, and evolve it over three unique sequential evolutions with a total of 14 rounds of screening to generate a series of enzyme variants. The native enzyme has a low level of promiscuous activity toward p-nitrophenyl acetate but almost undetectable activity toward larger p-nitrophenyl esters. Using p-nitrophenyl acetate as an evolutionary intermediate, we have generated variants with altered specificity and catalytic activity up to 3 orders of magnitude higher than the native enzyme toward the larger nonphysiological p-nitrophenyl ester substrates. Several variants also possess increased stability resulting from the multidimensional approach to screening. Crystal structure analysis and substrate docking show how the enzyme active site changes over the course of the evolutions as either a direct or an indirect result of mutations.

Global and disease-associated genetic variation in the human Fanconi anemia gene family

PubMed Central

Rogers, Kai J.; Fu, Wenqing; Akey, Joshua M.; Monnat, Raymond J.

2014-01-01

Fanconi anemia (FA) is a human recessive genetic disease resulting from inactivating mutations in any of 16 FANC (Fanconi) genes. Individuals with FA are at high risk of developmental abnormalities, early bone marrow failure and leukemia. These are followed in the second and subsequent decades by a very high risk of carcinomas of the head and neck and anogenital region, and a small continuing risk of leukemia. In order to characterize base pair-level disease-associated (DA) and population genetic variation in FANC genes and the segregation of this variation in the human population, we identified 2948 unique FANC gene variants including 493 FA DA variants across 57 240 potential base pair variation sites in the 16 FANC genes. We then analyzed the segregation of this variation in the 7578 subjects included in the Exome Sequencing Project (ESP) and the 1000 Genomes Project (1KGP). There was a remarkably high frequency of FA DA variants in ESP/1KGP subjects: at least 1 FA DA variant was identified in 78.5% (5950 of 7578) individuals included in these two studies. Six widely used functional prediction algorithms correctly identified only a third of the known, DA FANC missense variants. We also identified FA DA variants that may be good candidates for different types of mutation-specific therapies. Our results demonstrate the power of direct DNA sequencing to detect, estimate the frequency of and follow the segregation of deleterious genetic variation in human populations. PMID:25104853

Bioenergetics in human evolution and disease: implications for the origins of biological complexity and the missing genetic variation of common diseases.

PubMed

Wallace, Douglas C

2013-07-19

Two major inconsistencies exist in the current neo-Darwinian evolutionary theory that random chromosomal mutations acted on by natural selection generate new species. First, natural selection does not require the evolution of ever increasing complexity, yet this is the hallmark of biology. Second, human chromosomal DNA sequence variation is predominantly either neutral or deleterious and is insufficient to provide the variation required for speciation or for predilection to common diseases. Complexity is explained by the continuous flow of energy through the biosphere that drives the accumulation of nucleic acids and information. Information then encodes complex forms. In animals, energy flow is primarily mediated by mitochondria whose maternally inherited mitochondrial DNA (mtDNA) codes for key genes for energy metabolism. In mammals, the mtDNA has a very high mutation rate, but the deleterious mutations are removed by an ovarian selection system. Hence, new mutations that subtly alter energy metabolism are continuously introduced into the species, permitting adaptation to regional differences in energy environments. Therefore, the most phenotypically significant gene variants arise in the mtDNA, are regional, and permit animals to occupy peripheral energy environments where rarer nuclear DNA (nDNA) variants can accumulate, leading to speciation. The neutralist-selectionist debate is then a consequence of mammals having two different evolutionary strategies: a fast mtDNA strategy for intra-specific radiation and a slow nDNA strategy for speciation. Furthermore, the missing genetic variation for common human diseases is primarily mtDNA variation plus regional nDNA variants, both of which have been missed by large, inter-population association studies.

