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Sample records for repeat protein involved

  1. 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.

  2. XAB2, a novel tetratricopeptide repeat protein involved in transcription-coupled DNA repair and transcription.

    PubMed

    Nakatsu, Y; Asahina, H; Citterio, E; Rademakers, S; Vermeulen, W; Kamiuchi, S; Yeo, J P; Khaw, M C; Saijo, M; Kodo, N; Matsuda, T; Hoeijmakers, J H; Tanaka, K

    2000-11-10

    Nucleotide excision repair is a highly versatile DNA repair system responsible for elimination of a wide variety of lesions from the genome. It is comprised of two subpathways: transcription-coupled repair that accomplishes efficient removal of damage blocking transcription and global genome repair. Recently, the basic mechanism of global genome repair has emerged from biochemical studies. However, little is known about transcription-coupled repair in eukaryotes. Here we report the identification of a novel protein designated XAB2 (XPA-binding protein 2) that was identified by virtue of its ability to interact with XPA, a factor central to both nucleotide excision repair subpathways. The XAB2 protein of 855 amino acids consists mainly of 15 tetratricopeptide repeats. In addition to interacting with XPA, immunoprecipitation experiments demonstrated that a fraction of XAB2 is able to interact with the transcription-coupled repair-specific proteins CSA and CSB as well as RNA polymerase II. Furthermore, antibodies against XAB2 inhibited both transcription-coupled repair and transcription in vivo but not global genome repair when microinjected into living fibroblasts. These results indicate that XAB2 is a novel component involved in transcription-coupled repair and transcription.

  3. 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.

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

    PubMed Central

    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

    2016-01-01

    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 S7cp and S7i were aborted in S7ai/S7cp and S7ai/S7i, respectively, whereas they were normal in both N22 and Dular possessing a neutral allele, S7n. 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 S7ai in heterozygote S7ai/S7cp; sterility occurred in the transformants Cpslo17-S7ai. Our results may provide implications for overcoming hybrid embryo sac sterility in intersubspecific hybrid rice and utilization of hybrid heterosis for cultivated rice improvement. PMID:27182946

  5. 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. Copyright © 2016 by the Genetics Society of America.

  6. 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. © 2015 The Authors.

  7. 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

  8. 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.

  9. 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.

  10. 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

  11. MLT-10 Defines a Family of DUF644 and Proline-rich Repeat Proteins Involved in the Molting Cycle of Caenorhabditis elegans

    PubMed Central

    Meli, Vijaykumar S.; Osuna, Beatriz; Ruvkun, Gary

    2010-01-01

    The molting cycle of nematodes involves the periodic synthesis and removal of a collagen-rich exoskeleton, but the underlying molecular mechanisms are not well understood. Here, we describe the mlt-10 gene of Caenorhabditis elegans, which emerged from a genetic screen for molting-defective mutants sensitized by low cholesterol. MLT-10 defines a large family of nematode-specific proteins comprised of DUF644 and tandem P-X2-L-(S/T)-P repeats. Conserved nuclear hormone receptors promote expression of the mlt-10 gene in the hypodermis whenever the exoskeleton is remade. Further, a MLT-10::mCherry fusion protein is released from the hypodermis to the surrounding matrices and fluids during molting. The fusion protein is also detected in strands near the surface of animals. Both loss-of-function and gain-of-function mutations of mlt-10 impede the removal of old cuticles. However, the substitution mutation mlt-10(mg364), which disrupts the proline-rich repeats, causes the most severe phenotype. Mutations of mlt-10 are also associated with abnormalities in the exoskeleton and improper development of the epidermis. Thus, mlt-10 encodes a secreted protein involved in three distinct but interconnected aspects of the molting cycle. We propose that the molting cycle of C. elegans involves the dynamic assembly and disassembly of MLT-10 and possibly the paralogs of MLT-10. PMID:20335506

  12. 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.

  13. 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

  14. OsPPR6, a pentatricopeptide repeat protein involved in editing and splicing chloroplast RNA, is required for chloroplast biogenesis in rice.

    PubMed

    Tang, Jianpeng; Zhang, Wenwei; Wen, Kai; Chen, Gaoming; Sun, Juan; Tian, Yunlu; Tang, Weijie; Yu, Jun; An, Hongzhou; Wu, Tingting; Kong, Fei; Terzaghi, William; Wang, Chunming; Wan, Jianmin

    2017-08-30

    OsPPR6, a pentatricopeptide repeat protein involved in editing and splicing chloroplast RNA, is required for chloroplast biogenesis in rice. The chloroplast has its own genetic material and genetic system, but it is also regulated by nuclear-encoded genes. However, little is known about nuclear-plastid regulatory mechanisms underlying early chloroplast biogenesis in rice. In this study, we isolated and characterized a mutant, osppr6, that showed early chloroplast developmental defects leading to albino leaves and seedling death. We found that the osppr6 mutant failed to form thylakoid membranes. Using map-based cloning and complementation tests, we determined that OsPPR6 encoded a new Pentatricopeptide Repeat (PPR) protein localized in plastids. In the osppr6 mutants, mRNA levels of plastidic genes transcribed by the plastid-encoded RNA polymerase decreased, while those of genes transcribed by the nuclear-encoded RNA polymerase increased. Western blot analyses validated these expression results. We further investigated plastidic RNA editing and splicing in the osppr6 mutants and found that the ndhB transcript was mis-edited and the ycf3 transcript was mis-spliced. Therefore, we demonstrate that OsPPR6, a PPR protein, regulates early chloroplast biogenesis and participates in editing of ndhB and splicing of ycf3 transcripts in rice.

  15. Mitochondrial Function and Maize Kernel Development Requires Dek2, a Pentatricopeptide Repeat Protein Involved in nad1 mRNA Splicing.

    PubMed

    Qi, Weiwei; Yang, Yang; Feng, Xuzhen; Zhang, Mingliang; Song, Rentao

    2017-01-01

    In flowering plants, many respiration-related proteins are encoded by the mitochondrial genome and the splicing of mitochondrion-encoded messenger RNA (mRNA) involves a complex collaboration with nuclear-encoded proteins. Pentatricopeptide repeat (PPR) proteins have been implicated in these RNA-protein interactions. Maize defective kernel 2 (dek2) is a classic mutant with small kernels and delayed development. Through positional cloning and allelic confirmation, we found Dek2 encodes a novel P-type PPR protein that targets mitochondria. Mitochondrial transcript analysis indicated that dek2 mutation causes reduced splicing efficiency of mitochondrial nad1 intron 1. Mitochondrial complex analysis in dek2 immature kernels showed severe deficiency of complex I assembly. Dramatically up-regulated expression of alternative oxidases (AOXs), transcriptome data, and TEM analysis results revealed that proper splicing of nad1 is critical for mitochondrial functions and inner cristaes morphology. This study indicated that Dek2 is a new PPR protein that affects the splicing of mitochondrial nad1 intron 1 and is required for mitochondrial function and kernel development. Copyright © 2017 by the Genetics Society of America.

  16. 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

  17. Seedling Lethal1, a pentatricopeptide repeat protein lacking an E/E+ or DYW domain in Arabidopsis, is involved in plastid gene expression and early chloroplast development.

    PubMed

    Pyo, Young Jae; Kwon, Kwang-Chul; Kim, Anna; Cho, Myeon Haeng

    2013-12-01

    Chloroplasts are the site of photosynthesis and the biosynthesis of essential metabolites, including amino acids, fatty acids, and secondary metabolites. It is known that many seedling-lethal mutants are impaired in chloroplast function or development, indicating the development of functional chloroplast is essential for plant growth and development. Here, we isolated a novel transfer DNA insertion mutant, dubbed sel1 (for seedling lethal1), that exhibited a pigment-defective and seedling-lethal phenotype with a disrupted pentatricopeptide repeat (PPR) gene. Sequence analysis revealed that SEL1 is a member of the PLS subgroup, which is lacking known E/E(+) or DYW domains at the C terminus, in the PLS subfamily of the PPR protein family containing a putative N-terminal transit peptide and 14 putative PPR or PPR-like motifs. Confocal microscopic analysis showed that the SEL1-green fluorescent protein fusion protein is localized in chloroplasts. Transmission electron microscopic analysis revealed that the sel1 mutant is impaired in the etioplast, as well as in chloroplast development. In sel1 mutants, plastid-encoded proteins involved in photosynthesis were rarely detected due to the lack of the corresponding transcripts. Furthermore, transcript profiles of plastid genes revealed that, in sel1 mutants, the transcript levels of plastid-encoded RNA polymerase-dependent genes were greatly reduced, but those of nuclear-encoded RNA polymerase-dependent genes were increased or not changed. Additionally, the RNA editing of two editing sites of the acetyl-CoA carboxylase beta subunit gene transcripts in the sel1 mutant was compromised, though it is not directly connected with the sel1 mutant phenotype. Our results demonstrate that SEL1 is involved in the regulation of plastid gene expression required for normal chloroplast development.

  18. 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.

  19. 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

  20. 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.

  1. Identifying tandem Ankyrin repeats in protein structures.

    PubMed

    Chakrabarty, Broto; Parekh, Nita

    2014-12-30

    Tandem repetition of structural motifs in proteins is frequently observed across all forms of life. Topology of repeating unit and its frequency of occurrence are associated to a wide range of structural and functional roles in diverse proteins, and defects in repeat proteins have been associated with a number of diseases. It is thus desirable to accurately identify specific repeat type and its copy number. Weak evolutionary constraints on repeat units and insertions/deletions between them make their identification difficult at the sequence level and structure based approaches are desired. The proposed graph spectral approach is based on protein structure represented as a graph for detecting one of the most frequently observed structural repeats, Ankyrin repeat. It has been shown in a large number of studies that 3-dimensional topology of a protein structure is well captured by a graph, making it possible to analyze a complex protein structure as a mathematical entity. In this study we show that eigen spectra profile of a protein structure graph exhibits a unique repetitive profile for contiguous repeating units enabling the detection of the repeat region and the repeat type. The proposed approach uses a non-redundant set of 58 Ankyrin proteins to define rules for the detection of Ankyrin repeat motifs. It is evaluated on a set of 370 proteins comprising 125 known Ankyrin proteins and remaining non-solenoid proteins and the prediction compared with UniProt annotation, sequence-based approach, RADAR, and structure-based approach, ConSole. To show the efficacy of the approach, we analyzed the complete PDB structural database and identified 641 previously unrecognized Ankyrin repeat proteins. We observe a unique eigen spectra profile for different repeat types and show that the method can be easily extended to detect other repeat types. It is implemented as a web server, AnkPred. It is freely available at 'bioinf.iiit.ac.in/AnkPred'. AnkPred provides an elegant and

  2. 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.

  3. Secretion of CyaA-PrtB and HlyA-PrtB fusion proteins in Escherichia coli: involvement of the glycine-rich repeat domain of Erwinia chrysanthemi protease B.

    PubMed Central

    Létoffé, S; Wandersman, C

    1992-01-01

    Protease B from Erwinia chrysanthemi was shown previously to have a C-terminal secretion signal located downstream of a domain that contains six glycine-rich repeats. This domain is conserved in all known bacterial proteins secreted by the signal peptide-independent pathway. The role of these repeats in the secretion process is controversial. We compared the secretion processes of various heterologous polypeptides fused either directly to the signal or separated from it by the glycine-rich domain. Although the repeats are not involved in the secretion of small truncated protease B carboxy-terminal peptides, they are required for the secretion of higher-molecular-weight fusion proteins. Secretion efficiency was also dependent on the size of the passenger polypeptide. Images PMID:1629152

  4. 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.

  5. 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.

  6. 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

  7. 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).

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

    PubMed

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

    2017-01-04

    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. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  9. 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

  10. Exploring the repeat protein universe through computational protein design

    SciTech Connect

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

    2015-12-16

    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. In this paper, 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. Finally, 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.

  11. Exploring the repeat protein universe through computational protein design

    DOE PAGES

    Brunette, TJ; Parmeggiani, Fabio; Huang, Po-Ssu; ...

    2015-12-16

    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. In this paper, 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 unrelatedmore » 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. Finally, 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.« less

  12. 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.

  13. TFIID TAF6-TAF9 complex formation involves the HEAT repeat-containing C-terminal domain of TAF6 and is modulated by TAF5 protein.

    PubMed

    Scheer, Elisabeth; Delbac, Frédéric; Tora, Laszlo; Moras, Dino; Romier, Christophe

    2012-08-10

    The general transcription factor TFIID recognizes specifically the core promoter of genes transcribed by eukaryotic RNA polymerase II, nucleating the assembly of the preinitiation complex at the transcription start site. However, the understanding in molecular terms of TFIID assembly and function remains poorly understood. Histone fold motifs have been shown to be extremely important for the heterodimerization of many TFIID subunits. However, these subunits display several evolutionary conserved noncanonical features when compared with histones, including additional regions whose role is unknown. Here we show that the conserved additional C-terminal region of TFIID subunit TAF6 can be divided into two domains: a small middle domain (TAF6M) and a large C-terminal domain (TAF6C). Our crystal structure of the TAF6C domain from Antonospora locustae at 1.9 Å resolution reveals the presence of five conserved HEAT repeats. Based on these data, we designed several mutants that were introduced into full-length human TAF6. Surprisingly, the mutants affect the interaction between TAF6 and TAF9, suggesting that the formation of the complex between these two TFIID subunits do not only depend on their histone fold motifs. In addition, the same mutants affect even more strongly the interaction between TAF6 and TAF9 in the context of a TAF5-TAF6-TAF9 complex. Expression of these mutants in HeLa cells reveals that most of them are unstable, suggesting their poor incorporation within endogenous TFIID. Taken together, our results suggest that the conserved additional domains in histone fold-containing subunits of TFIID and of co-activator SAGA are important for the assembly of these complexes.

  14. TFIID TAF6-TAF9 Complex Formation Involves the HEAT Repeat-containing C-terminal Domain of TAF6 and Is Modulated by TAF5 Protein*

    PubMed Central

    Scheer, Elisabeth; Delbac, Frédéric; Tora, Laszlo; Moras, Dino; Romier, Christophe

    2012-01-01

    The general transcription factor TFIID recognizes specifically the core promoter of genes transcribed by eukaryotic RNA polymerase II, nucleating the assembly of the preinitiation complex at the transcription start site. However, the understanding in molecular terms of TFIID assembly and function remains poorly understood. Histone fold motifs have been shown to be extremely important for the heterodimerization of many TFIID subunits. However, these subunits display several evolutionary conserved noncanonical features when compared with histones, including additional regions whose role is unknown. Here we show that the conserved additional C-terminal region of TFIID subunit TAF6 can be divided into two domains: a small middle domain (TAF6M) and a large C-terminal domain (TAF6C). Our crystal structure of the TAF6C domain from Antonospora locustae at 1.9 Å resolution reveals the presence of five conserved HEAT repeats. Based on these data, we designed several mutants that were introduced into full-length human TAF6. Surprisingly, the mutants affect the interaction between TAF6 and TAF9, suggesting that the formation of the complex between these two TFIID subunits do not only depend on their histone fold motifs. In addition, the same mutants affect even more strongly the interaction between TAF6 and TAF9 in the context of a TAF5-TAF6-TAF9 complex. Expression of these mutants in HeLa cells reveals that most of them are unstable, suggesting their poor incorporation within endogenous TFIID. Taken together, our results suggest that the conserved additional domains in histone fold-containing subunits of TFIID and of co-activator SAGA are important for the assembly of these complexes. PMID:22696218

  15. Seedling Lethal1, a Pentatricopeptide Repeat Protein Lacking an E/E+ or DYW Domain in Arabidopsis, Is Involved in Plastid Gene Expression and Early Chloroplast Development1[C][W

    PubMed Central

    Pyo, Young Jae; Kwon, Kwang-Chul; Kim, Anna; Cho, Myeon Haeng

    2013-01-01

    Chloroplasts are the site of photosynthesis and the biosynthesis of essential metabolites, including amino acids, fatty acids, and secondary metabolites. It is known that many seedling-lethal mutants are impaired in chloroplast function or development, indicating the development of functional chloroplast is essential for plant growth and development. Here, we isolated a novel transfer DNA insertion mutant, dubbed sel1 (for seedling lethal1), that exhibited a pigment-defective and seedling-lethal phenotype with a disrupted pentatricopeptide repeat (PPR) gene. Sequence analysis revealed that SEL1 is a member of the PLS subgroup, which is lacking known E/E+ or DYW domains at the C terminus, in the PLS subfamily of the PPR protein family containing a putative N-terminal transit peptide and 14 putative PPR or PPR-like motifs. Confocal microscopic analysis showed that the SEL1-green fluorescent protein fusion protein is localized in chloroplasts. Transmission electron microscopic analysis revealed that the sel1 mutant is impaired in the etioplast, as well as in chloroplast development. In sel1 mutants, plastid-encoded proteins involved in photosynthesis were rarely detected due to the lack of the corresponding transcripts. Furthermore, transcript profiles of plastid genes revealed that, in sel1 mutants, the transcript levels of plastid-encoded RNA polymerase-dependent genes were greatly reduced, but those of nuclear-encoded RNA polymerase-dependent genes were increased or not changed. Additionally, the RNA editing of two editing sites of the acetyl-CoA carboxylase beta subunit gene transcripts in the sel1 mutant was compromised, though it is not directly connected with the sel1 mutant phenotype. Our results demonstrate that SEL1 is involved in the regulation of plastid gene expression required for normal chloroplast development. PMID:24144791

  16. Nanostructured functional films from engineered repeat proteins

    PubMed Central

    Grove, Tijana Z.; Regan, Lynne; Cortajarena, Aitziber L.

    2013-01-01

    Fundamental advances in biotechnology, medicine, environment, electronics and energy require methods for precise control of spatial organization at the nanoscale. Assemblies that rely on highly specific biomolecular interactions are an attractive approach to form materials that display novel and useful properties. Here, we report on assembly of films from the designed, rod-shaped, superhelical, consensus tetratricopeptide repeat protein (CTPR). We have designed three peptide-binding sites into the 18 repeat CTPR to allow for further specific and non-covalent functionalization of films through binding of fluorescein labelled peptides. The fluorescence signal from the peptide ligand bound to the protein in the solid film is anisotropic, demonstrating that CTPR films can impose order on otherwise isotropic moieties. Circular dichroism measurements show that the individual protein molecules retain their secondary structure in the film, and X-ray scattering, birefringence and atomic force microscopy experiments confirm macroscopic alignment of CTPR molecules within the film. This work opens the door to the generation of innovative biomaterials with tailored structure and function. PMID:23594813

  17. 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.

  18. 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.

  19. TPRpred: a tool for prediction of TPR-, PPR- and SEL1-like repeats from protein sequences.

    PubMed

    Karpenahalli, Manjunatha R; Lupas, Andrei N; Söding, Johannes

    2007-01-03

    Solenoid repeat proteins of the Tetratrico Peptide Repeat (TPR) family are involved as scaffolds in a broad range of protein-protein interactions. Several resources are available for the prediction of TPRs, however, they often fail to detect divergent repeat units. We have developed TPRpred, a profile-based method which uses a P-value-dependent score offset to include divergent repeat units and which exploits the tendency of repeats to occur in tandem. TPRpred detects not only TPR-like repeats, but also the related Pentatrico Peptide Repeats (PPRs) and SEL1-like repeats. The corresponding profiles were generated through iterative searches, by varying the threshold parameters for inclusion of repeat units into the profiles, and the best profiles were selected based on their performance on proteins of known structure. We benchmarked the performance of TPRpred in detecting TPR-containing proteins and in delineating the individual repeats therein, against currently available resources. TPRpred performs significantly better in detecting divergent repeats in TPR-containing proteins, and finds more individual repeats than the existing methods. The web server is available at http://tprpred.tuebingen.mpg.de, and the C++ and Perl sources of TPRpred along with the profiles can be downloaded from ftp://ftp.tuebingen.mpg.de/ebio/protevo/TPRpred/.

  20. 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

  1. Downregulation of a barley (Hordeum vulgare) leucine-rich repeat, non-arginine-aspartate receptor-like protein kinase reduces expression of numerous genes involved in plant pathogen defense.

    PubMed

    Parrott, David L; Huang, Li; Fischer, Andreas M

    2016-03-01

    Pattern recognition receptors represent a first line of plant defense against pathogens. Comparing the flag leaf transcriptomes of barley (Hordeum vulgare L.) near-isogenic lines varying in the allelic state of a locus controlling senescence, we have previously identified a leucine-rich repeat receptor-like protein kinase gene (LRR-RLK; GenBank accession: AK249842), which was strongly upregulated in leaves of early-as compared to late-senescing germplasm. Bioinformatic analysis indicated that this gene codes for a subfamily XII, non-arginine-aspartate (non-RD) LRR-RLK. Virus-induced gene silencing resulted in a two-fold reduction of transcript levels as compared to controls. Transcriptomic comparison of leaves from untreated plants, from plants treated with virus only without any plant sequences (referred to as 'empty virus' control), and from plants in which AK249842 expression was knocked down identified numerous genes involved in pathogen defense. These genes were strongly induced in 'empty virus' as compared to untreated controls, but their expression was significantly reduced (again compared to 'empty virus' controls) when AK249842 was knocked down, indicating that their expression partially depends on the LRR-RLK investigated here. Expression analysis, using datasets from BarleyBase/PLEXdb, demonstrated that AK249842 transcript levels are heavily influenced by the allelic state of the well-characterized mildew resistance a (Mla) locus, and that the gene is induced after powdery mildew and stem rust infection. Together, our data suggest that AK249842 is a barley pattern recognition receptor with a tentative role in defense against fungal pathogens, setting the stage for its full functional characterization.

  2. 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.

  3. A theoretical model for the mechanical unfolding of repeat proteins.

    PubMed

    Makarov, Dmitrii E

    2009-03-18

    We consider the mechanical stretching of a polypeptide chain formed by multiple interacting repeats. The folding thermodynamics and the interactions among the repeats are described by the Ising model. Unfolded repeats act as soft entropic springs, whereas folded repeats respond to a force as stiffer springs. We show that the resulting force-extension curve may exhibit a pronounced force maximum corresponding to the unfolding of the first repeat. This event is followed by the unfolding of the remaining repeats, which takes place at a lower force. As the protein extension is increased, the force-extension curve of a sufficiently long repeat protein displays a plateau, where the force remains nearly constant and the protein unfolds sequentially so that the number of unfolded repeats is proportional to the extension. Such a sequential mechanical unfolding mechanism is displayed even by the repeat proteins whose thermal denaturation is highly cooperative, provided that they are long enough. By contrast, the unfolding of short repeat progressions can be cooperative.

  4. A Theoretical Model for the Mechanical Unfolding of Repeat Proteins

    PubMed Central

    Makarov, Dmitrii E.

    2009-01-01

    We consider the mechanical stretching of a polypeptide chain formed by multiple interacting repeats. The folding thermodynamics and the interactions among the repeats are described by the Ising model. Unfolded repeats act as soft entropic springs, whereas folded repeats respond to a force as stiffer springs. We show that the resulting force-extension curve may exhibit a pronounced force maximum corresponding to the unfolding of the first repeat. This event is followed by the unfolding of the remaining repeats, which takes place at a lower force. As the protein extension is increased, the force-extension curve of a sufficiently long repeat protein displays a plateau, where the force remains nearly constant and the protein unfolds sequentially so that the number of unfolded repeats is proportional to the extension. Such a sequential mechanical unfolding mechanism is displayed even by the repeat proteins whose thermal denaturation is highly cooperative, provided that they are long enough. By contrast, the unfolding of short repeat progressions can be cooperative. PMID:19289042

  5. 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/.

  6. A novel ankyrin repeat-rich gene in potato, Star, involved in response to late blight.

    PubMed

    Wu, Tian; Tian, Zhendong; Liu, Jun; Yao, Chunguang; Xie, Conghua

    2009-06-01

    The Solanum tuberosum ankyrin repeat gene (Star) is a novel gene from potato leaves challenged by Phytophthora infestans, a pathogen causing late blight disease. The gene was isolated, based on the reported expressed sequence tag, by the rapid amplification of cDNA ends. Star contains a maximum open reading frame of 1542 bp encoding a peptide with 514 amino acids, and it encodes a RING finger ankyrin repeat protein, a putative E3 ubiquitin ligase. To the authors' knowledge, it is the first RING finger ankyrin repeat gene isolated from the potato. The gene is highly expressed in roots, stems, and flowers at the transcript level. Star mRNA was strongly expressed from 24 to 72 h in potato leaves inoculated with P. infestans. The results suggested that Star may be involved in the development of organs and may play a role in late blight resistance.

  7. 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.

  8. 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.

  9. Tandem-repeat protein domains across the tree of life

    PubMed Central

    Jernigan, Kristin K.

    2015-01-01

    Tandem-repeat protein domains, composed of repeated units of conserved stretches of 20–40 amino acids, are required for a wide array of biological functions. Despite their diverse and fundamental functions, there has been no comprehensive assessment of their taxonomic distribution, incidence, and associations with organismal lifestyle and phylogeny. In this study, we assess for the first time the abundance of armadillo (ARM) and tetratricopeptide (TPR) repeat domains across all three domains in the tree of life and compare the results to our previous analysis on ankyrin (ANK) repeat domains in this journal. All eukaryotes and a majority of the bacterial and archaeal genomes analyzed have a minimum of one TPR and ARM repeat. In eukaryotes, the fraction of ARM-containing proteins is approximately double that of TPR and ANK-containing proteins, whereas bacteria and archaea are enriched in TPR-containing proteins relative to ARM- and ANK-containing proteins. We show in bacteria that phylogenetic history, rather than lifestyle or pathogenicity, is a predictor of TPR repeat domain abundance, while neither phylogenetic history nor lifestyle predicts ARM repeat domain abundance. Surprisingly, pathogenic bacteria were not enriched in TPR-containing proteins, which have been associated within virulence factors in certain species. Taken together, this comparative analysis provides a newly appreciated view of the prevalence and diversity of multiple types of tandem-repeat protein domains across the tree of life. A central finding of this analysis is that tandem repeat domain-containing proteins are prevalent not just in eukaryotes, but also in bacterial and archaeal species. PMID:25653910

  10. A General Computational Approach for Repeat Protein Design

    PubMed Central

    Parmeggiani, Fabio; Huang, Po-Ssu; Vorobiev, Sergey; Xiao, Rong; Park, Keunwan; Caprari, Silvia; Su, Min; Jayaraman, Seetharaman; Mao, Lei; Janjua, Haleema; Montelione, Gaetano T.; Hunt, John; Baker, David

    2014-01-01

    Repeat proteins have considerable potential for use as modular binding reagents or biomaterials in biomedical and nanotechnology applications. Here we describe a general computational method for building idealized repeats that integrates available family sequences and structural information with Rosetta de novo protein design calculations. Idealized designs from six different repeat families were generated and experimentally characterized; 80% of the proteins were expressed and soluble and more than 40% were folded and monomeric with high thermal stability. Crystal structures determined for members of three families are within 1 Å root-mean-square deviation to the design models. The method provides a general approach for fast and reliable generation of stable modular repeat protein scaffolds. PMID:25451037

  11. 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

  12. Functional insights from the distribution and role of homopeptide repeat-containing proteins.

    PubMed

    Faux, Noel G; Bottomley, Stephen P; Lesk, Arthur M; Irving, James A; Morrison, John R; de la Banda, Maria Garcia; Whisstock, James C

    2005-04-01

    Expansion of "low complex" repeats of amino acids such as glutamine (Poly-Q) is associated with protein misfolding and the development of degenerative diseases such as Huntington's disease. The mechanism by which such regions promote misfolding remains controversial, the function of many repeat-containing proteins (RCPs) remains obscure, and the role (if any) of repeat regions remains to be determined. Here, a Web-accessible database of RCPs is presented. The distribution and evolution of RCPs that contain homopeptide repeats tracts are considered, and the existence of functional patterns investigated. Generally, it is found that while polyamino acid repeats are extremely rare in prokaryotes, several eukaryote putative homologs of prokaryote RCP-involved in important housekeeping processes-retain the repetitive region, suggesting an ancient origin for certain repeats. Within eukarya, the most common uninterrupted amino acid repeats are glutamine, asparagines, and alanine. Interestingly, while poly-Q repeats are found in vertebrates and nonvertebrates, poly-N repeats are only common in more primitive nonvertebrate organisms, such as insects and nematodes. We have assigned function to eukaryote RCPs using Online Mendelian Inheritance in Man (OMIM), the Human Reference Protein Database (HRPD), FlyBase, and Wormpep. Prokaryote RCPs were annotated using BLASTp searches and Gene Ontology. These data reveal that the majority of RCPs are involved in processes that require the assembly of large, multiprotein complexes, such as transcription and signaling.

  13. 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.

  14. The Analysis of Repeated Measures Designs Involving Multiple Dependent Variables.

    ERIC Educational Resources Information Center

    Schutz, Robert W.; Gessaroli, Marc E.

    1987-01-01

    The article discusses the concepts and interpretations for four methods of testing differences among means in a mixed model repeated measures design. The four methods discussed are: traditional ANOVA and the MANOVA methods for the single dependent variable case, and a Multivariate Mixed Model analysis and a Doubly Multivariate analysis for the…

  15. 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

  16. History Repeats Itself: Parental Involvement in Children's Career Exploration

    ERIC Educational Resources Information Center

    Levine, Kathryn A.; Sutherland, Dawn

    2013-01-01

    Parent involvement in children's education remains one of the most significant predictors for children's academic achievement. This finding generally holds across the range of social group categories including race, culture, class, and family structure. However, relatively little research has been conducted on parental involvement in children's…

  17. History Repeats Itself: Parental Involvement in Children's Career Exploration

    ERIC Educational Resources Information Center

    Levine, Kathryn A.; Sutherland, Dawn

    2013-01-01

    Parent involvement in children's education remains one of the most significant predictors for children's academic achievement. This finding generally holds across the range of social group categories including race, culture, class, and family structure. However, relatively little research has been conducted on parental involvement in children's…

  18. The evolution of filamin-a protein domain repeat perspective.

    PubMed

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

    2012-09-01

    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. Copyright © 2012 Elsevier Inc. All rights reserved.

  19. 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

  20. REPPER—repeats and their periodicities in fibrous proteins

    PubMed Central

    Gruber, Markus; Söding, Johannes; Lupas, Andrei N.

    2005-01-01

    REPPER (REPeats and their PERiodicities) is an integrated server that detects and analyzes regions with short gapless repeats in protein sequences or alignments. It finds periodicities by Fourier Transform (FTwin) and internal similarity analysis (REPwin). FTwin assigns numerical values to amino acids that reflect certain properties, for instance hydrophobicity, and gives information on corresponding periodicities. REPwin uses self-alignments and displays repeats that reveal significant internal similarities. Both programs use a sliding window to ensure that different periodic regions within the same protein are detected independently. FTwin and REPwin are complemented by secondary structure prediction (PSIPRED) and coiled coil prediction (COILS), making the server a versatile analysis tool for sequences of fibrous proteins. REPPER is available at . PMID:15980460

  1. ITN1, a novel gene encoding an ankyrin-repeat protein that affects the ABA-mediated production of reactive oxygen species and is involved in salt-stress tolerance in Arabidopsis thaliana.

