Science.gov

Sample records for armadillo repeat proteins

  1. Structures of designed armadillo-repeat proteins show propagation of inter-repeat interface effects

    PubMed Central

    Reichen, Christian; Madhurantakam, Chaithanya; Hansen, Simon; Grütter, Markus G.; Plückthun, Andreas; Mittl, Peer R. E.

    2016-01-01

    The armadillo repeat serves as a scaffold for the development of modular peptide-recognition modules. In order to develop such a system, three crystal structures of designed armadillo-repeat proteins with third-generation N-caps (YIII-type), four or five internal repeats (M-type) and second-generation C-caps (AII-type) were determined at 1.8 Å (His-YIIIM4AII), 2.0 Å (His-YIIIM5AII) and 1.95 Å (YIIIM5AII) resolution and compared with those of variants with third-generation C-caps. All constructs are full consensus designs in which the internal repeats have exactly the same sequence, and hence identical conformations of the internal repeats are expected. The N-cap and internal repeats M1 to M3 are indeed extremely similar, but the comparison reveals structural differences in internal repeats M4 and M5 and the C-cap. These differences are caused by long-range effects of the C-cap, contacting molecules in the crystal, and the intrinsic design of the repeat. Unfortunately, the rigid-body movement of the C-terminal part impairs the regular arrangement of internal repeats that forms the putative peptide-binding site. The second-generation C-cap improves the packing of buried residues and thereby the stability of the protein. These considerations are useful for future improvements of an armadillo-repeat-based peptide-recognition system. PMID:26894544

  2. Gudu, an Armadillo repeat-containing protein, is required for spermatogenesis in Drosophila.

    PubMed

    Cheng, Wei; Ip, Y Tony; Xu, Zuoshang

    2013-12-01

    The Drosophila annotated gene CG5155 encodes a protein that contains 10 Armadillo-repeats and has an unknown function. To fill this gap, we performed loss-of-function studies using RNAi. By analysis of four independent Drosophila RNAi lines targeting two non-overlapping regions of the CG5155 transcript, we demonstrate that this gene is required for male fertility. Therefore, we have named this gene Gudu. The transcript of Gudu is highly enriched in adult testes. Knockdown of Gudu by a ubiquitous driver leads to defects in the formation of the individualization complex that is required for spermatid maturation, thereby impairing spermatogenesis. Furthermore, testis-specific knockdown of Gudu by crossing the RNAi lines with the bam-Gal4 driver is sufficient to cause the infertility and defective spermatogenesis. Since Gudu is highly homologous to vertebrate ARMC4, also an Armadillo-repeat-containing protein enriched in testes, our results suggest that Gudu and ARMC4 are a subfamily of Armadillo-repeat containing proteins that may have an evolutionarily conserved function in spermatogenesis.

  3. Spontaneous self-assembly of engineered armadillo repeat protein fragments into a folded structure.

    PubMed

    Watson, Randall P; Christen, Martin T; Ewald, Christina; Bumbak, Fabian; Reichen, Christian; Mihajlovic, Maja; Schmidt, Elena; Güntert, Peter; Caflisch, Amedeo; Plückthun, Andreas; Zerbe, Oliver

    2014-07-08

    Repeat proteins are built of modules, each of which constitutes a structural motif. We have investigated whether fragments of a designed consensus armadillo repeat protein (ArmRP) recognize each other. We examined a split ArmRP consisting of an N-capping repeat (denoted Y), three internal repeats (M), and a C-capping repeat (A). We demonstrate that the C-terminal MA fragment adopts a fold similar to the corresponding part of the entire protein. In contrast, the N-terminal YM2 fragment constitutes a molten globule. The two fragments form a 1:1 YM2:MA complex with a nanomolar dissociation constant essentially identical to the crystal structure of the continuous YM3A protein. Molecular dynamics simulations show that the complex is structurally stable over a 1 μs timescale and reveal the importance of hydrophobic contacts across the interface. We propose that the existence of a stable complex recapitulates possible intermediates in the early evolution of these repeat proteins.

  4. The microtubule plus-end tracking protein ARMADILLO-REPEAT KINESIN1 promotes microtubule catastrophe in Arabidopsis.

    PubMed

    Eng, Ryan Christopher; Wasteneys, Geoffrey O

    2014-08-01

    Microtubule dynamics are critically important for plant cell development. Here, we show that Arabidopsis thaliana ARMADILLO-REPEAT KINESIN1 (ARK1) plays a key role in root hair tip growth by promoting microtubule catastrophe events. This destabilizing activity appears to maintain adequate free tubulin concentrations in order to permit rapid microtubule growth, which in turn is correlated with uniform tip growth. Microtubules in ark1-1 root hairs exhibited reduced catastrophe frequency and slower growth velocities, both of which were restored by low concentrations of the microtubule-destabilizing drug oryzalin. An ARK1-GFP (green fluorescent protein) fusion protein expressed under its endogenous promoter localized to growing microtubule plus ends and rescued the ark1-1 root hair phenotype. Transient overexpression of ARK1-RFP (red fluorescent protein) increased microtubule catastrophe frequency. ARK1-fusion protein constructs lacking the N-terminal motor domain still labeled microtubules, suggesting the existence of a second microtubule binding domain at the C terminus of ARK1. ARK1-GFP was broadly expressed in seedlings, but mutant phenotypes were restricted to root hairs, indicating that ARK1's function is redundant in cells other than those forming root hairs.

  5. ND9P, a novel protein with armadillo-like repeats involved in exocytosis: physiological studies using allelic mutants in paramecium.

    PubMed Central

    Froissard, M; Keller, A M; Cohen, J

    2001-01-01

    In Paramecium, a number of mutants affected in the exocytotic membrane fusion step of the regulated secretory pathway have been obtained. Here, we report the isolation of one of the corresponding genes, ND9, previously suspected to encode a soluble protein interacting with both plasma and trichocyst membranes. Nd9p is a novel polypeptide that contains C-terminal Armadillo-like repeats. Point mutations were found in the first N-terminal quarter of the molecule and in the last putative Armadillo repeat, respectively, for the two thermosensitive mutants, nd9-1 and nd9-2. The different behaviors of these mutants in recovery experiments upon temperature shifts suggest that the N-terminal domain of the molecule may be involved in membrane binding activity, whereas the C-terminal domain is a candidate for protein-protein interactions. The nonsense nd9-3 mutation that produces a short N-terminal peptide has a dominant negative effect on the nd9-1 allele. We show here that, when overexpressed, the dominant negative effect can be produced even on the wild-type allele, suggesting competition for a common target. We suggest that Nd9p could act, like some SNARE proteins, at the membrane-cytosol interface to promote membrane fusion. PMID:11156983

  6. 3D model for Cancerous Inhibitor of Protein Phosphatase 2A armadillo domain unveils highly conserved protein-protein interaction characteristics.

    PubMed

    Dahlström, Käthe M; Salminen, Tiina A

    2015-12-07

    Cancerous Inhibitor of Protein Phosphatase 2A (CIP2A) is a human oncoprotein, which exerts its cancer-promoting function through interaction with other proteins, for example Protein Phosphatase 2A (PP2A) and MYC. The lack of structural information for CIP2A significantly prevents the design of anti-cancer therapeutics targeting this protein. In an attempt to counteract this fact, we modeled the three-dimensional structure of the N-terminal domain (CIP2A-ArmRP), analyzed key areas and amino acids, and coupled the results to the existing literature. The model reliably shows a stable armadillo repeat fold with a positively charged groove. The fact that this conserved groove highly likely binds peptides is corroborated by the presence of a conserved polar ladder, which is essential for the proper peptide-binding mode of armadillo repeat proteins and, according to our results, several known CIP2A interaction partners appropriately possess an ArmRP-binding consensus motif. Moreover, we show that Arg229Gln, which has been linked to the development of cancer, causes a significant change in charge and surface properties of CIP2A-ArmRP. In conclusion, our results reveal that CIP2A-ArmRP shares the typical fold, protein-protein interaction site and interaction patterns with other natural armadillo proteins and that, presumably, several interaction partners bind into the central groove of the modeled CIP2A-ArmRP. By providing essential structural characteristics of CIP2A, the present study significantly increases our knowledge on how CIP2A interacts with other proteins in cancer progression and how to develop new therapeutics targeting CIP2A.

  7. Crystal structures of the armadillo repeat domain of adenomatous polyposis coli and its complex with the tyrosine-rich domain of Sam68.

    PubMed

    Morishita, Ella Czarina; Murayama, Kazutaka; Kato-Murayama, Miyuki; Ishizuka-Katsura, Yoshiko; Tomabechi, Yuri; Hayashi, Tomoatsu; Terada, Takaho; Handa, Noriko; Shirouzu, Mikako; Akiyama, Tetsu; Yokoyama, Shigeyuki

    2011-10-12

    Adenomatous polyposis coli (APC) is a tumor suppressor protein commonly mutated in colorectal tumors. APC plays important roles in Wnt signaling and other cellular processes. Here, we present the crystal structure of the armadillo repeat (Arm) domain of APC, which facilitates the binding of APC to various proteins. APC-Arm forms a superhelix with a positively charged groove. We also determined the structure of the complex of APC-Arm with the tyrosine-rich (YY) domain of the Src-associated in mitosis, 68 kDa protein (Sam68), which regulates TCF-1 alternative splicing. Sam68-YY forms numerous interactions with the residues on the groove and is thereby fixed in a bent conformation. We assessed the effects of mutations and phosphorylation on complex formation between APC-Arm and Sam68-YY. Structural comparisons revealed different modes of ligand recognition between the Arm domains of APC and other Arm-containing proteins.

  8. Cytokine responses to Mycobacterium leprae unique proteins differentiate between Mycobacterium leprae infected and naive armadillos.

    PubMed

    Pena, Maria; Geluk, Annemieke; Van Der Ploeg-Van Schip, Jolien J; Franken, Kees L M C; Sharma, Rahul; Truman, Richard

    2011-12-01

    New diagnostic tools for early detection of leprosy are necessary to help reduce its transmission and severity. M. leprae unique proteins have been used to assess differences in human T-cell responses in leprosy patients, household contacts and endemic controls. In this study, we examined the response of M. leprae-infected armadillos to a variety of M. leprae recombinant antigen candidates currently being examined for diagnostic efficacy in humans. Among recently M. leprae infected armadillos, IFN-gamma expression was enhanced after stimulation of PBMC with all M. leprae recombinant proteins except for ML2283 (mean: 2.65 Relative Quantification (RQ)). The group mean stimulation index for M. leprae proteins ML0009, ML1601, ML2478 and ML2531 averaged 35.2 RQ and was significantly higher (P < 0.05) than that measured among the non-infected, naive group (mean 6.2 RQ). Although ML0840 tended to enhance IFN-gamma levels, the mean IFN-gamma transcript levels of the currently experimentally inoculated group (20.1 RQ) was not significantly different statistically (P = 0.10) from the mean of the naive group (7.5 RQ). Also no statistically significant differences were observed in IFN-gamma transcript levels between the resistant and currently experimentally inoculated group (P > 0.05) or between the resistant and the naive group (P > 0.05) after stimulation of PBMCs with all M. leprae recombinant proteins. Only low levels of TNF-alpha were observed across all groups after in vitro stimulation with all the antigens examined. These data suggest that armadillos can be used effectively to help identify M. leprae specific proteins that may be applied for monitoring T-cell responses in M. leprae infected hosts as their disease progresses as well as for the early diagnosis of leprosy.

  9. The Armadillo Repeat Gene ZAK IXIK Promotes Arabidopsis Early Embryo and Endosperm Development through a Distinctive Gametophytic Maternal Effect[C][W][OA

    PubMed Central

    Ngo, Quy A.; Baroux, Celia; Guthörl, Daniela; Mozerov, Peter; Collinge, Margaret A.; Sundaresan, Venkatesan; Grossniklaus, Ueli

    2012-01-01

    The proper balance of parental genomic contributions to the fertilized embryo and endosperm is essential for their normal growth and development. The characterization of many gametophytic maternal effect (GME) mutants affecting seed development indicates that there are certain classes of genes with a predominant maternal contribution. We present a detailed analysis of the GME mutant zak ixik (zix), which displays delayed and arrested growth at the earliest stages of embryo and endosperm development. ZIX encodes an Armadillo repeat (Arm) protein highly conserved across eukaryotes. Expression studies revealed that ZIX manifests a GME through preferential maternal expression in the early embryo and endosperm. This parent-of-origin–dependent expression is regulated by neither the histone and DNA methylation nor the DNA demethylation pathways known to regulate some other GME mutants. The ZIX protein is localized in the cytoplasm and nucleus of cells in reproductive tissues and actively dividing root zones. The maternal ZIX allele is required for the maternal expression of MINISEED3. Collectively, our results reveal a reproductive function of plant Arm proteins in promoting early seed growth, which is achieved through a distinct GME of ZIX that involves mechanisms for maternal allele-specific expression that are independent of the well-established pathways. PMID:23064319

  10. Macronodular Adrenal Hyperplasia due to Mutations in an Armadillo Repeat Containing 5 (ARMC5) Gene: A Clinical and Genetic Investigation

    PubMed Central

    Faucz, Fabio R.; Zilbermint, Mihail; Lodish, Maya B.; Szarek, Eva; Trivellin, Giampaolo; Sinaii, Ninet; Berthon, Annabel; Libé, Rossella; Assié, Guillaume; Espiard, Stéphanie; Drougat, Ludivine; Ragazzon, Bruno; Bertherat, Jerome

    2014-01-01

    Context: Inactivating germline mutations of the probable tumor suppressor gene, armadillo repeat containing 5 (ARMC5), have recently been identified as a genetic cause of macronodular adrenal hyperplasia (MAH). Objective: We searched for ARMC5 mutations in a large cohort of patients with MAH. The clinical phenotype of patients with and without ARMC5 mutations was compared. Methods: Blood DNA from 34 MAH patients was genotyped using Sanger sequencing. Diurnal serum cortisol measurements, plasma ACTH levels, urinary steroids, 6-day Liddle's test, adrenal computed tomography, and weight of adrenal glands at adrenalectomy were assessed. Results: Germline ARMC5 mutations were found in 15 of 34 patients (44.1%). In silico analysis of the mutations indicated that seven (20.6%) predicted major implications for gene function. Late-night cortisol levels were higher in patients with ARMC5-damaging mutations compared with those without and/or with nonpathogenic mutations (14.5 ± 5.6 vs 6.7 ± 4.3, P < .001). All patients carrying a pathogenic ARMC5 mutation had clinical Cushing's syndrome (seven of seven, 100%) compared with 14 of 27 (52%) of those without or with mutations that were predicted to be benign (P = .029). Repeated-measures analysis showed overall higher urinary 17-hydroxycorticosteroids and free cortisol values in the patients with ARMC5-damaging mutations during the 6-day Liddle's test (P = .0002). Conclusions: ARMC5 mutations are implicated in clinically severe Cushing's syndrome associated with MAH. Knowledge of a patient's ARMC5 status has important clinical implications for the diagnosis of Cushing's syndrome and genetic counseling of patients and their families. PMID:24601692

  11. All repeats are not equal: a module-based approach to guide repeat protein design.

    PubMed

    Sawyer, Nicholas; Chen, Jieming; Regan, Lynne

    2013-05-27

    Repeat proteins composed of tandem arrays of a short structural motif often mediate protein-protein interactions. Past efforts to design repeat protein-based molecular recognition tools have focused on the creation of templates from the consensus of individual repeats, regardless of their natural context. Such an approach assumes that all repeats are essentially equivalent. In this study, we present the results of a "module-based" approach in which modules composed of tandem repeats are aligned to identify repeat-specific features. Using this approach to analyze tetratricopeptide repeat modules that contain three tandem repeats (3TPRs), we identify two classes of 3TPR modules with distinct structural signatures that are correlated with different sets of functional residues. Our analyses also reveal a high degree of correlation between positions across the entire ligand-binding surface, indicative of a coordinated, coevolving binding surface. Extension of our analyses to different repeat protein modules reveals more examples of repeat-specific features, especially in armadillo repeat modules. In summary, the module-based analyses that we present effectively capture key repeat-specific features that will be important to include in future repeat protein design templates.

  12. A novel human gene (SARM) at chromosome 17q11 encodes a protein with a SAM motif and structural similarity to Armadillo/beta-catenin that is conserved in mouse, Drosophila, and Caenorhabditis elegans.

    PubMed

    Mink, M; Fogelgren, B; Olszewski, K; Maroy, P; Csiszar, K

    2001-06-01

    A novel human gene, SARM, encodes the orthologue of a Drosophila protein (CG7915) and contains a unique combination of the sterile alpha (SAM) and the HEAT/Armadillo motifs. The SARM gene was identified on chromosome 17q11, between markers D17S783 and D17S841 on BAC clone AC002094, which also included a HERV repeat and keratin-18-like, MAC30, TNFAIP1, HSPC017, and vitronectin genes in addition to three unknown genes. The mouse SARM gene was located on a mouse chromosome 11 BAC clone (AC002324). The SARM gene is 1.8 kb centromeric to the vitronectin gene, and the two genes share a promoter region that directs a high level of liver-specific expression of both the SARM and the vitronectin genes. In addition to the liver, the SARM gene was highly expressed in the kidney. A 0.4-kb antisense transcript was coordinately expressed with the SARM gene in the kidney and liver, while in the brain and malignant cell lines, it appeared independent of SARM gene transcription. The SARM gene encodes a protein of 690 amino acids. Based on amino acid sequence homology, we have identified a SAM motif within this derived protein. Structure modeling and protein folding recognition studies confirmed the presence of alpha-alpha right-handed superhelix-like folds consistent with the structure of the Armadillo and HEAT repeats of the beta-catenin and importin protein families. Both motifs are known to be involved in protein-protein interactions promoting the formation of diverse protein complexes. We have identified the same conserved SAM/Armadillo motif combination in the mouse, Drosophila, and Caenorhabditis elegans SARM proteins.

  13. Expansion and Function of Repeat Domain Proteins During Stress and Development in Plants

    PubMed Central

    Sharma, Manisha; Pandey, Girdhar K.

    2016-01-01

    The recurrent repeats having conserved stretches of amino acids exists across all domains of life. Subsequent repetition of single sequence motif and the number and length of the minimal repeating motifs are essential characteristics innate to these proteins. The proteins with tandem peptide repeats are essential for providing surface to mediate protein–protein interactions for fundamental biological functions. Plants are enriched in tandem repeat containing proteins typically distributed into various families. This has been assumed that the occurrence of multigene repeats families in plants enable them to cope up with adverse environmental conditions and allow them to rapidly acclimatize to these conditions. The evolution, structure, and function of repeat proteins have been studied in all kingdoms of life. The presence of repeat proteins is particularly profuse in multicellular organisms in comparison to prokaryotes. The precipitous expansion of repeat proteins in plants is presumed to be through internal tandem duplications. Several repeat protein gene families have been identified in plants. Such as Armadillo (ARM), Ankyrin (ANK), HEAT, Kelch-like repeats, Tetratricopeptide (TPR), Leucine rich repeats (LRR), WD40, and Pentatricopeptide repeats (PPR). The structure and functions of these repeat proteins have been extensively studied in plants suggesting a critical role of these repeating peptides in plant cell physiology, stress and development. In this review, we illustrate the structural, functional, and evolutionary prospects of prolific repeat proteins in plants. PMID:26793205

  14. Rapid Identification of a Natural Knockout Allele of ARMADILLO REPEAT-CONTAINING KINESIN1 That Causes Root Hair Branching by Mapping-By-Sequencing1[C][W

    PubMed Central

    Rishmawi, Louai; Sun, Hequan; Schneeberger, Korbinian; Hülskamp, Martin; Schrader, Andrea

    2014-01-01

    In Arabidopsis (Arabidopsis thaliana), branched root hairs are an indicator of defects in root hair tip growth. Among 62 accessions, one accession (Heiligkreuztal2 [HKT2.4]) displayed branched root hairs, suggesting that this accession carries a mutation in a gene of importance for tip growth. We determined 200- to 300-kb mapping intervals using a mapping-by-sequencing approach of F2 pools from crossings of HKT2.4 with three different accessions. The intersection of these mapping intervals was 80 kb in size featuring not more than 36 HKT2.4-specific single nucleotide polymorphisms, only two of which changed the coding potential of genes. Among them, we identified the causative single nucleotide polymorphism changing a splicing site in ARMADILLO REPEAT-CONTAINING KINESIN1. The applied strategies have the potential to complement statistical methods in high-throughput phenotyping studies using different natural accessions to identify causative genes for distinct phenotypes represented by only one or a few accessions. PMID:25248719

  15. Protein Repeats from First Principles.

    PubMed

    Turjanski, Pablo; Parra, R Gonzalo; Espada, Rocío; Becher, Verónica; Ferreiro, Diego U

    2016-04-05

    Some natural proteins display recurrent structural patterns. Despite being highly similar at the tertiary structure level, repeating patterns within a single repeat protein can be extremely variable at the sequence level. We use a mathematical definition of a repetition and investigate the occurrences of these in sequences of different protein families. We found that long stretches of perfect repetitions are infrequent in individual natural proteins, even for those which are known to fold into structures of recurrent structural motifs. We found that natural repeat proteins are indeed repetitive in their families, exhibiting abundant stretches of 6 amino acids or longer that are perfect repetitions in the reference family. We provide a systematic quantification for this repetitiveness. We show that this form of repetitiveness is not exclusive of repeat proteins, but also occurs in globular domains. A by-product of this work is a fast quantification of the likelihood of a protein to belong to a family.

  16. Protein Repeats from First Principles

    PubMed Central

    Turjanski, Pablo; Parra, R. Gonzalo; Espada, Rocío; Becher, Verónica; Ferreiro, Diego U.

    2016-01-01

    Some natural proteins display recurrent structural patterns. Despite being highly similar at the tertiary structure level, repeating patterns within a single repeat protein can be extremely variable at the sequence level. We use a mathematical definition of a repetition and investigate the occurrences of these in sequences of different protein families. We found that long stretches of perfect repetitions are infrequent in individual natural proteins, even for those which are known to fold into structures of recurrent structural motifs. We found that natural repeat proteins are indeed repetitive in their families, exhibiting abundant stretches of 6 amino acids or longer that are perfect repetitions in the reference family. We provide a systematic quantification for this repetitiveness. We show that this form of repetitiveness is not exclusive of repeat proteins, but also occurs in globular domains. A by-product of this work is a fast quantification of the likelihood of a protein to belong to a family. PMID:27044676

  17. Learning with "Armadillo Ray"

    ERIC Educational Resources Information Center

    Hubbard, Kathy; Terrell, Chelsea

    2009-01-01

    "Armadillo Ray," by John Beifuss, is the tale of a young, curious armadillo who wants to know what the moon is. He is joined in his quest by snakes, prairie dogs, sage grouse, and owls. The beauty of the book is its simplicity, illustrations and landscapes, and its potential links to reading, geography, science, and mathematics. In this…

  18. Pentapeptide Repeat Proteins and Cyanobacteria

    SciTech Connect

    Buchko, Garry W.

    2009-10-16

    Cyanobacteria are unique in many ways and one unusual feature is the presence of a suite of proteins that contain at least one domain with a minimum of eight tandem repeated five-residues (Rfr) of the general consensus sequence A[N/D]LXX. The function of such pentapeptide repeat proteins (PRPs) are still unknown, however, their prevalence in cyanobacteria suggests that they may play some role in the unique biological activities of cyanobacteria. As part of an inter-disciplinary Membrane Biology Grand Challenge at the Environmental Molecular Sciences Laboratory (Pacific Northwest National Laboratory) and Washington University in St. Louis, the genome of Cyanothece 51142 was sequenced and its molecular biology studied with relation to circadian rhythms. The genome of Cyanothece encodes for 35 proteins that contain at least one PRP domain. These proteins range in size from 105 (Cce_3102) to 930 (Cce_2929) kDa with the PRP domains ranging in predicted size from 12 (Cce_1545) to 62 (cce_3979) tandem pentapeptide repeats. Transcriptomic studies with 29 out of the 35 genes showed that at least three of the PRPs in Cyanothece 51142 (cce_0029, cce_3083, and cce_3272) oscillated with repeated periods of light and dark, further supporting a biological function for PRPs. Using X-ray diffraction crystallography, the structure for two pentapeptide repeat proteins from Cyanothece 51142 were determined, cce_1272 (aka Rfr32) and cce_4529 (aka Rfr23). Analysis of their molecular structures suggests that all PRP may share the same structural motif, a novel type of right-handed quadrilateral β-helix, or Rfr-fold, reminiscent of a square tower with four distinct faces. Each pentapeptide repeat occupies one face of the Rfr-fold with four consecutive pentapeptide repeats completing a coil that, in turn, stack upon each other to form “protein skyscrapers”. Details of the structural features of the Rfr-fold are reviewed here together with a discussion for the possible role of end

  19. Structural and biophysical properties of h-FANCI ARM repeat protein.

    PubMed

    Siddiqui, Mohd Quadir; Choudhary, Rajan Kumar; Thapa, Pankaj; Kulkarni, Neha; Rajpurohit, Yogendra S; Misra, Hari S; Gadewal, Nikhil; Kumar, Satish; Hasan, Syed K; Varma, Ashok K

    2016-11-10

    Fanconi anemia complementation groups - I (FANCI) protein facilitates DNA ICL (Inter-Cross-link) repair and plays a crucial role in genomic integrity. FANCI is a 1328 amino acids protein which contains armadillo (ARM) repeats and EDGE motif at the C-terminus. ARM repeats are functionally diverse and evolutionarily conserved domain that plays a pivotal role in protein-protein and protein-DNA interactions. Considering the importance of ARM repeats, we have explored comprehensive in silico and in vitro approach to examine folding pattern. Size exclusion chromatography, dynamic light scattering (DLS) and glutaraldehyde crosslinking studies suggest that FANCI ARM repeat exist as monomer as well as in oligomeric forms. Circular dichroism (CD) and fluorescence spectroscopy results demonstrate that protein has predominantly α- helices and well-folded tertiary structure. DNA binding was analysed using electrophoretic mobility shift assay by autoradiography. Temperature-dependent CD, Fluorescence spectroscopy and DLS studies concluded that protein unfolds and start forming oligomer from 30°C. The existence of stable portion within FANCI ARM repeat was examined using limited proteolysis and mass spectrometry. The normal mode analysis, molecular dynamics and principal component analysis demonstrated that helix-turn-helix (HTH) motif present in ARM repeat is highly dynamic and has anti-correlated motion. Furthermore, FANCI ARM repeat has HTH structural motif which binds to double-stranded DNA.

  20. RepeatsDB: a database of tandem repeat protein structures

    PubMed Central

    Di Domenico, Tomás; Potenza, Emilio; Walsh, Ian; Gonzalo Parra, R.; Giollo, Manuel; Minervini, Giovanni; Piovesan, Damiano; Ihsan, Awais; Ferrari, Carlo; Kajava, Andrey V.; Tosatto, Silvio C.E.

    2014-01-01

    RepeatsDB (http://repeatsdb.bio.unipd.it/) is a database of annotated tandem repeat protein structures. Tandem repeats pose a difficult problem for the analysis of protein structures, as the underlying sequence can be highly degenerate. Several repeat types haven been studied over the years, but their annotation was done in a case-by-case basis, thus making large-scale analysis difficult. We developed RepeatsDB to fill this gap. Using state-of-the-art repeat detection methods and manual curation, we systematically annotated the Protein Data Bank, predicting 10 745 repeat structures. In all, 2797 structures were classified according to a recently proposed classification schema, which was expanded to accommodate new findings. In addition, detailed annotations were performed in a subset of 321 proteins. These annotations feature information on start and end positions for the repeat regions and units. RepeatsDB is an ongoing effort to systematically classify and annotate structural protein repeats in a consistent way. It provides users with the possibility to access and download high-quality datasets either interactively or programmatically through web services. PMID:24311564

  1. Evolution of Protein Domain Repeats in Metazoa

    PubMed Central

    Schüler, Andreas; Bornberg-Bauer, Erich

    2016-01-01

    Repeats are ubiquitous elements of proteins and they play important roles for cellular function and during evolution. Repeats are, however, also notoriously difficult to capture computationally and large scale studies so far had difficulties in linking genetic causes, structural properties and evolutionary trajectories of protein repeats. Here we apply recently developed methods for repeat detection and analysis to a large dataset comprising over hundred metazoan genomes. We find that repeats in larger protein families experience generally very few insertions or deletions (indels) of repeat units but there is also a significant fraction of noteworthy volatile outliers with very high indel rates. Analysis of structural data indicates that repeats with an open structure and independently folding units are more volatile and more likely to be intrinsically disordered. Such disordered repeats are also significantly enriched in sites with a high functional potential such as linear motifs. Furthermore, the most volatile repeats have a high sequence similarity between their units. Since many volatile repeats also show signs of recombination, we conclude they are often shaped by concerted evolution. Intriguingly, many of these conserved yet volatile repeats are involved in host-pathogen interactions where they might foster fast but subtle adaptation in biological arms races. Key Words: protein evolution, domain rearrangements, protein repeats, concerted evolution. PMID:27671125

  2. Dynamic combinatorial libraries of artificial repeat proteins.

    PubMed

    Eisenberg, Margarita; Shumacher, Inbal; Cohen-Luria, Rivka; Ashkenasy, Gonen

    2013-06-15

    Repeat proteins are found in almost all cellular systems, where they are involved in diverse molecular recognition processes. Recent studies have suggested that de novo designed repeat proteins may serve as universal binders, and might potentially be used as practical alternative to antibodies. We describe here a novel chemical methodology for producing small libraries of repeat proteins, and screening in parallel the ligand binding of library members. The first stage of this research involved the total synthesis of a consensus-based three-repeat tetratricopeptide (TPR) protein (~14 kDa), via sequential attachment of the respective peptides. Despite the effectiveness of the synthesis and ligation steps, this method was found to be too demanding for the production of proteins containing variable number of repeats. Additionally, the analysis of binding of the individual proteins was time consuming. Therefore, we designed and prepared novel dynamic combinatorial libraries (DCLs), and show that their equilibration can facilitate the formation of TPR proteins containing up to eight repeating units. Interestingly, equilibration of the library building blocks in the presence of the biologically relevant ligands, Hsp90 and Hsp70, induced their oligomerization into forming more of the proteins with large recognition surfaces. We suggest that this work presents a novel simple and rapid tool for the simultaneous screening of protein mixtures with variable binding surfaces, and for identifying new binders for ligands of interest.

  3. Spectrin repeat proteins in the nucleus.

    PubMed

    Young, Kevin G; Kothary, Rashmi

    2005-02-01

    Spectrin repeat sequences are among the more common repeat elements identified in proteins, typically occurring in large structural proteins. Examples of spectrin repeat-containing proteins include dystrophin, alpha-actinin and spectrin itself--all proteins with well-demonstrated roles of establishing and maintaining cell structure. Over the past decade, it has become clear that, although these proteins display a cytoplasmic and plasma membrane distribution, several are also found both at the nuclear envelope, and within the intranuclear space. In this review, we provide an overview of recent work regarding various spectrin repeat-containing structural proteins in the nucleus. As well, we hypothesize about the regulation of their nuclear localization and possible nuclear functions based on domain architecture, known interacting proteins and evolutionary relationships. Given their large size, and their potential for interacting with multiple proteins and with chromatin, spectrin repeat-containing proteins represent strong candidates for important organizational proteins within the nucleus. Supplementary material for this article can be found on the BioEssays website (http://www.interscience.wiley.com/jpages/0265-9247/suppmat/index.html).

  4. A Large Complement of the Predicted Arabidopsis ARM Repeat Proteins Are Members of the U-Box E3 Ubiquitin Ligase Family1[w

    PubMed Central

    Mudgil, Yashwanti; Shiu, Shin-Han; Stone, Sophia L.; Salt, Jennifer N.; Goring, Daphne R.

    2004-01-01

    The Arabidopsis genome was searched to identify predicted proteins containing armadillo (ARM) repeats, a motif known to mediate protein-protein interactions in a number of different animal proteins. Using domain database predictions and models generated in this study, 108 Arabidopsis proteins were identified that contained a minimum of two ARM repeats with the majority of proteins containing four to eight ARM repeats. Clustering analysis showed that the 108 predicted Arabidopsis ARM repeat proteins could be divided into multiple groups with wide differences in their domain compositions and organizations. Interestingly, 41 of the 108 Arabidopsis ARM repeat proteins contained a U-box, a motif present in a family of E3 ligases, and these proteins represented the largest class of Arabidopsis ARM repeat proteins. In 14 of these U-box/ARM repeat proteins, there was also a novel conserved domain identified in the N-terminal region. Based on the phylogenetic tree, representative U-box/ARM repeat proteins were selected for further study. RNA-blot analyses revealed that these U-box/ARM proteins are expressed in a variety of tissues in Arabidopsis. In addition, the selected U-box/ARM proteins were found to be functional E3 ubiquitin ligases. Thus, these U-box/ARM proteins represent a new family of E3 ligases in Arabidopsis. PMID:14657406

  5. Litopenaeus vannamei sterile-alpha and armadillo motif containing protein (LvSARM) is involved in regulation of Penaeidins and antilipopolysaccharide factors.

    PubMed

    Wang, Pei-Hui; Gu, Zhi-Hua; Wan, Ding-Hui; Zhu, Wei-Bin; Qiu, Wei; Weng, Shao-Ping; Yu, Xiao-Qiang; He, Jian-Guo

    2013-01-01

    The Toll-like receptor (TLR)-mediated NF-κB pathway is tightly controlled because overactivation may result in severe damage to the host, such as in the case of chronic inflammatory diseases and cancer. In mammals, sterile-alpha and armadillo motif-containing protein (SARM) plays an important role in negatively regulating this pathway. While Caenorhabditis elegans SARM is crucial for an efficient immune response against bacterial and fungal infections, it is still unknown whether Drosophila SARM participates in immune responses. Here, Litopenaeus vannamei SARM (LvSARM) was cloned and functionally characterized. LvSARM shared signature domains with and exhibited significant similarities to mammalian SARM. Real-time quantitative PCR analysis indicated that the expression of LvSARM was responsive to Vibrio alginolyticus and white spot syndrome virus (WSSV) infections in the hemocyte, gill, hepatopancreas and intestine. In Drosophila S2 cells, LvSARM was widely distributed in the cytoplasm and could significantly inhibit the promoters of the NF-κB pathway-controlled antimicrobial peptide genes (AMPs). Silencing of LvSARM using dsRNA-mediated RNA interference increased the expression levels of Penaeidins and antilipopolysaccharide factors, which are L.vannamei AMPs, and increased the mortality rate after V. alginolyticus infection. Taken together, our results reveal that LvSARM may be a novel component of the shrimp Toll pathway that negatively regulates shrimp AMPs, particularly Penaeidins and antilipopolysaccharide factors.

  6. Exploring the repeat protein universe through computational protein design.

    PubMed

    Brunette, T J; Parmeggiani, Fabio; Huang, Po-Ssu; Bhabha, Gira; Ekiert, Damian C; Tsutakawa, Susan E; Hura, Greg L; Tainer, John A; Baker, David

    2015-12-24

    A central question in protein evolution is the extent to which naturally occurring proteins sample the space of folded structures accessible to the polypeptide chain. Repeat proteins composed of multiple tandem copies of a modular structure unit are widespread in nature and have critical roles in molecular recognition, signalling, and other essential biological processes. Naturally occurring repeat proteins have been re-engineered for molecular recognition and modular scaffolding applications. Here we use computational protein design to investigate the space of folded structures that can be generated by tandem repeating a simple helix-loop-helix-loop structural motif. Eighty-three designs with sequences unrelated to known repeat proteins were experimentally characterized. Of these, 53 are monomeric and stable at 95 °C, and 43 have solution X-ray scattering spectra consistent with the design models. Crystal structures of 15 designs spanning a broad range of curvatures are in close agreement with the design models with root mean square deviations ranging from 0.7 to 2.5 Å. Our results show that existing repeat proteins occupy only a small fraction of the possible repeat protein sequence and structure space and that it is possible to design novel repeat proteins with precisely specified geometries, opening up a wide array of new possibilities for biomolecular engineering.

  7. Armadillo/Pangolin regulates PCNA and DREF promoter activities.

    PubMed

    Kwon, Eunjeong; Hayashi, Yuko; Otsuki, Kyoko; Hirose, Fumiko; Nishida, Yasuyoshi; Yoo, Mi-Ae; Yamaguchi, Masamitsu

    2004-09-17

    Here we show that Armadillo and Pangolin (dTCF), downstream effectors of the Wingless (Wg) signal transduction pathway, activate transcription of the important DNA replication-related genes encoding Drosophila proliferating cell nuclear antigen (PCNA) and DNA replication-related element-binding factor (DREF). By transient luciferase expression assays and band mobility shift assays, we demonstrated the PCNA gene to be a direct target gene for the Armadillo/Pangolin complex. Using a GAL4-UAS system, stimulation of the PCNA gene by Armadillo/Pangolin was confirmed in adult females. From the published reports of an inhibitory role, we expected that Drosophila CREB-binding protein (dCBP) would interfere with activation. However, effects were only observed with the DREF but not the PCNA gene. In the latter case, as in mammals, dCBP could potentiate Armadillo-mediated activation. These results suggest that first, PCNA and DREF genes are targets of the Armadillo/Pangolin complex and second, dCBP modulates Wg signaling in a gene-specific manner.

  8. The S-Domain Receptor Kinase Arabidopsis Receptor Kinase2 and the U Box/Armadillo Repeat-Containing E3 Ubiquitin Ligase9 Module Mediates Lateral Root Development under Phosphate Starvation in Arabidopsis.

    PubMed

    Deb, Srijani; Sankaranarayanan, Subramanian; Wewala, Gayathri; Widdup, Ellen; Samuel, Marcus A

    2014-08-01

    When plants encounter nutrient-limiting conditions in the soil, the root architecture is redesigned to generate numerous lateral roots (LRs) that increase the surface area of roots, promoting efficient uptake of these deficient nutrients. Of the many essential nutrients, reduced availability of inorganic phosphate has a major impact on plant growth because of the requirement of inorganic phosphate for synthesis of organic molecules, such as nucleic acids, ATP, and phospholipids, that function in various crucial metabolic activities. In our screens to identify a potential role for the S-domain receptor kinase1-6 and its interacting downstream signaling partner, the Arabidopsis (Arabidopsis thaliana) plant U box/armadillo repeat-containing E3 ligase9 (AtPUB9), we identified a role for this module in regulating LR development under phosphate-starved conditions. Our results show that Arabidopsis double mutant plants lacking AtPUB9 and Arabidopsis Receptor Kinase2 (AtARK2; ark2-1/pub9-1) display severely reduced LRs when grown under phosphate-starved conditions. Under these starvation conditions, these plants accumulated very low to no auxin in their primary root and LR tips as observed through expression of the auxin reporter DR5::uidA transgene. Exogenous auxin was sufficient to rescue the LR developmental defects in the ark2-1/pub9-1 lines, indicating a requirement of auxin accumulation for this process. Our subcellular localization studies with tobacco (Nicotiana tabacum) suspension-cultured cells indicate that interaction between ARK2 and AtPUB9 results in accumulation of AtPUB9 in the autophagosomes. Inhibition of autophagy in wild-type plants resulted in reduction of LR development and auxin accumulation under phosphate-starved conditions, suggesting a role for autophagy in regulating LR development. Thus, our study has uncovered a previously unknown signaling module (ARK2-PUB9) that is required for auxin-mediated LR development under phosphate-starved conditions.

  9. Analysis of LRRK2 accessory repeat domains: prediction of repeat length, number and sites of Parkinson's disease mutations.

    PubMed

    Mills, Ryan D; Mulhern, Terrence D; Cheng, Heung-Chin; Culvenor, Janetta G

    2012-10-01

    Various investigators have identified the major domain organization of LRRK2 (leucine-rich repeat kinase 2), which includes a GTPase ROC (Ras of complex proteins) domain followed by a COR (C-terminal of ROC) domain and a protein kinase domain. In addition, there are four domains composed of structural repeat motifs likely to be involved in regulation and localization of this complex protein. In the present paper, we report our bioinformatic analyses of the human LRRK2 amino acid sequence to predict the repeat size, number and likely boundaries for the armadillo repeat, ankyrin repeat, the leucine-rich repeat and WD40 repeat regions of LRRK2. Homology modelling using known protein structures with similar domains was used to predict structures, exposed residues and location of mutations for these repeat regions. We predict that the armadillo repeats, ankyrin repeats and leucine-rich repeats together form an extended N-terminal flexible 'solenoid'-like structure composed of tandem repeat modules likely to be important in anchoring to the membrane and cytoskeletal structures as well as binding to other protein ligands. Near the C-terminus of LRRK2, the WD40 repeat region is predicted to form a closed propeller structure that is important for protein complex formation.

  10. Superfamily of ankyrin repeat proteins in tomato.

    PubMed

    Yuan, Xiaowei; Zhang, Shizhong; Qing, Xiaohe; Sun, Meihong; Liu, Shiyang; Su, Hongyan; Shu, Huairui; Li, Xinzheng

    2013-07-10

    The ankyrin repeat (ANK) protein family plays a crucial role in plant growth and development and in response to biotic and abiotic stresses. However, no detailed information concerning this family is available for tomato (Solanum lycopersicum) due to the limited information on whole genome sequences. In this study, we identified a total of 130 ANK genes in tomato genome (SlANK), and these genes were distributed across all 12 chromosomes at various densities. And chromosomal localizations of SlANK genes indicated 25 SlANK genes were involved in tandem duplications. Based on their domain composition, all of the SlANK proteins were grouped into 13 subgroups. A combined phylogenetic tree was constructed with the aligned SlANK protein sequences. This tree revealed that the SlANK proteins comprise five major groups. An analysis of the expression profiles of SlANK genes in tomato in different tissues and in response to stresses showed that the SlANK proteins play roles in plant growth, development and stress responses. To our knowledge, this is the first report of a genome-wide analysis of the tomato ANK gene family. This study provides valuable information regarding the classification and putative functions of SlANK genes in tomato.

  11. Biochemical analysis of oligomerization of expanded polyalanine repeat proteins.

    PubMed

    Nojima, Jun; Oma, Yoko; Futai, Eugene; Sasagawa, Noboru; Kuroda, Reiko; Turk, Boris; Ishiura, Shoichi

    2009-08-01

    Many human proteins contain amino acid repeats that can form homopolymeric amino acid (HPAA) tracts. HPAA tract proteins that contain polyalanine sequences promote diseases, including oculopharyngeal muscular dystrophy. The pathological properties of these proteins develop when the repeats match or exceed approximately 20 residues. We analyzed the oligomerization of yellow fluorescent protein (YFP) and GST fusion proteins containing >20 alanine repeats by using sucrose density gradient centrifugation. YFP and GST fusion proteins having 23 polyalanine residues sedimented readily in sucrose density gradients, suggesting instability and oligomerization of proteins with an excess of 20 alanine repeats. Moreover, GST fusion proteins were resistant to trypsin digestion after oligomerization. Oligomerized artificial proteins with long polyalanine repeats may be suitable models for studying polyalanine-related diseases.

  12. Diverse functions of WD40 repeat proteins in histone recognition

    PubMed Central

    Suganuma, Tamaki; Pattenden, Samantha G.; Workman, Jerry L.

    2008-01-01

    WD40 repeat proteins have been shown to bind the histone H3 tail at the center of their β-propeller structure. In contrast, in this issue of Genes & Development, Song and colleagues (pp. 1313–1318) demonstrate that the WD40 repeat protein p55 binds a structured region of H4 through a novel binding pocket on the side of β-propeller, illustrating a diversity of histone recognition by WD40 repeat proteins. PMID:18483215

  13. Sterile-α- and armadillo motif-containing protein inhibits the TRIF-dependent downregulation of signal regulatory protein α to interfere with intracellular bacterial elimination in Burkholderia pseudomallei-infected mouse macrophages.

    PubMed

    Baral, Pankaj; Utaisincharoen, Pongsak

    2013-09-01

    Burkholderia pseudomallei, the causative agent of melioidosis, evades macrophage killing by suppressing the TRIF-dependent pathway, leading to inhibition of inducible nitric oxide synthase (iNOS) expression. We previously demonstrated that virulent wild-type B. pseudomallei inhibits the TRIF-dependent pathway by upregulating sterile-α- and armadillo motif-containing protein (SARM) and by inhibiting downregulation of signal regulatory protein α (SIRPα); both molecules are negative regulators of Toll-like receptor signaling. In contrast, the less virulent lipopolysaccharide (LPS) mutant of B. pseudomallei is unable to exhibit these features and is susceptible to macrophage killing. However, the functional relationship of these two negative regulators in the evasion of macrophage defense has not been elucidated. We demonstrated here that SIRPα downregulation was observed after inhibition of SARM expression by small interfering RNA in wild-type-infected macrophages, indicating that SIRPα downregulation is regulated by SARM. Furthermore, this downregulation requires activation of the TRIF signaling pathway, as we observed abrogation of SIRPα downregulation as well as restricted bacterial growth in LPS mutant-infected TRIF-depleted macrophages. Although inhibition of SARM expression is correlated to SIRPα downregulation and iNOS upregulation in gamma interferon-activated wild-type-infected macrophages, these phenomena appear to bypass the TRIF-dependent pathway. Similar to live bacteria, the wild-type LPS is able to upregulate SARM and to prevent SIRPα downregulation, implying that the LPS of B. pseudomallei may play a crucial role in regulating the expression of these two negative regulators. Altogether, our findings show a previously unrecognized role of B. pseudomallei-induced SARM in inhibiting SIRPα downregulation-mediated iNOS upregulation, facilitating the ability of the bacterium to multiply in macrophages.

  14. Repeat proteins challenge the concept of structural domains.

    PubMed

    Espada, Rocío; Parra, R Gonzalo; Sippl, Manfred J; Mora, Thierry; Walczak, Aleksandra M; Ferreiro, Diego U

    2015-10-01

    Structural domains are believed to be modules within proteins that can fold and function independently. Some proteins show tandem repetitions of apparent modular structure that do not fold independently, but rather co-operate in stabilizing structural forms that comprise several repeat-units. For many natural repeat-proteins, it has been shown that weak energetic links between repeats lead to the breakdown of co-operativity and the appearance of folding sub-domains within an apparently regular repeat array. The quasi-1D architecture of repeat-proteins is crucial in detailing how the local energetic balances can modulate the folding dynamics of these proteins, which can be related to the physiological behaviour of these ubiquitous biological systems.

  15. PRIGSA: protein repeat identification by graph spectral analysis.

    PubMed

    Chakrabarty, Broto; Parekh, Nita

    2014-12-01

    Repetition of a structural motif within protein is associated with a wide range of structural and functional roles. In most cases the repeating units are well conserved at the structural level while at the sequence level, they are mostly undetectable suggesting the need for structure-based methods. Since most known methods require a training dataset, de novo approach is desirable. Here, we propose an efficient graph-based approach for detecting structural repeats in proteins. In a protein structure represented as a graph, interactions between inter- and intra-repeat units are well captured by the eigen spectra of adjacency matrix of the graph. These conserved interactions give rise to similar connections and a unique profile of the principal eigen spectra for each repeating unit. The efficacy of the approach is shown on eight repeat families annotated in UniProt, comprising of both solenoid and nonsolenoid repeats with varied secondary structure architecture and repeat lengths. The performance of the approach is also tested on other known benchmark datasets and the performance compared with two repeat identification methods. For a known repeat type, the algorithm also identifies the type of repeat present in the protein. A web tool implementing the algorithm is available at the URL http://bioinf.iiit.ac.in/PRIGSA/.

  16. RepeatsDB 2.0: improved annotation, classification, search and visualization of repeat protein structures

    PubMed Central

    Paladin, Lisanna; Hirsh, Layla; Piovesan, Damiano; Andrade-Navarro, Miguel A.; Kajava, Andrey V.; Tosatto, Silvio C.E.

    2017-01-01

    RepeatsDB 2.0 (URL: http://repeatsdb.bio.unipd.it/) is an update of the database of annotated tandem repeat protein structures. Repeat proteins are a widespread class of non-globular proteins carrying heterogeneous functions involved in several diseases. Here we provide a new version of RepeatsDB with an improved classification schema including high quality annotations for ∼5400 protein structures. RepeatsDB 2.0 features information on start and end positions for the repeat regions and units for all entries. The extensive growth of repeat unit characterization was possible by applying the novel ReUPred annotation method over the entire Protein Data Bank, with data quality is guaranteed by an extensive manual validation for >60% of the entries. The updated web interface includes a new search engine for complex queries and a fully re-designed entry page for a better overview of structural data. It is now possible to compare unit positions, together with secondary structure, fold information and Pfam domains. Moreover, a new classification level has been introduced on top of the existing scheme as an independent layer for sequence similarity relationships at 40%, 60% and 90% identity. PMID:27899671

  17. Ising Model Reprogramming of a Repeat Protein's Equilibrium Unfolding Pathway.

    PubMed

    Millership, C; Phillips, J J; Main, E R G

    2016-05-08

    Repeat proteins are formed from units of 20-40 aa that stack together into quasi one-dimensional non-globular structures. This modular repetitive construction means that, unlike globular proteins, a repeat protein's equilibrium folding and thus thermodynamic stability can be analysed using linear Ising models. Typically, homozipper Ising models have been used. These treat the repeat protein as a series of identical interacting subunits (the repeated motifs) that couple together to form the folded protein. However, they cannot describe subunits of differing stabilities. Here we show that a more sophisticated heteropolymer Ising model can be constructed and fitted to two new helix deletion series of consensus tetratricopeptide repeat proteins (CTPRs). This analysis, showing an asymmetric spread of stability between helices within CTPR ensembles, coupled with the Ising model's predictive qualities was then used to guide reprogramming of the unfolding pathway of a variant CTPR protein. The designed behaviour was engineered by introducing destabilising mutations that increased the thermodynamic asymmetry within a CTPR ensemble. The asymmetry caused the terminal α-helix to thermodynamically uncouple from the rest of the protein and preferentially unfold. This produced a specific, highly populated stable intermediate with a putative dimerisation interface. As such it is the first step in designing repeat proteins with function regulated by a conformational switch.

  18. Artificial leucine rich repeats as new scaffolds for protein design.

    PubMed

    Baabur-Cohen, Hemda; Dayalan, Subashini; Shumacher, Inbal; Cohen-Luria, Rivka; Ashkenasy, Gonen

    2011-04-15

    The leucine rich repeat (LRR) motif that participates in many biomolecular recognition events in cells was suggested as a general scaffold for producing artificial receptors. We describe here the design and first total chemical synthesis of small LRR proteins, and their structural analysis. When evaluating the tertiary structure as a function of different number of repeating units (1-3), we were able to find that the 3-repeats sequence, containing 90 amino acids, folds into the expected structure.

  19. Burkholderia pseudomallei-induced expression of a negative regulator, sterile-alpha and Armadillo motif-containing protein, in mouse macrophages: a possible mechanism for suppression of the MyD88-independent pathway.

    PubMed

    Pudla, M; Limposuwan, K; Utaisincharoen, P

    2011-07-01

    Burkholderia pseudomallei, a causative agent of melioidosis, is a Gram-negative facultative intracellular bacterium that can survive and multiply in macrophages. Previously, we demonstrated that B. pseudomallei failed to activate gene expression downstream of the MyD88-independent pathway, particularly the expression of beta interferon (IFN-β) and inducible nitric oxide synthase (iNOS), leading to the inability of macrophages to kill this bacterium. In the present report, we extended our study to show that B. pseudomallei was able to activate sterile-α and Armadillo motif (SARM)-containing protein, a known negative regulator of the MyD88-independent pathway. Both live B. pseudomallei and heat-killed B. pseudomallei were able to upregulate SARM expression in a time-dependent manner in mouse macrophage cell line RAW 264.7. The expression of SARM required bacterial internalization, as it could be inhibited by cytochalasin D. In addition, the intracellular survival of B. pseudomallei was suppressed in SARM-deficient macrophages. Increased expression of IFN-β and iNOS and degradation of IκBα correlated with enhanced macrophage killing capability. These results demonstrated that B. pseudomallei modulated macrophage defense mechanisms by upregulating SARM, thus leading to the suppression of IFN-β and iNOS needed for bacterial elimination.

  20. Were protein internal repeats formed by "bricolage"?

    PubMed

    Lavorgna, G; Patthy, L; Boncinelli, E

    2001-03-01

    Is evolution an engineer, or is it a tinkerer--a "bricoleur"--building up complex molecules in organisms by increasing and adapting the materials at hand? An analysis of completely sequenced genomes suggests the latter, showing that increasing repetition of modules within the proteins encoded by these genomes is correlated with increasing complexity of the organism.

  1. Expression and characterization of recombinant interferon gamma (IFN-gamma) from the nine-banded armadillo (Dasypus novemcinctus) and its effect on Mycobacterium leprae-infected macrophages.

    PubMed

    Peña, M T; Adams, J E; Adams, L B; Gillis, T P; Williams, D L; Spencer, J S; Krahenbuhl, J L; Truman, R W

    2008-08-01

    Armadillos (Dasypus novemcinctus) manifest the full histopathological spectrum of leprosy, and are hosts of choice for in vivo propagation of Mycobacterium leprae. Though potentially useful as a model of leprosy pathogenesis, few armadillo-specific reagents exist. We have identified a region of high homology to the interferon gamma (IFN-gamma) of other mammals within the recently published armadillo whole genomic sequence. cDNA was made from ConA-stimulated armadillo peripheral blood mononuclear cells (PBMC), amplified, and cloned into a pET expression vector for transformation and over-expression in Escherichia coli. The recombinant protein (rDnIFN-gamma) was characterized by western blot and its biological function confirmed with bioassays including intracellular killing of Toxoplasma gondii and induction of indoleamine 2, 3-dioxygenase activity. In using rIFN-gamma to activate macrophages from mice, humans or armadillos, similar to humans, rIFN-gamma-activated armadillo MPhi did not produce nitrite and or inhibit the viability of M. leprae in vitro. Conversely, murine rIFN-gamma-activated mouse MPhi produced high levels of nitrite and killed intracellular M. leprae in vitro. These data indicate that the response of armadillo MPhi to rDnIFN-gamma is similar to that which occurs in humans, and demonstrates a potentially important value of the armadillo as a model in leprosy research.

  2. Distribution and Evolution of Yersinia Leucine-Rich Repeat Proteins

    PubMed Central

    Hu, Yueming; Huang, He; Hui, Xinjie; Cheng, Xi; White, Aaron P.

    2016-01-01

    Leucine-rich repeat (LRR) proteins are widely distributed in bacteria, playing important roles in various protein-protein interaction processes. In Yersinia, the well-characterized type III secreted effector YopM also belongs to the LRR protein family and is encoded by virulence plasmids. However, little has been known about other LRR members encoded by Yersinia genomes or their evolution. In this study, the Yersinia LRR proteins were comprehensively screened, categorized, and compared. The LRR proteins encoded by chromosomes (LRR1 proteins) appeared to be more similar to each other and different from those encoded by plasmids (LRR2 proteins) with regard to repeat-unit length, amino acid composition profile, and gene expression regulation circuits. LRR1 proteins were also different from LRR2 proteins in that the LRR1 proteins contained an E3 ligase domain (NEL domain) in the C-terminal region or an NEL domain-encoding nucleotide relic in flanking genomic sequences. The LRR1 protein-encoding genes (LRR1 genes) varied dramatically and were categorized into 4 subgroups (a to d), with the LRR1a to -c genes evolving from the same ancestor and LRR1d genes evolving from another ancestor. The consensus and ancestor repeat-unit sequences were inferred for different LRR1 protein subgroups by use of a maximum parsimony modeling strategy. Structural modeling disclosed very similar repeat-unit structures between LRR1 and LRR2 proteins despite the different unit lengths and amino acid compositions. Structural constraints may serve as the driving force to explain the observed mutations in the LRR regions. This study suggests that there may be functional variation and lays the foundation for future experiments investigating the functions of the chromosomally encoded LRR proteins of Yersinia. PMID:27217422

  3. The evolution of filamin – A protein domain repeat perspective

    PubMed Central

    Light, Sara; Sagit, Rauan; Ithychanda, Sujay S.; Qin, Jun; Elofsson, Arne

    2013-01-01

    Particularly in higher eukaryotes, some protein domains are found in tandem repeats, performing broad functions often related to cellular organization. For instance, the eukaryotic protein filamin interacts with many proteins and is crucial for the cytoskeleton. The functional properties of long repeat domains are governed by the specific properties of each individual domain as well as by the repeat copy number. To provide better understanding of the evolutionary and functional history of repeating domains, we investigated the mode of evolution of the filamin domain in some detail. Among the domains that are common in long repeat proteins, sushi and spectrin domains evolve primarily through cassette tandem duplications while scavenger and immunoglobulin repeats appear to evolve through clustered tandem duplications. Additionally, immunoglobulin and filamin repeats exhibit a unique pattern where every other domain shows high sequence similarity. This pattern may be the result of tandem duplications, serve to avert aggregation between adjacent domains or it is the result of functional constraints. In filamin, our studies confirm the presence of interspersed integrin binding domains in vertebrates, while invertebrates exhibit more varied patterns, including more clustered integrin binding domains. The most notable case is leech filamin, which contains a 20 repeat expansion and exhibits unique dimerization topology. Clearly, invertebrate filamins are varied and contain examples of similar adjacent integrin-binding domains. Given that invertebrate integrin shows more similarity to the weaker filamin binder, integrin β3, it is possible that the distance between integrin-binding domains is not as crucial for invertebrate filamins as for vertebrates. PMID:22414427

  4. Methionine-rich repeat proteins: a family of membrane-associated proteins which contain unusual repeat regions.

    PubMed

    Weiss, Jamie L; Evans, Nicholas A; Ahmed, Tanweer; Wrigley, Jonathan D J; Khan, Shukria; Wright, Charles; Keen, Jeffrey N; Holzenburg, Andreas; Findlay, John B C

    2005-03-01

    We report the protein isolation, cloning and characterization of members of an unusual protein family, which comprise the most abundant proteins present in the squid eye. The proteins in this family have a range of molecular weights from 32 to 36 kDa. Electron microscopy and detergent solubilization demonstrate that these proteins are tightly associated with membrane structures where they may form tetramers. Despite this, these proteins have no stretches of hydrophobic residues that could form typical transmembrane domains. They share an unusual protein sequence rich in methionine, and contain multiple repeating motifs. We have therefore named these proteins Methionine-Rich Repeat Proteins (MRRPs). The use of structure prediction algorithms suggest very little recognized secondary structure elements. At the time of cloning no sequence or structural homologues have been found in any database. We have isolated three closely related cDNA clones from the MRRP family. Coupled in vitro transcription/translation of the MRRP clones shows that they encode proteins with molecular masses similar to components of native MRRPs. Immunoblot analysis of these proteins reveals that they are also present in squid brain, optic lobe, and heart, and also indicate that MRRP-like protein motifs may also exist in mammalian tissues. We propose that MRRPs define a family of important proteins that have an unusual mode of attachment or insertion into cell membranes and are found in evolutionarily diverse organisms.

  5. The first crystal structure of an archaeal helical repeat protein

    PubMed Central

    Yoneda, Kazunari; Sakuraba, Haruhiko; Tsuge, Hideaki; Katunuma, Nobuhiko; Kuramitsu, Seiki; Kawabata, Takeshi; Ohshima, Toshihisa

    2005-01-01

    The crystal structure of ST1625p, a protein encoded by a hypothetical open reading frame ST1625 in the genome of the hyperthermophilic archaeon Sulfolobus tokodaii, was determined at 2.2 Å resolution. The only sequence similarity exhibited by the amino-acid sequence of ST1625p was a 33% identity with the sequence of SSO0983p from S. solfataricus. The 19 kDa monomeric protein was observed to consist of a right-handed superhelix assembled from a tandem repeat of ten α-­helices. A structural homology search using the DALI and MATRAS algorithms indicates that this protein can be classified as a helical repeat protein. PMID:16511116

  6. Alanine repeats influence protein localization in splicing speckles and paraspeckles.

    PubMed

    Chang, Shuo-Hsiu; Chang, Wei-Lun; Lu, Chia-Chen; Tarn, Woan-Yuh

    2014-12-16

    Mammalian splicing regulatory protein RNA-binding motif protein 4 (RBM4) has an alanine repeat-containing C-terminal domain (CAD) that confers both nuclear- and splicing speckle-targeting activities. Alanine-repeat expansion has pathological potential. Here we show that the alanine-repeat tracts influence the subnuclear targeting properties of the RBM4 CAD in cultured human cells. Notably, truncation of the alanine tracts redistributed a portion of RBM4 to paraspeckles. The alanine-deficient CAD was sufficient for paraspeckle targeting. On the other hand, alanine-repeat expansion reduced the mobility of RBM4 and impaired its splicing activity. We further took advantage of the putative coactivator activator (CoAA)-RBM4 conjoined splicing factor, CoAZ, to investigate the function of the CAD in subnuclear targeting. Transiently expressed CoAZ formed discrete nuclear foci that emerged and subsequently separated-fully or partially-from paraspeckles. Alanine-repeat expansion appeared to prevent CoAZ separation from paraspeckles, resulting in their complete colocalization. CoAZ foci were dynamic but, unlike paraspeckles, were resistant to RNase treatment. Our results indicate that the alanine-rich CAD, in conjunction with its conjoined RNA-binding domain(s), differentially influences the subnuclear localization and biogenesis of RBM4 and CoAZ.

  7. The first crystal structure of an archaeal helical repeat protein

    SciTech Connect

    Yoneda, Kazunari; Sakuraba, Haruhiko; Tsuge, Hideaki; Katunuma, Nobuhiko; Kuramitsu, Seiki; Kawabata, Takeshi; Ohshima, Toshihisa

    2005-07-01

    The crystal structure of ST1625p, a protein encoded by a hypothetical open reading frame ST1625 in the genome of the hyperthermophilic archaeon Sulfolobus tokodaii, was determined at 2.2 Å resolution. The structure of ST1625p consists of a unique superhelix with a low-level structure resemblance to doamins from other proteins with known three-dimensional structures. The crystal structure of ST1625p, a protein encoded by a hypothetical open reading frame ST1625 in the genome of the hyperthermophilic archaeon Sulfolobus tokodaii, was determined at 2.2 Å resolution. The only sequence similarity exhibited by the amino-acid sequence of ST1625p was a 33% identity with the sequence of SSO0983p from S. solfataricus. The 19 kDa monomeric protein was observed to consist of a right-handed superhelix assembled from a tandem repeat of ten α-helices. A structural homology search using the DALI and MATRAS algorithms indicates that this protein can be classified as a helical repeat protein.

  8. Gibbs motif sampling: detection of bacterial outer membrane protein repeats.

    PubMed Central

    Neuwald, A. F.; Liu, J. S.; Lawrence, C. E.

    1995-01-01

    The detection and alignment of locally conserved regions (motifs) in multiple sequences can provide insight into protein structure, function, and evolution. A new Gibbs sampling algorithm is described that detects motif-encoding regions in sequences and optimally partitions them into distinct motif models; this is illustrated using a set of immunoglobulin fold proteins. When applied to sequences sharing a single motif, the sampler can be used to classify motif regions into related submodels, as is illustrated using helix-turn-helix DNA-binding proteins. Other statistically based procedures are described for searching a database for sequences matching motifs found by the sampler. When applied to a set of 32 very distantly related bacterial integral outer membrane proteins, the sampler revealed that they share a subtle, repetitive motif. Although BLAST (Altschul SF et al., 1990, J Mol Biol 215:403-410) fails to detect significant pairwise similarity between any of the sequences, the repeats present in these outer membrane proteins, taken as a whole, are highly significant (based on a generally applicable statistical test for motifs described here). Analysis of bacterial porins with known trimeric beta-barrel structure and related proteins reveals a similar repetitive motif corresponding to alternating membrane-spanning beta-strands. These beta-strands occur on the membrane interface (as opposed to the trimeric interface) of the beta-barrel. The broad conservation and structural location of these repeats suggests that they play important functional roles. PMID:8520488

  9. A designed repeat protein as an affinity capture reagent.

    PubMed

    Speltz, Elizabeth B; Brown, Rebecca S H; Hajare, Holly S; Schlieker, Christian; Regan, Lynne

    2015-10-01

    Repeat proteins are an attractive target for protein engineering and design. We have focused our attention on the design and engineering of one particular class: tetratricopeptide repeat (TPR) proteins. In previous work, we have shown that the structure and stability of TPR proteins can be manipulated in a rational fashion [Cortajarena (2011) Prot. Sci. 20: , 1042-1047; Main (2003) Structure 11: , 497-508]. Building on those studies, we have designed and characterized a number of different peptide-binding TPR modules and we have also assembled these modules into supramolecular arrays [Cortajarena (2009) ACS Chem. Biol. 5: , 545-552; Cortajarena (2008) ACS Chem. Biol. 3: , 161-166; Jackrel (2009) Prot. Sci. 18: , 762-774; Kajander (2007) Acta Crystallogr. D Biol. Crystallogr. 63: , 800-811]. Here we focus on the development of one such TPR-peptide interaction for a practical application, affinity purification. We illustrate the general utility of our designed protein interaction. Furthermore, this example highlights how basic research on protein-peptide interactions can lead to the development of novel reagents with important practical applications.

  10. Repeat-containing protein effectors of plant-associated organisms

    PubMed Central

    Mesarich, Carl H.; Bowen, Joanna K.; Hamiaux, Cyril; Templeton, Matthew D.

    2015-01-01

    Many plant-associated organisms, including microbes, nematodes, and insects, deliver effector proteins into the apoplast, vascular tissue, or cell cytoplasm of their prospective hosts. These effectors function to promote colonization, typically by altering host physiology or by modulating host immune responses. The same effectors however, can also trigger host immunity in the presence of cognate host immune receptor proteins, and thus prevent colonization. To circumvent effector-triggered immunity, or to further enhance host colonization, plant-associated organisms often rely on adaptive effector evolution. In recent years, it has become increasingly apparent that several effectors of plant-associated organisms are repeat-containing proteins (RCPs) that carry tandem or non-tandem arrays of an amino acid sequence or structural motif. In this review, we highlight the diverse roles that these repeat domains play in RCP effector function. We also draw attention to the potential role of these repeat domains in adaptive evolution with regards to RCP effector function and the evasion of effector-triggered immunity. The aim of this review is to increase the profile of RCP effectors from plant-associated organisms. PMID:26557126

  11. Tandem Repeats in Proteins: Prediction Algorithms and Biological Role

    PubMed Central

    Pellegrini, Marco

    2015-01-01

    Tandem repetitions in protein sequence and structure is a fascinating subject of research which has been a focus of study since the late 1990s. In this survey, we give an overview on the multi-faceted aspects of research on protein tandem repeats (PTR for short), including prediction algorithms, databases, early classification efforts, mechanisms of PTR formation and evolution, and synthetic PTR design. We also touch on the rather open issue of the relationship between PTR and flexibility (or disorder) in proteins. Detection of PTR either from protein sequence or structure data is challenging due to inherent high (biological) signal-to-noise ratio that is a key feature of this problem. As early in silico analytic tools have been key enablers for starting this field of study, we expect that current and future algorithmic and statistical breakthroughs will have a high impact on the investigations of the biological role of PTR. PMID:26442257

  12. The Armadillo as a Model for Peripheral Neuropathy in Leprosy

    PubMed Central

    Truman, Richard W.; Ebenezer, Gigi J.; Pena, Maria T.; Sharma, Rahul; Balamayooran, Gayathriy; Gillingwater, Thomas H.; Scollard, David M.; McArthur, Justin C.; Rambukkana, Anura

    2014-01-01

    Leprosy (also known as Hansen's Disease) is a chronic infectious disease caused by Mycobacterium leprae that primarily targets the peripheral nervous system; skin, muscle, and other tissues are also affected. Other than humans, nine-banded armadillos (Dasypus novemcinctus) are the only natural hosts of M. leprae, and they are the only laboratory animals that develop extensive neurological involvement with this bacterium. Infection in the armadillo closely recapitulates many of the structural, physiological, and functional aspects of leprosy seen in humans. Armadillos can be useful models of leprosy for basic scientific investigations into the pathogenesis of leprosy neuropathy and its associated myopathies, as well as for translational research studies in piloting new diagnostic methods or therapeutic interventions. Practical and ethical constraints often limit investigation into human neuropathies, but armadillos are an abundant source of leprotic neurologic fibers. Studies with these animals may provide new insights into the mechanisms involved in leprosy that also might benefit the understanding of other demyelinating neuropathies. Although there is only a limited supply of armadillo-specific reagents, the armadillo whole genomic sequence has been completed, and gene expression studies can be employed. Clinical procedures, such as electrophysiological nerve conduction testing, provide a functional assessment of armadillo nerves. A variety of standard histopathological and immunopathological procedures including Epidermal Nerve Fiber Density (ENFD) analysis, Schwann Cell Density, and analysis for other conserved cellular markers can be used effectively with armadillos and will be briefly reviewed in this text. PMID:24615444

  13. Deep conservation of human protein tandem repeats within the eukaryotes.

    PubMed

    Schaper, Elke; Gascuel, Olivier; Anisimova, Maria

    2014-05-01

    Tandem repeats (TRs) are a major element of protein sequences in all domains of life. They are particularly abundant in mammals, where by conservative estimates one in three proteins contain a TR. High generation-scale duplication and deletion rates were reported for nucleic TR units. However, it is not known whether protein TR units can also be frequently lost or gained providing a source of variation for rapid adaptation of protein function, or alternatively, tend to have conserved TR unit configurations over long evolutionary times. To obtain a systematic picture, we performed a proteome-wide analysis of the mode of evolution for human protein TRs. For this purpose, we propose a novel method for the detection of orthologous TRs based on circular profile hidden Markov models. For all detected TRs, we reconstructed bispecies TR unit phylogenies across 61 eukaryotes ranging from human to yeast. Moreover, we performed additional analyses to correlate functional and structural annotations of human TRs with their mode of evolution. Surprisingly, we find that the vast majority of human TRs are ancient, with TR unit number and order preserved intact since distant speciation events. For example, ≥ 61% of all human TRs have been strongly conserved at least since the root of all mammals, approximately 300 Ma. Further, we find no human protein TR that shows evidence for strong recent duplications and deletions. The results are in contrast to the high generation-scale mutability of nucleic TRs. Presumably, most protein TRs fold into stable and conserved structures that are indispensable for the function of the TR-containing protein. All of our data and results are available for download from http://www.atgc-montpellier.fr/TRE.

  14. Repeat-modulated population genetic effects in fungal proteins.

    PubMed

    Braun, F N; Liberles, D A

    2004-07-01

    A number of fungal lineages, notably N. crassa, have evolved a novel mechanism of processing genomic duplication events known as repeat-induced point (RIP) mutation. This mechanism appears, on the one hand, to act as a conservative genomic safeguard, by introducing stop codons into duplicated nucleotide sequences, thereby preempting consequences such as dosage effects. However, it also typically performs further nonsynonymous (i.e., amino acid-changing) nucleotide substitutions, the significance of which is unclear. We explore here the possibility that RIP-mutated genes which evade silencing may have some microevolutionary impact on functional sequences. Our approach focuses on structurally important hydrophobic/polar (HP) amino-acid substitutions effected by RIP. We exploit a simple generic protein folding model to predict the associated emergence of increased protein-structural stability and variance within a large population.

  15. A WD-Repeat Protein Stabilizes ORC Binding to Chromatin

    PubMed Central

    Shen, Zhen; Sathyan, Kizhakke M.; Geng, Yijie; Zheng, Ruiping; Chakraborty, Arindam; Freeman, Brian; Wang, Fei; Prasanth, Kannanganattu V.; Prasanth, Supriya G.

    2015-01-01

    SUMMARY Origin recognition complex (ORC) plays critical roles in the initiation of DNA replication and cell-cycle progression. In metazoans, ORC associates with origin DNA during G1 and with heterochromatin in postreplicated cells. However, what regulates the binding of ORC to chromatin is not understood. We have identified a highly conserved, leucine-rich repeats and WD40 repeat domain-containing protein 1 (LRWD1) or ORC-associated (ORCA) in human cells that interacts with ORC and modulates chromatin association of ORC. ORCA colocalizes with ORC and shows similar cell-cycle dynamics. We demonstrate that ORCA efficiently recruits ORC to chromatin. Depletion of ORCA in human primary cells and embryonic stem cells results in loss of ORC association to chromatin, concomitant reduction of MCM binding, and a subsequent accumulation in G1 phase. Our results suggest ORCA-mediated association of ORC to chromatin is critical to initiate preRC assembly in G1 and chromatin organization in post-G1 cells. PMID:20932478

  16. Ankyrin-repeat proteins from sponge symbionts modulate amoebal phagocytosis.

    PubMed

    Nguyen, Mary T H D; Liu, Michael; Thomas, Torsten

    2014-03-01

    Bacteria-eukaryote symbiosis occurs in all stages of evolution, from simple amoebae to mammals, and from facultative to obligate associations. Sponges are ancient metazoans that form intimate symbiotic interactions with complex communities of bacteria. The basic nutritional requirements of the sponge are in part satisfied by the phagocytosis of bacterial food particles from the surrounding water. How bacterial symbionts, which are permanently associated with the sponge, survive in the presence of phagocytic cells is largely unknown. Here, we present the discovery of a genomic fragment from an uncultured gamma-proteobacterial sponge symbiont that encodes for four proteins, whose closest known relatives are found in a sponge genome. Through recombinant approaches, we show that these four eukaryotic-like, ankyrin-repeat proteins (ARP) when expressed in Eschericha coli can modulate phagocytosis of amoebal cells and lead to accumulation of bacteria in the phagosome. Mechanistically, two ARPs appear to interfere with phagosome development in a similar way to reduced vacuole acidification, by blocking the fusion of the early phagosome with the lysosome and its digestive enzymes. Our results show that ARP from sponge symbionts can function to interfere with phagocytosis, and we postulate that this might be one mechanism by which symbionts can escape digestion in a sponge host.

  17. Human-armadillo interaction in Ceará, Brazil: Potential for transmission of Mycobacterium leprae.

    PubMed

    Kerr, Ligia; Kendall, Carl; Sousa, Cesar Augusto Barros de; Frota, Cristiane Cunha; Graham, Jove; Rodrigues, Laura; Fernandes, Rafael Lima; Barreto, Maurício Lima

    2015-12-01

    Several factors suggest that armadillos present an important risk for human leprosy infection. This study uses semi-structured interviews to better illustrate how human interaction with armadillos may increase the risk of leprosy transmission. The participants were all residents of the state of Ceará, in northeastern Brazil, all acknowledged contact with armadillos either through hunting, through cooking, or through consumption of its meat. This study raises important issues about contact between human beings and armadillos. The interviews provide evidence of numerous situations in which leprosy transmission via the armadillo is possible. At a minimum, people who hunt armadillos need to be made aware of the risk of infection.

  18. Molecular systematics of armadillos (Xenarthra, Dasypodidae): contribution of maximum likelihood and Bayesian analyses of mitochondrial and nuclear genes.

    PubMed

    Delsuc, Frédéric; Stanhope, Michael J; Douzery, Emmanuel J P

    2003-08-01

    The 30 living species of armadillos, anteaters, and sloths (Mammalia: Xenarthra) represent one of the three major clades of placentals. Armadillos (Cingulata: Dasypodidae) are the earliest and most speciose xenarthran lineage with 21 described species. The question of their tricky phylogeny was here studied by adding two mitochondrial genes (NADH dehydrogenase subunit 1 [ND1] and 12S ribosomal RNA [12S rRNA]) to the three protein-coding nuclear genes (alpha2B adrenergic receptor [ADRA2B], breast cancer susceptibility exon 11 [BRCA1], and von Willebrand factor exon 28 [VWF]) yielding a total of 6869 aligned nucleotide sites for thirteen xenarthran species. The two mitochondrial genes were characterized by marked excesses of transitions over transversions-with a strong bias toward CT transitions for the 12S rRNA-and exhibited two- to fivefold faster evolutionary rates than the fastest nuclear gene (ADRA2B). Maximum likelihood and Bayesian phylogenetic analyses supported the monophyly of Dasypodinae, Tolypeutinae, and Euphractinae, with the latter two armadillo subfamilies strongly clustering together. Conflicting branching points between individual genes involved relationships within the subfamilies Tolypeutinae and Euphractinae. Owing to a greater number of informative sites, the overall concatenation favored the mitochondrial topology with the classical grouping of Cabassous and Priodontes within Tolypeutinae, and a close relationship between Euphractus and Chaetophractus within Euphractinae. However, low statistical support values associated with almost equal distributions of apomorphies among alternatives suggested that two parallel events of rapid speciation occurred within these two armadillo subfamilies.

  19. A matrix protein silences transposons and repeats through interaction with retinoblastoma-associated proteins.

    PubMed

    Xu, Yifeng; Wang, Yizhong; Stroud, Hume; Gu, Xiaofeng; Sun, Bo; Gan, Eng-Seng; Ng, Kian-Hong; Jacobsen, Steven E; He, Yuehui; Ito, Toshiro

    2013-02-18

    Epigenetic regulation helps to maintain genomic integrity by suppressing transposable elements (TEs) and also controls key developmental processes, such as flowering time. To prevent TEs from causing rearrangements and mutations, TE and TE-like repetitive DNA sequences are usually methylated, whereas histones are hypoacetylated and methylated on specific residues (e.g., H3 lysine 9 dimethylation [H3K9me2]). TEs and repeats can also attenuate gene expression. However, how various histone modifiers are recruited to target loci is not well understood. Here we show that knockdown of the nuclear matrix protein with AT-hook DNA binding motifs TRANSPOSABLE ELEMENT SILENCING VIA AT-HOOK (TEK) in Arabidopsis Landsberg erecta results in robust activation of various TEs, the TE-like repeat-containing floral repressor genes FLOWERING LOCUS C (FLC) and FWA. This derepression is associated with chromatin conformational changes, increased histone acetylation, reduced H3K9me2, and even TE transposition. TEK directly binds to an FLC-repressive regulatory region and the silencing repeats of FWA and associates with Arabidopsis homologs of the Retinoblastoma-associated protein 46/48, FVE and MSI5, which mediate histone deacetylation. We propose that the nuclear matrix protein TEK acts in the maintenance of genome integrity by silencing TE and repeat-containing genes.

  20. Rational design of alpha-helical tandem repeat proteins with closed architectures

    PubMed Central

    Doyle, Lindsey; Hallinan, Jazmine; Bolduc, Jill; Parmeggiani, Fabio; Baker, David; Stoddard, Barry L.; Bradley, Philip

    2015-01-01

    Tandem repeat proteins, which are formed by repetition of modular units of protein sequence and structure, play important biological roles as macromolecular binding and scaffolding domains, enzymes, and building blocks for the assembly of fibrous materials1,2. The modular nature of repeat proteins enables the rapid construction and diversification of extended binding surfaces by duplication and recombination of simple building blocks3,4. The overall architecture of tandem repeat protein structures – which is dictated by the internal geometry and local packing of the repeat building blocks – is highly diverse, ranging from extended, super-helical folds that bind peptide, DNA, and RNA partners5–9, to closed and compact conformations with internal cavities suitable for small molecule binding and catalysis10. Here we report the development and validation of computational methods for de novo design of tandem repeat protein architectures driven purely by geometric criteria defining the inter-repeat geometry, without reference to the sequences and structures of existing repeat protein families. We have applied these methods to design a series of closed alpha-solenoid11 repeat structures (alpha-toroids) in which the inter-repeat packing geometry is constrained so as to juxtapose the N- and C-termini; several of these designed structures have been validated by X-ray crystallography. Unlike previous approaches to tandem repeat protein engineering12–20, our design procedure does not rely on template sequence or structural information taken from natural repeat proteins and hence can produce structures unlike those seen in nature. As an example, we have successfully designed and validated closed alpha-solenoid repeats with a left-handed helical architecture that – to our knowledge – is not yet present in the protein structure database21. PMID:26675735

  1. Equilibrium folding and stability of myotrophin: a model ankyrin repeat protein.

    PubMed

    Mosavi, Leila K; Williams, Suzanna; Peng Zy, Zheng-yu

    2002-07-05

    Proteins containing stretches of repeating amino acid sequences are prevalent throughout nature, yet little is known about the general folding and assembly mechanisms of these systems. Here we propose myotrophin as a model system to study the folding of ankyrin repeat proteins. Myotrophin is folded over a large pH range and is soluble at high concentrations. Thermal and urea denaturation studies show that the protein displays cooperative two-state folding properties despite its modular nature. Taken together with previous studies on other ankyrin repeat proteins, our data suggest that the two-state folding pathway may be characteristic of ankyrin repeat proteins and other integrated alpha-helical repeat proteins in general.