VarBin, a novel method for classifying true and false positive variants in NGS data

PubMed Central

2013-01-01

Background Variant discovery for rare genetic diseases using Illumina genome or exome sequencing involves screening of up to millions of variants to find only the one or few causative variant(s). Sequencing or alignment errors create "false positive" variants, which are often retained in the variant screening process. Methods to remove false positive variants often retain many false positive variants. This report presents VarBin, a method to prioritize variants based on a false positive variant likelihood prediction. Methods VarBin uses the Genome Analysis Toolkit variant calling software to calculate the variant-to-wild type genotype likelihood ratio at each variant change and position divided by read depth. The resulting Phred-scaled, likelihood-ratio by depth (PLRD) was used to segregate variants into 4 Bins with Bin 1 variants most likely true and Bin 4 most likely false positive. PLRD values were calculated for a proband of interest and 41 additional Illumina HiSeq, exome and whole genome samples (proband's family or unrelated samples). At variant sites without apparent sequencing or alignment error, wild type/non-variant calls cluster near -3 PLRD and variant calls typically cluster above 10 PLRD. Sites with systematic variant calling problems (evident by variant quality scores and biases as well as displayed on the iGV viewer) tend to have higher and more variable wild type/non-variant PLRD values. Depending on the separation of a proband's variant PLRD value from the cluster of wild type/non-variant PLRD values for background samples at the same variant change and position, the VarBin method's classification is assigned to each proband variant (Bin 1 to Bin 4). Results To assess VarBin performance, Sanger sequencing was performed on 98 variants in the proband and background samples. True variants were confirmed in 97% of Bin 1 variants, 30% of Bin 2, and 0% of Bin 3/Bin 4. Conclusions These data indicate that VarBin correctly classifies the majority of true variants as Bin 1 and Bin 3/4 contained only false positive variants. The "uncertain" Bin 2 contained both true and false positive variants. Future work will further differentiate the variants in Bin 2. PMID:24266885

DoEstRare: A statistical test to identify local enrichments in rare genomic variants associated with disease.

PubMed

Persyn, Elodie; Karakachoff, Matilde; Le Scouarnec, Solena; Le Clézio, Camille; Campion, Dominique; Consortium, French Exome; Schott, Jean-Jacques; Redon, Richard; Bellanger, Lise; Dina, Christian

2017-01-01

Next-generation sequencing technologies made it possible to assay the effect of rare variants on complex diseases. As an extension of the "common disease-common variant" paradigm, rare variant studies are necessary to get a more complete insight into the genetic architecture of human traits. Association studies of these rare variations show new challenges in terms of statistical analysis. Due to their low frequency, rare variants must be tested by groups. This approach is then hindered by the fact that an unknown proportion of the variants could be neutral. The risk level of a rare variation may be determined by its impact but also by its position in the protein sequence. More generally, the molecular mechanisms underlying the disease architecture may involve specific protein domains or inter-genic regulatory regions. While a large variety of methods are optimizing functionality weights for each single marker, few evaluate variant position differences between cases and controls. Here, we propose a test called DoEstRare, which aims to simultaneously detect clusters of disease risk variants and global allele frequency differences in genomic regions. This test estimates, for cases and controls, variant position densities in the genetic region by a kernel method, weighted by a function of allele frequencies. We compared DoEstRare with previously published strategies through simulation studies as well as re-analysis of real datasets. Based on simulation under various scenarios, DoEstRare was the sole to consistently show highest performance, in terms of type I error and power both when variants were clustered or not. DoEstRare was also applied to Brugada syndrome and early-onset Alzheimer's disease data and provided complementary results to other existing tests. DoEstRare, by integrating variant position information, gives new opportunities to explain disease susceptibility. DoEstRare is implemented in a user-friendly R package.

Identification of Alternative Splice Variants Using Unique Tryptic Peptide Sequences for Database Searches.