    PubMed

    Sakamoto, Hikaru; Matsuda, Osamu; Iba, Koh

    2008-11-01

    Salt stress and abscisic acid (ABA) induce accumulation of reactive oxygen species (ROS) in plant cells. ROS not only act as second messengers for the activation of salt-stress responses, but also have deleterious effects on plant growth due to their cytotoxicity. Therefore, the timing and degree of activation of ROS-producing or ROS-scavenging enzymes must be tightly regulated under salt-stress conditions. We identified a novel locus of Arabidopsis, designated itn1 (increased tolerance to NaCl1), whose disruption leads to increased salt-stress tolerance in vegetative tissues. ITN1 encodes a transmembrane protein with an ankyrin-repeat motif that has been implicated in diverse cellular processes such as signal transduction. Comparative microarray analysis between wild-type and the itn1 mutant revealed that induction of genes encoding the ROS-producing NADPH oxidases (RBOHC and RBOHD) under salt-stress conditions was suppressed in the mutant. This suppression was accompanied by a corresponding reduction in ROS accumulation. The ABA-induced expression of RBOHC and RBOHD was also suppressed in the mutant, as was the case for RD29A, an ABA-inducible marker gene. However, the ABA-induced expression of another marker gene, RD22, was not impaired in the mutant. These results suggest that the itn1 mutation partially impairs ABA signaling pathways, possibly leading to the reduction in ROS accumulation under salt-stress conditions. We discuss the possible mechanisms underlying the salt-tolerant phenotype of the itn1 mutant.

  2. 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.

  3. 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

  4. Protein binding to expanded telomere repeats in Tetrahymena thermophila.

    PubMed

    McGuire, Jennifer M; Gana, Joyce Ache; Petcherskaia, Marina; Kirk, Karen E

    2003-01-01

    The ends of eukaryotic chromosomes are protected by DNA-protein structures called telomeres. Telomeric DNA is highly conserved, usually consisting of long tracts of a repeating G-rich sequence. Tetrahymena thermophila telomeric DNA consists of alternating blocks of GGGG and TT sequences (i.e. a G4T2 repeat sequence). We examined the relative importance of the guanine and thymine elements of the repeat sequence in promoting in vitro binding by T. thermophila proteins. We identified single- and, for the first time, double-stranded telomere binding activities from a crude T. thermophila protein extract and tested the binding of these activities to altered telomere repeat sequences. All deletions or substitutions made to the guanine element virtually abolished binding, indicating that four G's are essential for recognition by the binding activity. However, G's alone are not sufficient for efficient binding, as elimination of the thymine element dramatically reduced binding. By contrast, substantial expansion of the thymine element was well tolerated, even though one such change, G4T4, is lethal in vivo. We tested up to a four-fold expansion of the thymine element and found that highly efficient binding was still achieved. These results suggest a minimal recognition sequence for T. thermophila proteins, with the T element providing an important spacer between essential G elements.

  5. 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.

  6. StaRProtein, A Web Server for Prediction of the Stability of Repeat Proteins

    PubMed Central

    Xu, Yongtao; Zhou, Xu; Huang, Meilan

    2015-01-01

    Repeat proteins have become increasingly important due to their capability to bind to almost any proteins and the potential as alternative therapy to monoclonal antibodies. In the past decade repeat proteins have been designed to mediate specific protein-protein interactions. The tetratricopeptide and ankyrin repeat proteins are two classes of helical repeat proteins that form different binding pockets to accommodate various partners. It is important to understand the factors that define folding and stability of repeat proteins in order to prioritize the most stable designed repeat proteins to further explore their potential binding affinities. Here we developed distance-dependant statistical potentials using two classes of alpha-helical repeat proteins, tetratricopeptide and ankyrin repeat proteins respectively, and evaluated their efficiency in predicting the stability of repeat proteins. We demonstrated that the repeat-specific statistical potentials based on these two classes of repeat proteins showed paramount accuracy compared with non-specific statistical potentials in: 1) discriminate correct vs. incorrect models 2) rank the stability of designed repeat proteins. In particular, the statistical scores correlate closely with the equilibrium unfolding free energies of repeat proteins and therefore would serve as a novel tool in quickly prioritizing the designed repeat proteins with high stability. StaRProtein web server was developed for predicting the stability of repeat proteins. PMID:25807112

  7. 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

  8. 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.

  9. 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.

  10. Characterization of muscle ankyrin repeat proteins in human skeletal muscle.

    PubMed

    Wette, Stefan G; Smith, Heather K; Lamb, Graham D; Murphy, Robyn M

    2017-09-01

    Muscle ankyrin repeat proteins (MARPs) are a family of titin-associated, stress-response molecules and putative transducers of stretch-induced signaling in skeletal muscle. In cardiac muscle, cardiac ankyrin repeat protein (CARP) and diabetes-related ankyrin repeat protein (DARP) reportedly redistribute from binding sites on titin to the nucleus following a prolonged stretch. However, it is unclear whether ankyrin repeat domain protein 2 (Ankrd 2) shows comparable stretch-induced redistribution to the nucleus. We measured the following in rested human skeletal muscle: 1) the absolute amount of MARPs and 2) the distribution of Ankrd 2 and DARP in both single fibers and whole muscle preparations. In absolute amounts, Ankrd 2 is the most abundant MARP in human skeletal muscle, there being ~3.1 µmol/kg, much greater than DARP and CARP (~0.11 and ~0.02 µmol/kg, respectively). All DARP was found to be tightly bound at cytoskeletal (or possibly nuclear) sites. In contrast, ~70% of the total Ankrd 2 is freely diffusible in the cytosol [including virtually all of the phosphorylated (p)Ankrd 2-Ser99 form], ~15% is bound to non-nuclear membranes, and ~15% is bound at cytoskeletal sites, likely at the N2A region of titin. These data are not consistent with the proposal that Ankrd 2, per se, or pAnkrd 2-Ser99 mediates stretch-induced signaling in skeletal muscle, dissociating from titin and translocating to the nucleus, because the majority of these forms of Ankrd 2 are already free in the cytosol. It will be necessary to show that the titin-associated Ankrd 2 is modified by stretch in some as-yet-unidentified way, distinct from the diffusible pool, if it is to act as a stretch-sensitive signaling molecule. Copyright © 2017 the American Physiological Society.

  11. 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

  12. 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

  13. 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.

  14. 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

  15. The evolution and function of protein tandem repeats in plants.

    PubMed

    Schaper, Elke; Anisimova, Maria

    2015-04-01

    Sequence tandem repeats (TRs) are abundant in proteomes across all domains of life. For plants, little is known about their distribution or contribution to protein function. We exhaustively annotated TRs and studied the evolution of TR unit variations for all Ensembl plants. Using phylogenetic patterns of TR units, we detected conserved TRs with unit number and order preserved during evolution, and those TRs that have diverged via recent TR unit gains/losses. We correlated the mode of evolution of TRs to protein function. TR number was strongly correlated with proteome size, with about one-half of all TRs recognized as common protein domains. The majority of TRs have been highly conserved over long evolutionary distances, some since the separation of red algae and green plants c. 1.6 billion yr ago. Conversely, recurrent recent TR unit mutations were rare. Our results suggest that the first TRs by far predate the first plants, and that TR appearance is an ongoing process with similar rates across the plant kingdom. Interestingly, the few detected highly mutable TRs might provide a source of variation for rapid adaptation. In particular, such TRs are enriched in leucine-rich repeats (LRRs) commonly found in R genes, where TR unit gain/loss may facilitate resistance to emerging pathogens. © 2014 The Authors. New Phytologist © 2014 New Phytologist Trust.

  16. 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

  17. 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.

  18. Inferring repeat-protein energetics from evolutionary information.

    PubMed

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

    2017-06-01

    Natural protein sequences contain a record of their history. A common constraint in a given protein family is the ability to fold to specific structures, and it has been shown possible to infer the main native ensemble by analyzing covariations in extant sequences. Still, many natural proteins that fold into the same structural topology show different stabilization energies, and these are often related to their physiological behavior. We propose a description for the energetic variation given by sequence modifications in repeat proteins, systems for which the overall problem is simplified by their inherent symmetry. We explicitly account for single amino acid and pair-wise interactions and treat higher order correlations with a single term. We show that the resulting evolutionary field can be interpreted with structural detail. We trace the variations in the energetic scores of natural proteins and relate them to their experimental characterization. The resulting energetic evolutionary field allows the prediction of the folding free energy change for several mutants, and can be used to generate synthetic sequences that are statistically indistinguishable from the natural counterparts.

  19. 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.

  20. 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.

  1. 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

  2. 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

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

    PubMed

    Albornos, Lucía; Martín, Ignacio; Iglesias, Rebeca; Jiménez, Teresa; Labrador, Emilia; Dopico, Berta

    2012-11-07

    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. 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. 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 described group of 20 to 40

  4. 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

  5. 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.

  6. Notch4-induced inhibition of endothelial sprouting requires the ankyrin repeats and involves signaling through RBP-Jkappa.

    PubMed

    MacKenzie, Farrell; Duriez, Patrick; Larrivée, Bruno; Chang, Linda; Pollet, Ingrid; Wong, Fred; Yip, Calvin; Karsan, Aly

    2004-09-15

    Notch proteins comprise a family of transmembrane receptors. Ligand activation of Notch releases the intracellular domain of the receptor that translocates to the nucleus and regulates transcription through the DNA-binding protein RBP-Jkappa. Previously, it has been shown that the Notch4 intracellular region (N4IC) can inhibit endothelial sprouting and angiogenesis. Here, N4IC deletion mutants were assessed for their ability to inhibit human microvascular endothelial cell (HMEC) sprouting with the use of a quantitative endothelial sprouting assay. Deletion of the ankyrin repeats, but not the RAM (RBP-Jkappa associated module) domain or C-terminal region (CT), abrogated the inhibition of fibroblast growth factor 2 (FGF-2)- and vascular endothelial growth factor (VEGF)-induced sprouting by Notch4, whereas the ankyrin repeats alone partially blocked sprouting. The ankyrin repeats were also the only domain required for up-regulation of RBP-Jkappa-dependent gene expression. Interestingly, enforced expression of the ankyrin domain alone was sufficient to up-regulate some, but not all, RBP-Jkappa-dependent genes. Although N4IC reduced VEGF receptor-2 (VEGFR-2) and vascular endothelial (VE)-cadherin expression, neither of these events is necessary and sufficient to explain N4IC-mediated inhibition of sprouting. A constitutively active RBP-Jkappa mutant significantly inhibited HMEC sprouting but not as strongly as N4IC. Thus, Notch4-induced inhibition of sprouting requires the ankyrin repeats and appears to involve RBP-Jkappa-dependent and -independent signaling.

  7. The WD-repeat protein superfamily in Arabidopsis: conservation and divergence in structure and function

    PubMed Central

    van Nocker, Steven; Ludwig, Philip

    2003-01-01

    Background The WD motif (also known as the Trp-Asp or WD40 motif) is found in a multitude of eukaryotic proteins involved in a variety of cellular processes. Where studied, repeated WD motifs act as a site for protein-protein interaction, and proteins containing WD repeats (WDRs) are known to serve as platforms for the assembly of protein complexes or mediators of transient interplay among other proteins. In the model plant Arabidopsis thaliana, members of this superfamily are increasingly being recognized as key regulators of plant-specific developmental events. Results We analyzed the predicted complement of WDR proteins from Arabidopsis, and compared this to those from budding yeast, fruit fly and human to illustrate both conservation and divergence in structure and function. This analysis identified 237 potential Arabidopsis proteins containing four or more recognizable copies of the motif. These were classified into 143 distinct families, 49 of which contained more than one Arabidopsis member. Approximately 113 of these families or individual proteins showed clear homology with WDR proteins from the other eukaryotes analyzed. Where conservation was found, it often extended across all of these organisms, suggesting that many of these proteins are linked to basic cellular mechanisms. The functional characterization of conserved WDR proteins in Arabidopsis reveals that these proteins help adapt basic mechanisms for plant-specific processes. Conclusions Our results show that most Arabidopsis WDR proteins are strongly conserved across eukaryotes, including those that have been found to play key roles in plant-specific processes, with diversity in function conferred at least in part by divergence in upstream signaling pathways, downstream regulatory targets and /or structure outside of the WDR regions. PMID:14672542

  8. 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

  9. A WD-repeat protein stabilizes ORC binding to chromatin.

    PubMed

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

    2010-10-08

    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. Copyright © 2010 Elsevier Inc. All rights reserved.

  10. Alternative conformations of the Tau repeat domain in complex with an engineered binding protein.

    PubMed

    Grüning, Clara S R; Mirecka, Ewa A; Klein, Antonia N; Mandelkow, Eckhard; Willbold, Dieter; Marino, Stephen F; Stoldt, Matthias; Hoyer, Wolfgang

    2014-08-15

    The aggregation of Tau into paired helical filaments is involved in the pathogenesis of several neurodegenerative diseases, including Alzheimer disease. The aggregation reaction is characterized by conformational conversion of the repeat domain, which partially adopts a cross-β-structure in the resulting amyloid-like fibrils. Here, we report the selection and characterization of an engineered binding protein, β-wrapin TP4, targeting the Tau repeat domain. TP4 was obtained by phage display using the four-repeat Tau construct K18ΔK280 as a target. TP4 binds K18ΔK280 as well as the longest isoform of human Tau, hTau40, with nanomolar affinity. NMR spectroscopy identified two alternative TP4-binding sites in the four-repeat domain, with each including two hexapeptide motifs with high β-sheet propensity. Both binding sites contain the aggregation-determining PHF6 hexapeptide within repeat 3. In addition, one binding site includes the PHF6* hexapeptide within repeat 2, whereas the other includes the corresponding hexapeptide Tau(337-342) within repeat 4, denoted PHF6**. Comparison of TP4-binding with Tau aggregation reveals that the same regions of Tau are involved in both processes. TP4 inhibits Tau aggregation at substoichiometric concentration, demonstrating that it interferes with aggregation nucleation. This study provides residue-level insight into the interaction of Tau with an aggregation inhibitor and highlights the structural flexibility of Tau. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  11. Folding of proteins with WD-repeats: comparison of six members of the WD-repeat superfamily to the G protein beta subunit.

    PubMed

    Garcia-Higuera, I; Fenoglio, J; Li, Y; Lewis, C; Panchenko, M P; Reiner, O; Smith, T F; Neer, E J

    1996-11-05

    The family of WD-repeat proteins comprises over 30 different proteins that share a highly conserved repeating motif [Neer, E. J., Schmidt, C. J., Nambudripad, R., & Smith, T. F. (1994) Nature 371, 297-300]. Members of this family include the signal-transducing G protein beta subunit, as well as other proteins that regulate signal transduction, transcription, pre-mRNA splicing, cytoskeletal organization, and vesicular fusion. The crystal structure of one WD-repeat protein (G beta) has now been solved (Wall et al., 1995; Sondek et al, 1996) and reveals that the seven repeating units form a circular, propeller-like structure with seven blades each made up of four beta strands. It is very likely that all WD-repeat proteins form a similar structure. If so, it will be possible to use information about important surface regions of one family member to predict properties of another. If WD proteins form structures similar to G beta, their hydrodynamic properties should be those of compact, globular proteins, and they should be resistant to cleavage by trypsin. However, the only studied example of a WD-repeat protein, G beta, synthesized in vitro in a rabbit reticulocyte lysate, is unable to fold into a native structure without its partner protein G gamma. The non-WD-repeat amino terminal alpha helix of G beta does not inhibit folding because G beta does not fold even when this region is removed. It is not known whether all WD-repeat proteins are unable to fold when synthesized in an in vitro system. We synthesized seven members of the family in a rabbit reticulocyte lysate, determined their Stokes radius, sedimentation coefficient, and frictional ratio, and assayed their stability to trypsin. Our working definition of folding was that the proteins from globular, trypsin-resistant structures because, except for G beta gamma, their functions are not known or cannot be assayed in reticulocyte lysates. We chose proteins that include amino and carboxyl extensions as well as

  12. Differential interaction and aggregation of 3-repeat and 4-repeat tau isoforms with 14-3-3{zeta} protein

    SciTech Connect

    Sadik, Golam; Tanaka, Toshihisa; Kato, Kiyoko; Yanagi, Kentaro; Kudo, Takashi; Takeda, Masatoshi

    2009-05-22

    Tau isoforms, 3-repeat (3R) and 4-repeat tau (4R), are differentially involved in neuronal development and in several tauopathies. 14-3-3 protein binds to tau and 14-3-3/tau association has been found both in the development and in tauopathies. To understand the role of 14-3-3 in the differential regulation of tau isoforms, we have performed studies on the interaction and aggregation of 3R-tau and 4R-tau, either phosphorylated or unphosphorylated, with 14-3-3{zeta}. We show by surface plasmon resonance studies that the interaction between unphosphorylated 3R-tau and 14-3-3{zeta} is {approx}3-folds higher than that between unphosphorylated 4R-tau and 14-3-3{zeta}. Phosphorylation of tau by protein kinase A (PKA) increases the affinity of both 3R- and 4R-tau for 14-3-3{zeta} to a similar level. An in vitro aggregation assay employing both transmission electron microscopy and fluorescence spectroscopy revealed the aggregation of unphosphorylated 4R-tau to be significantly higher than that of unphosphorylated 3R-tau following the induction of 14-3-3{zeta}. The filaments formed from 3R- and 4R-tau were almost similar in morphology. In contrast, the aggregation of both 3R- and 4R-tau was reduced to a similar low level after phosphorylation with PKA. Taken together, these results suggest that 14-3-3{zeta} exhibits a similar role for tau isoforms after PKA-phosphorylation, but a differential role for unphosphorylated tau. The significant aggregation of 4R-tau by 14-3-3{zeta} suggests that 14-3-3 may act as an inducer in the generation of 4R-tau-predominant neurofibrillary tangles in tauopathies.

  13. Sample size/power calculations for population pharmacodynamic experiments involving repeated-count measurements.

    PubMed

    Ogungbenro, Kayode; Aarons, Leon

    2010-09-01

    Repeated discrete outcome variables such as count measurements often arise in pharmacodynamic experiments. Count measurements can only take nonnegative integer values; this and correlation between repeated measurements from an individual make the design and analysis of repeated-count data special. Sample size/power calculation is an important part of clinical trial design to ensure adequate power for detecting significant effect, and it is often based on the procedure for analysis. This paper describes an approach for calculating sample size/power for population pharmacokinetic/pharmacodynamic experiments involving repeated-count measurements modeled as a Poisson process based on mixed-effects modeling technique. The noncentral version of the Wald chi(2) test is used for testing parameter/treatment significance. The approach was applied to two examples and the results were compared to results obtained from simulations in NONMEM. The first example involves calculating the power of a design to detect parameter significance between two groups: placebo and treatment group. The second example involves characterization of the dose-efficacy relationship of oxybutynin using a mixed-effects modeling approach. Weekly urge urinary incontinence episodes (a discrete count variable) is the primary efficacy variable and is modeled as a Poisson variable. A prospective study based on two different formulations of oxybutynin was designed using published population pharmacokinetic/pharmacodynamic model. The results of simulation studies showed good agreement between the proposed method and NONMEM simulations.

  14. 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.

  15. Predicted structures of two proteins involved in human diseases.

    PubMed

    Zhou, H X; Wang, G

    2001-01-01

    Structures of 79 proteins involved in human diseases were predicted by sequence alignments with structural templates. The predicted structures for ALDP and CSA, proteins responsible for adrenoleukodystrophy and the Cockayne syndrome, respectively, were analyzed to elucidate the molecular basis of disease mutations. In particular we positioned residue P484 of ALDP in the homodimer interface. This positioning is consistent with a recent experimental finding that the mutation P484R significantly decreases the self-interaction of ALDP and suggests that the disease mechanism of this mutation lies in the impaired ALDP dimerization. We identified two new WD repeats in CSA and suggest that one of these forms part of the interaction surface with other proteins.

  16. 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

  17. Two tetratricopeptide repeat proteins facilitate human aryl hydrocarbon receptor signalling in yeast.

    PubMed

    Miller, Charles A

    2002-07-01

    A human aryl hydrocarbon (Ah) receptor signalling pathway was constructed in yeast and used to identify regulatory proteins that may be related to those present in mammalian cells. The sequence similarity of human hepatitis B protein X-associated protein 2 (XAP2) protein to yeast Cpr7 and Cns1 proteins suggested that these proteins might be involved in Ah receptor signalling in this model system. Ah receptor signalling from a lacZ reporter gene was reduced by approximately 60% in cells that lacked Cpr7. In vitro interaction experiments indicated that a Cpr7-GST fusion protein and Ah receptor formed a complex. Expression of Cpr7, Cns1 and the isolated tetratricopeptide repeat (TPR) region of Cpr7 from plasmids restored Ah receptor signalling function in the Cpr7-deficient strain. Thus, Cpr7 and Cns1 proteins facilitate the signalling of human Ah receptor expressed in yeast, perhaps in the same manner as the TPR-containing XAP2 protein and related chaperone proteins in mammalian cells.

  18. Deletion of internal structured repeats increases the stability of a leucine-rich repeat protein, YopM

    PubMed Central

    Barrick, Doug

    2011-01-01

    Mapping the stability distributions of proteins in their native folded states provides a critical link between structure, thermodynamics, and function. Linear repeat proteins have proven more amenable to this kind of mapping than globular proteins. C-terminal deletion studies of YopM, a large, linear leucine-rich repeat (LRR) protein, show that stability is distributed quite heterogeneously, yet a high level of cooperativity is maintained [1]. Key components of this distribution are three interfaces that strongly stabilize adjacent sequences, thereby maintaining structural integrity and promoting cooperativity. To better understand the distribution of interaction energy around these critical interfaces, we studied internal (rather than terminal) deletions of three LRRs in this region, including one of these stabilizing interfaces. Contrary to our expectation that deletion of structured repeats should be destabilizing, we find that internal deletion of folded repeats can actually stabilize the native state, suggesting that these repeats are destabilizing, although paradoxically, they are folded in the native state. We identified two residues within this destabilizing segment that deviate from the consensus sequence at a position that normally forms a stacked leucine ladder in the hydrophobic core. Replacement of these nonconsensus residues with leucine is stabilizing. This stability enhancement can be reproduced in the context of nonnative interfaces, but it requires an extended hydrophobic core. Our results demonstrate that different LRRs vary widely in their contribution to stability, and that this variation is context-dependent. These two factors are likely to determine the types of rearrangements that lead to folded, functional proteins, and in turn, are likely to restrict the pathways available for the evolution of linear repeat proteins. PMID:21764506

  19. 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.

  20. 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.

  1. A combined NMR and computational approach to investigate peptide binding to a designed Armadillo repeat protein.

    PubMed

    Ewald, Christina; Christen, Martin T; Watson, Randall P; Mihajlovic, Maja; Zhou, Ting; Honegger, Annemarie; Plückthun, Andreas; Caflisch, Amedeo; Zerbe, Oliver

    2015-05-22

    The specific recognition of peptide sequences by proteins plays an important role both in biology and in diagnostic applications. Here we characterize the relatively weak binding of the peptide neurotensin (NT) to the previously developed Armadillo repeat protein VG_328 by a multidisciplinary approach based on solution NMR spectroscopy, mutational studies, and molecular dynamics (MD) simulations, totaling 20μs for all MD runs. We describe assignment challenges arising from the repetitive nature of the protein sequence, and we present novel approaches to address them. Partial assignments obtained for VG_328 in combination with chemical shift perturbations allowed us to identify the repeats not involved in binding. Their subsequent elimination resulted in a reduced-size binder with very similar affinity for NT, for which near-complete backbone assignments were achieved. A binding mode suggested by automatic docking and further validated by explicit solvent MD simulations is consistent with paramagnetic relaxation enhancement data collected using spin-labeled NT. Favorable intermolecular interactions are observed in the MD simulations for the residues that were previously shown to contribute to binding in an Ala scan of NT. We further characterized the role of residues within the N-cap for protein stability and peptide binding. Our multidisciplinary approach demonstrates that an initial low-resolution picture for a low-micromolar-peptide binder can be refined through the combination of NMR, protein design, docking, and MD simulations to establish its binding mode, even in the absence of crystallographic data, thereby providing valuable information for further design.

  2. 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.

  3. 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.

  4. Ehrlichia chaffeensis Tandem Repeat Proteins and Ank200 are Type 1 Secretion System Substrates Related to the Repeats-in-Toxin Exoprotein Family

    PubMed Central

    Wakeel, Abdul; den Dulk-Ras, Amke; Hooykaas, Paul J. J.; McBride, Jere W.

    2011-01-01

    Ehrlichia chaffeensis has type 1 and 4 secretion systems (T1SS and T4SS), but the substrates have not been identified. Potential substrates include secreted tandem repeat protein (TRP) 47, TRP120, and TRP32, and the ankyrin repeat protein, Ank200, that are involved in molecular host–pathogen interactions including DNA binding and a network of protein–protein interactions with host targets associated with signaling, transcriptional regulation, vesicle trafficking, and apoptosis. In this study we report that E. chaffeensis TRP47, TRP32, TRP120, and Ank200 were not secreted in the Agrobacterium tumefaciens Cre recombinase reporter assay routinely used to identify T4SS substrates. In contrast, all TRPs and the Ank200 proteins were secreted by the Escherichia coli complemented with the hemolysin secretion system (T1SS), and secretion was reduced in a T1SS mutant (ΔTolC), demonstrating that these proteins are T1SS substrates. Moreover, T1SS secretion signals were identified in the C-terminal domains of the TRPs and Ank200, and a detailed bioinformatic analysis of E. chaffeensis TRPs and Ank200 revealed features consistent with those described in the repeats-in-toxins (RTX) family of exoproteins, including glycine- and aspartate-rich tandem repeats, homology with ATP-transporters, a non-cleavable C-terminal T1SS signal, acidic pIs, and functions consistent with other T1SS substrates. Using a heterologous E. coli T1SS, this investigation has identified the first Ehrlichia T1SS substrates supporting the conclusion that the T1SS and corresponding substrates are involved in molecular host–pathogen interactions that contribute to Ehrlichia pathobiology. Further investigation of the relationship between Ehrlichia TRPs, Ank200, and the RTX exoprotein family may lead to a greater understanding of the importance of T1SS substrates and specific functions of T1SS in the pathobiology of obligately intracellular bacteria. PMID:22919588

  5. 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.

  6. A putative leucine-rich repeat receptor kinase involved in brassinosteroid signal transduction.

    PubMed

    Li, J; Chory, J

    1997-09-05

    Brassinosteroids are a class of growth-promoting regulators that play a key role throughout plant development. Despite their importance, nothing is known of the mechanism of action of these steroid hormones. We describe the identification of 18 Arabidopsis dwarf mutants that are unable to respond to exogenously added brassinosteroid, a phenotype that might be expected for brassinosteroid signaling mutants. All 18 mutations define alleles of a single previously described gene, BRI1. We cloned BRI1 and examined its expression pattern. It encodes a ubiquitously expressed putative receptor kinase. The extracellular domain contains 25 tandem leucine-rich repeats that resemble repeats found in animal hormone receptors, plant disease resistance genes, and genes involved in unknown signaling pathways controlling plant development.

  7. Bap, a Staphylococcus aureus Surface Protein Involved in Biofilm Formation

    PubMed Central

    Cucarella, Carme; Solano, Cristina; Valle, Jaione; Amorena, Beatriz; Lasa, Íñigo; Penadés, José R.

    2001-01-01

    Identification of new genes involved in biofilm formation is needed to understand the molecular basis of strain variation and the pathogenic mechanisms implicated in chronic staphylococcal infections. A biofilm-producing Staphylococcus aureus isolate was used to generate biofilm-negative transposon (Tn917) insertion mutants. Two mutants were found with a significant decrease in attachment to inert surfaces (early adherence), intercellular adhesion, and biofilm formation. The transposon was inserted at the same locus in both mutants. This locus (bap [for biofilm associated protein]) encodes a novel cell wall associated protein of 2,276 amino acids (Bap), which shows global organizational similarities to surface proteins of gram-negative (Pseudomonas aeruginosa and Salmonella enterica serovar Typhi) and gram-positive (Enteroccocus faecalis) microorganisms. Bap's core region represents 52% of the protein and consists of 13 successive nearly identical repeats, each containing 86 amino acids. bap was present in a small fraction of bovine mastitis isolates (5% of the 350 S. aureus isolates tested), but it was absent from the 75 clinical human S. aureus isolates analyzed. All staphylococcal isolates harboring bap were highly adherent and strong biofilm producers. In a mouse infection model bap was involved in pathogenesis, causing a persistent infection. PMID:11292810

  8. Yeast ABC proteins involved in multidrug resistance.

    PubMed

    Piecuch, Agata; Obłąk, Ewa

    2014-03-01

    Pleiotropic drug resistance is a complex phenomenon that involves many proteins that together create a network. One of the common mechanisms of multidrug resistance in eukaryotic cells is the active efflux of a broad range of xenobiotics through ATP-binding cassette (ABC) transporters. Saccharomyces cerevisiae is often used as a model to study such activity because of the functional and structural similarities of its ABC transporters to mammalian ones. Numerous ABC transporters are found in humans and some are associated with the resistance of tumors to chemotherapeutics. Efflux pump modulators that change the activity of ABC proteins are the most promising candidate drugs to overcome such resistance. These modulators can be chemically synthesized or isolated from natural sources (e.g., plant alkaloids) and might also be used in the treatment of fungal infections. There are several generations of synthetic modulators that differ in specificity, toxicity and effectiveness, and are often used for other clinical effects.

  9. 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.

  10. 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.

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

    PubMed Central

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

    2012-01-01

    Expansion of CAG/CTG repeats is the underlying cause of >fourteen 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 as to 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, repair efficiency at CAG/CTG repeats and at control DNA sequences was markedly reduced under the striatal conditions, likely due to the lower level of APE1, FEN1 and LIG1. Damage located towards the 5’ end of the repeat tract was poorly repaired accumulating incompletely processed intermediates as compared to an AP lesion in the centre or at the 3’ end of the repeats or within a control sequences. Moreover, repair of lesions at the 5’ end of CAG or CTG repeats involved multinucleotide synthesis, particularly under the cerebellar stoichiometry, suggesting that long-patch BER processes lesions at sequences susceptible to hairpin formation. Our results show that BER stoichiometry, nucleotide sequence and DNA damage position modulate repair outcome, and suggest that a suboptimal LP-BER activity promotes CAG/CTG repeat instability. PMID:22497302

  12. 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.

  13. 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.

  14. Rational design of α-helical tandem repeat proteins with closed architectures.

    PubMed

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

    2015-12-24

    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 materials. The modular nature of repeat proteins enables the rapid construction and diversification of extended binding surfaces by duplication and recombination of simple building blocks. 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 partners, to closed and compact conformations with internal cavities suitable for small molecule binding and catalysis. 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 α-solenoid repeat structures (α-toroids) in which the inter-repeat packing geometry is constrained so as to juxtapose the amino (N) and carboxy (C) termini; several of these designed structures have been validated by X-ray crystallography. Unlike previous approaches to tandem repeat protein engineering, 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 α-solenoid repeats with a left-handed helical architecture that--to our knowledge--is not yet present in the protein structure database.