  2. Ankyrin repeat and SOCS box protein 15 regulates protein synthesis in skeletal muscle.

    PubMed

    McDaneld, T G; Hannon, K; Moody, D E

    2006-06-01

    Ankyrin repeat and SOCS box protein 15 (ASB15) is an Asb family member expressed predominantly in skeletal muscle. We have previously reported that ASB15 mRNA abundance decreases after administration of beta-adrenergic receptor agonists. Because beta-adrenergic receptor agonists are known to stimulate muscle hypertrophy, the objective of this study was to determine whether ASB15 regulates cellular processes that contribute to muscle growth. Stable myoblast C2C12 cells expressing full-length ASB15 (ASB15-FL) and ASB15 lacking the ankyrin repeat (ASB15-Ank) or SOCS box (ASB15-SOCS) motifs were evaluated for changes in proliferation, differentiation, protein synthesis, and protein degradation. Expression of ASB15-FL caused a delay in differentiation, followed by an increase in protein synthesis of approximately 34% (P<0.05). A consistent effect of ASB15 overexpression was observed in vivo, where ectopic expression of ASB15 increased skeletal muscle fiber area (P<0.0001) after 9 days. Expression of ASB15-SOCS altered differentiation of myoblasts, resulting in detachment of cells from culture plates. Expression of ASB15-Ank increased protein degradation by 84 h of differentiation (P<0.05), and in vivo ectopic expression of an ASB15 construct lacking both the ankyrin repeat and SOCS box motifs decreased skeletal muscle fiber area (P<0.0001). Together, these results suggest ASB15 participates in the regulation of protein turnover and muscle cell development by stimulating protein synthesis and regulating differentiation of muscle cells. This is the first study to demonstrate a role for an Asb family member in skeletal muscle growth.

  3. Podocan-like protein: a novel small leucine-rich repeat matrix protein in bone.

    PubMed

    Mochida, Yoshiyuki; Kaku, Masaru; Yoshida, Keiko; Katafuchi, Michitsuna; Atsawasuwan, Phimon; Yamauchi, Mitsuo

    2011-07-01

    Recently, significant attention has been drawn to the biology of small leucine-rich repeat proteoglycans (SLRPs) due to their multiple functionalities in various cell types and tissues. Here, we characterize a novel SLRP member, "Podocan-like (Podnl) protein" identified by a bioinformatics approach. The Podnl protein has a signal peptide, a unique cysteine-rich N-terminal cluster, 21 leucine-rich repeat (LRR) motifs, and one putative N-glycosylation site. This protein is structurally similar to podocan in SLRPs. The gene was highly expressed in mineralized tissues and in osteoblastic cells and the high expression level was observed at and after matrix mineralization in vitro. Podnl was enriched in newly formed bones based on immunohistochemical analysis. When Podnl was transfected into osteoblastic cells, the protein with N-glycosylation was detected mainly in the cultured medium, indicating that Podnl is a secreted N-glycosylated protein. The endogenous Podnl protein was also present in bone matrix. These data provide a new insight into our understanding of the emerging SLRP functions in bone formation.

  4. The ankyrin repeat containing SOCS box protein 5: a novel protein associated with arteriogenesis.

    PubMed

    Boengler, Kerstin; Pipp, Frederic; Fernandez, Borja; Richter, Alexandra; Schaper, Wolfgang; Deindl, Elisabeth

    2003-02-28

    Arteriogenesis, the growth of pre-existing collateral arteries, can be induced in rabbit by occlusion of the femoral artery. In order to identify and characterize genes differentially expressed during the early phase of arteriogenesis, cDNA of collateral arteries 24h after femoral ligation or sham operation was subjected to suppression subtractive hybridization. We identified the ankyrin repeat containing SOCS box protein 5 (asb5) and cloned the rabbit full-length cDNA. Asb5 was demonstrated to be a single-copy gene. We localized the asb5 protein in vivo in endothelial and smooth muscle cells of collateral arteries as well as in satellite cells. Asb5 was significantly upregulated in growing collateral arteries on mRNA and protein level. The infusion of doxorubicin in rabbit led to a significant decrease of the asb5 mRNA. In summary, our data show that asb5 is a novel protein implicated in the initiation of arteriogenesis.

  5. Positive and strongly relaxed purifying selection drive the evolution of repeats in proteins

    PubMed Central

    Persi, Erez; Wolf, Yuri I.; Koonin, Eugene V

    2016-01-01

    Protein repeats are considered hotspots of protein evolution, associated with acquisition of new functions and novel phenotypic traits, including disease. Paradoxically, however, repeats are often strongly conserved through long spans of evolution. To resolve this conundrum, it is necessary to directly compare paralogous (horizontal) evolution of repeats within proteins with their orthologous (vertical) evolution through speciation. Here we develop a rigorous methodology to identify highly periodic repeats with significant sequence similarity, for which evolutionary rates and selection (dN/dS) can be estimated, and systematically characterize their evolution. We show that horizontal evolution of repeats is markedly accelerated compared with their divergence from orthologues in closely related species. This observation is universal across the diversity of life forms and implies a biphasic evolutionary regime whereby new copies experience rapid functional divergence under combined effects of strongly relaxed purifying selection and positive selection, followed by fixation and conservation of each individual repeat. PMID:27857066

  6. Modular mutagenesis of human placental ribonuclease inhibitor, a protein with leucine-rich repeats.

    PubMed Central

    Lee, F S; Vallee, B L

    1990-01-01

    Human placental ribonuclease inhibitor (PRI) is a potent protein inhibitor of pancreatic ribonucleases and the homologous blood vessel-inducing protein angiogenin. Although inhibition by PRI occurs with a 1:1 stoichiometry, its primary structure is composed predominantly of seven internal leucine-rich repeats. These internal repeats were systematically deleted either singly or in combination by "modular" mutagenesis. Deletion of repeat units 3 plus 4 or repeat unit 6 results in mutants that both bind to and inhibit ribonuclease A. Therefore, the angiogenin/ribonuclease binding site in PRI must reside primarily or entirely in repeats 1, 2, 5, or 7, the short N- or C-terminal segments, or a combination of these. Deletion of repeat units 3-5, 5-6, or 5 alone results in mutants that exhibit only binding activity. Hence, the binding site cannot reside exclusively in repeat 5. Other internal deletions or N- or C-terminal deletions of 6-86% of the protein all abolish activity. These results suggest that PRI has a modular structure, with one primary structural repeat constituting one module. The approach taken may be applicable to other proteins with repeat structures. Images PMID:2408043

  7. Origin of a folded repeat protein from an intrinsically disordered ancestor.

    PubMed

    Zhu, Hongbo; Sepulveda, Edgardo; Hartmann, Marcus D; Kogenaru, Manjunatha; Ursinus, Astrid; Sulz, Eva; Albrecht, Reinhard; Coles, Murray; Martin, Jörg; Lupas, Andrei N

    2016-09-13

    Repetitive proteins are thought to have arisen through the amplification of subdomain-sized peptides. Many of these originated in a non-repetitive context as cofactors of RNA-based replication and catalysis, and required the RNA to assume their active conformation. In search of the origins of one of the most widespread repeat protein families, the tetratricopeptide repeat (TPR), we identified several potential homologs of its repeated helical hairpin in non-repetitive proteins, including the putatively ancient ribosomal protein S20 (RPS20), which only becomes structured in the context of the ribosome. We evaluated the ability of the RPS20 hairpin to form a TPR fold by amplification and obtained structures identical to natural TPRs for variants with 2-5 point mutations per repeat. The mutations were neutral in the parent organism, suggesting that they could have been sampled in the course of evolution. TPRs could thus have plausibly arisen by amplification from an ancestral helical hairpin.

  8. De-coding and re-coding RNA recognition by PUF and PPR repeat proteins.

    PubMed

    Hall, Traci M Tanaka

    2016-02-01

    PUF and PPR proteins are two families of α-helical repeat proteins that recognize single-stranded RNA sequences. Both protein families hold promise as scaffolds for designed RNA-binding domains. A modular protein RNA recognition code was apparent from the first crystal structures of a PUF protein in complex with RNA, and recent studies continue to advance our understanding of natural PUF protein recognition (de-coding) and our ability to engineer specificity (re-coding). Degenerate recognition motifs make de-coding specificity of individual PPR proteins challenging. Nevertheless, re-coding PPR protein specificity using a consensus recognition code has been successful.

  9. The energy landscape of modular repeat proteins: topology determines folding mechanism in the ankyrin family.

    PubMed

    Ferreiro, Diego U; Cho, Samuel S; Komives, Elizabeth A; Wolynes, Peter G

    2005-12-02

    Proteins consisting of repeating amino acid motifs are abundant in all kingdoms of life, especially in higher eukaryotes. Repeat-containing proteins self-organize into elongated non-globular structures. Do the same general underlying principles that dictate the folding of globular domains apply also to these extended topologies? Using a simplified structure-based model capturing a perfectly funneled energy landscape, we surveyed the predicted mechanism of folding for ankyrin repeat containing proteins. The ankyrin family is one of the most extensively studied classes of non-globular folds. The model based only on native contacts reproduces most of the experimental observations on the folding of these proteins, including a folding mechanism that is reminiscent of a nucleation propagation growth. The confluence of simulation and experimental results suggests that the folding of non-globular proteins is accurately described by a funneled energy landscape, in which topology plays a determinant role in the folding mechanism.

  10. Repeat protein engineering: creating functional nanostructures/biomaterials from modular building blocks.

    PubMed

    Main, Ewan R G; Phillips, Jonathan J; Millership, Charlotte

    2013-10-01

    There is enormous interest in molecular self-assembly and the development of biological systems to form smart nanostructures for biotechnology (so-called 'bottom-up fabrications'). Repeat proteins are ideal choices for development of such systems as they: (i) possess a relatively simple relationship between sequence, structure and function; (ii) are modular and non-globular in structure; (iii) act as diverse scaffolds for the mediation of a diverse range of protein-protein interactions; and (iv) have been extensively studied and successfully engineered and designed. In the present review, we summarize recent advances in the use of engineered repeat proteins in the self-assembly of novel materials, nanostructures and biosensors. In particular, we show that repeat proteins are excellent monomeric programmable building blocks that can be triggered to associate into a range of morphologies and can readily be engineered as stimuli-responsive biofunctional materials.

  11. Differential Occurrence of Interactions and Interaction Domains in Proteins Containing Homopolymeric Amino Acid Repeats

    PubMed Central

    Pelassa, Ilaria; Fiumara, Ferdinando

    2015-01-01

    Homopolymeric amino acids repeats (AARs), which are widespread in proteomes, have often been viewed simply as spacers between protein domains, or even as “junk” sequences with no obvious function but with a potential to cause harm upon expansion as in genetic diseases associated with polyglutamine or polyalanine expansions, including Huntington disease and cleidocranial dysplasia. A growing body of evidence indicates however that at least some AARs can form organized, functional protein structures, and can regulate protein function. In particular, certain AARs can mediate protein-protein interactions, either through homotypic AAR-AAR contacts or through heterotypic contacts with other protein domains. It is still unclear however, whether AARs may have a generalized, proteome-wide role in shaping protein-protein interaction networks. Therefore, we have undertaken here a bioinformatics screening of the human proteome and interactome in search of quantitative evidence of such a role. We first identified the sets of proteins that contain repeats of any one of the 20 amino acids, as well as control sets of proteins chosen at random in the proteome. We then analyzed the connectivity between the proteins of the AAR-containing protein sets and we compared it with that observed in the corresponding control networks. We find evidence for different degrees of connectivity in the different AAR-containing protein networks. Indeed, networks of proteins containing polyglutamine, polyglutamate, polyproline, and other AARs show significantly increased levels of connectivity, whereas networks containing polyleucine and other hydrophobic repeats show lower degrees of connectivity. Furthermore, we observed that numerous protein-protein, -nucleic acid, and -lipid interaction domains are significantly enriched in specific AAR protein groups. These findings support the notion of a generalized, combinatorial role of AARs, together with conventional protein interaction domains, in

  12. Molecular tandem repeat strategy for elucidating mechanical properties of high-strength proteins

    PubMed Central

    Jung, Huihun; Pena-Francesch, Abdon; Saadat, Alham; Sebastian, Aswathy; Kim, Dong Hwan; Hamilton, Reginald F.; Albert, Istvan; Allen, Benjamin D.; Demirel, Melik C.

    2016-01-01

    Many globular and structural proteins have repetitions in their sequences or structures. However, a clear relationship between these repeats and their contribution to the mechanical properties remains elusive. We propose a new approach for the design and production of synthetic polypeptides that comprise one or more tandem copies of a single unit with distinct amorphous and ordered regions. Our designed sequences are based on a structural protein produced in squid suction cups that has a segmented copolymer structure with amorphous and crystalline domains. We produced segmented polypeptides with varying repeat number, while keeping the lengths and compositions of the amorphous and crystalline regions fixed. We showed that mechanical properties of these synthetic proteins could be tuned by modulating their molecular weights. Specifically, the toughness and extensibility of synthetic polypeptides increase as a function of the number of tandem repeats. This result suggests that the repetitions in native squid proteins could have a genetic advantage for increased toughness and flexibility. PMID:27222581

  13. Molecular tandem repeat strategy for elucidating mechanical properties of high-strength proteins.

    PubMed

    Jung, Huihun; Pena-Francesch, Abdon; Saadat, Alham; Sebastian, Aswathy; Kim, Dong Hwan; Hamilton, Reginald F; Albert, Istvan; Allen, Benjamin D; Demirel, Melik C

    2016-06-07

    Many globular and structural proteins have repetitions in their sequences or structures. However, a clear relationship between these repeats and their contribution to the mechanical properties remains elusive. We propose a new approach for the design and production of synthetic polypeptides that comprise one or more tandem copies of a single unit with distinct amorphous and ordered regions. Our designed sequences are based on a structural protein produced in squid suction cups that has a segmented copolymer structure with amorphous and crystalline domains. We produced segmented polypeptides with varying repeat number, while keeping the lengths and compositions of the amorphous and crystalline regions fixed. We showed that mechanical properties of these synthetic proteins could be tuned by modulating their molecular weights. Specifically, the toughness and extensibility of synthetic polypeptides increase as a function of the number of tandem repeats. This result suggests that the repetitions in native squid proteins could have a genetic advantage for increased toughness and flexibility.

  14. Intensification: A Resource for Amplifying Population-Genetic Signals with Protein Repeats.

    PubMed

    Chen, Jieming; Wang, Bo; Regan, Lynne; Gerstein, Mark

    2017-02-03

    Large-scale genome sequencing holds great promise for the interpretation of protein structures through the discovery of many, rare functional variants in the human population. However, because protein-coding regions are under high selective constraints, these variants occur at low frequencies, such that there is often insufficient statistics for downstream calculations. To address this problem, we develop the Intensification approach, which uses the modular structure of repeat protein domains to amplify signals of selection from population genetics and traditional interspecies conservation. In particular, we are able to aggregate variants at the codon level to identify important positions in repeat domains that show strong conservation signals. This allows us to compare conservation over different evolutionary timescales. It also enables us to visualize population-genetic measures on protein structures. We make available the Intensification results as an online resource (http://intensification.gersteinlab.org) and illustrate the approach through a case study on the tetratricopeptide repeat.

  15. Thorns in armadillo ears and noses and their role in the transmission of leprosy.

    PubMed

    Job, C K; Harris, E B; Allen, J L; Hastings, R C

    1986-11-01

    Both ears from 494 wild nine-banded armadillos (Dasypus novemcinctus) and nose specimens from 224 animals were collected and histopathologically studied. Lepromatous granulomas were present in the ear specimens of ten of 494 animals. There were thorns in the ears of 22.5% of animals, and in 36.6% of the nose specimens. In one armadillo, there was evidence to suggest that Mycobacterium leprae entered the tissue through the thorn pricks. In the normal habitat of the armadillo in Louisiana there are thorny bushes consisting mostly of the green briar and the southern dewberry. Thorn pricks as a means of transmission of leprosy in the wild armadillos is suggested.

  16. Structural diversity and nuclear protein binding sites in the long terminal repeats of feline leukemia virus.

    PubMed Central

    Fulton, R; Plumb, M; Shield, L; Neil, J C

    1990-01-01

    The long terminal repeat U3 sequences were determined for multiple feline leukemia virus proviruses isolated from naturally occurring T-cell tumors. Heterogeneity was evident, even among proviruses cloned from individual tumors. Proviruses with one, two, or three repeats of the long terminal repeat enhancer sequences coexisted in one tumor, while two proviruses with distinct direct repeats were found in another. The enhancer repeats are characteristic of retrovirus variants with accelerated leukemogenic potential and occur between -155 and -244 base pairs relative to the RNA cap site. The termini of the repeats occur at or near sequence features which have been recognized at other retrovirus recombinational junctions. In vitro footprint analysis of the feline leukemia virus enhancer revealed three major nuclear protein binding sites, located at consensus sequences for the simian virus 40 core enhancer, the nuclear factor 1 binding site, and an indirect repeat which is homologous to the PEA2 binding site in the polyomavirus enhancer. Only the simian virus 40 core enhancer sequence is present in all of the enhancer repeats. Cell type differences in binding activities to the three motifs may underlie the selective process which leads to outgrowth of viruses with specific sequence duplications. Images PMID:2157050

  17. HHrep: de novo protein repeat detection and the origin of TIM barrels

    PubMed Central

    Söding, Johannes; Remmert, Michael; Biegert, Andreas

    2006-01-01

    HHrep is a web server for the de novo identification of repeats in protein sequences, which is based on the pairwise comparison of profile hidden Markov models (HMMs). Its main strength is its sensitivity, allowing it to detect highly divergent repeat units in protein sequences whose repeats could as yet only be detected from their structures. Examples include sequences with β-propellor fold, ferredoxin-like fold, double psi barrels or (βα)8 (TIM) barrels. We illustrate this with proteins from four superfamilies of TIM barrels by revealing a clear 4- and 8-fold symmetry, which we detect solely from their sequences. This symmetry might be the trace of an ancient origin through duplication of a βαβα or βα unit. HHrep can be accessed at . PMID:16844977

  18. HHrep: de novo protein repeat detection and the origin of TIM barrels.

    PubMed

    Söding, Johannes; Remmert, Michael; Biegert, Andreas

    2006-07-01

    HHrep is a web server for the de novo identification of repeats in protein sequences, which is based on the pairwise comparison of profile hidden Markov models (HMMs). Its main strength is its sensitivity, allowing it to detect highly divergent repeat units in protein sequences whose repeats could as yet only be detected from their structures. Examples include sequences with beta-propellor fold, ferredoxin-like fold, double psi barrels or (betaalpha)8 (TIM) barrels. We illustrate this with proteins from four superfamilies of TIM barrels by revealing a clear 4- and 8-fold symmetry, which we detect solely from their sequences. This symmetry might be the trace of an ancient origin through duplication of a betaalphabetaalpha or betaalpha unit. HHrep can be accessed at http://hhrep.tuebingen.mpg.de.

  19. Malaria parasites possess a telomere repeat-binding protein that shares ancestry with transcription factor IIIA.

    PubMed

    Bertschi, Nicole L; Toenhake, Christa G; Zou, Angela; Niederwieser, Igor; Henderson, Rob; Moes, Suzette; Jenoe, Paul; Parkinson, John; Bartfai, Richard; Voss, Till S

    2017-03-13

    Telomere repeat-binding factors (TRFs) are essential components of the molecular machinery that regulates telomere function. TRFs are widely conserved across eukaryotes and bind duplex telomere repeats via a characteristic MYB-type domain. Here, we identified the telomere repeat-binding protein PfTRZ in the malaria parasite Plasmodium falciparum, a member of the Alveolate phylum for which TRFs have not been described so far. PfTRZ lacks an MYB domain and binds telomere repeats via a C2H2-type zinc finger domain instead. In vivo, PfTRZ binds with high specificity to the telomeric tract and to interstitial telomere repeats upstream of subtelomeric virulence genes. Conditional depletion experiments revealed that PfTRZ regulates telomere length homeostasis and is required for efficient cell cycle progression. Intriguingly, we found that PfTRZ also binds to and regulates the expression of 5S rDNA genes. Combined with detailed phylogenetic analyses, our findings identified PfTRZ as a remote functional homologue of the basic transcription factor TFIIIA, which acquired a new function in telomere maintenance early in the apicomplexan lineage. Our work sheds unexpected new light on the evolution of telomere repeat-binding proteins and paves the way for dissecting the presumably divergent mechanisms regulating telomere functionality in one of the most deadly human pathogens.

  20. Myotonin protein-kinase [AGC]n trinucleotide repeat in seven nonhuman primates

    SciTech Connect

    Novelli, G.; Sineo, L.; Pontieri, E. ||

    1994-09-01

    Myotonic dystrophy (DM) is due to a genomic instability of a trinucleotide [AGC]n motif, located at the 3{prime} UTR region of a protein-kinase gene (myotonin protein kinase, MT-PK). The [AGC] repeat is meiotically and mitotically unstable, and it is directly related to the manifestations of the disorder. Although a gene dosage effect of the MT-PK has been demonstrated n DM muscle, the mechanism(s) by which the intragenic repeat expansion leads to disease is largely unknown. This non-standard mutational event could reflect an evolutionary mechanism widespread among animal genomes. We have isolated and sequenced the complete 3{prime}UTR region of the MT-PK gene in seven primates (macaque, orangutan, gorilla, chimpanzee, gibbon, owl monkey, saimiri), and examined by comparative sequence nucleotide analysis the [AGC]n intragenic repeat and the surrounding nucleotides. The genomic organization, including the [AGC]n repeat structure, was conserved in all examined species, excluding the gibbon (Hylobates agilis), in which the [AGC]n upstream sequence (GGAA) is replaced by a GA dinucleotide. The number of [AGC]n in the examined species ranged between 7 (gorilla) and 13 repeats (owl monkeys), with a polymorphism informative content (PIC) similar to that observed in humans. These results indicate that the 3{prime}UTR [AGC] repeat within the MT-PK gene is evolutionarily conserved, supporting that this region has important regulatory functions.

  1. Phase variable DNA repeats in Neisseria gonorrhoeae influence transcription, translation, and protein sequence variation

    PubMed Central

    Zelewska, Marta A.; Pulijala, Madhuri; Spencer-Smith, Russell; Mahmood, Hiba-Tun-Noor A.; Norman, Billie; Churchward, Colin P.; Calder, Alan

    2016-01-01

    There are many types of repeated DNA sequences in the genomes of the species of the genus Neisseria, from homopolymeric tracts to tandem repeats of hundreds of bases. Some of these have roles in the phase-variable expression of genes. When a repeat mediates phase variation, reversible switching between tract lengths occurs, which in the species of the genus Neisseria most often causes the gene to switch between on and off states through frame shifting of the open reading frame. Changes in repeat tract lengths may also influence the strength of transcription from a promoter. For phenotypes that can be readily observed, such as expression of the surface-expressed Opa proteins or pili, verification that repeats are mediating phase variation is relatively straightforward. For other genes, particularly those where the function has not been identified, gathering evidence of repeat tract changes can be more difficult. Here we present analysis of the repetitive sequences that could mediate phase variation in the Neisseria gonorrhoeae strain NCCP11945 genome sequence and compare these results with other gonococcal genome sequences. Evidence is presented for an updated phase-variable gene repertoire in this species, including a class of phase variation that causes amino acid changes at the C-terminus of the protein, not previously described in N. gonorrhoeae. PMID:28348872

  2. DNA-protein interactions at the telomeric repeats of Schizosaccharomyces pombe.

    PubMed Central

    Duffy, M; Chambers, A

    1996-01-01

    Gel retardation assays using a probe containing the repeat region of a Schizosaccharomyces pombe chromosomal telomere identified four specific DNA- protein complexes in S. pombe total protein extracts (I, I', IIa and IIb). The proteins responsible for these complexes bound to the telomeric repeat region irrespective of whether or not the repeats were in close proximity to the end of a DNA molecule, and none of them bound strongly to single-stranded DNA. The protein responsible for complex I (TeRF I) was separated from the activity responsible for complexes IIa and IIb (TeRF II) using heparin-Sepharose chromatography. Both factors were efficiently cross-competed by an oligonucleotide containing the 18 bp sequence 5'-GGTTACAGGTTACAGGTT-3', which corresponds to two complete telomeric repeat units. Mutation of the T residues at positions 4 and 11 in the oligonucleotide dramatically reduced binding by TeRF II, but had no affect on binding by TeRF I. The protein responsible for complex I' did not bind strongly to either the wild-type or mutant oligonucleotide. PMID:8628672

  3. Primary structure of streptococcal Pep M5 protein: Absence of extensive sequence repeats

    PubMed Central

    Manjula, Belur N.; Mische, Sheenah M.; Fischetti, Vincent A.

    1983-01-01

    Extensive sequence repeats have been observed in a biologically active fragment of type 24 streptococcal M protein, namely Pep M24 [Beachey, E. H., Sayer, J. M. & Kang, A. H. (1978) Proc. Natl. Acad. Sci. USA 75, 3163-3167]. To determine whether such extensive repetition in sequence is a common characteristic of the antiphagocytic streptococcal M proteins, we have determined the sequences of the clostripain peptides of Pep M5, a biologically active fragment of the type 5 M protein that is analogous to Pep M24. These sequences, together with the amino-terminal sequence of the whole molecule, accounted for nearly two thirds of the Pep M5 molecule. However, extensive identical repeats of the kind observed in Pep M24 were not present in Pep M5. Preliminary study of the amino acid sequence analysis of the M protein from type 6 Streptococcus has also indicated the absence of sequence repeats within the regions of this molecule examined so far. These results suggest that extensive sequence repeats may not be a common characteristic of M-protein molecules. On the other hand, the seven-residue periodicity of the nonpolar residues, a characteristic of α-helical coiled-coil structures, appeared to extend over most of the Pep M5 molecule. This feature has been observed previously for the partial sequences of three M protein serotypes. Thus, the important element of the M-protein structure appears to be the seven-residue periodicity necessary for the maintenance of the coiled-coil structure rather than extensive identical amino acid sequence repeats. PMID:16593365

  4. RCPdb: An evolutionary classification and codon usage database for repeat-containing proteins

    PubMed Central

    Faux, Noel G.; Huttley, Gavin A.; Mahmood, Khalid; Webb, Geoffrey I.; Garcia de la Banda, Maria; Whisstock, James C.

    2007-01-01

    Over 3% of human proteins contain single amino acid repeats (repeat-containing proteins, RCPs). Many repeats (homopeptides) localize to important proteins involved in transcription, and the expansion of certain repeats, in particular poly-Q and poly-A tracts, can also lead to the development of neurological diseases. Previous studies have suggested that the homopeptide makeup is a result of the presence of G+C-rich tracts in the encoding genes and that expansion occurs via replication slippage. Here, we have performed a large-scale genomic analysis of the variation of the genes encoding RCPs in 13 species and present these data in an online database (http://repeats.med.monash.edu.au/genetic_analysis/). This resource allows rapid comparison and analysis of RCPs, homopeptides, and their underlying genetic tracts across the eukaryotic species considered. We report three major findings. First, there is a bias for a small subset of codons being reiterated within homopeptides, and there is no G+C or A+T bias relative to the organism’s transcriptome. Second, single base pair transversions from the homocodon are unusually common and may represent a mechanism of reducing the rate of homopeptide mutations. Third, homopeptides that are conserved across different species lie within regions that are under stronger purifying selection in contrast to nonconserved homopeptides. PMID:17567984

  5. Chlorovirus Skp1-Binding Ankyrin Repeat Protein Interplay and Mimicry of Cellular Ubiquitin Ligase Machinery

    PubMed Central

    Noel, Eric A.; Kang, Ming; Adamec, Jiri; Oyler, George A.

    2014-01-01

    ABSTRACT The ubiquitin-proteasome system is targeted by many viruses that have evolved strategies to redirect host ubiquitination machinery. Members of the genus Chlorovirus are proposed to share an ancestral lineage with a broader group of related viruses, nucleo-cytoplasmic large DNA viruses (NCLDV). Chloroviruses encode an Skp1 homolog and ankyrin repeat (ANK) proteins. Several chlorovirus-encoded ANK repeats contain C-terminal domains characteristic of cellular F-boxes or related NCLDV chordopox PRANC (pox protein repeats of ankyrin at C-terminal) domains. These observations suggested that this unique combination of Skp1 and ANK repeat proteins might form complexes analogous to the cellular Skp1-Cul1-F-box (SCF) ubiquitin ligase complex. We identified two ANK proteins from the prototypic chlorovirus Paramecium bursaria chlorella virus-1 (PBCV-1) that functioned as binding partners for the virus-encoded Skp1, proteins A682L and A607R. These ANK proteins had a C-terminal Skp1 interactional motif that functioned similarly to cellular F-box domains. A C-terminal motif of ANK protein A682L binds Skp1 proteins from widely divergent species. Yeast two-hybrid analyses using serial domain deletion constructs confirmed the C-terminal localization of the Skp1 interactional motif in PBCV-1 A682L. ANK protein A607R represents an ANK family with one member present in all 41 sequenced chloroviruses. A comprehensive phylogenetic analysis of these related ANK and viral Skp1 proteins suggested partnered function tailored to the host alga or common ancestral heritage. Here, we show protein-protein interaction between corresponding family clusters of virus-encoded ANK and Skp1 proteins from three chlorovirus types. Collectively, our results indicate that chloroviruses have evolved complementing Skp1 and ANK proteins that mimic cellular SCF-associated proteins. IMPORTANCE Viruses have evolved ways to direct ubiquitination events in order to create environments conducive to their

  6. Structural and functional discussion of the tetra-trico-peptide repeat, a protein interaction module.

    PubMed

    Zeytuni, Natalie; Zarivach, Raz

    2012-03-07

    Tetra-trico-peptide repeat (TPR) domains are found in numerous proteins, where they serve as interaction modules and multiprotein complex mediators. TPRs can be found in all kingdoms of life and regulate diverse biological processes, such as organelle targeting and protein import, vesicle fusion, and biomineralization. This review considers the structural features of TPR domains that permit the great ligand-binding diversity of this motif, given that TPR-interacting partners display variations in both sequence and secondary structure. In addition, tools for predicting TPR-interacting partners are discussed, as are the abilities of TPR domains to serve as protein-protein interaction scaffolds in biotechnology and therapeutics.

  7. Evidence that Armadillo transduces wingless by mediating nuclear export or cytosolic activation of Pangolin.

    PubMed

    Chan, Siu-Kwong; Struhl, Gary

    2002-10-18

    Secreted proteins of the Wnt family have profound organizing roles during animal development and are transduced via the activities of the Frizzled (Fz) class of transmembrane receptors and the TCF/LEF/Pangolin class of transcription factors. beta-catenins, including Drosophila Armadillo (Arm), link activation of Fz at the cell surface to transcriptional regulation by TCF in the nucleus. The consensus view is that Wnt signaling induces beta-catenin to enter the nucleus and combine with TCF to form a transcription factor complex in which TCF binds DNA and the C-terminal domain of beta-catenin activates transcription. Here, we present findings, which challenge this view and suggest instead that beta-catenin may transduce Wnt signals by exporting TCF from the nucleus or activating it in the cytoplasm.

  8. Identification of an attractant for the nine-banded armadillo, dasypus novemcinctus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The nine-banded armadillo, Dasypus novemcinctus, is considered by many to be one of the greatest nuisance wildlife species in the Southeastern U.S. Exclusion is laborious because armadillos are adept at both burrowing and climbing, no repellents, toxicants, or fumigants are currently registered for...

  9. A protective protein antigen of Rickettsia rickettsii has tandemly repeated, near-identical sequences.

    PubMed Central

    Anderson, B E; McDonald, G A; Jones, D C; Regnery, R L

    1990-01-01

    The nucleotide sequence of a Rickettsia rickettsii gene that encodes a high-molecular-mass surface antigen (190 kilodaltons), which elicits protective immunity, was determined. The 6,747-nucleotide gene coded for a 2,249-amino-acid protein with a calculated molecular weight of 224,321. A 3.8-kilobase PstI fragment proximal to the 5' end of the gene was found to consist of 13 highly related tandem repeats which constituted over 40% of the coding region. The repeated sequences could be divided into either a 225-nucleotide, 75-amino-acid unit (type I) or a 216-nucleotide, 72-amino-acid unit (type II), with extensive homology between the two types of repeating units. The deduced amino acid sequence for these repeat units, overall, was slightly hydrophobic with short hydrophilic domains. The carboxy-terminal (nonrepetitive) portion of the deduced protein sequence was hydrophilic, with potential surface-exposed epitopes. The full-length reading frame was reconstructed in Escherichia coli, and transient expression of the 190-kilodalton antigen was demonstrated; however, the protein appeared to be severely degraded by proteases and was apparently toxic to E. coli. The conservation of this unique repetitive gene structure, coupled with results from previous reports showing the protective properties of the 190-kilodalton antigen, suggests that this protein plays an important role in the pathogenesis of and immunity to Rocky Mountain spotted fever. Images PMID:2117568

  10. Origin of a folded repeat protein from an intrinsically disordered ancestor

    PubMed Central

    Zhu, Hongbo; Sepulveda, Edgardo; Hartmann, Marcus D; Kogenaru, Manjunatha; Ursinus, Astrid; Sulz, Eva; Albrecht, Reinhard; Coles, Murray; Martin, Jörg; Lupas, Andrei N

    2016-01-01

    Repetitive proteins are thought to have arisen through the amplification of subdomain-sized peptides. Many of these originated in a non-repetitive context as cofactors of RNA-based replication and catalysis, and required the RNA to assume their active conformation. In search of the origins of one of the most widespread repeat protein families, the tetratricopeptide repeat (TPR), we identified several potential homologs of its repeated helical hairpin in non-repetitive proteins, including the putatively ancient ribosomal protein S20 (RPS20), which only becomes structured in the context of the ribosome. We evaluated the ability of the RPS20 hairpin to form a TPR fold by amplification and obtained structures identical to natural TPRs for variants with 2–5 point mutations per repeat. The mutations were neutral in the parent organism, suggesting that they could have been sampled in the course of evolution. TPRs could thus have plausibly arisen by amplification from an ancestral helical hairpin. DOI: http://dx.doi.org/10.7554/eLife.16761.001 PMID:27623012

  11. The impact of CRISPR repeat sequence on structures of a Cas6 protein-RNA complex

    SciTech Connect

    Wang, Ruiying; Zheng, Han; Preamplume, Gan; Shao, Yaming; Li, Hong

    2012-03-15

    The repeat-associated mysterious proteins (RAMPs) comprise the most abundant family of proteins involved in prokaryotic immunity against invading genetic elements conferred by the clustered regularly interspaced short palindromic repeat (CRISPR) system. Cas6 is one of the first characterized RAMP proteins and is a key enzyme required for CRISPR RNA maturation. Despite a strong structural homology with other RAMP proteins that bind hairpin RNA, Cas6 distinctly recognizes single-stranded RNA. Previous structural and biochemical studies show that Cas6 captures the 5' end while cleaving the 3' end of the CRISPR RNA. Here, we describe three structures and complementary biochemical analysis of a noncatalytic Cas6 homolog from Pyrococcus horikoshii bound to CRISPR repeat RNA of different sequences. Our study confirms the specificity of the Cas6 protein for single-stranded RNA and further reveals the importance of the bases at Positions 5-7 in Cas6-RNA interactions. Substitutions of these bases result in structural changes in the protein-RNA complex including its oligomerization state.

  12. XSTREAM: A practical algorithm for identification and architecture modeling of tandem repeats in protein sequences

    PubMed Central

    Newman, Aaron M; Cooper, James B

    2007-01-01

    Background Biological sequence repeats arranged in tandem patterns are widespread in DNA and proteins. While many software tools have been designed to detect DNA tandem repeats (TRs), useful algorithms for identifying protein TRs with varied levels of degeneracy are still needed. Results To address limitations of current repeat identification methods, and to provide an efficient and flexible algorithm for the detection and analysis of TRs in protein sequences, we designed and implemented a new computational method called XSTREAM. Running time tests confirm the practicality of XSTREAM for analyses of multi-genome datasets. Each of the key capabilities of XSTREAM (e.g., merging, nesting, long-period detection, and TR architecture modeling) are demonstrated using anecdotal examples, and the utility of XSTREAM for identifying TR proteins was validated using data from a recently published paper. Conclusion We show that XSTREAM is a practical and valuable tool for TR detection in protein and nucleotide sequences at the multi-genome scale, and an effective tool for modeling TR domains with diverse architectures and varied levels of degeneracy. Because of these useful features, XSTREAM has significant potential for the discovery of naturally-evolved modular proteins with applications for engineering novel biostructural and biomimetic materials, and identifying new vaccine and diagnostic targets. PMID:17931424

  13. Nanoparticles Self-Assembly Driven by High Affinity Repeat Protein Pairing.

    PubMed

    Gurunatha, Kargal L; Fournier, Agathe C; Urvoas, Agathe; Valerio-Lepiniec, Marie; Marchi, Valérie; Minard, Philippe; Dujardin, Erik

    2016-03-22

    Proteins are the most specific yet versatile biological self-assembling agents with a rich chemistry. Nevertheless, the design of new proteins with recognition capacities is still in its infancy and has seldom been exploited for the self-assembly of functional inorganic nanoparticles. Here, we report on the protein-directed assembly of gold nanoparticles using purpose-designed artificial repeat proteins having a rigid but modular 3D architecture. αRep protein pairs are selected for their high mutual affinity from a library of 10(9) variants. Their conjugation onto gold nanoparticles drives the massive colloidal assembly of free-standing, one-particle thick films. When the average number of proteins per nanoparticle is lowered, the extent of self-assembly is limited to oligomeric particle clusters. Finally, we demonstrate that the aggregates are reversibly disassembled by an excess of one free protein. Our approach could be optimized for applications in biosensing, cell targeting, or functional nanomaterials engineering.

  14. The armadillo as an animal model and reservoir host for Mycobacterium leprae.

    PubMed

    Balamayooran, Gayathriy; Pena, Maria; Sharma, Rahul; Truman, Richard W

    2015-01-01

    Apart from humans, armadillos are the only known natural hosts of Mycobacterium leprae. They are well developed as hosts for in vivo propagation of M leprae and are advancing as models for studying the pathogenesis of leprosy and translational research. Armadillos are immunologically intact. They exhibit the full Ridley-Jopling spectrum of histopathologic responses to M leprae and uniquely manifest extensive neurological involvement that closely recapitulates human leprosy. In addition, free-ranging armadillos in some regions are known to harbor a naturally occurring infection with M leprae, and zoonotic transmission between armadillos and humans has been implicated in a large number of new case presentations. We review the role of the armadillo as a model for leprosy and reservoir for human infection.

  15. Tianeptine modulates amygdalar glutamate neurochemistry and synaptic proteins in rats subjected to repeated stress.

    PubMed

    Piroli, Gerardo G; Reznikov, Leah R; Grillo, Claudia A; Hagar, Janel M; Fadel, Jim R; Reagan, Lawrence P

    2013-03-01

    Stress is a common environmental factor associated with depressive illness and the amygdala is thought to be integral for this association. For example, repeated stress impairs amygdalar neuroplasticity in rodents and these defects parallel amygdalar deficits in depressive illness patients. Because the excitatory neurotransmitter glutamate is important in neuroplasticity, we hypothesized that alterations in amygdalar glutamatergic systems may serve as key players in depressive illness. Moreover, restoration of amygdalar glutamatergic systems may serve as important therapeutic targets in the successful management of multiple stress-related mood disorders. To address these hypotheses, we measured glutamate efflux in the basolateral and central amygdalar complexes via in vivo microdialysis, as well as the expression of synaptic proteins that regulate vesicular glutamate packaging and release, in rats subjected to repeated stress and treated daily with saline or the antidepressant tianeptine. Glutamate efflux was significantly reduced in the central amygdalar complex of animals subjected to repeated stress. In addition, repeated stress nearly eliminated amygdalar vGLUT2 expression, thereby proving a potential mechanism through which repeated stress impairs amygdalar glutamate neurochemistry. These stress-induced changes in glutamate efflux and vGLUT2 expression were inhibited by daily tianeptine administration. Moreover, tianeptine administration increased the vesicular localization of SNAP-25, which could account for the ability of tianeptine to modify glutamatergic tone in non-stressed control rats. Collectively, these results demonstrate that repeated stress differentially affects amygdalar glutamate systems and further supports our previous studies indicating that tianeptine's antidepressant efficacy may involve targeting amygdalar glutatamatergic systems.

  16. Structure Function Studies of Vaccinia Virus Host Range Protein K1 Reveal a Novel Functional Surface for Ankyrin Repeat Proteins

    SciTech Connect

    Li, Yongchao; Meng, Xiangzhi; Xiang, Yan; Deng, Junpeng

    2010-06-15

    Poxvirus host tropism at the cellular level is regulated by virus-encoded host range proteins acting downstream of virus entry. The functioning mechanisms of most host range proteins are unclear, but many contain multiple ankyrin (ANK) repeats, a motif that is known for ligand interaction through a concave surface. We report here the crystal structure of one of the ANK repeat-containing host range proteins, the vaccinia virus K1 protein. The structure, at a resolution of 2.3 {angstrom}, showed that K1 consists entirely of ANK repeats, including seven complete ones and two incomplete ones, one each at the N and C terminus. Interestingly, Phe82 and Ser83, which were previously shown to be critical for K1's function, are solvent exposed and located on a convex surface, opposite the consensus ANK interaction surface. The importance of this convex surface was further supported by our additional mutagenesis studies. We found that K1's host range function was negatively affected by substitution of either Asn51 or Cys47 and completely abolished by substitution of both residues. Cys47 and Asn51 are also exposed on the convex surface, spatially adjacent to Phe82 and Ser83. Altogether, our data showed that K1 residues on a continuous convex ANK repeat surface are critical for the host range function, suggesting that K1 functions through ligand interaction and does so with a novel ANK interaction surface.

  17. Fragile X mental retardation protein regulates synaptic and behavioral plasticity to repeated cocaine administration.

    PubMed

    Smith, Laura N; Jedynak, Jakub P; Fontenot, Miles R; Hale, Carly F; Dietz, Karen C; Taniguchi, Makoto; Thomas, Feba S; Zirlin, Benjamin C; Birnbaum, Shari G; Huber, Kimberly M; Thomas, Mark J; Cowan, Christopher W

    2014-05-07

    Repeated cocaine exposure causes persistent, maladaptive alterations in brain and behavior, and hope for effective therapeutics lies in understanding these processes. We describe here an essential role for fragile X mental retardation protein (FMRP), an RNA-binding protein and regulator of dendritic protein synthesis, in cocaine conditioned place preference, behavioral sensitization, and motor stereotypy. Cocaine reward deficits in FMRP-deficient mice stem from elevated mGluR5 (or GRM5) function, similar to a subset of fragile X symptoms, and do not extend to natural reward. We find that FMRP functions in the adult nucleus accumbens (NAc), a critical addiction-related brain region, to mediate behavioral sensitization but not cocaine reward. FMRP-deficient mice also exhibit several abnormalities in NAc medium spiny neurons, including reduced presynaptic function and premature changes in dendritic morphology and glutamatergic neurotransmission following repeated cocaine treatment. Together, our findings reveal FMRP as a critical mediator of cocaine-induced behavioral and synaptic plasticity.

  18. Trinucleotide repeats and protein folding and disease: the perspective from studies with the androgen receptor

    PubMed Central

    Orafidiya, Folake A; McEwan, Iain J

    2015-01-01

    The androgen receptor (AR), a ligand activated transcription factor plays a number of roles in reproduction, homeostasis and pathogenesis of disease. It has two major polymorphic sequences; a polyglutamine and a polyglycine repeat that determine the length of the protein and influence receptor folding, structure and function. Here, we review the role the folding of the AR plays in the pathogenesis of spinal-bulbar muscular atrophy (SBMA), a neuromuscular degenerative disease arising from expansion of the polyglutamine repeat. We discuss current management for SBMA patients and how research on AR structure function may lead to future drug treatments. PMID:28031874

  19. Control of Neural Circuit Formation by Leucine-Rich Repeat Proteins

    PubMed Central

    de Wit, Joris; Ghosh, Anirvan

    2014-01-01

    The function of neural circuits depends on the precise connectivity between populations of neurons. Increasing evidence indicates that disruptions in excitatory or inhibitory synapse formation or function lead to excitation/inhibition (E/I) imbalances and contribute to neurodevelopmental and psychiatric disorders. Leucine-rich repeat (LRR)-containing surface proteins have emerged as key organizers of excitatory and inhibitory synapses. Distinct LRR proteins are expressed in different cell types and interact with key pre- and postsynaptic proteins. These protein interaction networks allow LRR proteins to coordinate pre- and postsynaptic elements during synapse formation and differentiation, pathway-specific synapse development, and synaptic plasticity. LRR proteins thus play a critical role in organizing synaptic connections into functional neural circuits, and their dysfunction may contribute to neuropsychiatric disorders. PMID:25131359

  20. Designed ankyrin repeat proteins: a novel tool for testing epidermal growth factor receptor 2 expression in breast cancer.

    PubMed

    Theurillat, Jean-Philippe; Dreier, Birgit; Nagy-Davidescu, Gabriela; Seifert, Burkhardt; Behnke, Silvia; Zürrer-Härdi, Ursina; Ingold, Fabienne; Plückthun, Andreas; Moch, Holger

    2010-09-01

    Designed ankyrin repeat proteins are a novel class of specific binding molecules, which display increased thermodynamic stability, smaller size and at least equal target affinity compared to immunoglobulins, making them potentially powerful tools in diagnostic pathology and therapeutic oncology. Here, we investigated whether designed ankyrin repeat proteins can reliably identify the amplification status of the epidermal growth factor receptor 2 in breast cancer. Designed ankyrin repeat proteins specific for epidermal growth factor receptor 2 were tested in paraffin-embedded tissue sections. Detection using enzymatic biotinylation proved to be most specific and sensitive. The affinity of the designed ankyrin repeat proteins was found crucial, but for a picomolar binder no further gain was found by making it multivalent. The best designed ankyrin repeat protein, G3 (K(D) 90 pM) was compared on breast cancer tissue microarrays (n=792) to an FDA-approved rabbit monoclonal antibody against epidermal growth factor receptor 2 (clone 4B5; Ventana Medical Systems) and correlated with corresponding epidermal growth factor receptor 2 amplification status measured by fluorescent in situ hybridization. Amplification status and epidermal growth factor receptor 2 expression measured by designed ankyrin repeat protein and antibody correlated strongly with each other (P<0.0001 each), the correlation between designed ankyrin repeat protein and amplification status being the strongest (0.87 compared to 0.77 for the antibody, Kendall's tau-beta). Using a modified scoring system for the designed ankyrin repeat protein, we show that the designed ankyrin repeat protein detects a positive epidermal growth factor receptor 2 amplification status with similar sensitivity and significantly higher specificity than the antibody (P=0.0005). This study suggests that designed ankyrin repeat proteins provide a valuable alternative to antibodies for the detection of epidermal growth factor receptor

  1. Inhibition of eukaryotic translation by tetratricopeptide-repeat proteins of Orientia tsutsugamushi.

    PubMed

    Bang, Sunyoung; Min, Chan-Ki; Ha, Na-Young; Choi, Myung-Sik; Kim, Ik-Sang; Kim, Yeon-Sook; Cho, Nam-Hyuk

    2016-02-01

    Orientia tsutsugamushi, an obligate intracellular bacterium, is the causative agent of scrub typhus. The genome of Orientia tsutsugamushi has revealed multiple ORFs encoding tetratricopeptide-repeat (TPR) proteins. The TPR protein family has been shown to be involved in a diverse spectrum of cellular functions such as cell cycle control, transcription, protein transport, and protein folding, especially in eukaryotic cells. However, little is known about the function of the TPR proteins in O. tsutsugamushi. To investigate the potential role of TPR proteins in host-pathogen interaction, two oriential TPR proteins were expressed in E. coli and applied for GSTpull down assay. DDX3, a DEAD-box containing RNA helicase, was identified as a specific eukaryotic target of the TPR proteins. Since the RNA helicase is involved in multiple RNA-modifying processes such as initiation of translation reaction, we performed in vitro translation assay in the presence of GST-TPR fusion proteins by using rabbit reticulocyte lysate system. The TPR proteins inhibited in vitro translation of a reporter luciferase in a dose dependent manner whereas the GST control proteins did not. These results suggested TPR proteins of O. tsutsugamushi might be involved in the modulation of eukaryotic translation through the interaction with DDX3 RNA helicase after secretion into host cytoplasm.

  2. Induction of homologous recombination between sequence repeats by the activation induced cytidine deaminase (AID) protein.

    PubMed

    Buerstedde, Jean-Marie; Lowndes, Noel; Schatz, David G

    2014-07-08

    The activation induced cytidine deaminase (AID) protein is known to initiate somatic hypermutation, gene conversion or switch recombination by cytidine deamination within the immunoglobulin loci. Using chromosomally integrated fluorescence reporter transgenes, we demonstrate a new recombinogenic activity of AID leading to intra- and intergenic deletions via homologous recombination of sequence repeats. Repeat recombination occurs at high frequencies even when the homologous sequences are hundreds of bases away from the positions of AID-mediated cytidine deamination, suggesting DNA end resection before strand invasion. Analysis of recombinants between homeologous repeats yielded evidence for heteroduplex formation and preferential migration of the Holliday junctions to the boundaries of sequence homology. These findings broaden the target and off-target mutagenic potential of AID and establish a novel system to study induced homologous recombination in vertebrate cells.DOI: http://dx.doi.org/10.7554/eLife.03110.001.

  3. Telomere repeat binding proteins are functional components of Arabidopsis telomeres and interact with telomerase

    PubMed Central

    Procházková Schrumpfová, Petra; Vychodilová, Ivona; Dvořáčková, Martina; Majerská, Jana; Dokládal, Ladislav; Schořová, Šárka; Fajkus, Jiří

    2014-01-01

    Although telomere-binding proteins constitute an essential part of telomeres, in vivo data indicating the existence of a structure similar to mammalian shelterin complex in plants are limited. Partial characterization of a number of candidate proteins has not identified true components of plant shelterin or elucidated their functional mechanisms. Telomere repeat binding (TRB) proteins from Arabidopsis thaliana bind plant telomeric repeats through a Myb domain of the telobox type in vitro, and have been shown to interact with POT1b (Protection of telomeres 1). Here we demonstrate co-localization of TRB1 protein with telomeres in situ using fluorescence microscopy, as well as in vivo interaction using chromatin immunoprecipitation. Classification of the TRB1 protein as a component of plant telomeres is further confirmed by the observation of shortening of telomeres in knockout mutants of the trb1 gene. Moreover, TRB proteins physically interact with plant telomerase catalytic subunits. These findings integrate TRB proteins into the telomeric interactome of A. thaliana. PMID:24397874

  4. Structural basis for specific single-stranded RNA recognition by designer pentatricopeptide repeat proteins

    PubMed Central

    Shen, Cuicui; Zhang, Delin; Guan, Zeyuan; Liu, Yexing; Yang, Zhao; Yang, Yan; Wang, Xiang; Wang, Qiang; Zhang, QunXia; Fan, Shilong; Zou, Tingting; Yin, Ping

    2016-01-01

    As a large family of RNA-binding proteins, pentatricopeptide repeat (PPR) proteins mediate multiple aspects of RNA metabolism in eukaryotes. Binding to their target single-stranded RNAs (ssRNAs) in a modular and base-specific fashion, PPR proteins can serve as designable modules for gene manipulation. However, the structural basis for nucleotide-specific recognition by designer PPR (dPPR) proteins remains to be elucidated. Here, we report four crystal structures of dPPR proteins in complex with their respective ssRNA targets. The dPPR repeats are assembled into a right-handed superhelical spiral shell that embraces the ssRNA. Interactions between different PPR codes and RNA bases are observed at the atomic level, revealing the molecular basis for the modular and specific recognition patterns of the RNA bases U, C, A and G. These structures not only provide insights into the functional study of PPR proteins but also open a path towards the potential design of synthetic sequence-specific RNA-binding proteins. PMID:27088764

  5. RNA-binding specificity landscape of the pentatricopeptide repeat protein PPR10.

    PubMed

    Miranda, Rafael G; Rojas, Margarita; Montgomery, Michael P; Gribbin, Kyle P; Barkan, Alice

    2017-04-01

    Pentatricopeptide repeat (PPR) proteins comprise a large family of helical repeat proteins that influence gene expression in mitochondria and chloroplasts. PPR tracts can bind RNA via a modular one repeat-one nucleotide mechanism in which the nucleotide is specified by the identities of several amino acids in each repeat. This mode of recognition, the so-called PPR code, offers opportunities for the prediction of native PPR binding sites and the design of proteins to bind specified RNAs. However, a deep understanding of the parameters that dictate the affinity and specificity of PPR-RNA interactions is necessary to realize these goals. We report a comprehensive analysis of the sequence specificity of PPR10, a protein that binds similar RNA sequences of ∼18 nucleotides (nt) near the chloroplast atpH and psaJ genes in maize. We assessed the contribution of each nucleotide in the atpH binding site to PPR10 affinity in vitro by analyzing the effects of single-nucleotide changes at each position. In a complementary approach, the RNAs bound by PPR10 from partially randomized RNA pools were analyzed by deep sequencing. The results revealed three patches in which nucleotide identity has a major impact on binding affinity. These include 5 nt for which protein contacts were not observed in a PPR10-RNA crystal structure and 4 nt that are not explained by current views of the PPR code. These findings highlight aspects of PPR-RNA interactions that pose challenges for binding site prediction and design.

  6. DNA-binding proteins from marine bacteria expand the known sequence diversity of TALE-like repeats

    PubMed Central

    de Lange, Orlando; Wolf, Christina; Thiel, Philipp; Krüger, Jens; Kleusch, Christian; Kohlbacher, Oliver; Lahaye, Thomas

    2015-01-01

    Transcription Activator-Like Effectors (TALEs) of Xanthomonas bacteria are programmable DNA binding proteins with unprecedented target specificity. Comparative studies into TALE repeat structure and function are hindered by the limited sequence variation among TALE repeats. More sequence-diverse TALE-like proteins are known from Ralstonia solanacearum (RipTALs) and Burkholderia rhizoxinica (Bats), but RipTAL and Bat repeats are conserved with those of TALEs around the DNA-binding residue. We study two novel marine-organism TALE-like proteins (MOrTL1 and MOrTL2), the first to date of non-terrestrial origin. We have assessed their DNA-binding properties and modelled repeat structures. We found that repeats from these proteins mediate sequence specific DNA binding conforming to the TALE code, despite low sequence similarity to TALE repeats, and with novel residues around the BSR. However, MOrTL1 repeats show greater sequence discriminating power than MOrTL2 repeats. Sequence alignments show that there are only three residues conserved between repeats of all TALE-like proteins including the two new additions. This conserved motif could prove useful as an identifier for future TALE-likes. Additionally, comparing MOrTL repeats with those of other TALE-likes suggests a common evolutionary origin for the TALEs, RipTALs and Bats. PMID:26481363

  7. Translation of CGA codon repeats in yeast involves quality control components and ribosomal protein L1.

    PubMed

    Letzring, Daniel P; Wolf, Andrew S; Brule, Christina E; Grayhack, Elizabeth J

    2013-09-01

    Translation of CGA codon repeats in the yeast Saccharomyces cerevisiae is inefficient, resulting in dose-dependent reduction in expression and in production of an mRNA cleavage product, indicative of a stalled ribosome. Here, we use genetics and translation inhibitors to understand how ribosomes respond to CGA repeats. We find that CGA codon repeats result in a truncated polypeptide that is targeted for degradation by Ltn1, an E3 ubiquitin ligase involved in nonstop decay, although deletion of LTN1 does not improve expression downstream from CGA repeats. Expression downstream from CGA codons at residue 318, but not at residue 4, is improved by deletion of either ASC1 or HEL2, previously implicated in inhibition of translation by polybasic sequences. Thus, translation of CGA repeats likely causes ribosomes to stall and exploits known quality control systems. Expression downstream from CGA repeats at amino acid 4 is improved by paromomycin, an aminoglycoside that relaxes decoding specificity. Paromomycin has no effect if native tRNA(Arg(ICG)) is highly expressed, consistent with the idea that failure to efficiently decode CGA codons might occur in part due to rejection of the cognate tRNA(Arg(ICG)). Furthermore, expression downstream from CGA repeats is improved by inactivation of RPL1B, one of two genes encoding the universally conserved ribosomal protein L1. The effects of rpl1b-Δ and of either paromomycin or tRNA(Arg(ICG)) on CGA decoding are additive, suggesting that the rpl1b-Δ mutant suppresses CGA inhibition by means other than increased acceptance of tRNA(Arg(ICG)). Thus, inefficient decoding of CGA likely involves at least two independent defects in translation.

  8. Brucella suis in armadillos (Chaetophractus villosus) from La Pampa, Argentina.

    PubMed

    Kin, Marta S; Fort, Marcelo; de Echaide, Susana T; Casanave, Emma B

    2014-06-04

    Brucellosis is a zoonotic disease transmitted from an animal reservoir to humans. Both, wildlife and domestic animals, contribute to the spreading of these zoonosis. The surveillance of the animal health status is strictly regulated for domestic animals, whereas disease monitoring in wildlife does not exist. The aim of the present study was to provide data on the prevalence of anti-Brucella antibodies in Chaetophractus villosus from a region of La Pampa, Argentina to assess public health risks. The C. villosus is endemic to South America, and in Argentina it represents a food resource for human consumption. A total of 150 sera of armadillos bleeding between 2007 and 2010 were tested using buffered plate antigen test (BPAT), serum agglutination test (SAT), 2-mercaptoethanol (2-ME) and complement fixation test (CFT), for the detection of anti-Brucella antibodies. Antibodies to Brucella sp. were found in 16% (24:150) of the armadillos tested using the BPAT test. All 24 positive samples were confirmed by the SAT, 2-ME and CFT tests. Strain isolation was attempted from liver and spleen samples of two animals with positive serology. Isolates were characterized by conventional biotyping and identification of specific DNA using polymerase chain reaction (PCR). A total of 2 isolates were recovered from spleen and liver. Both of them were identified as Brucella suis biovar 1. This preliminary study provides the first report on the seroprevalence of brucellosis and describes the first isolate of B. suis biovar 1 in C. villosus in Argentina.

  9. pangolin encodes a Lef-1 homologue that acts downstream of Armadillo to transduce the Wingless signal in Drosophila.

    PubMed

    Brunner, E; Peter, O; Schweizer, L; Basler, K

    1997-02-27

    Members of the Wnt/Wingless (Wg) family of signalling proteins organize many aspects of animal development by regulating the expression of particular target genes in responding cells. Recent biochemical studies indicate that the vertebrate HMG-domain proteins Lef-1 and XTcf-3 can physically interact with beta-catenin, a homologue of Drosophila Armadillo (Arm), the most downstream component known in the Wnt signal transduction pathway. However, these studies do not address whether the endogenous Lef/Tcf family members are required in vivo to transduce Wnt signals. Using genetic methods in Drosophila, we define a new segment polarity gene, pangolin (pan), and show that its product is required in vivo for Wg signal transduction in embryos and in developing adult tissues. In addition, we show that pan encodes a Lef/Tcf homologue and provide evidence that its protein product binds to the beta-catenin homologue Armadillo in vivo. Finally, we demonstrate that Pan functions downstream of Arm to transduce the Wg signal. Thus, our results indicate that Pan is an essential component of the Wg transduction pathway and suggest that it acts directly to regulate gene transcription in response to Wg signalling.

  10. Structural and Functional Insights into Small, Glutamine-Rich, Tetratricopeptide Repeat Protein Alpha

    PubMed Central

    Roberts, Joanna D.; Thapaliya, Arjun; Martínez-Lumbreras, Santiago; Krysztofinska, Ewelina M.; Isaacson, Rivka L.

    2015-01-01

    The small glutamine-rich, tetratricopeptide repeat-containing protein alpha (SGTA) is an emerging player in the quality control of secretory and membrane proteins mislocalized to the cytosol, with established roles in tail-anchored (TA) membrane protein biogenesis. SGTA consists of three structural domains with individual functions, an N-terminal dimerization domain that assists protein sorting pathways, a central tetratricopeptide repeat (TPR) domain that mediates interactions with heat-shock proteins, proteasomal, and hormonal receptors, and viral proteins, and a C-terminal glutamine rich region that binds hydrophobic substrates. SGTA has been linked to viral lifecycles and hormone receptor signaling, with implications in the pathogenesis of various disease states. Thus far, a range of biophysical techniques have been employed to characterize SGTA structure in some detail, and to investigate its interactions with binding partners in different biological contexts. A complete description of SGTA structure, together with further investigation into its function as a co-chaperone involved quality control, could provide us with useful insights into its role in maintaining cellular proteostasis, and broaden our understanding of mechanisms underlying associated pathologies. This review describes how some structural features of SGTA have been elucidated, and what this has uncovered about its cellular functions. A brief background on the structure and function of SGTA is given, highlighting its importance to biomedicine and related fields. The current level of knowledge and what remains to be understood about the structure and function of SGTA is summarized, discussing the potential direction of future research. PMID:26734616

  11. Protein O-fucosyltransferase 2 adds O-fucose to thrombospondin type 1 repeats.

    PubMed

    Luo, Yi; Koles, Kate; Vorndam, Wendy; Haltiwanger, Robert S; Panin, Vladislav M

    2006-04-07

    O-Fucose is an unusual form of glycosylation found on epidermal growth factor-like (EGF) repeats and thrombospondin type 1 repeats (TSRs) in many secreted and transmembrane proteins. Recently O-fucose on EGF repeats was shown to play important roles in Notch signaling. In contrast, physiological roles for O-fucose on TSRs are unknown. In the accompanying paper (Luo, Y., Nita-Lazar, A., and Haltiwanger, R. S. (2006) J. Biol. Chem. 281, 9385-9392), we demonstrated that an enzyme distinct from protein O-fucosyltransferase 1 adds O-fucose to TSRs. A known homologue of O-fucosyltransferase 1 is putative protein O-fucosyltransferase 2. The cDNA sequence encoding O-fucosyltransferase 2 was originally identified during a data base search for fucosyltransferases in Drosophila. Like O-fucosyltransferase 1, O-fucosyltransferase 2 is conserved from Caenorhabditis elegans to humans. Although O-fucosyltransferase 2 was assumed to be another protein O-fucosyltransferase, no biochemical characterization existed supporting this contention. Here we show that RNAi-mediated reduction of the O-fucosyltransferase 2 message significantly decreased TSR-specific O-fucosyltransferase activity in Drosophila S2 cells. We also found that O-fucosyltransferase 2 is predominantly localized in the endoplasmic reticulum compartment of these cells. Furthermore, we expressed recombinant Drosophila O-fucosyltransferase 2 and showed that it O-fucosylates TSRs but not EGF repeats in vitro. These results demonstrate that O-fucosyltransferase 2 is in fact a TSR-specific O-fucosyltransferase.

  12. Polymorphic CAG Repeat and Protein Expression of Androgen Receptor Gene in Colorectal Cancer.

    PubMed

    Huang, Rui; Wang, Guiyu; Song, Yanni; Wang, Feng; Zhu, Bing; Tang, Qingchao; Liu, Zheng; Chen, Yinggang; Zhang, Qian; Muhammad, Shan; Wang, Xishan

    2015-04-01

    Although somatic alterations in CAG repeats in the androgen receptor (AR) gene have been suggested to predispose to colorectal cancer, less is known about AR in colorectal cancer carcinogenesis. Because of lack of relevant analysis on CAG repeat length and AR expression in colorectal cancer, we aimed to investigate the prognostic value of polymorphic CAG and protein expression of the AR gene in patients with colorectal cancer. A case-control study was carried out on 550 patients with colorectal cancer and 540 healthy controls to investigate whether polymorphic CAG within the AR gene is linked to increased risk for colorectal cancer. Polymorphic CAG and AR expression were analyzed to clarify their relationship with clinicopathologic and prognostic factors in patients with colorectal cancer. The study showed that the AR gene in patients with colorectal cancer had a longer CAG repeat sequence than those in the control group, as well as increased risk for colorectal cancer among females (P = 0.013), males (P = 0.002), and total colorectal cancer population (P < 0.001), respectively. AR expression exhibited a significant difference in long CAG repeat sequence among males (P < 0.001), females (P < 0.001), and total colorectal cancer study population (P < 0.001). Both long CAG repeat sequence and negative AR expression were associated with a short 5-year overall survival (OS) rate in colorectal cancer. Long CAG repeat sequences and the absence of AR expression were closely related to the development of colorectal cancer. Both long CAG and decreased AR expression were correlated with the poor 5-year OS in patients with colorectal cancer.

  13. Mechanism of RNA stabilization and translational activation by a pentatricopeptide repeat protein

    PubMed Central

    Prikryl, Jana; Rojas, Margarita; Schuster, Gadi; Barkan, Alice

    2011-01-01

    Pentatricopeptide repeat (PPR) proteins comprise a large family of helical repeat proteins that bind RNA and modulate organellar RNA metabolism. The mechanisms underlying the functions attributed to PPR proteins are unknown. We describe in vitro studies of the maize protein PPR10 that clarify how PPR10 modulates the stability and translation of specific chloroplast mRNAs. We show that recombinant PPR10 bound to its native binding site in the chloroplast atpI–atpH intergenic region (i) blocks both 5′→3′ and 3′→ 5 exoribonucleases in vitro; (ii) is sufficient to define the native processed atpH mRNA 5′-terminus in conjunction with a generic 5′→3′ exoribonuclease; and (iii) remodels the structure of the atpH ribosome-binding site in a manner that can account for PPR10’s ability to enhance atpH translation. In addition, we show that the minimal PPR10-binding site spans 17 nt. We propose that the site-specific barrier and RNA remodeling activities of PPR10 are a consequence of its unusually long, high-affinity interface with single-stranded RNA, that this interface provides a functional mimic to bacterial small RNAs, and that analogous activities underlie many of the biological functions that have been attributed to PPR proteins. PMID:21173259

  14. AdpC is a Prevotella intermedia 17 leucine-rich repeat internalin-like protein.

    PubMed

    Iyer, Divya; Anaya-Bergman, Cecilia; Jones, Kevin; Yanamandra, Sai; Sengupta, Dipanwita; Miyazaki, Hiroshi; Lewis, Janina P

    2010-06-01

    The oral bacterium Prevotella intermedia attaches to and invades gingival epithelial cells, fibroblasts, and endothelial cells. Several genes encoding proteins that mediate both the adhesion and invasion processes are carried on the genome of this bacterium. Here, we characterized one such protein, AdpC, belonging to the leucine-rich repeat (LRR) protein family. Bioinformatics analysis revealed that this protein shares similarity with the Treponema pallidum LRR (LRR(TP)) family of proteins and contains six LRRs. Despite the absence of a signal peptide, this protein is localized on the bacterial outer membrane, indicating that it is transported through an atypical secretion mechanism. The recombinant form of this protein (rAdpC) was shown to bind fibrinogen. In addition, the heterologous host strain Escherichia coli BL21 expressing rAdpC (V2846) invaded fibroblast NIH 3T3 cells at a 40-fold-higher frequency than control E. coli BL21 cells expressing a sham P. intermedia 17 protein. Although similar results were obtained by using human umbilical vein endothelial cells (HUVECs), only a 3-fold-increased invasion of V2846 into oral epithelial HN4 cells was observed. Thus, AdpC-mediated invasion is cell specific. This work demonstrated that AdpC is an important invasin protein of P. intermedia 17.

  15. Interaction between a plasma membrane-localized ankyrin-repeat protein ITN1 and a nuclear protein RTV1

    SciTech Connect

    Sakamoto, Hikaru; Sakata, Keiko; Kusumi, Kensuke; Kojima, Mikiko; Sakakibara, Hitoshi; Iba, Koh

    2012-06-29

    Highlights: Black-Right-Pointing-Pointer ITN1, a plasma membrane ankyrin protein, interacts with a nuclear DNA-binding protein RTV1. Black-Right-Pointing-Pointer The nuclear transport of RTV1 is partially inhibited by interaction with ITN1. Black-Right-Pointing-Pointer RTV1 can promote the nuclear localization of ITN1. Black-Right-Pointing-Pointer Both overexpression of RTV1 and the lack of ITN1 increase salicylic acids sensitivity in plants. -- Abstract: The increased tolerance to NaCl 1 (ITN1) protein is a plasma membrane (PM)-localized protein involved in responses to NaCl stress in Arabidopsis. The predicted structure of ITN1 is composed of multiple transmembrane regions and an ankyrin-repeat domain that is known to mediate protein-protein interactions. To elucidate the molecular functions of ITN1, we searched for interacting partners using a yeast two-hybrid assay, and a nuclear-localized DNA-binding protein, RTV1, was identified as a candidate. Bimolecular fluorescence complementation analysis revealed that RTV1 interacted with ITN1 at the PM and nuclei in vivo. RTV1 tagged with red fluorescent protein localized to nuclei and ITN1 tagged with green fluorescent protein localized to PM; however, both proteins localized to both nuclei and the PM when co-expressed. These findings suggest that RTV1 and ITN1 regulate the subcellular localization of each other.

  16. Assembly of Neuronal Connectivity by Neurotrophic Factors and Leucine-Rich Repeat Proteins

    PubMed Central

    Ledda, Fernanda; Paratcha, Gustavo

    2016-01-01

    Proper function of the nervous system critically relies on sophisticated neuronal networks interconnected in a highly specific pattern. The architecture of these connections arises from sequential developmental steps such as axonal growth and guidance, dendrite development, target determination, synapse formation and plasticity. Leucine-rich repeat (LRR) transmembrane proteins have been involved in cell-type specific signaling pathways that underlie these developmental processes. The members of this superfamily of proteins execute their functions acting as trans-synaptic cell adhesion molecules involved in target specificity and synapse formation or working in cis as cell-intrinsic modulators of neurotrophic factor receptor trafficking and signaling. In this review, we will focus on novel physiological mechanisms through which LRR proteins regulate neurotrophic factor receptor signaling, highlighting the importance of these modulatory events for proper axonal extension and guidance, tissue innervation and dendrite morphogenesis. Additionally, we discuss few examples linking this set of LRR proteins to neurodevelopmental and psychiatric disorders. PMID:27555809

  17. TolC-dependent secretion of an ankyrin repeat-containing protein of Rickettsia typhi.

    PubMed

    Kaur, Simran J; Rahman, M Sayeedur; Ammerman, Nicole C; Beier-Sexton, Magda; Ceraul, Shane M; Gillespie, Joseph J; Azad, Abdu F

    2012-09-01

    Rickettsia typhi, the causative agent of murine (endemic) typhus, is an obligate intracellular pathogen with a life cycle involving both vertebrate and invertebrate hosts. In this study, we characterized a gene (RT0218) encoding a C-terminal ankyrin repeat domain-containing protein, named Rickettsia ankyrin repeat protein 1 (RARP-1), and identified it as a secreted effector protein of R. typhi. RT0218 showed differential transcript abundance at various phases of R. typhi intracellular growth. RARP-1 was secreted by R. typhi into the host cytoplasm during in vitro infection of mammalian cells. Transcriptional analysis revealed that RT0218 was cotranscribed with adjacent genes RT0217 (hypothetical protein) and RT0216 (TolC) as a single polycistronic mRNA. Given one of its functions as a facilitator of extracellular protein secretion in some Gram-negative bacterial pathogens, we tested the possible role of TolC in the secretion of RARP-1. Using Escherichia coli C600 and an isogenic tolC insertion mutant as surrogate hosts, our data demonstrate that RARP-1 is secreted in a TolC-dependent manner. Deletion of either the N-terminal signal peptide or the C-terminal ankyrin repeats abolished RARP-1 secretion by wild-type E. coli. Importantly, expression of R. typhi tolC in the E. coli tolC mutant restored the secretion of RARP-1, suggesting that TolC has a role in RARP-1 translocation across the outer membrane. This work implies that the TolC component of the putative type 1 secretion system of R. typhi is involved in the secretion process of RARP-1.

  18. Rings and ribbons in protein structures: Characterization using helical parameters and Ramachandran plots for repeating dipeptides.

    PubMed

    Hayward, Steven; Leader, David P; Al-Shubailly, Fawzia; Milner-White, E James

    2014-02-01

    Helical parameters displayed on a Ramachandran plot allow peptide structures with successive residues having identical main chain conformations to be studied. We investigate repeating dipeptide main chain conformations and present Ramachandran plots encompassing the range of possible structures. Repeating dipeptides fall into the categories: rings, ribbons, and helices. Partial rings occur in the form of "nests" and "catgrips"; many nests are bridged by an oxygen atom hydrogen bonding to the main chain NH groups of alternate residues, an interaction optimized by the ring structure of the nest. A novel recurring feature is identified that we name unpleated β, often situated at the ends of a β-sheet strand. Some are partial rings causing the polypeptide to curve gently away from the sheet; some are straight. They lack β-pleat and almost all incorporate a glycine. An example is the first glycine in the GxxxxGK motif of P-loop proteins. Ribbons in repeating dipeptides can be either flat, as seen in repeated type II and type II' β-turns, or twisted, as in multiple type I and type I' β-turns. Hexa- and octa-peptides in such twisted ribbons occur frequently in proteins, predominantly with type I β-turns, and are the same as the "β-bend ribbons" hitherto identified only in short peptides. One is seen in the GTPase-activating protein for Rho in the active, but not the inactive, form of the enzyme. It forms a β-bend ribbon, which incorporates the catalytic arginine, allowing its side chain guanidino group to approach the active site and enhance enzyme activity.

  19. A small multifunctional pentatricopeptide repeat protein in the chloroplast of Chlamydomonas reinhardtii.

    PubMed

    Jalal, Abdullah; Schwarz, Christian; Schmitz-Linneweber, Christian; Vallon, Olivier; Nickelsen, Jörg; Bohne, Alexandra-Viola

    2015-03-01

    Organellar biogenesis is mainly regulated by nucleus-encoded factors, which act on various steps of gene expression including RNA editing, processing, splicing, stabilization, and translation initiation. Among these regulatory factors, pentatricopeptide repeat (PPR) proteins form the largest family of RNA binding proteins, with hundreds of members in flowering plants. In striking contrast, the genome of the unicellular green alga Chlamydomonas reinhardtii encodes only 14 such proteins. In this study, we analyzed PPR7, the smallest and most highly expressed PPR protein in C. reinhardtii. Green fluorescent protein-based localization and gel-filtration analysis revealed that PPR7 forms a part of a high-molecular-weight ribonucleoprotein complex in the chloroplast stroma. RIP-chip analysis of PPR7-bound RNAs demonstrated that the protein associates with a diverse set of chloroplast transcripts in vivo, i.e. rrnS, psbH, rpoC2, rbcL, atpA, cemA-atpH, tscA, and atpI-psaJ. Furthermore, the investigation of PPR7 RNAi strains revealed that depletion of PPR7 results in a light-sensitive phenotype, accompanied by altered levels of its target RNAs that are compatible with the defects in their maturation or stabilization. PPR7 is thus an unusual type of small multifunctional PPR protein, which interacts, probably in conjunction with other RNA binding proteins, with numerous target RNAs to promote a variety of post-transcriptional events.

  20. Identification of a Soybean Protein That Interacts with GAGA Element Dinucleotide Repeat DNA1

    PubMed Central

    Sangwan, Indu; O'Brian, Mark R.

    2002-01-01

    Dinucleotide repeat DNA with the pattern (GA)n/(TC)n, so-called GAGA elements, control gene expression in animals, and are recognized by a specific regulatory protein. Here, a yeast one-hybrid screen was used to isolate soybean (Glycine max) cDNA encoding a GAGA-binding protein (GBP) that binds to (GA)n/(CT)n DNA. Soybean GBP was dissimilar from the GAGA factor of Drosophila melanogaster. Recombinant GBP protein did not bind to dinucleotide repeat sequences other than (GA)n/(CT)n. GBP bound to the promoter of the heme and chlorophyll synthesis gene Gsa1, which contains a GAGA element. Removal of that GAGA element abrogated binding of GBP to the promoter. Furthermore, insertion of the GAGA element to a nonspecific DNA conferred GBP-binding activity on that DNA. Thus, the GAGA element of the Gsa1 promoter is both necessary and sufficient for GBP binding. Gbp mRNA was expressed in leaves and was induced in symbiotic root nodules elicited by the bacterium Bradyrhizobium japonicum. In addition, Gbp transcripts were much higher in leaves of dark-treated etiolated plantlets than in those exposed to light for 24 h. Homologs of GBP were found in other dicots and in the monocot rice (Oryza sativa), as well. We suggest that interaction between GAGA elements and GBP-like proteins is a regulatory feature in plants. PMID:12177492

  1. Electrostatic effect of H1-histone protein binding on nucleosome repeat length

    NASA Astrophysics Data System (ADS)

    Cherstvy, Andrey G.; Teif, Vladimir B.

    2014-08-01

    Within a simple biophysical model we describe the effect of electrostatic binding of H1 histone proteins on the nucleosome repeat length in chromatin. The length of wrapped DNA optimizes its binding energy to the histone core and the elastic energy penalty of DNA wrapping. The magnitude of the effect predicted from our model is in agreement with the systematic experimental data on the linear variation of nucleosome repeat lengths with H1/nucleosome ratio (Woodcock C L et al 2006 Chromos. Res. 14 17-25). We compare our model to the data for different cell types and organisms, with a widely varying ratio of bound H1 histones per nucleosome. We underline the importance of this non-specific histone-DNA charge-balance mechanism in regulating the positioning of nucleosomes and the degree of compaction of chromatin fibers in eukaryotic cells.

  2. A naturally occurring repeat protein with high internal sequence identity defines a new class of TPR-like proteins

    PubMed Central

    Marold, Jacob D.; Kavran, Jennifer M.; Bowman, Gregory D.; Barrick, Doug

    2016-01-01

    SUMMARY Linear repeat proteins often have high structural similarity and low (~25%) pairwise sequence identities (PSI) among modules. We identified a unique P. anserina (Pa) sequence with tetratricopeptide repeat (TPR) homology, which contains longer (42 residue) repeats (42PRs) with an average PSI >91%. We determined the crystal structure of five tandem Pa 42PRs to 1.6Å, and examined the stability and solution properties of constructs containing three to six Pa 42PRs. Compared to 34-residue TPRs (34PRs), Pa 42PRs have a one-turn extension of each helix, and bury more surface area. Unfolding transitions shift to higher denaturant concentration and become sharper as repeats are added. Fitted Ising models show Pa 42PRs to be more cooperative than consensus 34PRs, with increased magnitudes of intrinsic and interfacial free energies. These results demonstrate the tolerance of the TPR motif to length variation, and provide a basis to understand the effects of helix length on intrinsic/interfacial stability. PMID:26439765

  3. Imperfect DNA mirror repeats in E. coli TnsA and other protein-coding DNA.

    PubMed

    Lang, Dorothy M

    2005-09-01

    DNA imperfect mirror repeats (DNA-IMRs) are ubiquitous in protein-coding DNA. However, they overlap and often have different centers of symmetry, making it difficult to evaluate their relationship to each other and to specific DNA and protein motifs and structures. This paper describes a systematic method of determining a hierarchy for DNA-IMRs and evaluates their relationship to protein structural elements (PSEs)--helices, turns and beta-sheets. DNA-IMRs are identifed by two different methods--DNA-IMRs terminated by reverse dinucleotides (rd-IMRs) and DNA-IMRs terminated by a single (mono) matching nucleotide (m-IMRs). Both rd-IMRs and m-IMRs are evaluated in 17 proteins, and illustrated in detail for TnsA. For each of the proteins, Fisher's exact test (FET) is used to measure the coincidence between the terminal dinucleotides of rd-IMRs and the terminal amino acids of individual PSEs. A significant correlation over a span of about 3 nt was found for each protein. The correlation is robust and for most genes, all rd-IMRsprotein intervals translated by rd-IMRs>16 nt contain approximately 88% of the potential functional motifs. The protein translation of the longest rd- and m-IMRs span sequences important to the protein's structure and function. In all 17 proteins studied, the population of rd-IMRs is substantially less than the expected number and the population of m-IMRs greater than the expected number, indicating strong selective pressures. The association of rd-IMRs with PSEs restricts their spatial distribution, and therefore, their number. The greater than predicted number of m-IMRs indicates that DNA symmetry exists throughout the entire protein-coding region and may stabilize the sequence.