PubMed

Tran, Trung T; Bollineni, Ravi C; Strozynski, Margarita; Koehler, Christian J; Thiede, Bernd

2017-07-07

Alternative splicing is a mechanism in eukaryotes by which different forms of mRNAs are generated from the same gene. Identification of alternative splice variants requires the identification of peptides specific for alternative splice forms. For this purpose, we generated a human database that contains only unique tryptic peptides specific for alternative splice forms from Swiss-Prot entries. Using this database allows an easy access to splice variant-specific peptide sequences that match to MS data. Furthermore, we combined this database without alternative splice variant-1-specific peptides with human Swiss-Prot. This combined database can be used as a general database for searching of LC-MS data. LC-MS data derived from in-solution digests of two different cell lines (LNCaP, HeLa) and phosphoproteomics studies were analyzed using these two databases. Several nonalternative splice variant-1-specific peptides were found in both cell lines, and some of them seemed to be cell-line-specific. Control and apoptotic phosphoproteomes from Jurkat T cells revealed several nonalternative splice variant-1-specific peptides, and some of them showed clear quantitative differences between the two states.

Generation and Characterization of Monoclonal Antibodies against a Cyclic Variant of Hepatitis C Virus E2 Epitope 412-422

PubMed Central

Sandomenico, Annamaria; Leonardi, Antonio; Berisio, Rita; Sanguigno, Luca; Focà, Giuseppina; Focà, Annalia; Ruggiero, Alessia; Doti, Nunzianna; Muscariello, Livio; Barone, Daniela; Farina, Claudio; Owsianka, Ania; Vitagliano, Luigi

2016-01-01

ABSTRACT The hepatitis C virus (HCV) E2 envelope glycoprotein is crucial for virus entry into hepatocytes. A conserved region of E2 encompassing amino acids 412 to 423 (epitope I) and containing Trp420, a residue critical for virus entry, is recognized by several broadly neutralizing antibodies. Peptides embodying this epitope I sequence adopt a β-hairpin conformation when bound to neutralizing monoclonal antibodies (MAbs) AP33 and HCV1. We therefore generated new mouse MAbs that were able to bind to a cyclic peptide containing E2 residues 412 to 422 (C-epitope I) but not to the linear counterpart. These MAbs bound to purified E2 with affinities of about 50 nM, but they were unable to neutralize virus infection. Structural analysis of the complex between C-epitope I and one of our MAbs (C2) showed that the Trp420 side chain is largely buried in the combining site and that the Asn417 side chain, which is glycosylated in E2 and solvent exposed in other complexes, is slightly buried upon C2 binding. Also, the orientation of the cyclic peptide in the antibody-combining site is rotated by 180° compared to the orientations of the other complexes. All these structural features, however, do not explain the lack of neutralization activity. This is instead ascribed to the high degree of selectivity of the new MAbs for the cyclic epitope and to their inability to interact with the epitope in more flexible and extended conformations, which recent data suggest play a role in the mechanisms of neutralization escape. IMPORTANCE Hepatitis C virus (HCV) remains a major health care burden, affecting almost 3% of the global population. The conserved epitope comprising residues 412 to 423 of the viral E2 glycoprotein is a valid vaccine candidate because antibodies recognizing this region exhibit potent neutralizing activity. This epitope adopts a β-hairpin conformation when bound to neutralizing MAbs. We explored the potential of cyclic peptides mimicking this structure to elicit anti-HCV antibodies. MAbs that specifically recognize a cyclic variant of the epitope bind to soluble E2 with a lower affinity than other blocking antibodies and do not neutralize virus. The structure of the complex between one such MAb and the cyclic epitope, together with new structural data showing the linear peptide bound to neutralizing MAbs in extended conformations, suggests that the epitope displays a conformational flexibility that contributes to neutralization escape. Such features can be of major importance for the design of epitope-based anti-HCV vaccines. PMID:26819303

Structural and biochemical studies on ATP binding and hydrolysis by the Escherichia coli RNA chaperone Hfq.