  15. 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

  16. Analysis of repeat-protein folding using nearest-neighbor statistical mechanical models

    PubMed Central

    Aksel, Tural; Barrick, Doug

    2010-01-01

    The linear “Ising” model, which has been around for nearly a century, treats the behavior of linear arrays of repetitive, interacting subunits. Linear “repeat-proteins” have only been described in the last decade or so, and their folding energies have only been characterized very recently. Owing to their repetitive structures, linear repeat-proteins are particularly well suited for analysis by the nearest-neighbor Ising formalism. After briefly describing the historical origins and applications of the Ising model to biopolymers, and introducing repeat protein structure, this chapter will focus on the application of the linear Ising model to repeat proteins. When applied to homopolymers, the model can be represented and applied in a fairly simplified form. When applied to heteropolymers, where differences in energies among individual subunits (i.e. repeats) must be included, some (but not all) of this simplicity is lost. Derivations of the linear Ising model for both homopolymer and heteropolymer repeat-proteins will be presented. With the increased complexity required for analysis of heteropolymeric repeat proteins, the ability to resolve different energy terms from experimental data can be compromised. Thus, a simple matrix approach will be developed to help inform on the degree to which different thermodynamic parameters can be extracted from a particular set of unfolding curves. Finally, we will describe the application of these models to analyze repeat-protein folding equilibria, focusing on simplified repeat proteins based on “consensus” sequence information. PMID:19289204

  17. FG repeats facilitate integral protein trafficking to the inner nuclear membrane.

    PubMed

    Kerr, Alastair Rw; Schirmer, Eric C

    2011-09-01

    The mechanism for nucleo-cytoplasmic transport of integral membrane proteins is poorly understood compared to transport of soluble molecules. We recently demonstrated that at least four distinct mechanisms can contribute to transport of integral proteins through the peripheral channels of the nuclear pore complex. One of these requires having multiple phenylalanine-glycine (FG) pairings on the integral protein. It also requires the nuclear pore complex protein Nup35, which separately contains FG repeats. FG-repeats on nuclear pore complex proteins in the central channel have been proposed to interact with FGs on transport receptors to facilitate transport of soluble proteins. Here we show that FG repeats occur quite frequently in both transmembrane and soluble proteins identified in multiple separate proteomic analyses of nuclear envelopes. We postulate that the FG repeats enable these proteins to function as their own transport receptors.

  18. Curve-based multivariate distance matrix regression analysis: application to genetic association analyses involving repeated measures

    PubMed Central

    Salem, Rany M.; O'Connor, Daniel T.

    2010-01-01

    Most, if not all, human phenotypes exhibit a temporal, dosage-dependent, or age effect. Despite this fact, it is rare that data are collected over time or in sequence in relevant studies of the determinants of these phenotypes. The costs and organizational sophistication necessary to collect repeated measurements or longitudinal data for a given phenotype are clearly impediments to this, but greater efforts in this area are needed if insights into human phenotypic expression are to be obtained. Appropriate data analysis methods for genetic association studies involving repeated or longitudinal measures are also needed. We consider the use of longitudinal profiles obtained from fitted functions on repeated data collections from a set of individuals whose similarities are contrasted between sets of individuals with different genotypes to test hypotheses about genetic influences on time-dependent phenotype expression. The proposed approach can accommodate uncertainty of the fitted functions, as well as weighting factors across the time points, and is easily extended to a wide variety of complex analysis settings. We showcase the proposed approach with data from a clinical study investigating human blood vessel response to tyramine. We also compare the proposed approach with standard analytic procedures and investigate its robustness and power via simulation studies. The proposed approach is found to be quite flexible and performs either as well or better than traditional statistical methods. PMID:20423962

  19. 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

  20. 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.

  1. 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.

  2. Leucine-rich Repeat and WD Repeat-containing Protein 1 Is Recruited to Pericentric Heterochromatin by Trimethylated Lysine 9 of Histone H3 and Maintains Heterochromatin Silencing*

    PubMed Central

    Chan, Kui Ming; Zhang, Zhiguo

    2012-01-01

    Lrwd1, a protein containing a leucine-rich repeat and a WD40 repeat domain, interacts with the origin replication complex (ORC), a protein complex involved in both initiation of DNA replication and heterochromatin silencing. Lrwd1 and ORC are known to co-purify with repressive histone marks (trimethylated lysine 9 of histone H3 (H3K9me3) and trimethylated lysine 20 of histone H4 (H4K20me3)) and localize to pericentric heterochromatin. However, how the Lrwd1 is recruited to heterochromatin and the functional significance of the localization of Lrwd1 to the heterochromatin are not known. Here, we show that Lrwd1 preferentially binds to trimethylated repressive histone marks in vitro, which is dependent on an intact WD40 domain but independent of ORC proteins. The localization of Lrwd1 and Orc2 at pericentric heterochromatin in mouse cells is lost in cells lacking H3K9me3 but not in cells lacking H4K20me3. In addition, depletion of HP1α has little impact on the localization of Lrwd1 on pericentric heterochromatin. Finally, depletion of Lrwd1 and Orc2 in mouse cells leads to increased transcription of major satellite repeats. These results indicate that the Lrwd1 is recruited to pericentric heterochromatin through binding to H3K9me3 and that the association of Lrwd1 with pericentric heterochromatin is required for heterochromatin silencing and maintenance. PMID:22427655

  3. 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

  4. RRW: repeated random walks on genome-scale protein networks for local cluster discovery

    PubMed Central

    Macropol, Kathy; Can, Tolga; Singh, Ambuj K

    2009-01-01

    Background We propose an efficient and biologically sensitive algorithm based on repeated random walks (RRW) for discovering functional modules, e.g., complexes and pathways, within large-scale protein networks. Compared to existing cluster identification techniques, RRW implicitly makes use of network topology, edge weights, and long range interactions between proteins. Results We apply the proposed technique on a functional network of yeast genes and accurately identify statistically significant clusters of proteins. We validate the biological significance of the results using known complexes in the MIPS complex catalogue database and well-characterized biological processes. We find that 90% of the created clusters have the majority of their catalogued proteins belonging to the same MIPS complex, and about 80% have the majority of their proteins involved in the same biological process. We compare our method to various other clustering techniques, such as the Markov Clustering Algorithm (MCL), and find a significant improvement in the RRW clusters' precision and accuracy values. Conclusion RRW, which is a technique that exploits the topology of the network, is more precise and robust in finding local clusters. In addition, it has the added flexibility of being able to find multi-functional proteins by allowing overlapping clusters. PMID:19740439

  5. Predominant D1 Receptors Involvement in the Over-expression of CART Peptides after Repeated Cocaine Administration.

    PubMed

    Hu, Zhenzhen; Oh, Eun-Hye; Chung, Yeon Bok; Hong, Jin Tae; Oh, Ki-Wan

    2015-03-01

    The aim of this study was to investigate the involvement of dopaminergic receptors (DR) in behavioral sensitization, as measured by locomotor activity, and the over-expression of cocaine- and amphetamine-regulated transcript (CART) peptides after repeated administration of cocaine in mice. Repeated administrations of cocaine induced behavioral sensitization and CART over-expression in mice. The levels of striatal CART mRNA were significantly increased on the 3(rd) day. CART peptides were over-expressed on the 5(th) day in the striata of behaviorally sensitized mice. A higher proportion of CART(+) cells in the cocaine-treated mice were present in the nucleus accumbens (NAc) shell than in the dorsolateral (DL) part of caudate putamen (CP). The concomitant administration of both D1R and D2R antagonists, SCH 23390 (D1R selective) and raclopride (D2R selective), blocked cocaine induced-behavioral sensitization, CART over-expression, and cyclic adenosine 5'-monophosphate (cAMP)/protein kinase A (PKA)/phospho-cAMP response element-binding protein (pCREB) signal pathways. SCH 23390 more predominantly inhibited the locomotor activity, CART over-expression, pCREB and PKA activity than raclopride. Cocaine induced-behavioral sensitization was also attenuated in the both D1R and D2R knockout (KO) mice, respectively. CART over-expression and activated cAMP/PKA/pCREB signal pathways were inhibited in the D1R-KO mice, but not in the D2R-KO mice. It is suggested that behavioral sensitization, CART over-expression and activated cAMP/PKA/pCREB signal pathways induced by repeated administration of cocaine could be more predominantly mediated by D1R.

  6. Predominant D1 Receptors Involvement in the Over-expression of CART Peptides after Repeated Cocaine Administration

    PubMed Central

    Hu, Zhenzhen; Oh, Eun-Hye; Chung, Yeon Bok; Hong, Jin Tae

    2015-01-01

    The aim of this study was to investigate the involvement of dopaminergic receptors (DR) in behavioral sensitization, as measured by locomotor activity, and the over-expression of cocaine- and amphetamine-regulated transcript (CART) peptides after repeated administration of cocaine in mice. Repeated administrations of cocaine induced behavioral sensitization and CART over-expression in mice. The levels of striatal CART mRNA were significantly increased on the 3rd day. CART peptides were over-expressed on the 5th day in the striata of behaviorally sensitized mice. A higher proportion of CART+ cells in the cocaine-treated mice were present in the nucleus accumbens (NAc) shell than in the dorsolateral (DL) part of caudate putamen (CP). The concomitant administration of both D1R and D2R antagonists, SCH 23390 (D1R selective) and raclopride (D2R selective), blocked cocaine induced-behavioral sensitization, CART over-expression, and cyclic adenosine 5'-monophosphate (cAMP)/protein kinase A (PKA)/phospho-cAMP response element-binding protein (pCREB) signal pathways. SCH 23390 more predominantly inhibited the locomotor activity, CART over-expression, pCREB and PKA activity than raclopride. Cocaine induced-behavioral sensitization was also attenuated in the both D1R and D2R knockout (KO) mice, respectively. CART over-expression and activated cAMP/PKA/pCREB signal pathways were inhibited in the D1R-KO mice, but not in the D2R-KO mice. It is suggested that behavioral sensitization, CART over-expression and activated cAMP/PKA/pCREB signal pathways induced by repeated administration of cocaine could be more predominantly mediated by D1R. PMID:25729269

  7. 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

  8. Leucine-rich-repeat-containing variable lymphocyte receptors as modules to target plant-expressed proteins

    DOE PAGES

    Velásquez, André C.; Nomura, Kinya; Cooper, Max D.; ...

    2017-04-19

    The ability to target and manipulate protein-based cellular processes would accelerate plant research; yet, the technology to specifically and selectively target plant-expressed proteins is still in its infancy. Leucine-rich repeats (LRRs) are ubiquitously present protein domains involved in mediating protein–protein interactions. LRRs confer the binding specificity to the highly diverse variable lymphocyte receptor (VLR) antibodies (including VLRA, VLRB and VLRC types) that jawless vertebrates make as the functional equivalents of jawed vertebrate immunoglobulin-based antibodies. Here, VLRBs targeting an effector protein from a plant pathogen, HopM1, were developed by immunizing lampreys and using yeast surface display to select for high-affinity VLRBs.more » HopM1-specific VLRBs (VLRM1) were expressed in planta in the cytosol, the trans-Golgi network, and the apoplast. Expression of VLRM1 was higher when the protein localized to an oxidizing environment that would favor disulfide bridge formation (when VLRM1 was not localized to the cytoplasm), as disulfide bonds are necessary for proper VLR folding. VLRM1 specifically interacted in planta with HopM1 but not with an unrelated bacterial effector protein while HopM1 failed to interact with a non-specific VLRB. Later, VLRs may be used as flexible modules to bind proteins or carbohydrates of interest in planta, with broad possibilities for their use by binding directly to their targets and inhibiting their action, or by creating chimeric proteins with new specificities in which endogenous LRR domains are replaced by those present in VLRs.« less

  9. 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

  10. 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.

  11. Subcellular localization of WD40 repeat 1 protein in PC12 rat pheochromocytoma cells.

    PubMed

    Shin, Dong Hoon; Lee, Eunju; Chung, Yoon Hee; Mun, Ga Hee; Park, Ji yeong; Lomax, Margaret I; Oh, Seung Ha

    2004-09-09

    The dynamics of actin filament protein is crucial for various physiological processes of the cells. Among the proteins correlating with actin dynamics, a novel 67-kDa WD40 repeat protein 1 (WDR1) was the vertebrate homologue of actin-interacting protein 1 (Aip1). Even though previous studies have provided the clues on the function of WDR1 in specific organs under pathological conditions, the exact subcellular localization of WDR1 is not known. Therefore, in the present study, we undertook to determine the distribution of WDR1 within PC12 pheochromocytoma cells (PC12 cells) using light and electron microscopic techniques. Double immunocytochemistry clearly showed that WDR1 immunoreactivities (IRs) were co-localized with anti-actin antibody, suggesting the involvement of WDR1 in actin dynamics. WDR1 immunoreactivities (IRs) in PC12 cells showed different distribution patterns as nerve growth factor (NGF) concentrations varied. During active proliferation, the distribution of WDR1 IRs seemed to be similar to those found in cortical actin patches, whereas WDR1 IR was observed in cytoplasmic actin cables after PC12 cells were induced to differentiate by treating with NGF. Though further studies are necessary to determine the function of WDR1, the current data represents a first step towards the in vitro study of WDR1 protein.

  12. Life under tension: Computational studies of proteins involved in mechanotransduction

    NASA Astrophysics Data System (ADS)

    Sotomayor, Marcos Manuel

    Living organisms rely on macroscopic and microscopic structures that produce and transform force: from mechanical motion of our muscles and bones to sound transduction and cell volume regulation, handling of forces is essential to life. Investigation of the microscopic machinery behind force generation and transduction in the cell has only become possible with recent advances in x-ray crystallography, nuclear magnetic resonance spectroscopy, single-molecule force spectroscopy, and computer modeling. In this thesis, molecular dynamics simulations have been used to study proteins that transform forces into biochemical signals (mechanotransduction). The first protein studied is the mechanosensitive channel of small conductance MscS. This membrane channel has been proposed to act as a safety valve during osmotic shock, facilitating the release of ions and small solutes upon increase in membrane tension, thereby preventing bacterial cells from bursting. The second set of proteins studied are ankyrin and cadherin repeats, likely forming part of the transduction apparatus in hearing and other mechanical senses. Simulations of all these proteins went beyond the standard approach in which only equilibrium properties are monitored; we adopted and developed strategies in which external electric fields and forces are used to probe their response and function and at the same time produce verifiable predictions. The outcome of the simulations performed on MscS, in close collaborations with experimentalists, allowed us to establish conduction properties of different conformations and propose structural models of MscS's open and closed states. Simulations of ankyrin and cadherin repeats focused on their elastic properties, resulting in the discovery and prediction of ankyrin's tertiary and secondary structure elasticity (later on corroborated by atomic force microscopy experiments), and the discovery of a novel form of secondary structure elasticity mediated by calcium ions in

  13. The pentatricopeptide repeat MTSF1 protein stabilizes the nad4 mRNA in Arabidopsis mitochondria

    PubMed Central

    Haïli, Nawel; Arnal, Nadège; Quadrado, Martine; Amiar, Souad; Tcherkez, Guillaume; Dahan, Jennifer; Briozzo, Pierre; Colas des Francs-Small, Catherine; Vrielynck, Nathalie; Mireau, Hakim

    2013-01-01

    Gene expression in plant mitochondria involves a complex collaboration of transcription initiation and termination, as well as subsequent mRNA processing to produce mature mRNAs. In this study, we describe the function of the Arabidopsis mitochondrial stability factor 1 (MTSF1) gene and show that it encodes a pentatricopeptide repeat protein essential for the 3′-processing of mitochondrial nad4 mRNA and its stability. The nad4 mRNA is highly destabilized in Arabidopsis mtsf1 mutant plants, which consequently accumulates low amounts of a truncated form of respiratory complex I. Biochemical and genetic analyses demonstrated that MTSF1 binds with high affinity to the last 20 nucleotides of nad4 mRNA. Our data support a model for MTSF1 functioning in which its association with the last nucleotides of the nad4 3′ untranslated region stabilizes nad4 mRNA. Additionally, strict conservation of the MTSF1-binding sites strongly suggests that the protective function of MTSF1 on nad4 mRNA is conserved in dicots. These results demonstrate that the mRNA stabilization process initially identified in plastids, whereby proteins bound to RNA extremities constitute barriers to exoribonuclease progression occur in plant mitochondria to protect and concomitantly define the 3′ end of mature mitochondrial mRNAs. Our study also reveals that short RNA molecules corresponding to pentatricopeptide repeat-binding sites accumulate also in plant mitochondria. PMID:23658225

  14. Yeast telomere repeat sequence (TRS) improves circular plasmid segregation, and TRS plasmid segregation involves the RAP1 gene product.

    PubMed Central

    Longtine, M S; Enomoto, S; Finstad, S L; Berman, J

    1992-01-01

    Telomere repeat sequences (TRSs) can dramatically improve the segregation of unstable circular autonomously replicating sequence (ARS) plasmids in Saccharomyces cerevisiae. Deletion analysis demonstrated that yeast TRSs, which conform to the general sequence (C(1-3)A)n, are able to stabilize circular ARS plasmids. A number of TRS clones of different primary sequence and C(1-3)A tract length confer the plasmid stabilization phenotype. TRS sequences do not appear to improve plasmid replication efficiency, as determined by plasmid copy number analysis and functional assays for ARS activity. Pedigree analysis confirms that TRS-containing plasmids are missegregated at low frequency and that missegregated TRS-containing plasmids, like ARS plasmids, are preferentially retained by the mother cell. Plasmids stabilized by TRSs have properties that distinguish them from centromere-containing plasmids and 2 microns-based recombinant plasmids. Linear ARS plasmids, which include two TRS tracts at their termini, segregate inefficiently, while circular plasmids with one or two TRS tracts segregate efficiently, suggesting that plasmid topology or TRS accessibility interferes with TRS segregation function on linear plasmids. In strains carrying the temperature-sensitive mutant alleles rap1grc4 and rap1-5, TRS plasmids are not stable at the semipermissive temperature, suggesting that RAP1 protein is involved in TRS plasmid stability. In Schizosaccharomyces pombe, an ARS plasmid was stabilized by the addition of S. pombe telomere sequence, suggesting that the ability to improve the segregation of ARS plasmids is a general property of telomere repeats. PMID:1569937

  15. Identification of long-lived proteins retained in cells undergoing repeated asymmetric divisions

    PubMed Central

    Thayer, Nathaniel H.; Leverich, Christina K.; Fitzgibbon, Matthew P.; Nelson, Zara W.; Henderson, Kiersten A.; Gafken, Philip R.; Hsu, Jessica J.; Gottschling, Daniel E.

    2014-01-01

    Long-lived proteins have been implicated in age-associated decline in metazoa, but they have only been identified in extracellular matrices or postmitotic cells. However, the aging process also occurs in dividing cells undergoing repeated asymmetric divisions. It was not clear whether long-lived proteins exist in asymmetrically dividing cells or whether they are involved in aging. Here we identify long-lived proteins in dividing cells during aging using the budding yeast, Saccharomyces cerevisiae. Yeast mother cells undergo a limited number of asymmetric divisions that define replicative lifespan. We used stable-isotope pulse-chase and total proteome mass-spectrometry to identify proteins that were both long-lived and retained in aging mother cells after ∼18 cells divisions. We identified ∼135 proteins that we designate as long-lived asymmetrically retained proteins (LARPS). Surprisingly, the majority of LARPs appeared to be stable fragments of their original full-length protein. However, 15% of LARPs were full-length proteins and we confirmed several candidates to be long-lived and retained in mother cells by time-lapse microscopy. Some LARPs localized to the plasma membrane and remained robustly in the mother cell upon cell division. Other full-length LARPs were assembled into large cytoplasmic structures that had a strong bias to remain in mother cells. We identified age-associated changes to LARPs that include an increase in their levels during aging because of their continued synthesis, which is not balanced by turnover. Additionally, several LARPs were posttranslationally modified during aging. We suggest that LARPs contribute to age-associated phenotypes and likely exist in other organisms. PMID:25228775

  16. X-linked late-onset sensorineural deafness caused by a deletion involving OA1 and a novel gene containing WD-40 repeats.

    PubMed Central

    Bassi, M T; Ramesar, R S; Caciotti, B; Winship, I M; De Grandi, A; Riboni, M; Townes, P L; Beighton, P; Ballabio, A; Borsani, G

    1999-01-01

    We have identified a novel gene, transducin (beta)-like 1 (TBL1), in the Xp22.3 genomic region, that shows high homology with members of the WD-40-repeat protein family. The gene contains 18 exons spanning approximately 150 kb of the genomic region adjacent to the ocular albinism gene (OA1) on the telomeric side. However, unlike OA1, TBL1 is transcribed from telomere to centromere. Northern analysis indicates that TBL1 is ubiquitously expressed, with two transcripts of approximately 2.1 kb and 6.0 kb. The open reading frame encodes a 526-amino acid protein, which shows the presence of six beta-transducin repeats (WD-40 motif) in the C-terminal domain. The homology with known beta-subunits of G proteins and other WD-40-repeat containing proteins is restricted to the WD-40 motif. Genomic analysis revealed that the gene is either partly or entirely deleted in patients carrying Xp22.3 terminal deletions. The complexity of the contiguous gene-syndrome phenotype shared by these patients depends on the number of known disease genes involved in the deletions. Interestingly, one patient carrying a microinterstitial deletion involving the 3' portion of both TBL1 and OA1 shows the OA1 phenotype associated with X-linked late-onset sensorineural deafness. We postulate an involvement of TBL1 in the pathogenesis of the ocular albinism with late-onset sensorineural deafness phenotype. PMID:10330347

  17. 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.

  18. 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

  19. 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.

  20. Neural circuitry involved in quitting after repeated failures: role of the cingulate and temporal parietal junction

    PubMed Central

    Zhao, Weihua; Kendrick, Keith M; Chen, Fei; Li, Hong; Feng, Tingyong

    2016-01-01

    The more times people fail the more likely they are to give up, however little is known about the neural mechanisms underlying this impact of repeated failure on decision making. Here we have used a visual shape discrimination task with computer-controlled feedback combined with functional magnetic resonance imaging (fMRI) to investigate the neural circuits involved. The behavioral task confirmed that the more times subjects experienced failure the more likely they were to give up, with three successive failures being the key threshold and the majority of subjects reaching the point where they decided to quit and try a new stimulus set after three or four failures. The fMRI analysis revealed activity changes in frontal, parietal, temporal, limbic and striatal regions, especially anterior cingulate cortex (ACC), posterior cingulate cortex (PCC) and temporal parietal junction (TPJ) associated with the number of previous failures experienced. Furthermore, their parameter estimates were predictive of subjects’ quitting rate. Thus, subjects reach the point where they decide to quit after three/four failures and this is associated with differential changes in brain regions involved in error monitoring and reward which regulate both failure detection and changes in decision-making strategy. PMID:27097529

  1. Molecular characterization and analysis of a gene encoding the acidic repeat protein (Arp) of Treponema pallidum.

    PubMed

    Liu, Hsi; Rodes, Berta; George, Robert; Steiner, Bret

    2007-06-01

    The acidic repeat protein (arp) genes from three subspecies of the treponeme Treponema pallidum (T. pallidum subsp. pallidum, Nichols strain; T. pallidum subsp. pertenue, CDC-1 and CDC-2 strains; and T. pallidum subsp. endemicum, Bosnia A strain) were cloned and sequenced. The predicted protein sequence contained a high percentage of glutamic acid, hence the name acidic repeat protein, or Arp. The protein had a potential membrane-spanning domain and a signal peptidase I site. The gene from the Nichols strain of T. pallidum subsp. pallidum contained a set of 14 nearly identical repeats of a 60 bp sequence, which occupied approximately 51 % of the length of the gene. Analyses of arp from laboratory strains showed that the 5' and 3' ends of the genes were conserved, but there was considerable heterogeneity in the number of repeats of this 60 bp sequence. Based on amino acid variations, the 14 sequence repeats could be classified into three types, which were named type I, type II and type III repeats. The type II repeat was the most common in the strains examined. The arp gene of the Nichols strain was subsequently cloned into the expression vector pBAD/TOPO ThioFusion. The expressed protein was detected in a Western blot assay using rabbit immune sera produced against T. pallidum, or synthetic peptides derived from the repeat sequences. Using an ELISA, rapid plasma reagin (RPR) test-positive sera reacted with synthetic peptides derived from the repeat region but not with peptides derived from N and C termini of the Arp protein. These results show that the Arp protein is immunogenic and could prove to be a useful target for serological diagnosis of T. pallidum infection.

  2. 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.

  3. 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

  4. 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

  5. Constraints and consequences of the emergence of amino acid repeats in eukaryotic proteins.

    PubMed

    Chavali, Sreenivas; Chavali, Pavithra L; Chalancon, Guilhem; de Groot, Natalia Sanchez; Gemayel, Rita; Latysheva, Natasha S; Ing-Simmons, Elizabeth; Verstrepen, Kevin J; Balaji, Santhanam; Babu, M Madan

    2017-09-01

    Proteins with amino acid homorepeats have the potential to be detrimental to cells and are often associated with human diseases. Why, then, are homorepeats prevalent in eukaryotic proteomes? In yeast, homorepeats are enriched in proteins that are essential and pleiotropic and that buffer environmental insults. The presence of homorepeats increases the functional versatility of proteins by mediating protein interactions and facilitating spatial organization in a repeat-dependent manner. During evolution, homorepeats are preferentially retained in proteins with stringent proteostasis, which might minimize repeat-associated detrimental effects such as unregulated phase separation and protein aggregation. Their presence facilitates rapid protein divergence through accumulation of amino acid substitutions, which often affect linear motifs and post-translational-modification sites. These substitutions may result in rewiring protein interaction and signaling networks. Thus, homorepeats are distinct modules that are often retained in stringently regulated proteins. Their presence facilitates rapid exploration of the genotype-phenotype landscape of a population, thereby contributing to adaptation and fitness.

  6. 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.

  7. Ab initio detection of fuzzy amino acid tandem repeats in protein sequences

    PubMed Central

    2012-01-01

    Background Tandem repetitions within protein amino acid sequences often correspond to regular secondary structures and form multi-repeat 3D assemblies of varied size and function. Developing internal repetitions is one of the evolutionary mechanisms that proteins employ to adapt their structure and function under evolutionary pressure. While there is keen interest in understanding such phenomena, detection of repeating structures based only on sequence analysis is considered an arduous task, since structure and function is often preserved even under considerable sequence divergence (fuzzy tandem repeats). Results In this paper we present PTRStalker, a new algorithm for ab-initio detection of fuzzy tandem repeats in protein amino acid sequences. In the reported results we show that by feeding PTRStalker with amino acid sequences from the UniProtKB/Swiss-Prot database we detect novel tandemly repeated structures not captured by other state-of-the-art tools. Experiments with membrane proteins indicate that PTRStalker can detect global symmetries in the primary structure which are then reflected in the tertiary structure. Conclusions PTRStalker is able to detect fuzzy tandem repeating structures in protein sequences, with performance beyond the current state-of-the art. Such a tool may be a valuable support to investigating protein structural properties when tertiary X-ray data is not available. PMID:22536906

  8. Cytosolic events involved in chloroplast protein targeting.

    PubMed

    Lee, Dong Wook; Jung, Chanjin; Hwang, Inhwan

    2013-02-01

    Chloroplasts are unique organelles that are responsible for photosynthesis. Although chloroplasts contain their own genome, the majority of chloroplast proteins are encoded by the nuclear genome. These proteins are transported to the chloroplasts after translation in the cytosol. Chloroplasts contain three membrane systems (outer/inner envelope and thylakoid membranes) that subdivide the interior into three soluble compartments known as the intermembrane space, stroma, and thylakoid lumen. Several targeting mechanisms are required to deliver proteins to the correct chloroplast membrane or soluble compartment. These mechanisms have been extensively studied using purified chloroplasts in vitro. Prior to targeting these proteins to the various compartments of the chloroplast, they must be correctly sorted in the cytosol. To date, it is not clear how these proteins are sorted in the cytosol and then targeted to the chloroplasts. Recently, the cytosolic carrier protein AKR2 and its associated cofactor Hsp17.8 for outer envelope membrane proteins of chloroplasts were identified. Additionally, a mechanism for controlling unimported plastid precursors in the cytosol has been discovered. This review will mainly focus on recent findings concerning the possible cytosolic events that occur prior to protein targeting to the chloroplasts. This article is part of a Special Issue entitled: Protein Import and Quality Control in Mitochondria and Plastids. Copyright © 2012 Elsevier B.V. All rights reserved.

  9. 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

  10. Van der Waals Interactions Involving Proteins

    NASA Technical Reports Server (NTRS)

    Roth, Charles M.; Neal, Brian L.; Lenhoff, Abraham M.

    1996-01-01

    Van der Waals (dispersion) forces contribute to interactions of proteins with other molecules or with surfaces, but because of the structural complexity of protein molecules, the magnitude of these effects is usually estimated based on idealized models of the molecular geometry, e.g., spheres or spheroids. The calculations reported here seek to account for both the geometric irregularity of protein molecules and the material properties of the interacting media. Whereas the latter are found to fall in the generally accepted range, the molecular shape is shown to cause the magnitudes of the interactions to differ significantly from those calculated using idealized models. with important consequences. First, the roughness of the molecular surface leads to much lower average interaction energies for both protein-protein and protein-surface cases relative to calculations in which the protein molecule is approximated as a sphere. These results indicate that a form of steric stabilization may be an important effect in protein solutions. Underlying this behavior is appreciable orientational dependence, one reflection of which is that molecules of complementary shape are found to exhibit very strong attractive dispersion interactions. Although this has been widely discussed previously in the context of molecular recognition processes, the broader implications of these phenomena may also be important at larger molecular separations, e.g., in the dynamics of aggregation, precipitation, and crystal growth.