  4. The Arabidopsis ERECTA gene encodes a putative receptor protein kinase with extracellular leucine-rich repeats.

    PubMed Central

    Torii, K U; Mitsukawa, N; Oosumi, T; Matsuura, Y; Yokoyama, R; Whittier, R F; Komeda, Y

    1996-01-01

    Arabidopsis Landsberg erecta is one of the most popular ecotypes and is used widely for both molecular and genetic studies. It harbors the erecta (er) mutation, which confers a compact inflorescence, blunt fruits, and short petioles. We have identified five er mutant alleles from ecotypes Columbia and Wassilewskija. Phenotypic characterization of the mutant alleles suggests a role for the ER gene in regulating the shape of organs originating from the shoot apical meristem. We cloned the ER gene, and here, we report that it encodes a putative receptor protein kinases. The deduced ER protein contains a cytoplasmic protein kinase catalytic domain, a transmembrane region, and an extracellular domain consisting of leucine-rich repeats, which are thought to interact with other macromolecules. Our results suggest that cell-cell communication mediated by a receptor kinase has an important role in plant morphogenesis. PMID:8624444

  5. Leucine-Rich Repeat Transmembrane Proteins Instruct Discrete Dendrite Targeting in an Olfactory Map

    PubMed Central

    Hong, Weizhe; Zhu, Haitao; Potter, Christopher J.; Barsh, Gabrielle; Kurusu, Mitsuhiko; Zinn, Kai; Luo, Liqun

    2010-01-01

    Olfactory systems utilize discrete neural pathways to process and integrate odorant information. In Drosophila, axons of first-order olfactory receptor neurons (ORNs) and dendrites of second-order projection neurons (PNs) form class-specific synaptic connections at ∼50 glomeruli. The mechanisms underlying PN dendrite targeting to distinct glomeruli in a 3-dimensional discrete neural map are unclear. Here we show that the leucine-rich repeat (LRR) transmembrane protein Capricious (Caps) is differentially expressed in different classes of PNs. Loss- and gain-of-function studies indicate that Caps instructs the segregation of Caps-positive and negative PN dendrites to discrete glomerular targets. Moreover, Caps does not mediate homophilic interactions and regulates PN dendrite targeting independent of pre-synaptic ORNs. The closely related protein Tartan plays a partially redundant function with Capricious. These LRR proteins are likely part of a combinatorial cell-surface code that instructs discrete olfactory map formation. PMID:19915565

  6. Leucine-rich repeat transmembrane proteins instruct discrete dendrite targeting in an olfactory map.

    PubMed

    Hong, Weizhe; Zhu, Haitao; Potter, Christopher J; Barsh, Gabrielle; Kurusu, Mitsuhiko; Zinn, Kai; Luo, Liqun

    2009-12-01

    Olfactory systems utilize discrete neural pathways to process and integrate odorant information. In Drosophila, axons of first-order olfactory receptor neurons (ORNs) and dendrites of second-order projection neurons (PNs) form class-specific synaptic connections at approximately 50 glomeruli. The mechanisms underlying PN dendrite targeting to distinct glomeruli in a three-dimensional discrete neural map are unclear. We found that the leucine-rich repeat (LRR) transmembrane protein Capricious (Caps) was differentially expressed in different classes of PNs. Loss-of-function and gain-of-function studies indicated that Caps instructs the segregation of Caps-positive and Caps-negative PN dendrites to discrete glomerular targets. Moreover, Caps-mediated PN dendrite targeting was independent of presynaptic ORNs and did not involve homophilic interactions. The closely related protein Tartan was partially redundant with Caps. These LRR proteins are probably part of a combinatorial cell-surface code that instructs discrete olfactory map formation.

  7. Crystallization of a pentapeptide-repeat protein by reductive cyclic pentylation of free amines with glutaraldehyde.

    PubMed

    Vetting, Matthew W; Hegde, Subray S; Blanchard, John S

    2009-05-01

    The pentapeptide-repeat protein EfsQnr from Enterococcus faecalis protects DNA gyrase from inhibition by fluoroquinolones. EfsQnr was cloned and purified to homogeneity, but failed to produce diffraction-quality crystals in initial crystallization screens. Treatment of EfsQnr with glutaraldehyde and the strong reducing agent borane-dimethylamine resulted in a derivatized protein which produced crystals that diffracted to 1.6 A resolution; their structure was subsequently determined by single-wavelength anomalous dispersion. Analysis of the derivatized protein using Fourier transform ion cyclotron resonance mass spectrometry indicated a mass increase of 68 Da per free amino group. Electron-density maps about a limited number of structurally ordered lysines indicated that the modification was a cyclic pentylation of free amines, producing piperidine groups.

  8. ST proteins, a new family of plant tandem repeat proteins with a DUF2775 domain mainly found in Fabaceae and Asteraceae

    PubMed Central

    2012-01-01

    Background Many proteins with tandem repeats in their sequence have been described and classified according to the length of the repeats: I) Repeats of short oligopeptides (from 2 to 20 amino acids), including structural cell wall proteins and arabinogalactan proteins. II) Repeats that range in length from 20 to 40 residues, including proteins with a well-established three-dimensional structure often involved in mediating protein-protein interactions. (III) Longer repeats in the order of 100 amino acids that constitute structurally and functionally independent units. Here we analyse ShooT specific (ST) proteins, a family of proteins with tandem repeats of unknown function that were first found in Leguminosae, and their possible similarities to other proteins with tandem repeats. Results ST protein sequences were only found in dicotyledonous plants, limited to several plant families, mainly the Fabaceae and the Asteraceae. ST mRNAs accumulate mainly in the roots and under biotic interactions. Most ST proteins have one or several Domain(s) of Unknown Function 2775 (DUF2775). All deduced ST proteins have a signal peptide, indicating that these proteins enter the secretory pathway, and the mature proteins have tandem repeat oligopeptides that share a hexapeptide (E/D)FEPRP followed by 4 partially conserved amino acids, which could determine a putative N-glycosylation signal, and a fully conserved tyrosine. In a phylogenetic tree, the sequences clade according to taxonomic group. A possible involvement in symbiosis and abiotic stress as well as in plant cell elongation is suggested, although different STs could play different roles in plant development. Conclusions We describe a new family of proteins called ST whose presence is limited to the plant kingdom, specifically to a few families of dicotyledonous plants. They present 20 to 40 amino acid tandem repeat sequences with different characteristics (signal peptide, DUF2775 domain, conservative repeat regions) from the

  9. Methylation of C9orf72 expansion reduces RNA foci formation and dipeptide-repeat proteins expression in cells.

    PubMed

    Bauer, Peter O

    2016-01-26

    A hexanucleotide repeat expansion in the C9orf72 gene is the most common genetic cause of both frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS), together referred to as c9FTD/ALS. It has been suggested that a loss of C9orf72 protein expression, the formation of toxic RNA foci and dipeptide-repeat proteins contribute to C9orf72-related diseases. Interestingly, it has been shown that trimethylation of histones and methylation of CpG islands near the repeat expansion may play a role in the pathogenesis c9FTD/ALS. Recently, methylation of expanded repeat itself has been reported. To further elucidate the mechanisms underlying these diseases, the influence of epigenetic modification in the repeat expansion on its pathogenic effect was assessed. Here, a reduced formation of toxic RNA foci and dipeptide-repeat proteins upon methylation of the GGGGCC repeat in a cellular model of c9FTD/ALS is shown. Additionally, a novel methylcytosine-capture DNA hybridization immunoassay for semi-quantitative detection of the repeat methylation levels is presented, potentially usable for methylation analysis in patients carrying C9orf72 repeat expansion carriers as a diagnostic tool. Presented results suggest that increased level of pathogenic GGGGCC expansion methylation may be sufficient to alleviate the molecular pathology of the C9orf72-related diseases.

  10. Analysis of the equilibrium and kinetics of the ankyrin repeat protein myotrophin

    NASA Astrophysics Data System (ADS)

    Faccin, Mauro; Bruscolini, Pierpaolo; Pelizzola, Alessandro

    2011-02-01

    We apply the Wako-Saito-Muñoz-Eaton model to the study of myotrophin, a small ankyrin repeat protein, whose folding equilibrium and kinetics have been recently characterized experimentally. The model, which is a native-centric with binary variables, provides a finer microscopic detail than the Ising model that has been recently applied to some different repeat proteins, while being still amenable for an exact solution. In partial agreement with the experiments, our results reveal a weakly three-state equilibrium and a two-state-like kinetics of the wild-type protein despite the presence of a nontrivial free-energy profile. These features appear to be related to a careful "design" of the free-energy landscape, so that mutations can alter this picture, stabilizing some intermediates and changing the position of the rate-limiting step. Also, the experimental findings of two alternative pathways, an N-terminal and a C-terminal one, are qualitatively confirmed, even if the variations in the rates upon the experimental mutations cannot be quantitatively reproduced. Interestingly, the folding and unfolding pathways appear to be different, even if closely related: a property that is not generally considered in the phenomenological interpretation of the experimental data.

  11. The Role of Leucine-Rich Repeat Containing Protein 10 (LRRC10) in Dilated Cardiomyopathy

    PubMed Central

    Brody, Matthew J.; Lee, Youngsook

    2016-01-01

    Leucine-rich repeat containing protein 10 (LRRC10) is a cardiomyocyte-specific member of the Leucine-rich repeat containing (LRRC) protein superfamily with critical roles in cardiac function and disease pathogenesis. Recent studies have identified LRRC10 mutations in human idiopathic dilated cardiomyopathy (DCM) and Lrrc10 homozygous knockout mice develop DCM, strongly linking LRRC10 to the molecular etiology of DCM. LRRC10 localizes to the dyad region in cardiomyocytes where it can interact with actin and α-actinin at the Z-disc and associate with T-tubule components. Indeed, this region is becoming increasingly recognized as a signaling center in cardiomyocytes, not only for calcium cycling, excitation-contraction coupling, and calcium-sensitive hypertrophic signaling, but also as a nodal signaling hub where the myocyte can sense and respond to mechanical stress. Disruption of a wide range of critical structural and signaling molecules in cardiomyocytes confers susceptibility to cardiomyopathies in addition to the more classically studied mutations in sarcomeric proteins. However, the molecular mechanisms underlying DCM remain unclear. Here, we review what is known about the cardiomyocyte functions of LRRC10, lessons learned about LRRC10 and DCM from the Lrrc10 knockout mouse model, and discuss ongoing efforts to elucidate molecular mechanisms whereby mutation or absence of LRRC10 mediates cardiac disease. PMID:27536250

  12. SIP, a novel ankyrin repeat containing protein, sequesters steroid receptor coactivators in the cytoplasm.

    PubMed

    Zhang, Ying; Zhang, Hua; Liang, Jing; Yu, Wenhua; Shang, Yongfeng

    2007-06-06

    Steroid receptor coactivators (SRCs) exert profound effects on animal development and physiology. These coactivators are nuclear proteins and transcription co-regulators that function to facilitate the transcription initiation mediated by nuclear receptors, as well as by other well-known transcription factors. However, how these co-regulators are functionally regulated is poorly understood. During genome-wide screening for SRC-interacting proteins, we identified a novel ankyrin repeat containing protein, SIP (SRC-Interacting Protein), which interacts with SRC coactivators in the cytoplasm. We demonstrated that extracellular stimuli such as the addition of estrogen, induced phosphorylation of SIP in its PEST (Proline, Glutamate, Serine, and Threonine rich) domain by casein kinase II. The phosphorylation of SIP resulted in dissociation of SRC proteins from SIP in the cytoplasm and led to subsequent nuclear translocation of SRC proteins and gene coactivation. Both gain-of-function and loss-of-function experiments indicate that SIP functions to sequester SRC coactivators in the cytoplasm and buffer the availability of these coactivators, thus providing a mechanism for the regulation of the transcription regulators.

  13. Identifying proteins that bind to specific RNAs - focus on simple repeat expansion diseases

    PubMed Central

    Jazurek, Magdalena; Ciesiolka, Adam; Starega-Roslan, Julia; Bilinska, Katarzyna; Krzyzosiak, Wlodzimierz J.

    2016-01-01

    RNA–protein complexes play a central role in the regulation of fundamental cellular processes, such as mRNA splicing, localization, translation and degradation. The misregulation of these interactions can cause a variety of human diseases, including cancer and neurodegenerative disorders. Recently, many strategies have been developed to comprehensively analyze these complex and highly dynamic RNA–protein networks. Extensive efforts have been made to purify in vivo-assembled RNA–protein complexes. In this review, we focused on commonly used RNA-centric approaches that involve mass spectrometry, which are powerful tools for identifying proteins bound to a given RNA. We present various RNA capture strategies that primarily depend on whether the RNA of interest is modified. Moreover, we briefly discuss the advantages and limitations of in vitro and in vivo approaches. Furthermore, we describe recent advances in quantitative proteomics as well as the methods that are most commonly used to validate robust mass spectrometry data. Finally, we present approaches that have successfully identified expanded repeat-binding proteins, which present abnormal RNA–protein interactions that result in the development of many neurological diseases. PMID:27625393

  14. Nuclear magnetic resonance spectroscopy of mussel adhesive protein repeating peptide segment.

    PubMed

    Olivieri, M P; Wollman, R M; Alderfer, J L

    1997-12-01

    Mussel adhesive protein (MAP) is the adhesive agent used by the common blue sea mussel (Mytilus edulis) to attach the animal to various underwater surfaces. It is generally composed of 75 to 85 repeating decameric units with the reported primary sequence NH2-Ala(1)-Lyst(2)-Pro(3)-Ser(4)-Tyr(5)-Hyp(6)-Hyp(7)-Thr(8)-DOPA( 9)- Lys(10)-COOH. This study examines this peptide's solution-state conformation using proton nuclear magnetic resonance (NMR) spectroscopy. NMR and molecular modeling of the decamer before and after molecular dynamics calculations in water suggests a conformation that retains an overall bent helix.

  15. Selection and characterization of Her2 binding-designed ankyrin repeat proteins.

    PubMed

    Zahnd, Christian; Pecorari, Frédéric; Straumann, Nadine; Wyler, Emanuel; Plückthun, Andreas

    2006-11-17

    Designed ankyrin repeat proteins (DARPins) are a novel class of binding proteins that bind their target protein with high affinity and specificity and have very favorable expression and stability properties. We describe here the in vitro selection of DARPins against human epidermal growth factor receptor 2 (Her2), an important target for cancer therapy and diagnosis. Several DARPins bind to the same epitope as trastuzumab (Herceptin), but none were selected that bind to the epitope of pertuzumab (Omnitarg). Some of the selected DARPins bind with low nanomolar affinity (Kd=7.3 nm) to the target. Further analysis revealed that all DARPins are highly specific and do not cross-react with epidermal growth factor receptor I (EGFR1) or any other investigated protein. The selected DARPins specifically bind to strongly Her2-overexpressing cell lines such as SKBR-3 but also recognize small amounts of Her2 on weakly expressing cell lines such as MCF-7. Furthermore, the DARPins also lead to a highly specific and strong staining of plasma membranes of paraffinated sections of human mamma-carcinoma tissue. Thus, the selected DARPins might be used for the development of diagnostic tests for the status of Her2 overexpression in different adenocarcinomas, and they may be further evaluated for their potential in targeted therapy since their favorable expression properties make the construction of fusion proteins very convenient.

  16. Crystallization of a pentapeptide-repeat protein by reductive cyclic pentylation of free amines with glutaraldehyde

    SciTech Connect

    Vetting, Matthew W. Hegde, Subray S.; Blanchard, John S.

    2009-05-01

    A method to modify proteins with glutaraldehyde under reducing conditions is presented. Treatment with glutaraldehyde and dimethylaminoborane was found to result in cyclic pentylation of free amines and facilitated the structural determination of a protein previously recalcitrant to the formation of diffraction quality crystals. The pentapeptide-repeat protein EfsQnr from Enterococcus faecalis protects DNA gyrase from inhibition by fluoroquinolones. EfsQnr was cloned and purified to homogeneity, but failed to produce diffraction-quality crystals in initial crystallization screens. Treatment of EfsQnr with glutaraldehyde and the strong reducing agent borane–dimethylamine resulted in a derivatized protein which produced crystals that diffracted to 1.6 Å resolution; their structure was subsequently determined by single-wavelength anomalous dispersion. Analysis of the derivatized protein using Fourier transform ion cyclotron resonance mass spectrometry indicated a mass increase of 68 Da per free amino group. Electron-density maps about a limited number of structurally ordered lysines indicated that the modification was a cyclic pentylation of free amines, producing piperidine groups.

  17. In vivo interactions between procyanidins and human saliva proteins: effect of repeated exposures to procyanidins solution.

    PubMed

    Brandão, Elsa; Soares, Susana; Mateus, Nuno; de Freitas, Victor

    2014-10-01

    The general accepted mechanism for astringency arises from the interaction between tannins and salivary proteins (SP) resulting in (in)soluble aggregates. By HPLC analysis, it was observed that repeated sips of procyanidins (PC) solution practically depleted aPRPs (∼14%) and statherin (∼2%), and significantly reduced the amount of gPRPs. On the other hand, bPRPs were not significantly affected. In the analysis performed after the last exposure to PC solution, it was seen a significant recovering of the chromatographic peaks corresponding especially to aPRPs (∼74%) and statherin (∼80%). In vitro interaction between SP and PC results in the decrease of the chromatographic peaks of aPRPs and statherin, suggesting that these proteins were involved in the formation of a significant quantity of insoluble complexes. In general, the results suggest that the different families of SP can be involved in different stages of the development of astringency sensation.

  18. A designed ankyrin repeat protein evolved to picomolar affinity to Her2.

    PubMed

    Zahnd, Christian; Wyler, Emanuel; Schwenk, Jochen M; Steiner, Daniel; Lawrence, Michael C; McKern, Neil M; Pecorari, Frédéric; Ward, Colin W; Joos, Thomas O; Plückthun, Andreas

    2007-06-15

    Designed ankyrin repeat proteins (DARPins) are a novel class of binding molecules, which can be selected to recognize specifically a wide variety of target proteins. DARPins were previously selected against human epidermal growth factor receptor 2 (Her2) with low nanomolar affinities. We describe here their affinity maturation by error-prone PCR and ribosome display yielding clones with zero to seven (average 2.5) amino acid substitutions in framework positions. The DARPin with highest affinity (90 pM) carried four mutations at framework positions, leading to a 3000-fold affinity increase compared to the consensus framework variant, mainly coming from a 500-fold increase of the on-rate. This DARPin was found to be highly sensitive in detecting Her2 in human carcinoma extracts. We have determined the crystal structure of this DARPin at 1.7 A, and found that a His to Tyr mutation at the framework position 52 alters the inter-repeat H-bonding pattern and causes a significant conformational change in the relative disposition of the repeat subdomains. These changes are thought to be the reason for the enhanced on-rate of the mutated DARPin. The DARPin not bearing the residue 52 mutation has an unusually slow on-rate, suggesting that binding occurred via conformational selection of a relatively rare state, which was stabilized by this His52Tyr mutation, increasing the on-rate again to typical values. An analysis of the structural location of the framework mutations suggests that randomization of some framework residues either by error-prone PCR or by design in a future library could increase affinities and the target binding spectrum.

  19. Exaggerated phosphorylation of brain tau protein in CRH KO mice exposed to repeated immobilization stress.

    PubMed

    Kvetnansky, Richard; Novak, Petr; Vargovic, Peter; Lejavova, Katarina; Horvathova, Lubica; Ondicova, Katarina; Manz, George; Filipcik, Peter; Novak, Michal; Mravec, Boris

    2016-07-01

    Neuroendocrine and behavioral stress responses are orchestrated by corticotropin-releasing hormone (CRH) and norepinephrine (NE) synthesizing neurons. Recent findings indicate that stress may promote development of neurofibrillary pathology in Alzheimer's disease. Therefore, we investigated relationships among stress, tau protein phosphorylation, and brain NE using wild-type (WT) and CRH-knockout (CRH KO) mice. We assessed expression of phosphorylated tau (p-tau) at the PHF-1 epitope and NE concentrations in the locus coeruleus (LC), A1/C1 and A2/C2 catecholaminergic cell groups, hippocampus, amygdala, nucleus basalis magnocellularis, and frontal cortex of unstressed, singly stressed or repeatedly stressed mice. Moreover, gene expression and protein levels of tyrosine hydroxylase (TH) and CRH receptor mRNA were determined in the LC. Plasma corticosterone levels were also measured. Exposure to a single stress increases tau phosphorylation throughout the brain in WT mice when compared to singly stressed CRH KO animals. In contrast, repeatedly stressed CRH KO mice showed exaggerated tau phosphorylation relative to WT controls. We also observed differences in extent of tau phosphorylation between investigated structures, e.g. the LC and hippocampus. Moreover, CRH deficiency leads to different responses to stress in gene expression of TH, NE concentrations, CRH receptor mRNA, and plasma corticosterone levels. Our data indicate that CRH effects on tau phosphorylation are dependent on whether stress is single or repeated, and differs between brain regions. Our findings indicate that CRH attenuates mechanisms responsible for development of stress-induced tau neuropathology, particularly in conditions of chronic stress. However, the involvement of central catecholaminergic neurons in these mechanisms remains unclear and is in need of further investigation.

  20. Reduced stability of retinoblastoma protein by gankyrin, an oncogenic ankyrin-repeat protein overexpressed in hepatomas.

    PubMed

    Higashitsuji, H; Itoh, K; Nagao, T; Dawson, S; Nonoguchi, K; Kido, T; Mayer, R J; Arii, S; Fujita, J

    2000-01-01

    Hepatocellular carcinoma (HCC) is one of the most common cancers in Asia and Africa, where hepatitis virus infection and exposure to specific liver carcinogens are prevalent. Although inactivation of some tumor suppressor genes such as p53 and p16INK4Ahas been identified, no known oncogene is commonly activated in hepatocellular carcinomas. Here we have isolated genes overexpressed in hepatocellular carcinomas by cDNA subtractive hybridization, and identified an oncoprotein consisting of six ankyrin repeats (gankyrin). The expression of gankyrin was increased in all 34 hepatocellular carcinomas studied. Gankyrin induced anchorage-independent growth and tumorigenicity in NIH/3T3 cells. Gankyrin bound to the product of the retinoblastoma gene (RB1), increasing its phosphorylation and releasing the activity of the transcription factor E2F-1. Gankyrin accelerated the degradation of RB1 in vitro and in vivo, and was identical to or interacted with a subunit of the 26S proteasome. These results demonstrate the importance of ubiquitin-proteasome pathway in the regulation of cell growth and oncogenic transformation, and indicate that gankyrin overexpression contributes to hepatocarcinogenesis by destabilizing RB1.

  1. WD40-Repeat Proteins in Plant Cell Wall Formation: Current Evidence and Research Prospects

    PubMed Central

    Guerriero, Gea; Hausman, Jean-Francois; Ezcurra, Inés

    2015-01-01

    The metabolic complexity of living organisms relies on supramolecular protein structures which ensure vital processes, such as signal transduction, transcription, translation and cell wall synthesis. In eukaryotes WD40-repeat (WDR) proteins often function as molecular “hubs” mediating supramolecular interactions. WDR proteins may display a variety of interacting partners and participate in the assembly of complexes involved in distinct cellular functions. In plants, the formation of lignocellulosic biomass involves extensive synthesis of cell wall polysaccharides, a process that requires the assembly of large transmembrane enzyme complexes, intensive vesicle trafficking, interactions with the cytoskeleton, and coordinated gene expression. Because of their function as supramolecular hubs, WDR proteins could participate in each or any of these steps, although to date only few WDR proteins have been linked to the cell wall by experimental evidence. Nevertheless, several potential cell wall-related WDR proteins were recently identified using in silico approaches, such as analyses of co-expression, interactome and conserved gene neighborhood. Notably, some WDR genes are frequently genomic neighbors of genes coding for GT2-family polysaccharide synthases in eukaryotes, and this WDR-GT2 collinear microsynteny is detected in diverse taxa. In angiosperms, two WDR genes are collinear to cellulose synthase genes, CesAs, whereas in ascomycetous fungi several WDR genes are adjacent to chitin synthase genes, chs. In this Perspective we summarize and discuss experimental and in silico studies on the possible involvement of WDR proteins in plant cell wall formation. The prospects of biotechnological engineering for enhanced biomass production are discussed. PMID:26734023

  2. An update on polygalacturonase-inhibiting protein (PGIP), a leucine-rich repeat protein that protects crop plants against pathogens

    PubMed Central

    Kalunke, Raviraj M.; Tundo, Silvio; Benedetti, Manuel; Cervone, Felice; De Lorenzo, Giulia; D'Ovidio, Renato

    2015-01-01

    Polygalacturonase inhibiting proteins (PGIPs) are cell wall proteins that inhibit the pectin-depolymerizing activity of polygalacturonases secreted by microbial pathogens and insects. These ubiquitous inhibitors have a leucine-rich repeat structure that is strongly conserved in monocot and dicot plants. Previous reviews have summarized the importance of PGIP in plant defense and the structural basis of PG-PGIP interaction; here we update the current knowledge about PGIPs with the recent findings on the composition and evolution of pgip gene families, with a special emphasis on legume and cereal crops. We also update the information about the inhibition properties of single pgip gene products against microbial PGs and the results, including field tests, showing the capacity of PGIP to protect crop plants against fungal, oomycetes and bacterial pathogens. PMID:25852708

  3. A pollen-specific novel calmodulin-binding protein with tetratricopeptide repeats

    NASA Technical Reports Server (NTRS)

    Safadi, F.; Reddy, V. S.; Reddy, A. S.

    2000-01-01

    Calcium is essential for pollen germination and pollen tube growth. A large body of information has established a link between elevation of cytosolic Ca(2+) at the pollen tube tip and its growth. Since the action of Ca(2+) is primarily mediated by Ca(2+)-binding proteins such as calmodulin (CaM), identification of CaM-binding proteins in pollen should provide insights into the mechanisms by which Ca(2+) regulates pollen germination and tube growth. In this study, a CaM-binding protein from maize pollen (maize pollen calmodulin-binding protein, MPCBP) was isolated in a protein-protein interaction-based screening using (35)S-labeled CaM as a probe. MPCBP has a molecular mass of about 72 kDa and contains three tetratricopeptide repeats (TPR) suggesting that it is a member of the TPR family of proteins. MPCBP protein shares a high sequence identity with two hypothetical TPR-containing proteins from Arabidopsis. Using gel overlay assays and CaM-Sepharose binding, we show that the bacterially expressed MPCBP binds to bovine CaM and three CaM isoforms from Arabidopsis in a Ca(2+)-dependent manner. To map the CaM-binding domain several truncated versions of the MPCBP were expressed in bacteria and tested for their ability to bind CaM. Based on these studies, the CaM-binding domain was mapped to an 18-amino acid stretch between the first and second TPR regions. Gel and fluorescence shift assays performed with CaM and a CaM-binding synthetic peptide further confirmed MPCBP binding to CaM. Western, Northern, and reverse transcriptase-polymerase chain reaction analysis have shown that MPCBP expression is specific to pollen. MPCBP was detected in both soluble and microsomal proteins. Immunoblots showed the presence of MPCBP in mature and germinating pollen. Pollen-specific expression of MPCBP, its CaM-binding properties, and the presence of TPR motifs suggest a role for this protein in Ca(2+)-regulated events during pollen germination and growth.

  4. Serine-Aspartate Repeat Protein D Increases Staphylococcus aureus Virulence and Survival in Blood

    PubMed Central

    Uchiyama, Satoshi; Valderrama, J. Andrés; Ajayi, Clement; Sollid, Johanna U. E.; van Sorge, Nina M.; Nizet, Victor; van Strijp, Jos A. G.

    2016-01-01

    ABSTRACT Staphylococcus aureus expresses a panel of cell wall-anchored adhesins, including proteins belonging to the microbial surface components recognizing adhesive matrix molecule (MSCRAMM) family, exemplified by the serine-aspartate repeat protein D (SdrD), which serve key roles in colonization and infection. Deletion of sdrD from S. aureus subsp. aureus strain NCTC8325-4 attenuated bacterial survival in human whole blood ex vivo, which was associated with increased killing by human neutrophils. Remarkably, SdrD was able to inhibit innate immune-mediated bacterial killing independently of other S. aureus proteins, since addition of recombinant SdrD protein and heterologous expression of SdrD in Lactococcus lactis promoted bacterial survival in human blood. SdrD contributes to bacterial virulence in vivo, since fewer S. aureus subsp. aureus NCTC8325-4 ΔsdrD bacteria than bacteria of the parent strain were recovered from blood and several organs using a murine intravenous infection model. Collectively, our findings reveal a new property of SdrD as an important key contributor to S. aureus survival and the ability to escape the innate immune system in blood. PMID:27795358

  5. Ankyrin repeat-rich membrane spanning/Kidins220 protein interacts with mammalian Septin 5.

    PubMed

    Park, Han Jeong; Park, Hwan-Woo; Lee, Shin-Jae; Arevalo, Juan Carlos; Park, Young-Seok; Lee, Seung-Pyo; Paik, Ki-Suk; Chao, Moses V; Chang, Mi-Sook

    2010-08-01

    Neurotrophin receptors utilize specific adaptor proteins to activate signaling pathways involved in various neuronal functions, such as neurite outgrowth and cytoskeletal remodeling. The Ankyrin-Repeat Rich Membrane Spanning (ARMS)/kinase D-interacting substrate-220 kDa (Kidins220) serves as a unique downstream adaptor protein of Trk receptor tyrosine kinases. To gain insight into the role of ARMS/Kidins220, a yeast two-hybrid screen of a rat dorsal root ganglion library was performed using the C-terminal region of ARMS/Kidins220 as bait. The screen identified a mammalian septin, Septin 5 (Sept5), as an interacting protein. Co-immunoprecipitation using lysates from transiently transfected HEK-293 cells revealed the specific interaction between ARMS/Kidins220 and Sept5. Endogenous ARMS/Kidins220 and Sept5 proteins were colocalized in primary hippocampal neurons and were also predominantly expressed at the plasma membrane and in the tips of growing neurites in nerve growth factor-treated PC12 cells. Mapping of Sept5 domains important for ARMS/Kidins220 binding revealed a highly conserved N-terminal region of Sept5. The direct interaction between ARMS/Kidins220 and Sept5 suggests a possible role of ARMS/Kidins220 as a functional link between neurotrophin receptors and septins to mediate neurotrophin-induced intracellular signaling events, such as neurite outgrowth and cytoskeletal remodeling.

  6. Pentapeptide-repeat proteins that act as topoisomerase poison resistance factors have a common dimer interface

    PubMed Central

    Vetting, Matthew W.; Hegde, Subray S.; Zhang, Yong; Blanchard, John S.

    2011-01-01

    The protein AlbG is a self-resistance factor against albicidin, a nonribosomally encoded hybrid polyketide-peptide with antibiotic and phytotoxic properties produced by Xanthomonas albilineans. Primary-sequence analysis indicates that AlbG is a member of the pentapeptide-repeat family of proteins (PRP). The structure of AlbG from X. albilineans was determined at 2.0 Å resolution by SAD phasing using data collected from a single trimethyllead acetate derivative on a home source. AlbG folds into a right-handed quadrilateral β-helix composed of approximately eight semi-regular coils. The regularity of the β-­helix is blemished by a large loop/deviation in the β-helix between coils 4 and 5. The C-terminus of the β-helix is capped by a dimerization module, yielding a dimer with a 110 Å semi-collinear β-helical axis. This method of dimer formation appears to be common to all PRP proteins that confer resistance to topoisomerase poisons and contrasts with most PRP proteins, which are typically monomeric. PMID:21393830

  7. The effect of iron supplementation in the diet of Dasypus novemcinctus (Linnaeus, 1758) armadillos in captivity.

    PubMed

    Rosa, P S; Pinke, C A E; Pedrini, S C B; Silva, E A

    2009-02-01

    Armadillos of the species Dasypus novemcinctus have been used as an experimental model of leprosy. Besides non-human primates, they are the only species naturally infected with Mycobacterium leprae and when experimentally inoculated, reproduce the lepromatous form of the disease producing large quantities of bacilli. This species has been maintained in captivity by numerous researchers and specific housing and feeding requirements have been developed to guarantee their survival during long experimental periods. In the 'Lauro de Souza Lima' Institute, armadillos receive dog food, ground beef, boiled eggs and vitamin C. However, despite the balanced diet, anemia has been observed in some captive animals, especially in armadillos inoculated with M. leprae in advanced stages of infection. Thus, the objective of the present study was to evaluate the effect of iron sulfate supplementation in the feed provided for armadillos, both inoculated and non-inoculated with M. leprae, by means of the evaluation of their hematological profile. Fourteen armadillos received 10 mg/animal of iron sulfate (Hematofer(R)) diluted in sterile water mixed with their daily feed for 50 days. Hemograms and serum iron dosages for each armadillo were performed before and after supplementation. The hematocrit values increased significantly after iron supplementation, both in armadillos inoculated and non-inoculated with M. leprae. It is possible that the amount of iron in the feed is insufficient for the formation of hemoglobin, leading to microcytic anemia. Dietary supplementation with iron sulfate reversed this state, showing the importance of understanding the metabolism of exotic species for their maintenance in captivity, and thus ensuring their well-being.

  8. An isoform of microtubule-associated protein 2 (MAP2) containing four repeats of the tubulin-binding motif.

    PubMed

    Doll, T; Meichsner, M; Riederer, B M; Honegger, P; Matus, A

    1993-10-01

    Microtubule-associated protein 2 (MAP2) exists in both high- and low-molecular mass isoforms, each of which has a tubulin-binding domain consisting of 3 imperfect tandem repeats of 31 amino acids containing a more highly conserved 18 amino acid 'core' sequence. We describe here a novel form of low molecular mass MAP2 (MAP2c) that contains an additional 4th repeat of this tubulin-binding motif. Like the 3 previously known repeat sequences, this 4th copy is highly conserved between MAP2 and the two other known members of the same gene family, tau and MAP4. In each of these three genes the additional 4th repeat is inserted between the 1st and 2nd repeats of the 3-repeat form of the molecule. Experiments with brain cell cultures, in which the relative proportions of neurons and glia had been manipulated by drug treatment, showed that 4-repeat MAP2c is associated with glial cells whereas 3-repeat MAP2c is expressed in neurons. Whereas 3-repeat MAP2c is expressed early in development and then declines, the level of 4-repeat MAP2c increases later in development, corresponding to the relatively late differentiation of glial cells compared to neurons. When transfected into non-neuronal cells, the 4-repeat version of MAP2c behaved indistinguishably from the 3-repeat form in stabilising and rearranging cellular microtubules. The presence of an additional 4th repeat of the tubulin-binding motif in all three members of the MAP2 gene family suggests that this variant arose prior to their differentiation from an ancestral gene.

  9. Quantification of interaction strengths between chaperones and tetratricopeptide repeat domain-containing membrane proteins.

    PubMed

    Schweiger, Regina; Soll, Jürgen; Jung, Kirsten; Heermann, Ralf; Schwenkert, Serena

    2013-10-18

    The three tetratricopeptide repeat domain-containing docking proteins Toc64, OM64, and AtTPR7 reside in the chloroplast, mitochondrion, and endoplasmic reticulum of Arabidopsis thaliana, respectively. They are suggested to act during post-translational protein import by association with chaperone-bound preprotein complexes. Here, we performed a detailed biochemical, biophysical, and computational analysis of the interaction between Toc64, OM64, and AtTPR7 and the five cytosolic chaperones HSP70.1, HSP90.1, HSP90.2, HSP90.3, and HSP90.4. We used surface plasmon resonance spectroscopy in combination with Interaction Map® analysis to distinguish between chaperone oligomerization and docking protein-chaperone interactions and to calculate binding affinities for all tested interactions. Complementary to this, we applied pulldown assays as well as microscale thermophoresis as surface immobilization independent techniques. The data revealed that OM64 prefers HSP70 over HSP90, whereas Toc64 binds all chaperones with comparable affinities. We could further show that AtTPR7 is able to bind HSP90 in addition to HSP70. Moreover, differences between the HSP90 isoforms were detected and revealed a weaker binding for HSP90.1 to AtTPR7 and OM64, showing that slight differences in the amino acid composition or structure of the chaperones influence binding to the tetratricopeptide repeat domain. The combinatory approach of several methods provided a powerful toolkit to determine binding affinities of similar interaction partners in a highly quantitative manner.

  10. Bipartite Topology of Treponema pallidum Repeat Proteins C/D and I

    PubMed Central

    Anand, Arvind; LeDoyt, Morgan; Karanian, Carson; Luthra, Amit; Koszelak-Rosenblum, Mary; Malkowski, Michael G.; Puthenveetil, Robbins; Vinogradova, Olga; Radolf, Justin D.

    2015-01-01

    We previously identified Treponema pallidum repeat proteins TprC/D, TprF, and TprI as candidate outer membrane proteins (OMPs) and subsequently demonstrated that TprC is not only a rare OMP but also forms trimers and has porin activity. We also reported that TprC contains N- and C-terminal domains (TprCN and TprCC) orthologous to regions in the major outer sheath protein (MOSPN and MOSPC) of Treponema denticola and that TprCC is solely responsible for β-barrel formation, trimerization, and porin function by the full-length protein. Herein, we show that TprI also possesses bipartite architecture, trimeric structure, and porin function and that the MOSPC-like domains of native TprC and TprI are surface-exposed in T. pallidum, whereas their MOSPN-like domains are tethered within the periplasm. TprF, which does not contain a MOSPC-like domain, lacks amphiphilicity and porin activity, adopts an extended inflexible structure, and, in T. pallidum, is tightly bound to the protoplasmic cylinder. By thermal denaturation, the MOSPN and MOSPC-like domains of TprC and TprI are highly thermostable, endowing the full-length proteins with impressive conformational stability. When expressed in Escherichia coli with PelB signal sequences, TprC and TprI localize to the outer membrane, adopting bipartite topologies, whereas TprF is periplasmic. We propose that the MOSPN-like domains enhance the structural integrity of the cell envelope by anchoring the β-barrels within the periplasm. In addition to being bona fide T. pallidum rare outer membrane proteins, TprC/D and TprI represent a new class of dual function, bipartite bacterial OMP. PMID:25805501

  11. The nucleotide sequence, DNA damage location, and protein stoichiometry influence the base excision repair outcome at CAG/CTG repeats.

    PubMed

    Goula, Agathi-Vasiliki; Pearson, Christopher E; Della Maria, Julie; Trottier, Yvon; Tomkinson, Alan E; Wilson, David M; Merienne, Karine

    2012-05-08

    Expansion of CAG/CTG repeats is the underlying cause of >14 genetic disorders, including Huntington's disease (HD) and myotonic dystrophy. The mutational process is ongoing, with increases in repeat size enhancing the toxicity of the expansion in specific tissues. In many repeat diseases, the repeats exhibit high instability in the striatum, whereas instability is minimal in the cerebellum. We provide molecular insights into how base excision repair (BER) protein stoichiometry may contribute to the tissue-selective instability of CAG/CTG repeats by using specific repair assays. Oligonucleotide substrates with an abasic site were mixed with either reconstituted BER protein stoichiometries mimicking the levels present in HD mouse striatum or cerebellum, or with protein extracts prepared from HD mouse striatum or cerebellum. In both cases, the repair efficiency at CAG/CTG repeats and at control DNA sequences was markedly reduced under the striatal conditions, likely because of the lower level of APE1, FEN1, and LIG1. Damage located toward the 5' end of the repeat tract was poorly repaired, with the accumulation of incompletely processed intermediates as compared to an AP lesion in the center or at the 3' end of the repeats or within control sequences. Moreover, repair of lesions at the 5' end of CAG or CTG repeats involved multinucleotide synthesis, particularly at the cerebellar stoichiometry, suggesting that long-patch BER processes lesions at sequences susceptible to hairpin formation. Our results show that the BER stoichiometry, nucleotide sequence, and DNA damage position modulate repair outcome and suggest that a suboptimal long-patch BER activity promotes CAG/CTG repeat instability.

  12. A divergent Pumilio repeat protein family for pre-rRNA processing and mRNA localization

    DOE PAGES

    Qiu, Chen; McCann, Kathleen L.; Wine, Robert N.; ...

    2014-12-15

    Pumilio/feminization of XX and XO animals (fem)-3 mRNA-binding factor (PUF) proteins bind sequence specifically to mRNA targets using a single-stranded RNA-binding domain comprising eight Pumilio (PUM) repeats. PUM repeats have now been identified in proteins that function in pre-rRNA processing, including human Puf-A and yeast Puf6. This is a role not previously ascribed to PUF proteins. In this paper we present crystal structures of human Puf-A that reveal a class of nucleic acid-binding proteins with 11 PUM repeats arranged in an “L”-like shape. In contrast to classical PUF proteins, Puf-A forms sequence-independent interactions with DNA or RNA, mediated by conservedmore » basic residues. We demonstrate that equivalent basic residues in yeast Puf6 are important for RNA binding, pre-rRNA processing, and mRNA localization. Finally, PUM repeats can be assembled into alternative folds that bind to structured nucleic acids in addition to forming canonical eight-repeat crescent-shaped RNA-binding domains found in classical PUF proteins.« less

  13. A divergent Pumilio repeat protein family for pre-rRNA processing and mRNA localization

    SciTech Connect

    Qiu, Chen; McCann, Kathleen L.; Wine, Robert N.; Baserga, Susan J.; Hall, Traci M. Tanaka

    2014-12-15

    Pumilio/feminization of XX and XO animals (fem)-3 mRNA-binding factor (PUF) proteins bind sequence specifically to mRNA targets using a single-stranded RNA-binding domain comprising eight Pumilio (PUM) repeats. PUM repeats have now been identified in proteins that function in pre-rRNA processing, including human Puf-A and yeast Puf6. This is a role not previously ascribed to PUF proteins. In this paper we present crystal structures of human Puf-A that reveal a class of nucleic acid-binding proteins with 11 PUM repeats arranged in an “L”-like shape. In contrast to classical PUF proteins, Puf-A forms sequence-independent interactions with DNA or RNA, mediated by conserved basic residues. We demonstrate that equivalent basic residues in yeast Puf6 are important for RNA binding, pre-rRNA processing, and mRNA localization. Finally, PUM repeats can be assembled into alternative folds that bind to structured nucleic acids in addition to forming canonical eight-repeat crescent-shaped RNA-binding domains found in classical PUF proteins.

  14. Protection of Armadillo/β-Catenin by Armless, a Novel Positive Regulator of Wingless Signaling

    PubMed Central

    Reim, Gerlinde; Hruzova, Martina; Goetze, Sandra; Basler, Konrad

    2014-01-01

    The Wingless (Wg/Wnt) signaling pathway is essential for metazoan development, where it is central to tissue growth and cellular differentiation. Deregulated Wg pathway activation underlies severe developmental abnormalities, as well as carcinogenesis. Armadillo/β-Catenin plays a key role in the Wg transduction cascade; its cytoplasmic and nuclear levels directly determine the output activity of Wg signaling and are thus tightly controlled. In all current models, once Arm is targeted for degradation by the Arm/β-Catenin destruction complex, its fate is viewed as set. We identified a novel Wg/Wnt pathway component, Armless (Als), which is required for Wg target gene expression in a cell-autonomous manner. We found by genetic and biochemical analyses that Als functions downstream of the destruction complex, at the level of the SCF/Slimb/βTRCP E3 Ub ligase. In the absence of Als, Arm levels are severely reduced. We show by biochemical and in vivo studies that Als interacts directly with Ter94, an AAA ATPase known to associate with E3 ligases and to drive protein turnover. We suggest that Als antagonizes Ter94's positive effect on E3 ligase function and propose that Als promotes Wg signaling by rescuing Arm from proteolytic degradation, spotlighting an unexpected step where the Wg pathway signal is modulated. PMID:25369031

  15. Vaccinia virus K1L protein mediates host-range function in RK-13 cells via ankyrin repeat and may interact with a cellular GTPase-activating protein.

    PubMed

    Bradley, Ritu R; Terajima, Masanori

    2005-12-01

    The K1L protein of vaccinia virus is required for its growth in certain cell lines (RK-13 and human). The cowpox host-range protein CP77 has been shown to complement K1L function in RK-13 cells, despite a lack of homology between the two proteins except for ankyrin repeats. We investigated the role of ankyrin repeats of K1L protein in RK-13 cells. The growth of a recombinant vaccinia virus, with K1L gene mutated in the most conserved ankyrin repeat, was severely impaired. Infection with the mutant virus caused shutdown of cellular and viral protein synthesis early in infection. We also investigated the interaction of K1L protein with cellular proteins and found that K1L interacts with the rabbit homologue of human ACAP2, a GTPase-activating protein with ankyrin repeats. Our result suggests the importance of ankyrin repeat for host-range function of K1L in RK-13 cells and identifies ACAP2 as a cellular protein, which may be interacting with K1L.

  16. Destabilizing an interacting motif strengthens the association of a designed ankyrin repeat protein with tubulin

    PubMed Central

    Ahmad, Shoeb; Pecqueur, Ludovic; Dreier, Birgit; Hamdane, Djemel; Aumont-Nicaise, Magali; Plückthun, Andreas; Knossow, Marcel; Gigant, Benoît

    2016-01-01

    Affinity maturation by random mutagenesis and selection is an established technique to make binding molecules more suitable for applications in biomedical research, diagnostics and therapy. Here we identified an unexpected novel mechanism of affinity increase upon in vitro evolution of a tubulin-specific designed ankyrin repeat protein (DARPin). Structural analysis indicated that in the progenitor DARPin the C-terminal capping repeat (C-cap) undergoes a 25° rotation to avoid a clash with tubulin upon binding. Additionally, the C-cap appears to be involved in electrostatic repulsion with tubulin. Biochemical and structural characterizations demonstrated that the evolved mutants achieved a gain in affinity through destabilization of the C-cap, which relieves the need of a DARPin conformational change upon tubulin binding and removes unfavorable interactions in the complex. Therefore, this specific case of an order-to-disorder transition led to a 100-fold tighter complex with a subnanomolar equilibrium dissociation constant, remarkably associated with a 30% decrease of the binding surface. PMID:27380724

  17. Extended gene expression by medium exchange and repeated transient transfection for recombinant protein production enhancement.

    PubMed

    Cervera, Laura; Gutiérrez-Granados, Sonia; Berrow, Nicholas Simon; Segura, Maria Mercedes; Gòdia, Francesc

    2015-05-01

    Production of recombinant products in mammalian cell cultures can be achieved by stable gene expression (SGE) or transient gene expression (TGE). The former is based on the integration of a plasmid DNA into the host cell genome allowing continuous gene expression. The latter is based on episomal plasmid DNA expression. Conventional TGE is limited to a short production period of usually about 96 h, therefore limiting productivity. A novel gene expression approach termed extended gene expression (EGE) is explored in this study. The aim of EGE is to prolong the production period by the combination of medium exchange and repeated transfection of cell cultures with plasmid DNA to improve overall protein production. The benefit of this methodology was evaluated for the production of three model recombinant products: intracellular GFP, secreted GFP, and a Gag-GFP virus-like particles (VLPs). Productions were carried out in HEK 293 cell suspension cultures grown in animal-derived component free media using polyethylenimine (PEI) as transfection reagent. Transfections were repeated throughout the production process using different plasmid DNA concentrations, intervals of time, and culture feeding conditions in order to identify the best approach to achieve sustained high-level gene expression. Using this novel EGE strategy, the production period was prolonged between 192 and 240 h with a 4-12-fold increase in production levels, depending on the product type considered.

  18. Expression of Anaplasma marginale ankyrin repeat-containing proteins during infection of the mammalian host and tick vector

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Using searches of the NCBI conserved domain database and SMART genomic architecture analysis, we identified three ankyrin repeat-containing genes in Anaplasma marginale: AM705, AM926 and AM638. Recombinant protein was used to immunize mice and generate fusion hybridomas secreting protein-specific mo...

  19. Two novel transcripts encoding two Ankyrin repeat containing proteins have preponderant expression during the mouse spermatogenesis.

    PubMed

    Wang, Fei; Hu, Jiarui; Song, Ping; Gong, Wuming

    2007-12-01

    The clone 4921537P18 expressed preponderantly in mouse testis was identified by screening the Riken cDNA database, and two new full-length isoforms of this clone, which were named gsarp1 (Gonad Specific Ankyrin Repeat (ANK) Protein 1) and gsarp2, were found and isolated from mouse testis in the course of the research. Both of the GSARP1 and GSARP2 contain an ANK region circular composed by seven ANKs, and their structural feature is very similar to that of the IkappaB family proteins, while IkappaB proteins associate with the transcription factor NF-kappaB via their ANKs in the NF-kappaB pathway. We investigated the expression pattern at the mRNA level by Reverse transcription PCR. The gsarp1 has high expression level in mouse testis, while has low expression level in the ovary, and the gsarp2 is only expressed in mouse testis. The gsarp1 and gsarp2 begin to be detected at the early and later pachytene stage of meiosis separately, while both have high-expression level at the stage of MI and MII. The result of in situ hybridization reveals that the gsarp1 is primarily expressed in spermatocytes, while gsarp2 is expressed in spermatocytes and spermatids. In view of the structural feature and expression pattern of the GSARP1 and GSARP2, we speculate that they may play a certain role in a signal pathway of meiosis.

  20. reduced ocelli encodes the leucine rich repeat protein Pray For Elves in Drosophila melanogaster.

    PubMed

    Caldwell, Jason C; Fineberg, Sarah K; Eberl, Daniel F

    2007-01-01

    The ocelli are three simple photoreceptors on the vertex of the fruit fly head. We sought to identify the gene encoded by the classical ocellar mutant, reduced ocelli (rdo). Deficiency and inversion breakpoint mapping and P-element induced male recombination analyses were performed and Pray For Elves (PFE; CG15151; Fbgn0032661) emerged as a promising candidate for the rdo phenotype. The PFE locus maps to polytene region 36E on chromosome 2L between elfless (Fbgn0032660) and Arrestin 1 (Fbgn0000120). FlyBase annotation predicts that PFE encodes a serine/threonine kinase, yet protein prediction programs revealed no kinase domain. These analyses suggest that PFE simply encodes a leucine rich repeat molecule of unknown function, but presumably functions in nervous system protein-protein interaction. Two classical spontaneous alleles of rdo, rdo(1) and rdo(2), were characterized and the underlying mutations result from a small deletion spanning exon 1/intron 1 and a B104/roo insertion into the 3'UTR of PFE, respectively. Transposase-mediated excisions of several P-elements inserted into the PFE locus revert the rdo phenotype and a full-length PFE cDNA is sufficient to rescue rdo. A Gal4 enhancer trap reveals a broad adult neural expression pattern for PFE. Our identification and initial characterization of the rdo locus will contribute to the understanding of neurogenesis and neural development in the simple photoreceptors of the Drosophila visual system.

  1. TAPO: A combined method for the identification of tandem repeats in protein structures.

    PubMed

    Do Viet, Phuong; Roche, Daniel B; Kajava, Andrey V

    2015-09-14

    In recent years, there has been an emergence of new 3D structures of proteins containing tandem repeats (TRs), as a result of improved expression and crystallization strategies. Databases focused on structure classifications (PDB, SCOP, CATH) do not provide an easy solution for selection of these structures from PDB. Several approaches have been developed, but no best approach exists to identify the whole range of 3D TRs. Here we describe the TAndem PrOtein detector (TAPO) that uses periodicities of atomic coordinates and other types of structural representation, including strings generated by conformational alphabets, residue contact maps, and arrangements of vectors of secondary structure elements. The benchmarking shows the superior performance of TAPO over the existing programs. In accordance with our analysis of PDB using TAPO, 19% of proteins contain 3D TRs. This analysis allowed us to identify new families of 3D TRs, suggesting that TAPO can be used to regularly update the collection and classification of existing repetitive structures.

  2. The Leucine-rich Pentatricopeptide-Repeat Containing Protein Regulates Mitochondrial Transcription

    PubMed Central

    Sondheimer, Neal; Fang, Ji-Kang; Polyak, Erzsebet; Falk, Marni; Avadhani, Narayan G.

    2010-01-01

    Mitochondrial function depends upon the coordinated expression of the mitochondrial and nuclear genomes. Although the basal factors that carry out the process of mitochondrial transcription are known, the regulation of this process is incompletely understood. To further our understanding of mitochondrial gene regulation we identified proteins that bound to the previously described point of termination for the major mRNA-coding transcript H2. One was the leucine-rich pentatricopeptide-repeat containing protein (LRPPRC), which has been linked to the French-Canadian variant of Leigh syndrome. Cells with reduced expression of LRPPRC had a reduction in oxygen consumption. The expression of mitochondrial mRNA and tRNA was dependent upon LRPPRC levels, but reductions in LRPPRC did not affect the expression of mitochondrial rRNA. Reduction of LRPPRC levels interfered with mitochondrial transcription in vitro but did not affect the stability of mitochondrial mRNAs or alter the expression of nuclear genes responsible for mitochondrial transcription in vivo. These findings demonstrate the control of mitochondrial mRNA synthesis by a protein that has an established role in regulating nuclear transcription, and a link to mitochondrial disease. PMID:20677761

  3. Aurora A phosphorylation of WD40-repeat protein 62 in mitotic spindle regulation

    PubMed Central

    Lim, Nicholas R.; Yeap, Yvonne Y. C.; Ang, Ching-Seng; Williamson, Nicholas A.; Bogoyevitch, Marie A.; Quinn, Leonie M.; Ng, Dominic C. H.

    2016-01-01

    ABSTRACT Mitotic spindle organization is regulated by centrosomal kinases that potentiate recruitment of spindle-associated proteins required for normal mitotic progress including the microcephaly protein WD40-repeat protein 62 (WDR62). WDR62 functions underlie normal brain development as autosomal recessive mutations and wdr62 loss cause microcephaly. Here we investigate the signaling interactions between WDR62 and the mitotic kinase Aurora A (AURKA) that has been recently shown to cooperate to control brain size in mice. The spindle recruitment of WDR62 is closely correlated with increased levels of AURKA following mitotic entry. We showed that depletion of TPX2 attenuated WDR62 localization at spindle poles indicating that TPX2 co-activation of AURKA is required to recruit WDR62 to the spindle. We demonstrated that AURKA activity contributed to the mitotic phosphorylation of WDR62 residues Ser49 and Thr50 and phosphorylation of WDR62 N-terminal residues was required for spindle organization and metaphase chromosome alignment. Our analysis of several MCPH-associated WDR62 mutants (V65M, R438H and V1314RfsX18) that are mislocalized in mitosis revealed that their interactions and phosphorylation by AURKA was substantially reduced consistent with the notion that AURKA is a key determinant of WDR62 spindle recruitment. Thus, our study highlights the role of AURKA signaling in the spatiotemporal control of WDR62 at spindle poles where it maintains spindle organization. PMID:26713495

  4. Notchless encodes a novel WD40-repeat-containing protein that modulates Notch signaling activity.

    PubMed Central

    Royet, J; Bouwmeester, T; Cohen, S M

    1998-01-01

    Signaling by Notch family receptors is involved in many cell-fate decisions during development. Several modifiers of Notch activity have been identified, suggesting that regulation of Notch signaling is complex. In a genetic screen for modifiers of Notch activity, we identified a gene encoding a novel WD40-repeat protein. The gene is called Notchless, because loss-of-function mutant alleles dominantly suppress the wing notching caused by certain Notch alleles. Reducing Notchless activity increases Notch activity. Overexpression of Notchless in Xenopus or Drosophila appears to have a dominant-negative effect in that it also increases Notch activity. Biochemical studies show that Notchless binds to the cytoplasmic domain of Notch, suggesting that it serves as a direct regulator of Notch signaling activity. PMID:9857191

  5. Overexpression of NOTCH-regulated Ankyrin Repeat Protein is associated with papillary thyroid carcinoma progression

    PubMed Central

    Zhang, Mingdi; Qin, Yiyu; Zuo, Bin; Gong, Wei; Zhang, Shenglai; Gong, Yurong; Quan, Zhiwei; Chu, Bingfeng

    2017-01-01

    Papillary thyroid cancer (PTC) is one of the endocrine cancers with high clinical and genetic heterogeneity. NOTCH signaling and its downstream NOTCH-Regulated Ankyrin Repeat Protein (NRARP) have been implicated in oncogenesis of many cancers, but the roles in PTCs are less studied. In this study, we show that NRARP is frequently over-expressed in thyroid carcinoma. The over-activation of NRARP is highly and positively correlated with NOTCH genes. Moreover, we find that the expression of NRARP is highly associated with several epithelial mesenchymal transition (EMT) markers and contributes to poor survival outcomes. Therefore, these results indicate that NRARP is an important clinical biomarker in thyroid carcinoma and it promotes EMT induction as well as the progression of PTCs via NOTCH signaling activation. PMID:28207739

  6. A Conserved Region between the Heptad Repeats of Paramyxovirus Fusion Proteins is Critical for Proper F Protein Folding†

    PubMed Central

    Gardner, Amanda E.; Martin, Kimberly L.; Dutch, Rebecca E.

    2008-01-01

    Paramyxoviruses are a diverse family which utilizes a fusion (F) protein to enter cells via fusion of the viral lipid bilayer with a target cell membrane. Although certain regions of F are known to play critical roles in membrane fusion, the function of much of the protein remains unclear. Sequence alignment of a set of paramyxovirus F proteins and analysis utilizing Block Maker identified a region of conserved amino acid sequence in a large domain between the heptad repeats of F1, designated CBF1. We employed site-directed mutagenesis to analyze the function of completely conserved residues of CBF1 in both the simian virus 5 (SV5) and Hendra virus F proteins. The majority of CBF1 point mutants were deficient in homotrimer formation, proteolytic processing, and transport to the cell surface. For some SV5 F mutants, proteolytic cleavage and surface expression could be restored by expression at 30°C, and varying levels of fusion promotion were observed at this temperature. In addition, the mutant SV5 F V402A displayed a hyperfusogenic phenotype at both 30°C and 37°C, indicating this mutation allows for efficient fusion with only an extremely small amount of cleaved, active protein. The recently published prefusogenic structure of PIV5/SV5 F [Yin, H.S., et al. (2006) Nature 439, 38–44] indicates that residues within and flanking CBF1 interact with the fusion peptide domain. Together, these data suggest that CBF1-fusion peptide interactions are critical for the initial folding of paramyxovirus F proteins from across this important viral family, and can also modulate subsequent membrane fusion promotion. PMID:17417875

  7. Force Spectroscopy of the Plasmodium falciparum Vaccine Candidate Circumsporozoite Protein Suggests a Mechanically Pliable Repeat Region.

    PubMed

    Patra, Aditya Prasad; Sharma, Shobhona; Ainavarapu, Sri Rama Koti

    2017-02-10

    The most effective vaccine candidate of malaria is based on the Plasmodium falciparum circumsporozoite protein (CSP), a major surface protein implicated in the structural strength, motility, and immune evasion properties of the infective sporozoites. It is suspected that reversible conformational changes of CSP are required for infection of the mammalian host, but the detailed structure and dynamic properties of CSP remain incompletely understood, limiting our understanding of its function in the infection. Here, we report the structural and mechanical properties of the CSP studied using single-molecule force spectroscopy on several constructs, one including the central region of CSP, which is rich in NANP amino acid repeats (CSPrep), and a second consisting of a near full-length sequence without the signal and anchor hydrophobic domains (CSPΔHP). Our results show that the CSPrep is heterogeneous, with 40% of molecules requiring virtually no mechanical force to unfold (<10 piconewtons (pN)), suggesting that these molecules are mechanically compliant and perhaps act as entropic springs, whereas the remaining 60% are partially structured with low mechanical resistance (∼70 pN). CSPΔHP having multiple force peaks suggests specifically folded domains, with two major populations possibly indicating the open and collapsed forms. Our findings suggest that the overall low mechanical resistance of the repeat region, exposed on the outer surface of the sporozoites, combined with the flexible full-length conformations of CSP, may provide the sporozoites not only with immune evasion properties, but also with lubricating capacity required during its navigation through the mosquito and vertebrate host tissues. We anticipate that these findings would further assist in the design and development of future malarial vaccines.

  8. The armadillo: a model for the neuropathy of leprosy and potentially other neurodegenerative diseases.

    PubMed

    Sharma, Rahul; Lahiri, Ramanuj; Scollard, David M; Pena, Maria; Williams, Diana L; Adams, Linda B; Figarola, John; Truman, Richard W

    2013-01-01

    Leprosy (also known as Hansen's disease) is an infectious peripheral neurological disorder caused by Mycobacterium leprae that even today leaves millions of individuals worldwide with life-long disabilities. The specific mechanisms by which this bacterium induces nerve injury remain largely unknown, mainly owing to ethical and practical limitations in obtaining affected human nerve samples. In addition to humans, nine-banded armadillos (Dasypus novemcinctus) are the only other natural host of M. leprae, and they develop a systemically disseminated disease with extensive neurological involvement. M. leprae is an obligate intracellular parasite that cannot be cultivated in vitro. Because of the heavy burdens of bacilli they harbor, nine-banded armadillos have become the organism of choice for propagating large quantities of M. leprae, and they are now advancing as models of leprosy pathogenesis and nerve damage. Although armadillos are exotic laboratory animals, the recently completed whole genome sequence for this animal is enabling researchers to undertake more sophisticated molecular studies and to develop armadillo-specific reagents. These advances will facilitate the use of armadillos in piloting new therapies and diagnostic regimens, and will provide new insights into the oldest known infectious neurodegenerative disorder.

  9. The armadillo: a model for the neuropathy of leprosy and potentially other neurodegenerative diseases

    PubMed Central

    Sharma, Rahul; Lahiri, Ramanuj; Scollard, David M.; Pena, Maria; Williams, Diana L.; Adams, Linda B.; Figarola, John; Truman, Richard W.

    2013-01-01

    Leprosy (also known as Hansen’s disease) is an infectious peripheral neurological disorder caused by Mycobacterium leprae that even today leaves millions of individuals worldwide with life-long disabilities. The specific mechanisms by which this bacterium induces nerve injury remain largely unknown, mainly owing to ethical and practical limitations in obtaining affected human nerve samples. In addition to humans, nine-banded armadillos (Dasypus novemcinctus) are the only other natural host of M. leprae, and they develop a systemically disseminated disease with extensive neurological involvement. M. leprae is an obligate intracellular parasite that cannot be cultivated in vitro. Because of the heavy burdens of bacilli they harbor, nine-banded armadillos have become the organism of choice for propagating large quantities of M. leprae, and they are now advancing as models of leprosy pathogenesis and nerve damage. Although armadillos are exotic laboratory animals, the recently completed whole genome sequence for this animal is enabling researchers to undertake more sophisticated molecular studies and to develop armadillo-specific reagents. These advances will facilitate the use of armadillos in piloting new therapies and diagnostic regimens, and will provide new insights into the oldest known infectious neurodegenerative disorder. PMID:23223615

  10. Nuclear localization of MBNL1: splicing-mediated autoregulation and repression of repeat-derived aberrant proteins.

    PubMed

    Kino, Yoshihiro; Washizu, Chika; Kurosawa, Masaru; Oma, Yoko; Hattori, Nobutaka; Ishiura, Shoichi; Nukina, Nobuyuki

    2015-02-01

    In some neurological diseases caused by repeat expansions such as myotonic dystrophy, the RNA-binding protein muscleblind-like 1 (MBNL1) accumulates in intranuclear inclusions containing mutant repeat RNA. The interaction between MBNL1 and mutant RNA in the nucleus is a key event leading to loss of MBNL function, yet the details of this effect have been elusive. Here, we investigated the mechanism and significance of MBNL1 nuclear localization. We found that MBNL1 contains two classes of nuclear localization signal (NLS), a classical bipartite NLS and a novel conformational NLS. Alternative splicing of exon 7 acts as a switch between these NLS types and couples MBNL1 activity and intracellular localization. Depending on its nuclear localization, MBNL1 promoted nuclear accumulation of mutant RNA containing a CUG or CAG repeat, some of which produced proteins containing homopolymeric tracts such as polyglutamine. Furthermore, MBNL1 repressed the expression of these homopolymeric proteins including those presumably produced through repeat-associated non-ATG (RAN) translation. These results suggest that nuclear retention of expanded RNA reflects a novel role of MBNL proteins in repressing aberrant protein expression and may provide pathological and therapeutic implications for a wide range of repeat expansion diseases associated with nuclear RNA retention and/or RAN translation.

  11. RAP, the sole octotricopeptide repeat protein in Arabidopsis, is required for chloroplast 16S rRNA maturation.

    PubMed

    Kleinknecht, Laura; Wang, Fei; Stübe, Roland; Philippar, Katrin; Nickelsen, Jörg; Bohne, Alexandra-Viola

    2014-02-01

    The biogenesis and activity of chloroplasts in both vascular plants and algae depends on an intracellular network of nucleus-encoded, trans-acting factors that control almost all aspects of organellar gene expression. Most of these regulatory factors belong to the helical repeat protein superfamily, which includes tetratricopeptide repeat, pentatricopeptide repeat, and the recently identified octotricopeptide repeat (OPR) proteins. Whereas green algae express many different OPR proteins, only a single orthologous OPR protein is encoded in the genomes of most land plants. Here, we report the characterization of the only OPR protein in Arabidopsis thaliana, RAP, which has previously been implicated in plant pathogen defense. Loss of RAP led to a severe defect in processing of chloroplast 16S rRNA resulting in impaired chloroplast translation and photosynthesis. In vitro RNA binding and RNase protection assays revealed that RAP has an intrinsic and specific RNA binding capacity, and the RAP binding site was mapped to the 5' region of the 16S rRNA precursor. Nucleoid localization of RAP was shown by transient green fluorescent protein import assays, implicating the nucleoid as the site of chloroplast rRNA processing. Taken together, our data indicate that the single OPR protein in Arabidopsis is important for a basic process of chloroplast biogenesis.

  12. Mycobacterium leprae in six-banded (Euphractus sexcinctus) and nine-banded armadillos (Dasypus novemcinctus) in Northeast Brazil.

    PubMed

    Frota, Cristiane Cunha; Lima, Luana Nepomuceno Costa; Rocha, Adalgiza da Silva; Suffys, Philip Noel; Rolim, Benedito Neilson; Rodrigues, Laura Cunha; Barreto, Maurício Lima; Kendall, Carl; Kerr, Ligia Regina Sansigolo

    2012-12-01

    Human beings are the main reservoir of the causative agent of leprosy, Mycobacterium leprae. In the Americas, nine-banded armadillos (Dasypus novemcinctus) also act as a reservoir for the bacillus. In the state of Ceará (CE), which is located in Northeast Brazil and is an endemic area of leprosy, there are several species of armadillos, including D. novemcinctus and Euphractus sexcinctus (six-banded armadillo). Contact between humans and armadillos occur mainly through hunting, cleaning, preparing, cooking and eating. This study identified M. leprae DNA in the two main species of armadillos found in Northeast Brazil. A total of 29 wild armadillos (27 D. novemcinctus and 2 E. sexcinctus) were captured in different environments of CE countryside. Samples from the ear, nose, liver and spleen from each of these animals were tested by a nested M. leprae-specific repetitive element polymerase chain reaction assay. The samples that tested positive were confirmed by DNA sequencing. M. leprae was detected in 21% (6/29) of the animals, including five D. novemcinctus and one E. sexcinctus. This is the first Brazilian study to identify the presence of a biomarker of M. leprae in wild armadillos (D. novemcinctus and E. sexcinctus) in a leprosy hyperendemic area where there is continuous contact between humans and armadillos.

  13. The Protein Synthesis Inhibitor Blasticidin S Enters Mammalian Cells via Leucine-rich Repeat-containing Protein 8D

    PubMed Central

    Lee, Clarissa C.; Freinkman, Elizaveta; Sabatini, David M.; Ploegh, Hidde L.

    2014-01-01

    Leucine-rich repeat-containing 8 (LRRC8) proteins have been identified as putative receptors involved in lymphocyte development and adipocyte differentiation. They remain poorly characterized, and no specific function has been assigned to them. There is no consensus on how this family of proteins might function because homology searches suggest that members of the LRRC8 family act not as plasma membrane receptors, but rather as channels that mediate cell-cell signaling. Here we provide experimental evidence that supports a role for LRRC8s in the transport of small molecules. We show that LRRC8D is a mammalian protein required for the import of the antibiotic blasticidin S. We characterize localization and topology of LRRC8A and LRRC8D and demonstrate that LRRC8D interacts with LRRC8A, LRRC8B, and LRRC8C. Given the suggested involvement in solute transport, our results support a model in which LRRC8s form one or more complexes that may mediate cell-cell communication by transporting small solutes. PMID:24782309

  14. Frameshift Mutation Confers Function as Virulence Factor to Leucine-Rich Repeat Protein from Acidovorax avenae

    PubMed Central

    Kondo, Machiko; Hirai, Hiroyuki; Furukawa, Takehito; Yoshida, Yuki; Suzuki, Aika; Kawaguchi, Takemasa; Che, Fang-Sik

    2017-01-01

    Many plant pathogens inject type III (T3SS) effectors into host cells to suppress host immunity and promote successful infection. The bacterial pathogen Acidovorax avenae causes brown stripe symptom in many species of monocotyledonous plants; however, individual strains of each pathogen infect only one host species. T3SS-deleted mutants of A. avenae K1 (virulent to rice) or N1141 (virulent to finger millet) caused no symptom in each host plant, suggesting that T3SS effectors are involved in the symptom formation. To identify T3SS effectors as virulence factors, we performed whole-genome and predictive analyses. Although the nucleotide sequence of the novel leucine-rich repeat protein (Lrp) gene of N1141 had high sequence identity with K1 Lrp, the amino acid sequences of the encoded proteins were quite different due to a 1-bp insertion within the K1 Lrp gene. An Lrp-deleted K1 strain (KΔLrp) did not cause brown stripe symptom in rice (host plant for K1); by contrast, the analogous mutation in N1141 (NΔLrp) did not interfere with infection of finger millet. In addition, NΔLrp retained the ability to induce effector-triggered immunity (ETI), including hypersensitive response cell death and expression of ETI-related genes. These data indicated that K1 Lrp functions as a virulence factor in rice, whereas N1141 Lrp does not play a similar role in finger millet. Yeast two-hybrid screening revealed that K1 Lrp interacts with oryzain α, a pathogenesis-related protein of the cysteine protease family, whereas N1141 Lrp, which contains LRR domains, does not. This specific interaction between K1 Lrp and oryzain α was confirmed by Bimolecular fluorescence complementation assay in rice cells. Thus, K1 Lrp protein may have acquired its function as virulence factor in rice due to a frameshift mutation. PMID:28101092

  15. Histology, histochemistry and fine structure of the lacrimal and nictitans gland in the South American armadillo Chaetophractus villosus (Xenarthra, Mammalia).

    PubMed

    Aldana Marcos, Hernán Javier; Cintia Ferrari, Carina; Cervino, Claudio; Affanni, Jorge Mario

    2002-12-01

    The anatomical, histological, histochemical and ultrastructural characteristics of the lacrimal gland (LG) and nictitans gland (NG) of the armadillo Chaetophractus villosus were described. The histochemical and histological features of both glands in male and female adult animals were compared. The tissues were processed with conventional techniques for light and transmission electron microscopy. Fixed specimens were submitted to a battery of tests for glycans, glycosaminglycans, glycoconjugates, proteins, and lipids. The LG of the armadillo may be considered within the set of glandulae lacrimales superior in which primates, carnivores, perisodactyls and artiodactyls are included. The localization of the NG was similar to that of other mammals. Lacrimal and NG were histologically and histochemically identical. The secretory endpieces consisted of three cell types: (1) Mucous cells (MC) with different types of mucous secretory granules with neutral and sialic acid-containing glycoconjugates (GCs). (2)Seromucous cells (SMC) showing a variety of moderately electron dense secretory granules with flocculent material with carboxylated acidic, neutral, and sialic acid-containing GCs. Intercellular canaliculi with junctional complexes and basolateral intercellular spaces were frequent. (3) Serous cells (SC) with electron dense secretory granules. Histochemically, they showed the strongest reaction for proteins and neutral, weakly acid and carboxylated acidic GCs. The epithelium of the intra- and inter-lobular excretory ducts showed secretory activity, junctional complexes, and wide basolateral intercellular spaces with lateral folds. The endpieces and ducts were surrounded by myoepithelial cells. The stroma was characterized by fenestrated endothelium, unmyelinated axons, and abundant plasma cells. MC, SMC, and the duct system were richly innervated by hypolemmal nerve terminals.

  16. Spermatogenesis is seasonal in the large hairy armadillo, Chaetophractus villosus (Dasypodidae, Xenarthra, Mammalia).

    PubMed

    Luaces, Juan P; Rossi, Luis F; Merico, Valeria; Zuccotti, Maurizio; Redi, Carlo A; Solari, Alberto J; Merani, Maria S; Garagna, Silvia

    2013-01-01

    Very little is known about the distinct reproductive biology of armadillos. Very few studies have investigated armadillo spermatogenesis, with data available only for Euphractus sexcinctus and Dasypus novemcinctus. In the present study, we analysed male germ cell differentiation in the large hairy armadillo Chaetophractus villosus throughout the year, describing a cycle of the seminiferous epithelium made of eight different stages. Evaluation of the testis/body mass ratio, analysis of the architecture of the seminiferous epithelium and the frequency of defective seminiferous tubules allowed identification of a temporal interruption of spermatogenesis during the period between mid-May to July (mid-end autumn) in correlation with very low testosterone levels. Overall, these results suggest that spermatogenesis is seasonal in C. villosus.

  17. Ultrastructural characterisation of the olfactory mucosa of the armadillo Dasypus hybridus (Dasypodidae, Xenarthra)

    PubMed Central

    FERRARI, C. C.; CARMANCHAHI, P. D.; ALDANA MARCOS, H. J.; AFFANNI, J. M.

    2000-01-01

    The ultrastructure of the olfactory mucosa of the armadillo Dasypus hybridus was studied. A comparison with the olfactory mucosa of another armadillo (Chaetophractus villosus) was made. The olfactory mucosa of D. hybridus shows many features which are similar to those of other mammals. Interestingly, it differs from the olfactory mucosa of the armadillo C. villosus. A suggestion is made that these differences may be due to differences in the digging habits of these species. In Dasypus, the supporting cells (SCs) showed dense vacuoles, multivesicular bodies and lysosome-like bodies probably related with the endocytotic system. The SCs show a dense network of SER presumably associated with xenobiotic mechanisms. The olfactory receptor neurons exhibit lysosome-like bodies and multivesicular bodies in their perikarya. These organelles suggest the presence of an endocytotic system. Duct cells of Bowman's glands exhibit secretory activities. Bowman's glands are compound-branched tubulo-acinar mixed glands with merocrine secretory mechanisms. PMID:10739023

  18. Intestinal parasites of Tolypeutes matacus, the most frequently consumed armadillo in the Chaco region.

    PubMed

    Ríos, T A; Ezquiaga, M C; Abba, A M; Navone, G T

    2016-12-01

    The southern three-banded armadillo Tolypeutes matacus (Desmarest, 1804) is distributed from eastern Bolivia, south-west Brazil, the Gran Chaco of Paraguay and Argentina, and lives in areas with dry vegetation. This armadillo is one of the most frequently consumed species by people in this area. The objective of this work was test for zoonotic species among helminths in 12 intestinal tracts of T. matacus in a locality from the Argentinean Chaco (Chamical, La Rioja province). The parasites were studied with conventional parasite morphology and morphometrics, and prevalence, mean intensity and mean abundance were calculated for each species encountered. In the small intestine, seven species of nematodes and two species of cestodes were identified. In the large intestine, two species of nematodes were recorded. We did not find zoonotic species but have added new host records. This study in the Chaco region thus contributes to growing knowledge of the parasite fauna associated with armadillo species in this region.

  19. The tetratricopeptide repeat-containing protein slow green1 is required for chloroplast development in Arabidopsis.

    PubMed

    Hu, Zhihong; Xu, Fan; Guan, Liping; Qian, Pingping; Liu, Yaqiong; Zhang, Huifang; Huang, Yan; Hou, Suiwen

    2014-03-01

    A new gene, SG1, was identified in a slow-greening mutant (sg1) isolated from an ethylmethanesulphonate-mutagenized population of Arabidopsis thaliana. The newly formed leaves of sg1 were initially albino, but gradually became pale green. After 3 weeks, the leaves of the mutant were as green as those of the wild-type plants. Transmission electron microscopic observations revealed that the mutant displayed delayed proplastid to chloroplast transition. The results of map-based cloning showed that SG1 encodes a chloroplast-localized tetratricopeptide repeat-containing protein. Quantitative real-time reverse transcription-PCR data demonstrated the presence of SG1 gene expression in all tissues, particularly young green tissues. The sg1 mutation disrupted the expression levels of several genes associated with chloroplast development, photosynthesis, and chlorophyll biosynthesis. The results of genetic analysis indicated that gun1 and gun4 partially restored the expression patterns of the previously detected chloroplast-associated genes, thereby ameliorating the slow-greening phenotype of sg1. Taken together, the results suggest that the newly identified protein, SG1, is required for chloroplast development in Arabidopsis.

  20. Functional analysis of a RING domain ankyrin repeat protein that is highly expressed during flower senescence.

    PubMed

    Xu, Xinjia; Jiang, Cai-Zhong; Donnelly, Linda; Reid, Michael S

    2007-01-01

    A gene encoding a RING zinc finger ankyrin repeat protein (MjXB3), a putative E3 ubiquitin ligase, is highly expressed in petals of senescing four o'clock (Mirabilis jalapa) flowers, increasing >40,000-fold during the onset of visible senescence. The gene has homologues in many other species, and the Petunia homologue is strongly up-regulated in senescing Petunia corollas. Silencing the expression of this gene in Petunia, using virus-induced gene silencing, resulted in a 2 d extension in flower life. In Mirabilis, a 2 kb promoter region, 5' upstream of the MjXB3 gene, was isolated. The promoter sequence included putative binding sites for many DNA-binding proteins, including the bZIP, Myb, homeodomain-leucine zipper (HD-Zip), MADS-box, and WRKY transcription factors. The construct containing a 1 kb promoter region immediately upstream of the MjXB3 gene drove the strongest expression of the beta-glucuronidase (GUS) reporter gene in a transient expression assay. In Petunia, GUS expression under the control of this heterologous promoter fragment was specific to senescing flowers. The Mirabilis promoter GUS construct was tested in other flower species; while GUS activity in carnation petals was high during senescence, no expression was detected in three monocotyledonous flowers--daylily (Hemerocallis 'Stella d'Oro'), daffodil (Narcissus pseudonarcissus 'King Alfred'), and orchid (Dendrobium 'Emma White').

  1. Ribosome-associated pentatricopeptide repeat proteins function as translational activators in mitochondria of trypanosomes

    PubMed Central

    Aphasizheva, Inna; Maslov, Dmitri A.; Qian, Yu; Huang, Lan; Wang, Qi; Costello, Catherine E.; Aphasizhev, Ruslan

    2016-01-01

    Summary Mitochondrial ribosomes of Trypanosoma brucei are composed of 9S and 12S rRNAs, eubacterial-type ribosomal proteins, polypeptides lacking discernible motifs and approximately 20 pentatricopeptide repeat (PPR) RNA binding proteins. Several PPRs also populate the polyadenylation complex; among these, KPAF1 and KPAF2 function as general mRNA 3′ adenylation/uridylation factors. The A/U-tail enables mRNA binding to the small ribosomal subunit and is essential for translation. The presence of A/U-tail also correlates with requirement for translation of certain mRNAs in mammalian and insect parasite stages. Here, we inquired whether additional PPRs activate translation of individual mRNAs. Proteomic analysis identified KRIPP1 and KRIPP8 as components of the small ribosomal subunit in mammalian and insect forms, but also revealed their association with the polyadenylation complex in the latter. RNAi knockdowns demonstrated essential functions of KRIPP1 and KRIPP8 in the actively respiring insect stage, but not in the mammalian stage. In the KRIPP1 knockdown, A/U-tailed mRNA encoding cytochrome c oxidase subunit 1 declined concomitantly with the de novo synthesis of this subunit whereas polyadenylation and translation of cyb mRNA were unaffected. In contrast, the KRIPP8 knockdown inhibited A/U-tailing and translation of both CO1 and cyb mRNAs. Our findings indicate that ribosome-associated PPRs may selectively activate mRNAs for translation. PMID:26713541

  2. Overexpression of MIP2, a novel WD-repeat protein, promotes proliferation of H9c2 cells

    SciTech Connect

    Wei, Xing; Song, Lan; Jiang, Lei; Wang, Guiliang; Luo, Xinjing; Zhang, Bin; Xiao, Xianzhong

    2010-03-19

    WD40 repeat proteins have a wide range of diverse biological functions including signal transduction, cell cycle regulation, RNA splicing, and transcription. Myocardial ischemic preconditioning up-regulated protein 2 (MIP2) is a novel member of the WD40 repeat proteins superfamily that contains five WD40 repeats. Little is known about its biological role, and the purpose of this study was to determine the role of MIP2 in regulating cellular proliferation. Transfection and constitutive expression of MIP2 in the rat cardiomyoblast cell line H9c2 results in enhanced growth of those cells as measured by cell number and is proportional to the amount of MIP2 expressed. Overexpression of MIP2 results in a shorter cell cycle, as measured by flow cytometry. Collectively, these data suggest that MIP2 may participate in the progression of cell proliferation in H9c2 cells.