PubMed

Hämmerle, Hermann; Beich-Frandsen, Mads; Večerek, Branislav; Rajkowitsch, Lukas; Carugo, Oliviero; Djinović-Carugo, Kristina; Bläsi, Udo

2012-01-01

In Escherichia coli the RNA chaperone Hfq is involved in riboregulation by assisting base-pairing between small regulatory RNAs (sRNAs) and mRNA targets. Several structural and biochemical studies revealed RNA binding sites on either surface of the donut shaped Hfq-hexamer. Whereas sRNAs are believed to contact preferentially the YKH motifs present on the proximal site, poly(A)(15) and ADP were shown to bind to tripartite binding motifs (ARE) circularly positioned on the distal site. Hfq has been reported to bind and to hydrolyze ATP. Here, we present the crystal structure of a C-terminally truncated variant of E. coli Hfq (Hfq(65)) in complex with ATP, showing that it binds to the distal R-sites. In addition, we revisited the reported ATPase activity of full length Hfq purified to homogeneity. At variance with previous reports, no ATPase activity was observed for Hfq. In addition, FRET assays neither indicated an impact of ATP on annealing of two model oligoribonucleotides nor did the presence of ATP induce strand displacement. Moreover, ATP did not lead to destabilization of binary and ternary Hfq-RNA complexes, unless a vast stoichiometric excess of ATP was used. Taken together, these studies strongly suggest that ATP is dispensable for and does not interfere with Hfq-mediated RNA transactions.

Tendon and ligament injuries: the genetic component

PubMed Central

September, Alison V; Schwellnus, Martin P; Collins, Malcolm

2007-01-01

Tendons and ligaments within the upper and lower limbs are some of the more common sites of musculoskeletal injuries during physical activity. Several extrinsic and intrinsic factors have been shown to be associated with these injuries. More recently, studies have suggested that there is also, at least in part, a genetic component to the Achilles tendon, rotator cuff and anterior cruciate ligament injuries. However, specific genes have not been suggested to be associated with rotator cuff or anterior cruciate ligament injuries. Sequence variants of the tenascin C (TNC) gene, on the other hand, have been shown to be associated with Achilles tendinopathies and Achilles tendon ruptures, whereas a variant of the collagen V α 1 (COL5A1) gene has also been shown to be associated with Achilles tendinopathies. Both genes encode for important structural components of tendons and ligaments. The COL5A1 gene encodes for a component of type V collagen, which has an important role in regulating collagen fibre assembly and fibre diameters. The TNC gene, on the other hand, encodes for TNC, which regulates the tissue's response to mechanical load. To date, only variants in two genes have been shown to be associated with Achilles tendon injuries. In addition, although specific genes have not been identified, investigators have suggested that there is also a genetic component to both rotator cuff and anterior cruciate ligament injuries. In future, specific genotypes associated with increased risk of injury to specific tendons and ligaments can prevent these injuries by identifying individuals at higher risk. PMID:17261551

High resolution mapping of the binding site on human IgG1 for Fc gamma RI, Fc gamma RII, Fc gamma RIII, and FcRn and design of IgG1 variants with improved binding to the Fc gamma R.

PubMed

Shields, R L; Namenuk, A K; Hong, K; Meng, Y G; Rae, J; Briggs, J; Xie, D; Lai, J; Stadlen, A; Li, B; Fox, J A; Presta, L G

2001-03-02

Immunoglobulin G (IgG) Fc receptors play a critical role in linking IgG antibody-mediated immune responses with cellular effector functions. A high resolution map of the binding site on human IgG1 for human Fc gamma RI, Fc gamma RIIA, Fc gamma RIIB, Fc gamma RIIIA, and FcRn receptors has been determined. A common set of IgG1 residues is involved in binding to all Fc gamma R; Fc gamma RII and Fc gamma RIII also utilize residues outside this common set. In addition to residues which, when altered, abrogated binding to one or more of the receptors, several residues were found that improved binding only to specific receptors or simultaneously improved binding to one type of receptor and reduced binding to another type. Select IgG1 variants with improved binding to Fc gamma RIIIA exhibited up to 100% enhancement in antibody-dependent cell cytotoxicity using human effector cells; these variants included changes at residues not found at the binding interface in the IgG/Fc gamma RIIIA co-crystal structure (Sondermann, P., Huber, R., Oosthuizen, V., and Jacob, U. (2000) Nature 406, 267-273). These engineered antibodies may have important implications for improving antibody therapeutic efficacy.