  11. Van der Waals interactions involving proteins.

    PubMed Central

    Roth, C M; Neal, B L; Lenhoff, A M

    1996-01-01

    Van der Waals (dispersion) forces contribute to interactions of proteins with other molecules or with surfaces, but because of the structural complexity of protein molecules, the magnitude of these effects is usually estimated based on idealized models of the molecular geometry, e.g., spheres or spheroids. The calculations reported here seek to account for both the geometric irregularity of protein molecules and the material properties of the interacting media. Whereas the latter are found to fall in the generally accepted range, the molecular shape is shown to cause the magnitudes of the interactions to differ significantly from those calculated using idealized models, with important consequences. First, the roughness of the molecular surface leads to much lower average interaction energies for both protein-protein and protein-surface cases relative to calculations in which the protein molecule is approximated as a sphere. These results indicate that a form of steric stabilization may be an important effect in protein solutions. Underlying this behavior is appreciable orientational dependence, one reflection of which is that molecules of complementary shape are found to exhibit very strong attractive dispersion interactions. Although this has been widely discussed previously in the context of molecular recognition processes, the broader implications of these phenomena may also be important at larger molecular separations, e.g., in the dynamics of aggregation, precipitation, and crystal growth. Images FIGURE 3 PMID:8789115

  12. The design and structural characterization of a synthetic pentatricopeptide repeat protein.

    PubMed

    Gully, Benjamin S; Shah, Kunal R; Lee, Mihwa; Shearston, Kate; Smith, Nicole M; Sadowska, Agata; Blythe, Amanda J; Bernath-Levin, Kalia; Stanley, Will A; Small, Ian D; Bond, Charles S

    2015-02-01

    Proteins of the pentatricopeptide repeat (PPR) superfamily are characterized by tandem arrays of a degenerate 35-amino-acid α-hairpin motif. PPR proteins are typically single-stranded RNA-binding proteins with essential roles in organelle biogenesis, RNA editing and mRNA maturation. A modular, predictable code for sequence-specific binding of RNA by PPR proteins has recently been revealed, which opens the door to the de novo design of bespoke proteins with specific RNA targets, with widespread biotechnological potential. Here, the design and production of a synthetic PPR protein based on a consensus sequence and the determination of its crystal structure to 2.2 Å resolution are described. The crystal structure displays helical disorder, resulting in electron density representing an infinite superhelical PPR protein. A structural comparison with related tetratricopeptide repeat (TPR) proteins, and with native PPR proteins, reveals key roles for conserved residues in directing the structure and function of PPR proteins. The designed proteins have high solubility and thermal stability, and can form long tracts of PPR repeats. Thus, consensus-sequence synthetic PPR proteins could provide a suitable backbone for the design of bespoke RNA-binding proteins with the potential for high specificity.

  13. 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.

  14. 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.

  15. The involvement of collagen triple helix repeat containing 1 in muscular dystrophies.

    PubMed

    Spector, Itai; Zilberstein, Yael; Lavy, Adi; Genin, Olga; Barzilai-Tutsch, Hila; Bodanovsky, Ana; Halevy, Orna; Pines, Mark

    2013-03-01

    Fibrosis is the main complication of muscular dystrophies. We identified collagen triple helix repeat containing 1 (Cthrc1) in skeletal and cardiac muscles of mice, representing Duchenne and congenital muscle dystrophies (DMD and CMD, respectively), and dysferlinopathy. In all of the mice, Cthrc1 was associated with high collagen type I levels; no Cthrc1 or collagen was observed in muscles of control mice. High levels of Cthrc1 were also observed in biopsy specimens from patients with DMD, in whom they were reversibly correlated with that of β-dystroglycan, whereas collagen type I levels were elevated in all patients with DMD. At the muscle sites where collagen and Cthrc1 were adjacent, collagen fibers appeared smaller, suggesting involvement of Cthrc1 in collagen turnover. Halofuginone, an inhibitor of Smad3 phosphorylation downstream of the transforming growth factor-β signaling, reduced Cthrc1 levels in skeletal and cardiac muscles of mice, representing DMD, CMD, and dysferlinopathy. The myofibroblasts infiltrating the dystrophic muscles of the murine models of DMD, CMD, and dysferlinopathy were the source of Cthrc1. Transforming growth factor-β did not affect Cthrc1 levels in the mdx fibroblasts but decreased them in the control fibroblasts, in association with increased migration of mdx fibroblasts and dystrophic muscle invasion by myofibroblasts. To our knowledge, this is the first demonstration of Cthrc1 as a marker of the severity of the disease progression in the dystrophic muscles, and as a possible target for therapy.

  16. 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.

  17. Multiple intermediates on the energy landscape of a 15-HEAT-repeat protein

    PubMed Central

    Tsytlonok, Maksym; Craig, Patricio O.; Sivertsson, Elin; Serquera, David; Perrett, Sarah; Best, Robert B.; Wolynes, Peter G.; Itzhaki, Laura S.

    2014-01-01

    Repeat proteins are a special class of modular, non-globular proteins composed of small structural motifs arrayed to form elongated architectures and stabilised solely by short-range contacts. We find a remarkable complexity in the unfolding of the large HEAT repeat protein PR65/A. In contrast to what has been seen for small repeat proteins in which unfolding propagates from one end, the HEAT array of PR65/A ruptures at multiple distant sites, leading to intermediate states with non-contiguous folded subdomains. Kinetic analysis allows us to define a network of intermediates and to delineate the pathways that connect them. There is a dominant sequence of unfolding, reflecting a non-uniform distribution of stability across the repeat array; however the unfolding of certain intermediates is competitive, leading to parallel pathways. Theoretical models accounting for the heterogeneous contact density in the folded structure are able to rationalize the variation in stability across the array. This variation in stability also suggests how folding may direct function in a large repeat protein: The stability distribution enables certain regions to present rigid motifs for molecular recognition while affording others flexibility to broaden the search area as in a fly-casting mechanism. Thus PR65/A uses the two ends of the repeat array to bind diverse partners and thereby coordinate the dephosphorylation of many different substrates and of multiple sites within hyperphosphorylated substrates. PMID:24120762

  18. Direct Observation of Parallel Folding Pathways Revealed Using a Symmetric Repeat Protein System

    PubMed Central

    Aksel, Tural; Barrick, Doug

    2014-01-01

    Although progress has been made to determine the native fold of a polypeptide from its primary structure, the diversity of pathways that connect the unfolded and folded states has not been adequately explored. Theoretical and computational studies predict that proteins fold through parallel pathways on funneled energy landscapes, although experimental detection of pathway diversity has been challenging. Here, we exploit the high translational symmetry and the direct length variation afforded by linear repeat proteins to directly detect folding through parallel pathways. By comparing folding rates of consensus ankyrin repeat proteins (CARPs), we find a clear increase in folding rates with increasing size and repeat number, although the size of the transition states (estimated from denaturant sensitivity) remains unchanged. The increase in folding rate with chain length, as opposed to a decrease expected from typical models for globular proteins, is a clear demonstration of parallel pathways. This conclusion is not dependent on extensive curve-fitting or structural perturbation of protein structure. By globally fitting a simple parallel-Ising pathway model, we have directly measured nucleation and propagation rates in protein folding, and have quantified the fluxes along each path, providing a detailed energy landscape for folding. This finding of parallel pathways differs from results from kinetic studies of repeat-proteins composed of sequence-variable repeats, where modest repeat-to-repeat energy variation coalesces folding into a single, dominant channel. Thus, for globular proteins, which have much higher variation in local structure and topology, parallel pathways are expected to be the exception rather than the rule. PMID:24988356

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

    USDA-ARS?s Scientific Manuscript database

    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...

  20. 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

  1. Solute carriers involved in energy transfer of mitochondria form a homologous protein family.

    PubMed

    Aquila, H; Link, T A; Klingenberg, M

    1987-02-09

    The sequences of three mitochondrial carriers involved in energy transfer, the ADP/ATP carrier, phosphate carrier and uncoupling carrier, are analyzed. Similarly to what has been previously reported for the ADP/ATP carrier and the uncoupling protein, now also the phosphate carrier is found to have a tripartite structure comprising three similar repeats of approx. 100 residues each. The three sequences show a fair overall homology with each other. More significant homologies are found by comparing the repeats within and between the carriers in a scheme where the sequences are spliced into repeats, which are arranged for maximum homology by allowing possible insertions or deletions. A striking conservation of critical residues, glycine, proline, of charged and of aromatic residues is found throughout all nine repeats. This is indicative of a similar structural principle in the repeats. Hydropathy profiles of the three proteins and a search for amphipathic alpha-spans reveal six membrane-spanning segments for each carrier, providing further support for the basic structural identity of the repeats. The proposed folding pattern of the carriers in the membrane is exemplified with the phosphate carrier. A possible tertiary arrangement of the repeats and the membrane-spanning helices is shown. The emergence of a mitochondrial carrier family by triplication and by divergent evolution from a common gene of about 100 residues is discussed.

  2. Mitogen-activated protein kinase is required for the behavioural desensitization that occurs after repeated injections of angiotensin II.

    PubMed

    Vento, Peter J; Daniels, Derek

    2012-12-01

    Angiotensin II (Ang II) acts on central angiotensin type 1 (AT(1)) receptors to increase water and saline intake. Prolonged exposure to Ang II in cell culture models results in a desensitization of the AT(1) receptor that is thought to involve receptor internalization, and a behavioural correlate of this desensitization has been shown in rats after repeated central injections of Ang II. Specifically, rats given repeated injections of Ang II drink less water than control animals after a subsequent test injection of Ang II. In the same conditions, however, repeated injections of Ang II have no effect on Ang II-induced saline intake. Given earlier studies indicating that separate intracellular signalling pathways mediate Ang II-induced water and saline intake, we hypothesized that the desensitization observed in rats may be incomplete, leaving the receptor able to activate mitogen-activated protein (MAP) kinases (ERK1/2), which play a role in Ang II-induced saline intake without affecting water intake. In support of this hypothesis, we found no difference in MAP kinase phosphorylation after an Ang II test injection in rats given prior treatment with repeated injections of vehicle, Ang II or Sar(1),Ile(4),Ile(8)-Ang II (SII), an Ang II analogue that activates MAP kinase without G protein coupling. In addition, we found that pretreatment with the MAP kinase inhibitor U0126 completely blocked the desensitizing effect of repeated Ang II injections on water intake. Furthermore, Ang II-induced water intake was reduced to a similar extent by repeated injections of Ang II or SII. The results suggest that G protein-independent signalling is sufficient to produce behavioural desensitization of the angiotensin system and that the desensitization requires MAP kinase activation.

  3. 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

  4. 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.

  5. 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.

  6. 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

  7. 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.

  8. DNA CTG triplet repeats involved in dynamic mutations of neurologically related gene sequences form stable duplexes

    NASA Technical Reports Server (NTRS)

    Smith, G. K.; Jie, J.; Fox, G. E.; Gao, X.

    1995-01-01

    DNA triplet repeats, 5'-d(CTG)n and 5'-d(CAG)n, are present in genes which have been implicated in several neurodegenerative disorders. To investigate possible stable structures formed by these repeating sequences, we have examined d(CTG)n, d(CAG)n and d(CTG).d(CAG)n (n = 2 and 3) using NMR and UV optical spectroscopy. These studies reveal that single stranded (CTG)n (n > 2) forms stable, antiparallel helical duplexes, while the single stranded (CAG)n requires at least three repeating units to form a duplex. NMR and UV melting experiments show that the Tm increases in the order of [(CAG)3]2 < [(CTG)3]2 << (CAG)3.(CTG)3. The (CTG)3 duplex is stable and exhibits similar NMR spectra in solutions containing 0.1-4 M NaCl and at a pH range from 4.6 to 8.8. The (CTG)3 duplex, which contains multiple-T.T mismatches, displays many NMR spectral characteristics similar to those of B-form DNA. However, unique NOE and 1H-31P coupling patterns associated with the repetitive T.T mismatches in the CTG repeats are discerned. These results, in conjunction with recent in vitro studies suggest that longer CTG repeats may form hairpin structures, which can potentially cause interruption in replication, leading to dynamic expansion or deletion of triplet repeats.

  9. DNA CTG triplet repeats involved in dynamic mutations of neurologically related gene sequences form stable duplexes.

    PubMed Central

    Smith, G K; Jie, J; Fox, G E; Gao, X

    1995-01-01

    DNA triplet repeats, 5'-d(CTG)n and 5'-d(CAG)n, are present in genes which have been implicated in several neurodegenerative disorders. To investigate possible stable structures formed by these repeating sequences, we have examined d(CTG)n, d(CAG)n and d(CTG).d(CAG)n (n = 2 and 3) using NMR and UV optical spectroscopy. These studies reveal that single stranded (CTG)n (n > 2) forms stable, antiparallel helical duplexes, while the single stranded (CAG)n requires at least three repeating units to form a duplex. NMR and UV melting experiments show that the Tm increases in the order of [(CAG)3]2 < [(CTG)3]2 << (CAG)3.(CTG)3. The (CTG)3 duplex is stable and exhibits similar NMR spectra in solutions containing 0.1-4 M NaCl and at a pH range from 4.6 to 8.8. The (CTG)3 duplex, which contains multiple-T.T mismatches, displays many NMR spectral characteristics similar to those of B-form DNA. However, unique NOE and 1H-31P coupling patterns associated with the repetitive T.T mismatches in the CTG repeats are discerned. These results, in conjunction with recent in vitro studies suggest that longer CTG repeats may form hairpin structures, which can potentially cause interruption in replication, leading to dynamic expansion or deletion of triplet repeats. PMID:7501450

  10. DNA CTG triplet repeats involved in dynamic mutations of neurologically related gene sequences form stable duplexes

    NASA Technical Reports Server (NTRS)

    Smith, G. K.; Jie, J.; Fox, G. E.; Gao, X.

    1995-01-01

    DNA triplet repeats, 5'-d(CTG)n and 5'-d(CAG)n, are present in genes which have been implicated in several neurodegenerative disorders. To investigate possible stable structures formed by these repeating sequences, we have examined d(CTG)n, d(CAG)n and d(CTG).d(CAG)n (n = 2 and 3) using NMR and UV optical spectroscopy. These studies reveal that single stranded (CTG)n (n > 2) forms stable, antiparallel helical duplexes, while the single stranded (CAG)n requires at least three repeating units to form a duplex. NMR and UV melting experiments show that the Tm increases in the order of [(CAG)3]2 < [(CTG)3]2 << (CAG)3.(CTG)3. The (CTG)3 duplex is stable and exhibits similar NMR spectra in solutions containing 0.1-4 M NaCl and at a pH range from 4.6 to 8.8. The (CTG)3 duplex, which contains multiple-T.T mismatches, displays many NMR spectral characteristics similar to those of B-form DNA. However, unique NOE and 1H-31P coupling patterns associated with the repetitive T.T mismatches in the CTG repeats are discerned. These results, in conjunction with recent in vitro studies suggest that longer CTG repeats may form hairpin structures, which can potentially cause interruption in replication, leading to dynamic expansion or deletion of triplet repeats.

  11. Is Asp-His-Ser/Thr-Trp tetrad hydrogen-bond network important to WD40-repeat proteins: a statistical and theoretical study.

    PubMed

    Wu, Xian-Hui; Zhang, Hui; Wu, Yun-Dong

    2010-04-01

    WD40-repeat proteins are abundant and play important roles in forming protein complexes. The domain usually has seven WD40 repeats, which folds into a seven beta-sheet propeller with each beta-sheet in a four-strand structure. An analysis of 20 available WD40-repeat proteins in Protein Data Bank reveals that each protein has at least one Asp-His-Ser/Thr-Trp (D-H-S/T-W) hydrogen-bonded tetrad, and some proteins have up to six or seven such tetrads. The relative positions of the four residues in the tetrads are also found to be conserved. A sequence alignment analysis of 560 WD40-repeat protein sequences in human reveals very similar features, indicating that such tetrad may be a general feature of WD40-repeat proteins. We carried out density functional theory and found that these tetrads can lead to significant stabilization including hydrogen-bonding cooperativity. The hydrogen bond involving Trp is significant. These results lead us to propose that the tetrads may be critical to the stability and the mechanism of folding of these proteins.

  12. 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

  13. Variation of Serine-Aspartate Repeats in Membrane Proteins Possibly Contributes to Staphylococcal Microevolution

    PubMed Central

    Zhao, Xin

    2012-01-01

    Tandem repeats (either as microsatellites or minisatellites) in eukaryotic and prokaryotic organisms are mutation-prone DNA. While minisatellites in prokaryotic genomes are underrepresented, the cell surface adhesins of bacteria often contain the minisatellite SD repeats, encoding the amino acid pair of serine-asparatate, especially in Staphylococcal strains. However, their relationship to biological functions is still elusive. In this study, effort was made to uncover the copy number variations of SD repeats by bioinformatic analysis and to detect changes in SD repeats during a plasmid-based assay, as a first step to understand its biological functions. The SD repeats were found to be mainly present in the cell surface proteins. The SD repeats were genetically unstable and polymorphic in terms of copy numbers and sequence compositions. Unlike SNPs, the change of its copy number was reversible, without frame shifting. More significantly, a rearrangement hot spot, the ATTC/AGRT site, was found to be mainly responsible for the instability and reversibility of SD repeats. These characteristics of SD repeats may facilitate bacteria to respond to environmental changes, with low cost, low risk and high efficiency. PMID:22509353

  14. Development of repeatable arrays of proteins using immobilized DNA microplate (RAPID-M) technology.

    PubMed

    Ashaari, Nur Suhanawati; Ramarad, Suganti; Khairuddin, Dzulaikha; Akhir, Nor Azurah Mat; Hara, Yuka; Mahadi, Nor Muhammad; Mohamed, Rahmah; Nathan, Sheila

    2015-11-12

    Protein microarrays have enormous potential as in vitro diagnostic tools stemming from the ability to miniaturize whilst generating maximum evaluation of diagnostically relevant information from minute amounts of sample. In this report, we present a method known as repeatable arrays of proteins using immobilized DNA microplates (RAPID-M) for high-throughput in situ protein microarray fabrication. The RAPID-M technology comprises of cell-free expression using immobilized DNA templates and in situ protein purification onto standard microarray slides. To demonstrate proof-of-concept, the repeatable protein arrays developed using our RAPID-M technology utilized green fluorescent protein (GFP) and a bacterial outer membrane protein (OmpA) as the proteins of interest for microarray fabrication. Cell-free expression of OmpA and GFP proteins using beads-immobilized DNA yielded protein bands with the expected molecular sizes of 27 and 30 kDa, respectively. We demonstrate that the beads-immobilized DNA remained stable for at least four cycles of cell-free expression. The OmpA and GFP proteins were still functional after in situ purification on the Ni-NTA microarray slide. The RAPID-M platform for protein microarray fabrication of two different representative proteins was successfully developed.

  15. 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

  16. 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.

  17. Intermediates in the folding equilibrium of repeat proteins from the TPR family.

    PubMed

    González-Charro, Vicente; Rey, Antonio

    2014-09-01

    In recent decades, advances in computational methods and experimental biophysical techniques have improved our understanding of protein folding. Although some of these advances have been remarkable, the structural variability of globular proteins usually encountered makes it difficult to extract general features of their folding processes. To overcome this difficulty, experimental and computational studies of the folding of repeat (or modular) proteins are of interest. Because their native structures can be described as linear arrays of the same, repeated, supersecondary structure unit, it is possible to seek a possibly independent behavior of the different modules without taking into account the intrinsic stability associated with different secondary structure motifs. In this work we have used a Monte Carlo-based simulation to study the folding equilibrium of four repeat proteins belonging to the tetratricopeptide repeat family. Our studies provide new insights into their energy profiles, enabling investigation about the existence of intermediate states and their relative stabilities. We have also performed structural analyses to describe the structure of these intermediates, going through the vast number of conformations obtained from the simulations. In this way, we have tried to identify the regions of each protein in which the modular structure yields a different behavior and, more specifically, regions of the proteins that can stay folded when the rest of the chain has been thermally denatured.

  18. Gonosomal mosaicism in myotonic dystrophy patients: Involvement of mitotic events in (CTG)[sub n] repeat variation and selection against extreme expansion in sperm

    SciTech Connect

    Jansen, G.; Coerwinkel, M.; Wieringa, B.; Nillesen, W.; Smeets, H.; Brunner, H.; Wieringa, B. ); Willems, P.; Vits, L. ); Hoeweler, C. )

    1994-04-01

    Myotonic dystrophy (DM) is caused by abnormal expansion of a polymorphic (CTG)[sub n] repeat, located in the DM protein kinase gene. The authors determined the (CTG)[sub n] repeat lengths in a broad range of tissue DNAs from patients with mild, classical, or congenital manifestation of DM. Differences in the repeat length were seen in somatic tissues from single DM individuals and twins. Repeats appeared to expand to a similar extent in tissues originating from the same embryonal origin. In most male patients carrying intermediate- or small-sized expansions in blood, the repeat lengths covered a markedly wider range in sperm. In contrast, male patients with large allele expansions in blood (>700 CTGs) had similar or smaller repeats in sperm, when detectable. Sperm alleles with >1,000 CTGs were not seen. The authors conclude that DM patients can be considered gonosomal mosaics, i.e., combined somatic and germ-line tissue mosaics. Most remarkably, they observed multiple cases where the length distributions of intermediate- or small-sized alleles in fathers' sperm were significantly different from that in their offspring's blood. The combined findings indicate that intergenerational length changes in the unstable CTG repeat are most likely to occur during early embryonic mitotic divisions in both somatic and germ-line tissue formation. Both the initial CTG length, the overall number of cell divisions involved in tissue formation, and perhaps a specific selection process in spermatogenesis may influence the dynamics of this process. A model explaining mitotic instability and sex-dependent segregation phenomena in DM manifestation is discussed. 59 refs., 5 figs.

  19. A tandem-repeat galectin-9 involved in immune response of yellow catfish, Pelteobagrus fulvidraco, against Aeromonas hydrophila.

    PubMed

    Wang, Yun; Ke, Fei; Ma, Jingjing; Zhou, Shuaibang

    2016-04-01

    Galectins exclusively recognize and bind β-galactoside on cell surface by carbohydrate recognition domain (CRD). In spite of extensive study of mammalian galectin importance in immune system, little is known about that of fish. To study the immune response of yellow catfish to pathogens, a tandem-repeat galectin-9 from yellow catfish was identified and named PfGAL9. Its full-length cDNA was 1314 bp, including a 117 bp of 5' untranslated region (UTR), a 951 bp of open reading frame (ORF), and a 246 bp of 3' UTR. The ORF encoded 316 amino acids (35.12 KDa), shared the highest 78% identity with the predicted galectin-9 of Ictalurus punctatus. This protein possessed two distinct CRDs with two highly conserved sugar binding motifs. Quantitative PCR showed that PfGAL9 was lowly expressed in skin, gill, fin, muscle, heart, and intestine, highly expressed in tested immune tissues (head kidney, trunk kidney, liver, spleen, and blood) in normal body. After inactivated Aeromonas hydrophila challenge, PfGAL9 was remarkably increased in head kidney and liver in a time-dependent manner. The recombinant protein was expressed in Escherichia coli, which not only agglutinated but also bond all examined bacteria. The binding activities are consistent with the size of aggregates formed by agglutinated bacteria. The agglutination must depend on its direct interaction with bacteria. These results suggested that PfGAL9 was involved in the innate immune response against bacterial infection and clearance of pathogens in yellow catfish.

  20. 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.

  1. Proteins involved in meiotic recombination: a role in male infertility?

    PubMed

    Sanderson, Matthew L; Hassold, Terry J; Carrell, Douglas T

    2008-01-01

    Meiotic recombination results in the formation of crossovers, by which genetic information is exchanged between homologous chromosomes during prophase I of meiosis. Recombination is a complex process involving many proteins. Alterations in the genes involved in recombination may result in infertility. Molecular studies have improved our understanding of the roles and mechanisms of the proteins and protein complexes involved in recombination, some of which have function in mitotic cells as well as meiotic cells. Human gene sequencing studies have been performed for some of these genes and have provided further information on the phenotypes observed in some infertile individuals. However, further studies are needed to help elucidate the particular role(s) of a given protein and to increase our understanding of these protein systems. This review will focus on our current understanding of proteins involved in meiotic recombination from a genomic perspective, summarizing our current understanding of known mutations and single nucleotide polymorphisms that may affect male fertility by altering meiotic recombination.

  2. The Repeat Region of the Circumsporozoite Protein is Critical for Sporozoite Formation and Maturation in Plasmodium

    PubMed Central

    Patzewitz, Eva-Maria; Wall, Richard J.; Hopp, Christine S.; Poulin, Benoit; Mohmmed, Asif; Malhotra, Pawan; Coppi, Alida; Sinnis, Photini; Tewari, Rita

    2014-01-01

    The circumsporozoite protein (CSP) is the major surface protein of the sporozoite stage of malaria parasites and has multiple functions as the parasite develops and then migrates from the mosquito midgut to the mammalian liver. The overall structure of CSP is conserved among Plasmodium species, consisting of a species-specific central tandem repeat region flanked by two conserved domains: the NH2-terminus and the thrombospondin repeat (TSR) at the COOH-terminus. Although the central repeat region is an immunodominant B-cell epitope and the basis of the only candidate malaria vaccine in Phase III clinical trials, little is known about its functional role(s). We used the rodent malaria model Plasmodium berghei to investigate the role of the CSP tandem repeat region during sporozoite development. Here we describe two mutant parasite lines, one lacking the tandem repeat region (ΔRep) and the other lacking the NH2-terminus as well as the repeat region (ΔNΔRep). We show that in both mutant lines oocyst formation is unaffected but sporozoite development is defective. PMID:25438048

  3. 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

  4. 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.

  5. 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.

  6. Upregulated Expression of B-Cell Antigen Family Tandem Repeat Proteins by Leishmania Amastigotes ▿ †

    PubMed Central

    Goto, Yasuyuki; Carter, Darrick; Guderian, Jeffrey; Inoue, Noboru; Kawazu, Shin-Ichiro; Reed, Steven G.

    2010-01-01

    Proteins with tandem repeat (TR) domains have been found in various protozoan parasites, and they are often targets of B-cell responses. Through systematic analyses of whole proteomes, we recently demonstrated that two trypanosomatid parasites, Leishmania infantum and Trypanosoma cruzi, are rich in antigenic proteins with large TR domains. However, the reason that these proteins are antigenic was unclear. Here, by performing molecular, immunological, and bioinformatic characterizations of Leishmania TR proteins, we found two possible factors affecting the antigenicity of these proteins; one factor is their fundamental composition as TR proteins, and the other is regulation of their expression by parasites. Enzyme-linked immunosorbent assays (ELISAs) using recombinant proteins revealed that the copy number of the repeat affects the affinity of binding between antigens and antibodies, as expected based on thermodynamic binding kinetics. Other than containing TR domains, the TR proteins do not share characteristics, such as sequence similarity or biased cellular location predicted by the presence of a signal sequence(s) and/or a transmembrane domain(s). However, the TR proteome contained a higher percentage of proteins upregulated in amastigotes than the whole proteome, and upregulated expression of a TR protein seemed to affect its antigenicity. These results indicate that Leishmania parasites actively utilize the TR protein family for parasitism in mammalian hosts. PMID:20160013

  7. 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

  8. 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

  9. 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.

  10. The contribution of entropy, enthalpy, and hydrophobic desolvation to cooperativity in repeat-protein folding

    PubMed Central

    Aksel, Tural; Majumdar, Ananya; Barrick, Doug

    2011-01-01

    Summary Cooperativity is a defining feature of protein folding, but its thermodynamic and structural origins are not completely understood. By constructing consensus ankyrin repeat protein arrays that have nearly identical sequences, we quantify cooperativity by resolving stability into intrinsic and interfacial components. Heteronuclear NMR and CD spectroscopy show that these constructs adopt ankyrin repeat structures. Applying a one-dimensional Ising model to a series of constructs chosen to maximize information content in unfolding transitions, we quantify stabilities of the terminal capping repeats, and resolve the effects of denaturant into intrinsic and interfacial components. Reversible thermal denaturation resolves interfacial and intrinsic free energies into enthalpic, entropic, and heat capacity terms. Intrinsic folding is entropically disfavored, whereas interfacial interaction is entropically favored and attends a decrease in heat capacity. These results suggest that helix formation and backbone ordering occurs upon intrinsic folding, whereas hydrophobic desolvation occurs upon interfacial interaction, contributing to cooperativity. PMID:21397186

  11. Tandem repeating modular proteins avoid aggregation in single molecule force spectroscopy experiments.

    PubMed

    Dougan, Lorna; Fernandez, Julio M

    2007-12-13

    We have used single molecule force spectroscopy to explore the unfolding and refolding behavior of the immunoglobulin-like I27 protein in aqueous 2,2,2-trifluoroethanol (TFE). In bulk solution experiments, a 28% v/v TFE solution has previously been observed to enhance intermolecular attractions and lead to misfolding and aggregation of tandem modular proteins of high sequence identity. In our single molecule experiments, however, we measure successful refolding of the polyprotein I27(8) in all TFE solutions up to 35% v/v. Using a single molecule micromanipulation technique, we have shown that refolding of a polyprotein with identical repeats is not hindered by the presence of this cosolvent. These experimental results provide new insight into the properties of tandem repeating proteins and raise interesting questions as to the evolutionary success of such proteins in avoiding misfolding and aggregation.

  12. Expression and clinical role of small glutamine-rich tetratricopeptide repeat (TPR)-containing protein alpha (SGTA) as a novel cell cycle protein in NSCLC.

    PubMed

    Xue, Qun; Lv, Liting; Wan, Chunhua; Chen, Buyou; Li, Mei; Ni, Tingting; Liu, Yifei; Liu, Yanhua; Cong, Xia; Zhou, Yiqun; Ni, Runzhou; Mao, Guoxin

    2013-09-01

    A small glutamine-rich tetratricopeptide repeat-containing protein alpha (SGTA) is a 35 kDa protein involved in a number of biological processes. However, the role of SGTA in non-small-cell lung cancer (NSCLC) tumorigenesis has never been elucidated. The purpose of this study was to determine whether SGTA could serve as a biomarker for stratification and prediction of prognosis in NSCLC. Small glutamine-rich tetratricopeptide repeat-containing protein alpha expression was evaluated by Western blot in 8 paired fresh lung cancer tissues and immunohistochemistry on 83 paraffin-embedded sections. The effect of SGTA was assessed by RNA interference in A549 cells. Serum starvation and refeeding, flow cytometry, CCK-8, and tunnel assays were performed. Small glutamine-rich tetratricopeptide repeat-containing protein alpha was highly expressed in NSCLC and significantly correlated with NSCLC histological differentiation, clinical stage, and Ki-67. Multivariate analysis indicated that SGTA was an independent prognostic factor for NSCLC patients' survival. The present investigation demonstrated that suppression of SGTA expression resulted in a significant decline of proliferation in A549 cells. Besides, SGTA could abolish the toxicity of cisplatin in A549 cells. These findings suggested that SGTA might play an important role in promoting the tumorigenesis of NSCLC, and thus be a promising therapeutic target to prevent NSCLC progression.

  13. 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

  14. 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

  15. FUNCTIONAL ANALYSIS OF A RING DOMAIN ANKYRIN REPEAT PROTEIN THAT IS HIGHLY EXPRESSED DURING FLOWER SENESCENCE

    USDA-ARS?s Scientific Manuscript database

    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 t...

  16. 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

  17. 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.

  18. Fibronectin-binding protein of Streptococcus pyogenes: sequence of the binding domain involved in adherence of streptococci to epithelial cells.