  3. SdrI, a serine-aspartate repeat protein identified in Staphylococcus saprophyticus strain 7108, is a collagen-binding protein.

    PubMed

    Sakinc, Türkan; Kleine, Britta; Gatermann, Sören G

    2006-08-01

    A gene encoding a serine-aspartate repeat protein of Staphylococcus saprophyticus, an important cause of urinary tract infections in young women, has been cloned and sequenced. In contrast to other SD repeat proteins, SdrI carries 21 additional N-terminal repeats with a consensus sequence of (P/A)ATKE(K/E)A(A/V)(T/I)(A/T/S)EE and has the longest SD(AD)(1-5) repetitive region (854 amino acids) described so far. This highly repetitive sequence contains only the amino acids serine, asparagine, and a distinctly greater amount of alanine (37%) than all other known SD repeat proteins (2.3 to 4.4%). In addition, it is a collagen-binding protein of S. saprophyticus and the second example in this organism of a surface protein carrying the LPXTG motif. We constructed an isogenic sdrI knockout mutant that showed decreased binding to immobilized collagen compared with wild-type S. saprophyticus strain 7108. Binding could be reconstituted by complementation. Collagen binding is specifically caused by SdrI, and the recently described UafA protein, the only LPXTG-containing protein in the genome sequence of the type strain, is not involved in this trait. Our experiments suggest that, as in other staphylococci, the presence of different LPXTG-anchored cell wall proteins is common in S. saprophyticus and support the notion that the presence of matrix-binding surface proteins is common in staphylococci.

  4. Structural determinants for improved stability of designed ankyrin repeat proteins with a redesigned C-capping module.

    PubMed

    Kramer, Michaela A; Wetzel, Svava K; Plückthun, Andreas; Mittl, Peer R E; Grütter, Markus G

    2010-12-03

    Designed ankyrin repeat proteins (DARPins) that specifically bind to almost any target can be obtained by ribosome display or phage display from combinatorial libraries. Although DARPins are already very stable molecules, molecular dynamics simulations, equilibrium denaturation experiments, structural studies, and recent NMR experiments suggested that the unfolding of the original C-terminal capping repeat (C-cap), taken from a natural ankyrin repeat protein, limits the stability of the initial DARPin design. Several point mutations had been introduced to optimize the C-cap and were shown to indeed further increase the stability of DARPins. We now determined crystal structures of DARPins with one or three full-consensus internal repeats (NI(1)C or NI(3)C) between an N-terminal capping repeat and mutants of the C-cap. An NI(1)C mutant, in which the C-cap was only extended by three additional helix-forming residues, showed no structural change but reduced B-factors in the C-cap. An NI(3)C C-cap mutant carrying five additional mutations in the interface to the preceding repeat, previously designed by using the consensus sequence as a guide, showed a rigid-body movement of the C-cap towards the internal repeat. This movement results in an increased buried surface area and a superior surface complementarity and explains the improved stability in equilibrium unfolding, compared to the original C-cap. A C-cap mutant with three additional mutations introducing suitably spaced charged residues did not show formation of salt bridges, explaining why its stability was not increased further. These structural studies underline the importance of repeat coupling for stability and help in the further design of this protein family.

  5. A repeat protein links Rubisco to form the eukaryotic carbon-concentrating organelle

    PubMed Central

    Mackinder, Luke C. M.; Meyer, Moritz T.; Mettler-Altmann, Tabea; Chen, Vivian K.; Mitchell, Madeline C.; Caspari, Oliver; Freeman Rosenzweig, Elizabeth S.; Pallesen, Leif; Reeves, Gregory; Itakura, Alan; Roth, Robyn; Sommer, Frederik; Geimer, Stefan; Mühlhaus, Timo; Schroda, Michael; Goodenough, Ursula; Stitt, Mark; Griffiths, Howard; Jonikas, Martin C.

    2016-01-01

    Biological carbon fixation is a key step in the global carbon cycle that regulates the atmosphere's composition while producing the food we eat and the fuels we burn. Approximately one-third of global carbon fixation occurs in an overlooked algal organelle called the pyrenoid. The pyrenoid contains the CO2-fixing enzyme Rubisco and enhances carbon fixation by supplying Rubisco with a high concentration of CO2. Since the discovery of the pyrenoid more that 130 y ago, the molecular structure and biogenesis of this ecologically fundamental organelle have remained enigmatic. Here we use the model green alga Chlamydomonas reinhardtii to discover that a low-complexity repeat protein, Essential Pyrenoid Component 1 (EPYC1), links Rubisco to form the pyrenoid. We find that EPYC1 is of comparable abundance to Rubisco and colocalizes with Rubisco throughout the pyrenoid. We show that EPYC1 is essential for normal pyrenoid size, number, morphology, Rubisco content, and efficient carbon fixation at low CO2. We explain the central role of EPYC1 in pyrenoid biogenesis by the finding that EPYC1 binds Rubisco to form the pyrenoid matrix. We propose two models in which EPYC1’s four repeats could produce the observed lattice arrangement of Rubisco in the Chlamydomonas pyrenoid. Our results suggest a surprisingly simple molecular mechanism for how Rubisco can be packaged to form the pyrenoid matrix, potentially explaining how Rubisco packaging into a pyrenoid could have evolved across a broad range of photosynthetic eukaryotes through convergent evolution. In addition, our findings represent a key step toward engineering a pyrenoid into crops to enhance their carbon fixation efficiency. PMID:27166422

  6. Tandem repeat recombinant proteins as potential antigens for the sero-diagnosis of Schistosoma mansoni infection.

    PubMed

    Kalenda, Yombo Dan Justin; Kato, Kentaro; Goto, Yasuyuki; Fujii, Yoshito; Hamano, Shinjiro

    2015-12-01

    The diagnosis of schistosome infection, followed by effective treatment and/or mass drug administration, is crucial to reduce the disease burden. Suitable diagnostic tests and field-applicable tools are required to sustain schistosomiasis control programs. We therefore assessed the potential of tandem repeat (TR) proteins for sero-diagnosis of Schistosoma mansoni infection using an experimental mouse model. TR genes in the genome of S. mansoni were searched in silico and 7 candidates, named SmTR1, 3, 8, 9, 10, 11 and 15, were selected. Total RNA was extracted from S. mansoni adult worms and eggs. Target TR genes were amplified, cloned, and the proteins were expressed in Escherichia coli competent cells. Female BALB/c mice were infected with 100 S. mansoni cercariae and sera were collected each week post-infection for 18 weeks. The levels of IgG antibodies to SmTR antigens were compared to those to soluble egg antigen (SEA) and to soluble worm antigen preparation (SWAP). Sera of infected mice reacted to all the antigens whereas those of naïve mice did not. IgG responses to SmTR1, 3, 9 and 10 were detected at the early stage of infection. Interestingly, antibodies reacting to SmTR3, 9, 10 and 15 dramatically decreased 4 weeks after treatment with praziquantel, while those against SEA and SWAP remained elevated. Our study suggests that TR proteins, especially SmTR10, may be suitable antigens for sero-diagnosis of infection by S. mansoni and are potential markers for monitoring and surveillance of schistosomiasis, including re-infection after treatment with praziquantel.

  7. Brain tumor specifies intermediate progenitor cell identity by attenuating β-catenin/Armadillo activity.

    PubMed

    Komori, Hideyuki; Xiao, Qi; McCartney, Brooke M; Lee, Cheng-Yu

    2014-01-01

    During asymmetric stem cell division, both the daughter stem cell and the presumptive intermediate progenitor cell inherit cytoplasm from their parental stem cell. Thus, proper specification of intermediate progenitor cell identity requires an efficient mechanism to rapidly extinguish the activity of self-renewal factors, but the mechanisms remain unknown in most stem cell lineages. During asymmetric division of a type II neural stem cell (neuroblast) in the Drosophila larval brain, the Brain tumor (Brat) protein segregates unequally into the immature intermediate neural progenitor (INP), where it specifies INP identity by attenuating the function of the self-renewal factor Klumpfuss (Klu), but the mechanisms are not understood. Here, we report that Brat specifies INP identity through its N-terminal B-boxes via a novel mechanism that is independent of asymmetric protein segregation. Brat-mediated specification of INP identity is critically dependent on the function of the Wnt destruction complex, which attenuates the activity of β-catenin/Armadillo (Arm) in immature INPs. Aberrantly increasing Arm activity in immature INPs further exacerbates the defects in the specification of INP identity and enhances the supernumerary neuroblast mutant phenotype in brat mutant brains. By contrast, reducing Arm activity in immature INPs suppresses supernumerary neuroblast formation in brat mutant brains. Finally, reducing Arm activity also strongly suppresses supernumerary neuroblasts induced by overexpression of klu. Thus, the Brat-dependent mechanism extinguishes the function of the self-renewal factor Klu in the presumptive intermediate progenitor cell by attenuating Arm activity, balancing stem cell maintenance and progenitor cell specification.

  8. Brain tumor specifies intermediate progenitor cell identity by attenuating β-catenin/Armadillo activity

    PubMed Central

    Komori, Hideyuki; Xiao, Qi; McCartney, Brooke M.; Lee, Cheng-Yu

    2014-01-01

    During asymmetric stem cell division, both the daughter stem cell and the presumptive intermediate progenitor cell inherit cytoplasm from their parental stem cell. Thus, proper specification of intermediate progenitor cell identity requires an efficient mechanism to rapidly extinguish the activity of self-renewal factors, but the mechanisms remain unknown in most stem cell lineages. During asymmetric division of a type II neural stem cell (neuroblast) in the Drosophila larval brain, the Brain tumor (Brat) protein segregates unequally into the immature intermediate neural progenitor (INP), where it specifies INP identity by attenuating the function of the self-renewal factor Klumpfuss (Klu), but the mechanisms are not understood. Here, we report that Brat specifies INP identity through its N-terminal B-boxes via a novel mechanism that is independent of asymmetric protein segregation. Brat-mediated specification of INP identity is critically dependent on the function of the Wnt destruction complex, which attenuates the activity of β-catenin/Armadillo (Arm) in immature INPs. Aberrantly increasing Arm activity in immature INPs further exacerbates the defects in the specification of INP identity and enhances the supernumerary neuroblast mutant phenotype in brat mutant brains. By contrast, reducing Arm activity in immature INPs suppresses supernumerary neuroblast formation in brat mutant brains. Finally, reducing Arm activity also strongly suppresses supernumerary neuroblasts induced by overexpression of klu. Thus, the Brat-dependent mechanism extinguishes the function of the self-renewal factor Klu in the presumptive intermediate progenitor cell by attenuating Arm activity, balancing stem cell maintenance and progenitor cell specification. PMID:24257623

  9. The human hnRNP M proteins: identification of a methionine/arginine-rich repeat motif in ribonucleoproteins.

    PubMed Central

    Datar, K V; Dreyfuss, G; Swanson, M S

    1993-01-01

    Recent reports indicate that proteins which directly bind to nascent RNA polymerase II transcripts, the heterogeneous nuclear ribonucleoproteins (hnRNPs), play an important role in both transcript-specific packaging and alternative splicing of pre-mRNAs. Here we describe the isolation and characterization of a group of abundant hnRNPs, the M1-M4 proteins, which appear as a cluster of four proteins of 64,000-68,000 daltons by two-dimensional electrophoresis. The M proteins are pre-mRNA binding proteins in vivo, and they bind avidly to poly(G) and poly(U) RNA homopolymers in vitro. Covalently associated polyadenylated RNA-protein complexes, generated by irradiating living HeLa cells with UV light, were purified and used to elicit antibodies in mice. The resulting antisera were then employed to isolate cDNA clones for the largest M protein, M4, by immunological screening. The deduced amino acid sequence of M4 indicates that the M proteins are members of the ribonucleoprotein consensus sequence family of RNA-binding proteins with greatest similarity to a hypothetical RNA-binding protein from Saccharomyces cerevisiae. The M proteins also possess an unusual hexapeptide-repeat region rich in methionine and arginine residues (MR repeat motif) that resembles a repeat in the 64,000 dalton subunit of cleavage stimulation factor, which is involved in 3'-end maturation of pre-mRNAs. Proteins immunologically related to M exist in divergent eukaryotes ranging from human to yeast. Images PMID:8441656

  10. Absence of FMR1 protein in two mentally retarded fragile X males without CGG repeat expansion

    SciTech Connect

    Lugenbeel, K.A.; Nelson, D.L.; Carson, N.L.; Chudley, A.E.

    1994-09-01

    Fragile X syndrome is characterized by absence of the product of the FMR1 gene due to an expansion and abnormal methylation of a CGG repeat located in exon 1. While the vast majority of fragile X patients demonstrate this common mutation, a small number of non-CGG mutations have been identified among patients exhibiting features of fragile X syndrome. Three patients with large deletions ablating all or a portion of FMR1 have been previously reported. A fourth patient has been described with a point mutation resulting in an Ile367 Asn substitution. While this last individual suggests that FMR1 is directly responsible for fragile X syndrome, the severe phenotype observed suggests a gain of function mutation. Our long-term goal is to understand both the normal function of the FMR1 gene product and the consequences of its absence. Using Western blot analysis of protein extracts prepared from transformed lymphoblastoid cell lines derived from individuals suspected of fragile X syndrome without CGG expansion, we have identified two fragile X males who display no FMR1 protein. In order to facilitate identification of small-scale mutations in these patients, primers have been designed which allow amplification of each exon of the FMR1 gene along with their intron boundaries. Exons 2 through 17 of FMR1 have been analyzed by amplification of patient genomic DNA using these primers. Each patient shows normal length amplification product from each exon as assayed by agarose gel electrophoresis, suggesting the absence of insertions, deletions, or other rearrangements. Sequence analysis of exons 8, 9, 10, 11, and 12 has shown no alteration from the normal FMR1 sequence. Current analysis has focused on the use of mutation detection electrophoresis (MDE) in order to identify candidate exons for mutations. RT-PCR analysis is also under way to determine if FMR1 mRNA is present and to offer an alternative approach to mutation detection.

  11. Knowledge-based design of reagentless fluorescent biosensors from a designed ankyrin repeat protein.

    PubMed

    Brient-Litzler, Elodie; Plückthun, Andreas; Bedouelle, Hugues

    2010-04-01

    Designed ankyrin repeat proteins (DARPins) can be selected from combinatorial libraries to bind any target antigen. They show high levels of recombinant expression, solubility and stability, and contain no cysteine residue. The possibility of obtaining, from any DARPin and at high yields, fluorescent conjugates which respond to the binding of the antigen by a variation of fluorescence, would have numerous applications in micro- and nano-analytical sciences. This possibility was explored with Off7, a DARPin directed against the maltose binding protein (MalE) from Escherichia coli, with known crystal structure of the complex. Eight residues of Off7, whose solvent accessible surface area varies on association with the antigen but which are not in direct contact with the antigen, were individually mutated into cysteine and then chemically coupled with a fluorophore. The conjugates were ranked according to their relative sensitivities. All of them showed an increase in their fluorescence intensity on antigen binding by >1.7-fold. The best conjugate retained the same affinity as the parental DARPin. Its signal increased linearly and specifically with the concentration of antigen, up to 15-fold in buffer and 3-fold in serum when fully saturated, the difference being mainly due to the absorption of light by serum. Its lower limit of detection was equal to 0.3 nM with a standard spectrofluorometer. Titrations with potassium iodide indicated that the fluorescence variation was due to a shielding of the fluorescent group from the solvent by the antigen. These results suggest rules for the design of reagentless fluorescent biosensors from any DARPin.

  12. Chemosensory regulation of a HEAT-repeat protein couples aggregation and sporulation in Myxococcus xanthus.

    PubMed

    Darnell, Cynthia L; Wilson, Janet M; Tiwari, Nitija; Fuentes, Ernesto J; Kirby, John R

    2014-09-01

    Chemosensory systems are complex, highly modified two-component systems (TCS) used by bacteria to control various biological functions ranging from motility to sporulation. Chemosensory systems and TCS both modulate phosphorelays comprised of histidine kinases and response regulators, some of which are single-domain response regulators (SD-RRs) such as CheY. In this study, we have identified and characterized the Che7 chemosensory system of Myxococcus xanthus, a common soil bacterium which displays multicellular development in response to stress. Both genetic and biochemical analyses indicate that the Che7 system regulates development via a direct interaction between the SD-RR CheY7 and a HEAT repeat domain-containing protein, Cpc7. Phosphorylation of the SD-RR affects the interaction with its target, and residues within the α4-β5-α5 fold of the REC domain govern this interaction. The identification of the Cpc7 interaction with CheY7 extends the diversity of known targets for SD-RRs in biological systems.

  13. Designed ankyrin repeat proteins: a new approach to mimic complex antigens for diagnostic purposes?

    PubMed

    Hausammann, Stefanie; Vogel, Monique; Kremer Hovinga, Johanna A; Lacroix-Desmazes, Sebastien; Stadler, Beda M; Horn, Michael P

    2013-01-01

    Inhibitory antibodies directed against coagulation factor VIII (FVIII) can be found in patients with acquired and congenital hemophilia A. Such FVIII-inhibiting antibodies are routinely detected by the functional Bethesda Assay. However, this assay has a low sensitivity and shows a high inter-laboratory variability. Another method to detect antibodies recognizing FVIII is ELISA, but this test does not allow the distinction between inhibitory and non-inhibitory antibodies. Therefore, we aimed at replacing the intricate antigen FVIII by Designed Ankyrin Repeat Proteins (DARPins) mimicking the epitopes of FVIII inhibitors. As a model we used the well-described inhibitory human monoclonal anti-FVIII antibody, Bo2C11, for the selection on DARPin libraries. Two DARPins were selected binding to the antigen-binding site of Bo2C11, which mimic thus a functional epitope on FVIII. These DARPins inhibited the binding of the antibody to its antigen and restored FVIII activity as determined in the Bethesda assay. Furthermore, the specific DARPins were able to recognize the target antibody in human plasma and could therefore be used to test for the presence of Bo2C11-like antibodies in a large set of hemophilia A patients. These data suggest, that our approach might be used to isolate epitopes from different sets of anti-FVIII antibodies in order to develop an ELISA-based screening assay allowing the distinction of inhibitory and non-inhibitory anti-FVIII antibodies according to their antibody signatures.

  14. Group B streptococcal serine-rich repeat proteins promote interaction with fibrinogen and vaginal colonization.

    PubMed

    Wang, Nai-Yu; Patras, Kathryn A; Seo, Ho Seong; Cavaco, Courtney K; Rösler, Berenice; Neely, Melody N; Sullam, Paul M; Doran, Kelly S

    2014-09-15

    Group B streptococcus (GBS) can cause severe disease in susceptible hosts, including newborns, pregnant women, and the elderly. GBS serine-rich repeat (Srr) surface glycoproteins are important adhesins/invasins in multiple host tissues, including the vagina. However, exact molecular mechanisms contributing to their importance in colonization are unknown. We have recently determined that Srr proteins contain a fibrinogen-binding region (BR) and hypothesize that Srr-mediated fibrinogen binding may contribute to GBS cervicovaginal colonization. In this study, we observed that fibrinogen enhanced wild-type GBS attachment to cervical and vaginal epithelium, and that this was dependent on Srr1. Moreover, purified Srr1-BR peptide bound directly to host cells, and peptide administration in vivo reduced GBS recovery from the vaginal tract. Furthermore, a GBS mutant strain lacking only the Srr1 "latching" domain exhibited decreased adherence in vitro and decreased persistence in a mouse model of GBS vaginal colonization, suggesting the importance of Srr-fibrinogen interactions in the female reproductive tract.

  15. Hybrid Sterility in Rice (Oryza sativa L.) Involves the Tetratricopeptide Repeat Domain Containing Protein.

    PubMed

    Yu, Yang; Zhao, Zhigang; Shi, Yanrong; Tian, Hua; Liu, Linglong; Bian, Xiaofeng; Xu, Yang; Zheng, Xiaoming; Gan, Lu; Shen, Yumin; Wang, Chaolong; Yu, Xiaowen; Wang, Chunming; Zhang, Xin; Guo, Xiuping; Wang, Jiulin; Ikehashi, Hiroshi; Jiang, Ling; Wan, Jianmin

    2016-07-01

    Intersubspecific hybrid sterility is a common form of reproductive isolation in rice (Oryza sativa L.), which significantly hampers the utilization of heterosis between indica and japonica varieties. Here, we elucidated the mechanism of S7, which specially causes Aus-japonica/indica hybrid female sterility, through cytological and genetic analysis, map-based cloning, and transformation experiments. Abnormal positioning of polar nuclei and smaller embryo sac were observed in F1 compared with male and female parents. Female gametes carrying S7(cp) and S7(i) were aborted in S7(ai)/S7(cp) and S7(ai)/S7(i), respectively, whereas they were normal in both N22 and Dular possessing a neutral allele, S7(n) S7 was fine mapped to a 139-kb region in the centromere region on chromosome 7, where the recombination was remarkably suppressed due to aggregation of retrotransposons. Among 16 putative open reading frames (ORFs) localized in the mapping region, ORF3 encoding a tetratricopeptide repeat domain containing protein was highly expressed in the pistil. Transformation experiments demonstrated that ORF3 is the candidate gene: downregulated expression of ORF3 restored spikelet fertility and eliminated absolutely preferential transmission of S7(ai) in heterozygote S7(ai)/S7(cp); sterility occurred in the transformants Cpslo17-S7(ai) Our results may provide implications for overcoming hybrid embryo sac sterility in intersubspecific hybrid rice and utilization of hybrid heterosis for cultivated rice improvement.

  16. Chemosensory Regulation of a HEAT-Repeat Protein Couples Aggregation and Sporulation in Myxococcus xanthus

    PubMed Central

    Darnell, Cynthia L.; Wilson, Janet M.; Tiwari, Nitija; Fuentes, Ernesto J.

    2014-01-01

    Chemosensory systems are complex, highly modified two-component systems (TCS) used by bacteria to control various biological functions ranging from motility to sporulation. Chemosensory systems and TCS both modulate phosphorelays comprised of histidine kinases and response regulators, some of which are single-domain response regulators (SD-RRs) such as CheY. In this study, we have identified and characterized the Che7 chemosensory system of Myxococcus xanthus, a common soil bacterium which displays multicellular development in response to stress. Both genetic and biochemical analyses indicate that the Che7 system regulates development via a direct interaction between the SD-RR CheY7 and a HEAT repeat domain-containing protein, Cpc7. Phosphorylation of the SD-RR affects the interaction with its target, and residues within the α4-β5-α5 fold of the REC domain govern this interaction. The identification of the Cpc7 interaction with CheY7 extends the diversity of known targets for SD-RRs in biological systems. PMID:24957622

  17. A designed ankyrin repeat protein selected to bind to tubulin caps the microtubule plus end.

    PubMed

    Pecqueur, Ludovic; Duellberg, Christian; Dreier, Birgit; Jiang, Qiyang; Wang, Chunguang; Plückthun, Andreas; Surrey, Thomas; Gigant, Benoît; Knossow, Marcel

    2012-07-24

    Microtubules are cytoskeleton filaments consisting of αβ-tubulin heterodimers. They switch between phases of growth and shrinkage. The underlying mechanism of this property, called dynamic instability, is not fully understood. Here, we identified a designed ankyrin repeat protein (DARPin) that interferes with microtubule assembly in a unique manner. The X-ray structure of its complex with GTP-tubulin shows that it binds to the β-tubulin surface exposed at microtubule (+) ends. The details of the structure provide insight into the role of GTP in microtubule polymerization and the conformational state of tubulin at the very microtubule end. They show in particular that GTP facilitates the tubulin structural switch that accompanies microtubule assembly but does not trigger it in unpolymerized tubulin. Total internal reflection fluorescence microscopy revealed that the DARPin specifically blocks growth at the microtubule (+) end by a selective end-capping mechanism, ultimately favoring microtubule disassembly from that end. DARPins promise to become designable tools for the dissection of microtubule dynamic properties selective for either of their two different ends.

  18. Genomic evidence for rod monochromacy in sloths and armadillos suggests early subterranean history for Xenarthra

    PubMed Central

    Emerling, Christopher A.; Springer, Mark S.

    2015-01-01

    Rod monochromacy is a rare condition in vertebrates characterized by the absence of cone photoreceptor cells. The resulting phenotype is colourblindness and low acuity vision in dim-light and blindness in bright-light conditions. Early reports of xenarthrans (armadillos, sloths and anteaters) suggest that they are rod monochromats, but this has not been tested with genomic data. We searched the genomes of Dasypus novemcinctus (nine-banded armadillo), Choloepus hoffmanni (Hoffmann's two-toed sloth) and Mylodon darwinii (extinct ground sloth) for retinal photoreceptor genes and examined them for inactivating mutations. We performed PCR and Sanger sequencing on cone phototransduction genes of 10 additional xenarthrans to test for shared inactivating mutations and estimated the timing of inactivation for photoreceptor pseudogenes. We concluded that a stem xenarthran became an long-wavelength sensitive-cone monochromat following a missense mutation at a critical residue in SWS1, and a stem cingulate (armadillos, glyptodonts and pampatheres) and stem pilosan (sloths and anteaters) independently acquired rod monochromacy early in their evolutionary history following the inactivation of LWS and PDE6C, respectively. We hypothesize that rod monochromacy in armadillos and pilosans evolved as an adaptation to a subterranean habitat in the early history of Xenarthra. The presence of rod monochromacy has major implications for understanding xenarthran behavioural ecology and evolution. PMID:25540280

  19. Genomic evidence for rod monochromacy in sloths and armadillos suggests early subterranean history for Xenarthra.

    PubMed

    Emerling, Christopher A; Springer, Mark S

    2015-02-07

    Rod monochromacy is a rare condition in vertebrates characterized by the absence of cone photoreceptor cells. The resulting phenotype is colourblindness and low acuity vision in dim-light and blindness in bright-light conditions. Early reports of xenarthrans (armadillos, sloths and anteaters) suggest that they are rod monochromats, but this has not been tested with genomic data. We searched the genomes of Dasypus novemcinctus (nine-banded armadillo), Choloepus hoffmanni (Hoffmann's two-toed sloth) and Mylodon darwinii (extinct ground sloth) for retinal photoreceptor genes and examined them for inactivating mutations. We performed PCR and Sanger sequencing on cone phototransduction genes of 10 additional xenarthrans to test for shared inactivating mutations and estimated the timing of inactivation for photoreceptor pseudogenes. We concluded that a stem xenarthran became an long-wavelength sensitive-cone monochromat following a missense mutation at a critical residue in SWS1, and a stem cingulate (armadillos, glyptodonts and pampatheres) and stem pilosan (sloths and anteaters) independently acquired rod monochromacy early in their evolutionary history following the inactivation of LWS and PDE6C, respectively. We hypothesize that rod monochromacy in armadillos and pilosans evolved as an adaptation to a subterranean habitat in the early history of Xenarthra. The presence of rod monochromacy has major implications for understanding xenarthran behavioural ecology and evolution.

  20. The ankyrin repeat domain of the TRPA protein painless is important for thermal nociception but not mechanical nociception.

    PubMed

    Hwang, Richard Y; Stearns, Nancy A; Tracey, W Daniel

    2012-01-01

    The Drosophila TRPA channel Painless is required for the function of polymodal nociceptors which detect noxious heat and noxious mechanical stimuli. These functions of Painless are reminiscent of mammalian TRPA channels that have also been implicated in thermal and mechanical nociception. A popular hypothesis to explain the mechanosensory functions of certain TRP channels proposes that a string of ankyrin repeats at the amino termini of these channels acts as an intracellular spring that senses force. Here, we describe the identification of two previously unknown Painless protein isoforms which have fewer ankyrin repeats than the canonical Painless protein. We show that one of these Painless isoforms, that essentially lacks ankyrin repeats, is sufficient to rescue mechanical nociception phenotypes of painless mutant animals but does not rescue thermal nociception phenotypes. In contrast, canonical Painless, which contains Ankyrin repeats, is sufficient to largely rescue thermal nociception but is not capable of rescuing mechanical nociception. Thus, we propose that in the case of Painless, ankryin repeats are important for thermal nociception but not for mechanical nociception.

  1. Hexanucleotide repeats in ALS/FTD form length-dependent RNA foci, sequester RNA binding proteins, and are neurotoxic.

    PubMed

    Lee, Youn-Bok; Chen, Han-Jou; Peres, João N; Gomez-Deza, Jorge; Attig, Jan; Stalekar, Maja; Troakes, Claire; Nishimura, Agnes L; Scotter, Emma L; Vance, Caroline; Adachi, Yoshitsugu; Sardone, Valentina; Miller, Jack W; Smith, Bradley N; Gallo, Jean-Marc; Ule, Jernej; Hirth, Frank; Rogelj, Boris; Houart, Corinne; Shaw, Christopher E

    2013-12-12

    The GGGGCC (G4C2) intronic repeat expansion within C9ORF72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Intranuclear neuronal RNA foci have been observed in ALS and FTD tissues, suggesting that G4C2 RNA may be toxic. Here, we demonstrate that the expression of 38× and 72× G4C2 repeats form intranuclear RNA foci that initiate apoptotic cell death in neuronal cell lines and zebrafish embryos. The foci colocalize with a subset of RNA binding proteins, including SF2, SC35, and hnRNP-H in transfected cells. Only hnRNP-H binds directly to G4C2 repeats following RNA immunoprecipitation, and only hnRNP-H colocalizes with 70% of G4C2 RNA foci detected in C9ORF72 mutant ALS and FTD brain tissues. We show that expanded G4C2 repeats are potently neurotoxic and bind hnRNP-H and other RNA binding proteins. We propose that RNA toxicity and protein sequestration may disrupt RNA processing and contribute to neurodegeneration.

  2. Behavioral responses of three armadillo species (Mammalia: Xenarthra) to an environmental enrichment program in Villavicencio, Colombia.

    PubMed

    Cortés Duarte, Alexandra; Trujillo, Fernando; Superina, Mariella

    2016-07-01

    Enrichment is a powerful tool to improve the welfare of animals under human care. Stress-related health and behavioral problems, as well as reproductive failure, are frequent in armadillos (Xenarthra, Cingulata, Dasypodidae) under human care, which hinders the development of successful ex situ conservation programs. Nevertheless, scientific studies on the effect of enrichment programs on armadillos are virtually non-existent. The objective of this study was to assess the impact of an enrichment program on the behavior of armadillos under human care. The behavior of 12 individuals of three species (Dasypus novemcinctus, D. sabanicola, and Cabassous unicinctus) maintained at Finca El Turpial, Villavicencio, Colombia, was recorded using scan sampling during three daily time blocks of 2 hr each before (4 weeks) and after (4 weeks) implementing an enrichment program. Enrichment did not stimulate the armadillos to change or extend their activity period. In general, activity levels were low during the entire study, and virtually no activity was recorded in the morning in any species, neither without nor with enrichment. The latter did, however, improve welfare by reducing abnormal and increasing natural foraging behaviors. All species were attracted by artificial termite mounds. Dasypus spp. showed special interest in cardboard boxes with food, while Cabassous was mainly attracted to hollow plastic balls filled with food. Our results suggest that separate enrichment programs need to be developed for different armadillo species, and that they should be applied during the time of day at which they are most active. Zoo Biol. 35:304-312, 2016. © 2016 Wiley Periodicals, Inc.

  3. Arabidopsis Acyl-CoA-binding protein ACBP2 interacts with an ethylene-responsive element-binding protein, AtEBP, via its ankyrin repeats.

    PubMed

    Li, Hong-Ye; Chye, Mee-Len

    2004-01-01

    Cytosolic acyl-CoA-binding proteins (ACBP) bind long-chain acyl-CoAs and act as intracellular acyl-CoA transporters and maintain acyl-CoA pools. Arabidopsis thaliana ACBP2 shows conservation at the acyl-CoA-binding domain to cytosolic ACBPs but is distinct by the presence of an N-terminal transmembrane domain and C-terminal ankyrin repeats. The function of the acyl-CoA-binding domain in ACBP2 has been confirmed by site-directed mutagenesis and four conserved residues crucial for palmitoyl-CoA binding have been identified. Results from ACBP2:GFP fusions transiently expressed in onion epidermal cells have demonstrated that the transmembrane domain functions in plasma membrane targeting, suggesting that ACBP2 transfers acyl-CoA esters to this membrane. In this study, we investigated the significance of its ankyrin repeats in mediating protein-protein interactions by yeast two-hybrid analysis and in vitro protein-binding assays; we showed that ACBP2 interacts with the A. thaliana ethylene-responsive element-binding protein AtEBP via its ankyrin repeats. This interaction was lacking in yeast two-hybrid analysis upon removal of the ankyrin repeats. When the subcellular localizations of ACBP2 and AtEBP were further investigated using autofluorescent protein fusions in transient expression by agroinfiltration of tobacco leaves, the DsRed:ACBP2 fusion protein was localized to the plasma membrane while the GFP:AtEBP fusion protein was targeted to the nucleus and plasma membrane. Co-expression of DsRed:ACBP2 and GFP:AtEBP showed a common localization of both proteins at the plasma membrane, suggesting that ACBP2 likely interacts with AtEBP at the plasma membrane.

  4. The protein network surrounding the human telomere repeat binding factors TRF1, TRF2, and POT1

    SciTech Connect

    Giannone, Richard J; McDonald, W Hayes; Hurst, Gregory {Greg} B; Shen, Rong-Fong; Wang, Yisong; Liu, Yie

    2010-01-01

    Telomere integrity (including telomere length and capping) is critical in overall genomic stability. Telomere repeat binding factors and their associated proteins play vital roles in telomere length regulation and end protection. In this study, we explore the protein network surrounding telomere repeat binding factors, TRF1, TRF2, and POT1 using dual-tag affinity purification in combination with multidimensional protein identification technology liquid chromatography - tandem mass spectrometry (MudPIT LC-MS/MS). After control subtraction and data filtering, we found that TRF2 and POT1 co-purified all six members of the telomere protein complex, while TRF1 identified five of six components at frequencies that lend evidence towards the currently accepted telomere architecture. Many of the known TRF1 or TRF2 interacting proteins were also identified. Moreover, putative associating partners identified for each of the three core components fell into functional categories such as DNA damage repair, ubiquitination, chromosome cohesion, chromatin modification/remodeling, DNA replication, cell cycle and transcription regulation, nucleotide metabolism, RNA processing, and nuclear transport. These putative protein-protein associations may participate in different biological processes at telomeres or, intriguingly, outside telomeres.

  5. A New Aspergillus fumigatus Typing Method Based on Hypervariable Tandem Repeats Located within Exons of Surface Protein Coding Genes (TRESP)

    PubMed Central

    Garcia-Rubio, Rocio; Gil, Horacio; Monteiro, Maria Candida; Pelaez, Teresa; Mellado, Emilia

    2016-01-01

    Aspergillus fumigatus is a saprotrophic mold fungus ubiquitously found in the environment and is the most common species causing invasive aspergillosis in immunocompromised individuals. For A. fumigatus genotyping, the short tandem repeat method (STRAf) is widely accepted as the first choice. However, difficulties associated with PCR product size and required technology have encouraged the development of novel typing techniques. In this study, a new genotyping method based on hypervariable tandem repeats within exons of surface protein coding genes (TRESP) was designed. A. fumigatus isolates were characterized by PCR amplification and sequencing with a panel of three TRESP encoding genes: cell surface protein A; MP-2 antigenic galactomannan protein; and hypothetical protein with a CFEM domain. The allele sequence repeats of each of the three targets were combined to assign a specific genotype. For the evaluation of this method, 126 unrelated A. fumigatus strains were analyzed and 96 different genotypes were identified, showing a high level of discrimination [Simpson’s index of diversity (D) 0.994]. In addition, 49 azole resistant strains were analyzed identifying 26 genotypes and showing a lower D value (0.890) among them. This value could indicate that these resistant strains are closely related and share a common origin, although more studies are needed to confirm this hypothesis. In summary, a novel genotyping method for A. fumigatus has been developed which is reproducible, easy to perform, highly discriminatory and could be especially useful for studying outbreaks. PMID:27701437

  6. Crystal structure of the dimeric protein core of decorin, the archetypal small leucine-rich repeat proteoglycan.

    PubMed

    Scott, Paul G; McEwan, Paul A; Dodd, Carole M; Bergmann, Ernst M; Bishop, Paul N; Bella, Jordi

    2004-11-02

    Decorin is a ubiquitous extracellular matrix proteoglycan with a variety of important biological functions that are mediated by its interactions with extracellular matrix proteins, cytokines, and cell surface receptors. Decorin is the prototype of the family of small leucine-rich repeat proteoglycans and proteins (SLRPs), characterized by a protein core composed of leucine-rich repeats (LRRs), flanked by two cysteine-rich regions. We report here the crystal structure of the dimeric protein core of decorin, the best characterized member of the SLRP family. Each monomer adopts the curved solenoid fold characteristic of LRR domains, with a parallel beta-sheet on the inside interwoven with loops containing short segments of beta-strands, 3(10) helices, and polyproline II helices on the outside. Two main features are unique to this structure. First, decorin dimerizes through the concave surfaces of the LRR domains, which have been implicated previously in protein-ligand interactions. The amount of surface buried in this dimer rivals the buried surfaces of some of the highest-affinity macromolecular complexes reported to date. Second, the C-terminal region adopts an unusual capping motif that involves a laterally extended LRR and a disulfide bond. This motif seems to be unique to SLRPs and has not been observed in any other LRR protein structure to date. Possible implications of these features for decorin ligand binding and SLRP function are discussed.

  7. Leucine-rich repeats and carboxyl terminus are required for interaction of yeast adenylate cyclase with RAS proteins.

    PubMed Central

    Suzuki, N; Choe, H R; Nishida, Y; Yamawaki-Kataoka, Y; Ohnishi, S; Tamaoki, T; Kataoka, T

    1990-01-01

    A Saccharomyces cerevisiae gene encoding adenylate cyclase has been analyzed by deletion and insertion mutagenesis to localize regions required for activation by the Sa. cerevisiae RAS2 protein. The NH2-terminal 657 amino acids were found to be dispensable for the activation. However, almost all 2-amino acid insertions in the middle 600 residues comprising leucine-rich repeats and deletions in the COOH-terminal 66 residues completely abolished activation by the RAS2 protein, whereas insertion mutations in the other regions generally had no effect. Chimeric adenylate cyclases were constructed by swapping the upstream and downstream portions surrounding the catalytic domains between the Sa. cerevisiae and Schizosaccharomyces pombe adenylate cyclases and examined for activation by the RAS2 protein. We found that the fusion containing both the NH2-terminal 1600 residues and the COOH-terminal 66 residues of the Sa. cerevisiae cyclase rendered the catalytic domain of the Sc. pombe cyclase, which otherwise did not respond to RAS proteins, activatable by the RAS2 protein. Thus the leucine-rich repeats and the COOH terminus of the Sa. cerevisiae adenylate cyclase appear to be required for interaction with RAS proteins. Images PMID:2247439

  8. Nuclear magnetic resonance assignment and secondary structure of an ankyrin-like repeat-bearing protein: myotrophin.

    PubMed

    Yang, Y; Rao, N S; Walker, E; Sen, S; Qin, J

    1997-06-01

    Multidimensional heteronuclear NMR has been applied to the structural analysis of myotrophin, a novel protein identified from spontaneously hypertensive rat hearts and hypertrophic human hearts. Myotrophin has been shown to stimulate protein synthesis in myocytes and likely plays an important role in the initiation of cardiac hypertrophy, a major cause of mortality in humans. Recent cDNA cloning revealed that myotrophin has 11B amino acids containing 2.5 contiguous ANK repeats, a motif known to be involved in a wide range of macromolecular recognition. A series of two- and three-dimensional heteronuclear bond correlation NMR experiments have been performed on uniformly 15N-labeled or uniformly 15N/13C-labeled protein to obtain the 1H, 15N, and 13C chemical shift assignments. The secondary structure of myotrophin has been determined by a combination of NOEs, NH exchange data, 3JHN alpha coupling constants, and chemical shifts of 1H alpha, 13C alpha, and 13 C beta. The protein has been found to consist of seven helices, all connected by turns or loops. Six of the seven helices (all but the C-terminal helix) form three separate helix-turn-helix motifs. The two full ANK repeats in myotrophin are characteristic of multiple turns followed by a helix-turn-helix motif. A hairpin-like turn involving L32-R36 in ANK repeat #1 exhibits slow conformational averaging on the NMR time scale and appears dynamically different from the corresponding region (D65-169) of ANK repeat #2.

  9. Chromosomal localization of the telomeric (TTAGGG)n sequence in four species of Armadillo (Dasypodidae) from Argentina: an approach to explaining karyotype evolution in the Xenarthra.

    PubMed

    Lizarralde, M S; Bolzán, A D; Poljak, S; Pigozzi, M I; Bustos, J; Merani, M S

    2005-01-01

    The distribution of the vertebrate telomeric sequence (TTAGGG)(n) in four species of armadillos (Dasypodidae, Xenarthra), i.e. Chaetophractus villosus (2n = 60), Chaetophractus vellerosus (2n = 62), Dasypus hybridus (2n = 64) and Zaedyus pichiy (2n = 62) was examined by FISH with a peptide nucleic acid (PNA) probe. Besides the expected telomeric hybridization, interstitial (centromeric) locations of the (TTAGGG)n sequence were observed in one chromosome pair of Chaetophractus vellerosus and Zaedyus pichiy, suggesting chromosome fusion of ancestral chromosomes occurring during the evolution of Dasypodidae. In addition, all the species analysed showed one to four apparently telocentric chromosomes, exhibiting only two telomeric signals. However, the immunodetection study of kinetochore proteins on synaptonemal complex spreads from C. villosus showed that the apparently telocentric chromosomes have a tiny short arm that can be resolved only in the more elongated pachytene bivalents. This finding suggests that none of the species of armadillos possess true telocentric chromosomes. Our present results support a reduction in the diploid number by fusion of acrocentrics with loss of chromosome material as a tendency in Dasypodidae.

  10. Limits of cooperativity in a structurally modular protein: response of the Notch ankyrin domain to analogous alanine substitutions in each repeat.

    PubMed

    Bradley, Christina Marchetti; Barrick, Doug

    2002-11-22

    To determine the limits of cooperativity in a structurally modular protein, we characterized the structure and stability of glycine variants of the ankyrin repeat domain from the Drosophila melangaster Notch receptor. The substitutions are of analogous alanine residues to glycine in each repeat, and allow the same perturbation to be examined at different positions in the protein. The ankyrin domain is insensitive to substitution in repeat one, suggesting that the first repeat is not fully-folded. Glycine substitutions in repeat two through seven are strongly destabilizing, but the variants retain their overall secondary and tertiary structures. Spectroscopic and calorimetric data are consistent with two-state unfolding transitions for the repeat-two through repeat-five glycine variants, and for the wild-type protein. These data indicate that, despite its modular structure, the Notch ankyrin domain unfolds as a cooperative unit consisting of the six C-terminal repeats, and that this cooperativity is maintained in the presence of severely destabilizing substitutions in the N-terminal and central repeats. In contrast, glycine substitution in repeat six leads to a multi-state unfolding transition, suggesting that the coupling that gives rise to long-range cooperativity in the wild-type protein may have a weak link in the C-terminal region. Such behavior is captured by a simple statistical thermodynamic model in which an unstable C-terminal region is coupled to a stable N-terminal region through a strongly stabilizing interface.

  11. PATTERNS OF MYCOBACTERIUM LEPRAE INFECTION IN WILD NINE-BANDED ARMADILLOS (DASYPUS NOVEMCINCTUS) IN MISSISSIPPI, USA.

    PubMed

    Perez-Heydrich, Carolina; Loughry, W J; Anderson, Corey Devin; Oli, Madan K

    2016-07-01

    The nine-banded armadillo ( Dasypus novemcinctus ) is the only known nonhuman reservoir of Mycobacterium leprae , the causative agent of Hansen's disease or leprosy. We conducted a 6-yr study on a wild population of armadillos in western Mississippi that was exposed to M. leprae to evaluate the importance of demographic and spatial risk factors on individual antibody status. We found that spatially derived covariates were not predictive of antibody status. Furthermore, analyses revealed no evidence of clustering by antibody-positive individuals. Lactating females and adult males had higher odds of being antibody positive than did nonlactating females. No juveniles or yearlings were antibody positive. Results of these analyses support the hypothesis that M. leprae infection patterns are spatially homogeneous within this armadillo population. Further research related to movement patterns, contact among individuals, antibody status, and environmental factors could help address hypotheses related to the role of environmental transmission on M. leprae infection and the mechanisms underlying the differential infection patterns among demographic groups.

  12. The repeat domain of the type III effector protein PthA shows a TPR-like structure and undergoes conformational changes upon DNA interaction.

    PubMed

    Murakami, Mário Tyago; Sforça, Mauricio Luis; Neves, Jorge Luiz; Paiva, Joice Helena; Domingues, Mariane Noronha; Pereira, André Luiz Araujo; Zeri, Ana Carolina de Mattos; Benedetti, Celso Eduardo

    2010-12-01

    Many plant pathogenic bacteria rely on effector proteins to suppress defense and manipulate host cell mechanisms to cause disease. The effector protein PthA modulates the host transcriptome to promote citrus canker. PthA possesses unusual protein architecture with an internal region encompassing variable numbers of near-identical tandem repeats of 34 amino acids termed the repeat domain. This domain mediates protein-protein and protein-DNA interactions, and two polymorphic residues in each repeat unit determine DNA specificity. To gain insights into how the repeat domain promotes protein-protein and protein-DNA contacts, we have solved the structure of a peptide corresponding to 1.5 units of the PthA repeat domain by nuclear magnetic resonance (NMR) and carried out small-angle X-ray scattering (SAXS) and spectroscopic studies on the entire 15.5-repeat domain of PthA2 (RD2). Consistent with secondary structure predictions and circular dichroism data, the NMR structure of the 1.5-repeat peptide reveals three α-helices connected by two turns that fold into a tetratricopeptide repeat (TPR)-like domain. The NMR structure corroborates the theoretical TPR superhelix predicted for RD2, which is also in agreement with the elongated shape of RD2 determined by SAXS. Furthermore, RD2 undergoes conformational changes in a pH-dependent manner and upon DNA interaction, and shows sequence similarities to pentatricopeptide repeat (PPR), a nucleic acid-binding motif structurally related to TPR. The results point to a model in which the RD2 structure changes its compactness as it embraces the DNA with the polymorphic diresidues facing the interior of the superhelix oriented toward the nucleotide bases.

  13. An Ehrlichia chaffeensis tandem repeat protein interacts with multiple host targets involved in cell signaling, transcriptional regulation, and vesicle trafficking.

    PubMed

    Wakeel, Abdul; Kuriakose, Jeeba A; McBride, Jere W

    2009-05-01

    Ehrlichia chaffeensis is an obligately intracellular bacterium that exhibits tropism for mononuclear phagocytes forming cytoplasmic membrane-bound microcolonies called morulae. To survive and replicate within phagocytes, E. chaffeensis exploits the host cell by modulating a number of host cell processes, but the ehrlichial effector proteins involved are unknown. In this study, we determined that p47, a secreted, differentially expressed, tandem repeat (TR) protein, interacts with multiple host proteins associated with cell signaling, transcriptional regulation, and vesicle trafficking. Yeast two-hybrid analysis revealed that p47 interacts with polycomb group ring finger 5 (PCGF5) protein, Src protein tyrosine kinase FYN (FYN), protein tyrosine phosphatase non-receptor type 2 (PTPN2), and adenylate cyclase-associated protein 1 (CAP1). p47 interaction with these proteins was further confirmed by coimmunoprecipitation assays and colocalization in HeLa cells transfected with p47-green fluorescent fusion protein (AcGFP1-p47). Moreover, confocal microscopy demonstrated p47-expressing dense-cored (DC) ehrlichiae colocalized with PCGF5, FYN, PTPN2, and CAP1. An amino-terminally truncated form of p47 containing TRs interacted only with PCGF5 and not with FYN, PTPN2, and CAP1, indicating differences in p47 domains that are involved in these interactions. These results demonstrate that p47 is involved in a complex network of interactions involving numerous host cell proteins. Furthermore, this study provides a new insight into the molecular and functional distinction of DC ehrlichiae, as well as the effector proteins involved in facilitating ehrlichial survival in mononuclear phagocytes.

  14. Leucine-rich repeats containing protein functions in the antibacterial immune reaction in stomach of kuruma shrimp Marsupenaeus japonicus.

    PubMed

    Shi, Xiu-Zhen; Feng, Xiao-Wu; Sun, Jie-Jie; Zhao, Xiao-Fan; Wang, Jin-Xing

    2017-02-01

    Leucine rich repeat (LRR) motif exists in many immune receptors of animals and plants. Most LRR containing (LRRC) proteins are involved in protein-ligand and protein-protein interaction, but the exact functions of most LRRC proteins were not well-studied. In this study, an LRRC protein was identified from kuruma shrimp Marsupenaeus japonicus, and named as MjLRRC1. MjLRRC1 was consistently expressed in different tissues of normal shrimp with higher expression in gills and stomach. At the transcriptional level, there were no significant changes of MjLRRC1 after injection of Vibrio anguillarum or Staphylococcus aureus in gills and hepatopancreas. While in V. anguillarum oral infection, MjLRRC1 was upregulated in stomach but not in intestine. The recombinant MjLRRC1 protein could bind to Gram-positive and Gram-negative bacteria, bacterial cell wall components including peptidoglycan, lipoteichoic acid, and lipopolysaccharide. MjLRRC1 regulated the expression of some antimicrobial peptide (AMP) genes and participated in bacteria clearance of stomach. All these results suggested that MjLRRC1 might play important roles in antibacterial immune response of kuruma shrimp.

  15. The inhibitor of DNA replication encoded by the Drosophila gene plutonium is a small, ankyrin repeat protein.

    PubMed Central

    Axton, J M; Shamanski, F L; Young, L M; Henderson, D S; Boyd, J B; Orr-Weaver, T L

    1994-01-01

    The plutonium (plu) gene product controls DNA replication early in Drosophila development. plu mutant females lay unfertilized eggs that have undergone extensive DNA synthesis. In fertilized embryos from plu mutant mothers, S-phase is uncoupled from mitosis. The gene is expressed only in ovaries and embryos, null alleles are strict maternal effect mutations, and the phenotype of inappropriate DNA replication is the consequence of loss-of-gene function. plu therefore negatively regulates S-phase at a time in early development when commitment to S-phase does not depend on cyclic transcription. plu encodes a protein with two ankyrin-like repeats, a domain for protein-protein interaction. plu is immediately adjacent to, but distinct from, the PCNA gene. Images PMID:8313891

  16. Genome-Wide Analysis of Arabidopsis Pentatricopeptide Repeat Proteins Reveals Their Essential Role in Organelle BiogenesisW⃞

    PubMed Central

    Lurin, Claire; Andrés, Charles; Aubourg, Sébastien; Bellaoui, Mohammed; Bitton, Frédérique; Bruyère, Clémence; Caboche, Michel; Debast, Cédrig; Gualberto, José; Hoffmann, Beate; Lecharny, Alain; Le Ret, Monique; Martin-Magniette, Marie-Laure; Mireau, Hakim; Peeters, Nemo; Renou, Jean-Pierre; Szurek, Boris; Taconnat, Ludivine; Small, Ian

    2004-01-01

    The complete sequence of the Arabidopsis thaliana genome revealed thousands of previously unsuspected genes, many of which cannot be ascribed even putative functions. One of the largest and most enigmatic gene families discovered in this way is characterized by tandem arrays of pentatricopeptide repeats (PPRs). We describe a detailed bioinformatic analysis of 441 members of the Arabidopsis PPR family plus genomic and genetic data on the expression (microarray data), localization (green fluorescent protein and red fluorescent protein fusions), and general function (insertion mutants and RNA binding assays) of many family members. The basic picture that arises from these studies is that PPR proteins play constitutive, often essential roles in mitochondria and chloroplasts, probably via binding to organellar transcripts. These results confirm, but massively extend, the very sparse observations previously obtained from detailed characterization of individual mutants in other organisms. PMID:15269332

  17. RAG-1 interacts with the repeated amino acid motif of the human homologue of the yeast protein SRP1.

    PubMed Central

    Cortes, P; Ye, Z S; Baltimore, D

    1994-01-01

    Genes for immunoglobulins and T-cell receptor are generated by a process known as V(D)J recombination. This process is highly regulated and mediated by the recombination activating proteins RAG-1 and RAG-2. By the use of the two-hybrid protein interaction system, we isolated a human protein that specifically interacts with RAG-1. This protein is the human homologue of the yeast SRP1 (suppressor of a temperature-sensitive RNA polymerase I mutation). The SRP1-1 mutation is an allele-specific dominant suppressor of a temperature-sensitive mutation in the zinc binding domain of the 190-kDa subunit of Saccharomyces cerevisiae RNA polymerase I. The human SRP cDNA clone was used to screen a mouse cDNA library. We obtained a 3.9-kbp cDNA clone encoding the mouse SRP1. The open reading frame of this cDNA encodes a 538-amino acid protein with eight degenerate repeats of 40-45 amino acids each. The mouse and human SRP1 are 98% identical, while the mouse and yeast SRP1 have 48% identity. After cotransfection of the genes encoding RAG-1 and human SRP1 into 293T cells, a stable complex was evident. Deletion analysis indicated that the region of the SRP1 protein interacting with RAG-1 involved four repeats. The domain of RAG-1 that associates with SRP1 mapped N-terminal to the zinc finger domain. Because this region of RAG-1 is not required for recombination and SRP1 appears to be bound to the nuclear envelope, we suggest that this interaction helps to localize RAG-1. Images PMID:8052633

  18. NUC-2, a component of the phosphate-regulated signal transduction pathway in Neurospora crassa, is an ankyrin repeat protein.

    PubMed

    Poleg, Y; Aramayo, R; Kang, S; Hall, J G; Metzenberg, R L

    1996-10-28

    In response to phosphorus limitation, the fungus Neurospora crassa synthesizes a number of enzymes that function to bring more phosphate into the cell. The NUC-2 protein appears to sense the availability of phosphate and transmits the signal downstream to the regulatory pathway. The nuc-2+ gene has been cloned by its ability to restore growth of a nuc-2 mutant under restrictive conditions of high pH and low phosphate concentration. We mapped the cloned gene to the right arm of linkage group II, consistent with the chromosomal position of the nuc-2 mutation as determined by classical genetic mapping. The nuc-2' open reading frame is interrupted by five introns and codes for a protein of 1066 amino acid residues. Its predicted amino acid sequence has high similarity to that of its homolog in Saccharomyces cerevisiae, PHO81. Both proteins contain six ankyrin repeats, which have been implicated in the cyclin-dependent kinase inhibitory activity of PHO81. The phenotypes of a nuc-2 mutant generated by repeat-induced point mutation and of a strain harboring a UV-induced nuc-2 allele are indistinguishable. Both are unable to grow under the restrictive conditions, a phenotype which is to some degree temperature dependent. The nuc-2+ gene is transcriptionally regulated. A 15-fold increase in the level of the nuc-2+ transcript occurs in response to a decrease in exogenous phosphate concentration.

  19. The La-related protein 1-specific domain repurposes HEAT-like repeats to directly bind a 5'TOP sequence

    SciTech Connect

    Lahr, Roni M.; Mack, Seshat M.; Heroux, Annie; Blagden, Sarah P.; Bousquet-Antonelli, Cecile; Deragon, Jean -Marc; Berman, Andrea J.

    2015-07-22

    La-related protein 1 (LARP1) regulates the stability of many mRNAs. These include 5'TOPs, mTOR-kinase responsive mRNAs with pyrimidine-rich 5' UTRs, which encode ribosomal proteins and translation factors. We determined that the highly conserved LARP1-specific C-terminal DM15 region of human LARP1 directly binds a 5'TOP sequence. The crystal structure of this DM15 region refined to 1.86 Å resolution has three structurally related and evolutionarily conserved helix-turn-helix modules within each monomer. These motifs resemble HEAT repeats, ubiquitous helical protein-binding structures, but their sequences are inconsistent with consensus sequences of known HEAT modules, suggesting this structure has been repurposed for RNA interactions. A putative mTORC1-recognition sequence sits within a flexible loop C-terminal to these repeats. We also present modelling of pyrimidine-rich single-stranded RNA onto the highly conserved surface of the DM15 region. Ultimately, these studies lay the foundation necessary for proceeding toward a structural mechanism by which LARP1 links mTOR signalling to ribosome biogenesis.

  20. The La-related protein 1-specific domain repurposes HEAT-like repeats to directly bind a 5'TOP sequence

    DOE PAGES

    Lahr, Roni M.; Mack, Seshat M.; Heroux, Annie; ...

    2015-07-22

    La-related protein 1 (LARP1) regulates the stability of many mRNAs. These include 5'TOPs, mTOR-kinase responsive mRNAs with pyrimidine-rich 5' UTRs, which encode ribosomal proteins and translation factors. We determined that the highly conserved LARP1-specific C-terminal DM15 region of human LARP1 directly binds a 5'TOP sequence. The crystal structure of this DM15 region refined to 1.86 Å resolution has three structurally related and evolutionarily conserved helix-turn-helix modules within each monomer. These motifs resemble HEAT repeats, ubiquitous helical protein-binding structures, but their sequences are inconsistent with consensus sequences of known HEAT modules, suggesting this structure has been repurposed for RNA interactions. Amore » putative mTORC1-recognition sequence sits within a flexible loop C-terminal to these repeats. We also present modelling of pyrimidine-rich single-stranded RNA onto the highly conserved surface of the DM15 region. Ultimately, these studies lay the foundation necessary for proceeding toward a structural mechanism by which LARP1 links mTOR signalling to ribosome biogenesis.« less

  1. The ankyrin repeat protein Diego mediates Frizzled-dependent planar polarization.

    PubMed

    Feiguin, F; Hannus, M; Mlodzik, M; Eaton, S

    2001-07-01

    During planar polarization of the Drosophila wing epithelium, the homophilic adhesion molecule Flamingo localizes to proximal/distal cell boundaries in response to Frizzled signaling; perturbing Frizzled signaling alters Flamingo distribution, many cell diameters distant, by a mechanism that is not well understood. This work identifies a tissue polarity gene, diego, that comprises six ankyrin repeats and colocalizes with Flamingo at proximal/distal boundaries. Diego is specifically required for polarized accumulation of Flamingo and drives ectopic clustering of Flamingo when overexpressed. Our data suggest that Frizzled acts through Diego to promote local clustering of Flamingo, and that clustering of Diego and Flamingo in one cell nonautonomously propagates to others.

  2. A repeat sequence domain of the ring-exported protein-1 of Plasmodium falciparum controls export machinery architecture and virulence protein trafficking.

    PubMed

    McHugh, Emma; Batinovic, Steven; Hanssen, Eric; McMillan, Paul J; Kenny, Shannon; Griffin, Michael D W; Crawford, Simon; Trenholme, Katharine R; Gardiner, Donald L; Dixon, Matthew W A; Tilley, Leann

    2015-12-01

    The malaria parasite Plasmodium falciparum dramatically remodels its host red blood cell to enhance its own survival, using a secretory membrane system that it establishes outside its own cell. Cisternal organelles, called Maurer's clefts, act as a staging point for the forward trafficking of virulence proteins to the red blood cell (RBC) membrane. The Ring-EXported Protein-1 (REX1) is a Maurer's cleft resident protein. We show that inducible knockdown of REX1 causes stacking of Maurer's cleft cisternae without disrupting the organization of the knob-associated histidine-rich protein at the RBC membrane. Genetic dissection of the REX1 sequence shows that loss of a repeat sequence domain results in the formation of giant Maurer's cleft stacks. The stacked Maurer's clefts are decorated with tether-like structures and retain the ability to dock onto the RBC membrane skeleton. The REX1 mutant parasites show deficient export of the major virulence protein, PfEMP1, to the red blood cell surface and markedly reduced binding to the endothelial cell receptor, CD36. REX1 is predicted to form a largely α-helical structure, with a repetitive charge pattern in the repeat sequence domain, providing potential insights into the role of REX1 in Maurer's cleft sculpting.

  3. MNF, an ankyrin repeat protein of myxoma virus, is part of a native cellular SCF complex during viral infection.

    PubMed

    Blanié, Sophie; Gelfi, Jacqueline; Bertagnoli, Stéphane; Camus-Bouclainville, Christelle

    2010-03-08

    Myxoma virus (MYXV), a member of the Poxviridae family, is the agent responsible for myxomatosis, a fatal disease in the European rabbit (Oryctolagus cuniculus). Like all poxviruses, MYXV is known for encoding multiple proteins that regulate cellular signaling pathways. Among them, four proteins share the same ANK/PRANC structure: M148R, M149R, MNF (Myxoma Nuclear factor) and M-T5, all of them described as virulence factors. This family of poxvirus proteins, recently identified, has drawn considerable attention for its potential role in modulating the host ubiquitin-proteasome system during viral infection. To date, many members of this novel protein family have been shown to interact with SCF components, in vitro. Here, we focus on MNF gene, which has been shown to express a nuclear protein presenting nine ANK repeats, one of which has been identified as a nuclear localization signal. In transfection, MNF has been shown to colocalise with the transcription factor NF-kappaB in the nucleus of TNFalpha-stimulated cells. Functionally, MNF is a critical virulence factor since its deletion generates an almost apathogenic virus. In this study, to pursue the investigation of proteins interacting with MNF and of its mechanism of action, we engineered a recombinant MYXV expressing a GFP-linked MNF under the control of MNF native promoter. Infection of rabbits with MYXV-GFPMNF recombinant virus provided the evidence that the GFP fusion does not disturb the main function of MNF. Hence, cells were infected with MYXV-GFPMNF and immunoprecipitation of the GFPMNF fusion protein was performed to identify MNF's partners. For the first time, endogenous components of SCF (Cullin-1 and Skp1) were co-precipitated with an ANK myxoma virus protein, expressed in an infectious context, and without over-expression of any protein.

  4. Repeated exposures to roadside particulate matter extracts suppresses pulmonary defense mechanisms, resulting in lipid and protein oxidative damage.

    PubMed

    Pardo, Michal; Porat, Ziv; Rudich, Assaf; Schauer, James J; Rudich, Yinon

    2016-03-01

    Exposure to particulate matter (PM) pollution in cities and urban canyons can be harmful to the exposed population. However, the underlying mechanisms that lead to health effects are not yet elucidated. It is postulated that exposure to repeated, small, environmentally relevant concentrations can affect lung homeostasis. This study examines the impact of repeated exposures to urban PM on mouse lungs with focus on inflammatory and oxidative stress parameters. Aqueous extracts from collected urban PM were administered to mice by 5 repeated intra-tracheal instillations (IT). Multiple exposures, led to an increase in cytokine levels in both bronchoalveolar lavage fluid and in the blood serum, indicating a systemic reaction. Lung mRNA levels of antioxidant/phase II detoxifying enzymes decreased by exposure to the PM extract, but not when metals were removed by chelation. Finally, disruption of lung tissue oxidant-inflammatory/defense balance was evidenced by increased levels of lipid and protein oxidation. Unlike response to a single IT exposure to the same dose and source of extract, multiple exposures result in lung oxidative damage and a systemic inflammatory reaction. These could be attributed to compromised capacity to activate the protective Nrf2 tissue defense system. It is suggested that water-soluble metals present in urban PM, potentially from break and tire wear, may constitute major drivers of the pulmonary and systemic responses to multiple exposure to urban PM.

  5. 6-alkynyl fucose is a bioorthogonal analog for O-fucosylation of epidermal growth factor-like repeats and thrombospondin type-1 repeats by protein O-fucosyltransferases 1 and 2.

    PubMed

    Al-Shareffi, Esam; Chaubard, Jean-Luc; Leonhard-Melief, Christina; Wang, Sheng-Kai; Wong, Chi-Huey; Haltiwanger, Robert S

    2013-02-01

    Protein O-fucosyltransferase 1 (Pofut1) and protein O-fucosyltransferase 2 (Pofut2) add O-linked fucose at distinct consensus sequences in properly folded epidermal growth factor (EGF)-like repeats and thrombospondin type-1 (TSR) repeats, respectively. Glycan chain elongation past O-fucose can occur to yield a tetrasaccharide on EGF repeats and a disaccharide on TSRs. Elimination of Pofut1 in mice causes embryonic lethality with Notch-like phenotypes demonstrating that O-fucosylation of Notch is essential for its function. Similarly, elimination of Pofut2 results in an early embryonic lethal phenotype in mice, although the molecular mechanism for the lethality is unknown. The recent development of sugar analogs has revolutionized the study of glycans by providing a convenient method for labeling and tracking glycosylation. In order to study O-fucosylation, we took advantage of the recently developed reporter, 6-alkynyl fucose. Using the Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC), or "click" reaction, azido-biotin allows tagging and detection of 6AF-modified proteins. Here we examine whether proteins containing EGF repeats or TSRs with O-fucose consensus sequences are specifically modified with 6AF in cell culture. Using mass spectrometry (MS), we demonstrate that 6AF is efficiently incorporated onto the appropriate consensus sequences on EGF repeats and TSRs. Furthermore, the elongation of the O-fucose monosaccharide on EGF repeats and TSRs is not hampered when 6AF is used. These results show that 6AF is efficiently utilized in a truly bioorthogonal manner by Pofut1, Pofut2 and the enzymes that elongate O-fucose, providing evidence that 6AF is a significant new tool in the study of protein O-fucosylation.

  6. Genome-Wide Screening of Retroviral Envelope Genes in the Nine-Banded Armadillo (Dasypus novemcinctus, Xenarthra) Reveals an Unfixed Chimeric Endogenous Betaretrovirus Using the ASCT2 Receptor

    PubMed Central

    Malicorne, Sébastien; Vernochet, Cécile; Cornelis, Guillaume; Mulot, Baptiste; Delsuc, Frédéric; Heidmann, Odile

    2016-01-01

    ABSTRACT Retroviruses enter host cells through the interaction of their envelope (Env) protein with a cell surface receptor, which triggers the fusion of viral and cellular membranes. The sodium-dependent neutral amino acid transporter ASCT2 is the common receptor of the large RD114 retrovirus interference group, whose members display frequent env recombination events. Germ line retrovirus infections have led to numerous inherited endogenous retroviruses (ERVs) in vertebrate genomes, which provide useful insights into the coevolutionary history of retroviruses and their hosts. Rare ERV-derived genes display conserved viral functions, as illustrated by the fusogenic syncytin env genes involved in placentation. Here, we searched for functional env genes in the nine-banded armadillo (Dasypus novemcinctus) genome and identified dasy-env1.1, which clusters with RD114 interference group env genes and with two syncytin genes sharing ASCT2 receptor usage. Using ex vivo pseudotyping and cell-cell fusion assays, we demonstrated that the Dasy-Env1.1 protein is fusogenic and can use both human and armadillo ASCT2s as receptors. This gammaretroviral env gene belongs to a provirus with betaretrovirus-like features, suggesting acquisition through recombination. Provirus insertion was found in several Dasypus species, where it has not reached fixation, whereas related family members integrated before diversification of the genus Dasypus >12 million years ago (Mya). This newly described ERV lineage is potentially useful as a population genetic marker. Our results extend the usage of ASCT2 as a retrovirus receptor to the mammalian clade Xenarthra and suggest that the acquisition of an ASCT2-interacting env gene is a major selective force driving the emergence of numerous chimeric viruses in vertebrates. IMPORTANCE Retroviral infection is initiated by the binding of the viral envelope glycoprotein to a host cell receptor(s), triggering membrane fusion. Ancient germ line infections

  7. Structural Model for the Interaction of a Designed Ankyrin Repeat Protein with the Human Epidermal Growth Factor Receptor 2

    PubMed Central

    Epa, V. Chandana; Dolezal, Olan; Doughty, Larissa; Xiao, Xiaowen; Jost, Christian; Plückthun, Andreas; Adams, Timothy E.

    2013-01-01

    Designed Ankyrin Repeat Proteins are a class of novel binding proteins that can be selected and evolved to bind to targets with high affinity and specificity. We are interested in the DARPin H10-2-G3, which has been evolved to bind with very high affinity to the human epidermal growth factor receptor 2 (HER2). HER2 is found to be over-expressed in 30% of breast cancers, and is the target for the FDA-approved therapeutic monoclonal antibodies trastuzumab and pertuzumab and small molecule tyrosine kinase inhibitors. Here, we use computational macromolecular docking, coupled with several interface metrics such as shape complementarity, interaction energy, and electrostatic complementarity, to model the structure of the complex between the DARPin H10-2-G3 and HER2. We analyzed the interface between the two proteins and then validated the structural model by showing that selected HER2 point mutations at the putative interface with H10-2-G3 reduce the affinity of binding up to 100-fold without affecting the binding of trastuzumab. Comparisons made with a subsequently solved X-ray crystal structure of the complex yielded a backbone atom root mean square deviation of 0.84–1.14 Ångstroms. The study presented here demonstrates the capability of the computational techniques of structural bioinformatics in generating useful structural models of protein-protein interactions. PMID:23527120

  8. Structural model for the interaction of a designed Ankyrin Repeat Protein with the human epidermal growth factor receptor 2.

    PubMed

    Epa, V Chandana; Dolezal, Olan; Doughty, Larissa; Xiao, Xiaowen; Jost, Christian; Plückthun, Andreas; Adams, Timothy E

    2013-01-01

    Designed Ankyrin Repeat Proteins are a class of novel binding proteins that can be selected and evolved to bind to targets with high affinity and specificity. We are interested in the DARPin H10-2-G3, which has been evolved to bind with very high affinity to the human epidermal growth factor receptor 2 (HER2). HER2 is found to be over-expressed in 30% of breast cancers, and is the target for the FDA-approved therapeutic monoclonal antibodies trastuzumab and pertuzumab and small molecule tyrosine kinase inhibitors. Here, we use computational macromolecular docking, coupled with several interface metrics such as shape complementarity, interaction energy, and electrostatic complementarity, to model the structure of the complex between the DARPin H10-2-G3 and HER2. We analyzed the interface between the two proteins and then validated the structural model by showing that selected HER2 point mutations at the putative interface with H10-2-G3 reduce the affinity of binding up to 100-fold without affecting the binding of trastuzumab. Comparisons made with a subsequently solved X-ray crystal structure of the complex yielded a backbone atom root mean square deviation of 0.84-1.14 Ångstroms. The study presented here demonstrates the capability of the computational techniques of structural bioinformatics in generating useful structural models of protein-protein interactions.

  9. A redundant nuclear protein binding site contributes to negative regulation of the mouse mammary tumor virus long terminal repeat.

    PubMed

    Bramblett, D; Hsu, C L; Lozano, M; Earnest, K; Fabritius, C; Dudley, J

    1995-12-01

    The tissue specificity of mouse mammary tumor virus (MMTV) expression is controlled by regulatory elements in the MMTV long terminal repeat (LTR). These regulatory elements include the hormone response element, located approximately between -200 and -75, as well as binding sites for NF-1, Oct-1 (OTF-1), and mammary gland enhancer factors. Naturally occurring MMTV deletion variants isolated from T-cell and kidney tumors, transgenic-mouse experiments with MMTV LTR deletions, and transient transfection assays with LTR constructs indicate that there are additional transcription regulatory elements, including a negative regulatory element (NRE), located upstream of the hormone response element. To further define this regulatory region, we have constructed a series of BAL 31 deletion mutants in the MMTV LTR for use in transient transfection assays. These assays indicated that deletion of two regions (referred to as promoter-distal and -proximal NREs) between -637 and -201 elevated basal MMTV promoter activity in the absence of glucocorticoids. The region between -637 and -264 was surveyed for the presence of nuclear protein binding sites by gel retardation assays. Only one type of protein complex (referred to as NRE-binding protein or NBP) bound exclusively to sites that mapped to the promoter-distal and -proximal NREs identified by BAL 31 mutations. The promoter-proximal binding site was mapped further by linker substitution mutations and transfection assays. Mutations that mapped to a region containing an inverted repeat beginning at -287 relative to the start of transcription elevated basal expression of a reporter gene driven by the MMTV LTR. A 59-bp DNA fragment from the distal NRE also bound the NBP complex. Gel retardation assays showed that mutations within both inverted repeats of the proximal NRE eliminated NBP binding and mutations within single repeats altered NBP binding. Intriguingly, the NBP complex was detected in extracts from T cells and lung cells but

  10. A redundant nuclear protein binding site contributes to negative regulation of the mouse mammary tumor virus long terminal repeat.

    PubMed Central

    Bramblett, D; Hsu, C L; Lozano, M; Earnest, K; Fabritius, C; Dudley, J

    1995-01-01

    The tissue specificity of mouse mammary tumor virus (MMTV) expression is controlled by regulatory elements in the MMTV long terminal repeat (LTR). These regulatory elements include the hormone response element, located approximately between -200 and -75, as well as binding sites for NF-1, Oct-1 (OTF-1), and mammary gland enhancer factors. Naturally occurring MMTV deletion variants isolated from T-cell and kidney tumors, transgenic-mouse experiments with MMTV LTR deletions, and transient transfection assays with LTR constructs indicate that there are additional transcription regulatory elements, including a negative regulatory element (NRE), located upstream of the hormone response element. To further define this regulatory region, we have constructed a series of BAL 31 deletion mutants in the MMTV LTR for use in transient transfection assays. These assays indicated that deletion of two regions (referred to as promoter-distal and -proximal NREs) between -637 and -201 elevated basal MMTV promoter activity in the absence of glucocorticoids. The region between -637 and -264 was surveyed for the presence of nuclear protein binding sites by gel retardation assays. Only one type of protein complex (referred to as NRE-binding protein or NBP) bound exclusively to sites that mapped to the promoter-distal and -proximal NREs identified by BAL 31 mutations. The promoter-proximal binding site was mapped further by linker substitution mutations and transfection assays. Mutations that mapped to a region containing an inverted repeat beginning at -287 relative to the start of transcription elevated basal expression of a reporter gene driven by the MMTV LTR. A 59-bp DNA fragment from the distal NRE also bound the NBP complex. Gel retardation assays showed that mutations within both inverted repeats of the proximal NRE eliminated NBP binding and mutations within single repeats altered NBP binding. Intriguingly, the NBP complex was detected in extracts from T cells and lung cells but

  11. Diabetes-Related Ankyrin Repeat Protein (DARP/Ankrd23) Modifies Glucose Homeostasis by Modulating AMPK Activity in Skeletal Muscle.

    PubMed

    Shimoda, Yoshiaki; Matsuo, Kiyonari; Kitamura, Youhei; Ono, Kazunori; Ueyama, Tomomi; Matoba, Satoaki; Yamada, Hiroyuki; Wu, Tongbin; Chen, Ju; Emoto, Noriaki; Ikeda, Koji

    2015-01-01

    Skeletal muscle is the major site for glucose disposal, the impairment of which closely associates with the glucose intolerance in diabetic patients. Diabetes-related ankyrin repeat protein (DARP/Ankrd23) is a member of muscle ankyrin repeat proteins, whose expression is enhanced in the skeletal muscle under diabetic conditions; however, its role in energy metabolism remains poorly understood. Here we report a novel role of DARP in the regulation of glucose homeostasis through modulating AMP-activated protein kinase (AMPK) activity. DARP is highly preferentially expressed in skeletal muscle, and its expression was substantially upregulated during myotube differentiation of C2C12 myoblasts. Interestingly, DARP-/- mice demonstrated better glucose tolerance despite similar body weight, while their insulin sensitivity did not differ from that in wildtype mice. We found that phosphorylation of AMPK, which mediates insulin-independent glucose uptake, in skeletal muscle was significantly enhanced in DARP-/- mice compared to that in wildtype mice. Gene silencing of DARP in C2C12 myotubes enhanced AMPK phosphorylation, whereas overexpression of DARP in C2C12 myoblasts reduced it. Moreover, DARP-silencing increased glucose uptake and oxidation in myotubes, which was abrogated by the treatment with AICAR, an AMPK activator. Of note, improved glucose tolerance in DARP-/- mice was abolished when mice were treated with AICAR. Mechanistically, gene silencing of DARP enhanced protein expression of LKB1 that is a major upstream kinase for AMPK in myotubes in vitro and the skeletal muscle in vivo. Together with the altered expression under diabetic conditions, our data strongly suggest that DARP plays an important role in the regulation of glucose homeostasis under physiological and pathological conditions, and thus DARP is a new therapeutic target for the treatment of diabetes mellitus.

  12. Structure of Minimal Tetratricopeptide Repeat Domain Protein Tah1 Reveals Mechanism of Its Interaction with Pih1 and Hsp90*

    PubMed Central

    Jiménez, Beatriz; Ugwu, Francisca; Zhao, Rongmin; Ortí, Leticia; Makhnevych, Taras; Pineda-Lucena, Antonio; Houry, Walid A.

    2012-01-01

    Tah1 and Pih1 are novel Hsp90 interactors. Tah1 acts as a cofactor of Hsp90 to stabilize Pih1. In yeast, Hsp90, Tah1, and Pih1 were found to form a complex that is required for ribosomal RNA processing through their effect on box C/D small nucleolar ribonucleoprotein assembly. Tah1 is a minimal tetratricopeptide repeat protein of 111 amino acid residues that binds to the C terminus of the Hsp90 molecular chaperone, whereas Pih1 consists of 344 residues of unknown fold. The NMR structure of Tah1 has been solved, and this structure shows the presence of two tetratricopeptide repeat motifs followed by a C helix and an unstructured region. The binding of Tah1 to Hsp90 is mediated by the EEVD C-terminal residues of Hsp90, which bind to a positively charged channel formed by Tah1. Five highly conserved residues, which form a two-carboxylate clamp that tightly interacts with the ultimate Asp-0 residue of the bound peptide, are also present in Tah1. Tah1 was found to bind to the C terminus of Pih1 through the C helix and the unstructured region. The C terminus of Pih1 destabilizes the protein in vitro and in vivo, whereas the binding of Tah1 to Pih1 allows for the formation of a stable complex. Based on our data, a model for an Hsp90-Tah1-Pih1 ternary complex is proposed. PMID:22179618

  13. The Hexapeptide Repeated Segment LIAGY is a Hot Spot of Aggregation of the Pseudomonas syringae Ice Nucleation Protein.

    PubMed

    Di Martino, Patrick

    2016-01-01

    Ice nucleation proteins (INPs) form oligomeric structures by self-assembly and aggregation. We looked for the presence of potential aggregating sequences inside the INP from Pseudomonas syringae by a computational approach with the AGGRESCAN, FOMDAMYLOID and TANGO softwares. A total of 38 hot spots of aggregation were predicted in the INP sequence: 7 localized in the Nterminal domain, 2 in the C-terminal region, 28 in the highly repetitive central (HRC) region and 1 shared between the HRC and the Carboxyl-terminus regions of the protein. All the hot spots of aggregation identified in the HRC domain overlapped a 8-residue low fidelity repeat including a LIAGYrelated sequence. We confirmed the predictions by an experimental approach using synthetic peptides corresponding to different parts of the INP central sequence, absorbance spectroscopy and fluorescence spectroscopy in the presence of Congo red (CR) or Thioflavin T (ThT), respectively. Peptide 620-SFIIAGYG-627 predicted to aggregate by the three softwares induced an increase in fluorescence of ThT. Peptide 729-GFKSILTAGY-738 predicted to aggregate by AGGRESCAN and FOLDAMYLOID induced a shift in the maximum of absorbance of CR. Peptide 1124-SVLTAGA-1130 predicted to aggregate only by TANGO did not interfere with CR absorbance or ThT fluorescence. In conclusion, the use of three aggregation prediction algorithms and two biochemical assays showed that the hexapeptide repeated segment LIAGY, previously shown to form a hairpin loop may be involved in the aggregation of the P. syringae INP.

  14. Programmable RNA-binding protein composed of repeats of a single modular unit

    PubMed Central

    Adamala, Katarzyna P.; Martin-Alarcon, Daniel A.; Boyden, Edward S.

    2016-01-01

    The ability to monitor and perturb RNAs in living cells would benefit greatly from a modular protein architecture that targets unmodified RNA sequences in a programmable way. We report that the RNA-binding protein PumHD (Pumilio homology domain), which has been widely used in native and modified form for targeting RNA, can be engineered to yield a set of four canonical protein modules, each of which targets one RNA base. These modules (which we call Pumby, for Pumilio-based assembly) can be concatenated in chains of varying composition and length, to bind desired target RNAs. The specificity of such Pumby–RNA interactions was high, with undetectable binding of a Pumby chain to RNA sequences that bear three or more mismatches from the target sequence. We validate that the Pumby architecture can perform RNA-directed protein assembly and enhancement of translation of RNAs. We further demonstrate a new use of such RNA-binding proteins, measurement of RNA translation in living cells. Pumby may prove useful for many applications in the measurement, manipulation, and biotechnological utilization of unmodified RNAs in intact cells and systems. PMID:27118836

  15. Identification of CHIP, a novel tetratricopeptide repeat-containing protein that interacts with heat shock proteins and negatively regulates chaperone functions.

    PubMed

    Ballinger, C A; Connell, P; Wu, Y; Hu, Z; Thompson, L J; Yin, L Y; Patterson, C

    1999-06-01

    The chaperone function of the mammalian 70-kDa heat shock proteins Hsc70 and Hsp70 is modulated by physical interactions with four previously identified chaperone cofactors: Hsp40, BAG-1, the Hsc70-interacting protein Hip, and the Hsc70-Hsp90-organizing protein Hop. Hip and Hop interact with Hsc70 via a tetratricopeptide repeat domain. In a search for additional tetratricopeptide repeat-containing proteins, we have identified a novel 35-kDa cytoplasmic protein, carboxyl terminus of Hsc70-interacting protein (CHIP). CHIP is highly expressed in adult striated muscle in vivo and is expressed broadly in vitro in tissue culture. Hsc70 and Hsp70 were identified as potential interaction partners for this protein in a yeast two-hybrid screen. In vitro binding assays demonstrated direct interactions between CHIP and both Hsc70 and Hsp70, and complexes containing CHIP and Hsc70 were identified in immunoprecipitates of human skeletal muscle cells in vivo. Using glutathione S-transferase fusions, we found that CHIP interacted with the carboxy-terminal residues 540 to 650 of Hsc70, whereas Hsc70 interacted with the amino-terminal residues 1 to 197 (containing the tetratricopeptide domain and an adjacent charged domain) of CHIP. Recombinant CHIP inhibited Hsp40-stimulated ATPase activity of Hsc70 and Hsp70, suggesting that CHIP blocks the forward reaction of the Hsc70-Hsp70 substrate-binding cycle. Consistent with this observation, both luciferase refolding and substrate binding in the presence of Hsp40 and Hsp70 were inhibited by CHIP. Taken together, these results indicate that CHIP decreases net ATPase activity and reduces chaperone efficiency, and they implicate CHIP in the negative regulation of the forward reaction of the Hsc70-Hsp70 substrate-binding cycle.

  16. The four-transmembrane protein IP39 of Euglena forms strands by a trimeric unit repeat.

    PubMed

    Suzuki, Hiroshi; Ito, Yasuyuki; Yamazaki, Yuji; Mineta, Katsuhiko; Uji, Masami; Abe, Kazuhiro; Tani, Kazutoshi; Fujiyoshi, Yoshinori; Tsukita, Sachiko

    2013-01-01

    Euglenoid flagellates have striped surface structures comprising pellicles, which allow the cell shape to vary from rigid to flexible during the characteristic movement of the flagellates. In Euglena gracilis, the pellicular strip membranes are covered with paracrystalline arrays of a major integral membrane protein, IP39, a putative four-membrane-spanning protein with the conserved sequence motif of the PMP-22/EMP/MP20/Claudin superfamily. Here we report the three-dimensional structure of Euglena IP39 determined by electron crystallography. Two-dimensional crystals of IP39 appear to form a striated pattern of antiparallel double-rows in which trimeric IP39 units are longitudinally polymerised, resulting in continuously extending zigzag-shaped lines. Structural analysis revealed an asymmetric molecular arrangement in the trimer, and suggested that at least four different interactions between neighbouring protomers are involved. A combination of such multiple interactions would be important for linear strand formation of membrane proteins in a lipid bilayer.

  17. Association of the "IUCN vulnerable" spiny rat Clyomys bishopi (Rodentia: Echimyidae) with palm trees and armadillo burrows in southeastern Brazil.

    PubMed

    Bueno, Adriana A; Lapenta, Marina J; Oliveira, Fátima; Motta-Junior, José C

    2004-12-01

    The globally vulnerable Clyomys bishopi, a semi-fossorial and colonial rodent, is apparently limited to cerrado (savannah-like vegetation) physiognomies in São Paulo State, Brazil. The aim of the study was to verify whether the presence of C. bishopi is associated to the occurrence of palm trees (Attalea gearensis, Syagrus loefgrenii) and armadillo burrows. Thirty six quadrats were placed in different physiognomies of cerrado vegetation at Itirapina Ecological Station, southeastern Brazil to survey the number of C. bishopi burrows of individuals of palm trees and burrows of armadillos. There was a strong dependence and association between the number of C. bishopi burrows and all measured variables (Contingency tables and Spearman rank correlations). It is suggested that this rodent can be found in great numbers where palm trees are abundant. The use of armadillo burrows possibly makes the movement of the rodents easier inside their own galleries.

  18. The VP1 structural protein of enterovirus 71 interacts with human ornithine decarboxylase and gene trap ankyrin repeat.

    PubMed

    Yeo, Wee M; Chow, Vincent T K

    2007-04-01

    Enterovirus 71 (EV71) is a major etiological agent of hand, foot and mouth disease (HFMD). Several outbreaks in East Asia were associated with neurological complications and numerous deaths. EV71 possesses four structural proteins VP1-VP4 that are necessary in the formation of the pentameric icosahedral capsid. The viral capsid contributes to virulence, and VP1 is a prime target for EV71 vaccine development. Using yeast two-hybrid analysis, we demonstrated binding affinity between VP1 and three human proteins, i.e. ornithine decarboxylase (ODC1), gene trap ankyrin repeat (GTAR), and KIAA0697 expressed in brain tissue. These interactions were authenticated by co-immunoprecipitation experiments, and by indirect immunofluorescent confocal microscopy of transfected and EV71-infected Vero cells. The significant interaction between VP1 and ODC1 may compromise the latter's activity, and interfere with polyamine biosynthesis, growth and proliferation of EV71-infected cells. The interaction between VP1 and GTAR is noteworthy, since ankyrin proteins are associated with certain neural cell adhesion molecules and with the CRASH neurological syndrome. Given that VP1 is synthesized in large amounts during productive infection, these viral-host protein interactions may provide insights into the role of VP1 in the pathogenesis of EV71 disease and its neurological complications such as acute flaccid paralysis and encephalitis.

  19. MORF9 increases the RNA-binding activity of PLS-type pentatricopeptide repeat protein in plastid RNA editing.

    PubMed

    Yan, Junjie; Zhang, Qunxia; Guan, Zeyuan; Wang, Qiang; Li, Li; Ruan, Fengying; Lin, Rongcheng; Zou, Tingting; Yin, Ping

    2017-04-10

    RNA editing is a post-transcriptional process that modifies the genetic information on RNA molecules. In flowering plants, RNA editing usually alters cytidine to uridine in plastids and mitochondria. The PLS-type pentatricopeptide repeat (PPR) protein and the multiple organellar RNA editing factor (MORF, also known as RNA editing factor interacting protein (RIP)) are two types of key trans-acting factors involved in this process. However, how they cooperate with one another remains unclear. Here, we have characterized the interactions between a designer PLS-type PPR protein (PLS)3PPR and MORF9, and found that RNA-binding activity of (PLS)3PPR is drastically increased on MORF9 binding. We also determined the crystal structures of (PLS)3PPR, MORF9 and the (PLS)3PPR-MORF9 complex. MORF9 binding induces significant compressed conformational changes of (PLS)3PPR, revealing the molecular mechanisms by which MORF9-bound (PLS)3PPR has increased RNA-binding activity. Similarly, increased RNA-binding activity is observed for the natural PLS-type PPR protein, LPA66, in the presence of MORF9. These findings significantly expand our understanding of MORF function in plant organellar RNA editing.

  20. Organellar RNA editing and plant-specific extensions of pentatricopeptide repeat proteins in jungermanniid but not in marchantiid liverworts.

    PubMed

    Rüdinger, Mareike; Polsakiewicz, Monika; Knoop, Volker

    2008-07-01

    The pyrimidine exchange type of RNA editing in land plant (embryophyte) organelles has largely remained an enigma with respect to its biochemical mechanisms, the underlying specificities, and its raison d'être. Apparently arising with the earliest embryophytes, RNA editing is conspicuously absent in one clade of liverworts, the complex thalloid Marchantiidae. Several lines of evidence suggest that the large gene family of organelle-targeted RNA-binding pentatricopeptide repeat (PPR) proteins plays a fundamental role in the sequence-specific editing of organelle transcripts. We here describe the identification of PPR protein genes with plant-specific carboxyterminal (C-terminal) sequence signatures (E, E+, and DYW domains) in ferns, lycopodiophytes, mosses, hornworts, and jungermanniid liverworts, one subclass of the basal most clade of embryophytes, on DNA and cDNA level. In contrast, we were unable to identify these genes in a wide sampling of marchantiid liverworts (including the phylogenetic basal genus Blasia)--taxa for which no RNA editing is observed in the organelle transcripts. On the other hand, we found significant diversity of this type of PPR proteins also in Haplomitrium, a genus with an extremely high rate of RNA editing and a phylogenetic placement basal to all other liverworts. Although the presence of modularly extended PPR proteins correlates well with organelle RNA editing, the now apparent complete loss of an entire gene family from one clade of embryophytes, the marchantiid liverworts, remains puzzling.

  1. Albino Leaf1 That Encodes the Sole Octotricopeptide Repeat Protein Is Responsible for Chloroplast Development1[OPEN

    PubMed Central

    Tan, Jianjie; Xing, Yi; Liu, Changhong; Chen, Qiaoling; Zhu, Haitao; Wang, Jiang; Zhang, Jingliu; Zhang, Guiquan

    2016-01-01

    Chloroplast, the photosynthetic organelle in plants, plays a crucial role in plant development and growth through manipulating the capacity of photosynthesis. However, the regulatory mechanism of chloroplast development still remains elusive. Here, we characterized a mutant with defective chloroplasts in rice (Oryza sativa), termed albino leaf1 (al1), which exhibits a distinct albino phenotype in leaves, eventually leading to al1 seedling lethality. Electronic microscopy observation demonstrated that the number of thylakoids was reduced and the structure of thylakoids was disrupted in the al1 mutant during rice development, which eventually led to the breakdown of chloroplast. Molecular cloning revealed that AL1 encodes the sole octotricopeptide repeat protein (RAP) in rice. Genetic complementation of Arabidopsis (Arabidopsis thaliana) rap mutants indicated that the AL1 protein is a functional RAP. Further analysis illustrated that three transcript variants were present in the AL1 gene, and the altered splices occurred at the 3′ untranslated region of the AL1 transcript. In addition, our results also indicate that disruption of the AL1 gene results in an altered expression of chloroplast-associated genes. Consistently, proteomic analysis demonstrated that the abundance of photosynthesis-associated proteins is altered significantly, as is that of a group of metabolism-associated proteins. More specifically, we found that the loss of AL1 resulted in altered abundances of ribosomal proteins, suggesting that RAP likely also regulates the homeostasis of ribosomal proteins in rice in addition to the ribosomal RNA. Taken together, we propose that AL1, particularly the AL1a and AL1c isoforms, plays an essential role in chloroplast development in rice. PMID:27208287

  2. Recombinant expression of TLR5 proteins by ligand supplementation and a leucine-rich repeat hybrid technique.

    PubMed

    Hong, Minsun; Yoon, Sung-Il; Wilson, Ian A

    2012-10-12

    Vertebrate TLR5 directly binds bacterial flagellin proteins and activates innate immune responses against pathogenic flagellated bacteria. Structural and biochemical studies on the TLR5/flagellin interaction have been challenging due to the technical difficulty in obtaining active recombinant proteins of TLR5 ectodomain (TLR5-ECD). We recently succeeded in production of the N-terminal leucine rich repeats (LRRs) of Danio rerio (dr) TLR5-ECD in a hybrid with another LRR protein, hagfish variable lymphocyte receptor (VLR), and determined the crystal structure of its complex with flagellin D1-D2-D3 domains. Although the structure provides valuable information about the interaction, it remains to be revealed how the C-terminal region of TLR5-ECD contributes to the interaction. Here, we present two methods to obtain recombinant TLR5 proteins that contain the C-terminal region in a baculovirus expression system. First, production of biologically active full-length drTLR5-ECD was substantially enhanced by supplementation of expression culture with purified flagellin proteins. Second, we designed TLR5-VLR hybrids using an LRR hybrid technology by single and double LRR fusions and were able to express diverse regions of drTLR5-ECD, allowing us to detect a previously unidentified TLR5/flagellin interaction. The drTLR5-VLR hybrid technique was also successfully applied to human TLR5-ECD whose expression has been highly problematic. These alternative TLR5 expression strategies provide an opportunity to obtain a complete view of the TLR5/flagellin interaction and can be applied to other LRR proteins.

  3. Coccidioidomycosis in armadillo hunters from the state of Ceará, Brazil.

    PubMed

    Brillhante, Raimunda Sâmia Nogueira; Moreira Filho, Renato Evando; Rocha, Marcos Fábio Gadelha; Castelo-Branco, Débora de Souza Collares Maia; Fechine, Maria Auxiliadora Bezerra; Lima, Rita Amanda Chaves de; Picanço, Yuri Vieira Cunha; Cordeiro, Rossana de Aguiar; Camargo, Zoilo Pires de; Queiroz, José Ajax Nogueira; Araujo, Roberto Wagner Bezerra de; Mesquita, Jacó Ricarte Lima de; Sidrim, José Júlio Costa

    2012-09-01

    Coccidioidomycosis is a systemic mycosis with a variable clinical presentation. Misdiagnosis of coccidioidomycosis as bacterial pneumopathy leads to inappropriate prescription of antibiotics and delayed diagnosis. This report describes an outbreak among armadillo hunters in northeastern Brazil in which an initial diagnosis of bacterial pneumonia was later confirmed as coccidioidomycosis caused by Coccidioides posadasii. Thus, this mycosis should be considered as an alternative diagnosis in patients reporting symptoms of pneumonia, even if these symptoms are only presented for a short period, who are from areas considered endemic for this disease.

  4. Drosha Inclusions Are New Components of Dipeptide-Repeat Protein Aggregates in FTLD-TDP and ALS C9orf72 Expansion Cases

    PubMed Central

    Porta, Sílvia; Kwong, Linda K.; Trojanowski, John Q.; Lee, Virginia M.-Y.

    2015-01-01

    Abstract Frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS) are 2 neurodegenerative disorders that share clinical, genetic, and neuropathologic features. The presence of abnormal expansions of GGGGCC repeats (G4C2 repeats) in a noncoding region of the Chromosome 9 open reading frame 72 (C9orf72) gene is the major genetic cause of both FTLD and ALS. Transcribed G4C2 repeats can form nuclear RNA foci and recruit RNA-binding proteins, thereby inhibiting their normal function. Moreover, through a repeat-associated non-ATG translation mechanism, G4C2 repeats translation leads to dipeptide-repeat protein aggregation in the cytoplasm of neurons. Here, we identify Drosha protein as a new component of these dipeptide-repeat aggregates. In C9orf72 mutation cases of FTLD-TDP (c9FTLD-TDP) and ALS (c9ALS), but not in FTLD or ALS cases without C9orf72 mutation, Drosha is mislocalized to form neuronal cytoplasmic inclusions in the hippocampus, frontal cortex, and cerebellum. Further characterization of Drosha-positive neuronal cytoplasmic inclusions in the hippocampus, frontal cortex, and cerebellum revealed colocalization with p62 and ubiquilin-2, 2 pathognomonic signatures of c9FTLD-TDP and c9ALS cases; however, Drosha inclusions rarely colocalized with TDP-43 pathology. We conclude that Drosha may play a unique pathogenic role in the onset or progression of FTLD-TDP/ALS in patients with the C9orf72 mutation. PMID:25756586

  5. The Influenza A Virus Non-structural Protein NS1 Upregulates The Expression of Collagen Triple Helix Repeat Containing 1 Protein.

    PubMed

    Zhu, C; Peng, G; Yi, W; Song, H; Liu, F; Liu, X

    2016-12-01

    Influenza A virus (IAV) infection induces a strong immune response and regulates the expression of many host proteins. The collagen triple helix repeat containing 1 (CTHRC1) protein is a secreted protein that exhibits increased expression during the viral infection process. However, the regulatory function of IAV on CTHRC1 expression is obscure. In this study, we investigated the effect of IAV on CTHRC1 expression and its regulatory mechanism. A total of 106 serum specimens from healthy people and 80 serum specimens from patients infected with IAV were collected. The CTHRC1 levels in the sera from the IVA patients and healthy individuals were measured using an enzyme-linked immunosorbent assay (ELISA), and the differences were statistically analysed. A549 cells were infected with the IAV or delNS1 virus. Additionally, A549 cells were cotransfected with a eukaryotic non-structural NS1 protein gene expression plasmid and the CTHRC1 gene promoter reporter plasmid (pCTHRC1-Luc), and, the luciferase activities were assessed. The CTHRC1 mRNA and protein expression were detected using reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting, respectively. The serum CTHRC1 level was significantly higher in the IAV patients than in the healthy individuals. IAV upregulated the CTHRC1 mRNA and protein expression. The non-structural NS1 protein specifically activated CTHRC1 gene promoter activity and upregulated CTHRC1 mRNA and protein expression. The activation function had a dose-dependent effect, indicating that influenza virus upregulated CTHRC1 expression through its NS1 protein.

  6. An evolutionary comparison of leucine-rich repeat containing G protein-coupled receptors reveals a novel LGR subtype.

    PubMed

    Van Hiel, Matthias B; Vandersmissen, Hans Peter; Van Loy, Tom; Vanden Broeck, Jozef

    2012-03-01

    Leucine-rich repeat containing G protein-coupled receptors or LGRs are receptors with important functions in development and reproduction. Belonging to this evolutionarily conserved group of receptors are the well-studied glycoprotein hormone receptors and relaxin receptors in mammals, as well as the bursicon receptor, which triggers cuticle hardening and tanning in freshly enclosed insects. In this study, the numerous LGR sequences in different animal phyla are analyzed and compared. Based on these data a phylogenetic tree was generated. This information sheds new light on structural and evolutionary aspects regarding this receptor group. Apart from vertebrates and insects, LGRs are also present in early chordates (Urochordata, Cephalochordata and Hyperoartia) and other arthropods (Arachnida and Branchiopoda) as well as in Mollusca, Echinodermata, Hemichordata, Nematoda, and even in ancient animal life forms, such as Cnidaria and Placozoa. Three distinct types of LGR exist, distinguishable by their number of leucine-rich repeats (LRRs), their type-specific hinge region and the presence or absence of an LDLa motif. Type C LGRs containing only one LDLa (C1 subtype) appear to be present in nearly all animal phyla. We here describe a second subtype, C2, containing multiple LDLa motifs, which was discovered in echinoderms, mollusks and in one insect species (Pediculus humanis corporis). In addition, eight putative LGRs can be predicted from the genome data of the placozoan species Trichoplax adhaerens. They may represent an ancient form of the LGRs, however, more genomic data will be required to confirm this hypothesis.

  7. Tetratricopeptide repeat protein protects photosystem I from oxidative disruption during assembly

    PubMed Central

    Heinnickel, Mark; Kim, Rick G.; Wittkopp, Tyler M.; Yang, Wenqiang; Walters, Karim A.; Herbert, Stephen K.; Grossman, Arthur R.

    2016-01-01

    A Chlamydomonas reinhardtii mutant lacking CGL71, a thylakoid membrane protein previously shown to be involved in photosystem I (PSI) accumulation, exhibited photosensitivity and highly reduced abundance of PSI under photoheterotrophic conditions. Remarkably, the PSI content of this mutant declined to nearly undetectable levels under dark, oxic conditions, demonstrating that reduced PSI accumulation in the mutant is not strictly the result of photodamage. Furthermore, PSI returns to nearly wild-type levels when the O2 concentration in the medium is lowered. Overall, our results suggest that the accumulation of PSI in the mutant correlates with the redox state of the stroma rather than photodamage and that CGL71 functions under atmospheric O2 conditions to allow stable assembly of PSI. These findings may reflect the history of the Earth’s atmosphere as it transitioned from anoxic to highly oxic (1–2 billion years ago), a change that required organisms to evolve mechanisms to assist in the assembly and stability of proteins or complexes with O2-sensitive cofactors. PMID:26903622

  8. Wound induced Beta vulgaris polygalacturonase-inhibiting protein genes encode a longer leucine-rich repeat domain and inhibit fungal polygalacturonases

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Polygalacturonase-inhibiting proteins (PGIPs) are leucine-rich repeat (LRR) proteins involved in plant defense. Sugar beet (Beta vulgaris L.) PGIP genes, BvPGIP1, BvPGIP2 and BvPGIP3, were isolated from two breeding lines, F1016 and F1010. Full-length cDNA sequences of the three BvPGIP genes encod...

  9. Expansion of Lysine-rich Repeats in Plasmodium Proteins Generates Novel Localization Sequences That Target the Periphery of the Host Erythrocyte*

    PubMed Central

    Davies, Heledd M.; Thalassinos, Konstantinos; Osborne, Andrew R.

    2016-01-01

    Repetitive low complexity sequences, mostly assumed to have no function, are common in proteins that are exported by the malaria parasite into its host erythrocyte. We identify a group of exported proteins containing short lysine-rich tandemly repeated sequences that are sufficient to localize to the erythrocyte periphery, where key virulence-related modifications to the plasma membrane and the underlying cytoskeleton are known to occur. Efficiency of targeting is dependent on repeat number, indicating that novel targeting modules could evolve by expansion of short lysine-rich sequences. Indeed, analysis of fragments of GARP from different species shows that two novel targeting sequences have arisen via the process of repeat expansion in this protein. In the protein Hyp12, the targeting function of a lysine-rich sequence is masked by a neighboring repetitive acidic sequence, further highlighting the importance of repetitive low complexity sequences. We show that sequences capable of targeting the erythrocyte periphery are present in at least nine proteins from Plasmodium falciparum and one from Plasmodium knowlesi. We find these sequences in proteins known to be involved in erythrocyte rigidification and cytoadhesion as well as in previously uncharacterized exported proteins. Together, these data suggest that expansion and contraction of lysine-rich repeats could generate targeting sequences de novo as well as modulate protein targeting efficiency and function in response to selective pressure. PMID:27777305

  10. Ankyrin repeat-rich membrane spanning (ARMS)/Kidins220 scaffold protein regulates neuroblastoma cell proliferation through p21.

    PubMed

    Jung, Heekyung; Shin, Joo-Hyun; Park, Young-Seok; Chang, Mi-Sook

    2014-12-31

    Cell proliferation is tightly controlled by the cell-cycle regulatory proteins, primarily by cyclins and cyclin-dependent kinases (CDKs) in the G1 phase. The ankyrin repeat-rich membrane spanning (ARMS) scaffold protein, also known as kinase D-interacting substrate of 220 kDa (Kidins 220), has been previously identified as a prominent downstream target of neurotrophin and ephrin receptors. Many studies have reported that ARMS/Kidins220 acts as a major signaling platform in organizing the signaling complex to regulate various cellular responses in the nervous and vascular systems. However, the role of ARMS/Kidins220 in cell proliferation and cell-cycle progression has never been investigated. Here we report that knockdown of ARMS/Kidins220 inhibits mouse neuroblastoma cell proliferation by inducing slowdown of cell cycle in the G1 phase. This effect is mediated by the upregulation of a CDK inhibitor p21, which causes the decrease in cyclin D1 and CDK4 protein levels and subsequent reduction of pRb hyperphosphorylation. Our results suggest a new role of ARMS/Kidins220 as a signaling platform to regulate tumor cell proliferation in response to the extracellular stimuli.

  11. Conserved Ankyrin Repeat Proteins and Their NIMA Kinase Partners Regulate Extracellular Matrix Remodeling and Intracellular Trafficking in Caenorhabditis elegans.

    PubMed

    Lažetić, Vladimir; Fay, David S

    2017-01-01

    Molting is an essential developmental process in nematodes during which the epidermal apical extracellular matrix, the cuticle, is remodeled to accommodate further growth. Using genetic approaches, we identified a requirement for three conserved ankyrin repeat-rich proteins, MLT-2/ANKS6, MLT-3/ANKS3, and MLT-4/INVS, in Caenorhabditis elegans molting. Loss of mlt function resulted in severe defects in the ability of larvae to shed old cuticle and led to developmental arrest. Genetic analyses demonstrated that MLT proteins functionally cooperate with the conserved NIMA kinase family members NEKL-2/NEK8 and NEKL-3/NEK6/NEK7 to promote cuticle shedding. MLT and NEKL proteins were specifically required within the hyp7 epidermal syncytium, and fluorescently tagged mlt and nekl alleles were expressed in puncta within this tissue. Expression studies further showed that NEKL-2-MLT-2-MLT-4 and NEKL-3-MLT-3 colocalize within largely distinct assemblies of apical foci. MLT-2 and MLT-4 were required for the normal accumulation of NEKL-2 at the hyp7-seam cell boundary, and loss of mlt-2 caused abnormal nuclear accumulation of NEKL-2 Correspondingly, MLT-3, which bound directly to NEKL-3, prevented NEKL-3 nuclear localization, supporting the model that MLT proteins may serve as molecular scaffolds for NEKL kinases. Our studies additionally showed that the NEKL-MLT network regulates early steps in clathrin-mediated endocytosis at the apical surface of hyp7, which may in part account for molting defects observed in nekl and mlt mutants. This study has thus identified a conserved NEKL-MLT protein network that regulates remodeling of the apical extracellular matrix and intracellular trafficking, functions that may be conserved across species.

  12. The ankyrin repeat-containing protein Akr1p is required for the endocytosis of yeast pheromone receptors.

    PubMed Central

    Givan, S A; Sprague, G F

    1997-01-01

    The Saccharomyces cerevisiae a-factor receptor (Ste3p) requires its C-terminal cytoplasmic tail for endocytosis. Wild-type receptor is delivered to the cell surface via the secretory pathway but remains there only briefly before being internalized and delivered to the vacuole for degradation. Receptors lacking all or part of the cytoplasmic tail are not subject to this constitutive endocytosis. We used the cytoplasmic tail of Ste3p as bait in the two-hybrid system in an effort to identify other proteins involved in endocytosis. One protein identified was Akr1p, an ankyrin repeat-containing protein. We applied three criteria to demonstrate that Akr1p is involved in the constitutive endocytosis of Ste3p. First, when receptor synthesis is shut off, akr1 delta cells retain the ability to mate longer than do AKR1 cells. Second, Ste3p half-life is increased by greater than 5-fold in akr1 delta cells compared with AKR1 cells. Third, after a pulse of synthesis, newly synthesized receptor remains at the cell surface in akr1 delta mutants, whereas it is rapidly internalized in AKR1 cells. Specifically, in akr1 delta mutants, newly synthesized receptor is accessible to exogenous protease, and by indirect immunofluorescence, the receptor is located at the cell surface. akr1 delta cells are also defective for endocytosis of the alpha-factor receptor (Ste2p). Despite the block to constitutive endocytosis exhibited by akr1 delta cells, they are competent to carry out ligand-mediated endocytosis of Ste3p. In contrast, akr1 delta cells cannot carry out ligand-mediated endocytosis of Ste2p. We discuss the implications for Akr1p function in endocytosis and suggest a link to the regulation of ADP-ribosylation proteins (Arf proteins). Images PMID:9243510

  13. The Role of Slr0151, a Tetratricopeptide Repeat Protein from Synechocystis sp. PCC 6803, during Photosystem II Assembly and Repair

    PubMed Central

    Rast, Anna; Rengstl, Birgit; Heinz, Steffen; Klingl, Andreas; Nickelsen, Jörg

    2016-01-01

    The assembly and repair of photosystem II (PSII) is facilitated by a variety of assembly factors. Among those, the tetratricopeptide repeat (TPR) protein Slr0151 from Synechocystis sp. PCC 6803 (hereafter Synechocystis) has previously been assigned a repair function under high light conditions (Yang et al., 2014). Here, we show that inactivation of slr0151 affects thylakoid membrane ultrastructure even under normal light conditions. Moreover, the level and localization of Slr0151 are affected in a variety of PSII-related mutants. In particular, the data suggest a close functional relationship between Slr0151 and Sll0933, which interacts with Ycf48 during PSII assembly and is homologous to PAM68 in Arabidopsis thaliana. Immunofluorescence analysis revealed a punctate distribution of Slr0151 within several different membrane types in Synechocystis cells. PMID:27200072

  14. Structures and Polymorphic Interactions of Two Heptad-Repeat Regions of the SARS Virus S2 Protein

    SciTech Connect

    Deng,Y.; Liu, J.; Zheng, Q.; Yong, W.; Lu, M.

    2006-01-01

    Entry of SARS coronavirus into its target cell requires large-scale structural transitions in the viral spike (S) glycoprotein in order to induce fusion of the virus and cell membranes. Here we describe the identification and crystal structures of four distinct a-helical domains derived from the highly conserved heptad-repeat (HR) regions of the S2 fusion subunit. The four domains are an antiparallel four-stranded coiled coil, a parallel trimeric coiled coil, a four-helix bundle, and a six-helix bundle that is likely the final fusogenic form of the protein. When considered together, the structural and thermodynamic features of the four domains suggest a possible mechanism whereby the HR regions, initially sequestered in the native S glycoprotein spike, are released and refold sequentially to promote membrane fusion. Our results provide a structural framework for understanding the control of membrane fusion and should guide efforts to intervene in the SARS coronavirus entry process.

  15. Anatomy, histology, histochemistry and fine structure of the Harderian gland in the South American armadillo Chaetophractus villosus (Xenarthra, Mammalia).

    PubMed

    Aldana Marcos, Hernán Javier; Affanni, Jorge Mario

    2005-06-01

    The anatomical, histological, histochemical and ultrastructural characteristics of the Harderian gland of the armadillo Chaetophractus villosus were described. The gland is the largest structure in the bony orbit. It is situated in the anteroventral region of the orbit. Obvious structural differences are not observed between males and females. The gland is compound-branched tubulo-alveolar, being characterized by a single layer of columnar cells surrounded by myoepithelial cells. It possesses a single excretory duct opened into the inner canthus. All glandular cells show yellow-green autofluorescence and additionally some glandular lumen may contain dense autofluorescent solid accretions. There are two peculiar and outstanding cytoplasmic features. One is represented by the smooth endoplasmic reticulum (SER), forming a closely woven meshwork. The other one is represented by "membranous bodies" apparently derived from the SER, RER and cytoskeleton with a "Star of David" configuration situated in the supranuclear region. Three types of vesicles are detected in the cytoplasm. Histochemical staining methods reveal lipids, proteins, neutral and acidic containing glycoconjugates in secretory vesicles. The mechanism of secretion appears either merocrine or apocrine. The epithelium of the intra- and inter-lobular excretory ducts suggests secretory activity. Tubulo-acinar glands similar to those seen in the lacrimal gland and nictitans glands are found related to the intralobular and main excretory ducts. The capillary network is characterized by fenestrated endothelium. The stroma possesses unmyelinated axons and plasma cells. The normal secretion of the secretory endpieces, particularly lipids, proteins and glycoconjugates, is complemented by mucous and serous secretions released by ductal cells and glands associated to the ducts.

  16. Effects of acute versus repeated cocaine exposure on the expression of endocannabinoid signaling-related proteins in the mouse cerebellum

    PubMed Central

    Palomino, Ana; Pavón, Francisco-Javier; Blanco-Calvo, Eduardo; Serrano, Antonia; Arrabal, Sergio; Rivera, Patricia; Alén, Francisco; Vargas, Antonio; Bilbao, Ainhoa; Rubio, Leticia; Rodríguez de Fonseca, Fernando; Suárez, Juan

    2014-01-01

    Growing awareness of cerebellar involvement in addiction is based on the cerebellum’s intermediary position between motor and reward, potentially acting as an interface between motivational and cognitive functions. Here, we examined the impact of acute and repeated cocaine exposure on the two main signaling systems in the mouse cerebellum: the endocannabinoid (eCB) and glutamate systems. To this end, we investigated whether eCB signaling-related gene and protein expression {cannabinoid receptor type 1 receptors and enzymes that produce [diacylglycerol lipase alpha/beta (DAGLα/β) and N-acyl phosphatidylethanolamine phospholipase D (NAPE-PLD)] and degrade [monoacylglycerol lipase (MAGL) and fatty acid amino hydrolase (FAAH)] eCB} were altered. In addition, we analyzed the gene expression of relevant components of the glutamate signaling system [glutamate synthesizing enzymes liver-type glutaminase isoform (LGA) and kidney-type glutaminase isoform (KGA), metabotropic glutamatergic receptor (mGluR3/5), NMDA-ionotropic glutamatergic receptor (NR1/2A/2B/2C) and AMPA-ionotropic receptor subunits (GluR1/2/3/4)] and the gene expression of tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis, because noradrenergic terminals innervate the cerebellar cortex. Results indicated that acute cocaine exposure decreased DAGLα expression, suggesting a down-regulation of 2-arachidonylglycerol (2-AG) production, as well as gene expression of TH, KGA, mGluR3 and all ionotropic receptor subunits analyzed in the cerebellum. The acquisition of conditioned locomotion and sensitization after repeated cocaine exposure were associated with an increased NAPE-PLD/FAAH ratio, suggesting enhanced anandamide production, and a decreased DAGLβ/MAGL ratio, suggesting decreased 2-AG generation. Repeated cocaine also increased LGA gene expression but had no effect on glutamate receptors. These findings indicate that acute cocaine modulates the expression of the eCB and

  17. Effects of acute versus repeated cocaine exposure on the expression of endocannabinoid signaling-related proteins in the mouse cerebellum.

    PubMed

    Palomino, Ana; Pavón, Francisco-Javier; Blanco-Calvo, Eduardo; Serrano, Antonia; Arrabal, Sergio; Rivera, Patricia; Alén, Francisco; Vargas, Antonio; Bilbao, Ainhoa; Rubio, Leticia; Rodríguez de Fonseca, Fernando; Suárez, Juan

    2014-01-01

    Growing awareness of cerebellar involvement in addiction is based on the cerebellum's intermediary position between motor and reward, potentially acting as an interface between motivational and cognitive functions. Here, we examined the impact of acute and repeated cocaine exposure on the two main signaling systems in the mouse cerebellum: the endocannabinoid (eCB) and glutamate systems. To this end, we investigated whether eCB signaling-related gene and protein expression {cannabinoid receptor type 1 receptors and enzymes that produce [diacylglycerol lipase alpha/beta (DAGLα/β) and N-acyl phosphatidylethanolamine phospholipase D (NAPE-PLD)] and degrade [monoacylglycerol lipase (MAGL) and fatty acid amino hydrolase (FAAH)] eCB} were altered. In addition, we analyzed the gene expression of relevant components of the glutamate signaling system [glutamate synthesizing enzymes liver-type glutaminase isoform (LGA) and kidney-type glutaminase isoform (KGA), metabotropic glutamatergic receptor (mGluR3/5), NMDA-ionotropic glutamatergic receptor (NR1/2A/2B/2C) and AMPA-ionotropic receptor subunits (GluR1/2/3/4)] and the gene expression of tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis, because noradrenergic terminals innervate the cerebellar cortex. Results indicated that acute cocaine exposure decreased DAGLα expression, suggesting a down-regulation of 2-arachidonylglycerol (2-AG) production, as well as gene expression of TH, KGA, mGluR3 and all ionotropic receptor subunits analyzed in the cerebellum. The acquisition of conditioned locomotion and sensitization after repeated cocaine exposure were associated with an increased NAPE-PLD/FAAH ratio, suggesting enhanced anandamide production, and a decreased DAGLβ/MAGL ratio, suggesting decreased 2-AG generation. Repeated cocaine also increased LGA gene expression but had no effect on glutamate receptors. These findings indicate that acute cocaine modulates the expression of the eCB and

  18. The hypersensitive induced reaction and leucine-rich repeat proteins regulate plant cell death associated with disease and plant immunity.

    PubMed

    Choi, Hyong Woo; Kim, Young Jin; Hwang, Byung Kook

    2011-01-01

    Pathogen-induced programmed cell death (PCD) is intimately linked with disease resistance and susceptibility. However, the molecular components regulating PCD, including hypersensitive and susceptible cell death, are largely unknown in plants. In this study, we show that pathogen-induced Capsicum annuum hypersensitive induced reaction 1 (CaHIR1) and leucine-rich repeat 1 (CaLRR1) function as distinct plant PCD regulators in pepper plants during Xanthomonas campestris pv. vesicatoria infection. Confocal microscopy and protein gel blot analyses revealed that CaLRR1 and CaHIR1 localize to the extracellular matrix and plasma membrane (PM), respectively. Bimolecular fluorescent complementation and coimmunoprecipitation assays showed that the extracellular CaLRR1 specifically binds to the PM-located CaHIR1 in pepper leaves. Overexpression of CaHIR1 triggered pathogen-independent cell death in pepper and Nicotiana benthamiana plants but not in yeast cells. Virus-induced gene silencing (VIGS) of CaLRR1 and CaHIR1 distinctly strengthened and compromised hypersensitive and susceptible cell death in pepper plants, respectively. Endogenous salicylic acid levels and pathogenesis-related gene transcripts were elevated in CaHIR1-silenced plants. VIGS of NbLRR1 and NbHIR1, the N. benthamiana orthologs of CaLRR1 and CaHIR1, regulated Bax- and avrPto-/Pto-induced PCD. Taken together, these results suggest that leucine-rich repeat and hypersensitive induced reaction proteins may act as cell-death regulators associated with plant immunity and disease.

  19. Regulation of the Nucleosome Repeat Length In Vivo by the DNA Sequence, Protein Concentrations and Long-Range Interactions

    PubMed Central

    Beshnova, Daria A.; Cherstvy, Andrey G.; Vainshtein, Yevhen; Teif, Vladimir B.

    2014-01-01

    The nucleosome repeat length (NRL) is an integral chromatin property important for its biological functions. Recent experiments revealed several conflicting trends of the NRL dependence on the concentrations of histones and other architectural chromatin proteins, both in vitro and in vivo, but a systematic theoretical description of NRL as a function of DNA sequence and epigenetic determinants is currently lacking. To address this problem, we have performed an integrative biophysical and bioinformatics analysis in species ranging from yeast to frog to mouse where NRL was studied as a function of various parameters. We show that in simple eukaryotes such as yeast, a lower limit for the NRL value exists, determined by internucleosome interactions and remodeler action. For higher eukaryotes, also the upper limit exists since NRL is an increasing but saturating function of the linker histone concentration. Counterintuitively, smaller H1 variants or non-histone architectural proteins can initiate larger effects on the NRL due to entropic reasons. Furthermore, we demonstrate that different regimes of the NRL dependence on histone concentrations exist depending on whether DNA sequence-specific effects dominate over boundary effects or vice versa. We consider several classes of genomic regions with apparently different regimes of the NRL variation. As one extreme, our analysis reveals that the period of oscillations of the nucleosome density around bound RNA polymerase coincides with the period of oscillations of positioning sites of the corresponding DNA sequence. At another extreme, we show that although mouse major satellite repeats intrinsically encode well-defined nucleosome preferences, they have no unique nucleosome arrangement and can undergo a switch between two distinct types of nucleosome positioning. PMID:24992723

  20. Leucine-rich repeat-containing G-protein-coupled receptor 5 is associated with invasion, metastasis, and could be a potential therapeutic target in human gastric cancer

    PubMed Central

    Xi, H Q; Cai, A Z; Wu, X S; Cui, J X; Shen, W S; Bian, S B; Wang, N; Li, J Y; Lu, C R; Song, Z; Wei, B; Chen, L

    2014-01-01

    Background: Leucine-rich repeat-containing G-protein-coupled receptor 5 (Lgr5), which is identified as a novel intestinal stem cell marker, is overexpressed in various tumours. In this study, we explore Lgr5 expression in gastric carcinoma and analyse its role in invasion, metastasis, and prognosis in carcinoma. Methods: A combination of immunohistochemistry, western blotting, and quantitative reverse transcription–polymerase chain reaction were used to detect mRNA and protein expression levels of Lgr5 and matrix metalloproteinase 2 (MMP2). Small interfering RNA against Lgr5 was designed, synthesised, and transfected into AGS cells. The effects of Lgr5 siRNA on cell invasion were detected by transwell invasion chamber assay and wound healing assay. Results: Leucine-rich repeat-containing G-protein-coupled receptor 5 expression was significantly higher in gastric carcinomas than in normal mucosa. Leucine-rich repeat-containing G-protein-coupled receptor 5 expression positively correlated with the depth of invasion, lymph node metastasis, distance of metastasis, and MMP2 expression levels. Multivariate analysis showed that Lgr5 had an independent effect on survival, and that it positively correlated with MMP2. Leucine-rich repeat-containing G-protein-coupled receptor 5 siRNAs inhibited Lgr5 mRNA and protein expression. Transwell assays indicated that these siRNAs resulted in significantly fewer cells migrating through the polycarbonate membrane, and wound healing assay also indicated that siRNAs decreased the migration of cells. Inhibition of Lgr5 resulted in a significant decrease in MMP2 and β-catenin levels compared with those in controls. Conclusions: Leucine-rich repeat-containing G-protein-coupled receptor 5 was correlated with invasion and metastasis. Leucine-rich repeat-containing G-protein-coupled receptor 5 inhibition could serve as a novel therapeutic approach. PMID:24594994

  1. Distribution of dipeptide repeat proteins in cellular models and C9orf72 mutation cases suggests link to transcriptional silencing.

    PubMed

    Schludi, Martin H; May, Stephanie; Grässer, Friedrich A; Rentzsch, Kristin; Kremmer, Elisabeth; Küpper, Clemens; Klopstock, Thomas; Arzberger, Thomas; Edbauer, Dieter

    2015-10-01

    A massive expansion of a GGGGCC repeat upstream of the C9orf72 coding region is the most common known cause of amyotrophic lateral sclerosis and frontotemporal dementia. Despite its intronic localization and lack of a canonical start codon, both strands are translated into aggregating dipeptide repeat (DPR) proteins: poly-GA, poly-GP, poly-GR, poly-PR and poly-PA. To address conflicting findings on the predominant toxicity of the different DPR species in model systems, we compared the expression pattern of the DPR proteins in rat primary neurons and postmortem brain and spinal cord of C9orf72 mutation patients. Only poly-GA overexpression closely mimicked the p62-positive neuronal cytoplasmic inclusions commonly observed for all DPR proteins in patients. In contrast, overexpressed poly-GR and poly-PR formed nucleolar p62-negative inclusions. In patients, most of the less common neuronal intranuclear DPR inclusions were para-nucleolar and p62 positive. Neuronal nucleoli in C9orf72 cases showed normal size and morphology regardless of the presence of poly-GR and poly-PR inclusions arguing against widespread nucleolar stress, reported in cellular models. Colocalization of para-nucleolar DPR inclusions with heterochromatin and a marker of transcriptional repression (H3K9me2) indicates a link to gene transcription. In contrast, we detected numerous intranuclear DPR inclusions not associated with nucleolar structures in ependymal and subependymal cells. In patients, neuronal inclusions of poly-GR, poly-GP and the poly-GA interacting protein Unc119 were less abundant than poly-GA inclusions, but showed similar regional and subcellular distribution. Regardless of neurodegeneration, all inclusions were most abundant in neocortex, hippocampus and thalamus, with few inclusions in brain stem and spinal cord. In the granular cell layer of the cerebellum, poly-GA and Unc119 inclusions were significantly more abundant in cases with FTLD than in cases with MND and FTLD/MND. Poly

  2. Reliability of plasma lipopolysaccharide-binding protein (LBP) from repeated measures in healthy adults.

    PubMed

    Citronberg, Jessica S; Wilkens, Lynne R; Lim, Unhee; Hullar, Meredith A J; White, Emily; Newcomb, Polly A; Le Marchand, Loïc; Lampe, Johanna W

    2016-09-01

    Plasma lipopolysaccharide-binding protein (LBP), a measure of internal exposure to bacterial lipopolysaccharide, has been associated with several chronic conditions and may be a marker of chronic inflammation; however, no studies have examined the reliability of this biomarker in a healthy population. We examined the temporal reliability of LBP measured in archived samples from participants in two studies. In Study one, 60 healthy participants had blood drawn at two time points: baseline and follow-up (either three, six, or nine months). In Study two, 24 individuals had blood drawn three to four times over a seven-month period. We measured LBP in archived plasma by ELISA. Test-retest reliability was estimated by calculating the intraclass correlation coefficient (ICC). Plasma LBP concentrations showed moderate reliability in Study one (ICC 0.60, 95 % CI 0.43-0.75) and Study two (ICC 0.46, 95 % CI 0.26-0.69). Restricting the follow-up period improved reliability. In Study one, the reliability of LBP over a three-month period was 0.68 (95 % CI: 0.41-0.87). In Study two, the ICC of samples taken ≤seven days apart was 0.61 (95 % CI 0.29-0.86). Plasma LBP concentrations demonstrated moderate test-retest reliability in healthy individuals with reliability improving over a shorter follow-up period.

  3. Tandem repeat sequence analysis of staphylococcal protein A (spa) gene in methicillin-resistant Staphylococcus pseudintermedius.

    PubMed

    Moodley, Arshnee; Stegger, Marc; Ben Zakour, Nouri L; Fitzgerald, J Ross; Guardabassi, Luca

    2009-03-30

    A putative staphylococcal protein A (spa) gene was discovered in the genome of Staphylococcus pseudintermedius and used for developing a species-specific spa typing protocol. Thirty-one clinical methicillin-resistant S. pseudintermedius (MRSP) isolates from dogs and cats in four countries were characterized by spa typing, pulsed-field gel electrophoresis (PFGE) and staphylococcal cassette chromosome (SCCmec) typing. The results indicated the occurrence of two MRSP clones that acquired distinct SCCmec elements in Europe (t02, PFGE type A, SCCmec type III,) and California (t06, PFGE type B, SCCmec type V). Sequence analysis of mecA revealed the occurrence of four alleles (mecA1 to mecA4), which correlated with the geographical origin of the isolates and enabled discrimination of two distinct subtypes within the European clone. The newly developed spa typing method appeared to be a promising tool for easy and rapid typing of MRSP, either alone or in combination with SCCmec and mecA typing for fine-structure epidemiological analysis.

  4. Osteoderm morphology and development in the nine-banded armadillo, Dasypus novemcinctus (Mammalia, Xenarthra, Cingulata).

    PubMed

    Vickaryous, Matthew K; Hall, Brian K

    2006-11-01

    Among modern mammals, armadillos (Xenarthra, Cingulata) are the only group that possesses osteoderms, bony inclusions within the integument. Along the body, osteoderms are organized into five discrete assemblages: the head, pectoral, banded, pelvic, and tail shields. The pectoral, banded, and pelvic shields articulate to form the carapace. We examined osteoderm skeletogenesis in the armadillo Dasypus novemcinctus using serial and whole-mount histochemistry. Compared with the rest of the skeleton, osteoderms have a delayed onset of development. Skeletogenesis begins as condensations of osteoblasts secreting osteoid, localized within the papillary layer of the dermis. Osteoderm formation is asynchronous both within each shield and across the body. The first osteoderms to mineralize are situated within the pectoral shield of the carapace, followed by elements within the banded, head, pelvic, and tail shields. In general, within each shield ossification begins craniomedially and proceeds caudally and laterally, except over the head, where the earliest elements form over the frontal and parietal bones. The absence of cartilage precursors indicates that osteoderms are dermal elements, possibly related to the all-encompassing vertebrate dermal skeleton (exoskeleton). The mode of development of D. novemcinctus osteoderms is unlike that described for squamate osteoderms, which arise via bone metaplasia, and instead is comparable with intramembranously derived elements of the skull.

  5. Pelvic peritoneum in male armadillo and anteater (Xenarthra, Mammalia): a comparative survey.

    PubMed

    Rezende, Lorenna Cardoso; Ferreira, Jussara Rocha

    2013-01-01

    The literature supports the hypothesis that the pelvic excavation is the bottom of the abdominal cavity, which is covered by the peritoneal serous membrane in order to promote visceral dynamics. We studied the peritoneum in eight specimens of Xenarthra (Euphractus sexcinctus, Myrmecophaga tridactyla and Tamandua tetradactyla). The animals were fixed in formaldehyde (10%). For description and analyzes of the pelvic peritoneum, dissection and photo documentation were performed. We saw that the parietal serous membrane reflected, involving the pelvic viscera. The urorectal septum is the floor of the higher pelvis as a serosa reflection between the bladder and the rectum. The bladder and gonads are completely peritonized in adult armadillo. In anteaters and young armadillos, the testicles are in a position analogous to the uterus, joined by the conjunctive septum at the midline and along with the bladder, they partially project to the higher and lower pelvis. In Myrmecophagidae, vesicogenital, rectogenital and sacrorectal recesses were observed. In Dasypodidae, the recesses are similar to those of other recent vertebrates.

  6. Finite Element Analysis of the Cingulata Jaw: An Ecomorphological Approach to Armadillo's Diets.

    PubMed

    Serrano-Fochs, Sílvia; De Esteban-Trivigno, Soledad; Marcé-Nogué, Jordi; Fortuny, Josep; Fariña, Richard A

    2014-01-01

    Finite element analyses (FEA) were applied to assess the lower jaw biomechanics of cingulate xenarthrans: 14 species of armadillos as well as one Pleistocene pampathere (11 extant taxa and the extinct forms Vassallia, Eutatus and Macroeuphractus). The principal goal of this work is to comparatively assess the biomechanical capabilities of the mandible based on FEA and to relate the obtained stress patterns with diet preferences and variability, in extant and extinct species through an ecomorphology approach. The results of FEA showed that omnivorous species have stronger mandibles than insectivorous species. Moreover, this latter group of species showed high variability, including some similar biomechanical features of the insectivorous Tolypeutes matacus and Chlamyphorus truncatus to those of omnivorous species, in agreement with reported diets that include items other than insects. It remains unclear the reasons behind the stronger than expected lower jaw of Dasypus kappleri. On the other hand, the very strong mandible of the fossil taxon Vassallia maxima agrees well with the proposed herbivorous diet. Moreover, Eutatus seguini yielded a stress pattern similar to Vassalia in the posterior part of the lower jaw, but resembling that of the stoutly built Macroeuphractus outesi in the anterior part. The results highlight the need for more detailed studies on the natural history of extant armadillos. FEA proved a powerful tool for biomechanical studies in a comparative framework.

  7. In vitro genetic selection analysis of alfalfa mosaic virus coat protein binding to 3'-terminal AUGC repeats in the viral RNAs.

    PubMed Central

    Houser-Scott, F; Ansel-McKinney, P; Cai, J M; Gehrke, L

    1997-01-01

    The coat proteins of alfalfa mosaic virus (AMV) and the related ilarviruses bind specifically to the 3' untranslated regions of the viral RNAs, which contain conserved repeats of the tetranucleotide sequence AUGC. The purpose of this study was to develop a more detailed understanding of RNA sequence and/or structural determinants required for coat protein binding by characterizing the role of the AUGC repeats. Starting with a complex pool of 39-nucleotide RNA molecules containing random substitutions in the AUGC repeats, in vitro genetic selection was used to identify RNAs that bound coat protein. After six iterative rounds of selection, amplification, and reselection, 25% of the RNAs selected from the randomized pool were wild type; that is, they contained all four AUGC sequences. Among the 31 clones analyzed, AUGC was clearly the preferred selected sequence at the four repeats, but some nucleotide sequence variability was observed at AUGC(865-868) if the other three AUGC repeats were present. Variant RNAs that bound coat protein with affinities equal to or greater than that of the wild-type molecule were not selected. To extend the in vitro selection results, RNAs containing specific nucleotide substitutions were transcribed in vitro and tested in coat protein and peptide binding assays. The data strongly suggest that the AUGC repeats provide sequence-specific determinants and contribute to a structural platform for specific coat protein binding. Coat protein may function in maintaining the 3' ends of the genomic RNAs during replication by stabilizing an RNA structure that defines the 3' terminus as the initiation site for minus-strand synthesis. PMID:9032367

  8. The N-terminal repeat and the ligand binding domain A of SdrI protein is involved in hydrophobicity of S. saprophyticus.

    PubMed

    Kleine, Britta; Ali, Liaqat; Wobser, Dominique; Sakιnç, Türkân

    2015-03-01

    Staphylococcus saprophyticus is an important cause of urinary tract infection, and its cell surface hydrophobicity may contribute to virulence by facilitating adherence of the organism to uroepithelia. S. saprophyticus expresses the surface protein SdrI, a member of the serine-aspartate repeat (SD) protein family, which has multifunctional properties. The SdrI knock out mutant has a reduced hydrophobicity index (HPI) of 25%, and expressed in the non-hydrophobic Staphylococcus carnosus strain TM300 causes hydrophobicity. Using hydrophobic interaction chromatography (HIC), we confined the hydrophobic site of SdrI to the N-terminal repeat region. S. saprophyticus strains carrying different plasmid constructs lacking either the N-terminal repeats, both B or SD-repeats were less hydrophobic than wild type and fully complemented SdrI mutant (HPI: 51%). The surface hydrophobicity and HPI of both wild type and the complemented strain were also influenced by calcium (Ca(2+)) and were reduced from 81.3% and 82.4% to 10.9% and 12.3%, respectively. This study confirms that the SdrI protein of S. saprophyticus is a crucial factor for surface hydrophobicity and also gives a first significant functional description of the N-terminal repeats, which in conjunction with the B-repeats form an optimal hydrophobic conformation.

  9. Instability of the expanded (CTG){sub n} repeats in the myotonin protein kinase gene in cultured lymphoblastoid cell lines from patients with myotonic dystrophy

    SciTech Connect

    Ashizawa, Tetsuo; Patel, B.J.; Monckton, D.G.

    1996-08-15

    The mutation associated with myotonic dystrophy (DM) is the expansion of an unstable trinucleotide repeat, (CTG){sub n}, in the 3{prime}-untranslated region of the myotonin protein kinase gene. Although expanded repeats show both germline and somatic instability, the mechanisms of the instability are poorly understood. To establish a model system in which somatic instability of the DM repeat could be studied in more detail, we established lymphoblastoid cell lines (LBCL) from DM patients. Analysis of the DNA from DM LBCL using Southern blotting showed that the (CTG). repeats were apparently stable up to 29 passages in culture. To study infrequent repeat size mutations that are undetectable due to the size heterogeneity, we established LBCL of single-cell origins by cloning using multiple steps of limiting dilution. After expansion to approximately 10{sup 6} cells (equivalent to approximately 20 cell cycles), the DNAs of these cell lines were analyzed by the small pool PCR technique using primers flanking the (CTG), repeat region. Two types of mutations of the expanded (CTG){sub n} repeat alleles were detected: (1) frequent mutations that show small changes of the (CTG){sub n} repeat size, resulting in alleles in a normal distribution around the progenitor allele, and (2) relatively rare mutations with large changes of the (CTG){sub n} repeat size, with a bias toward contraction. The former may represent the mechanism responsible for the so matic heterogeneity of the (CTG), repeat size observe in blood cells of DM patients. This in vitro experimental system will be useful for further studies on mechanisms involved in the regulation of the somatic stability of the (CTG). repeats in DM. 24 refs., 4 figs.

  10. Small Glutamine-Rich Tetratricopeptide Repeat-Containing Protein Alpha (SGTA) Ablation Limits Offspring Viability and Growth in Mice

    PubMed Central

    Philp, Lisa K.; Day, Tanya K.; Butler, Miriam S.; Laven-Law, Geraldine; Jindal, Shalini; Hickey, Theresa E.; Scher, Howard I.; Butler, Lisa M.; Tilley, Wayne D.

    2016-01-01

    Small glutamine-rich tetratricopeptide repeat-containing protein α (SGTA) has been implicated as a co-chaperone and regulator of androgen and growth hormone receptor (AR, GHR) signalling. We investigated the functional consequences of partial and full Sgta ablation in vivo using Cre-lox Sgta-null mice. Sgta+/− breeders generated viable Sgta−/− offspring, but at less than Mendelian expectancy. Sgta−/− breeders were subfertile with small litters and higher neonatal death (P < 0.02). Body size was significantly and proportionately smaller in male and female Sgta−/− (vs WT, Sgta+/− P < 0.001) from d19. Serum IGF-1 levels were genotype- and sex-dependent. Food intake, muscle and bone mass and adiposity were unchanged in Sgta−/−. Vital and sex organs had normal relative weight, morphology and histology, although certain androgen-sensitive measures such as penis and preputial size, and testis descent, were greater in Sgta−/−. Expression of AR and its targets remained largely unchanged, although AR localisation was genotype- and tissue-dependent. Generally expression of other TPR-containing proteins was unchanged. In conclusion, this thorough investigation of SGTA-null mutation reports a mild phenotype of reduced body size. The model’s full potential likely will be realised by genetic crosses with other models to interrogate the role of SGTA in the many diseases in which it has been implicated. PMID:27358191

  11. MASK, a large ankyrin repeat and KH domain-containing protein involved in Drosophila receptor tyrosine kinase signaling.

    PubMed

    Smith, Rachel K; Carroll, Pamela M; Allard, John D; Simon, Michael A

    2002-01-01

    The receptor tyrosine kinases Sevenless (SEV) and the Epidermal growth factor receptor (EGFR) are required for the proper development of the Drosophila eye. The protein tyrosine phosphatase Corkscrew (CSW) is a common component of many RTK signaling pathways, and is required for signaling downstream of SEV and EGFR. In order to identify additional components of these signaling pathways, mutations that enhanced the phenotype of a dominant negative form of Corkscrew were isolated. This genetic screen identified the novel signaling molecule MASK, a large protein that contains two blocks of ankyrin repeats as well as a KH domain. MASK genetically interacts with known components of these RTK signaling pathways. In the developing eye imaginal disc, loss of MASK function generates phenotypes similar to those generated by loss of other components of the SEV and EGFR pathways. These phenotypes include compromised photoreceptor differentiation, cell survival and proliferation. Although MASK is localized predominantly in the cellular cytoplasm, it is not absolutely required for MAPK activation or nuclear translocation. Based on our results, we propose that MASK is a novel mediator of RTK signaling, and may act either downstream of MAPK or transduce signaling through a parallel branch of the RTK pathway.

  12. XNAP, a conserved ankyrin repeat-containing protein with a role in the Notch pathway during Xenopus primary neurogenesis.

    PubMed

    Lahaye, Katia; Kricha, Sadia; Bellefroid, Eric J

    2002-01-01

    The Notch signaling pathway plays an important role in many cell-fate decisions during development. Here we investigate the regulation and function of the conserved gene XNAP, which is a member of the Delta-Notch synexpression group in Xenopus. XNAP encodes a small protein with two C-terminal tandem ankyrin repeats which is expressed in the neurectoderm and in the presomitic mesoderm in a pattern that resembles that of other component of the Notch pathway. When a myc-tag form of XNAP is overexpressed in Xenopus or Hela cells, XNAP protein is detected both in the nucleus and the cytoplasm. In embryos and in animal cap assays, XNAP expression is activated, perhaps directly, by the Notch pathway and this activation appears to be Su(H) dependent. Overexpression of XNAP in embryos decreases Notch signaling, which leads to an increase in the number of primary neurons that form within the domains of the neural plate where neurogenesis normally occurs. In culture Hela cells, XNAP overexpression interferes with ICD activation of a Notch regulated reporter gene. Together, these data indicate that XNAP is a novel target of the Notch pathway that may, in a feedback loop, modulate its activity.

  13. The Molecular Chaperone Hsp70 Activates Protein Phosphatase 5 (PP5) by Binding the Tetratricopeptide Repeat (TPR) Domain*

    PubMed Central

    Connarn, Jamie N.; Assimon, Victoria A.; Reed, Rebecca A.; Tse, Eric; Southworth, Daniel R.; Zuiderweg, Erik R. P.; Gestwicki, Jason E.; Sun, Duxin

    2014-01-01

    Protein phosphatase 5 (PP5) is auto-inhibited by intramolecular interactions with its tetratricopeptide repeat (TPR) domain. Hsp90 has been shown to bind PP5 to activate its phosphatase activity. However, the functional implications of binding Hsp70 to PP5 are not yet clear. In this study, we find that both Hsp90 and Hsp70 bind to PP5 using a luciferase fragment complementation assay. A fluorescence polarization assay shows that Hsp90 (MEEVD motif) binds to the TPR domain of PP5 almost 3-fold higher affinity than Hsp70 (IEEVD motif). However, Hsp70 binding to PP5 stimulates higher phosphatase activity of PP5 than the binding of Hsp90. We find that PP5 forms a stable 1:1 complex with Hsp70, but the interaction appears asymmetric with Hsp90, with one PP5 binding the dimer. Solution NMR studies reveal that Hsc70 and PP5 proteins are dynamically independent in complex, tethered by a disordered region that connects the Hsc70 core and the IEEVD-TPR contact area. This tethered binding is expected to allow PP5 to carry out multi-site dephosphorylation of Hsp70-bound clients with a range of sizes and shapes. Together, these results demonstrate that Hsp70 recruits PP5 and activates its phosphatase activity which suggests dual roles for PP5 that might link chaperone systems with signaling pathways in cancer and development. PMID:24327656

  14. Double-stranded endonuclease activity in Bacillus halodurans clustered regularly interspaced short palindromic repeats (CRISPR)-associated Cas2 protein.

    PubMed

    Nam, Ki Hyun; Ding, Fran; Haitjema, Charles; Huang, Qingqiu; DeLisa, Matthew P; Ke, Ailong

    2012-10-19

    The CRISPR (clustered regularly interspaced short palindromic repeats) system is a prokaryotic RNA-based adaptive immune system against extrachromosomal genetic elements. Cas2 is a universally conserved core CRISPR-associated protein required for the acquisition of new spacers for CRISPR adaptation. It was previously characterized as an endoribonuclease with preference for single-stranded (ss)RNA. Here, we show using crystallography, mutagenesis, and isothermal titration calorimetry that the Bacillus halodurans Cas2 (Bha_Cas2) from the subtype I-C/Dvulg CRISPR instead possesses metal-dependent endonuclease activity against double-stranded (ds)DNA. This activity is consistent with its putative function in producing new spacers for insertion into the 5'-end of the CRISPR locus. Mutagenesis and isothermal titration calorimetry studies revealed that a single divalent metal ion (Mg(2+) or Mn(2+)), coordinated by a symmetric Asp pair in the Bha_Cas2 dimer, is involved in the catalysis. We envision that a pH-dependent conformational change switches Cas2 into a metal-binding competent conformation for catalysis. We further propose that the distinct substrate preferences among Cas2 proteins may be determined by the sequence and structure in the β1-α1 loop.

  15. LRT, a tendon-specific leucine-rich repeat protein, promotes muscle-tendon targeting through its interaction with Robo.

    PubMed

    Wayburn, Bess; Volk, Talila

    2009-11-01

    Correct muscle migration towards tendon cells, and the adhesion of these two cell types, form the basis for contractile tissue assembly in the Drosophila embryo. While molecules promoting the attraction of muscles towards tendon cells have been described, signals involved in the arrest of muscle migration following the arrival of myotubes at their corresponding tendon cells have yet to be elucidated. Here, we describe a novel tendon-specific transmembrane protein, which we named LRT due to the presence of a leucine-rich repeat domain (LRR) in its extracellular region. Our analysis suggests that LRT acts non-autonomously to better target the muscle and/or arrest its migration upon arrival at its corresponding tendon cell. Muscles in embryos lacking LRT exhibited continuous formation of membrane extensions despite arrival at their corresponding tendon cells, and a partial failure of muscles to target their correct tendon cells. In addition, overexpression of LRT in tendon cells often stalled muscles located close to the tendon cells. LRT formed a protein complex with Robo, and we detected a functional genetic interaction between Robo and LRT at the level of muscle migration behavior. Taken together, our data suggest a novel mechanism by which muscles are targeted towards tendon cells as a result of LRT-Robo interactions. This mechanism may apply to the Robo-dependent migration of a wide variety of cell types.

  16. Sfr1, a Tetrahymena thermophila Sfi1 Repeat Protein, Modulates the Production of Cortical Row Basal Bodies

    PubMed Central

    Heydeck, Westley; Stemm-Wolf, Alexander J.; Knop, Janin; Poh, Christina C.

    2016-01-01

    ABSTRACT Basal bodies are essential microtubule-based structures that template, anchor, and orient cilia at the cell surface. Cilia act primarily in the generation of directional fluid flow and sensory reception, both of which are utilized for a broad spectrum of cellular processes. Although basal bodies contribute to vital cell functions, the molecular contributors of their assembly and maintenance are poorly understood. Previous studies of the ciliate Tetrahymena thermophila revealed important roles for two centrin family members in basal body assembly, separation of new basal bodies, and stability. Here, we characterize the basal body function of a centrin-binding protein, Sfr1, in Tetrahymena. Sfr1 is part of a large family of 13 proteins in Tetrahymena that contain Sfi1 repeats (SFRs), a motif originally identified in Saccharomyces cerevisiae Sfi1 that binds centrin. Sfr1 is the only SFR protein in Tetrahymena that localizes to all cortical row and oral apparatus basal bodies. In addition, Sfr1 resides predominantly at the microtubule scaffold from the proximal cartwheel to the distal transition zone. Complete genomic knockout of SFR1 (sfr1Δ) causes a significant increase in both cortical row basal body density and the number of cortical rows, contributing to an overall overproduction of basal bodies. Reintroduction of Sfr1 into sfr1Δ mutant cells leads to a marked reduction of cortical row basal body density and the total number of cortical row basal bodies. Therefore, Sfr1 directly modulates cortical row basal body production. This study reveals an inhibitory role for Sfr1, and potentially centrins, in Tetrahymena basal body production. IMPORTANCE Basal bodies and centrioles are structurally similar and, when rendered dysfunctional as a result of improper assembly or maintenance, are associated with human diseases. Centrins are conserved and abundant components of both structures whose basal body and centriolar functions remain incompletely understood

  17. Btk29A-mediated tyrosine phosphorylation of armadillo/β-catenin promotes ring canal growth in Drosophila oogenesis.

    PubMed

    Hamada-Kawaguchi, Noriko; Nishida, Yasuyoshi; Yamamoto, Daisuke

    2015-01-01

    Drosophila Btk29A is the ortholog of mammalian Btk, a Tec family nonreceptor tyrosine kinase whose deficit causes X-linked agammaglobulinemia in humans. The Btk29AficP mutation induces multiple abnormalities in oogenesis, including the growth arrest of ring canals, large intercellular bridges that allow the flow of cytoplasm carrying maternal products essential for embryonic development from the nurse cells to the oocyte during oogenesis. In this study, inactivation of Parcas, a negative regulator of Btk29A, was found to promote Btk29A accumulation on ring canals with a concomitant increase in the ring canal diameter, counteracting the Btk29AficP mutation. This mutation markedly reduced the accumulation of phosphotyrosine on ring canals and in the regions of cell-cell contact, where adhesion-supporting proteins such as DE-cadherin and β-catenin ortholog Armadillo (Arm) are located. Our previous in vitro and in vivo analyses revealed that Btk29A directly phosphorylates Arm, leading to its release from DE-cadherin. In the present experiments, immunohistological analysis revealed that phosphorylation at tyrosine 150 (Y150) and Y667 of Arm was diminished in Btk29AficP mutant ring canals. Overexpression of an Arm mutant with unphosphorylatable Y150 inhibited ring canal growth. Thus Btk29A-induced Y150 phosphorylation is necessary for the normal growth of ring canals. We suggest that the dissociation of tyrosine-phosphorylated Arm from DE-cadherin allows dynamic actin to reorganize, leading to ring canal expansion and cell shape changes during the course of oogenesis.

  18. Head-to-Head Comparison of Soluble vs. Qβ VLP Circumsporozoite Protein Vaccines Reveals Selective Enhancement of NANP Repeat Responses

    PubMed Central

    Schwenk, Robert; DeBot, Margot; Saudan, Philippe; Dutta, Sheetij

    2015-01-01

    Circumsporozoite protein (CSP) of Plasmodium falciparum is a promising malaria vaccine target. RTS,S, the most advanced malaria vaccine candidate consists of the central NANP repeat and carboxy-terminal region of CSP displayed on a hepatitis B virus-like particle (VLP). To build upon the success of RTS,S, we produced a near full-length Plasmodium falciparum CSP that also includes the conserved amino-terminal region of CSP. We recently showed that this soluble CSP, combined with a synthetic Toll-like-receptor-4 (TLR4) agonist in stable oil-in-water emulsion (GLA/SE), induces a potent and protective immune response in mice against transgenic parasite challenge. Here we have investigated whether the immunogenicity of soluble CSP could be further augmented by presentation on a VLP. Bacteriophage Qβ VLPs can be readily produced in E.coli, they have a diameter of 25 nm and contain packaged E. coli RNA which serves as a built in adjuvant through the activation of TLR7/8. CSP was chemically conjugated to Qβ and the CSP-Qβ vaccine immunogenicity and efficacy were compared to adjuvanted soluble CSP in the C57Bl/6 mouse model. When formulated with adjuvants lacking a TLR4 agonist (Alum, SE and Montanide) the Qβ-CSP induced higher anti-NANP repeat titers, higher levels of cytophilic IgG2b/c antibodies and a trend towards higher protection against transgenic parasite challenge as compared to soluble CSP formulated in the same adjuvant. The VLP and soluble CSP immunogenicity difference was most pronounced at low antigen dose, and within the CSP molecule, the titers against the NANP repeats were preferentially enhanced by Qβ presentation. While a TLR4 agonist enhanced the immunogenicity of soluble CSP to levels comparable to the VLP vaccine, the TLR4 agonist did not further improve the immunogenicity of the Qβ-CSP vaccine. The data presented here pave the way for further improvement in the Qβ conjugation chemistry and evaluation of both the Qβ-CSP and soluble CSP vaccines

  19. CArG, CCAAT, and CCAAT-like protein binding sites in avian retrovirus long terminal repeat enhancers.

    PubMed Central

    Zachow, K R; Conklin, K F

    1992-01-01

    A strong enhancer element is located within the long terminal repeats (LTRs) of exogenous, oncogenic avian retroviruses, such as Rous sarcoma virus (RSV) and the avian leukosis viruses. The LTRs of a second class of avian retroviruses, the endogenous viruses (evs), lack detectable enhancer function, a property that correlates with major sequence differences between the LTRs of these two virus groups. Despite this lack of independent enhancer activity, we previously identified sequences in ev LTRs that were able to functionally replace essential enhancer domains from the RSV enhancer with which they share limited sequence similarity. To identify candidate enhancer domains in ev LTRs that are functionally equivalent to those in RSV LTRs, we analyzed and compared ev and RSV LTR-specific DNA-protein interactions. Using this approach, we identified two candidate enhancer domains and one deficiency in ev LTRs. One of the proposed ev enhancer domains was identified as a CArG box, a motif also found upstream of several muscle-specific genes, and as the core sequence of the c-fos serum response element. The RSV LTR contains two CArG motifs, one at a previously identified site and one identified in this report at the same relative location as the ev CArG motif. A second factor binding site that interacts with a heat-stable protein was also identified in ev LTRs and, contrary to previous suggestions, appears to be different from previously described exogenous virus enhancer binding proteins. Finally, a deficiency in factor binding was found within the one inverted CCAAT box in ev LTRs, affirming the importance of sequences that flank CCAAT motifs in factor binding and providing a candidate defect in the ev enhancer. Images PMID:1312613

  20. Suppression of C9orf72 RNA repeat-induced neurotoxicity by the ALS-associated RNA-binding protein Zfp106

    PubMed Central

    Celona, Barbara; von Dollen, John; Vatsavayai, Sarat C; Kashima, Risa; Johnson, Jeffrey R; Tang, Amy A; Hata, Akiko; Miller, Bruce L; Huang, Eric J; Krogan, Nevan J; Seeley, William W; Black, Brian L

    2017-01-01

    Expanded GGGGCC repeats in the first intron of the C9orf72 gene represent the most common cause of familial amyotrophic lateral sclerosis (ALS), but the mechanisms underlying repeat-induced disease remain incompletely resolved. One proposed gain-of-function mechanism is that repeat-containing RNA forms aggregates that sequester RNA binding proteins, leading to altered RNA metabolism in motor neurons. Here, we identify the zinc finger protein Zfp106 as a specific GGGGCC RNA repeat-binding protein, and using affinity purification-mass spectrometry, we show that Zfp106 interacts with multiple other RNA binding proteins, including the ALS-associated factors TDP-43 and FUS. We also show that Zfp106 knockout mice develop severe motor neuron degeneration, which can be suppressed by transgenic restoration of Zfp106 specifically in motor neurons. Finally, we show that Zfp106 potently suppresses neurotoxicity in a Drosophila model of C9orf72 ALS. Thus, these studies identify Zfp106 as an RNA binding protein with important implications for ALS. DOI: http://dx.doi.org/10.7554/eLife.19032.001 PMID:28072389

  1. Complementary Activities of TELOMERE REPEAT BINDING Proteins and Polycomb Group Complexes in Transcriptional Regulation of Target Genes[OPEN

    PubMed Central

    Hartwig, Benjamin; James, Geo Velikkakam

    2016-01-01

    In multicellular organisms, Polycomb Repressive Complex 1 (PRC1) and PRC2 repress target genes through histone modification and chromatin compaction. Arabidopsis thaliana mutants strongly compromised in the pathway cannot develop differentiated organs. LIKE HETEROCHROMATIN PROTEIN1 (LHP1) is so far the only known plant PRC1 component that directly binds to H3K27me3, the histone modification set by PRC2, and also associates genome-wide with trimethylation of lysine 27 of histone H3 (H3K27me3). Surprisingly, lhp1 mutants show relatively mild phenotypic alterations. To explain this paradox, we screened for genetic enhancers of lhp1 mutants to identify novel components repressing target genes together with, or in parallel to, LHP1. Two enhancing mutations were mapped to TELOMERE REPEAT BINDING PROTEIN1 (TRB1) and its paralog TRB3. We show that TRB1 binds to thousands of genomic sites containing telobox or related cis-elements with a significant increase of sites and strength of binding in the lhp1 background. Furthermore, in combination with lhp1, but not alone, trb1 mutants show increased transcription of LHP1 targets, such as floral meristem identity genes, which are more likely to be bound by TRB1 in the lhp1 background. By contrast, expression of a subset of LHP1-independent TRB1 target genes, many involved in primary metabolism, is decreased in the absence of TRB1 alone. Thus, TRB1 is a bivalent transcriptional modulator that maintains downregulation of Polycomb Group (PcG) target genes in lhp1 mutants, while it sustains high expression of targets that are regulated independently of PcG. PMID:26721861

  2. Copper complex species within a fragment of the N-terminal repeat region in opossum PrP protein.

    PubMed

    Vagliasindi, Laura I; Arena, Giuseppe; Bonomo, Raffaele P; Pappalardo, Giuseppe; Tabbì, Giovanni

    2011-03-21

    A spectroscopic (UV-Vis, CD and EPR), thermodynamic and voltammetric study of the copper(ii) complexes with the Ac-PHPGGSNWGQ-NH(2) polypeptide (L), a fragment of the opossum PrP protein N-terminal four-repeat region, was carried out in aqueous solution. It suggests the formation of a highly distorted [Cu(L)H(-2)] complex species in the neutral region, the stereochemistry of which is ascribable to a square base pyramid and a CuN(3)O(2) chromophore, resulting from the coordination of a histidine imidazole and two peptide nitrogen atoms and probably oxygen atoms from water molecules. At basic pH values a [Cu(L)H(-3)](-) species with a pseudo-octahedral geometry was also obtained, with four nitrogen donor atoms in its equatorial plane, coming from the histidine residue and from peptidic nitrogen atoms. Interestingly, at pH values relatively higher than the neutrality, the coordination sphere of the copper complex in the [Cu(L)H(-2)] species changes its stereochemistry towards a pseudo-octahedron, as suggested by the change in the parallel copper hyperfine coupling constant of the EPR spectra at low temperature. A slight difference in the redox potentials between this two-faced [Cu(L)H(-2)] complex species seems to confirm this behaviour. Both potentiometric and spectroscopic data were compared with the analogous species obtained with the Ac-PHGGGWGQ-NH(2) peptide, belonging to the octarepeat domain of the human prion protein (hPrP) N-terminal region. The [Cu(L)H(-2)] species formed by the Ac-PHPGGSNWGQ-NH(2) decapeptide, having a slightly lower stability, turned out to be less abundant and to exist within a narrow pH range.

  3. Biological and biochemical characterization of mice expressing prion protein devoid of the octapeptide repeat region after infection with prions.

    PubMed

    Yamaguchi, Yoshitaka; Miyata, Hironori; Uchiyama, Keiji; Ootsuyama, Akira; Inubushi, Sachiko; Mori, Tsuyoshi; Muramatsu, Naomi; Katamine, Shigeru; Sakaguchi, Suehiro

    2012-01-01

    Accumulating lines of evidence indicate that the N-terminal domain of prion protein (PrP) is involved in prion susceptibility in mice. In this study, to investigate the role of the octapeptide repeat (OR) region alone in the N-terminal domain for the susceptibility and pathogenesis of prion disease, we intracerebrally inoculated RML scrapie prions into tg(PrPΔOR)/Prnp(0/0) mice, which express mouse PrP missing only the OR region on the PrP-null background. Incubation times of these mice were not extended. Protease-resistant PrPΔOR, or PrP(Sc)ΔOR, was easily detectable but lower in the brains of these mice, compared to that in control wild-type mice. Consistently, prion titers were slightly lower and astrogliosis was milder in their brains. However, in their spinal cords, PrP(Sc)ΔOR and prion titers were abundant and astrogliosis was as strong as in control wild-type mice. These results indicate that the role of the OR region in prion susceptibility and pathogenesis of the disease is limited. We also found that the PrP(Sc)ΔOR, including the pre-OR residues 23-50, was unusually protease-resistant, indicating that deletion of the OR region could cause structural changes to the pre-OR region upon prion infection, leading to formation of a protease-resistant structure for the pre-OR region.

  4. CARP, a cardiac ankyrin repeat protein, is up-regulated during wound healing and induces angiogenesis in experimental granulation tissue.

    PubMed

    Shi, Yubin; Reitmaier, Birgit; Regenbogen, Johannes; Slowey, R Michael; Opalenik, Susan R; Wolf, Eckhard; Goppelt, Andreas; Davidson, Jeffrey M

    2005-01-01

    Cardiac ankyrin repeat protein (CARP) was identified by subtractive hybridization as one of a group of genes that are rapidly modulated by acute wounding of mouse skin. Quantitative RT-PCR showed that CARP was strongly induced during the first day after wounding (157.1-fold), and the high level persisted for up to 14 days. Immunohistochemistry and in situ hybridization revealed that CARP was expressed in skeletal muscle, vessel wall, hair follicle, inflammatory cells, and epidermis in the wound area. To examine the effects of CARP on wound healing, we developed an adenoviral CARP vector to treat subcutaneously implanted sponges in either rats or Flk-1(LacZ) knock-in mice. Four days after infection, CARP-infected sponges in rats showed a remarkable increase in the vascular component in granulation tissue as compared to Ad-LacZ controls. This result was confirmed by CD34 immunostaining. By 7 days post-infection of sponge implants in Flk-1(LacZ) knock-in mice, granulation tissue showed many more LacZ-positive cells in Ad-CARP-infected sponges than in virus controls. Ad-CARP treatment also induced neovascularization and increased blood perfusion in rabbit excisional wounds in and ischemic rat wounds. These findings indicate that CARP could play a unique role in therapeutic angiogenesis during wound healing.