    PubMed Central

    Talay, S R; Valentin-Weigand, P; Jerlström, P G; Timmis, K N; Chhatwal, G S

    1992-01-01

    The sequence of the fibronectin-binding domain of the fibronectin-binding protein of Streptococcus pyogenes (Sfb protein) was determined, and its role in streptococcal adherence was investigated by use of an Sfb fusion protein in adherence studies. A 1-kb DNA fragment coding for the binding domain of Sfb protein was cloned into the expression vector pEX31 to produce an Sfb fusion protein consisting of the N-terminal part of MS2 polymerase and a C-terminal fragment of the streptococcal protein. Induction of the vector promoter resulted in hyperexpression of fibronectin-binding fusion protein in the cytoplasm of the recombinant Escherichia coli cells. Sequence determination of the cloned 1-kb fragment revealed an in-frame reading frame for a 268-amino-acid peptide composed of a 37-amino-acid sequence which is completely repeated three times and incompletely repeated a fourth time. Cloning of one repeat into pEX31 resulted in expression of small fusion peptides that show fibronectin-binding activity, indicating that one repeat contains at least one binding domain. Each repeat exhibits two charged domains and shows high homology with the 38-amino-acid D3 repeat of the fibronectin-binding protein of Staphylococcus aureus. Sequence comparison with other streptococcal ligand-binding surface proteins, including M protein, failed to reveal significant homology, which suggests that Sfb protein represents a novel type of functional protein in S. pyogenes. The Sfb fusion protein isolated from the cytoplasm of recombinant cells was purified by fast protein liquid chromatography. It showed a strong competitive inhibition of fibronectin binding to S. pyogenes and of the adherence of bacteria to cultured epithelial cells. In contrast, purified streptococcal lipoteichoic acid showed only a weak inhibition of fibronectin binding and streptococcal adherence. These results demonstrate that Sfb protein is directly involved in the fibronectin-mediated adherence of S. pyogenes to

  19. Differential Impact of Tetratricopeptide Repeat Proteins on the Steroid Hormone Receptors

    PubMed Central

    Schülke, Jan-Philip; Wochnik, Gabriela Monika; Lang-Rollin, Isabelle; Gassen, Nils Christian; Knapp, Regina Theresia; Berning, Barbara; Yassouridis, Alexander; Rein, Theo

    2010-01-01

    Background Tetratricopeptide repeat (TPR) motif containing co-chaperones of the chaperone Hsp90 are considered control modules that govern activity and specificity of this central folding platform. Steroid receptors are paradigm clients of Hsp90. The influence of some TPR proteins on selected receptors has been described, but a comprehensive analysis of the effects of TPR proteins on all steroid receptors has not been accomplished yet. Methodology and Principal Findings We compared the influence of the TPR proteins FK506 binding proteins 51 and 52, protein phosphatase-5, C-terminus of Hsp70 interacting protein, cyclophillin 40, hepatitis-virus-B X-associated protein-2, and tetratricopeptide repeat protein-2 on all six steroid hormone receptors in a homogeneous mammalian cell system. To be able to assess each cofactor's effect on the transcriptional activity of on each steroid receptor we employed transient transfection in a reporter gene assay. In addition, we evaluated the interactions of the TPR proteins with the receptors and components of the Hsp90 chaperone heterocomplex by coimmunoprecipitation. In the functional assays, corticosteroid and progesterone receptors displayed the most sensitive and distinct reaction to the TPR proteins. Androgen receptor's activity was moderately impaired by most cofactors, whereas the Estrogen receptors' activity was impaired by most cofactors only to a minor degree. Second, interaction studies revealed that the strongly receptor-interacting co-chaperones were all among the inhibitory proteins. Intriguingly, the TPR-proteins also differentially co-precipitated the heterochaperone complex components Hsp90, Hsp70, and p23, pointing to differences in their modes of action. Conclusion and Significance The results of this comprehensive study provide important insight into chaperoning of diverse client proteins via the combinatorial action of (co)-chaperones. The differential effects of the TPR proteins on steroid receptors bear on

  20. 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.

  1. RNA Binding Proteins Posttranscriptionally Regulate Genes Involved In Oncogenesis

    DTIC Science & Technology

    2010-06-01

    Cloning and characterization of HuR, a ubiquitously expressed Elav-like protein . J Biol Chem 1996, 271(14):8144-8151. 21. Meisner NC, Hackermuller J...Hauptmann S: Expression of the ELAV-like protein HuR is associated with higher tumor grade and increased cyclooxygenase-2 expression in human breast...SH3 domain, ankyrin repeat and pH domain 3 tumor microarray reveals 47 annotated genes up regulated in the HA-HuR overexpressing tumors as compared to

  2. Novel protein-protein interaction family proteins involved in chloroplast movement response.

    PubMed

    Kodama, Yutaka; Suetsugu, Noriyuki; Wada, Masamitsu

    2011-04-01

    To optimize photosynthetic activity, chloroplasts change their intracellular location in response to ambient light conditions; chloroplasts move toward low intensity light to maximize light capture, and away from high intensity light to avoid photodamage. Although several proteins have been reported to be involved in the chloroplast photorelocation movement response, any physical interaction among them was not found so far. We recently found a physical interaction between two plant-specific coiled-coil proteins, WEB1 (Weak Chloroplast Movement under Blue Light 1) and PMI2 (Plastid Movement Impaired 2), that were identified to regulate chloroplast movement velocity. Since the both coiled-coil regions of WEB1 and PMI2 were classified into an uncharacterized protein family having DUF827 (DUF: Domain of Unknown Function) domain, it was the first report that DUF827 proteins could mediate protein-protein interaction. In this mini-review article, we discuss regarding molecular function of WEB1 and PMI2, and also define a novel protein family composed of WEB1, PMI2 and WEB1/PMI2-like proteins for protein-protein interaction in land plants.

  3. Shifting transition states in the unfolding of a large ankyrin repeat protein

    PubMed Central

    Werbeck, Nicolas D.; Rowling, Pamela J. E.; Chellamuthu, Vasuki R.; Itzhaki, Laura S.

    2008-01-01

    The 33-amino-acid ankyrin motif comprises a β-turn followed by two anti-parallel α-helices and a loop and tandem arrays of the motif pack in a linear fashion to produce elongated structures characterized by short-range interactions. In this article we use site-directed mutagenesis to investigate the kinetic unfolding mechanism of D34, a 426-residue, 12-ankyrin repeat fragment of the protein ankyrinR. The data are consistent with a model in which the N-terminal half of the protein unfolds first by unraveling progressively from the start of the polypeptide chain to form an intermediate; in the next step, the C-terminal half of the protein unfolds via two pathways whose transition states have either the early or the late C-terminal ankyrin repeats folded. We conclude that the two halves of the protein unfold by different mechanisms because the N-terminal moiety folds and unfolds in the context of a folded C-terminal moiety, which therefore acts as a “seed” and confers a unique directionality on the process, whereas the C-terminal moiety folds and unfolds in the context of an unfolded N-terminal moiety and therefore behaves like a single-domain ankyrin repeat protein, having a high degree of symmetry and consequently more than one unfolding pathway accessible to it. PMID:18632570

  4. Repeated formaldehyde inhalation impaired olfactory function and changed SNAP25 proteins in olfactory bulb.

    PubMed

    Zhang, Qi; Yan, Weiqun; Bai, Yang; Zhu, Yingqiao; Ma, Jie

    2014-10-01

    Formaldehyde inhalation exposure, which can occur through occupational exposure, can lead to sensory irritation, neurotoxicity, mood disorders, and learning and memory impairment. However, its influence on olfactory function is unclear. To investigate the mechanism and the effect of repeated formaldehyde inhalation exposure on olfactory function. Rats were treated with formaldehyde inhalation (13·5±1·5 ppm, twice 30 minutes/day) for 14 days. Buried food pellet and locomotive activity tests were used to detect olfactory function and locomotion. Western blots were used to evaluate synaptosomal-associated protein 25 (SNAP25) protein levels in the olfactory bulb (OB) lysate and synaptosome, as well as mature and immature olfactory sensory neuron markers, olfactory marker protein (OMP), and Tuj-1. Real-time polymerase chain reaction (PCR) was used to detect SNAP25 mRNA amounts. Repeated formaldehyde inhalation exposure impaired olfactory function, whereas locomotive activities were unaffected. SNAP25 protein decreased significantly in the OB, but not in the occipital lobe. SNAP25 also decreased in the OB synaptosome when synaptophysin did not change after formaldehyde treatment. mRNA levels of SNAP25A and SNAP25B were unaffected. Mature and immature olfactory sensory neuron marker, OMP, and Tuj-1, did not change after formaldehyde treatment. Repeated formaldehyde exposure impaired olfactory function by disturbing SNAP25 protein in the OB.

  5. Anchoring skeletal muscle development and disease: the role of ankyrin repeat domain containing proteins in muscle physiology

    PubMed Central

    Tee, Jin-Ming; Peppelenbosch, Maikel P.

    2010-01-01

    The ankyrin repeat is a protein module with high affinity for other ankyrin repeats based on strong Van der Waals forces. The resulting dimerization is unusually resistant to both mechanical forces and alkanization, making this module exceedingly useful for meeting the extraordinary demands of muscle physiology. Many aspects of muscle function are controlled by the superfamily ankyrin repeat domain containing proteins, including structural fixation of the contractile apparatus to the muscle membrane by ankyrins, the archetypical member of the family. Additionally, other ankyrin repeat domain containing proteins critically control the various differentiation steps during muscle development, with Notch and developmental stage-specific expression of the members of the Ankyrin repeat and SOCS box (ASB) containing family of proteins controlling compartment size and guiding the various steps of muscle specification. Also, adaptive responses in fully formed muscle require ankyrin repeat containing proteins, with Myotrophin/V-1 ankyrin repeat containing proteins controlling the induction of hypertrophic responses following excessive mechanical load, and muscle ankyrin repeat proteins (MARPs) acting as protective mechanisms of last resort following extreme demands on muscle tissue. Knowledge on mechanisms governing the ordered expression of the various members of superfamily of ankyrin repeat domain containing proteins may prove exceedingly useful for developing novel rational therapy for cardiac disease and muscle dystrophies. PMID:20515317

  6. 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.

  7. 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

  8. Measuring the Activity of Leucine-Rich Repeat Kinase 2: A Kinase Involved in Parkinson's Disease

    PubMed Central

    Lee, Byoung Dae; Li, Xiaojie; Dawson, Ted M.; Dawson, Valina L.

    2015-01-01

    Mutations in the LRRK2 (Leucine-Rich Repeat Kinase 2) gene are the most common cause of autosomal dominant Parkinson's disease. LRRK2 has multiple functional domains including a kinase domain. The kinase activity of LRRK2 is implicated in the pathogenesis of Parkinson's disease. Developing an assay to understand the mechanisms of LRRK2 kinase activity is important for the development of pharmacologic and therapeutic applications. Here, we describe how to measure in vitro LRRK2 kinase activity and its inhibition. PMID:21960214

  9. 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

  10. Involvement of PCH family proteins in cytokinesis and actin distribution.

    PubMed

    Lippincott, J; Li, R

    2000-04-15

    Pombe Cdc15 homology (PCH) proteins constitute an extensive protein family whose members have been found in diverse eukaryotic organisms. These proteins are characterized by the presence of several conserved sequence and structural motifs. Recent studies in yeast and mammalian cultured cells have implicated these proteins in actin-based processes, in particular, cytokinesis. Here we review the recent findings on the in vivo localization, function, and binding partners of PCH family members. We also provide new microscopy data regarding the in vivo dynamics of a budding yeast PCH protein involved in cytokinesis.

  11. Ciliate pellicular proteome identifies novel protein families with characteristic repeat motifs that are common to alveolates.

    PubMed

    Gould, Sven B; Kraft, Lesleigh G K; van Dooren, Giel G; Goodman, Christopher D; Ford, Kristina L; Cassin, Andrew M; Bacic, Antony; McFadden, Geoffrey I; Waller, Ross F

    2011-03-01

    The pellicles of alveolates (ciliates, apicomplexans, and dinoflagellates) share a common organization, yet perform very divergent functions, including motility, host cell invasion, and armor. The alveolate pellicle consists of a system of flattened membrane sacs (alveoli, which are the defining feature of the group) below the plasma membrane that is supported by a membrane skeleton as well as a network of microtubules and other filamentous elements. We recently showed that a family of proteins, alveolins, are common and unique to this pellicular structure in alveolates. To identify additional proteins that contribute to this structure, a pellicle proteome study was conducted for the ciliate Tetrahymena thermophila. We found 1,173 proteins associated with this structure, 45% (529 proteins) of which represented novel proteins without matches to other functionally characterized proteins. Expression of four newly identified T. thermophila pellicular proteins as green fluorescent protein-fusion constructs confirmed pellicular location, and one new protein located in the oral apparatus. Bioinformatic analysis revealed that 21% of the putative pellicular proteins, predominantly the novel proteins, contained highly repetitive regions with strong amino acid biases for particular residues (K, E, Q, L, I, and V). When the T. thermophila novel proteins were compared with apicomplexan genomic data, 278 proteins with high sequence similarity were identified, suggesting that many of these putative pellicular components are shared between the alveolates. Of these shared proteins, 126 contained the distinctive repeat regions. Localization of two such proteins in Toxoplasma gondii confirmed their role in the pellicle and in doing so identified two new proteins of the apicomplexan invasive structure--the apical complex. Screening broadly for these repetitive domains in genomic data revealed large and actively evolving families of such proteins in alveolates, suggesting that these

  12. 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

  13. 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

  14. Halibut mitochondrial genomes contain extensive heteroplasmic tandem repeat arrays involved in DNA recombination

    PubMed Central

    Mjelle, Kenneth A; Karlsen, Bård O; Jørgensen, Tor E; Moum, Truls; Johansen, Steinar D

    2008-01-01

    Background Halibuts are commercially important flatfish species confined to the North Pacific and North Atlantic Oceans. We have determined the complete mitochondrial genome sequences of four specimens each of Atlantic halibut (Hippoglossus hippoglossus), Pacific halibut (Hippoglossus stenolepis) and Greenland halibut (Reinhardtius hippoglossoides), and assessed the nucleotide variability within and between species. Results About 100 variable positions were identified within the four specimens in each halibut species, with the control regions as the most variable parts of the genomes (10 times that of the mitochondrial ribosomal DNA). Due to tandem repeat arrays, the control regions have unusually large sizes compared to most vertebrate mtDNAs. The arrays are highly heteroplasmic in size and consist mainly of different variants of a 61-bp motif. Halibut mitochondrial genomes lacking arrays were also detected. Conclusion The complexity, distribution, and biological role of the heteroplasmic tandem repeat arrays in halibut mitochondrial control regions are discussed. We conclude that the most plausible explanation for array maintenance includes both the slipped-strand mispairing and DNA recombination mechanisms. PMID:18186947

  15. Methods for Mapping of Interaction Networks Involving Membrane Proteins

    SciTech Connect

    Hooker, Brian S.; Bigelow, Diana J.; Lin, Chiann Tso

    2007-11-23

    Numerous approaches have been taken to study protein interactions, such as tagged protein complex isolation followed by mass spectrometry, yeast two-hybrid methods, fluorescence resonance energy transfer, surface plasmon resonance, site-directed mutagenesis, and crystallography. Membrane protein interactions pose significant challenges due to the need to solubilize membranes without disrupting protein-protein interactions. Traditionally, analysis of isolated protein complexes by high-resolution 2D gel electrophoresis has been the main method used to obtain an overall picture of proteome constituents and interactions. However, this method is time consuming, labor intensive, detects only abundant proteins and is not suitable for the coverage required to elucidate large interaction networks. In this review, we discuss the application of various methods to elucidate interactions involving membrane proteins. These techniques include methods for the direct isolation of single complexes or interactors as well as methods for characterization of entire subcellular and cellular interactomes.

  16. Protein Ser/Thr phosphatases with kelch-like repeat domains.

    PubMed

    Kutuzov, Mikhail A; Andreeva, Alexandra V

    2002-09-01

    This report describes the presence in plants of protein Ser/Thr phosphatases of the PPP family, homologous to PfPPalpha phosphatase from Plasmodium falciparum. Like PfPPalpha, they possess large N-terminal domains and catalytic domains that are more closely related to the protein phosphatase 1 group. The N-terminal domains of PfPPalpha and its plant homologues contain tandem kelch-like repeats, not previously identified in any protein phosphatases, suggesting that the N-terminal domains may form beta-propeller structures mediating protein-protein interactions. We therefore suggest that this novel phosphatase group be designated as PPKLs for protein phosphatases with kelch-like repeat domains. Four PPKL isoforms are encoded in the Arabidopsis thaliana genome, of which at least three are expressed. PPKLs appear to be ubiquitous in Viridiplantae. The existence of a protein phosphatase group shared by Viridiplantae and Apicomplexa, but not other eukaryotes, is in line with the theory of the origin of Apicomplexa by endosymbiosis of nonphotosynthetic eukaryotes with red algae.

  17. 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

  18. 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

  19. Molecular Simulation Studies of Proteins Involved in Parkinson's Disease

    NASA Astrophysics Data System (ADS)

    Carloni, Paolo

    2007-12-01

    This contribution describes two recent computational studies related to proteins involved in Parkinson's Disease (PD). The first focuses on the interplay between dopamine and α-synuclein (AS), which plays a central role in PD (unpublished results). The second deals with the protein DJ-1, whose mutations are present in patients suffering from familiar PD [1]. Computational methods are used to investigate the relationship between such mutations and the protein oligomeric state, which may be important for the progression of the disease.

  20. An essential yeast gene encoding a TTAGGG repeat-binding protein

    SciTech Connect

    Brigati, C. Istituto Nazionale per la Ricerca sul Cancro, Genoa ); Kurtz, S.; Balderes, D.; Shore, D. ); Vidali, G. )

    1993-02-01

    Among all eukaryotes examined to date, telomere is a highly conserved structure. It is designed to protect chromosomes from degradation and fusion. Telomeres are composed of multiple repeats of short sequence elements and range in length from a few repeat units to > kb. The repeated sequence TTAGGG is found at telomeres in all vertebrates, certain slime molds, and trypanosomes. Because sequence TTAGGG is present at the telomere of all of these divergent organisms, it is likely that it constitutes a binding site for highly conserved proteins with important roles in chromosomal structure and function. The occurrence of a TTAGGG-binding activity in Saccharomyces cerevisiae and the presence of TTAGGG sequences at telomere junctions raise the possibility that there is a related factor with a functional role at telomeres in S. cervisiae. The research in this paper tests this hypothesis. 33 refs., 6 figs., 1 tab.

  1. 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.

  2. 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.

  3. 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. © 2017 Miranda et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  4. 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

  5. The crystal structure of the thiocyanate-forming protein from Thlaspi arvense, a kelch protein involved in glucosinolate breakdown.

    PubMed

    Gumz, Frauke; Krausze, Joern; Eisenschmidt, Daniela; Backenköhler, Anita; Barleben, Leif; Brandt, Wolfgang; Wittstock, Ute

    2015-09-01

    Kelch repeat-containing proteins are involved in diverse cellular processes, but only a small subset of plant kelch proteins has been functionally characterized. Thiocyanate-forming protein (TFP) from field-penny cress, Thlaspi arvense (Brassicaceae), is a representative of specifier proteins, a group of kelch proteins involved in plant specialized metabolism. As components of the glucosinolate-myrosinase system of the Brassicaceae, specifier proteins determine the profile of bioactive products formed when plant tissue is disrupted and glucosinolates are hydrolyzed by myrosinases. Here, we describe the crystal structure of TaTFP at a resolution of 1.4 Å. TaTFP crystallized as homodimer. Each monomer forms a six-blade β-propeller with a wide "top" and a narrower "bottom" opening with distinct strand-connecting loops protruding far beyond the lower propeller surface. Molecular modeling and mutational analysis identified residues for glucosinolate aglucone and Fe(2+) cofactor binding within these loops. As the first experimentally determined structure of a plant kelch protein, the crystal structure of TaTFP not only enables more detailed mechanistic studies on glucosinolate breakdown product formation, but also provides a new basis for research on the diverse roles and mechanisms of other kelch proteins in plants.

  6. The BC component of ABC toxins is an RHS-repeat-containing protein encapsulation device.

    PubMed

    Busby, Jason N; Panjikar, Santosh; Landsberg, Michael J; Hurst, Mark R H; Lott, J Shaun

    2013-09-26

    The ABC toxin complexes produced by certain bacteria are of interest owing to their potent insecticidal activity and potential role in human disease. These complexes comprise at least three proteins (A, B and C), which must assemble to be fully toxic. The carboxy-terminal region of the C protein is the main cytotoxic component, and is poorly conserved between different toxin complexes. A general model of action has been proposed, in which the toxin complex binds to the cell surface via the A protein, is endocytosed, and subsequently forms a pH-triggered channel, allowing the translocation of C into the cytoplasm, where it can cause cytoskeletal disruption in both insect and mammalian cells. Toxin complexes have been visualized using single-particle electron microscopy, but no high-resolution structures of the components are available, and the role of the B protein in the mechanism of toxicity remains unknown. Here we report the three-dimensional structure of the complex formed between the B and C proteins, determined to 2.5 Å by X-ray crystallography. These proteins assemble to form an unprecedented, large hollow structure that encapsulates and sequesters the cytotoxic, C-terminal region of the C protein like the shell of an egg. The shell is decorated on one end by a β-propeller domain, which mediates attachment of the B-C heterodimer to the A protein in the native complex. The structure reveals how C auto-proteolyses when folded in complex with B. The C protein is the first example, to our knowledge, of a structure that contains rearrangement hotspot (RHS) repeats, and illustrates a marked structural architecture that is probably conserved across both this widely distributed bacterial protein family and the related eukaryotic tyrosine-aspartate (YD)-repeat-containing protein family, which includes the teneurins. The structure provides the first clues about the function of these protein repeat families, and suggests a generic mechanism for protein

  7. 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.

  8. Key proteins involved in insulin vesicle exocytosis and secretion

    PubMed Central

    Xiong, Qian-Yin; Yu, Cui; Zhang, Yao; Ling, Liefeng; Wang, Lizhuo; Gao, Jia-Lin

    2017-01-01

    In vivo insulin secretion is predominantly affected by blood glucose concentration, blood concentration of amino acids, gastrointestinal hormones and free nerve functional status, in addition to other factors. Insulin is one of the most important hormones in the body, and its secretion is precisely controlled by nutrients, neurotransmitters and hormones. The insulin exocytosis process is similar to the neurotransmitter release mechanism. There are various types of proteins and lipids that participate in the insulin secretory vesicle fusion process, such as soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein, Ras-related proteins and vacuolar-type H+-ATPase (V-ATPase). Notably, the SNARE protein is the molecular basis of exocytotic activity. In the current review, the role of the vesicle membrane proteins (synaptobrevins, vesicle associated membrane proteins and target membrane proteins) and auxiliary proteins (Rab proteins and Munc-18 proteins) in vesicle fusion activity were summarized. A summary of these key proteins involved in insulin granule secretion will facilitate understanding of the pathogenesis of diabetes. PMID:28357064

  9. Novel identification of matrix proteins involved in calcitic biomineralization.

    PubMed

    Rose-Martel, Megan; Smiley, Sandy; Hincke, Maxwell T

    2015-02-26

    Calcitic biomineralization is essential for otoconia formation in vertebrates. This process is characterized by protein-crystal interactions that modulate crystal growth on an extracellular matrix. An excellent model for the study of calcitic biomineralization is the avian eggshell, the fastest known biomineralization process. The objective of this study is to identify and characterize matrix proteins associated with the eggshell mammillary cones, which are hypothesized to regulate the earliest stage of eggshell calcification. Mammillary cones were isolated from 2 models, fertilized and unfertilized, and the released proteins were identified by RP-nanoLC and ES-MS/MS proteomics. Proteomics analysis identified 49 proteins associated with the eggshell membrane fibers and, importantly, 18 mammillary cone-specific proteins with an additional 18 proteins identified as enriched in the mammillary cones. Among the most promising candidates for modulating protein-crystal interactions were extracellular matrix proteins, including ABI family member 3 (NESH) binding protein (ABI3BP), tiarin-like, hyaluronan and proteoglycan link protein 3 (HAPLN3), collagen alpha-1(X), collagen alpha-1(II) and fibronectin, in addition to the calcium binding proteins calumenin, EGF-like repeats and discoidin 1-like domains 3 (EDIL3), nucleobindin-2 and SPARC. In conclusion, we identified several cone-resident proteins that are candidates to regulate initiation of eggshell calcification. Further study of these proteins will determine their roles in modulating calcitic biomineralization and lead to insight into the process of otoconia formation/regeneration. Biomineralization is essential for the development of hard tissues in vertebrates, which includes both calcium phosphate and calcium carbonate structures. Calcitic mineralization by calcium carbonate is an important process in the formation of otoconia, which are gravity receptor organs located in the inner ear and are responsible for balance

  10. Repeated ischaemic isometric exercise increases muscle fibre conduction velocity in humans: involvement of Na+-K+-ATPase

    PubMed Central

    Rongen, G A; van Dijk, J P; van Ginneken, E E; Stegeman, D F; Smits, P; Zwarts, M J

    2002-01-01

    This study was performed to test two hypotheses: (1) ischaemic preconditioning (development of tolerance to ischaemia) influences muscle fibre conduction velocity (MFCV) during repeated ischaemic isometric exercise and (2) the increase in MFCV to supranormal levels during recovery from ischaemic exercise is caused by activation of Na+−K+-ATPase. For this purpose, MFCV was measured with surface electromyography (sEMG) during repeated ischaemic isometric exercise of the brachioradial muscle (2 min at 30 % of maximal voluntary contraction). The involvement of ischaemic preconditioning was tested by changing the duration of ischaemia and by intra-arterial infusion of adenosine (brachial artery, 50 μg min−1 dl−1). The role of Na+−K+-ATPase was explored using ouabain (0.2 μg min−1 dl−1). During the exercise, MFCV decreased from 4.4 ± 0.2 m s−1 to 3.7 ± 0.2 m s−1 (P < 0.01, n = 13). Similar reductions in MFCV were observed during repeated exercise, irrespective of the reperfusion time (10 min vs. 18 min) or duration of the ischaemia (2 vs. 10 min). However, initial MFCV gradually increased for each subsequent contraction when contractions were repeated at 10 min intervals (4.4 ± 0.2 m s−1vs. 4.9 ± 0.2 m s−1 for the first and fourth contraction respectively; P < 0.01; n = 13). This increase was not observed when contractions were performed at 18 min intervals, nor when additional ischaemia was applied. Intra-arterial adenosine did not affect MFCV. Intra-arterial ouabain did not affect the reduction in MFCV during exercise but completely prevented the increase in MFCV during recovery: from 4.7 ± 0.2 m s−1 to 5.2 ± 0.2 m s−1vs. 4.5 ± 0.1 m s−1 to 4.5 ± 0.1 m s−1 in the absence and presence of ouabain respectively (P < 0.05 for ouabain effect; n = 6). In conclusion, ischaemic preconditioning is not involved in changes in MFCV during repeated ischaemic isometric exercise. The increase in MFCV during recovery from repeated ischaemic

  11. Keeping History from Repeating Itself: Involving Parents about Retention Decisions to Support Student Achievement

    ERIC Educational Resources Information Center

    Akmal, Tariq T.; Larsen, Donald E.

    2004-01-01

    Collaborative ventures between families and schools can result in children being successful both academically and in life (Henderson & Berla, 1994; Jackson & Davis, 2000; Mapp, 1997). The most successful predictor of student achievement is an encouraging home environment, high expectations from parents, and parental involvement (Epstein, 2001;…

  12. 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

  13. 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.

  14. When does repeated search in scenes involve memory? Looking AT versus looking FOR objects in scenes

    PubMed Central

    Võ, Melissa L.-H.; Wolfe, Jeremy M.

    2014-01-01

    One might assume that familiarity with a scene or previous encounters with objects embedded in a scene would benefit subsequent search for those items. However, in a series of experiments we show that this is not the case: When participants were asked to subsequently search for multiple objects in the same scene, search performance remained essentially unchanged over the course of searches despite increasing scene familiarity. Similarly, looking at target objects during previews, which included letter search, 30 seconds of free viewing, or even 30 seconds of memorizing a scene, also did not benefit search for the same objects later on. However, when the same object was searched for again memory for the previous search was capable of producing very substantial speeding of search despite many different intervening searches. This was especially the case when the previous search engagement had been active rather than supported by a cue. While these search benefits speak to the strength of memory-guided search when the same search target is repeated, the lack of memory guidance during initial object searches – despite previous encounters with the target objects - demonstrates the dominance of guidance by generic scene knowledge in real-world search. PMID:21688939

  15. Posttraumatic regeneration involves differential expression of long terminal repeat (LTR) retrotransposons.

    PubMed

    Mashanov, Vladimir S; Zueva, Olga R; García-Arrarás, José E

    2012-10-01

    Retrotransposons are mobile genetic elements that constitute a sizable proportion of eukaryote genomes. Although retroelements are known to play significant roles in embryogenesis, stress reactions, and disease progression, they have never been studied in the context of animal regeneration. In this study, high-throughput transcriptome analysis revealed unexpectedly large-scale changes in transcriptional activity of retrotransposons in regenerating radial organs of the sea cucumber Holothuria glaberrima. In particular, we identified 36 long terminal repeat (LTR) retroelements, of which 20 showed significant changes in their expression during regeneration (11 up-regulated, 8 down-regulated, and one was initially up-regulated, but later down-regulated). We then studied in detail the most significantly up-regulated element, Gypsy-1_Hg. This transposon showed a drastic (>50-fold) increase in expression in regeneration and started to return to the normal levels only after the anatomical organization of the injured tissues was restored. All cells expressing Gypsy-1_Hg were located in the vicinity of the wound and included glia and neurons of the radial nerve. The retrotransposon-expressing cells survived programmed cell death and contributed to regeneration. Our findings demonstrate considerable changes in transcriptional activity of retrotransposons (both over-expression and down-regulation) associated with posttraumatic regeneration in an echinoderm. Copyright © 2012 Wiley Periodicals, Inc.

  16. Atomic model of human Rcd-1 reveals an armadillo-like-repeat protein with in vitro nucleic acid binding properties.

    PubMed

    Garces, Robert G; Gillon, Wanda; Pai, Emil F

    2007-02-01

    Rcd-1, a protein highly conserved across eukaryotes, was initially identified as a factor essential for nitrogen starvation-invoked differentiation in fission yeast, and its Saccharomyces cerevisiae homolog, CAF40, has been identified as part of the CCR4-NOT transcription complex, where it interacts with the NOT1 protein. Mammalian homologs are involved in various cellular differentiation processes including retinoic acid-induced differentiation and hematopoetic cell development. Here, we present the 2.2 A X-ray structure of the highly conserved region of human Rcd-1 and investigate possible functional abilities of this and the full-length protein. The monomer is made up of six armadillo repeats forming a solvent-accessible, positively-charged cleft 21-22 A wide that, in contrast to other armadillo proteins, stays fully exposed in the dimer. Prompted by this finding, we established that Rcd-1 can bind to single- and double-stranded oligonucleotides in vitro with the affinity of G/C/T > A. Mutation of an arginine residue within the cleft strongly reduced or abolished oligonucleotide binding. Rcd-1's ability to bind to nucleic acids, in addition to the previously reported protein-protein interaction with NOT1, suggests a new feature in Rcd-1's role in regulation of overall cellular differentiation processes.