  5. A repeat protein-based DNA polymerase inhibitor for an efficient and accurate gene amplification by PCR.

    PubMed

    Hwang, Da-Eun; Shin, Yong-Keol; Munashingha, Palinda Ruvan; Park, So-Yeon; Seo, Yeon-Soo; Kim, Hak-Sung

    2016-12-01

    A polymerase chain reaction (PCR) using a thermostable DNA polymerase is the most widely applied method in many areas of research, including life sciences, biotechnology, and medical sciences. However, a conventional PCR incurs an amplification of undesired genes mainly owing to non-specifically annealed primers and the formation of a primer-dimer complex. Herein, we present the development of a Taq DNA polymerase-specific repebody, which is a small-sized protein binder composed of leucine rich repeat (LRR) modules, as a thermolabile inhibitor for a precise and accurate gene amplification by PCR. We selected a repebody that specifically binds to the DNA polymerase through a phage display, and increased its affinity to up to 10 nM through a modular evolution approach. The repebody was shown to effectively inhibit DNA polymerase activity at low temperature and undergo thermal denaturation at high temperature, leading to a rapid and full recovery of the polymerase activity, during the initial denaturation step of the PCR. The performance and utility of the repebody was demonstrated through an accurate and efficient amplification of a target gene without nonspecific gene products in both conventional and real-time PCRs. The repebody is expected to be effectively utilized as a thermolabile inhibitor in a PCR. Biotechnol. Bioeng. 2016;113: 2544-2552. © 2016 Wiley Periodicals, Inc.

  6. Behavior of leucine-rich repeat-containing G-protein coupled receptor 5-expressing cells in the reprogramming process.

    PubMed

    Arioka, Yuko; Ito, Hiroyasu; Hirata, Akihiro; Semi, Katsunori; Yamada, Yasuhiro; Seishima, Mitsuru

    2017-02-04

    It remains unclear what cells are proper for the generation of induced pluripotent stem cells (iPSCs). Leucine-rich repeat-containing G-protein coupled receptor 5 (Lgr5) is well known as a tissue stem cell and progenitor marker, both of which are reported to be sensitive to reprogramming. In the present study, we examined the reprogramming behavior of Lgr5-expressing cells (Lgr5+ cells). First, we compared reprogramming behavior using mouse Lgr5+ and Lgr5 negative (Lgr5-) hair follicles (HFs). The number of alkaline phosphatase staining-positive cells was lesser in a well of Lgr5+ HFs than in Lgr5- HFs; however, the ratio of Nanog+ SSEA1+ cells in the cell mixture derived from Lgr5+ HFs was much higher than that from Lgr5- HFs. Lgr5+ cells could be induced from mouse embryonic fibroblasts (MEFs) after transduction with Yamanaka factors. As shown in HFs, the progeny of Lgr5+ cells arising from MEFs highly converted into Nanog+ cells and did not form Nanog- colonies. The progeny represented the status of the late reprogramming phase to a higher degree than the nonprogeny. We also confirmed this using human Lg5+ cells. Our findings suggest that the use of Lgr5+ cells will minimize sorting efforts for obtaining superior iPSCs.

  7. Ankyrin repeat-rich membrane spanning protein (kidins220) is required for neurotrophin and ephrin receptor-dependent dendrite development.

    PubMed

    Chen, Yu; Fu, Wing-Yu; Ip, Jacque P K; Ye, Tao; Fu, Amy K Y; Chao, Moses V; Ip, Nancy Y

    2012-06-13

    Dendrites are the primary sites on neurons for receiving and integrating inputs from their presynaptic partners. Defects in dendrite development perturb the formation of neural circuitry and impair information processing in the brain. Extracellular cues are important for shaping the dendritic morphogenesis, but the underlying molecular mechanisms are not well understood. In this study, we examined the role of ARMS (ankyrin repeat-rich membrane spanning protein), also known as Kidins220 (kinase D-interacting substrate of 220 kDa), previously identified as a downstream target of neurotrophin and ephrin receptors, in dendrite development. We report here that knockdown of ARMS/Kidins220 by in utero electroporation impairs dendritic branching in mouse cerebral cortex, and silencing of ARMS/Kidins220 in primary rat hippocampal neurons results in a significant decrease in the length, number, and complexity of the dendritic arbors. Overexpression of cell surface receptor tyrosine kinases, including TrkB and EphB2, in ARMS/Kidins220-deficient neurons can partially rescue the defective dendritic phenotype. More importantly, we show that PI3K (phosphoinositide-3-kinase)- and Akt-mediated signaling pathway is crucial for ARMS/Kidins220-dependent dendrite development. Furthermore, loss of ARMS/Kidins220 significantly reduced the clustering of EphB2 receptor signaling complex in neurons. Our results collectively suggest that ARMS/Kidins220 is a key player in organizing the signaling complex to transduce the extracellular stimuli to cellular responses during dendrite development.

  8. A prefoldin-associated WD-repeat protein (WDR92) is required for the correct architectural assembly of motile cilia

    PubMed Central

    Patel-King, Ramila S.; King, Stephen M.

    2016-01-01

    WDR92 is a highly conserved WD-repeat protein that has been proposed to be involved in apoptosis and also to be part of a prefoldin-like cochaperone complex. We found that WDR92 has a phylogenetic signature that is generally compatible with it playing a role in the assembly or function of specifically motile cilia. To test this hypothesis, we performed an RNAi-based knockdown of WDR92 gene expression in the planarian Schmidtea mediterranea and were able to achieve a robust reduction in mRNA expression to levels undetectable under our standard RT-PCR conditions. We found that this treatment resulted in a dramatic reduction in the rate of organismal movement that was caused by a switch in the mode of locomotion from smooth, cilia-driven gliding to muscle-based, peristaltic contractions. Although the knockdown animals still assembled cilia of normal length and in similar numbers to controls, these structures had reduced beat frequency and did not maintain hydrodynamic coupling. By transmission electron microscopy we observed that many cilia had pleiomorphic defects in their architecture, including partial loss of dynein arms, incomplete closure of the B-tubule, and occlusion or replacement of the central pair complex by accumulated electron-dense material. These observations suggest that WDR92 is part of a previously unrecognized cytoplasmic chaperone system that is specifically required to fold key components necessary to build motile ciliary axonemes. PMID:26912790

  9. A previously uncharacterized tetratricopeptide-repeat-containing protein is involved in cell envelope function in Rhizobium leguminosarum.

    PubMed

    Neudorf, Kara D; Vanderlinde, Elizabeth M; Tambalo, Dinah D; Yost, Christopher K

    2015-01-01

    Rhizobium leguminosarum is a soil bacterium that is an intracellular symbiont of leguminous plants through the formation of nitrogen-fixing root nodules. Due to the changing environments that rhizobia encounter, the cell is often faced with a variety of cell altering stressors that can compromise the cell envelope integrity. A previously uncharacterized operon (RL3499-RL3502) has been linked to proper cell envelope function, and mutants display pleiotropic phenotypes including an inability to grow on peptide-rich media. In order to identify functional partners to the operon, suppressor mutants capable of growth on complex, peptide-rich media were isolated. A suppressor mutant of a non-polar mutation to RL3500 was chosen for further characterization. Transposon mutagenesis, screening for loss of the suppressor phenotype, led to the identification of a Tn5 insertion in an uncharacterized tetratricopeptide-repeat-containing protein RL0936. Furthermore, RL0936 had a 3.5-fold increase in gene expression in the suppressor strain when compared with the WT and a 1.5-fold increase in the original RL3500 mutant. Mutation of RL0936 decreased desiccation tolerance and lowered the ability to form biofilms when compared with the WT strain. This work has identified a potential interaction between RL0936 and the RL3499-RL3502 operon that is involved in cell envelope development in R. leguminosarum, and has described phenotypic activities to a previously uncharacterized conserved hypothetical gene.

  10. Leucine-rich repeat-containing G-protein-coupled receptors as markers of adult stem cells.

    PubMed

    Barker, Nick; Clevers, Hans

    2010-05-01

    Molecular markers are used to characterize and track adult stem cells. Colon cancer research has led to the identification of 2 related receptors, leucine-rich repeat-containing, G-protein-coupled receptors (Lgr)5 and Lgr6, that are expressed by small populations of cells in a variety of adult organs. Genetic mouse models have allowed the visualization, isolation, and genetic marking of Lgr5(+ve) and Lgr6(+ve) cells and provided evidence that they are stem cells. The Lgr5(+ve) cells were found to occupy locations not commonly associated with stem cells in the stomach, small intestine, colon, and hair follicles. A multipotent population of skin stem cells express Lgr6. Single Lgr5(+ve) stem cells from the small intestine and the stomach can be cultured into long-lived organoids. Further studies of these markers might reveal adult stem cell populations in additional tissues. Identification of the ligands for Lgr5 and 6 will help elucidate stem cell functions and modes of intracellular signaling.

  11. An armadillo-like sphagesaurid crocodyliform from the Late Cretaceous of Brazil

    NASA Astrophysics Data System (ADS)

    Marinho, Thiago S.; Carvalho, Ismar S.

    2009-02-01

    The Sphagesauridae is a family of Crocodyliformes exclusively known for the Brazilian Late Cretaceous Bauru Basin. This lineage reveals how diverse was the morphology and ecology of terrestrial Crocodyliformes during the Late Cretaceous of Gondwana. Here is described Armadillosuchus arrudai gen. et sp. nov., a sphagesaurid that presents some mammal-like morphological features, such as propalinal and alternate unilateral jaw occlusion pattern and heavy body armor, composed of a rigid shield and mobile-banded section as in extant armadillos (Xenarthra, Dasypodidae). These unusual morphological features contrast to the double row of osteoderms observed on the closest relatives of A. arrudai. As its mammal analogs, A. arrudai presents some evidence of fossoriality and an exclusive terrestrial life style in contrast to the extant alligatorids and crocodylids.

  12. Liat1, an arginyltransferase-binding protein whose evolution among primates involved changes in the numbers of its 10-residue repeats.

    PubMed

    Brower, Christopher S; Rosen, Connor E; Jones, Richard H; Wadas, Brandon C; Piatkov, Konstantin I; Varshavsky, Alexander

    2014-11-18

    The arginyltransferase Ate1 is a component of the N-end rule pathway, which recognizes proteins containing N-terminal degradation signals called N-degrons, polyubiquitylates these proteins, and thereby causes their degradation by the proteasome. At least six isoforms of mouse Ate1 are produced through alternative splicing of Ate1 pre-mRNA. We identified a previously uncharacterized mouse protein, termed Liat1 (ligand of Ate1), that interacts with Ate1 but does not appear to be its arginylation substrate. Liat1 has a higher affinity for the isoforms Ate1(1A7A) and Ate1(1B7A). Liat1 stimulated the in vitro N-terminal arginylation of a model substrate by Ate1. All examined vertebrate and some invertebrate genomes encode proteins sequelogous (similar in sequence) to mouse Liat1. Sequelogs of Liat1 share a highly conserved ∼30-residue region that is shown here to be required for the binding of Liat1 to Ate1. We also identified non-Ate1 proteins that interact with Liat1. In contrast to Liat1 genes of nonprimate mammals, Liat1 genes of primates are subtelomeric, a location that tends to confer evolutionary instability on a gene. Remarkably, Liat1 proteins of some primates, from macaques to humans, contain tandem repeats of a 10-residue sequence, whereas Liat1 proteins of other mammals contain a single copy of this motif. Quantities of these repeats are, in general, different in Liat1 of different primates. For example, there are 1, 4, 13, 13, 17, and 17 repeats in the gibbon, gorilla, orangutan, bonobo, neanderthal, and human Liat1, respectively, suggesting that repeat number changes in this previously uncharacterized protein may contribute to evolution of primates.

  13. The Cell Signaling Adaptor Protein EPS-8 Is Essential for C. elegans Epidermal Elongation and Interacts with the Ankyrin Repeat Protein VAB-19

    PubMed Central

    Ding, Mei; King, Ryan S.; Berry, Emily C.; Wang, Ying; Hardin, Jeff; Chisholm, Andrew D.

    2008-01-01

    Background The epidermal cells of the C. elegans embryo undergo coordinated cell shape changes that result in the morphogenetic process of elongation. The cytoskeletal ankyrin repeat protein VAB-19 is required for cell shape changes and localizes to cell-matrix attachment structures. The molecular functions of VAB-19 in this process are obscure, as no previous interactors for VAB-19 have been described. Methodology/Principal Findings In screens for VAB-19 binding proteins we identified the signaling adaptor EPS-8. Within C. elegans epidermal cells, EPS-8 and VAB-19 colocalize at cell-matrix attachment structures. The central domain of EPS-8 is necessary and sufficient for its interaction with VAB-19. eps-8 null mutants, like vab-19 mutants, are defective in epidermal elongation and in epidermal-muscle attachment. The eps-8 locus encodes two isoforms, EPS-8A and EPS-8B, that appear to act redundantly in epidermal elongation. The function of EPS-8 in epidermal development involves its N-terminal PTB and central domains, and is independent of its C-terminal SH3 and actin-binding domains. VAB-19 appears to act earlier in the biogenesis of attachment structures and may recruit EPS-8 to these structures. Conclusions/Significance EPS-8 and VAB-19 define a novel pathway acting at cell-matrix attachments to regulate epithelial cell shape. This is the first report of a role for EPS-8 proteins in cell-matrix attachments. The existence of EPS-8B-like isoforms in Drosophila suggests this function of EPS-8 proteins could be conserved among other organisms. PMID:18833327

  14. beta-Catenin associates with the actin-bundling protein fascin in a noncadherin complex

    PubMed Central

    1996-01-01

    Catenins were first characterized as linking the cytoplasmic domains of cadherin cell-cell adhesion molecules to the cortical actin cytoskeleton. In addition to their essential role in modulating cadherin adhesivity, catenins have more recently been indicated to participate in cell and developmental signaling pathways. beta-Catenin, for example, associates directly with at least two receptor tyrosine kinases and transduces developmental signals within the Wnt pathway. Catenins also complex with the tumor suppressor protein adenomatous polyposis coli (APC), which appears to have a role in regulating cell proliferation. We have used the yeast two-hybrid method to reveal that fascin, a bundler of actin filaments, binds to beta-catenin's central Armadillo repeat domain. Western blotting of immunoprecipitates from cell line and mouse and rat brain extracts indicate that this interaction exists in vivo. Fascin and beta-catenin's association was further substantiated in vitro using purified proteins isolated from recombinant bacterial and baculoviral sources. Immunoprecipitation analysis indicates that fascin additionally binds to plakoglobin, which is highly homologous to beta-catenin but not to p120cas, a newly described catenin which contains a more divergent Armadillo-repeat domain. Immunoprecipitation, in vitro competition, and domain-mapping experiments demonstrate that fascin and E-cadherin utilize a similar binding site within beta-catenin, such that they form mutually exclusive complexes with beta-catenin. Immunofluorescence microscopy reveals that fascin and beta-catenin colocalize at cell-cell borders and dynamic cell leading edges of epithelial and endothelial cells. In addition to cell-cell borders, cadherins were unexpectedly observed to colocalize with fascin and beta-catenin at cell leading edges. It is conceivable that beta-catenin participates in modulating cytoskeletal dynamics in association with the microfilament-bundling protein fascin, perhaps in a

  15. Characterisation of glycoconjugate sugar residues in the vomeronasal organ of the armadillo Chaetophractus villosus (Mammalia, xenarthra)

    PubMed Central

    CARMANCHAHI, P. D.; FERRARI, C. C.; ALDANA MARCOS, H. J.; AFFANNI, J. M.; SONEZ, C. A.; PAZ, D. A.

    2000-01-01

    Conventional carbohydrate histochemistry and the binding patterns of 21 lectins were analysed to characterise the glycoconjugate content in the components of the vomeronasal organ of the armadillo Chaetophractus villosus. The mucomicrovillous complex of the sensory epithelium bound most of the lectins studied. No reaction was observed with Con A, PSA, S-Con A and SBA, and the sustentacular cells were stained with UEA-I, DSL, LEL, STL and Con A. The vomeronasal receptor neurons were labelled with S-WGA, WGA, PNA, UEA-I, STL, Con A, S-Con A, ECL and RCA120. The basal cell layer reacted with S-WGA, WGA, LCA, UEA-I, DSL, LEL, STL, Con A, JAC and VVA. The nonsensory epithelium exhibited a differential staining in relation to the different components. The mucociliary complex stained with ECL, DBA, JAC, RCA120, STL, LCA, PHA-E, PHA-L, LEL, BSL-I and VVA. However, SJA and UEA-I stained the mucus complex lining a subpopulation of columnar cells. The cytoplasm and cell membranes of columnar cells was labelled with DBA, DSL and LCA. The apical region of these cells exhibited moderate reactivity with LEL and SJA. None of the lectins bound specifically to secretory granules of the nonsecretory cells. Basal cells of the nonsensory epithelium were labelled with DSL, LEL, LCA, BSL-I and STL. The vomeronasal glands showed a positive reaction with WGA, DSL, LEL, LCA, DBA, PNA, RCA120 and SBA. Subpopulations of acinar cells were observed with ECL, S-WGA, Con A, S-Con A and DBA. PNA and RCA120 stained the cells lining the glandular ducts. In comparison with previous results obtained in the olfactory mucosa of the same group of armadillos, the carbohydrate composition of the vomeronasal organ sensory epithelium differed from the olfactory sensory epithelium. This is probably related to the different nature of molecules involved in the perireceptor processes. PMID:10853958

  16. The chicken FMR1 gene is highly conserved with a CCT 5{prime} - untranslated repeat and encodes an RNA-binding protein

    SciTech Connect

    Price, D.K.; Zhang, F.; Ashley, C.T. Jr.; Warren, S.T.

    1996-01-01

    The transcriptional silencing of the human gene, fragile X metal retardation 1 (FMR1), is due to abnormal methylation in response to an expanded 5{prime}-untranslated CGG trinucleotide repeat and accounts for most cases of fragile X syndrome, a frequent inherited form of metal retardation. Although the encoded fragile X mental retardation protein (FMRP) is known to have properties of a RNA-binding protein, the precise function of FMRP remains to be elucidated. We report the cloning of the chicken homolog of FMR1 and show strong evolutionary conservation, with nucleotide and amino acid identities of 85 and 92%, respectively, between chicken and human. In place of the mammalian CGG trinucleotide repeat, a 99-nt tripartite repetitive element containing a CCT trinucleotide repeat flanked on both sides by dinucleotide repeats was identified. Blocks of highly conserved 3{prime}-untranslated sequence were also found. Within the coding region, two copies each of the highly conserved K homology motif and the Arg-Gly-Gly (RGG) box motif, both ribonucleotide particle family domains implicated in RNA binding, were identified. Chicken FMRP was found to bind RNA in vitro, and this activity correlated with the presence of the carboxy-terminal portion of the protein that includes the RGG motifs. 49 refs., 7 figs.

  17. Repeated immobilization stress increases the binding of c-Fos-like proteins to a rat dopamine beta-hydroxylase promoter enhancer sequence.

    PubMed

    Nankova, B; Devlin, D; Kvetnanský, R; Kopin, I J; Sabban, E L

    1993-08-01

    Repeated immobilization stress elicits a large elevation in adrenal dopamine beta-hydroxylase (DBH) mRNA levels. This study attempts to analyze the molecular mechanism of increased DBH gene expression in stress. Adrenomedullary nuclear proteins were prepared from controls and rats exposed to various intervals of immobilization stress. Electrophoretic mobility shift assays showed that repeated stress led to increased binding of adrenomedullary nuclear factors to a cis-acting regulatory element in the rat DBH promoter (DBH-1). One of the partners in the DNA-protein complex is c-Fos or a Fos-related protein. There was a correlation between promoter binding activity and elevated steady-state levels of DBH mRNA. Our data indicate that this cis regulatory element in the rat DBH promoter is functional in vivo, and increased binding of AP1-like transcription factors to this motif is induced by immobilization stress.

  18. Ankyrin Repeat-rich Membrane Spanning/Kidins220 protein regulates dendritic branching and spine stability in vivo.

    PubMed

    Wu, Synphen H; Arévalo, Juan Carlos; Sarti, Federica; Tessarollo, Lino; Gan, Wen-Biao; Chao, Moses V

    2009-08-01

    The development of nervous system connectivity depends upon the arborization of dendritic fields and the stabilization of dendritic spine synapses. It is well established that neuronal activity and the neurotrophin BDNF modulate these correlated processes. However, the downstream mechanisms by which these extrinsic signals regulate dendritic development and spine stabilization are less well known. Here we report that a substrate of BDNF signaling, the Ankyrin Repeat-rich Membrane Spanning (ARMS) protein or Kidins220, plays a critical role in the branching of cortical and hippocampal dendrites and in the turnover of cortical spines. In the barrel somatosensory cortex and the dentate gyrus, regions where ARMS/Kidins220 is highly expressed, no difference in the complexity of dendritic arbors was observed in 1-month-old adolescent ARMS/Kidins220(+/-) mice compared to wild-type littermates. However, at 3 months of age, young adult ARMS/Kidins220(+/-) mice exhibited decreased dendritic complexity. This suggests that ARMS/Kidins220 does not play a significant role in the initial formation of dendrites but, rather, is involved in the refinement or stabilization of the arbors later in development. In addition, at 1 month of age, the rate of spine elimination was higher in ARMS/Kidins220(+/-) mice than in wild-type mice, suggesting that ARMS/Kidins220(+/-) levels regulate spine stability. Taken together, these data suggest that ARMS/Kidins220 is important for the growth of dendritic arbors and spine stability during an activity- and BDNF-dependent period of development.

  19. Bispecific designed ankyrin repeat proteins (DARPins) targeting epidermal growth factor receptor inhibit A431 cell proliferation and receptor recycling.

    PubMed

    Boersma, Ykelien L; Chao, Ginger; Steiner, Daniel; Wittrup, K Dane; Plückthun, Andreas

    2011-12-02

    The EGF receptor (EGFR) has been implicated in the development and progression of many tumors. Although monoclonal antibodies directed against EGFR have been approved for the treatment of cancer in combination with chemotherapy, there are limitations in their clinical efficacy, necessitating the search for robust targeting molecules that can be equipped with new effector functions or show a new mechanism of action. Designed ankyrin repeat proteins (DARPins) may provide the targeting component for such novel reagents. Previously, four DARPins were selected against EGFR with (sub)nanomolar affinity. As any targeting module should preferably be able to inhibit EGFR-mediated signaling, their effect on A431 cells overexpressing EGFR was examined: three of them were shown to inhibit proliferation by inducing G(1) arrest, as seen for the Food and Drug Administration-approved antibody cetuximab. To understand this inhibitory mechanism, we mapped the epitopes of the DARPins using yeast surface display. The epitopes for the biologically active DARPins overlapped with the EGF-binding site, whereas the fourth DARPin bound to a different domain, explaining the lack of a biological effect. To optimize the biological activity of the DARPins, we combined two DARPins binding to different epitopes with a flexible linker or with a leucine zipper, leading to a homodimer. The latter DARPin was able to reduce surface EGFR by inhibiting receptor recycling, leading to a dramatic decrease in cell viability. These results indicate that multispecific EGFR-specific DARPins are superior to cetuximab and may form the basis of new opportunities in tumor targeting and tumor therapy.

  20. Cloning and characterization of the genes encoding the ankyrin repeat and SOCS box-containing proteins Asb-1, Asb-2, Asb-3 and Asb-4.

    PubMed

    Kile, B T; Viney, E M; Willson, T A; Brodnicki, T C; Cancilla, M R; Herlihy, A S; Croker, B A; Baca, M; Nicola, N A; Hilton, D J; Alexander, W S

    2000-11-27

    Members of the suppressor of cytokine signalling (SOCS) family of proteins have been shown to inhibit cytokine signalling via direct interactions with JAK kinases or activated cytokine receptors. In addition to their novel amino-terminal regions and SH2 domains that mediate these interactions, the SOCS proteins also contain carboxy-terminal regions of homology called the SOCS box. The SOCS box serves to couple SOCS proteins and their binding partners with the elongin B and C complex, possibly targeting them for degradation. Several other families of proteins also contain SOCS boxes but differ from the SOCS proteins in the type of domain or motif they contain upstream of the SOCS box. We report here the cloning, characterization, mapping and expression analysis of four members of the ankyrin repeat and SOCS box-containing (Asb) protein family.

  1. Gross Osteology, Radiographic and Computed Tomographic Morphology of the Axial Skeleton of the Nine-Banded Armadillo (Dasypus novemcinctus).

    PubMed

    Alves, L S; Midon, M; Filadelpho, A L; Vulcano, L C

    2017-04-01

    This study used 20 nine-banded armadillo, four in vivo and 16 cadavers, to describe the osteoarticular anatomy of the axial skeleton by means of digital radiography and computed tomography. Vertebral formula obtained in this sample specimens was seven cervical, 10 thoracic, five lumbar, nine vertebrae related to the synsacrum and 20-27 free caudal vertebrae. Peculiar features of this species were noted as the presence of xenarthrous processes in the caudal thoracic vertebra to the last lumbar vertebra, with prominent mammillary processes in the thoracolumbar segment, fused cervical vertebra from the second to fourth vertebra and the presence of synsacrum related to sacral and caudal vertebra fused to the pelvis. They are homodont animals presenting only molariform teeth with formula of 8/8, totalling 32 teeth. There was no complexity in the execution of radiographic and tomographic examinations, concluding that execution can be carried out in nine-banded armadillos during the clinical routine for wild animals.

  2. Reassessment of the hairy long-nosed armadillo "Dasypus" pilosus (Xenarthra, Dasypodidae) and revalidation of the genus Cryptophractus Fitzinger, 1856.

    PubMed

    Castro, Mariela C; Ciancio, Martín R; Pacheco, Víctor; Salas-Gismondi, Rodolfo M; Bostelmann, J Enrique; Carlini, Alfredo A

    2015-04-14

    The hairy long-nosed armadillo, currently referred as Dasypus (Cryptophractus) pilosus, is an enigmatic species endemic to montane cloud forests and subparamo of Peruvian Andes. Its strikingly different external features, which include the carapace concealed by abundant hair, the presence of more movable bands, and a slender skull, have raised questions regarding its taxonomic status as subgenus or as genus. This paper assesses this issue based on a cladistic study and provides a detailed comparative description of the species, including the first account on the distinctive ornamentation of its osteoderms. Based on several unique characters in the carapace, skull, mandible, and teeth, as well as on the external phylogenetic position relative to other Dasypus, we favor the assignment of the hairy long-nosed armadillo to other genus. As result, we revalidate the original generic epithet, so that the valid name of the species is Cryptophractus pilosus Fitzinger, 1856.

  3. Loss of helminth species diversity in the large hairy armadillo Chaetophractus villosus on the Tierra del Fuego Island, Argentina.

    PubMed

    Ezquiaga, M C; Abba, A M; Navone, G T

    2016-03-01

    The aim of this work is to compare the taxonomic diversity of parasite species of the large hairy armadillo Chaetophractus villosus in its native range and in another recently introduced population (Tierra del Fuego island), and to evaluate whether the isolation of the latter determines a decrease in its parasitic diversity. Forty specimens from Buenos Aires and Tierra del Fuego Provinces were collected and examined for helminths. Eleven parasite species were found in the native population, and only one species was present in Tierra del Fuego (Trichohelix tuberculata). This may be explained because isolation and climatic conditions prevent encounters between potential host species and infective forms of parasites. Further sampling will be needed throughout the entire Patagonia steppe to confirm how the characteristic parasitic fauna of C. villosus behaves across the armadillo's southern distribution.

  4. Ancient DNA from the extinct South American giant glyptodont Doedicurus sp. (Xenarthra: Glyptodontidae) reveals that glyptodonts evolved from Eocene armadillos.

    PubMed

    Mitchell, Kieren J; Scanferla, Agustin; Soibelzon, Esteban; Bonini, Ricardo; Ochoa, Javier; Cooper, Alan

    2016-07-01

    Glyptodonts were giant (some of them up to ~2400 kg), heavily armoured relatives of living armadillos, which became extinct during the Late Pleistocene/early Holocene alongside much of the South American megafauna. Although glyptodonts were an important component of Cenozoic South American faunas, their early evolution and phylogenetic affinities within the order Cingulata (armoured New World placental mammals) remain controversial. In this study, we used hybridization enrichment and high-throughput sequencing to obtain a partial mitochondrial genome from Doedicurus sp., the largest (1.5 m tall, and 4 m long) and one of the last surviving glyptodonts. Our molecular phylogenetic analyses revealed that glyptodonts fall within the diversity of living armadillos. Reanalysis of morphological data using a molecular 'backbone constraint' revealed several morphological characters that supported a close relationship between glyptodonts and the tiny extant fairy armadillos (Chlamyphorinae). This is surprising as these taxa are among the most derived cingulates: glyptodonts were generally large-bodied and heavily armoured, while the fairy armadillos are tiny (~9-17 cm) and adapted for burrowing. Calibration of our phylogeny with the first appearance of glyptodonts in the Eocene resulted in a more precise timeline for xenarthran evolution. The osteological novelties of glyptodonts and their specialization for grazing appear to have evolved rapidly during the Late Eocene to Early Miocene, coincident with global temperature decreases and a shift from wet closed forest towards drier open woodland and grassland across much of South America. This environmental change may have driven the evolution of glyptodonts, culminating in the bizarre giant forms of the Pleistocene.

  5. Caenorhabditis elegans Kettin, a Large Immunoglobulin-like Repeat Protein, Binds to Filamentous Actin and Provides Mechanical Stability to the Contractile Apparatuses in Body Wall Muscle

    PubMed Central

    Ono, Kanako; Yu, Robinson; Mohri, Kurato

    2006-01-01

    Kettin is a large actin-binding protein with immunoglobulin-like (Ig) repeats, which is associated with the thin filaments in arthropod muscles. Here, we report identification and functional characterization of kettin in the nematode Caenorhabditis elegans. We found that one of the monoclonal antibodies that were raised against C. elegans muscle proteins specifically reacts with kettin (Ce-kettin). We determined the entire cDNA sequence of Ce-kettin that encodes a protein of 472 kDa with 31 Ig repeats. Arthropod kettins are splice variants of much larger connectin/titin-related proteins. However, the gene for Ce-kettin is independent of other connectin/titin-related genes. Ce-kettin localizes to the thin filaments near the dense bodies in both striated and nonstriated muscles. The C-terminal four Ig repeats and the adjacent non-Ig region synergistically bind to actin filaments in vitro. RNA interference of Ce-kettin caused weak disorganization of the actin filaments in body wall muscle. This phenotype was suppressed by inhibiting muscle contraction by a myosin mutation, but it was enhanced by tetramisole-induced hypercontraction. Furthermore, Ce-kettin was involved in organizing the cytoplasmic portion of the dense bodies in cooperation with α-actinin. These results suggest that kettin is an important regulator of myofibrillar organization and provides mechanical stability to the myofibrils during contraction. PMID:16597697

  6. When xenarthrans had enamel: insights on the evolution of their hypsodonty and paleontological support for independent evolution in armadillos

    NASA Astrophysics Data System (ADS)

    Ciancio, Martín R.; Vieytes, Emma C.; Carlini, Alfredo A.

    2014-09-01

    All xenarthrans known to date are characterized by having permanent teeth that are both high crowned and open rooted, i.e., euhypsodont, and with a type of hypsodonty different from that of the rest of Placentalia: dentine hypsodonty. Also, most xenarthrans lack enamel; however, its presence has been reported in the fossil armadillo Utaetus buccatus and in living Dasypus. Considering the divergence of Xenarthra from other eutherians that possessed enameled teeth, the absence of enamel is a derived character. Diverse specializations are known in the dentition of xenarthrans, but the primitive pattern of their teeth and dentitions is still unknown. Here, we describe the mandible and teeth of a fossil armadillo, Astegotherium dichotomus (Astegotheriini, Dasypodidae), from the early Middle Eocene of Argentine Patagonia, with teeth showing both true enamel and closed roots. It is the oldest xenarthran with mandibular remains exhibiting protohypsodonty and is therefore likely representative of ancestral cingulates and xenarthrans generally. Astegotherium supports a recent hypothesis based on molecular data that enamel loss occurred independently not only within xenarthrans but also within dasypodid armadillos.

  7. High-temperature protein G is essential for activity of the Escherichia coli clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system.

    PubMed

    Yosef, Ido; Goren, Moran G; Kiro, Ruth; Edgar, Rotem; Qimron, Udi

    2011-12-13

    Prokaryotic DNA arrays arranged as clustered regularly interspaced short palindromic repeats (CRISPR), along with their associated proteins, provide prokaryotes with adaptive immunity by RNA-mediated targeting of alien DNA or RNA matching the sequences between the repeats. Here, we present a thorough screening system for the identification of bacterial proteins participating in immunity conferred by the Escherichia coli CRISPR system. We describe the identification of one such protein, high-temperature protein G (HtpG), a homolog of the eukaryotic chaperone heat-shock protein 90. We demonstrate that in the absence of htpG, the E. coli CRISPR system loses its suicidal activity against λ prophage and its ability to provide immunity from lysogenization. Transcomplementation of htpG restores CRISPR activity. We further show that inactivity of the CRISPR system attributable to htpG deficiency can be suppressed by expression of Cas3, a protein that is essential for its activity. Accordingly, we also find that the steady-state level of overexpressed Cas3 is significantly enhanced following HtpG expression. We conclude that HtpG is a newly identified positive modulator of the CRISPR system that is essential for maintaining functional levels of Cas3.

  8. Repeated nightmares

    MedlinePlus

    ... different from night terrors . Alternative Names Nightmares - repeated; Dream anxiety disorder References American Academy of Family Physicians. Information from your family doctor. Nightmares and night terrors in children. ...

  9. The carriage of the serine-aspartate repeat protein-encoding sdr genes among Staphylococcus aureus lineages.

    PubMed

    Liu, Huanle; Lv, Jingnan; Qi, Xiuqin; Ding, Yu; Li, Dan; Hu, Longhua; Wang, Liangxing; Yu, Fangyou

    2015-01-01

    The serine-aspartate repeat proteins (Sdr) are members of a family of surface proteins and contribute to the pathogenicity of Staphylococcus aureus. Among 288 S. aureus isolates including 158 and 130 associated with skin and soft tissue infections and bloodstream infection, respectively; 275 (95.5%) were positive for at least one of three sdr genes tested. The positivity rates for sdrC, sdrD, and sdrE among S. aureus isolates were 87.8% (253/288), 63.9% (184/288), and 68.1% (196/288), respectively. 224 (77.8%) of 288 isolates were concomitantly positive for two or three sdr genes. There was an association between carriage of sdrE and methicillin-resistant S. aureus (MRSA) isolates, while the carriage rates of sdrC and sdrD in MRSA isolates were similar to those in methicillin-sensitive S. aureus (MSSA) isolates. The prevalence of co-existence of sdrC and sdrE among MRSA isolates was significantly higher than that among MSSA isolates (p<0.05). All ST1, ST5, ST7, and ST25 isolates were positive for sdrD. While all ST121 and ST398 isolates were negative for sdrD. All ST59 and ST88 isolates were positive for sdrE. All ST1 isolates were concomitantly positive for sdrC and sdrD. Concomitant carriage of sdrC, sdrD, and sdrE was found among all ST5, 75.0% (9/12) of ST1, 69.2% (9/13) of ST6, 78.6% (11/14) of ST25, and 90.9% (20/22) of ST88 isolates. sdrD was linked to CC5, CC7 and CC88 isolates, especially CC88 isolates. There was a strong association between the presence of sdrE and CC59, CC88, and CC5 isolates. A significant correlation between concomitant carriage of sdrC, sdrD, and sdrE and CC88 isolates was found. sdrC-positive, sdrD-positive and sdrE-negative gene profile was significantly associated with CC7 clone. There was an association between sdrC-positive, sdrD-negative, and sdrE-positive gene profile and CC59 isolates. A correlation between sdrC-positive, sdrD-negative, and sdrE-negative gene profile and CC121 clone was found. More CC59 isolates carried sdr

  10. Repeated Exposure to D-Amphetamine Decreases Global Protein Synthesis and Regulates the Translation of a Subset of mRNAs in the Striatum

    PubMed Central

    Biever, Anne; Boubaker-Vitre, Jihane; Cutando, Laura; Gracia-Rubio, Irene; Costa-Mattioli, Mauro; Puighermanal, Emma; Valjent, Emmanuel

    2017-01-01

    Repeated psychostimulant exposure induces persistent gene expression modifications that contribute to enduring changes in striatal GABAergic spiny projecting neurons (SPNs). However, it remains unclear whether changes in the control of mRNA translation are required for the establishment of these durable modifications. Here we report that repeated exposure to D-amphetamine decreases global striatal mRNA translation. This effect is paralleled by an enhanced phosphorylation of the translation factors, eIF2α and eEF2, and by the concomitant increased translation of a subset of mRNAs, among which the mRNA encoding for the activity regulated cytoskeleton-associated protein, also known as activity regulated gene 3.1 (Arc/Arg3.1). The enrichment of Arc/Arg3.1 mRNA in the polysomal fraction is accompanied by a robust increase of Arc/Arg3.1 protein levels within the striatum. Immunofluorescence analysis revealed that this increase occurred preferentially in D1R-expressing SPNs localized in striosome compartments. Our results suggest that the decreased global protein synthesis following repeated exposure to D-amphetamine favors the translation of a specific subset of mRNAs in the striatum. PMID:28119566

  11. Instability of the CGG repeat and expression of the FMRI protein in a male fragile X patient with a lung tumor

    SciTech Connect

    Graaff, E. de; Willemsen, R.; Zhong, N.

    1995-09-01

    The molecular mechanism of the fragile X syndrome is based on the expansion of an CGG repeat in the 5{prime} UTR of the FMR1 gene in the majority of fragile X patients. This repeat displays instability both between individuals and within an individual. We studied the instability of the CGG repeat and the expression of the FMR1 protein (FMRP) in several different tissues derived from a male fragile X patient. Using Southern blot analysis, only a full mutation is detected in 9 of the 11 tissues tested. The lung tumor contains a methylated premutation of 160 repeats, whereas in the testis, besides the full mutation, a premutation of 60 CGG repeats is detected. Immunohistochemistry of the testis revealed expression of FMR1 in the spermatogonia only, confirming the previous finding that, in the sperm cells of fragile X patients with a full mutation in their blood cells, only a premutation is present. Immunohistochemistry of brain and lung tissue revealed that 1% of the cells are expressing the FMRP. PCR analysis demonstrated the presence of a premutation of 160 repeats in these FMR1-expressing cells. This indicates that the tumor was derived from a lung cell containing a premutation. Remarkably, despite the methylation of the EagI and BssHII sites, FMRP expression is detected in the tumor. Methylation of both restriction sites has thus far resulted in a 100% correlation with the lack of FMR1 expression, but the results found in the tumor suggest that the CpGs in these restriction sites are not essential for regulation of FMR1 expression. This indicates a need for a more accurate study of the exact promoter of FMR1. 54 refs., 4 figs.

  12. Altered expression of an ankyrin-repeat protein results in leaf abnormalities, necrotic lesions, and the elaboration of a systemic signal.

    PubMed

    Wirdnam, Corina; Motoyama, Andrea; Arn-Bouldoires, Estelle; van Eeden, Sjoerd; Iglesias, Alejandro; Meins, Frederick

    2004-11-01

    The PR-like proteins, class I beta-1,3-glucanase (GLU I) and chitinase (CHN I), are induced as part of a stereotypic response that can provide protection against viral, bacterial, and fungal pathogens. We have identified two Nicotiana plumbaginifolia ankyrin-repeat proteins, designated Glucanohydrolase Binding Proteins (GBP) 1 and 2, that bind GLU I and CHN I both in vitro and when expressed in yeast cells. Sense as well as antisense transformants of tobacco carrying the GBP1 gene elaborated graft-transmissible acropetally moving signals that induced the downward curling of young leaves. This phenotype was associated with reduced starch, sucrose, and fructose accumulation; the formation of necrotic lesions; and, the induction of markers for the hypersensitive response. GBP1/2 are members of a conserved Plant- Specific Ankyrin- repeat (PANK) family that includes proteins implicated in carbohydrate allocation, reactive oxygen metabolism, hypersensitive cell death, rapid elicitor responses, virus pathogenesis, and auxin signaling. The similarity in phenotype of PANK transformants and transformants altered in carbohydrate metabolism leads us to propose that PANK family members are multifunctional proteins involved in linking plant defense responses and carbohydrate metabolism.

  13. Oldest cingulate skulls provide congruence between morphological and molecular scenarios of armadillo evolution.

    PubMed

    Billet, Guillaume; Hautier, Lionel; de Muizon, Christian; Valentin, Xavier

    2011-09-22

    The cingulates of the mammalian order Xenarthra present a typical case of disagreement between molecular and morphological phylogenetic studies. We report here the discovery of two new skulls from the Late Oligocene Salla Beds of Bolivia (approx. 26 Ma), which are the oldest known well-preserved cranial remains of the group. A new taxon is described: Kuntinaru boliviensis gen. et sp. nov. A phylogenetic analysis clusters K. boliviensis together with the armadillo subfamily Tolypeutinae. These skulls document an early spotty occurrence for the Tolypeutinae at 26 Ma, in agreement with the temporal predictions of previous molecular studies. The fossil record of tolypeutines is now characterized by a unique occurrence in the Late Oligocene, and a subsequent 12 Myr lack in the fossil record. It is noteworthy that the tolypeutines remain decidedly marginal in the Late Palaeogene and Early Neogene deposits, whereas other cingulate groups diversify. Also, the anatomical phylogenetic analysis herein, which includes K. boliviensis, is congruent with recent molecular phylogenetic analyses. Kuntinaru boliviensis is the oldest confident calibration point available for the whole Cingulata.

  14. Case study: ancient DNA recovered from pleistocene-age remains of a Florida armadillo.

    PubMed

    Letts, Brandon; Shapiro, Beth

    2012-01-01

    Warm, humid regions are not ideal for long-term DNA preservation. Consequently, little ancient DNA research has been carried out involving taxa that lived in, for example, tropical and subtropical regions. Those studies that have isolated ancient DNA from warm environments have mostly been limited to the most recent several thousand years. Here, we discuss an ancient DNA experiment in which we attempt to amplify mitochondrial DNA from remains of armadillo, glyptodont, and pampathere from sites in Florida, USA, all believed to be around 10,000-12,000 years old. We were successful in recovering DNA from only one of these samples. However, based on the amount and distribution of DNA damage, the ancient DNA recovered was well-preserved despite the age and preservation environment. In this case study chapter, we discuss the experimental procedure we used to characterize the DNA from the Floridian samples, focusing on challenges of working with ancient specimens from warm environments and steps taken to confirm the authenticity of the recovered sequence.

  15. Identification and localisation of glycoconjugates in the olfactory mucosa of the armadillo Chaetophractus villosus

    PubMed Central

    FERRARI, C. C.; CARMANCHAHI, P. D.; ALDANA MARCOS, H. J.; MUGNAINI, M. T.; AFFANNI, J. M.; PAZ, D. A.

    1999-01-01

    Conventional histochemistry and the binding patterns of 22 biotinylated lectins were examined for characterisation of glycoconjugates in the components of the olfactory mucosa of the armadillo Chaetophractus villosus. The mucous lining the olfactory epithelium showed binding sites for DSL, WGA, STL, LEL, PHA-E and JAC. Only the basilar processes of the supporting cells stained for Con-A and S-Con A. The olfactory receptor neurons stained with LEL, LCA, Con A, S-Con A, JAC and PNA. The layer of basal cells did not react with any of the lectins studied. Bowman's glands in the lamina propria showed subpopulations of acinar cells reacting with SBA, S-WGA, WGA, STL, Con A, PSA, PNA, SJA, VVA, JAC and S-Con A, but in our optical studies with lectins we were unable to differentiate between mucous and serous cells in the way that is possible on electron microscopy. The ducts of Bowman's glands were labelled with S-WGA, STL, LEL, PHA-E, BSL-I and JAC. This histochemical study on the glycoconjugates of the olfactory mucosa in the order Xenarthra provides a basis for further experimental investigations. PMID:10386777

  16. Improvement of LysM-Mediated Surface Display of Designed Ankyrin Repeat Proteins (DARPins) in Recombinant and Nonrecombinant Strains of Lactococcus lactis and Lactobacillus Species

    PubMed Central

    Zadravec, Petra; Štrukelj, Borut

    2015-01-01

    Safety and probiotic properties make lactic acid bacteria (LAB) attractive hosts for surface display of heterologous proteins. Protein display on nonrecombinant microorganisms is preferred for therapeutic and food applications due to regulatory requirements. We displayed two designed ankyrin repeat proteins (DARPins), each possessing affinity for the Fc region of human IgG, on the surface of Lactococcus lactis by fusing them to the Usp45 secretion signal and to the peptidoglycan-binding C terminus of AcmA, containing lysine motif (LysM) repeats. Growth medium containing a secreted fusion protein was used to test its heterologous binding to 10 strains of species of the genus Lactobacillus, using flow cytometry, whole-cell enzyme-linked immunosorbent assay (ELISA), and fluorescence microscopy. The fusion proteins bound to the surfaces of all lactobacilli; however, binding to the majority of bacteria was only 2- to 5-fold stronger than that of the control. Lactobacillus salivarius ATCC 11741 demonstrated exceptionally strong binding (32- to 55-fold higher than that of the control) and may therefore be an attractive host for nonrecombinant surface display. Genomic comparison of the species indicated the exopolysaccharides of Lb. salivarius as a possible reason for the difference. Additionally, a 15-fold concentration-dependent increase in nonrecombinant surface display on L. lactis was demonstrated by growing bacteria with sublethal concentrations of the antibiotics chloramphenicol and erythromycin. Nonrecombinant surface display on LAB, based on LysM repeats, was optimized by selecting Lactobacillus salivarius ATCC 11741 as the optimal host and by introducing antibiotics as additives for increasing surface display on L. lactis. Additionally, effective display of DARPins on the surfaces of nonrecombinant LAB has opened up several new therapeutic possibilities. PMID:25576617

  17. Improvement of LysM-mediated surface display of designed ankyrin repeat proteins (DARPins) in recombinant and nonrecombinant strains of Lactococcus lactis and Lactobacillus Species.

    PubMed

    Zadravec, Petra; Štrukelj, Borut; Berlec, Aleš

    2015-03-01

    Safety and probiotic properties make lactic acid bacteria (LAB) attractive hosts for surface display of heterologous proteins. Protein display on nonrecombinant microorganisms is preferred for therapeutic and food applications due to regulatory requirements. We displayed two designed ankyrin repeat proteins (DARPins), each possessing affinity for the Fc region of human IgG, on the surface of Lactococcus lactis by fusing them to the Usp45 secretion signal and to the peptidoglycan-binding C terminus of AcmA, containing lysine motif (LysM) repeats. Growth medium containing a secreted fusion protein was used to test its heterologous binding to 10 strains of species of the genus Lactobacillus, using flow cytometry, whole-cell enzyme-linked immunosorbent assay (ELISA), and fluorescence microscopy. The fusion proteins bound to the surfaces of all lactobacilli; however, binding to the majority of bacteria was only 2- to 5-fold stronger than that of the control. Lactobacillus salivarius ATCC 11741 demonstrated exceptionally strong binding (32- to 55-fold higher than that of the control) and may therefore be an attractive host for nonrecombinant surface display. Genomic comparison of the species indicated the exopolysaccharides of Lb. salivarius as a possible reason for the difference. Additionally, a 15-fold concentration-dependent increase in nonrecombinant surface display on L. lactis was demonstrated by growing bacteria with sublethal concentrations of the antibiotics chloramphenicol and erythromycin. Nonrecombinant surface display on LAB, based on LysM repeats, was optimized by selecting Lactobacillus salivarius ATCC 11741 as the optimal host and by introducing antibiotics as additives for increasing surface display on L. lactis. Additionally, effective display of DARPins on the surfaces of nonrecombinant LAB has opened up several new therapeutic possibilities.

  18. Influence of training intensity on adaptations in acid/base transport proteins, muscle buffer capacity, and repeated-sprint ability in active men.

    PubMed

    McGinley, Cian; Bishop, David J

    2016-12-01

    McGinley C, Bishop DJ. Influence of training intensity on adaptations in acid/base transport proteins, muscle buffer capacity, and repeated-sprint ability in active men. J Appl Physiol 121: 1290-1305, 2016. First published October 14, 2016; doi:10.1152/japplphysiol.00630.2016-This study measured the adaptive response to exercise training for each of the acid-base transport protein families, including providing isoform-specific evidence for the monocarboxylate transporter (MCT)1/4 chaperone protein basigin and for the electrogenic sodium-bicarbonate cotransporter (NBCe)1. We investigated whether 4 wk of work-matched, high-intensity interval training (HIIT), performed either just above the lactate threshold (HIITΔ20; n = 8), or close to peak aerobic power (HIITΔ90; n = 8), influenced adaptations in acid-base transport protein abundance, nonbicarbonate muscle buffer capacity (βmin vitro), and exercise capacity in active men. Training intensity did not discriminate between adaptations for most proteins measured, with abundance of MCT1, sodium/hydrogen exchanger (NHE) 1, NBCe1, carbonic anhydrase (CA) II, and CAXIV increasing after 4 wk, whereas there was little change in CAIII and CAIV abundance. βmin vitro also did not change. However, MCT4 protein content only increased for HIITΔ20 [effect size (ES): 1.06, 90% confidence limits × / ÷ 0.77], whereas basigin protein content only increased for HIITΔ90 (ES: 1.49, × / ÷ 1.42). Repeated-sprint ability (5 × 6-s sprints; 24 s passive rest) improved similarly for both groups. Power at the lactate threshold only improved for HIITΔ20 (ES: 0.49; 90% confidence limits ± 0.38), whereas peak O2 uptake did not change for either group. Detraining was characterized by the loss of adaptations for all of the proteins measured and for repeated-sprint ability 6 wk after removing the stimulus of HIIT. In conclusion, 4 wk of HIIT induced improvements in each of the acid-base transport protein families, but, remarkably, a 40

  19. Over-expression of rice leucine-rich repeat protein results in activation of defense response, thereby enhancing resistance to bacterial soft rot in Chinese cabbage.

    PubMed

    Park, Young Ho; Choi, Changhyun; Park, Eun Mi; Kim, Hyo Sun; Park, Hong Jae; Bae, Shin Cheol; Ahn, Ilpyung; Kim, Min Gab; Park, Sang Ryeol; Hwang, Duk-Ju

    2012-10-01

    Pectobacterium carotovorum subsp. carotovorum causes soft rot disease in various plants, including Chinese cabbage. The simple extracellular leucine-rich repeat (eLRR) domain proteins have been implicated in disease resistance. Rice leucine-rich repeat protein (OsLRP), a rice simple eLRR domain protein, is induced by pathogens, phytohormones, and salt. To see whether OsLRP enhances disease resistance to bacterial soft rot, OsLRP was introduced into Chinese cabbage by Agrobacterium-mediated transformation. Two independent transgenic lines over-expressing OsLRP were generated and further analyzed. Transgenic lines over-expressing OsLRP showed enhanced disease resistance to bacterial soft rot compared to non-transgenic control. Bacterial growth was retarded in transgenic lines over-expressing OsLRP compared to non-transgenic controls. We propose that OsLRP confers enhanced resistance to bacterial soft rot. Monitoring expression of defense-associated genes in transgenic lines over-expressing OsLRP, two different glucanases and Brassica rapa polygalacturonase inhibiting protein 2, PDF1 were constitutively activated in transgenic lines compared to non-transgenic control. Taken together, heterologous expression of OsLRP results in the activation of defense response and enhanced resistance to bacterial soft rot.

  20. Paxillin LD4 motif binds PAK and PIX through a novel 95-kD ankyrin repeat, ARF-GAP protein: A role in cytoskeletal remodeling.

    PubMed

    Turner, C E; Brown, M C; Perrotta, J A; Riedy, M C; Nikolopoulos, S N; McDonald, A R; Bagrodia, S; Thomas, S; Leventhal, P S

    1999-05-17

    Paxillin is a focal adhesion adaptor protein involved in the integration of growth factor- and adhesion-mediated signal transduction pathways. Repeats of a leucine-rich sequence named paxillin LD motifs (Brown M.C., M.S. Curtis, and C.E. Turner. 1998. Nature Struct. Biol. 5:677-678) have been implicated in paxillin binding to focal adhesion kinase (FAK) and vinculin. Here we demonstrate that the individual paxillin LD motifs function as discrete and selective protein binding interfaces. A novel scaffolding function is described for paxillin LD4 in the binding of a complex of proteins containing active p21 GTPase-activated kinase (PAK), Nck, and the guanine nucleotide exchange factor, PIX. The association of this complex with paxillin is mediated by a new 95-kD protein, p95PKL (paxillin-kinase linker), which binds directly to paxillin LD4 and PIX. This protein complex also binds to Hic-5, suggesting a conservation of LD function across the paxillin superfamily. Cloning of p95PKL revealed a multidomain protein containing an NH2-terminal ARF-GAP domain, three ankyrin-like repeats, a potential calcium-binding EF hand, calmodulin-binding IQ motifs, a myosin homology domain, and two paxillin-binding subdomains (PBS). Green fluorescent protein- (GFP-) tagged p95PKL localized to focal adhesions/complexes in CHO.K1 cells. Overexpression in neuroblastoma cells of a paxillin LD4 deletion mutant inhibited lamellipodia formation in response to insulin-like growth fac- tor-1. Microinjection of GST-LD4 into NIH3T3 cells significantly decreased cell migration into a wound. These data implicate paxillin as a mediator of p21 GTPase-regulated actin cytoskeletal reorganization through the recruitment to nascent focal adhesion structures of an active PAK/PIX complex potentially via interactions with p95PKL.

  1. The 1.7 Å resolution structure of At2g44920, a pentapeptide-repeat protein in the thylakoid lumen of Arabidopsis thaliana

    SciTech Connect

    Ni, Shuisong; McGookey, Michael E.; Tinch, Stuart L.; Jones, Alisha N.; Jayaraman, Seetharaman; Tong, Liang; Kennedy, Michael A.

    2012-01-09

    At2g44920 belongs to a diverse family (Pfam PF00805) of pentapeptide-repeat proteins (PRPs) that are present in all known organisms except yeast. PRPs contain at least eight tandem-repeating sequences of five amino acids with an approximate consensus sequence (STAV)(D/N)(L/F)(S/T/R)(X). Recent crystal structures show that PRPs adopt a highly regular four-sided right-handed {beta}-helical structure consisting mainly of type II and type IV {beta}-turns, sometimes referred to as a repeated five-residue (or Rfr) fold. Among sequenced genomes, PRP genes are most abundant in cyanobacteria, leading to speculation that PRPs play an important role in the unique lifestyle of photosynthetic cyanobacteria. Despite the recent structural characterization of several cyanobacterial PRPs, most of their functions remain unknown. Plants, whose chloroplasts are of cyanobacterial origin, have only four PRP genes in their genomes. At2g44920 is one of three PRPs located in the thylakoid lumen. Here, the crystal structure of a double methionine mutant of residues 81-224 of At2g44920, the naturally processed fragment of one of its full-length isoforms, is reported at 1.7 {angstrom} resolution. The structure of At2g44920 consists of the characteristic Rfr fold with five uninterrupted coils made up of 25 pentapeptide repeats and {alpha}-helical elements capping both termini. A disulfide bridge links the two {alpha}-helices with a conserved loop between the helical elements at its C-terminus. This structure represents the first structure of a PRP protein whose subcellular location has been experimentally confirmed to be the thylakoid lumen in a plant species.

  2. Trypanosoma cruzi III from armadillos (Dasypus novemcinctus novemcinctus) from Northeastern Venezuela and its biological behavior in murine model. Risk of emergency of Chagas' disease.

    PubMed

    Morocoima, Antonio; Carrasco, Hernán J; Boadas, Johanna; Chique, José David; Herrera, Leidi; Urdaneta-Morales, Servio

    2012-11-01

    Trypanosoma cruzi, etiological agent of Chagas' disease, was isolated from armadillos (Dasypus novemcinctus novemcinctus) captured in rural communities Northeastern Venezuela from Nueva Esparta State (no endemic for Chagas' disease), Monagas and Anzoátegui States (endemics). The isolates, genetically typed by PCR-RFLP as belonging to the TcIII DTU, have demonstrated in murine model heterogenic parasitemia, mortality and histotropism with marked parasitism in cardiac, skeletal, and smooth myocytes that showed correlation with lymphobasophilic inflammatory infiltrates. Our finding of T. cruzi infected armadillos in Isla Margarita (Nueva Esparta State), together with reports of triatomine vectors in this region, the accentuated synanthropy of armadillos, intense economic activity, migration due to tourism and the lack of environmental education programs all of them represent risks that could cause the emergence of Chagas' disease in this area. This is the first report of the TcIII DTU in Northeastern Venezuela, thus widening the geographic distribution of this DTU.

  3. Retroposed elements and their flanking regions resolve the evolutionary history of xenarthran mammals (armadillos, anteaters, and sloths).

    PubMed

    Möller-Krull, Maren; Delsuc, Frédéric; Churakov, Gennady; Marker, Claudia; Superina, Mariella; Brosius, Jürgen; Douzery, Emmanuel J P; Schmitz, Jürgen

    2007-11-01

    Armadillos, anteaters, and sloths (Order Xenarthra) comprise 1 of the 4 major clades of placental mammals. Isolated in South America from the other continental landmasses, xenarthrans diverged over a period of about 65 Myr, leaving more than 200 extinct genera and only 31 living species. The presence of both ancestral and highly derived anatomical features has made morphoanatomical analyses of the xenarthran evolutionary history difficult, and previous molecular analyses failed to resolve the relationships within armadillo subfamilies. We investigated the presence/absence patterns of retroposons from approximately 7,400 genomic loci, identifying 35 phylogenetically informative elements and an additional 39 informative rare genomic changes (RGCs). DAS-short interspersed elements (SINEs), previously described only in the Dasypus novemcinctus genome, were found in all living armadillo genera, including the previously unsampled Chlamyphorus, but were noticeably absent in sloths. The presence/absence patterns of the phylogenetically informative retroposed elements and other RGCs were then compared with data from the DNA sequences of the more than 12-kb flanking regions of these retroposons. Together, these data provide the first fully resolved genus tree of xenarthrans. Interestingly, multiple evidence supports the grouping of Chaetophractus and Zaedyus as a sister group to Euphractus within Euphractinae, an association that was not previously demonstrated. Also, flanking sequence analyses favor a close phylogenetic relationship between Cabassous and Tolypeutes within Tolypeutinae. Finally, the phylogenetic position of the subfamily Chlamyphorinae is resolved by the noncoding sequence data set as the sister group of Tolypeutinae. The data provide a stable phylogenetic framework for further evolutionary investigations of xenarthrans and important information for defining conservation priorities to save the diversity of one of the most curious groups of mammals.

  4. The EMB 506 gene encodes a novel ankyrin repeat containing protein that is essential for the normal development of Arabidopsis embryos.

    PubMed

    Albert, S; Després, B; Guilleminot, J; Bechtold, N; Pelletier, G; Delseny, M; Devic, M

    1999-01-01

    The EMB 506 gene of Arabidopsis, required for the normal development of the embryo beyond the globular stage, has been cloned. The gene encodes a protein of predicted size 35 kDa that contains five ankyrin (ANK) repeats within the C terminal moiety. ANK repeats are conserved domains of 33 amino acids involved in specific recognition of protein partners. The EMB 506 protein was detected at different stages of silique development but accumulated preferentially in the mature cauline leaves. The rescue of homozygous emb 506 embryos by complementation with the wild-type sequence cDNA demonstrated that the emb mutation is a consequence of the T-DNA insertion and that integration and expression of the transgene occurred during gametogenesis and/or early embryo development. In addition to the drastic effect of the emb 506 mutation during embryo development, complementation experiments revealed another effect of the gene: emb 506 plants transformed with the wild-type EMB 506 sequence were able to produce viable seeds but showed a reduction of apical dominance and the presence of adventitious buds or bracts along the stem. This result supports the idea that genes essential for embryogenesis may also be required at other stages of the plant life cycle.

  5. The leucine-rich repeats of LINGO-1 are not required for self-interaction or interaction with the amyloid precursor protein.

    PubMed

    Stein, Thomas; Walmsley, Adrian Robert

    2012-02-10

    LINGO-1 (leucine rich repeat and Ig domain containing Nogo receptor interacting protein-1) is a central nervous system transmembrane protein which simultaneously interacts with the Nogo-66 receptor and p75(NTR) or TROY on neurons to form a receptor complex responsible for myelin-mediated neurite outgrowth inhibition. On oligodendroglial cells, LINGO-1 interacts with p75(NTR) to constitutively inhibit multiple aspects of oligodendrocyte differentiation. Recently, LINGO-1 was identified as an in vivo interacting partner of the amyloid precursor protein (APP) and, correspondingly, cellular LINGO-1 expression was found to augment the release of the Abeta peptide, the potential causative agent of Alzheimer's disease. In addition, the recombinant LINGO-1 ectodomain has been shown to self-interact in solution and after crystallisation. Here, we have used deletional mutagenesis to identify the regions on LINGO-1 that are involved in homo- and heterotypic interactions. We have found that the N-terminal region containing the leucine-rich repeats along with the transmembrane and cytoplasmic domains of LINGO-1 are not required for self-interaction or interaction with APP.

  6. A novel calmodulin-binding protein, belonging to the WD-repeat family, is localized in dendrites of a subset of CNS neurons

    PubMed Central

    1996-01-01

    A rat brain synaptosomal protein of 110,000 M(r) present in a fraction highly enriched in adenylyl cyclase activity was microsequenced (Castets, F., G. Baillat, S. Mirzoeva, K. Mabrouk, J. Garin, J. d'Alayer, and A. Monneron. 1994. Biochemistry. 33:5063-5069). Peptide sequences were used to clone a cDNA encoding a novel, 780-amino acid protein named striatin. Striatin is a member of the WD-repeat family (Neer, E.J., C.J. Schmidt, R. Nambudripad, and T.F. Smith. 1994. Nature (Lond.). 371:297-300), the first one known to bind calmodulin (CaM) in the presence of Ca++. Subcellular fractionation shows that striatin is a membrane-associated, Lubrol-soluble protein. As analyzed by Northern blots, in situ hybridization, and immunocytochemistry, striatin is localized in the central nervous system, where it is confined to a subset of neurons, many of which are associated with the motor system. In particular, striatin is conspicuous in the dorsal part of the striatum, as well as in motoneurons. Furthermore, striatin is essentially found in dendrites, but not in axons, and is most abundant in dendritic spines. We propose that striatin interacts, through its WD- repeat domain and in a CaM/Ca(++)-dependent manner, with one or several members of a surrounding cluster of molecules engaged in a Ca(++)- signaling pathway specific to excitatory synapses. PMID:8769426

  7. Structural Organization of the WD repeat protein-encoding gene HIRA in the DiGeorge syndrome critical region of human chromosome 22.

    PubMed

    Lorain, S; Demczuk, S; Lamour, V; Toth, S; Aurias, A; Roe, B A; Lipinski, M

    1996-01-01

    The human gene HIRA lies within the smallest critical region for the DiGeorge syndrome (DGS), a haploinsufficiency developmental disorder associated with instertitial deletions in most patients in a juxtacentromeric region of chromosome 22. The HIRA protein sequence can be aligned over its entire length with Hir1 and Hir2, two yeast proteins with a regulatory function in chromatin assembly. The HIRA transcription unit was found to spread over approximately 100 kb of the DGS critical region. The human transcript is encoded from 25 exons between 59 and 861 bp in size. Domains of highest conservation with Hir1 and Hir2 are encoded from exons 1-11 and 13-25, respectively. The amino- and carboxy-terminal regions of homology are separated from each other by a domain unique to HIRA that is encoded from a single exon. Seven WD repeats are conserved between yeast and man in the amino-terminal region of the HIR proteins. Individual repeats were found to be encoded from one, two, or three exons of the HIRA gene. End sequences have been obtained for all 24 introns, opening the way to PCR amplification of the entire coding sequence starting from genomic DNA. Point mutations can also be sought in 16 of the 24 introns that are readily PCR-amplifiable.

  8. Emerging role for leucine-rich repeat-containing G-protein-coupled receptors LGR5 and LGR4 in cancer stem cells

    PubMed Central

    Nakata, Susumu; Phillips, Emma; Goidts, Violaine

    2014-01-01

    The concept of cancer stem cells has gained considerable interest in the last few decades, partly because of their potential implication in therapy resistance. However, the lack of specific cellular surface markers for these cells has impeded their isolation, making the characterization of this cellular subpopulation technically challenging. Recent studies have indicated that leucine-rich repeat-containing G-protein-coupled receptor 4 and 5 (LGR4 and LGR5) expression in multiple organs may represent a global marker of adult stem cells. This review aims to give an overview of LGR4 and LGR5 as cancer stem cell markers and their function in development. PMID:24711713

  9. Emerging role for leucine-rich repeat-containing G-protein-coupled receptors LGR5 and LGR4 in cancer stem cells.

    PubMed

    Nakata, Susumu; Phillips, Emma; Goidts, Violaine

    2014-01-01

    The concept of cancer stem cells has gained considerable interest in the last few decades, partly because of their potential implication in therapy resistance. However, the lack of specific cellular surface markers for these cells has impeded their isolation, making the characterization of this cellular subpopulation technically challenging. Recent studies have indicated that leucine-rich repeat-containing G-protein-coupled receptor 4 and 5 (LGR4 and LGR5) expression in multiple organs may represent a global marker of adult stem cells. This review aims to give an overview of LGR4 and LGR5 as cancer stem cell markers and their function in development.

  10. Crystal structure of LGR4-Rspo1 complex: insights into the divergent mechanisms of ligand recognition by leucine-rich repeat G-protein-coupled receptors (LGRs).

    PubMed

    Xu, Jin-Gen; Huang, Chunfeng; Yang, Zhengfeng; Jin, Mengmeng; Fu, Panhan; Zhang, Ni; Luo, Jian; Li, Dali; Liu, Mingyao; Zhou, Yan; Zhu, Yongqun

    2015-01-23

    Leucine-rich repeat G-protein-coupled receptors (LGRs) are a unique class of G-protein-coupled receptors characterized by a large extracellular domain to recognize ligands and regulate many important developmental processes. Among the three groups of LGRs, group B members (LGR4-6) recognize R-spondin family proteins (Rspo1-4) to stimulate Wnt signaling. In this study, we successfully utilized the "hybrid leucine-rich repeat technique," which fused LGR4 with the hagfish VLR protein, to obtain two recombinant human LGR4 proteins, LGR415 and LGR49. We determined the crystal structures of ligand-free LGR415 and the LGR49-Rspo1 complex. LGR4 exhibits a twisted horseshoe-like structure. Rspo1 adopts a flat and β-fold architecture and is bound in the concave surface of LGR4 in the complex through electrostatic and hydrophobic interactions. All the Rspo1-binding residues are conserved in LGR4-6, suggesting that LGR4-6 bind R-spondins through an identical surface. Structural analysis of our LGR4-Rspo1 complex with the previously determined LGR4 and LGR5 structures revealed that the concave surface of LGR4 is the sole binding site for R-spondins, suggesting a one-site binding model of LGR4-6 in ligand recognition. The molecular mechanism of LGR4-6 is distinct from the two-step mechanism of group A receptors LGR1-3 and the multiple-interface binding model of group C receptors LGR7-8, suggesting LGRs utilize the divergent mechanisms for ligand recognition. Our structures, together with previous reports, provide a comprehensive understanding of the ligand recognition by LGRs.

  11. Human C9ORF72 Hexanucleotide Expansion Reproduces RNA Foci and Dipeptide Repeat Proteins but Not Neurodegeneration in BAC Transgenic Mice.

    PubMed

    Peters, Owen M; Cabrera, Gabriela Toro; Tran, Helene; Gendron, Tania F; McKeon, Jeanne E; Metterville, Jake; Weiss, Alexandra; Wightman, Nicholas; Salameh, Johnny; Kim, Juhyun; Sun, Huaming; Boylan, Kevin B; Dickson, Dennis; Kennedy, Zachary; Lin, Ziqiang; Zhang, Yong-Jie; Daughrity, Lillian; Jung, Chris; Gao, Fen-Biao; Sapp, Peter C; Horvitz, H Robert; Bosco, Daryl A; Brown, Solange P; de Jong, Pieter; Petrucelli, Leonard; Mueller, Christian; Brown, Robert H

    2015-12-02

    A non-coding hexanucleotide repeat expansion in the C9ORF72 gene is the most common mutation associated with familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). To investigate the pathological role of C9ORF72 in these diseases, we generated a line of mice carrying a bacterial artificial chromosome containing exons 1 to 6 of the human C9ORF72 gene with approximately 500 repeats of the GGGGCC motif. The mice showed no overt behavioral phenotype but recapitulated distinctive histopathological features of C9ORF72 ALS/FTD, including sense and antisense intranuclear RNA foci and poly(glycine-proline) dipeptide repeat proteins. Finally, using an artificial microRNA that targets human C9ORF72 in cultures of primary cortical neurons from the C9BAC mice, we have attenuated expression of the C9BAC transgene and the poly(GP) dipeptides. The C9ORF72 BAC transgenic mice will be a valuable tool in the study of ALS/FTD pathobiology and therapy.

  12. Interaction of FUN14 domain containing 1, a mitochondrial outer membrane protein, with kinesin light chain 1 via the tetratricopeptide repeat domain

    PubMed Central

    Jang, Won Hee; Jeong, Young Joo; Choi, Sun Hee; Urm, Sang-Hwa; Seog, Dae-Hyun

    2017-01-01

    Kinesin 1 is a member of the kinesin superfamily proteins (KIFs) of microtubule-dependent molecular motor proteins that transport organelles and protein complexes in cells. Kinesin 1 consists of a homo- or hetero-dimer of kinesin heavy chains (KHCs), often, although not always, associated with two kinesin light chains (KLCs). KLCs are non-motor proteins that associate with many different binding proteins and cargoes, but their binding partners have not yet been fully identified. In the present study, a yeast two-hybrid system was used to identify proteins that interact with the tetratricopeptide repeat (TPR) domain of KLC1. The results of the current study revealed an interaction between the TPR domain of KLC1 and FUN14 domain-containing protein 1 (FUNDC1), which is a mitochondrial outer membrane protein mediating hypoxia-induced mitophagy. FUNDC1 bound to the six TPR motif-containing regions of KLC1 and did not interact with KIF5B (a motor subunit of kinesin 1) and KIF3A (a motor subunit of kinesin 2) in the yeast two-hybrid assay. The cytoplasmic amino N-terminal domain of FUNDC1 is essential for interaction with KLC1. When co-expressed in HEK-293T cells, FUNDC1 co-localized with KLC1 and co-immunoprecipitated with KLC1, but not KIF5B. Collectively, these results indicate that KLC1 may potentially compete with LC3, a key component for autophagosome formation, to interact with FUNDC1. PMID:28123706

  13. Crystal Structure of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated Csn2 Protein Revealed Ca[superscript 2+]-dependent Double-stranded DNA Binding Activity

    SciTech Connect

    Nam, Ki Hyun; Kurinov, Igor; Ke, Ailong

    2012-05-22

    Clustered regularly interspaced short palindromic repeats (CRISPR) and their associated protein genes (cas genes) are widespread in bacteria and archaea. They form a line of RNA-based immunity to eradicate invading bacteriophages and malicious plasmids. A key molecular event during this process is the acquisition of new spacers into the CRISPR loci to guide the selective degradation of the matching foreign genetic elements. Csn2 is a Nmeni subtype-specific cas gene required for new spacer acquisition. Here we characterize the Enterococcus faecalis Csn2 protein as a double-stranded (ds-) DNA-binding protein and report its 2.7 {angstrom} tetrameric ring structure. The inner circle of the Csn2 tetrameric ring is {approx}26 {angstrom} wide and populated with conserved lysine residues poised for nonspecific interactions with ds-DNA. Each Csn2 protomer contains an {alpha}/{beta} domain and an {alpha}-helical domain; significant hinge motion was observed between these two domains. Ca{sup 2+} was located at strategic positions in the oligomerization interface. We further showed that removal of Ca{sup 2+} ions altered the oligomerization state of Csn2, which in turn severely decreased its affinity for ds-DNA. In summary, our results provided the first insight into the function of the Csn2 protein in CRISPR adaptation by revealing that it is a ds-DNA-binding protein functioning at the quaternary structure level and regulated by Ca{sup 2+} ions.

  14. The Potato Nucleotide-binding Leucine-rich Repeat (NLR) Immune Receptor Rx1 Is a Pathogen-dependent DNA-deforming Protein*

    PubMed Central

    Fenyk, Stepan; Townsend, Philip D.; Dixon, Christopher H.; Spies, Gerhard B.; de San Eustaquio Campillo, Alba; Slootweg, Erik J.; Westerhof, Lotte B.; Gawehns, Fleur K. K.; Knight, Marc R.; Sharples, Gary J.; Goverse, Aska; Pålsson, Lars-Olof; Takken, Frank L. W.; Cann, Martin J.

    2015-01-01

    Plant nucleotide-binding leucine-rich repeat (NLR) proteins enable cells to respond to pathogen attack. Several NLRs act in the nucleus; however, conserved nuclear targets that support their role in immunity are unknown. Previously, we noted a structural homology between the nucleotide-binding domain of NLRs and DNA replication origin-binding Cdc6/Orc1 proteins. Here we show that the NB-ARC (nucleotide-binding, Apaf-1, R-proteins, and CED-4) domain of the Rx1 NLR of potato binds nucleic acids. Rx1 induces ATP-dependent bending and melting of DNA in vitro, dependent upon a functional P-loop. In situ full-length Rx1 binds nuclear DNA following activation by its cognate pathogen-derived effector protein, the coat protein of potato virus X. In line with its obligatory nucleocytoplasmic distribution, DNA binding was only observed when Rx1 was allowed to freely translocate between both compartments and was activated in the cytoplasm. Immune activation induced by an unrelated NLR-effector pair did not trigger an Rx1-DNA interaction. DNA binding is therefore not merely a consequence of immune activation. These data establish a role for DNA distortion in Rx1 immune signaling and define DNA as a molecular target of an activated NLR. PMID:26306038

  15. Crystal structure of clustered regularly interspaced short palindromic repeats (CRISPR)-associated Csn2 protein revealed Ca2+-dependent double-stranded DNA binding activity.

    PubMed

    Nam, Ki Hyun; Kurinov, Igor; Ke, Ailong

    2011-09-02

    Clustered regularly interspaced short palindromic repeats (CRISPR) and their associated protein genes (cas genes) are widespread in bacteria and archaea. They form a line of RNA-based immunity to eradicate invading bacteriophages and malicious plasmids. A key molecular event during this process is the acquisition of new spacers into the CRISPR loci to guide the selective degradation of the matching foreign genetic elements. Csn2 is a Nmeni subtype-specific cas gene required for new spacer acquisition. Here we characterize the Enterococcus faecalis Csn2 protein as a double-stranded (ds-) DNA-binding protein and report its 2.7 Å tetrameric ring structure. The inner circle of the Csn2 tetrameric ring is ∼26 Å wide and populated with conserved lysine residues poised for nonspecific interactions with ds-DNA. Each Csn2 protomer contains an α/β domain and an α-helical domain; significant hinge motion was observed between these two domains. Ca(2+) was located at strategic positions in the oligomerization interface. We further showed that removal of Ca(2+) ions altered the oligomerization state of Csn2, which in turn severely decreased its affinity for ds-DNA. In summary, our results provided the first insight into the function of the Csn2 protein in CRISPR adaptation by revealing that it is a ds-DNA-binding protein functioning at the quaternary structure level and regulated by Ca(2+) ions.

  16. Cooperative control between AtRGS1 and AtHXK1 in a WD40-repeat protein pathway in Arabidopsis thaliana

    DOE PAGES

    Huang, Jian -Ping; Tunc-Ozdemir, Meral; Chang, Ying; ...

    2015-10-13

    HEXOKINASE 1 (AtHXK1) and Regulator of G-protein Signaling 1 (AtRGS1) pathways, mediate D-glucose signaling in Arabidopsis. However, it is not known the degree, if any, that these pathways overlap and how. We show modest signaling crosstalk between these pathways, albeit complex with both epistatic interactions and additive effects that may be indirect. The action of HXK1 on AtRGS1 signaling lies downstream of the primary step in G protein-mediated sugar signaling in which the WD-repeat protein, AGB1, is the propelling signaling element. RHIP1, a previously unknown protein predicted here to have a 3-stranded helical structure, interacts with both AtRGS1 and AtHXK1more » in planta and is required for some glucose-regulated gene expression, providing a physical connection between these two proteins in sugar signaling. The rhip1 null mutant displays similar seedling growth phenotypes as rgs1-2 in response to glucose, further suggesting a role for RHIP1 in glucose signaling. Lastly, glucose signaling is a complex hierarchical relationship which is specific to the target gene and sugar phenotype and suggests that there are two glycolysis-independent glucose signaling sensors: AtRGS1 and AtHXK1 that weakly communicate with each other via feed-back and feed-forward loops to fine tune the response to glucose.« less

  17. Cooperative control between AtRGS1 and AtHXK1 in a WD40-repeat protein pathway in Arabidopsis thaliana

    SciTech Connect

    Huang, Jian -Ping; Tunc-Ozdemir, Meral; Chang, Ying; Jones, Alan M.

    2015-10-13

    HEXOKINASE 1 (AtHXK1) and Regulator of G-protein Signaling 1 (AtRGS1) pathways, mediate D-glucose signaling in Arabidopsis. However, it is not known the degree, if any, that these pathways overlap and how. We show modest signaling crosstalk between these pathways, albeit complex with both epistatic interactions and additive effects that may be indirect. The action of HXK1 on AtRGS1 signaling lies downstream of the primary step in G protein-mediated sugar signaling in which the WD-repeat protein, AGB1, is the propelling signaling element. RHIP1, a previously unknown protein predicted here to have a 3-stranded helical structure, interacts with both AtRGS1 and AtHXK1 in planta and is required for some glucose-regulated gene expression, providing a physical connection between these two proteins in sugar signaling. The rhip1 null mutant displays similar seedling growth phenotypes as rgs1-2 in response to glucose, further suggesting a role for RHIP1 in glucose signaling. Lastly, glucose signaling is a complex hierarchical relationship which is specific to the target gene and sugar phenotype and suggests that there are two glycolysis-independent glucose signaling sensors: AtRGS1 and AtHXK1 that weakly communicate with each other via feed-back and feed-forward loops to fine tune the response to glucose.

  18. Cooperative control between AtRGS1 and AtHXK1 in a WD40-repeat protein pathway in Arabidopsis thaliana

    PubMed Central

    Huang, Jian-Ping; Tunc-Ozdemir, Meral; Chang, Ying; Jones, Alan M.

    2015-01-01

    HEXOKINASE 1 (AtHXK1) and Regulator of G-protein Signaling 1 (AtRGS1) pathways, mediate D-glucose signaling in Arabidopsis. However, it is not known the degree, if any, that these pathways overlap and how. We show modest signaling crosstalk between these pathways, albeit complex with both epistatic interactions and additive effects that may be indirect. The action of HXK1 on AtRGS1 signaling lies downstream of the primary step in G protein-mediated sugar signaling in which the WD-repeat protein, AGB1, is the propelling signaling element. RHIP1, a previously unknown protein predicted here to have a 3-stranded helical structure, interacts with both AtRGS1 and AtHXK1 in planta and is required for some glucose-regulated gene expression, providing a physical connection between these two proteins in sugar signaling. The rhip1 null mutant displays similar seedling growth phenotypes as rgs1-2 in response to glucose, further suggesting a role for RHIP1 in glucose signaling. In conclusion, glucose signaling is a complex hierarchical relationship which is specific to the target gene and sugar phenotype and suggests that there are two glycolysis-independent glucose signaling sensors: AtRGS1 and AtHXK1 that weakly communicate with each other via feed-back and feed-forward loops to fine tune the response to glucose. PMID:26528314

  19. The rice DUF1620-containing and WD40-like repeat protein is required for the assembly of the restoration of fertility complex.

    PubMed

    Qin, Xiaojian; Huang, Qi; Xiao, Haijun; Zhang, Qiannan; Ni, Chenzi; Xu, Yanghong; Liu, Gai; Yang, Daichang; Zhu, Yingguo; Hu, Jun

    2016-05-01

    Cytoplasmic male sterility (CMS) and restoration of fertility (Rf) are widely distributed in plant species utilized by humans. RF5 and GRP162 are subunits of the restoration of fertility complex (RFC) in Hong-Lian rice. Despite the fact that the RFC is 400-500 kDa in size, the other proteins or factors in the complex still remain unknown. Here, we identified RFC subunit 3, which encodes a DUF1620-containing and WD40-like repeat protein (RFC3) that is present in all tissues but highly expressed in leaves. We established that RFC3 interacts with both RF5 and GRP162 in vitro and in vivo, and is transported into the mitochondria as a membrane protein. Furthermore, CMS RNA (atp6-orfH79) and CMS cytotoxic protein (ORFH79) accumulate when RFC3 is silenced in restorer lines. We presented the analysis with blue-native polyacrylamide gel electrophoresis, indicating that RFC is disrupted in the RNAi line. We concluded that RCF3 is indispensable as a scaffold protein for the assembly of the RFC complex. We unveil a new molecular player of the RFC in the Rf pathway in rice and propose the model of RFC based on these data.

  20. Association study between CAG trinucleotide repeats in the PCQAP gene (PC2 glutamine/Q-rich-associated protein) and schizophrenia.

    PubMed

    De Luca, Alessandro; Conti, Emanuela; Grifone, Nicoletta; Amati, Francesca; Spalletta, Gianfranco; Caltagirone, Carlo; Bonaviri, Giuseppina; Pasini, Augusto; Gennarelli, Massimo; Stefano, Bignotti; Berti, Lucia; Mittler, Gerhard; Meisterernst, Michael; Dallapiccola, Bruno; Novelli, Giuseppe

    2003-01-01

    Schizophrenia or schizoaffective disorders are quite common features in patients with DiGeorge/velocardiofacial syndrome (DGS/VCFS) as a result of hemizygosity of chromosome 22q11.2. We evaluated the PCQAP gene, which maps within the DGS/VCFS interval, as a potential candidate for schizophrenia susceptibility. PCQAP encodes for a subunit of the large multiprotein complex PC2, which exhibits a coactivator function in RNA polymerase II mediated transcription. Using a case-control study, we searched association between schizophrenia and the intragenic coding trinucleotide polymorphism. The distribution of the CAG repeat alleles was significantly different between patients and controls with the Mann-Whitney test (z = -2.5694, P = 0.0051; schizophrenics: n = 378, W = 161,002.5, Mean rank = 425.9325; controls: n = 444, W = 177,250.5, Mean rank = 399.2128). This result may indicate a possible involvement of the multiprotein complex PC2 in schizophrenia susceptibility.

  1. Repeated triggering of sporulation in Bacillus subtilis selects against a protein that affects the timing of cell division.

    PubMed

    Siebring, Jeroen; Elema, Matthijs J H; Drubi Vega, Fátima; Kovács, Akos T; Haccou, Patsy; Kuipers, Oscar P

    2014-01-01

    Bacillus subtilis sporulation is a last-resort phenotypical adaptation in response to starvation. The regulatory network underlying this developmental pathway has been studied extensively. However, how sporulation initiation is concerted in relation to the environmental nutrient availability is poorly understood. In a fed-batch fermentation set-up, in which sporulation of ultraviolet (UV)-mutagenized B. subtilis is repeatedly triggered by periods of starvation, fitter strains with mutated tagE evolved. These mutants display altered timing of phenotypical differentiation. The substrate for the wall teichoic acid (WTA)-modifying enzyme TagE, UDP-glucose, has recently been shown to be an intracellular proxy for nutrient availability, and influences the timing of cell division. Here we suggest that UDP-glucose also influences timing of cellular differentiation.

  2. Expression of Anaplasma marginale ankyrin repeat-containing proteins during infection of the mammalian host and tick vector.

    PubMed

    Ramabu, Solomon S; Schneider, David A; Brayton, Kelly A; Ueti, Massaro W; Graça, Telmo; Futse, James E; Noh, Susan M; Baszler, Timothy V; Palmer, Guy H

    2011-07-01

    Transmission of tick-borne pathogens requires transition between distinct host environments with infection and replication in host-specific cell types. Anaplasma marginale illustrates this transition: in the mammalian host, the bacterium infects and replicates in mature (nonnucleated) erythrocytes, while in the tick vector, replication occurs in nucleated epithelial cells. We hypothesized that proteins containing ankyrin motifs would be expressed by A. marginale only in tick cells and would traffic to the infected host cell nucleus. A. marginale encodes three proteins containing ankyrin motifs, an AnkA orthologue (the AM705 protein), AnkB (the AM926 protein), and AnkC (the AM638 protein). All three A. marginale Anks were confirmed to be expressed during intracellular infection: AnkA is expressed at significantly higher levels in erythrocytes, AnkB is expressed equally by both infected erythrocytes and tick cells, and AnkC is expressed exclusively in tick cells. There was no evidence of any of the Ank proteins trafficking to the nucleus. Thus, the hypothesis that ankyrin-containing motifs were predictive of cell type expression and nuclear localization was rejected. In contrast, AnkA orthologues in the closely related A. phagocytophilum and Ehrlichia chaffeensis have been shown to localize to the host cell nucleus. This difference, together with the lack of a nuclear localization signal in any of the AnkA orthologues, suggests that trafficking may be mediated by a separate transporter rather than by endogenous signals. Selection for divergence in Ank function among Anaplasma and Ehrlichia spp. is supported by both locus and allelic analyses of genes encoding orthologous proteins and their ankyrin motif compositions.

  3. PPR8522 encodes a chloroplast-targeted pentatricopeptide repeat protein necessary for maize embryogenesis and vegetative development

    PubMed Central

    M. Rogowsky, Peter

    2012-01-01

    The pentatricopeptide repeat (PPR) domain is an RNA binding domain allowing members of the PPR superfamily to participate in post-transcriptional processing of organellar RNA. Loss of PPR8522 from maize (Zea mays) confers an embryo-specific (emb) phenotype. The emb8522 mutation was isolated in an active Mutator (Mu) population and co-segregation analysis revealed that it was tightly linked to a MuDR insertion in the first exon of PPR8522. Independent evidence that disruption of PPR8522 caused the emb phenotype was provided by fine mapping to a region of 116kb containing no other gene than PPR8522 and complementation of the emb8522 mutant by a PPR8522 cDNA. The deduced PPR8522 amino acid sequence of 832 amino acids contains 10 PPR repeats and a chloroplast target peptide, the function of which was experimentally demonstrated by transient expression in Nicotiana benthamiana. Whereas mutant endosperm is apparently normal, mutant embryos deviate from normal development as early as 3 days after pollination, are reduced in size, exhibit more or less severe morphological aberrations depending on the genetic background, and generally do not germinate. The emb8522 mutation is the first to associate the loss of a PPR gene with an embryo-lethal phenotype in maize. Analyses of mutant plantlets generated by embryo-rescue experiments indicate that emb8522 also affects vegetative plant growth and chloroplast development. The loss of chloroplast transcription dependent on plastid-encoded RNA polymerase is the likely cause for the lack of an organized thylakoid network and an albino, seedling-lethal phenotype. PMID:22945943

  4. PPR8522 encodes a chloroplast-targeted pentatricopeptide repeat protein necessary for maize embryogenesis and vegetative development.

    PubMed

    Sosso, Davide; Canut, Matthieu; Gendrot, Ghislaine; Dedieu, Annick; Chambrier, Pierre; Barkan, Alice; Consonni, Gabriella; Rogowsky, Peter M

    2012-10-01

    The pentatricopeptide repeat (PPR) domain is an RNA binding domain allowing members of the PPR superfamily to participate in post-transcriptional processing of organellar RNA. Loss of PPR8522 from maize (Zea mays) confers an embryo-specific (emb) phenotype. The emb8522 mutation was isolated in an active Mutator (Mu) population and co-segregation analysis revealed that it was tightly linked to a MuDR insertion in the first exon of PPR8522. Independent evidence that disruption of PPR8522 caused the emb phenotype was provided by fine mapping to a region of 116kb containing no other gene than PPR8522 and complementation of the emb8522 mutant by a PPR8522 cDNA. The deduced PPR8522 amino acid sequence of 832 amino acids contains 10 PPR repeats and a chloroplast target peptide, the function of which was experimentally demonstrated by transient expression in Nicotiana benthamiana. Whereas mutant endosperm is apparently normal, mutant embryos deviate from normal development as early as 3 days after pollination, are reduced in size, exhibit more or less severe morphological aberrations depending on the genetic background, and generally do not germinate. The emb8522 mutation is the first to associate the loss of a PPR gene with an embryo-lethal phenotype in maize. Analyses of mutant plantlets generated by embryo-rescue experiments indicate that emb8522 also affects vegetative plant growth and chloroplast development. The loss of chloroplast transcription dependent on plastid-encoded RNA polymerase is the likely cause for the lack of an organized thylakoid network and an albino, seedling-lethal phenotype.

  5. PM2, a group 3 LEA protein from soybean, and its 22-mer repeating region confer salt tolerance in Escherichia coli

    SciTech Connect

    Yun Liu; Zheng Yizhi . E-mail: yzzheng@szu.edu.cn

    2005-05-27

    To have knowledge of the effect of soybean PM2 protein in protecting dehydrated cells and its functional region, PM2 cDNA was isolated from soybean immature seeds. The recombinants expressing full-length PM2, truncated polypeptides of PM2A (aa 1-262) or PM2B (aa 129-262, 22-mer repeating region), or artificial polypeptide PM2C (duplication of 22-mer repeating region) were constructed. By using SDS-PAGE and mass spectrometry approaches, these fusion polypeptides were identified and proved to be hydrophilic and heat-stable. Spot assays of BL/PM2 and BL/pET28 (as control) showed that protein PM2 increased salt tolerance (500 mM NaCl or 500 mM KCl) of Escherichia coli, rather than osmotic tolerance (1100 mM sorbitol). In addition, comparing the survival ratios of the transformants under 500 mM NaCl or 500 mM KCl stresses, the results showed that: (1) the survival ratios of BL/PM2 and BL/PM2B were quite similar, both showing much higher values than those of BL/pET28. (2) The survival ratios of BL/PM2C were much higher than those of BL/PM2, BL/PM2A, and BL/PM2B. This provides the first experimental evidence that PM2 polypeptide enhances salt tolerance of E. coli cells, and the 22-mer repeat region is an important functional region.

  6. Structure and expression of fibulin-2, a novel extracellular matrix protein with multiple EGF-like repeats and consensus motifs for calcium binding

    PubMed Central

    1993-01-01

    A new protein, fibulin-2, was predicted from sequence analysis of cDNA clones obtained from a mouse fibroblast library. This protein consists of a 1195-residue polypeptide preceded by a 26-residue signal peptide. The COOH-terminal region of 787 amino acids contained three anaphylatoxin-related segments (domain I), 11 EGF-like repeats (domain II), 10 of which had a consensus motif for calcium-binding, and a 115- residue globular domain III. Except for two additional EGF-like repeats, this COOH-terminal region showed 43% sequence identity with the previously described fibulin-1 (BM-90). The NH2-terminal 408 residues, unique to fibulin-2, showed no sequence homology to other known proteins and presumably form two additional domains that differ in their cysteine content. Recombinant fibulin-2 was produced and secreted by human cell clones as a disulfide-bonded trimer. Rotary shadowing visualized the protein as three 40-45 nm long rods which are connected at one end in a globe-like structure. No significant immunological cross-reaction could be detected between fibulin-1 and fibulin-2. Production of the fibulin-2 was demonstrated by Northern blots and radioimmunoassay in fibroblasts but not in several tumor cell lines. Together with the observation that the serum level of fibulin-2 is 1,000-fold lower than that of fibulin-1, the data indicate that these two isoforms are not always coordinately expressed. This is also suggested by Northern blots of tissue mRNAs and by immunofluorescence localizations using mouse tissues. The latter studies also demonstrated an extracellular localization for fibulin-2 in basement membranes and other connective tissue compartments. PMID:8245130

  7. Molecular cloning and characterization of the human ASB-8 gene encoding a novel member of ankyrin repeat and SOCS box containing protein family.

    PubMed

    Liu, Yongzhong; Li, Jinjun; Zhang, Fengrui; Qin, Wenxin; Yao, Genfu; He, Xianghuo; Xue, Peng; Ge, Chao; Wan, Dafang; Gu, Jianren

    2003-01-24

    We have cloned a new member of human ankyrin repeat and SOCS box containing protein family (ASB), designed as hASB-8, from a human placental cDNA library and further extended by 5(') and 3(')-RACE. The full-length cDNA was 2545bp in length, with a predicted open reading frame encoding a protein of 288 amino acids, which was 96% identical to mouse ASB-8 protein. Computer analysis revealed that the deduced amino acid sequence of the human ASB-8 contained four Ankyrin repeats and one SOCS box. The gene had four exons separated by three introns and was mapped to human chromosome 12q13. Human ASB-8 mRNA was expressed at the highest level of expression in skeletal muscle and at a varied level of expression in heart, brain, placenta, liver, kidney, and pancreas. The transcript of hASB-8 was not detected in adult normal lung tissue, but found in lung carcinoma cell lines SPC-A1, A549, and NCI-H446. Subcellular localization analysis showed that the EGFP-tagged hASB-8 protein was localized at cytoplasm in human hepatocellular carcinoma cell line BEL-7402. We also provided evidence that hASB-8 could interact with Elongin B-C complex in vitro. Furthermore, transfection with the truncated mutant of hASB-8 cDNA lacking SOCS box could suppress cell growth of lung adenocarcinoma SPC-A1 cells in vitro, which suggests that this gene might be related to the development of lung cancer.

  8. Inhibition of the leucine-rich repeat protein LINGO-1 enhances survival, structure, and function of dopaminergic neurons in Parkinson's disease models.

    PubMed

    Inoue, Haruhisa; Lin, Ling; Lee, Xinhua; Shao, Zhaohui; Mendes, Shannon; Snodgrass-Belt, Pamela; Sweigard, Harry; Engber, Tom; Pepinsky, Blake; Yang, Lichuan; Beal, M Flint; Mi, Sha; Isacson, Ole

    2007-09-04

    The nervous system-specific leucine-rich repeat Ig-containing protein LINGO-1 is associated with the Nogo-66 receptor complex and is endowed with a canonical EGF receptor (EGFR)-like tyrosine phosphorylation site. Our studies indicate that LINGO-1 expression is elevated in the substantia nigra of Parkinson's disease (PD) patients compared with age-matched controls and in animal models of PD after neurotoxic lesions. LINGO-1 expression is present in midbrain dopaminergic (DA) neurons in the human and rodent brain. Therefore, the role of LINGO-1 in cell damage responses of DA neurons was examined in vitro and in experimental models of PD induced by either oxidative (6-hydroxydopamine) or mitochondrial (N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) toxicity. In LINGO-1 knockout mice, DA neuron survival was increased and behavioral abnormalities were reduced compared with WT. This neuroprotection was accompanied by increased Akt phosphorylation (p-Akt). Similar neuroprotective in vivo effects on midbrain DA neurons were obtained in WT mice by blocking LINGO-1 activity using LINGO-1-Fc protein. Neuroprotection and enhanced neurite growth were also demonstrated for midbrain DA neurons in vitro. LINGO-1 antagonists (LINGO-1-Fc, dominant negative LINGO-1, and anti-LINGO-1 antibody) improved DA neuron survival in response to MPP+ in part by mechanisms that involve activation of the EGFR/Akt signaling pathway through a direct inhibition of LINGO-1's binding to EGFR. These results show that inhibitory agents of LINGO-1 activity can protect DA neurons against degeneration and indicate a role for the leucine-rich repeat protein LINGO-1 and related classes of proteins in the pathophysiological responses of midbrain DA neurons in PD.

  9. Molecular characterization and different expression patterns of the muscle ankyrin repeat protein (MARP) family during porcine skeletal muscle development in vitro and in vivo.

    PubMed

    Wang, Linjie; Lei, Minggang; Xiong, Yuanzhu

    2011-04-01

    CARP, ANKRD2, and DARP belong to the ankyrin repeat protein (MARP) family and play a critical role in the integration of cytoskeletal architecture, stress response, and transcriptional regulation. In this study, we cloned the cDNA and promoter sequences of porcine ankyrin repeat protein (MARP) gene family. RT-PCR analysis revealed that porcine CARP gene was predominantly expressed in heart. ANKRD2 was widely expressed in many tissues, a high expression level was observed in the skeletal muscle and heart. DARP gene was expressed specifically in skeletal muscle and heart. Moreover, the expression of CARP and ANKRD2 was significantly different in porcine skeletal muscle among different developmental stages and between the two breeds. Expression analysis in porcine satellite cells showed that CARP and ANKRD2 were induced in differentiated porcine satellite cells, suggesting a role of them in myogenic differentiation. This result suggests that the MARP gene family may be important genes for skeletal muscle growth and provides useful information for further studies on their roles in porcine skeletal muscle.

  10. Microscopic features of tick-bite lesions in anteaters and armadillos: Emas National Park and the Pantanal region of Brazil.

    PubMed

    Lima e Silva, M F; Szabó, M P J; Bechara, G H

    2004-10-01

    The naturally occurring wildlife host associations between ticks and tick-borne pathogens found in the neotropics are poorly described. Understanding tick-bite lesions is important as these are the site of host reaction to and pathogen delivery by ticks. As part of a comprehensive study concerning established and emerging tick-host relationships. the present work describes some aspects of tick-bite lesions in anteaters and armadillos captured at the Emas National Park and the Pantanal region of Brazil. Biopsies were of skin were taken and examine. Tick feeding sites of all animals displayed an eosinophilic homogeneous mass, the cement cone, and, occasionally, a feeding cavity underneath the tick attachment site. At these locations the epidermis was usually thickened due to keratinocyte hyperplasia. The main dermal changes included tissue infiltration with a varying number of inflammatory cells, edema, hemorrhage. and vascular dilatation. Cellular infiltration of the dermis was predominantly composed of mononuclear cells, neutrophils. and eosinophils. Mast cells were also seen in both non-parasitized and parasitized skin but were found in higher numbers at perivascular sites and in parasitized skin. Basophils were not seen at tick attachment sites of anteaters or armadillos.

  11. Orientia tsutsugamushi Strain Ikeda Ankyrin Repeat-Containing Proteins Recruit SCF1 Ubiquitin Ligase Machinery via Poxvirus-Like F-Box Motifs

    PubMed Central

    Beyer, Andrea R.; VieBrock, Lauren; Rodino, Kyle G.; Miller, Daniel P.; Tegels, Brittney K.; Marconi, Richard T.

    2015-01-01

    ABSTRACT A rising theme among intracellular microbes is the delivery of ankyrin repeat-containing effectors (Anks) that interact with target proteins to co-opt host cell functions. Orientia tsutsugamushi, an obligate intracellular bacterium and the etiologic agent of scrub typhus, encodes one of the largest Ank repertoires of any sequenced microorganism. They have been previously identified as type 1 secretion system substrates. Here, in silico and manual sequence analyses revealed that a large proportion of O. tsutsugamushi strain Ikeda Anks bear a eukaryotic/poxvirus-like F-box motif, which is known to recruit host cell SCF1 ubiquitin ligase machinery. We assessed the Anks for the ability to serve as F-box proteins. Coimmunoprecipitation assays demonstrated that F-box-containing Anks interact with overexpressed and/or endogenous SCF1 components. When coexpressed with FLAG-Ank4_01 or FLAG-Ank9, a glutathione S-transferase (GST)-tagged version of the SCF1 component SKP1 localized to subcellular sites of FLAG-Ank accumulation. The abilities of recombinant Anks to interact and colocalize with SKP1 were F-box dependent. GST-SKP1 precipitated O. tsutsugamushi-derived Ank9 from infected host cells, verifying both that the pathogen expresses Ank9 during infection and the protein's capability to bind SKP1. Aligning O. tsutsugamushi, poxviral, and eukaryotic F-box sequences delineated three F-box residues that are highly conserved and likely to be functionally important. Substitution of these residues ablated the ability of GFP-Ank9 to interact with GST-SKP1. These results demonstrate that O. tsutsugamushi strain Ikeda Anks can co-opt host cell polyubiquitination machinery, provide the first evidence that an O. tsutsugamushi Ank does so during infection, and advance overall understanding of microbial F-box proteins. IMPORTANCE Ankyrin repeat-containing proteins (Anks) are important virulence factors of intracellular bacteria that mediate protein-protein interactions with

  12. CSL encodes a leucine-rich-repeat protein implicated in red/violet light signaling to the circadian clock in Chlamydomonas.

    PubMed

    Kinoshita, Ayumi; Niwa, Yoshimi; Onai, Kiyoshi; Yamano, Takashi; Fukuzawa, Hideya; Ishiura, Masahiro; Matsuo, Takuya

    2017-03-01

    The green alga Chlamydomonas reinhardtii shows various light responses in behavior and physiology. One such photoresponse is the circadian clock, which can be reset by external light signals to entrain its oscillation to daily environmental cycles. In a previous report, we suggested that a light-induced degradation of the clock protein ROC15 is a trigger to reset the circadian clock in Chlamydomonas. However, light signaling pathways of this process remained unclear. Here, we screened for mutants that show abnormal ROC15 diurnal rhythms, including the light-induced protein degradation at dawn, using a luciferase fusion reporter. In one mutant, ROC15 degradation and phase resetting of the circadian clock by light were impaired. Interestingly, the impairments were observed in response to red and violet light, but not to blue light. We revealed that an uncharacterized gene encoding a protein similar to RAS-signaling-related leucine-rich repeat (LRR) proteins is responsible for the mutant phenotypes. Our results indicate that a previously uncharacterized red/violet light signaling pathway is involved in the phase resetting of circadian clock in Chlamydomonas.

  13. Toll-like receptor 2-mediated interleukin-8 expression in gingival epithelial cells by the Tannerella forsythia leucine-rich repeat protein BspA.

    PubMed

    Onishi, Shinsuke; Honma, Kiyonobu; Liang, Shuang; Stathopoulou, Panagiota; Kinane, Denis; Hajishengallis, George; Sharma, Ashu

    2008-01-01

    Tannerella forsythia is a gram-negative anaerobe strongly associated with chronic human periodontitis. This bacterium expresses a cell surface-associated and secreted protein, designated BspA, which has been recognized as an important virulence factor. The BspA protein belongs to the leucine-rich repeat (LRR) and bacterial immunoglobulin-like protein families. BspA is, moreover, a multifunctional protein which interacts with a variety of host cells, including monocytes which appear to respond to BspA through Toll-like receptor (TLR) signaling. Since gingival epithelium forms a barrier against periodontal pathogens, this study was undertaken to determine if gingival epithelial cells respond to BspA challenge and if TLRs play any role in BspA recognition. This study was also directed towards identifying the BspA domains responsible for cellular activation. We provide direct evidence for BspA binding to TLR2 and demonstrate that the release of the chemokine interleukin-8 from human gingival epithelial cells by BspA is TLR2 dependent. Furthermore, the LRR domain of BspA is involved in activation of TLR2, while TLR1 serves as a signaling partner. Thus, our findings suggest that BspA is an important modulator of host innate immune responses through activation of TLR2 in cooperation with TLR1.

  14. CSL encodes a leucine-rich-repeat protein implicated in red/violet light signaling to the circadian clock in Chlamydomonas

    PubMed Central

    Kinoshita, Ayumi; Niwa, Yoshimi; Onai, Kiyoshi; Fukuzawa, Hideya; Ishiura, Masahiro

    2017-01-01

    The green alga Chlamydomonas reinhardtii shows various light responses in behavior and physiology. One such photoresponse is the circadian clock, which can be reset by external light signals to entrain its oscillation to daily environmental cycles. In a previous report, we suggested that a light-induced degradation of the clock protein ROC15 is a trigger to reset the circadian clock in Chlamydomonas. However, light signaling pathways of this process remained unclear. Here, we screened for mutants that show abnormal ROC15 diurnal rhythms, including the light-induced protein degradation at dawn, using a luciferase fusion reporter. In one mutant, ROC15 degradation and phase resetting of the circadian clock by light were impaired. Interestingly, the impairments were observed in response to red and violet light, but not to blue light. We revealed that an uncharacterized gene encoding a protein similar to RAS-signaling-related leucine-rich repeat (LRR) proteins is responsible for the mutant phenotypes. Our results indicate that a previously uncharacterized red/violet light signaling pathway is involved in the phase resetting of circadian clock in Chlamydomonas. PMID:28333924

  15. The Pentatricopeptide Repeat Protein EMB2654 Is Essential for Trans-Splicing of a Chloroplast Small Ribosomal Subunit Transcript1[OPEN

    PubMed Central

    Sanglard, Lilian Vincis Pereira; Bussell, John D.; Howell, Katharine A.

    2017-01-01

    We report the partial complementation and subsequent comparative molecular analysis of two nonviable mutants impaired in chloroplast translation, one (emb2394) lacking the RPL6 protein, and the other (emb2654) carrying a mutation in a gene encoding a P-class pentatricopeptide repeat protein. We show that EMB2654 is required for the trans-splicing of the plastid rps12 transcript and that therefore the emb2654 mutant lacks Rps12 protein and fails to assemble the small subunit of the plastid ribosome, explaining the loss of plastid translation and consequent embryo-lethal phenotype. Predictions of the EMB2654 binding site match a small RNA “footprint” located on the 5′ half of the trans-spliced intron that is almost absent in the partially complemented mutant. EMB2654 binds sequence specifically to this target sequence in vitro. Altered patterns in nuclease-protected small RNA fragments in emb2654 show that EMB2654 binding must be an early step in, or prior to, the formation of a large protein-RNA complex covering the free ends of the two rps12 intron halves. PMID:28011633

  16. A tetratricopeptide repeat domain-containing protein SSR1 located in mitochondria is involved in root development and auxin polar transport in Arabidopsis.

    PubMed

    Zhang, Min; Wang, Cuiping; Lin, Qingfang; Liu, Aihua; Wang, Ting; Feng, Xuanjun; Liu, Jie; Han, Huiling; Ma, Yan; Bonea, Diana; Zhao, Rongmin; Hua, Xuejun

    2015-08-01

    Auxin polar transport mediated by a group of Pin-formed (PIN) transporters plays important roles in plant root development. However, the mechanism underlying the PIN expression and targeting in response to different developmental and environmental stimuli is still not fully understood. Here, we report a previously uncharacterized gene SSR1, which encodes a mitochondrial protein with tetratricopeptide repeat (TPR) domains, and show its function in root development in Arabidopsis thaliana. In ssr1-2, a SSR1 knock-out mutant, the primary root growth was dramatically inhibited due to severely impaired cell proliferation and cell elongation. Significantly lowered level of auxin was found in ssr1-2 roots by auxin measurement and was further supported by reduced expression of DR5-driven reporter gene. As a result, the maintenance of the root stem cell niche is compromised in ssr1-2. It is further revealed that the expression level of several PIN proteins, namely, PIN1, PIN2, PIN3, PIN4 and PIN7, were markedly reduced in ssr1-2 roots. In particular, we showed that the reduced protein level of PIN2 on cell membrane in ssr1-2 is due to impaired retrograde trafficking, possibly resulting from a defect in retromer sorting system, which destines PIN2 for degradation in vacuoles. In conclusion, our results indicated that SSR1 is functioning in root development in Arabidopsis, possibly by affecting PIN protein expression and subcellular targeting.

  17. Anatomy of Shoulder Girdle Muscle Modifications and Walking Adaptation in the Scaly Chinese Pangolin (Manis Pentadactyla Pentadactyla: Pholidota) Compared with the Partially Osteoderm-Clad Armadillos (Dasypodidae).

    PubMed

    Kawashima, Tomokazu; Thorington, Richard W; Bohaska, Paula W; Chen, Yen-Jean; Sato, Fumi

    2015-07-01

    Because pangolins are unique mammals with a body and limbs almost entirely sheathed in hard keratinous overlapping scales and with digging and climbing abilities, the shoulder girdle muscles may differ significantly from those of other mammals including the partially osteoderm-clad armadillos. Therefore, we conducted a functional anatomical study of the shoulder girdle muscles in Chinese pangolins (Manis pentadactyla pentadactyla, Pholidota) and some armadillo species (Dasypodidae). Our CT scans revealed that the pangolin's overlapping scales are hard structures completely encasing the limbs. The armadillo's limbs, however, are covered with small relatively soft non-overlapping scales embedded in the skin, and articulate completely free of the hard osteodermal carapace. The attachments of some shoulder girdle muscles in the pangolin have moved from the surrounding edges of the scapula to the spine, and they, therefore, fully cover the scapula. In addition, some pangolin shoulder girdle muscles cross the shoulder joint to insert on the distal humerus, but this does not occur in armadillos. We cannot rule out the possibility that these muscle modifications represent adaptations for digging and/or climbing in pangolins. Our results and previous literature do not establish specific links between them and locomotive modes. However, we propose that the Chinese pangolin may use its derived muscular features when walking to move its armor-restricted forelimbs more effectively by swinging its head from side to side.

  18. A gamma-2 herpesvirus nucleocytoplasmic shuttle protein interacts with importin alpha 1 and alpha 5.

    PubMed

    Goodwin, D J; Whitehouse, A

    2001-06-08

    Herpesvirus saimiri (HVS) is the prototype gamma-2 herpesvirus. This is an increasing important subfamily of herpesviruses due to the identification of the first human gamma-2 herpesvirus, Kaposi's sarcoma-associated herpesvirus. The HVS open reading frame (ORF) 57 protein is a multifunctional trans-regulatory protein homologous to genes identified in all classes of herpesviruses. Recent analysis has demonstrated that ORF 57 has the ability to bind viral RNA and to shuttle between the nucleus and cytoplasm, and is required for efficient nuclear export of viral transcripts. Here we have investigated the nucleocytoplasmic shuttling mechanism utilized by the ORF 57 protein. The yeast two-hybrid system was employed to identify interacting cellular proteins using ORF 57 as bait. We demonstrate that ORF 57 interacts with importin alpha isoforms 1 and 5. In addition, the binding of ORF 57 to importin alpha was mediated by the importin alpha hydrophobic internal armadillo repeats. An ORF 57 amino-terminal arginine-rich sequence, which functions as a nuclear localization sequence, was also required for this interaction. Furthermore, the ORF 57 protein is responsible for the redistribution of importin alpha into the nucleoli. These results identify novel cellular interactions essential for the functioning of this important herpesvirus regulatory protein.

  19. Staphylococcus epidermidis serine--aspartate repeat protein G (SdrG) binds to osteoblast integrin alpha V beta 3.

    PubMed

    Claro, T; Kavanagh, N; Foster, T J; O'Brien, F J; Kerrigan, S W

    2015-06-01

    Staphylococcus epidermidis is the leading etiologic agent of orthopaedic implant infection. Contamination of the implanted device during insertion allows bacteria gain entry into the sterile bone environment leading to condition known as osteomyelitis. Osteomyelitis is characterised by weakened bones associated with progressive bone loss. The mechanism through which S. epidermidis interacts with bone cells to cause osteomyelitis is poorly understood. We demonstrate here that S. epidermidis can bind to osteoblasts in the absence of matrix proteins. S. epidermidis strains lacking the cell wall protein SdrG had a significantly reduced ability to bind to osteoblasts. Consistent with this, expression of SdrG in Lactococcus lactis resulted in significantly increased binding to the osteoblasts. Protein analysis identified that SdrG contains a potential integrin recognition motif. αVβ3 is a major integrin expressed on osteoblasts and typically recognises RGD motifs in its ligands. Our results demonstrate that S. epidermidis binds to recombinant purified αVβ3, and that a mutant lacking SdrG failed to bind. Blocking αVβ3 on osteoblasts significantly reduced binding to S. epidermidis. These studies are the first to identify a mechanism through which S. epidermidis binds to osteoblasts and potentially offers a mechanism through which implant infection caused by S. epidermidis leads to osteomyelitis.

  20. Bipartite Topology of Treponema pallidum Repeat Proteins C/D and I: OUTER MEMBRANE INSERTION, TRIMERIZATION, AND PORIN FUNCTION REQUIRE A C-TERMINAL β-BARREL DOMAIN.

    PubMed

    Anand, Arvind; LeDoyt, Morgan; Karanian, Carson; Luthra, Amit; Koszelak-Rosenblum, Mary; Malkowski, Michael G; Puthenveetil, Robbins; Vinogradova, Olga; Radolf, Justin D

    2015-05-08

    We previously identified Treponema pallidum repeat proteins TprC/D, TprF, and TprI as candidate outer membrane proteins (OMPs) and subsequently demonstrated that TprC is not only a rare OMP but also forms trimers and has porin activity. We also reported that TprC contains N- and C-terminal domains (TprC(N) and TprC(C)) orthologous to regions in the major outer sheath protein (MOSP(N) and MOSP(C)) of Treponema denticola and that TprC(C) is solely responsible for β-barrel formation, trimerization, and porin function by the full-length protein. Herein, we show that TprI also possesses bipartite architecture, trimeric structure, and porin function and that the MOSP(C)-like domains of native TprC and TprI are surface-exposed in T. pallidum, whereas their MOSP(N)-like domains are tethered within the periplasm. TprF, which does not contain a MOSP(C)-like domain, lacks amphiphilicity and porin activity, adopts an extended inflexible structure, and, in T. pallidum, is tightly bound to the protoplasmic cylinder. By thermal denaturation, the MOSP(N) and MOSP(C)-like domains of TprC and TprI are highly thermostable, endowing the full-length proteins with impressive conformational stability. When expressed in Escherichia coli with PelB signal sequences, TprC and TprI localize to the outer membrane, adopting bipartite topologies, whereas TprF is periplasmic. We propose that the MOSP(N)-like domains enhance the structural integrity of the cell envelope by anchoring the β-barrels within the periplasm. In addition to being bona fide T. pallidum rare outer membrane proteins, TprC/D and TprI represent a new class of dual function, bipartite bacterial OMP.

  1. Duplication and Divergence of Leucine-Rich Repeat Receptor-Like Protein Kinase (LRR-RLK) Genes in Basal Angiosperm Amborella trichopoda

    PubMed Central

    Liu, Ping-Li; Xie, Lu-Lu; Li, Peng-Wei; Mao, Jian-Feng; Liu, Hui; Gao, Shu-Min; Shi, Peng-Hao; Gong, Jun-Qing

    2016-01-01

    Leucine-rich repeat receptor-like protein kinases (LRR-RLKs) are the largest group of receptor-like kinases, which are one of the largest protein superfamilies in plants, and play crucial roles in development and stress responses. Although the evolution of LRR-RLK families has been investigated in some eudicot and monocot plants, no comprehensive evolutionary studies have been performed for these genes in basal angiosperms like Amborella trichopoda. In this study, we identified 94 LRR-RLK genes in the genome of A. trichopoda. The number of LRR-RLK genes in the genome of A. trichopoda is only 17–50% of that of several eudicot and monocot species. Tandem duplication and whole-genome duplication have made limited contributions to the expansion of LRR-RLK genes in A. trichopoda. According to the phylogenetic analysis, all A. trichopoda LRR-RLK genes can be organized into 18 subfamilies, which roughly correspond to the LRR-RLK subfamilies defined in Arabidopsis thaliana. Most LRR-RLK subfamilies are characterized by highly conserved protein structures, motif compositions, and gene structures. The unique gene structure, protein structures, and protein motif compositions of each subfamily provide evidence for functional divergence among LRR-RLK subfamilies. Moreover, the expression data of LRR-RLK genes provided further evidence for the functional diversification of them. In addition, selection analyses showed that most LRR-RLK protein sites are subject to purifying selection. Our results contribute to a better understanding of the evolution of LRR-RLK gene family in angiosperm and provide a framework for further functional investigation on A. trichopoda LRR-RLKs. PMID:28066499

  2. Duplication and Divergence of Leucine-Rich Repeat Receptor-Like Protein Kinase (LRR-RLK) Genes in Basal Angiosperm Amborella trichopoda.

    PubMed

    Liu, Ping-Li; Xie, Lu-Lu; Li, Peng-Wei; Mao, Jian-Feng; Liu, Hui; Gao, Shu-Min; Shi, Peng-Hao; Gong, Jun-Qing

    2016-01-01

    Leucine-rich repeat receptor-like protein kinases (LRR-RLKs) are the largest group of receptor-like kinases, which are one of the largest protein superfamilies in plants, and play crucial roles in development and stress responses. Although the evolution of LRR-RLK families has been investigated in some eudicot and monocot plants, no comprehensive evolutionary studies have been performed for these genes in basal angiosperms like Amborella trichopoda. In this study, we identified 94 LRR-RLK genes in the genome of A. trichopoda. The number of LRR-RLK genes in the genome of A. trichopoda is only 17-50% of that of several eudicot and monocot species. Tandem duplication and whole-genome duplication have made limited contributions to the expansion of LRR-RLK genes in A. trichopoda. According to the phylogenetic analysis, all A. trichopoda LRR-RLK genes can be organized into 18 subfamilies, which roughly correspond to the LRR-RLK subfamilies defined in Arabidopsis thaliana. Most LRR-RLK subfamilies are characterized by highly conserved protein structures, motif compositions, and gene structures. The unique gene structure, protein structures, and protein motif compositions of each subfamily provide evidence for functional divergence among LRR-RLK subfamilies. Moreover, the expression data of LRR-RLK genes provided further evidence for the functional diversification of them. In addition, selection analyses showed that most LRR-RLK protein sites are subject to purifying selection. Our results contribute to a better understanding of the evolution of LRR-RLK gene family in angiosperm and provide a framework for further functional investigation on A. trichopoda LRR-RLKs.

  3. TMPyP4 Porphyrin Distorts RNA G-quadruplex Structures of the Disease-associated r(GGGGCC)n Repeat of the C9orf72 Gene and Blocks Interaction of RNA-binding Proteins*

    PubMed Central

    Zamiri, Bita; Reddy, Kaalak; Macgregor, Robert B.; Pearson, Christopher E.

    2014-01-01

    Certain DNA and RNA sequences can form G-quadruplexes, which can affect genetic instability, promoter activity, RNA splicing, RNA stability, and neurite mRNA localization. Amyotrophic lateral sclerosis and frontotemporal dementia can be caused by expansion of a (GGGGCC)n repeat in the C9orf72 gene. Mutant r(GGGGCC)n- and r(GGCCCC)n-containing transcripts aggregate in nuclear foci, possibly sequestering repeat-binding proteins such as ASF/SF2 and hnRNPA1, suggesting a toxic RNA pathogenesis, as occurs in myotonic dystrophy. Furthermore, the C9orf72 repeat RNA was recently demonstrated to undergo the noncanonical repeat-associated non-AUG translation (RAN translation) into pathologic dipeptide repeats in patient brains, a process that is thought to depend upon RNA structure. We previously demonstrated that the r(GGGGCC)n RNA forms repeat tract length-dependent G-quadruplex structures that bind the ASF/SF2 protein. Here we show that the cationic porphyrin (5,10,15,20-tetra(N-methyl-4-pyridyl) porphyrin (TMPyP4)), which can bind some G-quadruplex-forming sequences, can bind and distort the G-quadruplex formed by r(GGGGCC)8, and this ablates the interaction of either hnRNPA1 or ASF/SF2 with the repeat. These findings provide proof of concept that nucleic acid binding small molecules, such as TMPyP4, can distort the secondary structure of the C9orf72 repeat, which may beneficially disrupt protein interactions, which may ablate either protein sequestration and/or RAN translation into potentially toxic dipeptides. Disruption of secondary structure formation of the C9orf72 RNA repeats may be a viable therapeutic avenue, as well as a means to test the role of RNA structure upon RAN translation. PMID:24371143

  4. Repeated, Intermittent Exposures to diisopropylfluorophosphate in Rats: Protracted Effects on Cholinergic Markers, Nerve Growth Factor-Related Proteins, and Cognitive Function

    PubMed Central

    Terry, Alvin V.; Buccafusco, Jerry J.; Gearhart, Debra A.; Beck, Wayne D.; Middlemore-Risher, Mary-Louise; Truan, Jacob N.; Schwarz, Gary M.; Xu, Meng; Bartlett, Michael G.; Kutiyanawala, Ammar; Pillai, Anilkumar

    2011-01-01

    Organophosphates (OPs) pose a constant threat to human health due to their widespread use as pesticides and their potential employment in military and terrorist attacks. The acute toxicity of OPs has been extensively studied; however, the consequences of prolonged or repeated exposure to levels of OPs that produce no overt signs of acute toxicity (i.e., subthreshold levels) are poorly understood. Further, there is clinical evidence that such repeated exposures to OPs lead to prolonged deficits in cognition, although the mechanism for this effect is unknown. In this study, the behavioral and neurochemical effects of repeated, intermittent, and subthreshold exposures to the alkyl OP, diisopropylfluorophosphate (DFP) were investigated. Rats were injected with DFP subcutaneously (dose range, 0.25-1.0 mg/kg) every other day over the course of 30 days, and then given a two week, DFP-free washout period. In behavioral experiments conducted at various times during the washout period, dose dependent decrements in a water maze hidden platform task and a spontaneous novel object recognition (NOR) procedure were observed, while prepulse inhibition of the acoustic startle response was unaffected. There were modest decreases in open field locomotor activity and grip strength (particularly during the DFP exposure period); however, rotarod performance and water maze swim speeds were not affected. After washout, DFP concentrations were minimal in plasma and brain, however, cholinesterase inhibition was still detectable in the brain. Moreover, the 1.0 mg/kg dose of DFP was associated with (brain region-dependent) alterations in nerve growth factor-related proteins and cholinergic markers. The results of this prospective animal study thus provide evidence to support two novel hypotheses: 1) that intermittent, subthreshold exposures to alkyl OPs can lead to protracted deficits in specific domains of cognition and 2) that such cognitive deficits may be related to persistent functional

  5. Molecular modeling of the elastomeric properties of repeating units and building blocks of resilin, a disordered elastic protein.

    PubMed

    Khandaker, Md Shahriar K; Dudek, Daniel M; Beers, Eric P; Dillard, David A; Bevan, David R

    2016-08-01

    The mechanisms responsible for the properties of disordered elastomeric proteins are not well known. To better understand the relationship between elastomeric behavior and amino acid sequence, we investigated resilin, a disordered rubber-like protein, found in specialized regions of the cuticle of insects. Resilin of Drosophila melanogaster contains Gly-rich repetitive motifs comprised of the amino acids, PSSSYGAPGGGNGGR, which confer elastic properties to resilin. The repetitive motifs of insect resilin can be divided into smaller partially conserved building blocks: PSS, SYGAP, GGGN and GGR. Using molecular dynamics (MD) simulations, we studied the relative roles of SYGAP, and its less common variants SYSAP and TYGAP, on the elastomeric properties of resilin. Results showed that SYGAP adopts a bent structure that is one-half to one-third the end-to-end length of the other motifs having an equal number of amino acids but containing SYSAP or TYGAP substituted for SYGAP. The bent structure of SYGAP forms due to conformational freedom of glycine, and hydrogen bonding within the motif apparently plays a role in maintaining this conformation. These structural features of SYGAP result in higher extensibility compared to other motifs, which may contribute to elastic properties at the macroscopic level. Overall, the results are consistent with a role for the SYGAP building block in the elastomeric properties of these disordered proteins. What we learned from simulating the repetitive motifs of resilin may be applicable to the biology and mechanics of other elastomeric biomaterials, and may provide us the deeper understanding of their unique properties.

  6. Vaccinia virus K1 ankyrin repeat protein inhibits NF-κB activation by preventing RelA acetylation.

    PubMed

    Bravo Cruz, Ariana G; Shisler, Joanna L

    2016-10-01

    The vaccinia virus (VACV) K1 protein inhibits dsRNA-dependent protein kinase (PKR) activation. A consequence of this function is that K1 inhibits PKR-induced NF-κB activation during VACV infection. However, transient expression of K1 also inhibits Toll-like receptor (TLR)-induced NF-κB activation. This suggests that K1 has a second NF-κB inhibitory mechanism that is PKR-independent. This possibility was explored by expressing K1 independently of infection and stimulating NF-κB under conditions that minimized or excluded PKR activation. K1 inhibited both TNF- and phorbol 12-myristate 13-acetate (PMA)-induced NF-κB activation, as detected by transcription of synthetic (e.g. luciferase) and natural (e.g. CXCL8) genes controlled by NF-κB. K1 also inhibited NF-κB activity in PKRkd cells, cells that have greatly decreased amounts of PKR. K1 no longer prevented IκBα degradation or NF-κB nuclear translocation in the absence of PKR, suggesting that K1 acted on a nuclear event. Indeed, K1 was present in the nucleus and cytoplasm of stimulated and unstimulated cells. K1 inhibited acetylation of the RelA (p65) subunit of NF-κB, a nuclear event known to be required for NF-κB activation. Moreover, p65-CBP (CREB-binding protein) interactions were blocked in the presence of K1. However, K1 did not preclude NF-κB binding to oligonucleotides containing κB-binding sites. The current interpretation of these data is that NF-κB-promoter interactions still occur in the presence of K1, but NF-κB cannot properly trigger transcriptional activation because K1 antagonizes acetylation of RelA. Thus, in comparison to all known VACV NF-κB inhibitory proteins, K1 acts at one of the most downstream events of NF-κB activation.

  7. The Arabidopsis thaliana AT PRP39-1 gene, encoding a tetratricopeptide repeat protein with similarity to the yeast pre-mRNA processing protein PRP39, affects flowering time.

    PubMed

    Wang, Cunxi; Tian, Qing; Hou, Zhenglin; Mucha, Mark; Aukerman, Milo; Olsen, Odd-Arne

    2007-08-01

    Flowering is regulated by a network integrated from four major pathways, including the photoperiod, vernalization, gibberellin, and autonomous pathways. RNA processing within the autonomous pathway is well known to regulate Arabidopsis thaliana flowering time. Here we identify a novel Arabidopsis gene, designated AT PRP39-1, that affects flowering time. Based on observations that homozygous at prp39-1 plants are late flowering under both long and short days and responsive to GA and vernalization treatment, we tentatively conclude that AT PRP39-1 may represent a new component of the autonomous pathway. Consistent with previous studies on genes of the autonomous pathway, knockout of AT PRP39-1 in Arabidopsis displays an upregulation of the steady state level of FLC, and simultaneous downregulation of FT and SOC1 transcript levels in adult tissues. AT PRP39-1 encodes a tetratricopeptide repeat protein with a similarity to a yeast mRNA processing protein Prp39p, suggesting that the involvement of these tetratricopeptide repeat proteins in RNA processing is conserved among yeast, human, and plants. Structure modeling suggests that AT PRP39-1 has two TPR superhelical domains suitable for target protein binding. We discuss how AT PRP39-1 may function in the control of flowering in the context of the autonomous pathway.

  8. Rhoptry-associated protein (rap-1) genes in the sheep pathogen Babesia sp. Xinjiang: Multiple transcribed copies differing by 3' end repeated sequences.

    PubMed

    Niu, Qingli; Marchand, Jordan; Yang, Congshan; Bonsergent, Claire; Guan, Guiquan; Yin, Hong; Malandrin, Laurence

    2015-07-30

    Sheep babesiosis occurs mainly in tropical and subtropical areas. The sheep parasite Babesia sp. Xinjiang is widespread in China, and our goal is to characterize rap-1 (rhoptry-associated protein 1) gene diversity and expression as a first step of a long term goal aiming at developing a recombinant subunit vaccine. Seven different rap-1a genes were amplified in Babesia sp. Xinjiang, using degenerate primers designed from conserved motifs. Rap-1b and rap-1c gene types could not be identified. In all seven rap-1a genes, the 5' regions exhibited identical sequences over 936 nt, and the 3' regions differed at 28 positions over 147 nt, defining two types of genes designated α and β. The remaining 3' part varied from 72 to 360 nt in length, depending on the gene. This region consists of a succession of two to ten 36 nt repeats, which explains the size differences. Even if the nucleotide sequences varied, 6 repeats encoded the same stretch of amino acids. Transcription of at least four α and two β genes was demonstrated by standard RT-PCR.

  9. Repeated Baclofen treatment ameliorates motor dysfunction, suppresses reflex activity and decreases the expression of signaling proteins in reticular nuclei and lumbar motoneurons after spinal trauma in rats.

    PubMed

    Kucharíková, Andrea; Schreiberová, Andrea; Závodská, Monika; Gedrová, Štefánia; Hricová, Ľudmila; Pavel, Jaroslav; Gálik, Ján; Maršala, Martin; Lukáčová, Nadežda

    2014-03-01

    The interruption of supraspinal input to the spinal cord leads to motor dysfunction and the development of spasticity. Clinical studies have shown that Baclofen (a GABAB agonist), while effective in modulating spasticity is associated with side-effects and the development of tolerance. The aim of the present study was to assess if discontinued Baclofen treatment and its repeated application leads antispasticity effects, and whether such changes affect neuronal nitric oxide synthase (nNOS) in the brainstem, nNOS and parvalbumin (PV) in lumbar α-motoneurons and glial fibrillary acidic protein in the ventral horn of the spinal cord. Adult male Wistar rats were exposed to Th9 spinal cord transection. Baclofen (30mg/b.w.) diluted in drinking water, was administered for 6 days, starting at week 1 after injury and then repeated till week 4 after injury. The behavior of the animals was tested (tail-flick test, BBB locomotor score) from 1 to 8 weeks. Our results clearly indicate the role of nitric oxide, produced by nNOS in the initiation and the maintenance of spasticity states 1, 6 and 8 weeks after spinal trauma. A considerable decrease of nNOS staining after Baclofen treatment correlates with improvement of motor dysfunction. The findings also show that parvalbumin and astrocytes participate in the regulation of ion concentrations in the sub-acute phase after the injury.

  10. Analysis of polyglutamine-coding repeats in the TATA-binding protein in different human populations and in patients with schizophrenia an bipolar affective disorder

    SciTech Connect

    Rubinsztein, D.C.; Leggo, J.; Crow, T.J.

    1996-09-20

    A new class of disease (including Huntington disease, Kennedy disease, and spinocerebellar ataxias types 1 and 3) results from abnormal expansions of CAG trinucleotides in the coding regions of genes. In all of these diseases the CAG repeats are thought to be translated into polyglutamine tracts. There is accumulating evidence arguing for CAG trinucleotide expansions as one of the causative disease mutations in schizophrenia and bipolar affective disorder. We and others believe that the TATA-binding protein (TBP) is an important candidate to investigate in these diseases as it contains a highly polymorphic stretch of glutamine codons, which are close to the threshold length where the polyglutamine tracts start to be associated with disease. Thus, we examined the lengths of this polyglutamine repeat in normal unrelated East Anglians, South African Blacks, sub-Saharan Africans mainly from Nigeria, and Asian Indians. We also examined 43 bipolar affective disorder patients and 65 schizophrenic patients. The range of polyglutamine tract-lengths that we found in humans was from 26-42 codons. No patients with bipolar affective disorder and schizophrenia had abnormal expansions at this locus. 22 refs., 1 tab.

  11. A B-cell-specific nuclear protein that binds to DNA sites 5' to immunoglobulin S alpha tandem repeats is regulated during differentiation.

    PubMed Central

    Waters, S H; Saikh, K U; Stavnezer, J

    1989-01-01

    Immunoglobulin heavy-chain switching is effected by recombination events between sites associated with tandemly repeated switch sequences located 5' to immunoglobulin heavy-chain genes. Using the band mobility shift assay, we have identified two distinct sites 5' to the alpha heavy-chain switch sequence with affinity for a single B-cell-specific DNA-binding protein, S alpha-BP. S alpha-BP was present in nuclear extracts from pre-B and B cells but was not detected in extracts from plasmacytomas, B-cell hybridomas, T-cell lymphomas, or a macrophage cell line. It was also not detectable in other nonlymphoid cells tested. Evidence suggests there are S alpha-BP-binding sites near other immunoglobulin switch sequences. As with the S alpha sites, these sites appear to be distinct from the consensus tandem repeats characteristic of immunoglobulin switch sequences. The possible functions of S alpha-BP on contacting its binding sites are discussed in the context of immunoglobulin heavy-chain switch recombination. Images PMID:2511438

  12. PEGylation enhances tumor targeting of plasmid DNA by an artificial cationized protein with repeated RGD sequences, Pronectin.

    PubMed

    Hosseinkhani, Hossein; Tabata, Yasuhiko

    2004-05-31

    The objective of this study is to investigate feasibility of a non-viral gene carrier with repeated RGD sequences (Pronectin F+) in tumor targeting for gene expression. The Pronectin F+ was cationized by introducing spermine (Sm) to the hydroxyl groups to allow to polyionically complex with plasmid DNA. The cationized Pronectin F+ prepared was additionally modified with poly(ethylene glycol) (PEG) molecules which have active ester and methoxy groups at the terminal, to form various PEG-introduced cationized Pronectin F+. The cationized Pronectin F+ with or without PEGylation at different extents was mixed with a plasmid DNA of LacZ to form respective cationized Pronectin F+-plasmid DNA complexes. The plasmid DNA was electrophoretically complexed with cationized Pronectin F+ and PEG-introduced cationized Pronectin F+, irrespective of the PEGylation extent, although the higher N/P ratio of complexes was needed for complexation with the latter Pronectin F+. The molecular size and zeta potential measurements revealed that the plasmid DNA was reduced in size to about 250 nm and the charge was changed to be positive by the complexation with cationized Pronectin F+. For the complexation with PEG-introduced cationized Pronectin F+, the charge of complex became neutral being almost 0 mV with the increasing PEGylation extents, while the molecular size was similar to that of cationized Pronectin F+. When cationized Pronectin F+-plasmid DNA complexes with or without PEGylation were intravenously injected to mice carrying a subcutaneous Meth-AR-1 fibrosarcoma mass, the PEG-introduced cationized Pronectin F+-plasmid DNA complex specifically enhanced the level of gene expression in the tumor, to a significantly high extent compared with the cationized Pronectin F+-plasmid DNA complexes and free plasmid DNA. The enhanced level of gene expression depended on the percentage of PEG introduced, the N/P ratio, and the plasmid DNA dose. A fluorescent microscopic study revealed that the

  13. Genome-wide identification, characterization and expression analysis of populus leucine-rich repeat receptor-like protein kinase genes

    PubMed Central

    2013-01-01

    Background Leucine-rich repeat receptor-like kinases (LRR-RLKs) comprise the largest group within the receptor-like kinase (RLK) superfamily in plants. This gene family plays critical and diverse roles in plant growth, development and stress response. Although the LRR-RLK families in Arabidopsis and rice have been previously analyzed, no comprehensive studies have been performed on this gene family in tree species. Results In this work, 379 LRR-RLK genes were retrieved from the Populus trichocarpa genome and further grouped into 14 subfamilies based on their structural and sequence similarities. Approximately 82% (312 out of 379) of the PtLRR-RLK genes are located in segmental duplication blocks indicating the role of duplication process in the expansion of this gene family. The conservation and variation in motif composition and intron/exon arrangement among PtLRR-RLK subfamilies were analyzed to provide additional support for their phylogenetic relationship and more importantly to indicate the potential divergence in their functions. Expression profiling of PtLRR-RLKs showed that they were differentially expressed in different organs and tissues and some PtLRR-RLKs were specifically expressed in meristem tissues, which indicated their potential involvement in tissue development and differentiation. For most AtLRR-RLKs with defined functions, Populus homologues exhibiting similar expression patterns could be identified, which might indicate the functional conservation during evolution. Among 12 types of environmental cues analyzed by the genome-wide microarray data, PtLRR-RLKs showed specific responses to shoot organogenesis, wounding, low ammonium feeding, hypoxia and seasonal dormancy, but not to drought, re-watering after drought, flooding, AlCl3 treatment and bacteria or fungi treatments. Conclusions This study provides the first comprehensive genomic analysis of the Populus LRR-RLK gene family. Segmental duplication contributes significantly to the expansion

  14. The pentatricopeptide repeat protein MTSF2 stabilizes a nad1 precursor transcript and defines the 3΄ end of its 5΄-half intron.

    PubMed

    Wang, Chuande; Aubé, Fabien; Planchard, Noelya; Quadrado, Martine; Dargel-Graffin, Céline; Nogué, Fabien; Mireau, Hakim

    2017-03-08

    RNA expression in plant mitochondria implies a large number of post-transcriptional events in which transcript processing and stabilization are essential. In this study, we analyzed the function of the Arabidopsis mitochondrial stability factor 2 gene (MTSF2) and show that the encoded pentatricopeptide repeat protein is essential for the accumulation of stable nad1 mRNA. The production of mature nad1 requires the assembly of three independent RNA precursors via two trans-splicing reactions. Genetic analyses revealed that the lack of nad1 in mtsf2 mutants results from the specific destabilization of the nad1 exons 2-3 precursor transcript. We further demonstrated that MTSF2 binds to its 3΄ extremity with high affinity, suggesting a protective action by blocking exoribonuclease progression. By defining the 3΄ end of nad1 exons 2-3 precursor, MTSF2 concomitantly determines the 3΄ extremity of the first half of the trans-intron found at the end of the transcript. Therefore, binding of the MTSF2 protein to nad1 exons 2-3 precursor evolved both to stabilize the transcript and to define a 3΄ extremity compatible with the trans-splicing reaction needed to reconstitute mature nad1. We thus reveal that the range of transcripts stabilized by association with protective protein on their 3΄ end concerns also mitochondrial precursor transcripts.

  15. Plasmodium falciparum AARP1, a giant protein containing repeated motifs rich in asparagine and aspartate residues, is associated with the infected erythrocyte membrane.

    PubMed Central

    Barale, J C; Candelle, D; Attal-Bonnefoy, G; Dehoux, P; Bonnefoy, S; Ridley, R; Pereira da Silva, L; Langsley, G

    1997-01-01

    During Plasmodium falciparum asexual intraerythrocytic development, the host's cell plasma membrane is modified by the insertion of parasite proteins. One or more of these modifications mediate the cytoadherence of infected erythrocytes to host vascular endothelium. However, these surface antigens can be the target of cytophilic antibodies which promote phagocytosis of the infected erythrocyte. It has been proposed that antibodies directed to epitopes rich in asparagine play an important role in this process, which has promoted efforts to isolate the corresponding gene(s). We describe here P. falciparum asparagine- and aspartate-rich protein 1 (PfAARP1), a new giant (circa 700-kDa) protein associated with the infected erythrocyte membrane which is rich in asparagine and aspartate residues due to the presence of nine blocks of repeats. Topology analysis predicts that PfAARP1 has multiple transmembrane domains and at least five external loops. Human antibodies immunopurified against a sequence composed exclusively of asparagine and aspartate amino acids derived from PfAARP1 label the surface of the infected erythrocyte, demonstrating that such motifs are exposed. Interestingly, external loop 4 of PfAARP1 contains repetitions of these residues, and their possible role as a target of cytophilic antibodies is discussed. PMID:9234746

  16. The Cochaperone SGTA (Small Glutamine-rich Tetratricopeptide Repeat-containing Protein Alpha) Demonstrates Regulatory Specificity for the Androgen, Glucocorticoid, and Progesterone Receptors*

    PubMed Central

    Paul, Atanu; Garcia, Yenni A.; Zierer, Bettina; Patwardhan, Chaitanya; Gutierrez, Omar; Hildenbrand, Zacariah; Harris, Diondra C.; Balsiger, Heather A.; Sivils, Jeffrey C.; Johnson, Jill L.; Buchner, Johannes; Chadli, Ahmed; Cox, Marc B.

    2014-01-01

    Steroid hormone receptors are ligand-dependent transcription factors that require the ordered assembly of multichaperone complexes for transcriptional activity. Although heat shock protein (Hsp) 90 and Hsp70 are key players in this process, multiple Hsp70- and Hsp90-associated cochaperones associate with receptor-chaperone complexes to regulate receptor folding and activation. Small glutamine-rich tetratricopeptide repeat-containing protein alpha (SGTA) was recently characterized as an Hsp70 and Hsp90-associated cochaperone that specifically regulates androgen receptor activity. However, the specificity of SGTA for additional members of the steroid hormone receptor superfamily and the mechanism by which SGTA regulates receptor activity remain unclear. Here we report that SGTA associates with and specifically regulates the androgen, glucocorticoid, and progesterone receptors and has no effect on the mineralocorticoid and estrogen receptors in both yeast and mammalian cell-based reporter assays. In both systems, SGTA knockdown/deletion enhances receptor activity, whereas SGTA overexpression suppresses receptor activity. We demonstrate that SGTA binds directly to Hsp70 and Hsp90 in vitro with similar affinities yet predominately precipitates with Hsp70 from cell lysates, suggesting a role for SGTA in early, Hsp70-mediated folding. Furthermore, SGTA expression completely abrogates the regulation of receptor function by FKBP52 (52-kDa FK506-binding protein), which acts at a later stage of the chaperone cycle. Taken together, our data suggest a role for SGTA at distinct steps in the chaperone-dependent modulation of androgen, glucocorticoid, and progesterone receptor activity. PMID:24753260

  17. The cochaperone SGTA (small glutamine-rich tetratricopeptide repeat-containing protein alpha) demonstrates regulatory specificity for the androgen, glucocorticoid, and progesterone receptors.

    PubMed

    Paul, Atanu; Garcia, Yenni A; Zierer, Bettina; Patwardhan, Chaitanya; Gutierrez, Omar; Hildenbrand, Zacariah; Harris, Diondra C; Balsiger, Heather A; Sivils, Jeffrey C; Johnson, Jill L; Buchner, Johannes; Chadli, Ahmed; Cox, Marc B

    2014-05-30

    Steroid hormone receptors are ligand-dependent transcription factors that require the ordered assembly of multichaperone complexes for transcriptional activity. Although heat shock protein (Hsp) 90 and Hsp70 are key players in this process, multiple Hsp70- and Hsp90-associated cochaperones associate with receptor-chaperone complexes to regulate receptor folding and activation. Small glutamine-rich tetratricopeptide repeat-containing protein alpha (SGTA) was recently characterized as an Hsp70 and Hsp90-associated cochaperone that specifically regulates androgen receptor activity. However, the specificity of SGTA for additional members of the steroid hormone receptor superfamily and the mechanism by which SGTA regulates receptor activity remain unclear. Here we report that SGTA associates with and specifically regulates the androgen, glucocorticoid, and progesterone receptors and has no effect on the mineralocorticoid and estrogen receptors in both yeast and mammalian cell-based reporter assays. In both systems, SGTA knockdown/deletion enhances receptor activity, whereas SGTA overexpression suppresses receptor activity. We demonstrate that SGTA binds directly to Hsp70 and Hsp90 in vitro with similar affinities yet predominately precipitates with Hsp70 from cell lysates, suggesting a role for SGTA in early, Hsp70-mediated folding. Furthermore, SGTA expression completely abrogates the regulation of receptor function by FKBP52 (52-kDa FK506-binding protein), which acts at a later stage of the chaperone cycle. Taken together, our data suggest a role for SGTA at distinct steps in the chaperone-dependent modulation of androgen, glucocorticoid, and progesterone receptor activity.

  18. The T=1 capsid protein of Penicillium chrysogenum virus is formed by a repeated helix-rich core indicative of gene duplication.

    PubMed

    Luque, Daniel; González, José M; Garriga, Damiá; Ghabrial, Said A; Havens, Wendy M; Trus, Benes; Verdaguer, Nuria; Carrascosa, José L; Castón, José R

    2010-07-01

    Penicillium chrysogenum virus (PcV), a member of the Chrysoviridae family, is a double-stranded RNA (dsRNA) fungal virus with a multipartite genome, with each RNA molecule encapsidated in a separate particle. Chrysoviruses lack an extracellular route and are transmitted during sporogenesis and cell fusion. The PcV capsid, based on a T=1 lattice containing 60 subunits of the 982-amino-acid capsid protein, remains structurally undisturbed throughout the viral cycle, participates in genome metabolism, and isolates the virus genome from host defense mechanisms. Using three-dimensional cryoelectron microscopy, we determined the structure of the PcV virion at 8.0 A resolution. The capsid protein has a high content of rod-like densities characteristic of alpha-helices, forming a repeated alpha-helical core indicative of gene duplication. Whereas the PcV capsid protein has two motifs with the same fold, most dsRNA virus capsid subunits consist of dimers of a single protein with similar folds. The spatial arrangement of the alpha-helical core resembles that found in the capsid protein of the L-A virus, a fungal totivirus with an undivided genome, suggesting a conserved basic fold. The encapsidated genome is organized in concentric shells; whereas the inner dsRNA shells are well defined, the outermost layer is dense due to numerous interactions with the inner capsid surface, specifically, six interacting areas per monomer. The outermost genome layer is arranged in an icosahedral cage, sufficiently well ordered to allow for modeling of an A-form dsRNA. The genome ordering might constitute a framework for dsRNA transcription at the capsid interior and/or have a structural role for capsid stability.

  19. Interaction of Prevotella intermedia strain 17 leucine-rich repeat domain protein AdpF with eukaryotic cells promotes bacterial internalization.

    PubMed

    Sengupta, Dipanwita; Kang, Dae-Joong; Anaya-Bergman, Cecilia; Wyant, Tiana; Ghosh, Arnab K; Miyazaki, Hiroshi; Lewis, Janina P

    2014-06-01

    Prevotella intermedia is an oral bacterium implicated in a variety of oral diseases. Although internalization of this bacterium by nonphagocytic host cells is well established, the molecular players mediating the process are not well known. Here, the properties of a leucine-rich repeat (LRR) domain protein, designated AdpF, are described. This protein contains a leucine-rich region composed of 663 amino acid residues, and molecular modeling shows that it folds into a classical curved solenoid structure. The cell surface localization of recombinant AdpF (rAdpF) was confirmed by electron and confocal microscopy analyses. The recombinant form of this protein bound fibronectin in a dose-dependent manner. Furthermore, the protein was internalized by host cells, with the majority of the process accomplished within 30 min. The internalization of rAdpF was inhibited by nystatin, cytochalasin, latrunculin, nocodazole, and wortmannin, indicating that microtubules, microfilaments, and signal transduction are required for the invasion. It is noteworthy that preincubation of eukaryotic cells with AdpF increased P. intermedia 17 internalization by 5- and 10-fold for HeLa and NIH 3T3 fibroblast cell lines, respectively. The addition of the rAdpF protein was also very effective in inducing bacterial internalization into the oral epithelial cell line HN4, as well as into primary cells, including human oral keratinocytes (HOKs) and human umbilical vein endothelial cells (HUVECs). Finally, cells exposed to P. intermedia 17 internalized the bacteria more readily upon reinfection. Taken together, our data demonstrate that rAdpF plays a role in the internalization of P. intermedia 17 by a variety of host cells.

  20. Interaction of Prevotella intermedia Strain 17 Leucine-Rich Repeat Domain Protein AdpF with Eukaryotic Cells Promotes Bacterial Internalization

    PubMed Central

    Sengupta, Dipanwita; Kang, Dae-Joong; Anaya-Bergman, Cecilia; Wyant, Tiana; Ghosh, Arnab K.; Miyazaki, Hiroshi

    2014-01-01

    Prevotella intermedia is an oral bacterium implicated in a variety of oral diseases. Although internalization of this bacterium by nonphagocytic host cells is well established, the molecular players mediating the process are not well known. Here, the properties of a leucine-rich repeat (LRR) domain protein, designated AdpF, are described. This protein contains a leucine-rich region composed of 663 amino acid residues, and molecular modeling shows that it folds into a classical curved solenoid structure. The cell surface localization of recombinant AdpF (rAdpF) was confirmed by electron and confocal microscopy analyses. The recombinant form of this protein bound fibronectin in a dose-dependent manner. Furthermore, the protein was internalized by host cells, with the majority of the process accomplished within 30 min. The internalization of rAdpF was inhibited by nystatin, cytochalasin, latrunculin, nocodazole, and wortmannin, indicating that microtubules, microfilaments, and signal transduction are required for the invasion. It is noteworthy that preincubation of eukaryotic cells with AdpF increased P. intermedia 17 internalization by 5- and 10-fold for HeLa and NIH 3T3 fibroblast cell lines, respectively. The addition of the rAdpF protein was also very effective in inducing bacterial internalization into the oral epithelial cell line HN4, as well as into primary cells, including human oral keratinocytes (HOKs) and human umbilical vein endothelial cells (HUVECs). Finally, cells exposed to P. intermedia 17 internalized the bacteria more readily upon reinfection. Taken together, our data demonstrate that rAdpF plays a role in the internalization of P. intermedia 17 by a variety of host cells. PMID:24711565

  1. Preliminary crystallographic analysis of the ankyrin-repeat domain of Arabidopsis thaliana AKT1: identification of the domain boundaries for protein crystallization

    PubMed Central

    Chaves-Sanjuán, Antonio; Sánchez-Barrena, María José; González-Rubio, Juana María; Albert, Armando

    2014-01-01

    The Arabidopsis thaliana K+ transporter 1 (AKT1) participates in the maintenance of an adequate cell potassium (K+) concentration. The CBL-interacting protein kinase 23 (CIPK23) activates AKT1 for K+ uptake under low-K+ conditions. This process is mediated by the interaction between the cytosolic ankyrin-repeat (AR) domain of AKT1 and the kinase domain of CIPK23. However, the precise boundaries of the AR domain and the residues responsible for the interaction are still unknown. Here, the optimization procedure to obtain an AR domain construct suitable for crystallization and the preliminary crystallographic analysis of the obtained crystals are reported. The crystals belonged to space group P21212, with unit-cell parameters a = 34.83, b = 65.89, c = 85.44 Å, and diffracted to 1.98 Å resolution. PMID:24699751

  2. Preliminary crystallographic analysis of the ankyrin-repeat domain of Arabidopsis thaliana AKT1: identification of the domain boundaries for protein crystallization.

    PubMed

    Chaves-Sanjuán, Antonio; Sánchez-Barrena, María José; González-Rubio, Juana María; Albert, Armando

    2014-04-01

    The Arabidopsis thaliana K(+) transporter 1 (AKT1) participates in the maintenance of an adequate cell potassium (K(+)) concentration. The CBL-interacting protein kinase 23 (CIPK23) activates AKT1 for K(+) uptake under low-K(+) conditions. This process is mediated by the interaction between the cytosolic ankyrin-repeat (AR) domain of AKT1 and the kinase domain of CIPK23. However, the precise boundaries of the AR domain and the residues responsible for the interaction are still unknown. Here, the optimization procedure to obtain an AR domain construct suitable for crystallization and the preliminary crystallographic analysis of the obtained crystals are reported. The crystals belonged to space group P21212, with unit-cell parameters a = 34.83, b = 65.89, c = 85.44 Å, and diffracted to 1.98 Å resolution.

  3. Lactococcus garvieae carries a chromosomally encoded pentapeptide repeat protein that confers reduced susceptibility to quinolones in Escherichia coli producing a cytotoxic effect.

    PubMed

    Gibello, Alicia; Díaz de Alba, Paula; Blanco, M Mar; Machuca, Jesus; Cutuli, M Teresa; Rodríguez-Martínez, José Manuel

    2014-09-01

    This study characterises a chromosomal gene of Lactococcus garvieae encoding a pentapeptide repeat protein designated as LgaQnr. This gene has been implicated in reduced susceptibility to quinolones in this bacterium, which is of relevance to both veterinary and human medicine. All of the L. garvieae isolates analysed were positive for the lgaqnr gene. The expression of lgaqnr in Escherichia coli reduced the susceptibility to quinolones, producing an adverse effect. The reduced susceptibility to ciprofloxacin was 16-fold in E. coli ATCC 25922 and 32-fold in E. coli DH10B, compared to the control strains. The minimum inhibitory concentration of nalidixic acid was also increased 4 or 5-fold. The effect of the expression of lgaqnr in E. coli was investigated by electron microscopy and was observed to affect the structure of the cell and the inner membrane of the recombinant cells.

  4. Down-regulation of an ankyrin repeat-containing protein, V-1, during skeletal muscle differentiation and its re-expression in the regenerative process of muscular dystrophy.

    PubMed

    Furukawa, Yuko; Hashimoto, Naohiro; Yamakuni, Tohru; Ishida, Yukisato; Kato, Chieko; Ogashiwa, Masayo; Kobayashi, Masumi; Kobayashi, Takayoshi; Nonaka, Ikuya; Mizusawa, Hidehiro; Song, Si Young

    2003-01-01

    Using Western blot analysis and immunohistochemical methods, we examined the expression of V-1, a member of the ankyrin repeat-containing protein family, during differentiation and regeneration of skeletal muscle. The expression of V-1 was high in cultured myoblasts and decreased during their differentiation into myotubes, while high expression was maintained when muscle differentiation was inhibited by treatment with basic fibroblast growth factor. Down-regulation of V-1 also occurred during in vivo muscle differentiation from embryonic to postnatal stages, reaching an undetectable level in mature skeletal muscle. In contrast, strong V-1 immunoreactivity was detected again in myoblasts and regenerating muscle fibers with a small diameter, which were observed in Duchenne muscular dystrophy and its animal model, mdx mouse. Thus, it seems that V-1 is a good marker for early stage of muscle regeneration and changes of its expression suggest that V-1 plays a role in prenatal muscle differentiation and postnatal muscle regeneration.

  5. Notch-regulated ankyrin-repeat protein inhibits Notch1 signaling: multiple Notch1 signaling pathways involved in T cell development.

    PubMed

    Yun, Theodore J; Bevan, Michael J

    2003-06-15

    We have characterized the function of Notch-regulated ankyrin-repeat protein (Nrarp) in mouse cell lines and in hematopoietic stem cells (HSCs). Nrarp overexpression is able to block Notch-induced activation of CBF-1. In AKR1010 thymoma cells, Nrarp overexpression blocks CBF-1-dependent transcriptional activation of Notch-responsive genes and inhibits phenotypic changes associated with Notch activation. Enforced expression of Nrarp in mouse HSCs results in a profound block in T lineage commitment and progression through early stages of thymocyte maturation. In contrast, Deltex-1 overexpression in HSCs can also block T lineage commitment but not progression through the early double negative stages of thymocyte maturation. The different effects of Deltex-1 and Nrarp overexpression suggest that alternate Notch signaling pathways mediate T vs B lineage commitment and thymocyte maturation.

  6. Specific binding of the replication protein of plasmid pPS10 to direct and inverted repeats is mediated by an HTH motif.

    PubMed Central

    García de Viedma, D; Serrano-López, A; Díaz-Orejas, R

    1995-01-01

    The initiator protein of the plasmid pPS10, RepA, has a putative helix-turn-helix (HTH) motif at its C-terminal end. RepA dimers bind to an inverted repeat at the repA promoter (repAP) to autoregulate RepA synthesis. [D. García de Viedma, et al. (1996) EMBO J. in press]. RepA monomers bind to four direct repeats at the origin of replication (oriV) to initiate pPS10 replication This report shows that randomly generated mutations in RepA, associated with defficiencies in autoregulation, map either at the putative HTH motif or in its vicinity. These mutant proteins do not promote pPS10 replication and are severely affected in binding to both the repAP and oriV regions in vitro. Revertants of a mutant that map in the vicinity of the HTH motif have been obtained and correspond to a second amino acid substitution far upstream of the motif. However, reversion of mutants that map in the helices of the motif occurs less frequently, at least by an order of magnitude. All these data indicate that the helices of the HTH motif play an essential role in specific RepA-DNA interactions, although additional regions also seem to be involved in DNA binding activity. Some mutations have slightly different effects in replication and autoregulation, suggesting that the role of the HTH motif in the interaction of RepA dimers or monomers with their respective DNA targets (IR or DR) is not the same. Images PMID:8559664

  7. Exploring mechanisms of fatigue during repeated exercise and the dose dependent effects of carbohydrate and protein ingestion: study protocol for a randomised controlled trial

    PubMed Central

    2014-01-01

    Background Muscle glycogen has been well established as the primary metabolic energy substrate during physical exercise of moderate- to high-intensity and has accordingly been implicated as a limiting factor when such activity is sustained for a prolonged duration. However, the role of this substrate during repeated exercise after limited recovery is less clear, with ongoing debate regarding how recovery processes can best be supported via nutritional intervention. The aim of this project is to examine the causes of fatigue during repeated exercise bouts via manipulation of glycogen availability through nutritional intervention, thus simultaneously informing aspects of the optimal feeding strategy for recovery from prolonged exercise. Methods/Design The project involves two phases with each involving two treatment arms administered in a repeated measures design. For each treatment, participants will be required to exercise to the point of volitional exhaustion on a motorised treadmill at 70% of previously determined maximal oxygen uptake, before a four hour recovery period in which participants will be prescribed solutions providing 1.2 grams of sucrose per kilogram of body mass per hour of recovery (g.kg-1.h-1) relative to either a lower rate of sucrose ingestion (that is, 0.3 g.kg-1. h-1; Phase I) or a moderate dose (that is, 0.8 g.kg-1.h-1) rendered isocaloric via the addition of 0.4 g.kg-1.h-1 whey protein hydrolysate (Phase II); the latter administered in a double blind manner as part of a randomised and counterbalanced design. Muscle biopsies will be sampled at the beginning and end of recovery for determination of muscle glycogen resynthesis rates, with further biopsies taken following a second bout of exhaustive exercise to determine differences in substrate availability relative to the initial sample taken following the first exercise bout. Discussion Phase I will inform whether a dose–response relationship exists between carbohydrate ingestion rate

  8. Recombinant protein of heptad-repeat HR212, a stable fusion inhibitor with potent anti-HIV action in vitro

    SciTech Connect

    Pang, Wei; Wang Ruirui; Yang Liumeng; Liu Changmei; Tien Po Zheng Yongtang

    2008-07-20

    HR212, a recombinant protein expressed in Escherichia coli, has been previously reported to inhibit HIV-1 membrane fusion at low nanomolar level. Here we report that HR212 is effective in blocking laboratory strain HIV-1{sub IIIB} entry and replication with EC{sub 50} values of 3.92 {+-} 0.62 and 6.59 {+-} 1.74 nM, respectively, and inhibiting infection by clinic isolate HIV-1{sub KM018} with EC{sub 50} values of 44.44 {+-} 10.20 nM, as well as suppressing HIV-1-induced cytopathic effect with an EC{sub 50} value of 3.04 {+-} 1.20 nM. It also inhibited HIV-2{sub ROD} and HIV-2{sub CBL-20} entry and replication in the {mu}M range. Notably, HR212 was highly effective against T20-resistant strains with EC{sub 50} values ranging from 5.09 to 7.75 nM. Unlike T20, HR212 showed stability sufficient to inhibit syncytia formation in a time-of-addition assay, and was insensitive to proteinase K digestion. These results suggest that HR212 has great potential to be further developed as novel HIV-1 fusion inhibitor for treatment of HIV/AIDS patients, particularly for those infected by T20-resistant variants.

  9. [Local species, the market, and transportation in embryological research: the study of polyembryony in armadillos in the early twentieth century].

    PubMed

    García, Susana V

    2008-01-01

    The article examines the general affirmation that communication networks and mobility are important to science by examining how these factors played a relevant role in one specific case, that is, research on the embryology of certain species of armadillos in the early twentieth century. As we show, access to pregnant females of the species and transport time from the wild to the laboratory were significant issues in the early days of such research. The strategies used to obtain field samples, the temporality of certain phenomena, the commercial circuits of which the organisms under study were a part, and the transformations they underwent in the laboratory all afford a glimpse at some aspects of how these scientists approached their work and what problems they faced in producing new knowledge.

  10. Purinergic receptor-mediated rapid depletion of nuclear phosphorylated Akt depends on pleckstrin homology domain leucine-rich repeat phosphatase, calcineurin, protein phosphatase 2A, and PTEN phosphatases.