  17. 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.

  18. 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

  19. 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.

  20. An essential yeast gene encoding a TTAGGG repeat-binding protein.

    PubMed Central

    Brigati, C; Kurtz, S; Balderes, D; Vidali, G; Shore, D

    1993-01-01

    A yeast gene encoding a DNA-binding protein that recognizes the telomeric repeat sequence TTAGGG found in multicellular eukaryotes was identified by screening a lambda gt11 expression library with a radiolabeled TTAGGG multimer. This gene, which we refer to as TBF1 (TTAGGG repeat-binding factor 1), encodes a polypeptide with a predicted molecular mass of 63 kDa. The TBF1 protein, produced in vitro by transcription and translation of the cloned gene, binds to (TTAGGG)n probes and to a yeast telomeric junction sequence that contains two copies of the sequence TTAGGG separated by 5 bp. TBF1 appears to be identical to a previously described yeast TTAGGG-repeat binding activity called TBF alpha. TBF1 produced in vitro yields protein-DNA complexes with (TTAGGG)n probes that have mobilities on native polyacrylamide gels identical to those produced by partially purified TBF alpha from yeast cells. Furthermore, when extracts are prepared from a strain containing a TBF1 gene with an antigen tag, we find that the antigen copurifies with the predominant (TTAGGG)n-binding activity in the extracts. The DNA sequence of TBF1 was determined. The predicted protein sequence suggests that TBF1 may contain a nucleotide-binding domain, but no significant similarities to any other known proteins were identified, nor was an obvious DNA-binding motif apparent. Diploid cells heterozygous for a tbf1::URA3 insertion mutation are viable but upon sporulation give rise to tetrads with only two viable spores, both of which are Ura-, indicating that the TBF1 gene is essential for growth. Possible functions of TBF1 (TFB alpha) are discussed in light of these new results. Images PMID:8423796

  1. 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.

  2. 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.

  3. A protein involved in the assembly of an extracellular calcium storage matrix.

    PubMed

    Glazer, Lilah; Shechter, Assaf; Tom, Moshe; Yudkovski, Yana; Weil, Simy; Aflalo, Eliahu David; Pamuru, Ramachandra Reddy; Khalaila, Isam; Bentov, Shmuel; Berman, Amir; Sagi, Amir

    2010-04-23

    Gastroliths, the calcium storage organs of crustaceans, consist of chitin-protein-mineral complexes in which the mineral component is stabilized amorphous calcium carbonate. To date, only three proteins, GAP 65, gastrolith matrix protein (GAMP), and orchestin, have been identified in gastroliths. Here, we report a novel protein, GAP 10, isolated from the gastrolith of the crayfish Cherax quadricarinatus and specifically expressed in its gastrolith disc. The encoding gene was cloned by partial sequencing of the protein extracted from the gastrolith matrix. Based on an assembled microarray cDNA chip, GAP 10 transcripts were found to be highly (12-fold) up-regulated in premolt gastrolith disc and significantly down-regulated in the hypodermis at the same molt stage. The deduced protein sequence of GAP 10 lacks chitin-binding domains and does not show homology to known proteins in the GenBank data base. It does, however, have an amino acid composition that has similarity to proteins extracted from invertebrate and ascidian-calcified extracellular matrices. The GAP 10 sequence contains a predicted signal peptide and predicted phosphorylation sites. In addition, the protein is phosphorylated and exhibits calcium-binding ability. Repeated daily injections of GAP 10 double strand RNA to premolt C. quadricarinatus resulted in a prolonged premolt stage and in the development of gastroliths with irregularly rough surfaces. These findings suggest that GAP 10 may be involved in the assembly of the gastrolith chitin-protein-mineral complex, particularly in the deposition of amorphous calcium carbonate.

  4. 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.

  5. Exploiting genomic data to identify proteins involved in abalone reproduction.

    PubMed

    Mendoza-Porras, Omar; Botwright, Natasha A; McWilliam, Sean M; Cook, Mathew T; Harris, James O; Wijffels, Gene; Colgrave, Michelle L

    2014-08-28

    Aside from their critical role in reproduction, abalone gonads serve as an indicator of sexual maturity and energy balance, two key considerations for effective abalone culture. Temperate abalone farmers face issues with tank restocking with highly marketable abalone owing to inefficient spawning induction methods. The identification of key proteins in sexually mature abalone will serve as the foundation for a greater understanding of reproductive biology. Addressing this knowledge gap is the first step towards improving abalone aquaculture methods. Proteomic profiling of female and male gonads of greenlip abalone, Haliotis laevigata, was undertaken using liquid chromatography-mass spectrometry. Owing to the incomplete nature of abalone protein databases, in addition to searching against two publicly available databases, a custom database comprising genomic data was used. Overall, 162 and 110 proteins were identified in females and males respectively with 40 proteins common to both sexes. For proteins involved in sexual maturation, sperm and egg structure, motility, acrosomal reaction and fertilization, 23 were identified only in females, 18 only in males and 6 were common. Gene ontology analysis revealed clear differences between the female and male protein profiles reflecting a higher rate of protein synthesis in the ovary and higher metabolic activity in the testis. A comprehensive mass spectrometry-based analysis was performed to profile the abalone gonad proteome providing the foundation for future studies of reproduction in abalone. Key proteins involved in both reproduction and energy balance were identified. Genomic resources were utilised to build a database of molluscan proteins yielding >60% more protein identifications than in a standard workflow employing public protein databases. Copyright © 2014 Elsevier B.V. All rights reserved.

  6. 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

  7. Atomic model of human Rcd-1 reveals an armadillo-like-repeat protein with in vitro nucleic acid binding properties

    PubMed Central

    Garces, Robert G.; Gillon, Wanda; Pai, Emil F.

    2007-01-01

    Rcd-1, a protein highly conserved across eukaryotes, was initially identified as a factor essential for nitrogen starvation-invoked differentiation in fission yeast, and its Saccharomyces cerevisiae homolog, CAF40, has been identified as part of the CCR4–NOT transcription complex, where it interacts with the NOT1 protein. Mammalian homologs are involved in various cellular differentiation processes including retinoic acid-induced differentiation and hematopoetic cell development. Here, we present the 2.2 Å X-ray structure of the highly conserved region of human Rcd-1 and investigate possible functional abilities of this and the full-length protein. The monomer is made up of six armadillo repeats forming a solvent-accessible, positively-charged cleft 21–22 Å wide that, in contrast to other armadillo proteins, stays fully exposed in the dimer. Prompted by this finding, we established that Rcd-1 can bind to single- and double-stranded oligonucleotides in vitro with the affinity of G/C/T ≫ A. Mutation of an arginine residue within the cleft strongly reduced or abolished oligonucleotide binding. Rcd-1’s ability to bind to nucleic acids, in addition to the previously reported protein–protein interaction with NOT1, suggests a new feature in Rcd-1’s role in regulation of overall cellular differentiation processes. PMID:17189474

  8. [A case of pulmonary malignant lymphoma with endobronchial involvement with repeated improvement or progress of atelectasis].

    PubMed

    Ohe, Miki; Kohno, Hidekazu; Deguti, Naoko; Kanda, Hibiki; Kondo, Keiichi; Isobe, Takeshi

    2008-12-01

    A 68-year-old woman, presented to the emergency department with dry cough and increased shortness of breath. Her chest X-ray showed complete atelectasis of the right upper lobe. Her chest examination was significant for diffuse wheezing, and she was treated with corticosteroids. Fourteen hours after her arrival, wheezing and atelectasis on the chest X-ray disappeared. However, 10 days after admission, her chest X-ray showed atelectasis of the right lower lobe, and right middle and lower lobes atelectasis on 17 days, then became complete atelectasis of the lung on 22 days after admission. Bronchoscopic findings showed severe stenosis of the right upper lobe bronchus and truncus intermedius. Pathologic examination of the transbronchoscopic biopsy specimen showed diffuse large B-cell lymphoma. We diagnosed primary pulmonary malignant lymphoma because she had no extrapulmonary diseases. An extremely rare case of pulmonary malignant lymphoma with endobronchial involvement in which the site of atelectasis changed rapidly was reported.

  9. Psychological impact and recovery after involvement in a patient safety incident: a repeated measures analysis

    PubMed Central

    Van Gerven, Eva; Bruyneel, Luk; Panella, Massimiliano; Euwema, Martin; Sermeus, Walter; Vanhaecht, Kris

    2016-01-01

    Objective To examine individual, situational and organisational aspects that influence psychological impact and recovery of a patient safety incident on physicians, nurses and midwives. Design Cross-sectional, retrospective surveys of physicians, midwives and nurses. Setting 33 Belgian hospitals. Participants 913 clinicians (186 physicians, 682 nurses, 45 midwives) involved in a patient safety incident. Main outcome measures The Impact of Event Scale was used to retrospectively measure psychological impact of the safety incident at the time of the event and compare it with psychological impact at the time of the survey. Results Individual, situational as well as organisational aspects influenced psychological impact and recovery of a patient safety incident. Psychological impact is higher when the degree of harm for the patient is more severe, when healthcare professionals feel responsible for the incident and among female healthcare professionals. Impact of degree of harm differed across clinicians. Psychological impact is lower among more optimistic professionals. Overall, impact decreased significantly over time. This effect was more pronounced for women and for those who feel responsible for the incident. The longer ago the incident took place, the stronger impact had decreased. Also, higher psychological impact is related with the use of a more active coping and planning coping strategy, and is unrelated to support seeking coping strategies. Rendered support and a support culture reduce psychological impact, whereas a blame culture increases psychological impact. No associations were found with job experience and resilience of the health professional, the presence of a second victim support team or guideline and working in a learning culture. Conclusions Healthcare organisations should anticipate on providing their staff appropriate and timely support structures that are tailored to the healthcare professional involved in the incident and to the specific

  10. Neuromyelitis optica spectrum disorder presenting with repeated hypersomnia due to involvement of the hypothalamus and hypothalamus-amygdala linkage.

    PubMed

    Kume, Kodai; Deguchi, Kazushi; Ikeda, Kazuyo; Takata, Tadayuki; Kokudo, Yohei; Kamada, Masaki; Touge, Tetsuo; Takahashi, Toshiyuki; Kanbayashi, Takashi; Masaki, Tsutomu

    2015-06-01

    We report the case of a 46-year-old Japanese woman with neuromyelitis optica spectrum disorder presenting with repeated hypersomnia accompanied by decreased CSF orexin level. First episode associated with hypothalamic-pituitary dysfunction showed bilateral hypothalamic lesions that can cause secondary damage to the orexin neurons. The second episode associated with impaired memory showed a left temporal lesion involving the amygdala. The mechanism remains unknown, but the reduced blood flow in the hypothalamus ipsilateral to the amygdala lesion suggested trans-synaptic hypothalamic dysfunction secondary to the impaired amygdala. A temporal lesion involving the amygdala and hypothalamus could be responsible for hypersomnia due to neuromyelitis optica spectrum disorder. © The Author(s), 2015.

  11. 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.

  12. 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

  13. 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.

  14. 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.

  15. Identification of Ankrd2, a novel skeletal muscle gene coding for a stretch-responsive ankyrin-repeat protein.

    PubMed

    Kemp, T J; Sadusky, T J; Saltisi, F; Carey, N; Moss, J; Yang, S Y; Sassoon, D A; Goldspink, G; Coulton, G R

    2000-06-15

    Mechanically induced hypertrophy of skeletal muscles involves shifts in gene expression leading to increases in the synthesis of specific proteins. Full characterization of the regulation of muscle hypertrophy is a prerequisite for the development of novel therapies aimed at treating muscle wasting (atrophy) in human aging and disease. Using suppression subtractive hybridization, cDNAs corresponding to mRNAs that increase in relative abundance in response to mechanical stretch of mouse skeletal muscles in vivo were identified. A novel 1100-bp transcript was detected exclusively in skeletal muscle. This exhibited a fourfold increase in expression after 7 days of stretch. The transcript had an open reading frame of 328 amino acids encoding an ATP/GTP binding domain, a nuclear localization signal, two PEST protein-destabilization motifs, and a 132-amino-acid ankyrin-repeat region. We have named this gene ankyrin-repeat domain 2 (stretch-responsive muscle) (Ankrd2). We hypothesize that Ankrd2 plays an important role in skeletal muscle hypertrophy. Copyright 2000 Academic Press.

  16. 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

  17. 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.

  18. 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

  19. Identification of a novel polymorphism involving a CGG repeat in the PTCH gene and a genome-wide screening of CGG-containing genes.

    PubMed

    Nagao, Kazuaki; Fujii, Katsunori; Yamada, Masao; Miyashita, Toshiyuki

    2004-01-01

    Mutations in the human homologue of the Drosophila patched gene (PTCH) are responsible for the hereditary disorder called nevoid basal cell carcinoma syndrome (NBCCS). PTCH has a CGG triplet repeat located 4 bp upstream of the first methionine codon. Here we report a novel polymorphism involving the number of the CGG-repeat. The major allele (86.3%) contained a repeat size of seven, whereas the minor allele contained eight. No significant difference in the distributions of genotypes was observed between normal and NBCCS individuals. However, when the repeat was inserted between a heterologous promoter and the luciferase gene, the longer repeats tended to induce higher luciferase activities, suggesting that the repeat length potentially affects the levels of gene expression. A genome-wide screening revealed that 68 and 146 genes contained a CGG/CCG repeat in the coding region and in the 5'-untranslated region (5'-UTR), respectively. None of the genes had this repeat in 3'-UTR. Interestingly, the number of genes with a CGG repeat in the 5'-UTR was significantly higher than that with a CCG repeat in the 5'-UTR. The localization of a CGG/CCG repeat in PTCH is quite unique in that only four other genes have been found in which the repeat is localized up to 4 bp upstream of the first methionine.

  20. Proteins involved in vesicular transport and membrane fusion.

    PubMed

    Waters, M G; Griff, I C; Rothman, J E

    1991-08-01

    In the past year, new information about proteins involved in vesicular transport has been plentiful. Particularly noteworthy are the complementary findings that Sec17p is required for vesicle consumption in endoplasmic reticulum-to-Golgi transport in yeast and that an analogous activity in mammalian cells, termed SNAP, is required for transport from the cis to the medial cisternae of the Golgi apparatus.

  1. Poly-dipeptides encoded by the C9ORF72 repeats block global protein translation.

    PubMed

    Kanekura, Kohsuke; Yagi, Takuya; Cammack, Alexander J; Mahadevan, Jana; Kuroda, Masahiko; Harms, Matthew B; Miller, Timothy M; Urano, Fumihiko

    2016-05-01

    The expansion of the GGGGCC hexanucleotide repeat in the non-coding region of the Chromosome 9 open-reading frame 72 (C9orf72) gene is the most common genetic cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). This genetic alteration leads to the accumulation of five types of poly-dipeptides translated from the GGGGCC hexanucleotide repeat. Among these, poly-proline-arginine (poly-PR) and poly-glycine-arginine (poly-GR) peptides are known to be neurotoxic. However, the mechanisms of neurotoxicity associated with these poly-dipeptides are not clear. A proteomics approach identified a number of interacting proteins with poly-PR peptide, including mRNA-binding proteins, ribosomal proteins, translation initiation factors and translation elongation factors. Immunostaining of brain sections from patients with C9orf72 ALS showed that poly-GR was colocalized with a mRNA-binding protein, hnRNPA1. In vitro translation assays showed that poly-PR and poly-GR peptides made insoluble complexes with mRNA, restrained the access of translation factors to mRNA, and blocked protein translation. Our results demonstrate that impaired protein translation mediated by poly-PR and poly-GR peptides plays a role in neurotoxicity and reveal that the pathways altered by the poly-dipeptides-mRNA complexes are potential therapeutic targets for treatment of C9orf72 FTD/ALS. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  2. Viral and host proteins involved in picornavirus life cycle.

    PubMed

    Lin, Jing-Yi; Chen, Tzu-Chun; Weng, Kuo-Feng; Chang, Shih-Cheng; Chen, Li-Lien; Shih, Shin-Ru

    2009-11-20

    Picornaviruses cause several diseases, not only in humans but also in various animal hosts. For instance, human enteroviruses can cause hand-foot-and-mouth disease, herpangina, myocarditis, acute flaccid paralysis, acute hemorrhagic conjunctivitis, severe neurological complications, including brainstem encephalitis, meningitis and poliomyelitis, and even death. The interaction between the virus and the host is important for viral replication, virulence and pathogenicity. This article reviews studies of the functions of viral and host factors that are involved in the life cycle of picornavirus. The interactions of viral capsid proteins with host cell receptors is discussed first, and the mechanisms by which the viral and host cell factors are involved in viral replication, viral translation and the switch from translation to RNA replication are then addressed. Understanding how cellular proteins interact with viral RNA or viral proteins, as well as the roles of each in viral infection, will provide insights for the design of novel antiviral agents based on these interactions.

  3. Proteomic analysis of proteins involved in spermiogenesis in mouse.

    PubMed

    Guo, Xuejiang; Shen, Jian; Xia, Zhengrong; Zhang, Rui; Zhang, Ping; Zhao, Chun; Xing, Jun; Chen, Ling; Chen, Wen; Lin, Min; Huo, Ran; Su, Bing; Zhou, Zuomin; Sha, Jiahao

    2010-03-05

    Spermiogenesis is a unique process in mammals during which haploid round spermatids mature into spermatozoa in the testis. Its successful completion is necessary for fertilization and its malfunction is an important cause of male infertility. Here, we report the high-confidence identification of 2116 proteins in mouse haploid germ cells undergoing spermiogenesis: 299 of these were testis-specific and 155 were novel. Analysis of these proteins showed many proteins possibly functioning in unique processes of spermiogenesis. Of the 84 proteins annotated to be involved in vesicle-related events, VAMP4 was shown to be important for acrosome biogenesis by in vivo knockdown experiments. Knockdown of VAMP4 caused defects of acrosomal vesicle fusion and significantly increased head abnormalities in spermatids from testis and sperm from the cauda epididymis. Analysis of chromosomal distribution of the haploid genes showed underrepresentation on the X chromosome and overrepresentation on chromosome 11, which were due to meiotic sex chromosome inactivation and expansion of testis-expressed gene families, respectively. Comparison with transcriptional data showed translational regulation during spermiogenesis. This characterization of proteins involved in spermiogenesis provides an inventory of proteins useful for understanding the mechanisms of male infertility and may provide candidates for drug targets for male contraception and male infertility.

  4. 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.

  5. 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

  6. 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.

  7. Disulfide bonds in a recombinant protein modeled after a core repeat in an aquatic insect's silk protein.

    PubMed

    Smith, S V; Correia, J J; Case, S T

    1995-05-01

    We constructed a gene encoding rCAS, recombinant constant and subrepeat protein, modeled after tandem repeats found in the major silk proteins synthesized by aquatic larvae of the midge, Chironomus tentans. Bacterially synthesized rCAS was purified to near homogeneity and characterized by several biochemical and biophysical methods including amino-terminal sequencing, amino acid compositional analysis, sedimentation equilibrium ultracentrifugation, and mass spectrometry. Complementing these techniques with quantitative sulfhydryl assays, we discovered that the four cysteines present in rCAS form two intramolecular disulfide bonds. Mapping studies revealed that the disulfide bonds are heterogeneous. When reduced and denatured rCAS was allowed to refold and its disulfide bonding state monitored, it again adopted a conformation with two intramolecular disulfide bonds. The inherent ability of rCAS to quantitatively form two intramolecular disulfide bonds may reflect a previously unknown feature of the in vivo silk proteins from which it is derived.

  8. Identification and characterization of GSRP-56, a novel Golgi-localized spectrin repeat-containing protein

    SciTech Connect

    Kobayashi, Yuko . E-mail: yu-kobayashi@kinran.ac.jp; Katanosaka, Yuki; Iwata, Yuko; Matsuoka, Masayuki; Shigekawa, Munekazu; Wakabayashi, Shigeo . E-mail: wak@ri.ncvc.go.jp

    2006-10-01

    Spectrin repeat (SR)-containing proteins are important for regulation of integrity of biomembranes, not only the plasma membrane but also those of intracellular organelles, such as the Golgi, nucleus, endo/lysosomes, and synaptic vesicles. We identified a novel SR-containing protein, named GSRP-56 (Golgi-localized SR-containing protein-56), by a yeast two-hybrid method, using a member of the transient receptor potential channel family, TRPV2, as bait. GSRP-56 is an isoform derived from a giant SR-containing protein, Syne-1 (synaptic nuclear envelope protein-1, also referred to as Nesprin-1 or Enaptin), predicted to be produced by alternative splicing. Immunological analysis demonstrated that this isoform is a 56-kDa protein, which is localized predominantly in the Golgi apparatus in cardiomyocytes and C2C12 myoblasts/myotubes, and we found that two SR domains were required both for Golgi targeting and for interaction with TRPV2. Interestingly, overexpression of GSRP-56 resulted in a morphological change in the Golgi structure, characterized by its enlargement of cis-Golgi marker antibody-staining area, which would result partly from fragmentation of Golgi membranes. Our findings indicate that GSRP-56 is a novel, particularly small Golgi-localized member of the spectrin family, which possibly play a role in maintenance of the Golgi structure.

  9. 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.

  10. 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.

  11. Protein Phosphatases Involved in Regulating Mitosis: Facts and Hypotheses.

    PubMed

    Kim, Hyun-Soo; Fernandes, Gary; Lee, Chang-Woo

    2016-09-01

    Almost all eukaryotic proteins are subject to post-translational modifications during mitosis and cell cycle, and in particular, reversible phosphorylation being a key event. The recent use of high-throughput experimental analyses has revealed that more than 70% of all eukaryotic proteins are regulated by phosphorylation; however, the mechanism of dephosphorylation, counteracting phosphorylation, is relatively unknown. Recent discoveries have shown that many of the protein phosphatases are involved in the temporal and spatial control of mitotic events, such as mitotic entry, mitotic spindle assembly, chromosome architecture changes and cohesion, and mitotic exit. This implies that certain phosphatases are tightly regulated for timely dephosphorylation of key mitotic phosphoproteins and are essential for control of various mitotic processes. This review describes the physiological and pathological roles of mitotic phosphatases, as well as the versatile role of various protein phosphatases in several mitotic events.

  12. Novel leucine rich repeat domains in proteins from unicellular eukaryotes and bacteria.

    PubMed

    Miyashita, Hiroki; Kuroki, Yoshio; Matsushima, Norio

    2014-03-01

    Leucine rich repeats (LRRs) are present in over 20,000 proteins from viruses to eukaryotes. Two to sixty-two LRRs occur in tandem. Each repeat is typically 20-30 residues long and can be divided into an HCS (Highly conserved segment) and a VS (Variable segment). The HCS part consists of an eleven or a twelve residue stretch, LxxLxLxxNx(x/-)L, in which "L" is Leu, Ile, Val, or Phe, "N" is Asn, Thr, Ser, or Cys, "x" is a non-conserved residue, and "-" is a possible deletion site. Eight classes have been recognized. However, there are many unclassified or unrecognized LRRs. Here we performed to search novel LRRs using protein sequence database. The novel LRR domains are present over three hundred proteins, which include fungal ECM33 protein and Monosiga brevicollis LRR receptor kinase, from unicellular eukaryotes and bacteria. The HCS part is clearly different from that of the known LRRs and consists of a twelve or a thirteen residue stretch, VxGx(L/F)x(L/C)xxNx(x/-)L, that is characterized by the addition of Gly between the first conserved Val and the second conserved Leu. The novel LRRs identified here form a new family. The novel LRR domains were classified into four classes. The VS parts of the two classes are consistent with those of known, normal "SDS22-like" and "IRREKO" classes, while the other two classes have unique VS parts. The structures, functions, and evolution of the novel LRR domains and their proteins are described. The present results should stimulate various experimental studies.

  13. 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.

  14. The ESCRT-II proteins are involved in shaping the sarcoplasmic reticulum in C. elegans.

    PubMed

    Lefebvre, Christophe; Largeau, Céline; Michelet, Xavier; Fourrage, Cécile; Maniere, Xavier; Matic, Ivan; Legouis, Renaud; Culetto, Emmanuel

    2016-04-01

    The sarcoplasmic reticulum is a network of tubules and cisternae localized in close association with the contractile apparatus, and regulates Ca(2+)dynamics within striated muscle cell. The sarcoplasmic reticulum maintains its shape and organization despite repeated muscle cell contractions, through mechanisms which are still under investigation. The ESCRT complexes are essential to organize membrane subdomains and modify membrane topology in multiple cellular processes. Here, we report for the first time that ESCRT-II proteins play a role in the maintenance of sarcoplasmic reticulum integrity inC. elegans ESCRT-II proteins colocalize with the sarcoplasmic reticulum marker ryanodine receptor UNC-68. The localization at the sarcoplasmic reticulum of ESCRT-II and UNC-68 are mutually dependent. Furthermore, the characterization of ESCRT-II mutants revealed a fragmentation of the sarcoplasmic reticulum network, associated with an alteration of Ca(2+)dynamics. Our data provide evidence that ESCRT-II proteins are involved in sarcoplasmic reticulum shaping.

  15. The αRep artificial repeat protein scaffold: a new tool for crystallization and live cell applications.

    PubMed

    Valerio-Lepiniec, Marie; Urvoas, Agathe; Chevrel, Anne; Guellouz, Asma; Ferrandez, Yann; Mesneau, Agnès; de la Sierra-Gallay, Ines Li; Aumont-Nicaise, Magali; Desmadril, Michel; van Tilbeurgh, Herman; Minard, Philippe

    2015-10-01

    We have designed a new family of artificial proteins, named αRep, based on HEAT (acronym for Huntingtin, elongation factor 3 (EF3), protein pphosphatase 2A (PP2A), yeast kinase Tor1) repeat proteins containing an α-helical repeated motif. The sequence of the repeated motifs, first identified in a thermostable archae protein was optimized using a consensus design strategy and used for the construction of a library of artificial proteins. All proteins from this library share the same general fold but differ both in the number of repeats and in five highly randomized amino acid positions within each repeat. The randomized side chains altogether provide a hypervariable surface on αRep variants. Sequences from this library are efficiently expressed as soluble, folded and very stable proteins. αRep binders with high affinity for various protein targets were selected by phage display. Low micromolar to nanomolar dissociation constants between partners were measured and the structures of several complexes (specific αRep/protein target) were solved by X-ray crystallography. Using GFP as a model target, it was demonstrated that αReps can be used as bait in pull-down experiments. αReps can be expressed in eukaryotic cells and specifically interact with their target addressed to different cell compartments. © 2015 Authors; published by Portland Press Limited.

  16. 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

  17. NEDD8 protein is involved in ubiquitinated inclusion bodies.

    PubMed

    Dil Kuazi, Afroz; Kito, Katsumi; Abe, Yasuhito; Shin, Ryong-Woon; Kamitani, Tetsu; Ueda, Norifumi

    2003-02-01

    Proteolysis by the ubiquitin-proteasome system is considered to play a pathological role in several degenerative diseases that involve ubiquitinated inclusion bodies. In recent years, several ubiquitin-like proteins have been isolated, but it is uncertain whether their roles are associated with protein degradation through the ubiquitin-proteasome system. NEDD8 (neural precursor cell-expressed and developmentally down-regulated gene), which consists of 81 amino acid residues, possesses the highest sequence similarity to ubiquitin. Recent studies have indicated that NEDD8 is covalently ligated to cullin family proteins, which are components of certain ubiquitin E3 ligases, by a pathway analogous to that of ubiquitin. Thus, by focusing on the structural and functional association between NEDD8 and ubiquitin, it would be of interest to know whether the NEDD8 system is involved in pathological disorders of the ubiquitin-proteasome system. This study has examined the immunohistochemical distribution of NEDD8 protein by using a highly purified antibody in normal tissues and in tissues known to contain ubiquitinated inclusions. NEDD8 protein expression was widely observed in most types of tissues. Furthermore, accumulation of the NEDD8 protein was commonly observed in ubiquitinated inclusion bodies, including Lewy bodies in Parkinson's disease, Mallory bodies in alcoholic liver disease, and Rosenthal fibres in astrocytoma. Two of ten cases of neurofibrillary tangles and senile plaques from patients with Alzheimer's disease showed intense staining for NEDD8 as well as for ubiquitin. These findings suggest the possibility that the NEDD8 system is involved in the metabolism of these inclusion bodies via the ubiquitin-proteasome system.

  18. Rearrangements involving repeated sequences within a P element preferentially occur between units close to the transposon extremities

    SciTech Connect

    Paques, F.; Bucheton, B.; Wegnez, M.

    1996-02-01

    In a previous report we described rearrangements occurring at a high rate (30% of the progeny of dysgenic flies) within a cluster of 5S genes internal to a P element. These events were characterized as precise amplifications and deletions of 5S units. Here we analyze recombination events within P elements containing two repeated arrays of 5S genes flanking a central white gene. Deletions (50%) and duplications (3%) of the white gene together with various amounts of flanking 5S genes were observed. These recombinations occur preferentially between the most external 5S units of P transposons. Such rearrangements could be favored by interactions between the proteins bound to the P terminal sequences. 39 refs., 7 figs.

  19. Protein kinase C-associated kinase (PKK), a novel membrane-associated, ankyrin repeat-containing protein kinase.

    PubMed

    Chen, L; Haider, K; Ponda, M; Cariappa, A; Rowitch, D; Pillai, S

    2001-06-15

    A novel murine membrane-associated protein kinase, PKK (protein kinase C-associated kinase), was cloned on the basis of its physical association with protein kinase Cbeta (PKCbeta). The regulated expression of PKK in mouse embryos is consistent with a role for this kinase in early embryogenesis. The human homolog of PKK has over 90% identity to its murine counterpart, has been localized to chromosome 21q22.3, and is identical to the PKCdelta-interacting kinase, DIK (Bahr, C., Rohwer, A., Stempka, L., Rincke, G., Marks, F., and Gschwendt, M. (2000) J. Biol. Chem. 275, 36350-36357). PKK comprises an N-terminal kinase domain and a C-terminal region containing 11 ankyrin repeats. PKK exhibits protein kinase activity in vitro and associates with cellular membranes. PKK exists in three discernible forms at steady state: an underphosphorylated form of 100 kDa; a soluble, cytosolic, phosphorylated form of 110 kDa; and a phosphorylated, detergent-insoluble form of 112 kDa. PKK is initially synthesized as an underphosphorylated soluble 100-kDa protein that is quantitatively converted to a detergent-soluble 110-kDa form. This conversion requires an active catalytic domain. Although PKK physically associates with PKCbeta, it does not phosphorylate this PKC isoform. However, PKK itself may be phosphorylated by PKCbeta. PKK represents a developmentally regulated protein kinase that can associate with membranes. The functional significance of its association with PKCbeta remains to be ascertained.

  20. Involvement of Inflammation and Adverse Vascular Remodelling in the Blood Pressure Raising Effect of Repeatedly Heated Palm Oil in Rats

    PubMed Central

    Ng, Chun-Yi; Kamisah, Yusof; Faizah, Othman; Jubri, Zakiah; Qodriyah, Hj Mohd Saad; Jaarin, Kamsiah

    2012-01-01

    Oil thermoxidation during deep frying generates harmful oxidative free radicals that induce inflammation and increase the risk of hypertension. This study aimed to investigate the effect of repeatedly heated palm oil on blood pressure, aortic morphometry, and vascular cell adhesion molecule-1 (VCAM-1) expression in rats. Male Sprague-Dawley rats were divided into five groups: control, fresh palm oil (FPO), one-time-heated palm oil (1HPO), five-time-heated palm oil (5HPO), or ten-time-heated palm oil (10HPO). Feeding duration was six months. Blood pressure was measured at baseline and monthly using tail-cuff method. After six months, the rats were sacrificed and the aortic arches were dissected for morphometric and immunohistochemical analyses. FPO group showed significantly lower blood pressure than all other groups. Blood pressure was increased significantly in 5HPO and 10HPO groups. The aortae of 5HPO and 10HPO groups showed significantly increased thickness and area of intima-media, circumferential wall tension, and VCAM-1 than other groups. Elastic lamellae were disorganised and fragmented in 5HPO- and 10HPO-treated rats. VCAM-1 expression showed a significant positive correlation with blood pressure. In conclusion, prolonged consumption of repeatedly heated palm oil causes blood pressure elevation, adverse remodelling, and increased VCAM-1, which suggests a possible involvement of inflammation. PMID:22778962

  1. Large-scale modelling of the divergent spectrin repeats in nesprins: giant modular proteins.

    PubMed

    Autore, Flavia; Pfuhl, Mark; Quan, Xueping; Williams, Aisling; Roberts, Roland G; Shanahan, Catherine M; Fraternali, Franca

    2013-01-01

    Nesprin-1 and nesprin-2 are nuclear envelope (NE) proteins characterized by a common structure of an SR (spectrin repeat) rod domain and a C-terminal transmembrane KASH [Klarsicht-ANC-Syne-homology] domain and display N-terminal actin-binding CH (calponin homology) domains. Mutations in these proteins have been described in Emery-Dreifuss muscular dystrophy and attributed to disruptions of interactions at the NE with nesprins binding partners, lamin A/C and emerin. Evolutionary analysis of the rod domains of the nesprins has shown that they are almost entirely composed of unbroken SR-like structures. We present a bioinformatical approach to accurate definition of the boundaries of each SR by comparison with canonical SR structures, allowing for a large-scale homology modelling of the 74 nesprin-1 and 56 nesprin-2 SRs. The exposed and evolutionary conserved residues identify important pbs for protein-protein interactions that can guide tailored binding experiments. Most importantly, the bioinformatics analyses and the 3D models have been central to the design of selected constructs for protein expression. 1D NMR and CD spectra have been performed of the expressed SRs, showing a folded, stable, high content α-helical structure, typical of SRs. Molecular Dynamics simulations have been performed to study the structural and elastic properties of consecutive SRs, revealing insights in the mechanical properties adopted by these modules in the cell.

  2. 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.

  3. Large-Scale Modelling of the Divergent Spectrin Repeats in Nesprins: Giant Modular Proteins

    PubMed Central

    Autore, Flavia; Pfuhl, Mark; Quan, Xueping; Williams, Aisling; Roberts, Roland G.; Shanahan, Catherine M.; Fraternali, Franca

    2013-01-01

    Nesprin-1 and nesprin-2 are nuclear envelope (NE) proteins characterized by a common structure of an SR (spectrin repeat) rod domain and a C-terminal transmembrane KASH [Klarsicht–ANC–Syne-homology] domain and display N-terminal actin-binding CH (calponin homology) domains. Mutations in these proteins have been described in Emery-Dreifuss muscular dystrophy and attributed to disruptions of interactions at the NE with nesprins binding partners, lamin A/C and emerin. Evolutionary analysis of the rod domains of the nesprins has shown that they are almost entirely composed of unbroken SR-like structures. We present a bioinformatical approach to accurate definition of the boundaries of each SR by comparison with canonical SR structures, allowing for a large-scale homology modelling of the 74 nesprin-1 and 56 nesprin-2 SRs. The exposed and evolutionary conserved residues identify important pbs for protein-protein interactions that can guide tailored binding experiments. Most importantly, the bioinformatics analyses and the 3D models have been central to the design of selected constructs for protein expression. 1D NMR and CD spectra have been performed of the expressed SRs, showing a folded, stable, high content α-helical structure, typical of SRs. Molecular Dynamics simulations have been performed to study the structural and elastic properties of consecutive SRs, revealing insights in the mechanical properties adopted by these modules in the cell. PMID:23671687

  4. 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.

  5. 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.

  6. 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.

  7. Identifying Unstable Regions of Proteins Involved in Misfolding Diseases

    NASA Astrophysics Data System (ADS)

    Guest, Will; Cashman, Neil; Plotkin, Steven

    2009-05-01

    Protein misfolding is a necessary step in the pathogenesis of many diseases, including Creutzfeldt-Jakob disease (CJD) and familial amyotrophic lateral sclerosis (fALS). Identifying unstable structural elements in their causative proteins elucidates the early events of misfolding and presents targets for inhibition of the disease process. An algorithm was developed to calculate the Gibbs free energy of unfolding for all sequence-contiguous regions of a protein using three methods to parameterize energy changes: a modified G=o model, changes in solvent-accessible surface area, and all-atoms molecular dynamics. The entropic effects of disulfide bonds and post-translational modifications are treated analytically. It incorporates a novel method for finding local dielectric constants inside a protein to accurately handle charge effects. We have predicted the unstable parts of prion protein and superoxide dismutase 1, the proteins involved in CJD and fALS respectively, and have used these regions as epitopes to prepare antibodies that are specific to the misfolded conformation and show promise as therapeutic agents.

  8. Identification of a unique TLR2-interacting peptide motif in a microbial leucine-rich-repeat protein

    PubMed Central

    Myneni, Srinivas R.; Settem, Rajendra P.; Sojar, Hakimuddin T.; Malone, James P.; Vuokko, Loimaranta; Nakajima, Takuma; Sharma, Ashu

    2012-01-01

    Pathogenesis of many bacterially-induced inflammatory diseases is driven by toll- like receptor (TLR) mediated immune responses following recognition of bacterial factors by different TLRs. Periodontitis is a chronic inflammation of the tooth supporting apparatus often leading to tooth loss, and is caused by a Gram-negative bacterial consortium that includes Tannerella forsythia. This bacterium expresses a virulence factor, the BspA, which drives periodontal inflammation by activating TLR2. The N- terminal portion of the BspA protein comprises a leucine-rich repeat (LRR) domain previously shown to be involved in the binding and activation of TLR2. The objective of the current study was to identify specific epitopes in the LRR domain of BspA that interact with TLR2. Our results demonstrate that a sequence motif GC(S/T)GLXSIT is involved in mediating the interaction of BspA with TLR2. Thus, our study has identified a peptide motif that mediates the binding of a bacterial protein to TLR2 and highlights the promiscuous nature of TLR2 with respect to ligand binding. This work could provide a structural basis for designing peptidomimetics to modulate the activity of TLR2 in order to block bacterially-induced inflammation. PMID:22695115

  9. First identification of proteins involved in motility of Mycoplasma gallisepticum.

    PubMed

    Indikova, Ivana; Vronka, Martin; Szostak, Michael P

    2014-10-17

    Mycoplasma gallisepticum, the most pathogenic mycoplasma in poultry, is able to glide over solid surfaces. Although this gliding motility was first observed in 1968, no specific protein has yet been shown to be involved in gliding. We examined M. gallisepticum strains and clonal variants for motility and found that the cytadherence proteins GapA and CrmA were required for gliding. Loss of GapA or CrmA resulted in the loss of motility and hemadsorption and led to drastic changes in the characteristic flask-shape of the cells. To identify further genes involved in motility, a transposon mutant library of M. gallisepticum was generated and screened for motility-deficient mutants, using a screening assay based on colony morphology. Motility-deficient mutants had transposon insertions in gapA and the neighbouring downstream gene crmA. In addition, insertions were seen in gene mgc2, immediately upstream of gapA, in two motility-deficient mutants. In contrast to the GapA/CrmA mutants, the mgc2 motility mutants still possessed the ability to hemadsorb. Complementation of these mutants with a mgc2-hexahistidine fusion gene restored the motile phenotype. This is the first report assigning specific M. gallisepticum proteins to involvement in gliding motility.

  10. Proteomic detection of proteins involved in perchlorate and chlorate metabolism.

    PubMed

    Bansal, Reema; Deobald, Lee A; Crawford, Ronald L; Paszczynski, Andrzej J

    2009-09-01

    Mass spectrometry and a time-course cell lysis method were used to study proteins involved in perchlorate and chlorate metabolism in pure bacterial cultures and environmental samples. The bacterial cultures used included Dechlorosoma sp. KJ, Dechloromonas hortensis, Pseudomonas chloritidismutans ASK-1, and Pseudomonas stutzeri. The environmental samples included an anaerobic sludge enrichment culture from a sewage treatment plant, a sample of a biomass-covered activated carbon matrix from a bioreactor used for treating perchlorate-contaminated drinking water, and a waste water effluent sample from a paper mill. The approach focused on detection of perchlorate (and chlorate) reductase and chlorite dismutase proteins, which are the two central enzymes in the perchlorate (or chlorate) reduction pathways. In addition, acetate-metabolizing enzymes in pure bacterial samples and housekeeping proteins from perchlorate (or chlorate)-reducing microorganisms in environmental samples were also identified.

  11. 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

  12. Molecular signaling involving intrinsically disordered proteins in prostate cancer

    PubMed Central

    Russo, Anna; Manna, Sara La; Novellino, Ettore; Malfitano, Anna Maria; Marasco, Daniela

    2016-01-01

    Investigations on cellular protein interaction networks (PINs) reveal that proteins that constitute hubs in a PIN are notably enriched in Intrinsically Disordered Proteins (IDPs) compared to proteins that constitute edges, highlighting the role of IDPs in signaling pathways. Most IDPs rapidly undergo disorder-to-order transitions upon binding to their biological targets to perform their function. Conformational dynamics enables IDPs to be versatile and to interact with a broad range of interactors under normal physiological conditions where their expression is tightly modulated. IDPs are involved in many cellular processes such as cellular signaling, transcriptional regulation, and splicing; thus, their high-specificity/low-affinity interactions play crucial roles in many human diseases including cancer. Prostate cancer (PCa) is one of the leading causes of cancer-related mortality in men worldwide. Therefore, identifying molecular mechanisms of the oncogenic signaling pathways that are involved in prostate carcinogenesis is crucial. In this review, we focus on the aspects of cellular pathways leading to PCa in which IDPs exert a primary role. PMID:27212129

  13. [Proteins of human milk involved in immunological processes].

    PubMed

    Lis, Jolanta; Orczyk-Pawiłowicz, Magdalena; Kątnik-Prastowska, Iwona

    2013-05-31

    Human milk contains a lot of components (i.e. proteins, carbohydrates, lipids, inorganic elements) which provide basic nutrients for infants during the first period of their lives. Qualitative composition of milk components of healthy mothers is similar, but their levels change during lactation stages. Colostrum is the fluid secreted during the first days postpartum by mammary epithelial cells. Colostrum is replaced by transitional milk during 5-15 days postpartum and from 15 days postpartum mature milk is produced. Human milk, apart from nutritional components, is a source of biologically active molecules, i.e. immunoglobulins, growth factors, cytokines, acute phase proteins, antiviral and antibacterial proteins. Such components of human milk are responsible for specific biological activities of human milk. This secretion plays an important role in growth and development of newborns. Bioactive molecules present in the milk support the immature immune system of the newborn and also protect against the development of infection. In this article we describe the pathways involved in the production and secretion of human milk, the state of knowledge on the proteome of human milk, and the contents of components of milk during lactation. Moreover, some growth factors and proteins involved in innate and specific immunity, intercellular communication, immunomodulation, and inflammatory processes have been characterized.

  14. Molecular characterization of cotton C-repeat/dehydration-responsive element binding factor genes that are involved in response to cold stress.

    PubMed

    Ma, Liu-Feng; Zhang, Jian-Min; Huang, Geng-Qing; Li, Yang; Li, Xue-Bao; Zheng, Yong

    2014-07-01

    Low temperature, drought and salinity are major abiotic stresses that influence survival, productivity and geographical distribution of many important crops across the globe. The C-repeat/dehydration-responsive element binding transcription factors (CBF/DREB) are important proteins involved in response to abiotic stresses in plants. In this study, twenty-one CBF genes were identified in cotton (Gossypium hirsutum) by bioinformatic approach. The twenty-one CBF genes (named as GhCBF1--GhCBF21) were characterized to encode proteins that share high similarity with those plant cold stress-related CBF proteins, which contain the classic AP2 domain of 58 amino acid residues. Phylogenetic analysis revealed that the isolated cotton CBF genes can be classified into 4 groups: GhCBF I, GhCBF II, GhCBF III and GhCBF IV. RT-PCR analysis indicated that GhCBF genes were up-regulated in cotton plants under cold stress. Furthermore, four GhCBF genes were up-regulated in cotton under salinity and drought treatments. Our data provided valuable information for further exploring the roles of the CBF genes in cotton development and in response to cold stress.

  15. Carbon and amide detect backbone assignment methods of a novel repeat protein from the staphylocoagulase in S. aureus.

    PubMed

    Voehler, Markus; Ashoka, Maddur Appajaiah; Meiler, Jens; Bock, Paul E

    2017-08-17

    The C-terminal repeat domain of staphylocoagulase that is secreted by the S. aureus is believed to play an important role interacting with fibrinogen and promotes blood clotting. To study this interaction by NMR, full assignment of each amide residue in the HSQC spectrum was required. Despite of the short sequence of the repeat construct, the HSQC spectrum contained a substantial amount of overlapped and exchange broadened resonances, indicating little secondary or tertiary structure. This caused severe problems while using the conventional, amide based NMR method for the backbone assignment. With the growing interest in small apparently disordered proteins, these issues are being faced more frequently. An alternative strategy to improve the backbone assignment capability involved carbon direct detection methods. Circumventing the amide proton detection offers a larger signal dispersion and more uniform signal intensity. For peptides with higher concentrations and in combination with the cold carbon channels of new cryoprobes, higher fields, and sufficiently long relaxation times, the disadvantage of the lower sensitivity of the (13)C nucleus can be overcome. Another advantage of this method is the assignment of the proline backbone residues. Complete assignment with the carbon-detected strategy was achieved with a set of only two 3D, one 2D, and a HNCO measurement, which was necessary to translate the information to the HSQC spectrum.

  16. 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.

  17. 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.

  18. 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

  19. 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

  20. A Wd Repeat Protein, Rec14, Essential for Meiotic Recombination in Schizosaccharomyces Pombe

    PubMed Central

    Evans, D. H.; Li, Y. F.; Fox, M. E.; Smith, C. R.

    1997-01-01

    Mutations in the Schizosaccharomyces pombe rec14 gene reduce meiotic recombination by as much as a factor of 1000 in the three intervals tested on chromosomes I and III. A DNA clone complementing the rec14 mutation was shown by genetic and physical analysis to contain the rec14 gene, which was functional in plasmid-borne inserts as small as 1.4 kb. The rec14 gene contains two exons separated by a 53-bp intron, which was confirmed by analysis of rec14 transcripts. The spliced transcript encodes a protein product of 302 amino acids, which contains six WD repeat motifs found in the G-beta transducin family of proteins and other proteins, including the Saccharomyces cerevisiae Ski8 (Rec103) protein. Although the rec14 transcripts were present in mitotically dividing cells, rec14 mutations had no detectable effect on mitotic recombination. The pattern of expression of rec14 differs from that of previously analyzed S. pombe rec genes. Based upon mutant phenotypes and amino acid sequence similarities, we propose that S. pombe Rec14 is a functional homologue of S. cerevisiae Rec103. PMID:9258671

  1. 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.

  2. 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.

  3. 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

  4. Cloning, characterization, and serodiagnostic evaluation of Leishmania infantum tandem repeat proteins.

    PubMed

    Goto, Yasuyuki; Coler, Rhea N; Guderian, Jeffrey; Mohamath, Raodoh; Reed, Steven G

    2006-07-01

    Visceral leishmaniasis (VL) is a form of leishmaniasis, which is caused by infection with the protozoan parasite Leishmania, and is often fatal unless it is treated. Rapid and accurate diagnosis of VL is important for effective treatment. Here we report the cloning of previously undescribed tandem repeat (TR) proteins of Leishmania infantum and an evaluation of VL patient antibody responses to the corresponding proteins. By screening an L. infantum expression library with sera from human VL patients or infected hamsters, we identified 43 genes encoding B-cell antigens. Surprisingly, 19 of the 43 genes (44%) were TR proteins, and that percentage was significantly higher than that for genes picked randomly from the database. We then expressed the TR regions of LinJ16.1750, LinJ22.1590, and LinJ33.2870 and the entire LinJ28.2310 protein. These recombinant proteins were all recognized by Sudanese VL patient sera in an enzyme-linked immunosorbent assay. Recombinant LinJ16.1750 (rLinJ16.1750) showed the best performance among these antigens in terms of both sensitivity and specificity. Serological evaluation revealed that 97% (34 of 35) of Sudanese VL patients had significantly elevated antibody levels to rLinJ16.1750. Furthermore, when eight of the patient sera which had low reactivities to rK39 were tested with the novel recombinant antigens, some of the sera showed stronger antibody responses to these antigens than to rK39. Our results suggest that TR regions from the novel L. infantum proteins identified in this study are immunodominant B-cell epitopes and may represent good candidates for serodiagnosis of VL.

  5. Analysis of proteins involved in biodegradation of crop biomass

    NASA Technical Reports Server (NTRS)

    Crawford, Kamau; Trotman, Audrey

    1998-01-01

    The biodegradation of crop biomass for re-use in crop production is part of the bioregenerative life support concept proposed by the National Aeronautics and Space Administration (NASA) for long duration, manned space exploration. The current research was conducted in the laboratory to evaluate the use of electrophoretic analysis as a means of rapidly assaying for constitutive and induced proteins associated with the bacterial degradation of crop residue. The proteins involved in crop biomass biodegradation are either constitutive or induced. As a result, effluent and cultures were examined to investigate the potential of using electrophoretic techniques as a means of monitoring the biodegradation process. Protein concentration for optimum banding patterns was determined using the Bio-Rad Protein Assay kit. Four bacterial soil isolates were obtained from the G.W. Carver research Farm at Tuskegee University and used in the decomposition of components of plant biomass. The culture, WDSt3A was inoculated into 500 mL of either Tryptic Soy Broth or Nutrient Broth. Incubation, with shaking of each flask was for 96 hours at 30 C. The cultures consistently gave unique banding patterns under denaturing protein electrophoresis conditions, The associated extracellular enzymes also yielded characteristic banding patterns over a 14-day period, when native electrophoresis techniques were used to examine effluent from batch culture bioreactors. The current study evaluated sample preparation and staining protocols to determine the ease of use, reproducibility and reliability, as well as the potential for automation.

  6. Analysis of proteins involved in biodegradation of crop biomass

    NASA Technical Reports Server (NTRS)

    Crawford, Kamau; Trotman, Audrey

    1998-01-01

    The biodegradation of crop biomass for re-use in crop production is part of the bioregenerative life support concept proposed by the National Aeronautics and Space Administration (NASA) for long duration, manned space exploration. The current research was conducted in the laboratory to evaluate the use of electrophoretic analysis as a means of rapidly assaying for constitutive and induced proteins associated with the bacterial degradation of crop residue. The proteins involved in crop biomass biodegradation are either constitutive or induced. As a result, effluent and cultures were examined to investigate the potential of using electrophoretic techniques as a means of monitoring the biodegradation process. Protein concentration for optimum banding patterns was determined using the Bio-Rad Protein Assay kit. Four bacterial soil isolates were obtained from the G.W. Carver research Farm at Tuskegee University and used in the decomposition of components of plant biomass. The culture, WDSt3A was inoculated into 500 mL of either Tryptic Soy Broth or Nutrient Broth. Incubation, with shaking of each flask was for 96 hours at 30 C. The cultures consistently gave unique banding patterns under denaturing protein electrophoresis conditions, The associated extracellular enzymes also yielded characteristic banding patterns over a 14-day period, when native electrophoresis techniques were used to examine effluent from batch culture bioreactors. The current study evaluated sample preparation and staining protocols to determine the ease of use, reproducibility and reliability, as well as the potential for automation.

  7. Important amino acid residues involved in folding and binding of protein-protein complexes.

    PubMed

    Kulandaisamy, A; Lathi, V; ViswaPoorani, K; Yugandhar, K; Gromiha, M Michael

    2017-01-01

    Protein-protein interactions perform diverse functions in living organism. The integrative analysis of binding and stabilizing residues will provide insights on the functions of protein-protein complexes. In this work, we constructed a non-redundant dataset of 261 protein-protein complexes and identified binding site residues, stabilizing residues and common to both binding and stabilizing, termed as "key residues". We found that 6.1% of residues are involved in binding and 6.8% of residues are important for folding and stability. Among them, only 2% are involved in both folding and binding, which shows the importance and specific roles played by these residues. The key residues have been analyzed based on protein function, binding affinity, rigid and flexible complexes, amino acid preference and structure based parameters. We found that high affinity complexes have more key residues than low affinity complexes. In addition, key residues are enriched with the combination of specific hydrophobic and charged/polar residues. Atomic contacts between interacting proteins have distinct preferences of polar-polar, nonpolar-nonpolar and polar-nonpolar contacts in different functional classes of protein-protein complexes. Further, the influence of sequence and structural parameters such as surrounding hydrophobicity, solvent accessibility, secondary structure, long-range order and conservation score has been discussed. The analysis can be used to comprehend the interplay between stability and binding in protein-protein complexes. Copyright © 2016 Elsevier B.V. All rights reserved.

  8. 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.

  9. Repeat organic dust exposure-induced monocyte inflammation is associated with protein kinase C activity.

    PubMed

    Poole, Jill A; Wyatt, Todd A; Von Essen, Susanna G; Hervert, John; Parks, Conrad; Mathisen, Tracy; Romberger, Debra J

    2007-08-01

    Organic dust exposure results in an inflammatory response that attenuates over time, but repetitive exposures can result in chronic respiratory diseases. Mechanisms underlying this modulated response are not clear. This study investigated the effects of repeat versus single organic dust exposure-induced inflammatory mediators and protein kinase C (PKC) activity in monocytes. Settled organic dust was obtained from swine confinement facilities. Promonocytic THP-1 cells and human peripheral blood monocytes were pretreated with or without dust extract and then restimulated. Culture supernatants were evaluated for TNF-alpha, IL-6, CXCL8, and IL-10. Responses were compared with endotoxin-depleted dust, LPS, and peptidoglycan. PKC isoform (alpha, delta, epsilon, zeta) activation was evaluated by direct kinase activity. PKC isoform inhibitors' effects on TNF-alpha secretion were studied. Single exposure to organic dust stimulated monocyte secretion of TNF-alpha, IL-6, CXCL8, and IL-10 compared with unstimulated cells. TNF-alpha and IL-6 were diminished in pretreated cells restimulated with dust. Secretion of CXCL8 and IL-10 remained persistently elevated. TNF-alpha responses were retained after marked depletion of endotoxin. Dust exposure induced significant PKC alpha, delta, epsilon, and zeta activation, peaking at 30 to 60 minutes. PKC isoform activation was attenuated in repeat exposed cells. Inhibition of PKCalpha and PKCepsilon reduced dust-induced TNF-alpha secretion. Repeat organic dust exposure modulated inflammatory mediator production in monocytes independent of endotoxin. The inability of PKC to be reactivated may account for this observation. Targeting PKC and specific mediators associated with repetitive organic dust exposure may result in novel therapeutic strategies.

  10. Functional roles of the pepper leucine-rich repeat protein and its interactions with pathogenesis-related and hypersensitive-induced proteins in plant cell death and immunity.

    PubMed

    Hong, Jeum Kyu; Hwang, In Sun; Hwang, Byung Kook

    2017-05-15

    Pepper leucine-rich repeat protein (CaLRR1) interacts with defense response proteins to regulate plant cell death and immunity. This review highlights the current understanding of the molecular functions of CaLRR1 and its interactor proteins. Plant cell death and immune responses to microbial pathogens are controlled by complex and tightly regulated molecular signaling networks. Xanthomonas campestris pv. vesicatoria (Xcv)-inducible pepper (Capsicum annuum) leucine-rich repeat protein 1 (CaLRR1) serves as a molecular marker for plant cell death and immunity signaling. In this review, we discuss recent advances in elucidating the functional roles of CaLRR1 and its interacting plant proteins, and understanding how they are involved in the cell death and defense responses. CaLRR1 physically interacts with pepper pathogenesis-related proteins (CaPR10 and CaPR4b) and hypersensitive-induced reaction protein (CaHIR1) to regulate plant cell death and defense responses. CaLRR1 is produced in the cytoplasm and trafficked to the extracellular matrix. CaLRR1 binds to CaPR10 in the cytoplasm and CaPR4b and CaHIR1 at the plasma membrane. CaLRR1 synergistically accelerates CaPR10-triggered hypersensitive cell death, but negatively regulates CaPR4b- and CaHIR1-triggered cell death. CaHIR1 interacts with Xcv filamentous hemagglutinin (Fha1) to trigger disease-associated cell death. The subcellular localization and cellular function of these CaLRR1 interactors during plant cell death and defense responses were elucidated by Agrobacterium-mediated transient expression, virus-induced gene silencing, and transgenic overexpression studies. CaPR10, CaPR4b, and CaHIR1 positively regulate defense signaling mediated by salicylic acid and reactive oxygen species, thereby activating hypersensitive cell death and disease resistance. A comprehensive understanding of the molecular functions of CaLRR1 and its interacting protein partners in cell death and defense responses will provide valuable

  11. 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

  12. 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.

  13. 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.

  14. 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. © 2014 Wiley Periodicals, Inc.

  15. 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

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

    USDA-ARS?s Scientific Manuscript database

    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...

  17. 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.

  18. 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

  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. 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.

  1. reduced ocelli Encodes the Leucine Rich Repeat Protein Pray For Elves in Drosophila melanogaster

    PubMed Central

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

    2009-01-01

    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, rdo1 and rdo2, 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. PMID:18820435

  2. 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

  3. [Clustered regularly interspaced short palindromic repeat associated protein genes cas1 and cas2 in Shigella].

    PubMed

    Xue, Zerun; Wang, Yingfang; Duan, Guangcai; Wang, Pengfei; Wang, Linlin; Guo, Xiangjiao; Xi, Yuanlin

    2014-05-01

    To detect the distribution of clustered regularly interspaced short palindromic repeat (CRISPR) associated protein genes cas1 and cas2 in Shigella and to understand the characteristics of CRISPR with relationship between CRISPR and related characteristics on drug resistance. CRISPR associated protein genes cas1 and cas2 in Shigella were detected by PCR, with its products sequenced and compared. The CRISPR-associated protein genes cas1 and cas2 were found in all the 196 Shigella isolates which were isolated at different times and locations in China. Consistencies showed through related sequencing appeared as follows: cas2, cas1 (a) and cas1 (b) were 96.44%, 97.61% and 96.97%, respectively. There were two mutations including 3177129 site(C→G)and 3177126 site (G→C) of cas1 (b) gene in 2003135 strain which were not found in the corresponding sites of Z23 and 2008113. showed that in terms of both susceptibility and antibiotic-resistance, strain 2003135 was stronger than Z23 and 2008113. CRISPR system widely existed in Shigella, with the level of drug resistance in cas1 (b) gene mutant strains higher than in wild strains. Cas1 (b) gene mutation might be one of the reasons causing the different levels of resistance.

  4. Improved haplotype analysis of human myelin basic protein short tandem repeat loci.

    PubMed

    Watanabe, G; Umetsu, K; Yuasa, I; Suzuki, T

    2000-06-01

    We report an improved haplotype analysis of the human myelin basic protein gene (MBP) short tandem repeat (STR) polymorphism. The polymorphic G-->A transition and 2 conventional STR polymorphisms, MBPA and MBPB, were simultaneously determined by an amplified product length polymorphism technique. After the MBPC fragments containing MBPA and MBPB were amplified, the linkage of these 2 STR loci was determined by a second amplification, using polymerase chain reaction (PCR) technique, of the isolated MBPC fragments. The present haplotype analysis dispensed with family studies for the haplotyping of MBPA and MBPB. Polymorphisms of the MBP loci studied in German and Japanese populations showed a high genomic variation. Haplotype analysis of the MBP loci showed distinct differences between the German and the Japanese populations. Consequently, haplotype analysis of the MBP loci promises to be useful in forensic identification and paternity testing.

  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. 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.

  7. Repeat use of human recombinant bone morphogenetic protein-2 for second level lumbar arthrodesis.

    PubMed

    Singh, Kern; Dumonski, Mark; Stanley, Tom; Ponnappan, Ravi; Phillips, Frank M

    2011-02-01

    Prospective randomized controlled animal model. The purpose of this study is to determine whether the readministration of human recombinant bone morphogenetic protein-2 (rhBMP-2) induces an immune response and inhibits successful fusion in repeat posterolateral spinal surgery. Little research has been performed on the effectiveness or immunoreactivity of rhBMP-2 (Infuse, Medtronic, Memphis, TN) in the context of its reuse in posterolateral fusion spinal surgery at adjacent levels. A total of 34 New Zealand White rabbits underwent posterior intertransverse process fusion with the use of rhBMP-2 delivered on an absorbable collagen sponge (rhBMP-2/ACS). Two rabbits were killed early leaving 32 total rabbits. Serologic studies (Type I bovine collagen and rhBMP-2 antibodies) were obtained at 2-week intervals throughout the experiment. At 10 weeks, posteroanterior radiographs confirmed solid fusion masses in all rabbits. The 32 rabbits were randomly separated into 2 groups of 16, and each group underwent an adjacent level, bilateral intertransverse process fusion with either rhBMP-2/ACS or iliac crest. There was no statistical difference in fusion rates with repeat use of rhBMP-2 (n = 15/16, 94%) or iliac crest (n = 11/16, 69%) (P = 0.17) at the adjacent level. Four rabbits (n = 4/32, 13%) developed rhBMP-2 antibodies. Of these 4 rabbits, 1 developed anti-rhBMP antibodies after the first exposure and 3 developed antibodies after the second surgery. Eight rabbits (n = 8/32, 25%) developed collagen antibodies with 7 rabbits developing antibodies after the first exposure and 1 rabbit developing antibodies after the second exposure. The development of antibodies did not effect fusion rates. No rabbit demonstrated evidence of a systemic or anaphylactic reaction to repeat exposure to rhBMP-2. rhBMP-2 appears to be successful in promoting intertransverse fusions when used in both primary and repeat fusion environments. The infrequent development of antibodies to rhBMP-2 after

  8. Involvement of heat shock proteins in gluten-sensitive enteropathy.

    PubMed

    Sziksz, Erna; Pap, Domonkos; Veres, Gábor; Fekete, Andrea; Tulassay, Tivadar; Vannay, Ádám

    2014-06-07

    Gluten-sensitive enteropathy, also known as coeliac disease (CD), is an autoimmune disorder occurring in genetically susceptible individuals that damages the small intestine and interferes with the absorption of other nutrients. As it is triggered by dietary gluten and related prolamins present in wheat, rye and barley, the accepted treatment for CD is a strict gluten-free diet. However, a complete exclusion of gluten-containing cereals from the diet is often difficult, and new therapeutic strategies are urgently needed. A class of proteins that have already emerged as drug targets for other autoimmune diseases are the heat shock proteins (HSPs), which are highly conserved stress-induced chaperones that protect cells against harmful extracellular factors. HSPs are expressed in several tissues, including the gastrointestinal tract, and their levels are significantly increased under stress circumstances. HSPs exert immunomodulatory effects, and also play a crucial role in the maintenance of epithelial cell structure and function, as they are responsible for adequate protein folding, influence the degradation of proteins and cell repair processes after damage, and modulate cell signalling, cell proliferation and apoptosis. The present review discusses the involvement of HSPs in the pathophysiology of CD. Furthermore, HSPs may represent a useful therapeutic target for the treatment of CD due to the cytoprotective, immunomodulatory, and anti-apoptotic effects in the intestinal mucosal barrier.