    PubMed

    Mistafa, Oras; Ghalali, Aram; Kadekar, Sandeep; Högberg, Johan; Stenius, Ulla

    2010-09-03

    Akt is an important oncoprotein, and data suggest a critical role for nuclear Akt in cancer development. We have previously described a rapid (3-5 min) and P2X7-dependent depletion of nuclear phosphorylated Akt (pAkt) and effects on downstream targets, and here we studied mechanisms behind the pAkt depletion. We show that cholesterol-lowering drugs, statins, or extracellular ATP, induced a complex and coordinated response in insulin-stimulated A549 cells leading to depletion of nuclear pAkt. It involved protein/lipid phosphatases PTEN, pleckstrin homology domain leucine-rich repeat phosphatase (PHLPP1 and -2), protein phosphatase 2A (PP2A), and calcineurin. We employed immunocytology, immunoprecipitation, and proximity ligation assay techniques and show that PHLPP and calcineurin translocated to the nucleus and formed complexes with Akt within 3 min. Also PTEN translocated to the nucleus and then co-localized with pAkt close to the nuclear membrane. An inhibitor of the scaffolding immunophilin FK506-binding protein 51 (FKBP51) and calcineurin, FK506, prevented depletion of nuclear pAkt. Furthermore, okadaic acid, an inhibitor of PP2A, prevented the nuclear pAkt depletion. Chemical inhibition and siRNA indicated that PHLPP, PP2A, and PTEN were required for a robust depletion of nuclear pAkt, and in prostate cancer cells lacking PTEN, transfection of PTEN restored the statin-induced pAkt depletion. The activation of protein and lipid phosphatases was paralleled by a rapid proliferating cell nuclear antigen (PCNA) translocation to the nucleus, a PCNA-p21(cip1) complex formation, and cyclin D1 degradation. We conclude that these effects reflect a signaling pathway for rapid depletion of pAkt that may stop the cell cycle.

  11. A Single B-repeat of Staphylococcus epidermidis accumulation-associated protein induces protective immune responses in an experimental biomaterial-associated infection mouse model.

    PubMed

    Yan, Lin; Zhang, Lei; Ma, Hongyan; Chiu, David; Bryers, James D

    2014-09-01

    Nosocomial infections are the fourth leading cause of morbidity and mortality in the United States, resulting in 2 million infections and ∼100,000 deaths each year. More than 60% of these infections are associated with some type of biomedical device. Staphylococcus epidermidis is a commensal bacterium of the human skin and is the most common nosocomial pathogen infecting implanted medical devices, especially those in the cardiovasculature. S. epidermidis antibiotic resistance and biofilm formation on inert surfaces make these infections hard to treat. Accumulation-associated protein (Aap), a cell wall-anchored protein of S. epidermidis, is considered one of the most important proteins involved in the formation of S. epidermidis biofilm. A small recombinant protein vaccine comprising a single B-repeat domain (Brpt1.0) of S. epidermidis RP62A Aap was developed, and the vaccine's efficacy was evaluated in vitro with a biofilm inhibition assay and in vivo in a murine model of biomaterial-associated infection. A high IgG antibody response against S. epidermidis RP62A was detected in the sera of the mice after two subcutaneous immunizations with Brpt1.0 coadministered with Freund's adjuvant. Sera from Brpt1.0-immunized mice inhibited in vitro S. epidermidis RP62A biofilm formation in a dose-dependent pattern. After receiving two immunizations, each mouse was surgically implanted with a porous scaffold disk containing 5 × 10(6) CFU of S. epidermidis RP62A. Weight changes, inflammatory markers, and histological assay results after challenge with S. epidermidis indicated that the mice immunized with Brpt1.0 exhibited significantly higher resistance to S. epidermidis RP62A implant infection than the control mice. Day 8 postchallenge, there was a significantly lower number of bacteria in scaffold sections and surrounding tissues and a lower residual inflammatory response to the infected scaffold disks for the Brpt1.0-immunized mice than for of the ovalbumin (Ova

  12. Inhibitor of apoptosis (IAP)-like protein lacks a baculovirus IAP repeat (BIR) domain and attenuates cell death in plant and animal systems.

    PubMed

    Kim, Woe Yeon; Lee, Sun Yong; Jung, Young Jun; Chae, Ho Byoung; Nawkar, Ganesh M; Shin, Mi Rim; Kim, Sun Young; Park, Jin Ho; Kang, Chang Ho; Chi, Yong Hun; Ahn, Il Pyung; Yun, Dae Jin; Lee, Kyun Oh; Kim, Young-Myeong; Kim, Min Gab; Lee, Sang Yeol

    2011-12-09

    A novel Arabidopsis thaliana inhibitor of apoptosis was identified by sequence homology to other known inhibitor of apoptosis (IAP) proteins. Arabidopsis IAP-like protein (AtILP) contained a C-terminal RING finger domain but lacked a baculovirus IAP repeat (BIR) domain, which is essential for anti-apoptotic activity in other IAP family members. The expression of AtILP in HeLa cells conferred resistance against tumor necrosis factor (TNF)-α/ActD-induced apoptosis through the inactivation of caspase activity. In contrast to the C-terminal RING domain of AtILP, which did not inhibit the activity of caspase-3, the N-terminal region, despite displaying no homology to known BIR domains, potently inhibited the activity of caspase-3 in vitro and blocked TNF-α/ActD-induced apoptosis. The anti-apoptotic activity of the AtILP N-terminal domain observed in plants was reproduced in an animal system. Transgenic Arabidopsis lines overexpressing AtILP exhibited anti-apoptotic activity when challenged with the fungal toxin fumonisin B1, an agent that induces apoptosis-like cell death in plants. In AtIPL transgenic plants, suppression of cell death was accompanied by inhibition of caspase activation and DNA fragmentation. Overexpression of AtILP also attenuated effector protein-induced cell death and increased the growth of an avirulent bacterial pathogen. The current results demonstrated the existence of a novel plant IAP-like protein that prevents caspase activation in Arabidopsis and showed that a plant anti-apoptosis gene functions similarly in plant and animal systems.

  13. The MTL1 Pentatricopeptide Repeat Protein Is Required for Both Translation and Splicing of the Mitochondrial NADH DEHYDROGENASE SUBUNIT7 mRNA in Arabidopsis.

    PubMed

    Haïli, Nawel; Planchard, Noelya; Arnal, Nadège; Quadrado, Martine; Vrielynck, Nathalie; Dahan, Jennifer; des Francs-Small, Catherine Colas; Mireau, Hakim

    2016-01-01

    Mitochondrial translation involves a complex interplay of ancient bacteria-like features and host-derived functionalities. Although the basic components of the mitochondrial translation apparatus have been recognized, very few protein factors aiding in recruiting ribosomes on mitochondria-encoded messenger RNA (mRNAs) have been identified in higher plants. In this study, we describe the identification of the Arabidopsis (Arabidopsis thaliana) MITOCHONDRIAL TRANSLATION FACTOR1 (MTL1) protein, a new member of the Pentatricopeptide Repeat family, and show that it is essential for the translation of the mitochondrial NADH dehydrogenase subunit7 (nad7) mRNA. We demonstrate that mtl1 mutant plants fail to accumulate the Nad7 protein, even though the nad7 mature mRNA is produced and bears the same 5' and 3' extremities as in wild-type plants. We next observed that polysome association of nad7 mature mRNA is specifically disrupted in mtl1 mutants, indicating that the absence of Nad7 results from a lack of translation of nad7 mRNA. These findings illustrate that mitochondrial translation requires the intervention of gene-specific nucleus-encoded PPR trans-factors and that their action does not necessarily involve the 5' processing of their target mRNA, as observed previously. Interestingly, a partial decrease in nad7 intron 2 splicing was also detected in mtl1 mutants, suggesting that MTL1 is also involved in group II intron splicing. However, this second function appears to be less essential for nad7 expression than its role in translation. MTL1 will be instrumental to understand the multifunctionality of PPR proteins and the mechanisms governing mRNA translation and intron splicing in plant mitochondria.

  14. The molecular chaperone TRiC/CCT binds to the Trp-Asp 40 (WD40) repeat protein WDR68 and promotes its folding, protein kinase DYRK1A binding, and nuclear accumulation.

    PubMed

    Miyata, Yoshihiko; Shibata, Takeshi; Aoshima, Masato; Tsubata, Takuichi; Nishida, Eisuke

    2014-11-28

    Trp-Asp (WD) repeat protein 68 (WDR68) is an evolutionarily conserved WD40 repeat protein that binds to several proteins, including dual specificity tyrosine phosphorylation-regulated protein kinase (DYRK1A), MAPK/ERK kinase kinase 1 (MEKK1), and Cullin4-damage-specific DNA-binding protein 1 (CUL4-DDB1). WDR68 affects multiple and diverse physiological functions, such as controlling anthocyanin synthesis in plants, tissue growth in insects, and craniofacial development in vertebrates. However, the biochemical basis and the regulatory mechanism of WDR68 activity remain largely unknown. To better understand the cellular function of WDR68, here we have isolated and identified cellular WDR68 binding partners using a phosphoproteomic approach. More than 200 cellular proteins with wide varieties of biochemical functions were identified as WDR68-binding protein candidates. Eight T-complex protein 1 (TCP1) subunits comprising the molecular chaperone TCP1 ring complex/chaperonin-containing TCP1 (TRiC/CCT) were identified as major WDR68-binding proteins, and phosphorylation sites in both WDR68 and TRiC/CCT were identified. Co-immunoprecipitation experiments confirmed the binding between TRiC/CCT and WDR68. Computer-aided structural analysis suggested that WDR68 forms a seven-bladed β-propeller ring. Experiments with a series of deletion mutants in combination with the structural modeling showed that three of the seven β-propeller blades of WDR68 are essential and sufficient for TRiC/CCT binding. Knockdown of cellular TRiC/CCT by siRNA caused an abnormal WDR68 structure and led to reduction of its DYRK1A-binding activity. Concomitantly, nuclear accumulation of WDR68 was suppressed by the knockdown of TRiC/CCT, and WDR68 formed cellular aggregates when overexpressed in the TRiC/CCT-deficient cells. Altogether, our results demonstrate that the molecular chaperone TRiC/CCT is essential for correct protein folding, DYRK1A binding, and nuclear accumulation of WDR68.

  15. Allelic Variation in a Simple Sequence Repeat Element of Neisserial pglB2 and Its Consequences for Protein Expression and Protein Glycosylation

    PubMed Central

    Viburiene, Raimonda; Vik, Åshild; Koomey, Michael

    2013-01-01

    Neisseria species express an O-linked glycosylation system in which functionally distinct proteins are elaborated with variable glycans. A major source of glycan diversity in N. meningitidis results from two distinct pglB alleles responsible for the synthesis of either N,N′-diacetylbacillosamine or glyceramido-acetamido trideoxyhexose that occupy the reducing end of the oligosaccharides. Alternative modifications at C-4 of the precursor UDP-4-amino are attributable to distinct C-terminal domains that dictate either acetyltransferase or glyceramidotransferase activity, encoded by pglB and pglB2, respectively. Naturally occurring alleles of pglB2 have homopolymeric tracts of either 7 or 8 adenosines (As) bridging the C-terminal open reading frame (ORF) and the ORF encompassing the conserved N-terminal domain associated with phosphoglycosyltransferase activity. In the work presented here, we explored the consequences of such pglB2 allele variation and found that, although both alleles are functional vis-à-vis glycosylation, the 7A form results in the expression of a single, multidomain protein, while the 8A variant elicits two single-domain proteins. We also found that the glyceramidotransferase activity-encoding domain is essential to protein glycosylation, showing the critical role of the C-4 modification of the precursor UDP-4-amino in the pathway. These findings were further extended and confirmed by examining the phenotypic consequences of extended poly(A) tract length variation. Although ORFs related to those of pglB2 are broadly distributed in eubacteria, they are primarily found as two distinct, juxtaposed ORFs. Thus, the neisserial pglB2 system provides novel insights into the potential influence of hypermutability on modular evolution of proteins by providing a unique snapshot of the progression of ongoing gene fusion. PMID:23729645

  16. The cell proliferation-associated antigen of antibody Ki-67: a very large, ubiquitous nuclear protein with numerous repeated elements, representing a new kind of cell cycle-maintaining proteins

    PubMed Central

    1993-01-01

    The antigen defined by mAb Ki-67 is a human nuclear protein the expression of which is strictly associated with cell proliferation and which is widely used in routine pathology as a "proliferation marker" to measure the growth fraction of cells in human tumors. Ki-67 detects a double band with apparent molecular weights of 395 and 345 kD in immunoblots of proteins from proliferating cells. We cloned and sequenced the full length cDNA, identified two differentially spliced isoforms of mRNA with open reading frames of 9,768 and 8,688 bp encoding for this cell proliferation-associated protein with calculated molecular weights of 358,761 D and 319,508 D, respectively. New mAbs against a bacterially expressed part and a synthetic polypeptide deduced from the isolated cDNA react with the native Ki-67 antigen, thus providing a circle of evidence that we have cloned the authentic Ki-67 antigen cDNA. The central part of the Ki-67 antigen cDNA contains a large 6,845-bp exon with 16 tandemly repeated 366-bp elements, the "Ki-67 repeats", each including a highly conserved new motif of 66 bp, the "Ki-67 motif", which encodes for the epitope detected by Ki-67. Computer analysis of the nucleic acid and the deduced amino acid sequence of the Ki-67 antigen confirmed that the cDNA encodes for a nuclear and short-lived protein without any significant homology to known sequences. Ki-67 antigen-specific antisense oligonucleotides inhibit the proliferation of IM-9 cell line cells, indicating that the Ki-67 antigen may be an absolute requirement for maintaining cell proliferation. We conclude that the Ki-67 antigen defines a new category of cell cycle-associated nuclear nonhistone proteins. PMID:8227122

  17. Recombinant myxoma virus lacking all poxvirus ankyrin-repeat proteins stimulates multiple cellular anti-viral pathways and exhibits a severe decrease in virulence.

    PubMed

    Lamb, Stephanie A; Rahman, Masmudur M; McFadden, Grant

    2014-09-01

    Although the production of single gene knockout viruses is a useful strategy to study viral gene functions, the redundancy of many host interactive genes within a complex viral genome can obscure their collective functions. In this study, a rabbit-specific poxvirus, myxoma virus (MYXV), was genetically altered to disrupt multiple members of the poxviral ankyrin-repeat (ANK-R) protein superfamily, M-T5, M148, M149 and M150. A particularly robust activation of the NF-κB pathway was observed in A549 cells following infection with the complete ANK-R knockout (vMyx-ANKsKO). Also, an increased release of IL-6 was only observed upon infection with vMyx-ANKsKO. In virus-infected rabbit studies, vMyx-ANKsKO was the most extensively attenuated and produced the smallest primary lesion of all ANK-R mutant constructs. This study provides the first insights into the shared functions of the poxviral ANK-R protein superfamily in vitro and in vivo.

  18. Arabidopsis Kelch Repeat F-Box Proteins Regulate Phenylpropanoid Biosynthesis via Controlling the Turnover of Phenylalanine Ammonia-Lyase[C][W][OPEN

    PubMed Central

    Zhang, Xuebin; Gou, Mingyue; Liu, Chang-Jun

    2013-01-01

    Phenylalanine ammonia-lyase (PAL) catalyzes the first rate-limiting step in the phenylpropanoid pathway, which controls carbon flux to a variety of bioactive small-molecule aromatic compounds, and to lignin, the structural component of the cell wall. PAL is regulated at both the transcriptional and translational levels. Our knowledge about the transcriptional regulation of PAL is relatively comprehensive, but our knowledge of the molecular basis of the posttranslational regulation of PAL remains limited. Here, we demonstrate that the Arabidopsis thaliana Kelch repeat F-box (KFB) proteins KFB01, KFB20, and KFB50 physically interact with four PAL isozymes and mediate their proteolytic turnover via the ubiquitination-26S proteasome pathway. The KFB genes are differentially expressed in Arabidopsis tissues and respond to developmental and environmental cues. Up- or downregulation of their expression reciprocally affects the stability of the PAL enzymes, consequently altering the levels of phenylpropanoids. These data suggest that the KFB-mediated protein ubiquitination and degradation regulates the proteolysis of PALs, thus posttranslationally regulating phenylpropanoid metabolism. Characterizing the KFB-mediated proteolysis of PAL enzymes may inform future strategies for manipulating the synthesis of bioactive phenolics. PMID:24363316

  19. Disruption of a rice pentatricopeptide repeat protein causes a seedling-specific albino phenotype and its utilization to enhance seed purity in hybrid rice production.

    PubMed

    Su, Ning; Hu, Mao-Long; Wu, Dian-Xing; Wu, Fu-Qing; Fei, Gui-Lin; Lan, Ying; Chen, Xiu-Ling; Shu, Xiao-Li; Zhang, Xin; Guo, Xiu-Ping; Cheng, Zhi-Jun; Lei, Cai-Lin; Qi, Cun-Kou; Jiang, Ling; Wang, Haiyang; Wan, Jian-Min

    2012-05-01

    The pentatricopeptide repeat (PPR) gene family represents one of the largest gene families in higher plants. Accumulating data suggest that PPR proteins play a central and broad role in modulating the expression of organellar genes in plants. Here we report a rice (Oryza sativa) mutant named young seedling albino (ysa) derived from the rice thermo/photoperiod-sensitive genic male-sterile line Pei'ai64S, which is a leading male-sterile line for commercial two-line hybrid rice production. The ysa mutant develops albino leaves before the three-leaf stage, but the mutant gradually turns green and recovers to normal green at the six-leaf stage. Further investigation showed that the change in leaf color in ysa mutant is associated with changes in chlorophyll content and chloroplast development. Map-based cloning revealed that YSA encodes a PPR protein with 16 tandem PPR motifs. YSA is highly expressed in young leaves and stems, and its expression level is regulated by light. We showed that the ysa mutation has no apparent negative effects on several important agronomic traits, such as fertility, stigma extrusion rate, selfed seed-setting rate, hybrid seed-setting rate, and yield heterosis under normal growth conditions. We further demonstrated that ysa can be used as an early marker for efficient identification and elimination of false hybrids in commercial hybrid rice production, resulting in yield increases by up to approximately 537 kg ha(-1).

  20. SPT5, an essential gene important for normal transcription in Saccharomyces cerevisiae, encodes an acidic nuclear protein with a carboxy-terminal repeat.

    PubMed Central

    Swanson, M S; Malone, E A; Winston, F

    1991-01-01

    Mutations in the SPT5 gene of Saccharomyces cerevisiae were isolated previously as suppressors of delta insertion mutations at HIS4 and LYS2. In this study we have shown that spt5 mutations suppress the his4-912 delta and lys2-128 delta alleles by altering transcription. We cloned the SPT5 gene and found that either an increase or a decrease in the copy number of the wild-type SPT5 gene caused an Spt- phenotype. Construction and analysis of an spt5 null mutation demonstrated that SPT5 is essential for growth, suggesting that SPT5 may be required for normal transcription of a large number of genes. The SPT5 DNA sequence was determined; it predicted a 116-kDa protein with an extremely acidic amino terminus and a novel six-amino-acid repeat at the carboxy terminus (consensus = S-T/A-W-G-G-A/Q). By indirect immunofluorescence microscopy we showed that a bifunctional SPT5-beta-galactosidase protein was located in the yeast nucleus. This molecular analysis of the SPT5 gene revealed a number of interesting similarities to the previously characterized SPT6 gene of S. cerevisiae. These results suggest that SPT5 and SPT6 act in a related fashion to influence essential transcriptional processes in S. cerevisiae. Images PMID:1840633

  1. Maize Homologs of Hydroxycinnamoyltransferase, a Key Enzyme in Lignin Biosynthesis, Bind the Nucleotide Binding Leucine-Rich Repeat Rp1 Proteins to Modulate the Defense Response1

    PubMed Central

    Wang, Guan-Feng; He, Yijian; Strauch, Renee; Olukolu, Bode A.; Nielsen, Dahlia; Li, Xu; Balint-Kurti, Peter J.

    2015-01-01

    In plants, most disease resistance genes encode nucleotide binding Leu-rich repeat (NLR) proteins that trigger a rapid localized cell death called a hypersensitive response (HR) upon pathogen recognition. The maize (Zea mays) NLR protein Rp1-D21 derives from an intragenic recombination between two NLRs, Rp1-D and Rp1-dp2, and confers an autoactive HR in the absence of pathogen infection. From a previous quantitative trait loci and genome-wide association study, we identified a single-nucleotide polymorphism locus highly associated with variation in the severity of Rp1-D21-induced HR. Two maize genes encoding hydroxycinnamoyltransferase (HCT; a key enzyme involved in lignin biosynthesis) homologs, termed HCT1806 and HCT4918, were adjacent to this single-nucleotide polymorphism. Here, we show that both HCT1806 and HCT4918 physically interact with and suppress the HR conferred by Rp1-D21 but not other autoactive NLRs when transiently coexpressed in Nicotiana benthamiana. Other maize HCT homologs are unable to confer the same level of suppression on Rp1-D21-induced HR. The metabolic activity of HCT1806 and HCT4918 is unlikely to be necessary for their role in suppressing HR. We show that the lignin pathway is activated by Rp1-D21 at both the transcriptional and metabolic levels. We derive a model to explain the roles of HCT1806 and HCT4918 in Rp1-mediated disease resistance. PMID:26373661

  2. ANKYRIN REPEAT-CONTAINING PROTEIN 2A is an essential molecular chaperone for peroxisomal membrane-bound ASCORBATE PEROXIDASE3 in Arabidopsis.

    PubMed

    Shen, Guoxin; Kuppu, Sundaram; Venkataramani, Sujatha; Wang, Jing; Yan, Juqiang; Qiu, Xiaoyun; Zhang, Hong

    2010-03-01

    Arabidopsis thaliana ANKYRIN REPEAT-CONTAINING PROTEIN 2A (AKR2A) interacts with peroxisomal membrane-bound ASCORBATE PEROXIDASE3 (APX3). This interaction involves the C-terminal sequence of APX3 (i.e., a transmembrane domain plus a few basic amino acid residues). The specificity of the AKR2A-APX3 interaction suggests that AKR2A may function as a molecular chaperone for APX3 because binding of AKR2A to the transmembrane domain can prevent APX3 from forming aggregates after translation. Analysis of three akr2a mutants indicates that these mutant plants have reduced steady state levels of APX3. Reduced expression of AKR2A using RNA interference also leads to reduced steady state levels of APX3 and reduced targeting of APX3 to peroxisomes in plant cells. Since AKR2A also binds specifically to the chloroplast OUTER ENVELOPE PROTEIN7 (OEP7) and is required for the biogenesis of OEP7, AKR2A may serve as a molecular chaperone for OEP7 as well. The pleiotropic phenotype of akr2a mutants indicates that AKR2A plays many important roles in plant cellular metabolism and is essential for plant growth and development.

  3. Blockade of ankyrin repeat-rich membrane spanning protein modulates extracellular signal-regulated kinase expression and inhibits allergic inflammation in ovalbumin-sensitized mice.

    PubMed

    Ni, Xiuqin; Li, Xing; Tao, Shuhua; Xu, Minghui; Ma, Hongmei; Wang, Xiuli

    2013-07-01

    Ankyrin repeat-rich membrane spanning protein (ARMS), also known as kinase D-interacting substrate of 220 kDa (Kidins220), is a transmembrane protein that has been reported to be involved in the pathogenesis of asthma through the nerve growth factor (NGF)/tyrosine kinase A (TrkA) receptor signaling pathway. To investigate whether NGF/TrkA-Kidins220/ARMS-extracellular signal-regulated kinase (ERK) signaling is activated in airway inflammation of asthma, BALB/c mice were sensitized and challenged with ovalbumin (OVA). The effects of Kidins220/ARMS on ERK, interleukin (IL)-1β, IL-4 and tumor necrosis factor (TNF)-α in lung tissues following the allergic airway challenge in mice were assessed by administering anti-ARMS antibody to the mice. Pathological changes in the bronchi and lung tissues were examined via hematoxylin and eosin staining. The phosphorylated ERK, IL-1β, IL-4 and TNF-α levels were determined using western blot analysis and ELISA and were found to be overexpressed in lung tissues following the allergen challenge. Moreover, after the mice were treated with anti-NGF, anti-TrkA or anti-ARMS, the levels of Kidins220/ARMS, phosphorylated ERK, IL-1β, IL-4, TNF-α and allergen-induced airway inflammation were downregulated. These results suggested that NGF/TrkA-Kidins220/ARMS-ERK signaling was activated in airway inflammation induced by the allergic airway challenge, possibly representing a new mechanism in asthma.

  4. SPRi-MALDI MS: characterization and identification of a kinase from cell lysate by specific interaction with different designed ankyrin repeat proteins.

    PubMed

    Anders, Ulrike; Schaefer, Jonas V; Hibti, Fatima-Ezzahra; Frydman, Chiraz; Suckau, Detlev; Plückthun, Andreas; Zenobi, Renato

    2017-03-01

    We report on the direct coupling of surface plasmon resonance imaging (SPRi) with matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) for the investigation of specific, non-covalent interactions, using the example of designed ankyrin repeat proteins (DARPins) and ribosomal protein S6 kinase 2 (RPS6KA2) directly from lysate of SH-SY5Y cells, derived from human bone marrow. Due to an array format, tracing of binding kinetics of numerous DARPins simultaneously and in real time becomes possible. By optimizing both the proteolytic digest directly on the SPRi chip (amount of trypsin, incubation time, and temperature) as well as the MALDI matrix application (concentration of matrix and number of spray cycles), we are able to identify the specific interaction with RPS6KA2 directly from the cell lysate at a surface coverage of only 0.8 fmol/mm(2). Graphical Abstract Workflow of the direct coupling of SPRi with MALDI mass spectrometry.

  5. Recombinant myxoma virus lacking all poxvirus ankyrin-repeat proteins stimulates multiple cellular anti-viral pathways and exhibits a severe decrease in virulence

    PubMed Central

    Lamb, Stephanie A.; Rahman, Masmudur M.; McFadden, Grant

    2014-01-01

    Although the production of single gene knockout viruses is a useful strategy to study viral gene functions, the redundancy of many host interactive genes within a complex viral genome can obscure their collective functions. In this study, a rabbit-specific poxvirus, myxoma virus (MYXV), was genetically altered to disrupt multiple members of the poxviral ankyrin-repeat (ANK-R) protein superfamily, M-T5, M148, M149 and M150. A particularly robust activation of the NF-κB pathway was observed in A549 cells following infection with the complete ANK-R knockout (vMyx-ANKsKO). Also, an increased release of IL-6 was only observed upon infection with vMyx-ANKsKO. In virus-infected rabbit studies, vMyx-ANKsKO was the most extensively attenuated and produced the smallest primary lesion of all ANK-R mutant constructs. This study provides the first insights into the shared functions of the poxviral ANK-R protein superfamily in vitro and in vivo. PMID:25068401

  6. Mutations in the GW-repeat protein SUO reveal a developmental function for microRNA-mediated translational repression in Arabidopsis

    PubMed Central

    Yang, Li; Wu, Gang; Poethig, R. Scott

    2012-01-01

    Plant microRNAs (miRNAs) typically mediate RNA cleavage, but examples of miRNA-mediated translational repression have also been reported. However, the functional significance of this latter process is unknown. We identified SUO in a screen for Arabidopsis mutations that increase the accumulation of the miR156-regulated gene SPL3. suo has a loss-of-function phenotype characteristic of plants with reduced Argonaute (AGO)1 activity. An analysis of RNA and protein levels in suo mutants demonstrated that this phenotype is a consequence of a defect in miRNA-mediated translational repression; the effect of suo on vegetative phase change is attributable to a reduction in miR156/miR157 activity. SUO encodes a large protein with N-terminal bromo-adjacent homology (BAH) and transcription elongation factor S-II (TFS2N) domains and two C-terminal GW (glycine and tryptophan) repeats. SUO is present in the nucleus, and colocalizes with the processing-body component DCP1 in the cytoplasm. Our results reveal that SOU is a component of the miRNA pathway in Arabidopsis and demonstrate that translational repression is a functionally important aspect of miRNA activity in plants. PMID:22184231

  7. Mutations in the GW-repeat protein SUO reveal a developmental function for microRNA-mediated translational repression in Arabidopsis.

    PubMed

    Yang, Li; Wu, Gang; Poethig, R Scott

    2012-01-03

    Plant microRNAs (miRNAs) typically mediate RNA cleavage, but examples of miRNA-mediated translational repression have also been reported. However, the functional significance of this latter process is unknown. We identified SUO in a screen for Arabidopsis mutations that increase the accumulation of the miR156-regulated gene SPL3. suo has a loss-of-function phenotype characteristic of plants with reduced Argonaute (AGO)1 activity. An analysis of RNA and protein levels in suo mutants demonstrated that this phenotype is a consequence of a defect in miRNA-mediated translational repression; the effect of suo on vegetative phase change is attributable to a reduction in miR156/miR157 activity. SUO encodes a large protein with N-terminal bromo-adjacent homology (BAH) and transcription elongation factor S-II (TFS2N) domains and two C-terminal GW (glycine and tryptophan) repeats. SUO is present in the nucleus, and colocalizes with the processing-body component DCP1 in the cytoplasm. Our results reveal that SOU is a component of the miRNA pathway in Arabidopsis and demonstrate that translational repression is a functionally important aspect of miRNA activity in plants.

  8. Brain distribution of dipeptide repeat proteins in frontotemporal lobar degeneration and motor neurone disease associated with expansions in C9ORF72.

    PubMed

    Davidson, Yvonne S; Barker, Holly; Robinson, Andrew C; Thompson, Jennifer C; Harris, Jenny; Troakes, Claire; Smith, Bradley; Al-Saraj, Safa; Shaw, Chris; Rollinson, Sara; Masuda-Suzukake, Masami; Hasegawa, Masato; Pickering-Brown, Stuart; Snowden, Julie S; Mann, David M

    2014-06-20

    A hexanucleotide (GGGGCC) expansion in C9ORF72 gene is the most common genetic change seen in familial Frontotemporal Lobar Degeneration (FTLD) and familial Motor Neurone Disease (MND). Pathologically, expansion bearers show characteristic p62 positive, TDP-43 negative inclusion bodies within cerebellar and hippocampal neurons which also contain dipeptide repeat proteins (DPR) formed from sense and antisense RAN (repeat associated non ATG-initiated) translation of the expanded repeat region itself. 'Inappropriate' formation, and aggregation, of DPR might therefore confer neurotoxicity and influence clinical phenotype. Consequently, we compared the topographic brain distribution of DPR in 8 patients with Frontotemporal dementia (FTD), 6 with FTD + MND and 7 with MND alone (all 21 patients bearing expansions in C9ORF72) using a polyclonal antibody to poly-GA, and related this to the extent of TDP-43 pathology in key regions of cerebral cortex and hippocampus. There were no significant differences in either the pattern or severity of brain distribution of DPR between FTD, FTD + MND and MND groups, nor was there any relationship between the distribution of DPR and TDP-43 pathologies in expansion bearers. Likewise, there were no significant differences in the extent of TDP-43 pathology between FTLD patients bearing an expansion in C9ORF72 and non-bearers of the expansion. There were no association between the extent of DPR pathology and TMEM106B or APOE genotypes. However, there was a negative correlation between the extent of DPR pathology and age at onset. Present findings therefore suggest that although the presence and topographic distribution of DPR may be of diagnostic relevance in patients bearing expansion in C9ORF72 this has no bearing on the determination of clinical phenotype. Because TDP-43 pathologies are similar in bearers and non-bearers of the expansion, the expansion may act as a major genetic risk factor for FTLD and MND by rendering the brain

  9. Biophysical Analysis of Anopheles gambiae Leucine-Rich Repeat Proteins APL1A1, APL1B and APL1C and Their Interaction with LRIM1

    PubMed Central

    Williams, Marni; Summers, Brady J.; Baxter, Richard H. G.

    2015-01-01

    Natural infection of Anopheles gambiae by malaria-causing Plasmodium parasites is significantly influenced by the APL1 genetic locus. The locus contains three closely related leucine-rich repeat (LRR) genes, APL1A, APL1B and APL1C. Multiple studies have reported the participation of APL1A—C in the immune response of A. gambiae to invasion by both rodent and human Plasmodium isolates. APL1C forms a heterodimer with the related LRR protein LRIM1 via a C-terminal coiled-coil domain that is also present in APL1A and APL1B. The LRIM1/APL1C heterodimer protects A. gambiae from infection by binding the complement-like protein TEP1 to form a stable and active immune complex. Here we report solution x-ray scatting data for the LRIM1/APL1C heterodimer, the oligomeric state of LRIM1/APL1 LRR domains in solution and the crystal structure of the APL1B LRR domain. The LRIM1/APL1C heterodimeric complex has a flexible and extended structure in solution. In contrast to the APL1A, APL1C and LRIM1 LRR domains, the APL1B LRR domain is a homodimer. The crystal structure of APL1B-LRR shows that the homodimer is formed by an N-terminal helix that complements for the absence of an N-terminal capping motif in APL1B, which is a unique distinction within the LRIM1/APL1 protein family. Full-length APL1A1 and APL1B form a stable complex with LRIM1. These results support a model in which APL1A1, APL1B and APL1C can all form an extended, flexible heterodimer with LRIM1, providing a repertoire of functional innate immune complexes to protect A. gambiae from a diverse array of pathogens. PMID:25775123

  10. Genetic Screen of a Library of Chimeric Poxviruses Identifies an Ankyrin Repeat Protein Involved in Resistance to the Avian Type I Interferon Response

    PubMed Central

    Buttigieg, Karen; Laidlaw, Stephen M.; Ross, Craig; Davies, Marc; Goodbourn, Stephen

    2013-01-01

    Viruses must be able to resist host innate responses, especially the type I interferon (IFN) response. They do so by preventing the induction or activity of IFN and/or by resisting the antiviral effectors that it induces. Poxviruses are no exception, with many mechanisms identified whereby mammalian poxviruses, notably, vaccinia virus (VACV), but also cowpox and myxoma viruses, are able to evade host IFN responses. Similar mechanisms have not been described for avian poxviruses (avipoxviruses). Restricted for permissive replication to avian hosts, they have received less attention; moreover, the avian host responses are less well characterized. We show that the prototypic avipoxvirus, fowlpox virus (FWPV), is highly resistant to the antiviral effects of avian IFN. A gain-of-function genetic screen identified fpv014 to contribute to increased resistance to exogenous recombinant chicken alpha IFN (ChIFN1). fpv014 is a member of the large family of poxvirus (especially avipoxvirus) genes that encode proteins containing N-terminal ankyrin repeats (ANKs) and C-terminal F-box-like motifs. By binding the Skp1/cullin-1 complex, the F box in such proteins appears to target ligands bound by the ANKs for ubiquitination. Mass spectrometry and immunoblotting demonstrated that tandem affinity-purified, tagged fpv014 was complexed with chicken cullin-1 and Skp1. Prior infection with an fpv014-knockout mutant of FWPV still blocked transfected poly(I·C)-mediated induction of the beta IFN (ChIFN2) promoter as effectively as parental FWPV, but the mutant was more sensitive to exogenous ChIFN1. Therefore, unlike the related protein fpv012, fpv014 does not contribute to the FWPV block to induction of ChIFN2 but does confer resistance to an established antiviral state. PMID:23427151

  11. Members of a Novel Protein Family Containing Microneme Adhesive Repeat Domains Act as Sialic Acid-binding Lectins during Host Cell Invasion by Apicomplexan Parasites*

    PubMed Central

    Friedrich, Nikolas; Santos, Joana M.; Liu, Yan; Palma, Angelina S.; Leon, Ester; Saouros, Savvas; Kiso, Makoto; Blackman, Michael J.; Matthews, Stephen; Feizi, Ten; Soldati-Favre, Dominique

    2010-01-01

    Numerous intracellular pathogens exploit cell surface glycoconjugates for host cell recognition and entry. Unlike bacteria and viruses, Toxoplasma gondii and other parasites of the phylum Apicomplexa actively invade host cells, and this process critically depends on adhesins (microneme proteins) released onto the parasite surface from intracellular organelles called micronemes (MIC). The microneme adhesive repeat (MAR) domain of T. gondii MIC1 (TgMIC1) recognizes sialic acid (Sia), a key determinant on the host cell surface for invasion by this pathogen. By complementation and invasion assays, we demonstrate that TgMIC1 is one important player in Sia-dependent invasion and that another novel Sia-binding lectin, designated TgMIC13, is also involved. Using BLAST searches, we identify a family of MAR-containing proteins in enteroparasitic coccidians, a subclass of apicomplexans, including T. gondii, suggesting that all these parasites exploit sialylated glycoconjugates on host cells as determinants for enteric invasion. Furthermore, this protein family might provide a basis for the broad host cell range observed for coccidians that form tissue cysts during chronic infection. Carbohydrate microarray analyses, corroborated by structural considerations, show that TgMIC13, TgMIC1, and its homologue Neospora caninum MIC1 (NcMIC1) share a preference for α2–3- over α2–6-linked sialyl-N-acetyllactosamine sequences. However, the three lectins also display differences in binding preferences. Intense binding of TgMIC13 to α2–9-linked disialyl sequence reported on embryonal cells and relatively strong binding to 4-O-acetylated-Sia found on gut epithelium and binding of NcMIC1 to 6′sulfo-sialyl Lewisx might have implications for tissue tropism. PMID:19901027

  12. Biophysical Analysis of Anopheles gambiae Leucine-Rich Repeat Proteins APL1A1, APL1B and APL1C and Their Interaction with LRIM1

    SciTech Connect

    Williams, Marni; Summers, Brady J.; Baxter, Richard H. G.; Kobe, Bostjan

    2015-03-16

    Natural infection of Anopheles gambiae by malaria-causing Plasmodium parasites is significantly influenced by the APL1 genetic locus. The locus contains three closely related leucine-rich repeat (LRR) genes, APL1A, APL1B and APL1C. Multiple studies have reported the participation of APL1A—C in the immune response of A. gambiae to invasion by both rodent and human Plasmodium isolates. APL1C forms a heterodimer with the related LRR protein LRIM1 via a C-terminal coiled-coil domain that is also present in APL1A and APL1B. The LRIM1/APL1C heterodimer protects A. gambiae from infection by binding the complement-like protein TEP1 to form a stable and active immune complex. We report solution x-ray scatting data for the LRIM1/APL1C heterodimer, the oligomeric state of LRIM1/APL1 LRR domains in solution and the crystal structure of the APL1B LRR domain. The LRIM1/APL1C heterodimeric complex has a flexible and extended structure in solution. In contrast to the APL1A, APL1C and LRIM1 LRR domains, the APL1B LRR domain is a homodimer. The crystal structure of APL1B-LRR shows that the homodimer is formed by an N-terminal helix that complements for the absence of an N-terminal capping motif in APL1B, which is a unique distinction within the LRIM1/APL1 protein family. Full-length APL1A1 and APL1B form a stable complex with LRIM1. Our results support a model in which APL1A1, APL1B and APL1C can all form an extended, flexible heterodimer with LRIM1, providing a repertoire of functional innate immune complexes to protect A. gambiae from a diverse array of pathogens.

  13. Orthogonal assembly of a designed ankyrin repeat protein-cytotoxin conjugate with a clickable serum albumin module for half-life extension.

    PubMed

    Simon, Manuel; Frey, Raphael; Zangemeister-Wittke, Uwe; Plückthun, Andreas

    2013-11-20

    The generation of drug conjugates for safe and effective tumor targeting requires binding proteins tolerant to functionalization by rational engineering. Here, we show that Designed Ankyrin Repeat Proteins (DARPins), a novel class of binding proteins not derived from antibodies, can be used as building blocks for facile orthogonal assembly of bioconjugates for tumor targeting with tailored properties. DARPin Ec1, which targets the Epithelial Cell Adhesion Molecule (EpCAM), was genetically modified with a C-terminal cysteine for conjugation of the small molecule cytotoxin monomethylauristatin F (MMAF). In addition, it was N-terminally functionalized by metabolic introduction of the non-natural amino acid azidohomoalanine to enable linkage of site-specifically dibenzocyclooctyne-modified mouse serum albumin (MSA) for half-life extension using Cu(I)-free click chemistry. The conjugate MSA-Ec1-MMAF was assembled to obtain high yields of a pure and stable drug conjugate as confirmed by various analytical methods and in functional assays. The orthogonality of the assembly led to a defined reaction product and preserved the functional properties of all modules, including EpCAM-specific binding and internalization, FcRn binding mediated by MSA, and cytotoxic potency. Linkage of MMAF to the DARPin increased receptor-specific uptake of the drug while decreasing nonspecific uptake, and further coupling of the conjugate to MSA enhanced this effect. In mice, albumin conjugation increased the serum half-life from 11 min to 17.4 h, resulting in a more than 22-fold increase in the area-under-the-curve (AUC). Our data demonstrate the promise of the DARPin format for facile modular assembly of drug conjugates with improved pharmacokinetic performance for tumor targeting.

  14. Structure of the fusion core and inhibition of fusion by a heptad repeat peptide derived from the S protein of Middle East respiratory syndrome coronavirus.

    PubMed

    Gao, Jing; Lu, Guangwen; Qi, Jianxun; Li, Yan; Wu, Ying; Deng, Yao; Geng, Heyuan; Li, Hongbin; Wang, Qihui; Xiao, Haixia; Tan, Wenjie; Yan, Jinghua; Gao, George F

    2013-12-01

    Middle East respiratory syndrome coronavirus (MERS-CoV) recently emerged as a severe worldwide public health concern. The virus is highly pathogenic, manifesting in infected patients with an approximately 50% fatality rate. It is known that the surface spike (S) proteins of coronaviruses mediate receptor recognition and membrane fusion, thereby playing an indispensable role in initiating infection. In this process, heptad repeats 1 and 2 (HR1 and HR2) of the S protein assemble into a complex called the fusion core, which represents a key membrane fusion architecture. To date, however, the MERS-CoV fusion core remains uncharacterized. In this study, we performed a series of biochemical and biophysical analyses characterizing the HR1/HR2 complexes of this novel virus. The HR sequences were variably truncated and then connected with a flexible amino acid linker. In each case, the recombinant protein automatically assembled into a trimer in solution, displaying a typical α-helical structure. One of these trimers was successfully crystallized, and its structure was solved at a resolution of 1.9 Å. A canonical 6-helix bundle, like those reported for other coronaviruses, was revealed, with three HR1 helices forming the central coiled-coil core and three HR2 chains surrounding the core in the HR1 side grooves. This demonstrates that MERS-CoV utilizes a mechanism similar to those of other class I enveloped viruses for membrane fusion. With this notion, we further identified an HR2-based peptide that could potently inhibit MERS-CoV fusion and entry by using a pseudotyped-virus system. These results lay the groundwork for future inhibitory peptidic drug design.

  15. Biophysical Analysis of Anopheles gambiae Leucine-Rich Repeat Proteins APL1A1, APL1B and APL1C and Their Interaction with LRIM1

    DOE PAGES

    Williams, Marni; Summers, Brady J.; Baxter, Richard H. G.; ...

    2015-03-16

    Natural infection of Anopheles gambiae by malaria-causing Plasmodium parasites is significantly influenced by the APL1 genetic locus. The locus contains three closely related leucine-rich repeat (LRR) genes, APL1A, APL1B and APL1C. Multiple studies have reported the participation of APL1A—C in the immune response of A. gambiae to invasion by both rodent and human Plasmodium isolates. APL1C forms a heterodimer with the related LRR protein LRIM1 via a C-terminal coiled-coil domain that is also present in APL1A and APL1B. The LRIM1/APL1C heterodimer protects A. gambiae from infection by binding the complement-like protein TEP1 to form a stable and active immune complex.more » We report solution x-ray scatting data for the LRIM1/APL1C heterodimer, the oligomeric state of LRIM1/APL1 LRR domains in solution and the crystal structure of the APL1B LRR domain. The LRIM1/APL1C heterodimeric complex has a flexible and extended structure in solution. In contrast to the APL1A, APL1C and LRIM1 LRR domains, the APL1B LRR domain is a homodimer. The crystal structure of APL1B-LRR shows that the homodimer is formed by an N-terminal helix that complements for the absence of an N-terminal capping motif in APL1B, which is a unique distinction within the LRIM1/APL1 protein family. Full-length APL1A1 and APL1B form a stable complex with LRIM1. Our results support a model in which APL1A1, APL1B and APL1C can all form an extended, flexible heterodimer with LRIM1, providing a repertoire of functional innate immune complexes to protect A. gambiae from a diverse array of pathogens.« less

  16. Structure of C-terminal Tandem BRCT Repeats of Rtt107 Protein Reveals Critical Role in Interaction with Phosphorylated Histone H2A during DNA Damage Repair*

    PubMed Central

    Li, Xinxin; Liu, Kaixian; Li, Fudong; Wang, Juncheng; Huang, Hongda; Wu, Jihui; Shi, Yunyu

    2012-01-01

    Rtt107 (regulator of Ty1 transposition 107; Esc4) is a DNA repair protein from Saccharomyces cerevisiae that can restore stalled replication forks following DNA damage. There are six BRCT (BRCA1 C-terminal) domains in Rtt107 that act as binding sites for other recruited proteins during DNA repair. Several Rtt107 binding partners have been identified, including Slx4, Rtt101, Rad55, and the Smc5/6 (structural maintenance of chromosome) protein complex. Rtt107 can reportedly be recruited to chromatin in the presence of Rtt101 and Rtt109 upon DNA damage, but the chromatin-binding site of Rtt107 has not been identified. Here, we report our investigation of the interaction between phosphorylated histone H2A (γH2A) and the C-terminal tandem BRCT repeats (BRCT5-BRCT6) of Rtt107. The crystal structures of BRCT5-BRCT6 alone and in a complex with γH2A reveal the molecular basis of the Rtt107-γH2A interaction. We used in vitro mutagenesis and a fluorescence polarization assay to confirm the location of the Rtt107 motif that is crucial for this interaction. In addition, these assays indicated that this interaction requires the phosphorylation of H2A. An in vivo phenotypic analysis in yeast demonstrated the critical role of BRCT5-BRCT6 and its interaction with γH2A during the DNA damage response. Our results shed new light on the molecular mechanism by which Rtt107 is recruited to chromatin in response to stalled DNA replication forks. PMID:22262834

  17. The Ancestral Gene for Transcribed, Low-Copy Repeats in the Prader-Willi/Angleman Region Encodes a Large Protein Implicated in Protein Trafficking that is Deficient in Mice with Neuromuscular and

    SciTech Connect

    Ji, Y.

    1999-01-01

    Transcribed, low-copy repeat elements are associated with the breakpoint regions of common deletions in Prader-Willi and Angelman syndromes. We report here the identification of the ancestral gene ( HERC2 ) and a family of duplicated, truncated copies that comprise these low-copy repeats. This gene encodes a highly conserved giant protein, HERC2, that is distantly related to p532 (HERC1), a guanine nucleotide exchange factor (GEF) implicated in vesicular trafficking. The mouse genome contains a single Herc2 locus, located in the jdf2 (juvenile development and fertility-2) interval of chromosome 7C. We have identified single nucleotide splice junction mutations in Herc2 in three independent N-ethyl-N-nitrosourea-induced jdf2 mutant alleles, each leading to exon skipping with premature termination of translation and/or deletion of conserved amino acids. Therefore, mutations in Herc2 lead to the neuromuscular secretory vesicle and sperm acrosome defects, other developmental abnormalities and juvenile lethality of jdf2 mice. Combined, these findings suggest that HERC2 is an important gene encoding a GEF involved in protein trafficking and degradation pathways in the cell.

  18. Dominant short repeated sequences in bacterial genomes.

    PubMed

    Avershina, Ekaterina; Rudi, Knut

    2015-03-01

    We use a novel multidimensional searching approach to present the first exhaustive search for all possible repeated sequences in 166 genomes selected to cover the bacterial domain. We found an overrepresentation of repeated sequences in all but one of the genomes. The most prevalent repeats by far were related to interspaced short palindromic repeats (CRISPRs)—conferring bacterial adaptive immunity. We identified a deep branching clade of thermophilic Firmicutes containing the highest number of CRISPR repeats. We also identified a high prevalence of tandem repeated heptamers. In addition, we identified GC-rich repeats that could potentially be involved in recombination events. Finally, we identified repeats in a 16322 amino acid mega protein (involved in biofilm formation) and inverted repeats flanking miniature transposable elements (MITEs). In conclusion, the exhaustive search for repeated sequences identified new elements and distribution of these, which has implications for understanding both the ecology and evolution of bacteria.

  19. Molecular Analysis of Alternative Transcripts of the Equine Cordon-Bleu WH2 Repeat Protein-Like 1 (COBLL1) Gene.

    PubMed

    Park, Jeong-Woong; Jang, Hyun-Jun; Shin, Sangsu; Cho, Hyun-Woo; Choi, Jae-Young; Kim, Nam-Young; Lee, Hak-Kyo; Do, Kyong-Tak; Song, Ki-Duk; Cho, Byung-Wook

    2015-06-01

    The purpose of this study was to investigate the alternative splicing in equine cordon-bleu WH2 repeat protein-like 1 (COBLL1) gene that was identified in horse muscle and blood leukocytes, and to predict functional consequences of alternative splicing by bioinformatics analysis. In a previous study, RNA-seq analysis predicted the presence of alternative spliced isoforms of equine COBLL1, namely COBLL1a as a long form and COBLL1b as a short form. In this study, we validated two isoforms of COBLL1 transcripts in horse tissues by the real-time polymerase chain reaction, and cloned them for Sanger sequencing. The sequencing results showed that the alternative splicing occurs at exon 9. Prediction of protein structure of these isoforms revealed three putative phosphorylation sites at the amino acid sequences encoded in exon 9, which is deleted in COBLL1b. In expression analysis, it was found that COBLL1b was expressed ubiquitously and equivalently in all the analyzed tissues, whereas COBLL1a showed strong expression in kidney, spinal cord and lung, moderate expression in heart and skeletal muscle, and low expression in thyroid and colon. In muscle, both COBLL1a and COBLL1b expression decreased after exercise. It is assumed that the regulation of COBLL1 expression may be important for regulating glucose level or switching of energy source, possibly through an insulin signaling pathway, in muscle after exercise. Further study is warranted to reveal the functional importance of COBLL1 on athletic performance in race horses.

  20. Proline/arginine-rich end leucine-rich repeat protein N-terminus is a novel osteoclast antagonist that counteracts bone loss.

    PubMed

    Rucci, Nadia; Capulli, Mattia; Ventura, Luca; Angelucci, Adriano; Peruzzi, Barbara; Tillgren, Viveka; Muraca, Maurizio; Heinegård, Dick; Teti, Anna

    2013-09-01

    (hbd) PRELP is a peptide corresponding to the N-terminal heparin binding domain of the matrix protein proline/arginine-rich end leucine-rich repeat protein (PRELP). (hbd) PRELP inhibits osteoclastogenesis entering pre-fusion osteoclasts through a chondroitin sulfate- and annexin 2-dependent mechanism and reducing the nuclear factor-κB transcription factor activity. In this work, we hypothesized that (hbd) PRELP could have a pharmacological relevance, counteracting bone loss in a variety of in vivo models of bone diseases induced by exacerbated osteoclast activity. In healthy mice, we demonstrated that the peptide targeted the bone and increased trabecular bone mass over basal level. In mice treated with retinoic acid to induce an acute increase of osteoclast formation, the peptide consistently antagonized osteoclastogenesis and prevented the increase of the serum levels of the osteoclast-specific marker tartrate-resistant acid phosphatase. In ovariectomized mice, in which osteoclast activity was chronically enhanced by estrogen deficiency, (hbd) PRELP counteracted exacerbated osteoclast activity and bone loss. In mice carrying osteolytic bone metastases, in which osteoclastogenesis and bone resorption were enhanced by tumor cell-derived factors, (hbd) PRELP reduced the incidence of osteolytic lesions, both preventively and curatively, with mechanisms involving impaired tumor cell homing to bone and tumor growth in the bone microenvironment. Interestingly, in tumor-bearing mice, (hbd) PRELP also inhibited breast tumor growth in orthotopic sites and development of metastatic disease in visceral organs, reducing cachexia and improving survival especially when administered preventively. (hbd) PRELP was retained in the tumor tissue and appeared to affect tumor growth by interacting with the microenvironment rather than by directly affecting the tumor cells. Because safety studies and high-dose treatments revealed no adverse effects, (hbd) PRELP could be employed as a

  1. Expression of ankyrin repeat and suppressor of cytokine signaling box protein 4 (Asb-4) in proopiomelanocortin neurons of the arcuate nucleus of mice produces a hyperphagic, lean phenotype.

    PubMed

    Li, Ji-Yao; Chai, Biao-Xin; Zhang, Weizhen; Wang, Hui; Mulholland, Michael W

    2010-01-01

    Ankyrin repeat and suppressor of cytokine signaling box-containing protein 4 (Asb-4) is specifically expressed in the energy homeostasis-related brain areas and colocalizes with proopiomelanocortin (POMC) neurons of the arcuate nucleus (ARC). Injection of insulin into the third ventricle of the rat brain increased Asb-4 mRNA expression in the paraventricular nucleus but not in the ARC of the hypothalamus, whereas injection of leptin (ip) increased Asb-4 expression in both mouse paraventricular nucleus and ARC. A transgenic mouse in which Myc-tagged Asb-4 is specifically expressed in POMC neurons of the ARC was made and used to study the effects of Asb-4 on ingestive behavior and metabolic rate. Animals with overexpression of Asb-4 in POMC neurons demonstrated an increase in food intake. However, POMC-Asb-4 transgenic animals gained significantly less weight from 6-30 wk of age. The POMC-Asb-4 mice had reduced fat mass and increased lean mass and lower levels of blood leptin. The transgenic animals were resistant to high-fat diet-induced obesity. Transgenic mice had significantly higher rates of oxygen consumption and carbon dioxide production than wild-type mice during both light and dark periods. The locomotive activity of transgenic mice was increased. The overexpression of Asb-4 in POMC neurons increased POMC mRNA expression in the ARC. The transgenic animals had no observed effect on peripheral glucose metabolism and the activity of the autonomic nervous system. These results indicate that Asb-4 is a key regulatory protein in the central nervous system, involved in the control of feeding behavior and metabolic rate.

  2. Blockade of ankyrin repeat-rich membrane spanning protein modulates extracellular signal-regulated kinase expression and inhibits allergic inflammation in ovalbumin-sensitized mice

    PubMed Central

    NI, XIUQIN; LI, XING; TAO, SHUHUA; XU, MINGHUI; MA, HONGMEI; WANG, XIULI

    2013-01-01

    Ankyrin repeat-rich membrane spanning protein (ARMS), also known as kinase D-interacting substrate of 220 kDa (Kidins220), is a transmembrane protein that has been reported to be involved in the pathogenesis of asthma through the nerve growth factor (NGF)/tyrosine kinase A (TrkA) receptor signaling pathway. To investigate whether NGF/TrkA-Kidins220/ARMS-extracellular signal-regulated kinase (ERK) signaling is activated in airway inflammation of asthma, BALB/c mice were sensitized and challenged with ovalbumin (OVA). The effects of Kidins220/ARMS on ERK, interleukin (IL)-1β, IL-4 and tumor necrosis factor (TNF)-α in lung tissues following the allergic airway challenge in mice were assessed by administering anti-ARMS antibody to the mice. Pathological changes in the bronchi and lung tissues were examined via hematoxylin and eosin staining. The phosphorylated ERK, IL-1β, IL-4 and TNF-α levels were determined using western blot analysis and ELISA and were found to be overexpressed in lung tissues following the allergen challenge. Moreover, after the mice were treated with anti-NGF, anti-TrkA or anti-ARMS, the levels of Kidins220/ARMS, phosphorylated ERK, IL-1β, IL-4, TNF-α and allergen-induced airway inflammation were downregulated. These results suggested that NGF/TrkA-Kidins220/ARMS-ERK signaling was activated in airway inflammation induced by the allergic airway challenge, possibly representing a new mechanism in asthma. PMID:24649008

  3. Small kernel 1 encodes a pentatricopeptide repeat protein required for mitochondrial nad7 transcript editing and seed development in maize (Zea mays) and rice (Oryza sativa).

    PubMed

    Li, Xiao-Jie; Zhang, Ya-Feng; Hou, Mingming; Sun, Feng; Shen, Yun; Xiu, Zhi-Hui; Wang, Xiaomin; Chen, Zong-Liang; Sun, Samuel S M; Small, Ian; Tan, Bao-Cai

    2014-09-01

    RNA editing modifies cytidines (C) to uridines (U) at specific sites in the transcripts of mitochondria and plastids, altering the amino acid specified by the DNA sequence. Here we report the identification of a critical editing factor of mitochondrial nad7 transcript via molecular characterization of a small kernel 1 (smk1) mutant in Zea mays (maize). Mutations in Smk1 arrest both the embryo and endosperm development. Cloning of Smk1 indicates that it encodes an E-subclass pentatricopeptide repeat (PPR) protein that is targeted to mitochondria. Loss of SMK1 function abolishes the C → U editing at the nad7-836 site, leading to the retention of a proline codon that is edited to encode leucine in the wild type. The smk1 mutant showed dramatically reduced complex-I assembly and NADH dehydrogenase activity, and abnormal biogenesis of the mitochondria. Analysis of the ortholog in Oryza sativa (rice) reveals that rice SMK1 has a conserved function in C → U editing of the mitochondrial nad7-836 site. T-DNA knock-out mutants showed abnormal embryo and endosperm development, resulting in embryo or seedling lethality. The leucine at NAD7-279 is highly conserved from bacteria to flowering plants, and analysis of genome sequences from many plants revealed a molecular coevolution between the requirement for C → U editing at this site and the existence of an SMK1 homolog. These results demonstrate that Smk1 encodes a PPR-E protein that is required for nad7-836 editing, and this editing is critical to NAD7 function in complex-I assembly in mitochondria, and hence to embryo and endosperm development in maize and rice.

  4. Orientia tsutsugamushi ankyrin repeat-containing protein family members are Type 1 secretion system substrates that traffic to the host cell endoplasmic reticulum

    PubMed Central

    VieBrock, Lauren; Evans, Sean M.; Beyer, Andrea R.; Larson, Charles L.; Beare, Paul A.; Ge, Hong; Singh, Smita; Rodino, Kyle G.; Heinzen, Robert A.; Richards, Allen L.; Carlyon, Jason A.

    2015-01-01

    Scrub typhus is an understudied, potentially fatal infection that threatens one billion persons in the Asia-Pacific region. How the causative obligate intracellular bacterium, Orientia tsutsugamushi, facilitates its intracellular survival and pathogenesis is poorly understood. Many intracellular bacterial pathogens utilize the Type 1 (T1SS) or Type 4 secretion system (T4SS) to translocate ankyrin repeat-containing proteins (Anks) that traffic to distinct subcellular locations and modulate host cell processes. The O. tsutsugamushi genome encodes one of the largest known bacterial Ank repertoires plus T1SS and T4SS components. Whether these potential virulence factors are expressed during infection, how the Anks are potentially secreted, and to where they localize in the host cell are not known. We determined that O. tsutsugamushi transcriptionally expresses 20 unique ank genes as well as genes for both T1SS and T4SS during infection of mammalian host cells. Examination of the Anks' C-termini revealed that the majority of them resemble T1SS substrates. Escherichia coli expressing a functional T1SS was able to secrete chimeric hemolysin proteins bearing the C-termini of 19 of 20 O. tsutsugamushi Anks in an HlyBD-dependent manner. Thus, O. tsutsugamushi Anks C-termini are T1SS-compatible. Conversely, Coxiella burnetii could not secrete heterologously expressed Anks in a T4SS-dependent manner. Analysis of the subcellular distribution patterns of 20 ectopically expressed Anks revealed that, while 6 remained cytosolic or trafficked to the nucleus, 14 localized to, and in some cases, altered the morphology of the endoplasmic reticulum. This study identifies O. tsutsugamushi Anks as T1SS substrates and indicates that many display a tropism for the host cell secretory pathway. PMID:25692099

  5. Leucine-rich repeat-mediated intramolecular interactions in nematode recognition and cell death signaling by the tomato resistance protein Mi.

    PubMed

    Hwang, Chin-Feng; Williamson, Valerie M

    2003-06-01

    The root-knot nematode resistance gene Mi from tomato encodes a nucleotide-binding/leucine-rich repeat (NB/LRR) protein with a novel amino-terminal domain compared to related disease-resistance genes. The closely linked paralog Mi-1.1, which does not confer nematode resistance, encodes a protein 91% identical to the functional copy, Mi-1.2. The chimeric construct Mi-DS3, which encodes the 161 amino-terminal residues from Mi-1.1 fused to the remainder of Mi-1.2, induces localized necrosis when transiently expressed in Nicotiana benthamiana leaves. We produced mutant constructs that exchanged sequences encoding each of the 40 amino acid differences from the Mi-1.1 LRR region into Mi-DS3 and into Mi-1.2. For 23 of the substitutions, necrosis was lost upon transient expression of the mutated Mi-DS3 in N. benthamiana, and nematode resistance was lost when the altered Mi-1.2 was expressed in the tomato roots. One substitution, R961D, failed to give Mi-DS3-induced necrosis, but produced a dominant lethal phenotype when introduced into Mi-1.2. This gain-of-function phenotype was suppressed by co-expression with the amino-terminal region of Mi-1.1, suggesting that residue 961 is critical for negative regulation by the corresponding N-terminal region. Substitutions of Mi-1.1 residues 984-986 retained the ability to cause necrosis in Mi-DS3, but resulted in loss-of-nematode resistance in Mi-1.2, suggesting that these residues are essential for nematode recognition. None of the loss-of-function mutations in Mi-1.2 had a dominant negative phenotype. These results indicate that the Mi-1.2 LRR is involved in regulation of the transmission of the resistance response as well as in recognition of the nematode.

  6. The E-Subgroup Pentatricopeptide Repeat Protein Family in Arabidopsis thaliana and Confirmation of the Responsiveness PPR96 to Abiotic Stresses

    PubMed Central

    Liu, Jia-Ming; Zhao, Juan-Ying; Lu, Pan-Pan; Chen, Ming; Guo, Chang-Hong; Xu, Zhao-Shi; Ma, You-Zhi

    2016-01-01

    Pentatricopeptide repeat (PPR) proteins are extensive in all eukaryotes. Their functions remain as yet largely unknown. Mining potential stress responsive PPRs, and checking whether known PPR editing factors are affected in the stress treatments. It is beneficial to elucidate the regulation mechanism of PPRs involved in biotic and abiotic stress. Here, we explored the characteristics and origin of the 105 E subgroup PPRs in Arabidopsis thaliana. Phylogenetic analysis categorized the E subgroup PPRs into five discrete groups (Cluster I to V), and they may have a common origin in both A. thaliana and rice. An in silico expression analysis of the 105 E subgroup PPRs in A. thaliana was performed using available microarray data. Thirty-four PPRs were differentially expressed during A. thaliana seed imbibition, seed development stage(s), and flowers development processes. To explore potential stress responsive PPRs, differential expression of 92 PPRs was observed in A. thaliana seedlings subjected to different abiotic stresses. qPCR data of E subgroup PPRs under stress conditions revealed that the expression of 5 PPRs was responsive to abiotic stresses. In addition, PPR96 is involved in plant responses to salt, abscisic acid (ABA), and oxidative stress. The T-DNA insertion mutation inactivating PPR96 expression results in plant insensitivity to salt, ABA, and oxidative stress. The PPR96 protein is localized in the mitochondria, and altered transcription levels of several stress-responsive genes under abiotic stress treatments. Our results suggest that PPR96 may important function in a role connecting the regulation of oxidative respiration and environmental responses in A. thaliana. PMID:27994613

  7. Development of a chimeric Plasmodium berghei strain expressing the repeat region of the P. vivax circumsporozoite protein for in vivo evaluation of vaccine efficacy.

    PubMed

    Espinosa, Diego A; Yadava, Anjali; Angov, Evelina; Maurizio, Paul L; Ockenhouse, Christian F; Zavala, Fidel

    2013-08-01

    The development of vaccine candidates against Plasmodium vivax-the most geographically widespread human malaria species-is challenged by technical difficulties, such as the lack of in vitro culture systems and availability of animal models. Chimeric rodent Plasmodium parasites are safe and useful tools for the preclinical evaluation of new vaccine formulations. We report the successful development and characterization of chimeric Plasmodium berghei parasites bearing the type I repeat region of P. vivax circumsporozoite protein (CSP). The P. berghei-P. vivax chimeric strain develops normally in mosquitoes and produces highly infectious sporozoites that produce patent infection in mice that are exposed to the bites of as few as 3 P. berghei-P. vivax-infected mosquitoes. Using this transgenic parasite, we demonstrate that monoclonal and polyclonal antibodies against P. vivax CSP strongly inhibit parasite infection and thus support the notion that these antibodies play an important role in protective immunity. The chimeric parasites we developed represent a robust model for evaluating protective immune responses against P. vivax vaccines based on CSP.

  8. Repeated administration of AC-5216, a ligand for the 18 kDa translocator protein, improves behavioral deficits in a mouse model of post-traumatic stress disorder.

    PubMed

    Qiu, Zhi-Kun; Zhang, Li-Ming; Zhao, Nan; Chen, Hong-Xia; Zhang, You-Zhi; Liu, Yan-Qin; Mi, Tian-Yue; Zhou, Wen-Wen; Li, Yang; Yang, Ri-Fang; Xu, Jiang-Ping; Li, Yun-Feng

    2013-08-01

    Post-traumatic stress disorder (PTSD) is a severely disabling anxiety disorder that may occur following exposure to a serious traumatic event. It is a psychiatric condition that can afflict anyone who has experienced a life-threatening or violent event. Previous studies have shown that changes in 18 kDa translocator protein (TSPO) expression (or function), a promising target for treating neurological disorders without benzodiazepine-like side effects, may correlate with PTSD. However, few studies have investigated the anti-PTSD effects of TSPO ligands. AC-5216, a ligand for TSPO, induces anxiolytic- and anti-depressant-like effects in animal models. The present study aimed to determine whether AC-5216 ameliorates PTSD behavior in mice. Following the training session consisting of exposure to inescapable electric foot shocks, animals were administered AC-5216 daily during the behavioral assessments, i.e., situational reminders (SRs), the open field (OF) test, the elevated plus-maze (EPM) test, and the staircase test (ST). The results indicated that exposure to foot shocks induced long-term behavioral deficiencies in the mice, including freezing and anxiety-like behavior, which were significantly ameliorated by repeated treatment with AC-5216 but without any effect on spontaneous locomotor activity or body weight. In summary, this study demonstrated the anti-PTSD effects of AC-5216 treatment, suggesting that TSPO may represent a therapeutic target for anti-PTSD drug discovery and that TSPO ligands may be a promising new class of drugs for the future treatment of PTSD.

  9. Effect of pistachio oil on gene expression of IFN-induced protein with tetratricopeptide repeats 2: a biomarker of inflammatory response.

    PubMed

    Zhang, Jun; Kris-Etherton, Penny M; Thompson, Jerry T; Vanden Heuvel, John P

    2010-05-01

    When incorporated into the diet, pistachios have a beneficial effect on lipid and lipoprotein profiles. However, little is known about potential anti-inflammatory properties. This study was conducted to determine whether pistachio oil and an organic extract from pistachio oil extract (PE) regulated expression of inflammation-related genes. A mouse macrophage cell line (RAW 264.7 cells) was treated with pistachio oil and gene expression microarray analyses were performed. Pistachio oil significantly affected genes involved in immune response, defense response to bacteria, and gene silencing, of which INF-induced protein with tetratricopeptide repeats 2 (Ifit-2) was the most dramatically reduced. PE reduced the LPS-induced Ifit-2 by 78% and the bioactive molecules contained in PE, linoleic acid, and beta-sitosterol recapitulated this inhibition. Promoter analysis identified two adjacent IFN-stimulated response elements, which lie between -110 and -85bp of the 5'-flanking region of the Ifit-2 promoter, as being responsive to LPS activation and inhibition by PE. Our results indicate that pistachio oil and bioactive molecules present therein decrease Ifit-2 expressions, and due to the sensitivity of this effect, this gene is a potential biomarker for monitoring diet-induced changes in inflammation.

  10. Comparison of leucine-rich repeat-containing G protein-coupled receptor 5 expression in different cancer and normal cell lines

    PubMed Central

    ALIZADEH-NAVAEI, REZA; RAFIEI, ALIREZA; ABEDIAN-KENARI, SAEID; ASGARIAN-OMRAN, HOSSEIN; VALADAN, REZA; HEDAYATIZADEH-OMRAN, AKBAR

    2016-01-01

    Evaluating the expression of leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5) may be useful for predicting the best models and achieving more accurate results in cancer research. Therefore, the aim of the present study was to analyze the LGR5 expression levels in different cell lines. Eight commonly used cell lines were assessed (COS-7, NIH3T3, HEK293, VERO, HeLa, BHK, HepG2 and AGS). All the cell lines were cultured in RPMI-1640 medium contain 10% fetal calf serum at 37°C in humidified conditions with 5% CO2. According to the western blotting results, LGR5 was expressed in all cell lines. Densitometry results of LGR5 expression in the different cell lines showed that high LGR5 expression levels were apparent in BHK, AGS, VERO and NIH3T3 cell lines compared with the other cell lines. The results indicate that for the normal and cancer cell lines, BNK and AGS may be a better choice, respectively, for in vitro cancer studies. PMID:27347416

  11. The clustered regularly interspaced short palindromic repeats/associated proteins system for the induction of gene mutations and phenotypic changes in Bombyx mori.

    PubMed

    Song, Jia; Che, Jiaqian; You, Zhengying; Ye, Xiaogang; Li, Jisheng; Ye, Lupeng; Zhang, Yuyu; Qian, Qiujie; Zhong, Boxiong

    2016-12-01

    To probe the general phenomena of gene mutations, Bombyx mori, the lepidopterous model organism, was chosen as the experimental model. To easily detect phenotypic variations, the piggyBac system was utilized to introduce two marker genes into the silkworm, and 23.4% transposition efficiency aided in easily breeding a new strain for the entire experiment. Then, the clustered regularly interspaced short palindromic repeats/an associated protein (Cas9) system was utilized. The results showed that the Cas9 system can induce efficient gene mutations and the base changes could be detected since the G0 individuals in B. mori; and that the mutation rates on different target sites were diverse. Next, the gRNA2-targeted site that generated higher mutation rate was chosen, and the experimental results were enumerated. First, the mutation proportion in G1 generation was 30.1%, and some gene mutations were not inherited from the G0 generation; second, occasionally, base substitutions did not lead to variation in the amino-acid sequence, which decreased the efficiency of phenotypic changes compared with that of genotypic changes. These results laid the foundation for better use of the Cas9 system in silkworm gene editing.

  12. Structural and biochemical analysis of nuclease domain of clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein 3 (Cas3).

    PubMed

    Mulepati, Sabin; Bailey, Scott

    2011-09-09

    RNA transcribed from clustered regularly interspaced short palindromic repeats (CRISPRs) protects many prokaryotes from invasion by foreign DNA such as viruses, conjugative plasmids, and transposable elements. Cas3 (CRISPR-associated protein 3) is essential for this CRISPR protection and is thought to mediate cleavage of the foreign DNA through its N-terminal histidine-aspartate (HD) domain. We report here the 1.8 Å crystal structure of the HD domain of Cas3 from Thermus thermophilus HB8. Structural and biochemical studies predict that this enzyme binds two metal ions at its active site. We also demonstrate that the single-stranded DNA endonuclease activity of this T. thermophilus domain is activated not by magnesium but by transition metal ions such as manganese and nickel. Structure-guided mutagenesis confirms the importance of the metal-binding residues for the nuclease activity and identifies other active site residues. Overall, these results provide a framework for understanding the role of Cas3 in the CRISPR system.

  13. Suppression among alleles encoding nucleotide-binding-leucine-rich repeat resistance proteins interferes with resistance in F1 hybrid and allele-pyramided wheat plants.

    PubMed

    Stirnweis, Daniel; Milani, Samira D; Brunner, Susanne; Herren, Gerhard; Buchmann, Gabriele; Peditto, David; Jordan, Tina; Keller, Beat

    2014-09-01

    The development of high-yielding varieties with broad-spectrum durable disease resistance is the ultimate goal of crop breeding. In plants, immune receptors of the nucleotide-binding-leucine-rich repeat (NB-LRR) class mediate race-specific resistance against pathogen attack. When employed in agriculture this type of resistance is often rapidly overcome by newly adapted pathogen races. The stacking of different resistance genes or alleles in F1 hybrids or in pyramided lines is a promising strategy for achieving more durable resistance. Here, we identify a molecular mechanism which can negatively interfere with the allele-pyramiding approach. We show that pairwise combinations of different alleles of the powdery mildew resistance gene Pm3 in F1 hybrids and stacked transgenic wheat lines can result in suppression of Pm3-based resistance. This effect is independent of the genetic background and solely dependent on the Pm3 alleles. Suppression occurs at the post-translational level, as levels of RNA and protein in the suppressed alleles are unaffected. Using a transient expression system in Nicotiana benthamiana, the LRR domain was identified as the domain conferring suppression. The results of this study suggest that the expression of closely related NB-LRR resistance genes or alleles in the same genotype can lead to dominant-negative interactions. These findings provide a molecular explanation for the frequently observed ineffectiveness of resistance genes introduced from the secondary gene pool into polyploid crop species and mark an important step in overcoming this limitation.

  14. The evolution of armadillos, anteaters and sloths depicted by nuclear and mitochondrial phylogenies: implications for the status of the enigmatic fossil Eurotamandua.

    PubMed

    Delsuc, F; Catzeflis, F M; Stanhope, M J; Douzery, E J

    2001-08-07

    The mammalian order Xenarthra (armadillos, anteaters and sloths) is one of the four major clades of placentals, but it remains poorly studied from the molecular phylogenetics perspective. We present here a study encompassing most of the order's diversity in order to establish xenarthrans' intra-ordinal relationships, discuss the evolution of their morphological characters, search for their extant sister group and specify the timing of their radiation with special emphasis on the status of the controversial fossil Eurotamandua. Sequences of three genes (nuclear exon 28 of the Von Willebrand factor and mitochondrial 12S and 16S rRNAs) are compared for eight of the 13 living genera. Phylogenetic analyses confirm the order's monophyly and that of its three major lineages: armadillos (Cingulata), anteaters (Vermilingua) and sloths ('Tardigrada', renamed in 'Folivora'), and our results strongly support the grouping of hairy xenarthrans (anteaters and sloths) into Pilosa. Within placentals, Afrotheria might be the first lineage to branch off, followed by Xenarthra. The morphological adaptative convergence between New World xenarthrans and Old World pangolins is confirmed. Molecular datings place the early emergence of armadillos around the Cretaceous/Tertiary boundary, followed by the divergence between anteaters and sloths in the Early Eocene era. These Tertiary dates contradict the concept of a very ancient origin of modern xenarthran lineages. They also question the placement of the purported fossil anteater (Eurotamandua) from the Middle Eocene period of Europe with the Vermilingua and instead suggest the independent and convergent evolution of this enigmatic taxon.

  15. The myxoma virus m-t5 ankyrin repeat host range protein is a novel adaptor that coordinately links the cellular signaling pathways mediated by Akt and Skp1 in virus-infected cells.

    PubMed

    Werden, Steven J; Lanchbury, Jerry; Shattuck, Donna; Neff, Chris; Dufford, Max; McFadden, Grant

    2009-12-01

    Most poxviruses express multiple proteins containing ankyrin (ANK) repeats accounting for a large superfamily of related but unique determinants of poxviral tropism. Recently, select members of this novel family of poxvirus proteins have drawn considerable attention for their potential roles in modulating intracellular signaling networks during viral infection. The rabbit-specific poxvirus, myxoma virus (MYXV), encodes four unique ANK repeat proteins, termed M-T5, M148, M149, and M150, all of which include a carboxy-terminal PRANC domain which closely resembles a cellular protein motif called the F-box domain. Here, we show that each MYXV-encoded ANK repeat protein, including M-T5, interacts directly with the Skp1 component of the host SCF ubiquitin ligase complex, and that the binding of M-T5 to cullin 1 is indirect via binding to Skp1 in the host SCF complex. To understand the significance of these virus-host protein interactions, the various binding domains of M-T5 were mapped. The N-terminal ANK repeats I and II were identified as being important for interaction with Akt, whereas the C-terminal PRANC/F-box-like domain was essential for binding to Skp1. We also report that M-T5 can bind Akt and the host SCF complex (via Skp1) simultaneously in MYXV-infected cells. Finally, we report that M-T5 specifically mediates the relocalization of Akt from the nucleus to the cytoplasm during infection with the wild-type MYXV, but not the M-T5 knockout version of the virus. These results indicate that ANK/PRANC proteins play a critical role in reprogramming disparate cellular signaling cascades to establish a new cellular environment more favorable for virus replication.

  16. Nanospring behaviour of ankyrin repeats.

    PubMed

    Lee, Gwangrog; Abdi, Khadar; Jiang, Yong; Michaely, Peter; Bennett, Vann; Marszalek, Piotr E

    2006-03-09

    Ankyrin repeats are an amino-acid motif believed to function in protein recognition; they are present in tandem copies in diverse proteins in nearly all phyla. Ankyrin repeats contain antiparallel alpha-helices that can stack to form a superhelical spiral. Visual inspection of the extrapolated structure of 24 ankyrin-R repeats indicates the possibility of spring-like behaviour of the putative superhelix. Moreover, stacks of 17-29 ankyrin repeats in the cytoplasmic domains of transient receptor potential (TRP) channels have been identified as candidates for a spring that gates mechanoreceptors in hair cells as well as in Drosophila bristles. Here we report that tandem ankyrin repeats exhibit tertiary-structure-based elasticity and behave as a linear and fully reversible spring in single-molecule measurements by atomic force microscopy. We also observe an unexpected ability of unfolded repeats to generate force during refolding, and report the first direct measurement of the refolding force of a protein domain. Thus, we show that one of the most common amino-acid motifs has spring properties that could be important in mechanotransduction and in the design of nanodevices.

  17. Serine 192 in the tiny RS repeat of the adenoviral L4-33K splicing enhancer protein is essential for function and reorganization of the protein to the periphery of viral replication centers.

    PubMed

    Östberg, Sara; Törmänen Persson, Heidi; Akusjärvi, Göran

    2012-11-25

    The adenovirus L4-33K protein is a key regulator involved in the temporal shift from early to late pattern of mRNA expression from the adenovirus major late transcription unit. L4-33K is a virus-encoded alternative splicing factor, which enhances processing of 3' splice sites with a weak sequence context. Here we show that L4-33K expressed from a plasmid is localized at the nuclear margin of uninfected cells. During an infection L4-33K is relocalized to the periphery of E2A-72K containing viral replication centers. We also show that serine 192 in the tiny RS repeat of the conserved carboxy-terminus of L4-33K, which is critical for the splicing enhancer function of L4-33K, is necessary for the nuclear localization and redistribution of the protein to viral replication sites. Collectively, our results show a good correlation between the activity of L4-33K as a splicing enhancer protein and its localization to the periphery of viral replication centers.

  18. Serine 192 in the tiny RS repeat of the adenoviral L4-33K splicing enhancer protein is essential for function and reorganization of the protein to the periphery of viral replication centers

    SciTech Connect

    Oestberg, Sara; Toermaenen Persson, Heidi; Akusjaervi, Goeran

    2012-11-25

    The adenovirus L4-33K protein is a key regulator involved in the temporal shift from early to late pattern of mRNA expression from the adenovirus major late transcription unit. L4-33K is a virus-encoded alternative splicing factor, which enhances processing of 3 Prime splice sites with a weak sequence context. Here we show that L4-33K expressed from a plasmid is localized at the nuclear margin of uninfected cells. During an infection L4-33K is relocalized to the periphery of E2A-72K containing viral replication centers. We also show that serine 192 in the tiny RS repeat of the conserved carboxy-terminus of L4-33K, which is critical for the splicing enhancer function of L4-33K, is necessary for the nuclear localization and redistribution of the protein to viral replication sites. Collectively, our results show a good correlation between the activity of L4-33K as a splicing enhancer protein and its localization to the periphery of viral replication centers.

  19. Unfolding a linker between helical repeats.

    PubMed

    Ortiz, Vanessa; Nielsen, Steven O; Klein, Michael L; Discher, Dennis E

    2005-06-10

    In many multi-repeat proteins, linkers between repeats have little secondary structure and place few constraints on folding or unfolding. However, the large family of spectrin-like proteins, including alpha-actinin, spectrin, and dystrophin, share three-helix bundle, spectrin repeats that appear in crystal structures to be linked by long helices. All of these proteins are regularly subjected to mechanical stress. Recent single molecule atomic force microscopy (AFM) experiments demonstrate not only forced unfolding but also simultaneous unfolding of tandem repeats at finite frequency, which suggests that the contiguous helix between spectrin repeats can propagate a cooperative helix-to-coil transition. Here, we address what happens atomistically to the linker under stress by steered molecular dynamics simulations of tandem spectrin repeats in explicit