  9. Identification of an additional protein involved in mannan biosynthesis

    PubMed Central

    Wang, Yan; Mortimer, Jennifer C; Davis, Jonathan; Dupree, Paul; Keegstra, Kenneth

    2013-01-01

    Galactomannans comprise a β-1,4-mannan backbone substituted with α-1,6-galactosyl residues. Genes encoding the enzymes that are primarily responsible for backbone synthesis and side-chain addition of galactomannans were previously identified and characterized. To identify additional genes involved in galactomannan biosynthesis, we previously performed deep EST profiling of fenugreek (Trigonella foenum-graecum L.) seed endosperm, which accumulates large quantities of galactomannans as a reserve carbohydrate during seed development. One of the candidate genes encodes a protein that is likely to be a glycosyltransferase. Because this protein is involved in mannan biosynthesis, we named it ‘mannan synthesis-related’ (MSR). Here, we report the characterization of a fenugreek MSR gene (TfMSR) and its two Arabidopsis homologs, AtMSR1 and AtMSR2. TfMSR was highly and specifically expressed in the endosperm. TfMSR, AtMSR1 and AtMSR2 proteins were all determined to be localized to the Golgi by fluorescence confocal microscopy. The level of mannosyl residues in stem glucomannans decreased by approximately 40% for Arabidopsis msr1 single T-DNA insertion mutants and by more than 50% for msr1 msr2 double mutants, but remained unchanged for msr2 single mutants. In addition, in vitro mannan synthase activity from the stems of msr1 single and msr1 msr2 double mutants also decreased. Expression of AtMSR1 or AtMSR2 in the msr1 msr2 double mutant completely or partially restored mannosyl levels. From these results, we conclude that the MSR protein is important for mannan biosynthesis, and offer some ideas about its role. PMID:22966747

  10. Genes and proteins involved in bacterial magnetic particle formation.

    PubMed

    Matsunaga, Tadashi; Okamura, Yoshiko

    2003-11-01

    Magnetic bacteria synthesize intracellular magnetosomes that impart a cellular swimming behaviour referred to as magnetotaxis. The magnetic structures aligned in chains are postulated to function as biological compass needles allowing the bacterium to migrate along redox gradients through the Earth's geomagnetic field lines. Despite the discovery of this unique group of microorganisms 28 years ago, the mechanisms of magnetic crystal biomineralization have yet to be fully elucidated. This review describes the current knowledge of the genes and proteins involved in magnetite formation in magnetic bacteria and the biotechnological applications of biomagnetites in the interdisciplinary fields of nanobiotechnology, medicine and environmental management.

  11. 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.

  12. RAP, the Sole Octotricopeptide Repeat Protein in Arabidopsis, Is Required for Chloroplast 16S rRNA Maturation[W

    PubMed Central

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

    2014-01-01

    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. PMID:24585838

  13. 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.

  14. 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

  15. 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

  16. 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.

  17. Double-stranded Endonuclease Activity in Bacillus halodurans Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated Cas2 Protein*

    PubMed Central

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

    2012-01-01

    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 (Mg2+ or Mn2+), 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. PMID:22942283

  18. 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.

  19. Cardiac Ankyrin Repeat Protein Attenuates Cardiac Hypertrophy by Inhibition of ERK1/2 and TGF-β Signaling Pathways

    PubMed Central

    Jia, Chunshi; Ma, Xiaowei; Zhang, Lei; Xie, Xiaojie; Zhang, Yong; Gao, Xiang; Zhang, Youyi; Zhu, Dahai

    2012-01-01

    Aims It has been reported that cardiac ankyrin repeat protein is associated with heart development and diseases. This study is aimed to investigate the role of CARP in heart hypertrophy in vivo. Methods and Results We generated a cardiac-specific CARP-overexpressing transgenic mouse. Although such animals did not display any overt physiological abnormality, they developed less cardiac hypertrophy in response to pressure overload than did wildtype mice, as indicated by heart weight/body weight ratios, echocardiographic and histological analyses, and expression of hypertrophic markers. These mice also exhibited less cardiac hypertrophy after infusion of isoproterenol. To gain a molecular insight into how CARP attenuated heart hypertrophy, we examined expression of the mitogen-activated protein kinase cascade and found that the concentrations of phosphorylated ERK1/2 and MEK were markedly reduced in the hearts of transgenic mice subjected to pressure overload. In addition, the expressions of TGF-β and phosphorylated Smad3 were significantly downregulated in the hearts of CARP Tg mice in response to pressure overload. Furthermore, addition of human TGF-β1 could reverse the inhibitory effect of CARP on the hypertrophic response induced by phenylephrine in cardiomyocytes. It was also evidenced that the inhibitory effect of CARP on cardiac hypertrophy was not attributed to apoptosis. Conclusion CARP attenuates cardiac hypertrophy, in which the ERK and TGF-β pathways may be involved. Our findings highlight the significance of CARP as an anti-hypertrophic factor in therapy of cardiac hypertrophy. PMID:23227174

  20. Altering a gene involved in nuclear distribution increases the repeat-induced point mutation process in the fungus Podospora anserina.

    PubMed Central

    Bouhouche, Khaled; Zickler, Denise; Debuchy, Robert; Arnaise, Sylvie

    2004-01-01

    Repeat-induced point mutation (RIP) is a homology-dependent gene-silencing mechanism that introduces C:G-to-T:A transitions in duplicated DNA segments. Cis-duplicated sequences can also be affected by another mechanism called premeiotic recombination (PR). Both are active over the sexual cycle of some filamentous fungi, e.g., Neurospora crassa and Podospora anserina. During the sexual cycle, several developmental steps require precise nuclear movement and positioning, but connections between RIP, PR, and nuclear distributions have not yet been established. Previous work has led to the isolation of ami1, the P. anserina ortholog of the Aspergillus nidulans apsA gene, which is required for nuclear positioning. We show here that ami1 is involved in nuclear distribution during the sexual cycle and that alteration of ami1 delays the fruiting-body development. We also demonstrate that ami1 alteration affects loss of transgene functions during the sexual cycle. Genetically linked multiple copies of transgenes are affected by RIP and PR much more frequently in an ami1 mutant cross than in a wild-type cross. Our results suggest that the developmental slowdown of the ami1 mutant during the period of RIP and PR increases time exposure to the duplication detection system and thus increases the frequency of RIP and PR. PMID:15166143

  1. 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.

  2. 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

  3. 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

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

    PubMed Central

    Hu, Zhihong; Xu, Fan; Hou, Suiwen

    2014-01-01

    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. PMID:24420572

  5. 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.

  6. 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').

  7. Identification of target proteins involved in cochlear otosclerosis.

    PubMed

    Richard, Céline; Doherty, Joni K; Fayad, Jose N; Cordero, Ana; Linthicum, Fred H

    2015-06-01

    Investigation of differential protein expression will provide clues to pathophysiology in otosclerosis. Otosclerosis is a bone remodeling disorder limited to the endochondral layer of the otic capsule within the temporal bone. Some authors have suggested an inflammatory etiology for otosclerosis resulting from persistent measles virus infection involving the otic capsule. Despite numerous genetic studies, implication of candidate genes in the otosclerotic process remains elusive. We employed liquid chromatography-mass spectrometry (LC-MS) analysis on formalin-fixed celloidin-embedded temporal bone tissues for postmortem investigation of otosclerosis. Proteomic analysis was performed using human temporal bones from a patient with severe otosclerosis and a control temporal bone. Sections were dissected under microscopy to remove otosclerotic lesions and normal otic capsule for proteomic analysis. Tandem 2D chromatography mass spectrometry was employed. Data analysis and peptide matching to FASTA human databases was done using SEQUEST and proteome discoverer software. TGFβ1 was identified in otosclerosis but not in the normal control temporal bone specimen. Aside from TGFβ1, many proteins and predicted cDNA-encoded proteins were observed, with implications in cell death and/or proliferation pathways, suggesting a possible role in otosclerotic bone remodeling. Immunostaining using TGFβ1 monoclonal revealed marked staining of the spongiotic otosclerotic lesions. Mechanisms involved in cochlear extension of otosclerosis are still unclear, but the implication of TGFβ1 is supported by the present proteomic data and immunostaining results. The established role of TGFβ1 in the chondrogenesis process supports the theory of a reaction targeting the globulae interossei within the otic capsule.

  8. Possible involvement of poly(A) in protein synthesis.

    PubMed Central

    Jacobson, A; Favreau, M

    1983-01-01

    The experiments of this paper have re-evaluated the possibility that poly(A) is involved in protein synthesis by testing whether purified poly(A) might competitively inhibit in vitro protein synthesis in rabbit reticulocyte extracts. We have found that poly(A) inhibits the rate of translation of many different poly(A)+ mRNAs and that comparable inhibition is not observed with other ribopolymers. Inhibition by poly(A) preferentially affects the translation of adenylated mRNAs and can be overcome by increased mRNA concentrations or by translating mRNPs instead of mRNA. The extent of inhibition is dependent on the size of the competitor poly(A) as well as on the translation activity which a lysate has for poly(A)+ RNA. In light of our results and numerous experiments in the literature, we propose that poly(A) has a function in protein synthesis and that any role in the determination of mRNA stability is indirect. Images PMID:6137807

  9. Structural Insights into Protein-Protein Interactions Involved in Bacterial Cell Wall Biogenesis.

    PubMed

    Laddomada, Federica; Miyachiro, Mayara M; Dessen, Andréa

    2016-04-28

    The bacterial cell wall is essential for survival, and proteins that participate in its biosynthesis have been the targets of antibiotic development efforts for decades. The biosynthesis of its main component, the peptidoglycan, involves the coordinated action of proteins that are involved in multi-member complexes which are essential for cell division (the "divisome") and/or cell wall elongation (the "elongasome"), in the case of rod-shaped cells. Our knowledge regarding these interactions has greatly benefitted from the visualization of different aspects of the bacterial cell wall and its cytoskeleton by cryoelectron microscopy and tomography, as well as genetic and biochemical screens that have complemented information from high resolution crystal structures of protein complexes involved in divisome or elongasome formation. This review summarizes structural and functional aspects of protein complexes involved in the cytoplasmic and membrane-related steps of peptidoglycan biosynthesis, with a particular focus on protein-protein interactions whereby disruption could lead to the development of novel antibacterial strategies.

  10. Structural Insights into Protein-Protein Interactions Involved in Bacterial Cell Wall Biogenesis

    PubMed Central

    Laddomada, Federica; Miyachiro, Mayara M.; Dessen, Andréa

    2016-01-01

    The bacterial cell wall is essential for survival, and proteins that participate in its biosynthesis have been the targets of antibiotic development efforts for decades. The biosynthesis of its main component, the peptidoglycan, involves the coordinated action of proteins that are involved in multi-member complexes which are essential for cell division (the “divisome”) and/or cell wall elongation (the “elongasome”), in the case of rod-shaped cells. Our knowledge regarding these interactions has greatly benefitted from the visualization of different aspects of the bacterial cell wall and its cytoskeleton by cryoelectron microscopy and tomography, as well as genetic and biochemical screens that have complemented information from high resolution crystal structures of protein complexes involved in divisome or elongasome formation. This review summarizes structural and functional aspects of protein complexes involved in the cytoplasmic and membrane-related steps of peptidoglycan biosynthesis, with a particular focus on protein-protein interactions whereby disruption could lead to the development of novel antibacterial strategies. PMID:27136593

  11. 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.

  12. 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

  13. 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

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

    PubMed

    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

    2016-05-24

    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.

  15. Preoperative C-reactive protein predicts the need for repeated intracerebral brain abscess drainage.

    PubMed

    Neidert, Marian C; Karlin, Kirill; Actor, Bertrand; Regli, Luca; Bozinov, Oliver; Burkhardt, Jan-Karl

    2015-04-01

    To determine predicting factors for repeated surgical drainage in patients with intracerebral brain abscesses. Patients operated between 01/2008 and 10/2013 with a single-burr-hole technique to drain an intracerebral brain abscess were included from our prospective database. Clinical and radiological characteristics were analyzed retrospectively and compared between patients requiring a single surgical abscess drainage (S group) vs. patients requiring multiple surgical abscess aspirations (M group). Thirty-five patients (mean age 42.6 years, 14 females) including 27 patients in the S group and 8 in the M group were included in this study. Age, gender, causing bacterial agent, surgical technique and abscess volume were comparable for both groups. Preoperative mean C-reactive protein (CRP) (13.9 mg/l vs. 56.1 mg/l, p=0.015) was significantly higher in the M group. Preoperative mean leukocyte count (12.3×10(9)/l vs. 8.9×10(9)/l, p=0.050) was borderline significantly higher in the M group. Although the origin in the overall population was cryptogenic in 43% of the cases, this was never the case in the patient population needing multiple surgeries. Patients with multiple intracerebral brain abscess aspirations showed significantly higher preoperative CRP values than patients who needed surgery only once. Patients with high CRP values at admission and obvious origin of infection might need closer radiographic as well as clinical and laboratory exams after surgery to earlier select patients, which need repeated surgery. Copyright © 2015 Elsevier B.V. All rights reserved.

  16. 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.

  17. 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.

  18. Interferon-inducible GTPase: a novel viral response protein involved in rabies virus infection.

    PubMed

    Li, Ling; Wang, Hualei; Jin, Hongli; Cao, Zengguo; Feng, Na; Zhao, Yongkun; Zheng, Xuexing; Wang, Jianzhong; Li, Qian; Zhao, Guoxing; Yan, Feihu; Wang, Lina; Wang, Tiecheng; Gao, Yuwei; Tu, Changchun; Yang, Songtao; Xia, Xianzhu

    2016-05-01

    Rabies virus infection is a major public health concern because of its wide host-interference spectrum and nearly 100 % lethality. However, the interactions between host and virus remain unclear. To decipher the authentic response in the central nervous system after rabies virus infection, a dynamic analysis of brain proteome alteration was performed. In this study, 104 significantly differentially expressed proteins were identified, and intermediate filament, interferon-inducible GTPases, and leucine-rich repeat-containing protein 16C were the three outstanding groups among these proteins. Interferon-inducible GTPases were prominent because of their strong upregulation. Moreover, quantitative real-time PCR showed distinct upregulation of interferon-inducible GTPases at the level of transcription. Several studies have shown that interferon-inducible GTPases are involved in many biological processes, such as viral infection, endoplasmic reticulum stress response, and autophagy. These findings indicate that interferon-inducible GTPases are likely to be a potential target involved in rabies pathogenesis or the antiviral process.

  19. Enteral delivery of proteins enhances the expression of proteins involved in the cytoskeleton and protein biosynthesis in human duodenal mucosa.

    PubMed

    Goichon, Alexis; Bertrand, Julien; Chan, Philippe; Lecleire, Stéphane; Coquard, Aude; Cailleux, Anne-Françoise; Vaudry, David; Déchelotte, Pierre; Coëffier, Moïse

    2015-08-01

    Amino acids are well known to be key effectors of gut protein turnover. We recently reported that enteral delivery of proteins markedly stimulated global duodenal protein synthesis in carbohydrate-fed healthy humans, but specifically affected proteins remain unknown. We aimed to assess the influence of an enteral protein supply on the duodenal mucosal proteome in carbohydrate-fed humans. Six healthy volunteers received for 5 h, on 2 occasions and in random order, either an enteral infusion of maltodextrins alone (0.25 g · kg⁻¹ · h⁻¹) mimicking the fed state or maltodextrins with a protein powder (0.14 g proteins · kg⁻¹ · h⁻¹). Endoscopic duodenal biopsy specimens were then collected and frozen until analysis. A 2-dimensional polyacrylamide gel electrophoresis-based comparative proteomics analysis was then performed, and differentially expressed proteins (at least ±1.5-fold change; Student's t test, P < 0.05) were identified by mass spectrometry. Protein expression changes were confirmed by Western blot analysis. Thirty-two protein spots were differentially expressed after protein delivery compared with maltodextrins alone: 28 and 4 spots were up- or downregulated, respectively. Among the 22 identified proteins, 11 upregulated proteins were involved either in the cytoskeleton (ezrin, moesin, plastin 1, lamin B1, vimentin, and β-actin) or in protein biosynthesis (glutamyl-prolyl-transfer RNA synthetase, glutaminyl-transfer RNA synthetase, elongation factor 2, elongation factor 1δ, and eukaryotic translation and initiation factor 3 subunit f). Enteral delivery of proteins altered the duodenal mucosal proteome and mainly stimulated the expression of proteins involved in cytoskeleton and protein biosynthesis. These results suggest that protein supply may affect intestinal morphology by stimulating actin cytoskeleton remodeling. © 2015 American Society for Nutrition.

  20. Widespread structural brain involvement in ALS is not limited to the C9orf72 repeat expansion

    PubMed Central

    Westeneng, Henk-Jan; Walhout, Renée; Straathof, Milou; Schmidt, Ruben; Hendrikse, Jeroen; Veldink, Jan H; van den Heuvel, Martijn P; van den Berg, Leonard H

    2016-01-01

    Background In patients with a C9orf72 repeat expansion (C9+), a neuroimaging phenotype with widespread structural cerebral changes has been found. We aimed to investigate the specificity of this neuroimaging phenotype in patients with amyotrophic lateral sclerosis (ALS). Methods 156 C9− and 14 C9+ patients with ALS underwent high-resolution T1-weighted MRI; a subset (n=126) underwent diffusion-weighted imaging. Cortical thickness, subcortical volumes and white matter integrity were compared between C9+ and C9− patients. Using elastic net logistic regression, a model defining the neuroimaging phenotype of C9+ was determined and applied to C9− patients with ALS. Results C9+ patients showed cortical thinning outside the precentral gyrus, extending to the bilateral pars opercularis, fusiform, lingual, isthmus-cingulate and superior parietal cortex, and smaller volumes of the right hippocampus and bilateral thalamus, and reduced white matter integrity of the inferior and superior longitudinal fasciculus compared with C9− patients (p<0.05). Among 128 C9− patients, we detected a subgroup of 27 (21%) with a neuroimaging phenotype congruent to C9+ patients, while 101 (79%) C9− patients showed cortical thinning restricted to the primary motor cortex. C9− patients with a ‘C9+’ neuroimaging phenotype had lower performance on the frontal assessment battery, compared with other C9− patients with ALS (p=0.004). Conclusions This study shows that widespread structural brain involvement is not limited to C9+ patients, but also presents in a subgroup of C9− patients with ALS and relates to cognitive deficits. Our neuroimaging findings reveal an intermediate phenotype that may provide insight into the complex relationship between genetic factors and clinical characteristics. PMID:27756805

  1. Lupine protein hydrolysates inhibit enzymes involved in the inflammatory pathway.

    PubMed

    Millán-Linares, María del Carmen; Yust, María del Mar; Alcaide-Hidalgo, Juan María; Millán, Francisco; Pedroche, Justo

    2014-05-15

    Lupine protein hydrolysates (LPHs) were obtained from a lupine protein isolate (LPI) by enzymatic hydrolysis using two proteases, Izyme AL and Alcalase 2.4 L, and their potential anti-inflammatory capacities were studied by determining their in vitro inhibition of the following enzymes that are involved in the inflammatory process: phospholipase A2 (PLA2), cyclooxygenase 2 (COX-2), thrombin, and transglutaminase (TG). The strongest inhibitory activities toward PLA2 and TG were found in the hydrolysates obtained by hydrolysis with Izyme and subsequently with Alcalase, with more than 70% inhibition obtained in some cases. All of the hydrolysates tested inhibited more than 60% of the COX-2 activity. In no case did the percentage of thrombin activity inhibition exceed 40%. The best inhibitory activities were found in the LPH obtained after 15 min of hydrolysis with Alcalase and in the LPH obtained after 60 min of hydrolysis with Izyme followed by 15 min of hydrolysis with Alcalase. Enzyme kinetic analyses were conducted to determine the Km and Vmax parameters of these two hydrolysates using the Lineweaver-Burk equation. Both hydrolysates competitively inhibited the thrombin and PLA2 activities. In the case of COX-2 and TG, the inhibition appeared to be the mixed type. Copyright © 2013 Elsevier Ltd. All rights reserved.

  2. Arabinogalactan proteins are involved in root hair development in barley

    PubMed Central

    Marzec, Marek; Szarejko, Iwona; Melzer, Michael

    2015-01-01

    The arabinogalactan proteins (AGPs) are involved in a range of plant processes, including cell differentiation and expansion. Here, barley root hair mutants and their wild-type parent cultivars were used, as a model system, to reveal the role of AGPs in root hair development. The treatment of roots with different concentrations of βGlcY (a reagent which binds to all classes of AGPs) inhibited or totally suppressed the development of root hairs in all of the cultivars. Three groups of AGP (recognized by the monoclonal antibodies LM2, LM14, and MAC207) were diversely localized in trichoblasts and atrichoblasts of root hair-producing plants. The relevant epitopes were present in wild-type trichoblast cell walls and cytoplasm, whereas in wild-type atrichoblasts and in all epidermal cells of a root hairless mutant, they were only present in the cytoplasm. In all of cultivars the higher expression of LM2, LM14, and MAC207 was observed in trichoblasts at an early stage of development. Additionally, the LM2 epitope was detected on the surface of primordia and root hair tubes in plants able to generate root hairs. The major conclusion was that the AGPs recognized by LM2, LM14, and MAC207 are involved in the differentiation of barley root epidermal cells, thereby implying a requirement for these AGPs for root hair development in barley. PMID:25465033

  3. Insulin regulation of a novel WD-40 repeat protein in adipocytes.

    PubMed

    Rodgers, B D; Levine, M A; Bernier, M; Montrose-Rafizadeh, C

    2001-02-01

    A 400 bp PCR product generated with degenerate primers derived from the glucagon-like peptide-1 receptor was used to screen a rat skeletal muscle cDNA library. The predicted amino acid sequence of the 978 bp open reading frame has a predicted M(r) of 35 804, an estimated isoelectric point (pI) of 5.31 and contains seven WD-40 repeats, which are common to G-protein beta subunits (Gbeta). Although chemically and structurally similar to Gbeta subunits, the predicted amino acid sequence, when compared with the previously cloned Gbeta isoforms, was found to be only 31-41% similar and thus was named Gbeta-like (GbetaL, 'Gable'). Western blotting of whole-cell lysates and immunoprecipitates of membrane and cytosolic fractions of HEK 293 cells stably overexpressing a carboxy-terminal His-tagged GbetaL indicates that the protein is cytosolic and that it migrates at 42 kDa. A 4 kb transcript was detected in all tissues surveyed by northern blotting; however, an additional 2 kb transcript was detected in testis. Expression of GbetaL mRNA was highest in the brain and testis, followed by lung, heart, kidney, skeletal muscle, spleen and liver. In addition, reverse transcriptase/PCR showed that several other tissues and cell lines express GbetaL. The ubiquitous nature of the tissue expression pattern of GbetaL is similar to that of the insulin receptor, which suggests that insulin may influence GbetaL expression. Indeed, GbetaL protein and mRNA levels, in fully differentiated 3T3-L1 adipocytes, were upregulated by insulin in a concentration-dependent fashion. These changes were highly sensitive to insulin stimulation, being minimally affected by doses as low as 0.1 nM and maximally elevated by 1 nM doses. These data suggest that insulin regulates GbetaL production and imply that some of the actions of insulin may be mediated, in part, by this novel intracellular protein.

  4. Involvement of HxuC Outer Membrane Protein in Utilization of Hemoglobin by Haemophilus influenzae

    PubMed Central

    Cope, Leslie D.; Love, Robert P.; Guinn, Sarah E.; Gilep, Andrei; Usanov, Sergei; Estabrook, Ronald W.; Hrkal, Zbynek; Hansen, Eric J.

    2001-01-01

    Haemophilus influenzae can utilize different protein-bound forms of heme for growth in vitro. A previous study from this laboratory indicated that nontypeable Haemophilus influenzae (NTHI) strain N182 expressed three outer membrane proteins, designated HgbA, HgbB, and HgbC, that bound hemoglobin or hemoglobin-haptoglobin and were encoded by open reading frames (ORFs) that contained a CCAA nucleotide repeat. Testing of mutants expressing the HgbA, HgbB, and HgbC proteins individually revealed that expression of any one of these proteins was sufficient to allow wild-type growth with hemoglobin. In contrast, mutants that expressed only HgbA or HgbC grew significantly better with hemoglobin-haptoglobin than did a mutant expressing only HgbB. Construction of an isogenic hgbA hgbB hgbC mutant revealed that the absence of these three gene products did not affect the ability of NTHI N182 to utilize hemoglobin as a source of heme, although this mutant was severely impaired in its ability to utilize hemoglobin-haptoglobin. The introduction of a tonB mutation into this triple mutant eliminated its ability to utilize hemoglobin, indicating that the pathway for hemoglobin utilization in the absence of HgbA, HgbB, and HgbC involved a TonB-dependent process. Inactivation in this triple mutant of the hxuC gene, which encodes a predicted TonB-dependent outer membrane protein previously shown to be involved in the utilization of free heme, resulted in loss of the ability to utilize hemoglobin. The results of this study reinforce the redundant nature of the heme acquisition systems expressed by H. influenzae. PMID:11254593

  5. 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.

  6. 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.

  7. 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.

  8. 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.

  9. 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

  10. 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.

  11. 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.

  12. Protein-Derived Acetaminophen-Cysteine Can Be Detected After Repeated Supratherapeutic Ingestion of Acetaminophen in the Absence of Hepatotoxicity.

    PubMed

    O'Malley, G F; Mizrahi, F; Giraldo, P; O'Malley, R N; Rollins, D; Wilkins, D

    2015-09-01

    Generation of protein-derived acetaminophen-cysteine (APAP-CYS) is reported after ingestion of large and therapeutic dosages of acetaminophen in healthy and in liver-damaged patients. The incidence of protein-derived APAP-CYS adducts in repeated supratherapeutic dosages of APAP is not known. for 12 months, a standardized and comprehensive questionnaire was used to interview every consecutive patient at a pain management clinic. Patients found to ingest more than 4 g of APAP per day for a minimum of 14 consecutive days at the time of the encounter were invited to have blood drawn for hepatic transaminases and APAP-CYS adduct levels. Twelve subjects out of 990 interviewees met inclusion criteria. Ten of the 12 had measurable protein-derived APAP-CYS, none had evidence of liver injury. Patients that ingest repeated supratherapeutic amounts of APAP over several weeks may generate APAP-CYS protein adducts in the absence of hepatic injury.

  13. 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.

  14. WD40-repeat protein 62 is a JNK-phosphorylated spindle pole protein required for spindle maintenance and timely mitotic progression

    PubMed Central

    Bogoyevitch, Marie A.; Yeap, Yvonne Y. C.; Qu, Zhengdong; Ngoei, Kevin R.; Yip, Yan Y.; Zhao, Teresa T.; Heng, Julian I.; Ng, Dominic C. H.

    2012-01-01

    Summary The impact of aberrant centrosomes and/or spindles on asymmetric cell division in embryonic development indicates the tight regulation of bipolar spindle formation and positioning that is required for mitotic progression and cell fate determination. WD40-repeat protein 62 (WDR62) was recently identified as a spindle pole protein linked to the neurodevelopmental defect of microcephaly but its roles in mitosis have not been defined. We report here that the in utero electroporation of neuroprogenitor cells with WDR62 siRNAs induced their cell cycle exit and reduced their proliferative capacity. In cultured cells, we demonstrated cell-cycle-dependent accumulation of WDR62 at the spindle pole during mitotic entry that persisted until metaphase–anaphase transition. Utilizing siRNA depletion, we revealed WDR62 function in stabilizing the mitotic spindle specifically during metaphase. WDR62 loss resulted in spindle orientation defects, decreased the integrity of centrosomes displaced from the spindle pole and delayed mitotic progression. Additionally, we revealed JNK phosphorylation of WDR62 is required for maintaining metaphase spindle organization during mitosis. Our study provides the first functional characterization of WDR62 and has revealed requirements for JNK/WDR62 signaling in mitotic spindle regulation that may be involved in coordinating neurogenesis. PMID:22899712

  15. WD40-repeat protein 62 is a JNK-phosphorylated spindle pole protein required for spindle maintenance and timely mitotic progression.

    PubMed

    Bogoyevitch, Marie A; Yeap, Yvonne Y C; Qu, Zhengdong; Ngoei, Kevin R; Yip, Yan Y; Zhao, Teresa T; Heng, Julian I; Ng, Dominic C H

    2012-11-01

    The impact of aberrant centrosomes and/or spindles on asymmetric cell division in embryonic development indicates the tight regulation of bipolar spindle formation and positioning that is required for mitotic progression and cell fate determination. WD40-repeat protein 62 (WDR62) was recently identified as a spindle pole protein linked to the neurodevelopmental defect of microcephaly but its roles in mitosis have not been defined. We report here that the in utero electroporation of neuroprogenitor cells with WDR62 siRNAs induced their cell cycle exit and reduced their proliferative capacity. In cultured cells, we demonstrated cell-cycle-dependent accumulation of WDR62 at the spindle pole during mitotic entry that persisted until metaphase-anaphase transition. Utilizing siRNA depletion, we revealed WDR62 function in stabilizing the mitotic spindle specifically during metaphase. WDR62 loss resulted in spindle orientation defects, decreased the integrity of centrosomes displaced from the spindle pole and delayed mitotic progression. Additionally, we revealed JNK phosphorylation of WDR62 is required for maintaining metaphase spindle organization during mitosis. Our study provides the first functional characterization of WDR62 and has revealed requirements for JNK/WDR62 signaling in mitotic spindle regulation that may be involved in coordinating neurogenesis.

  16. 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

  17. What induces pocket openings on protein surface patches involved in protein-protein interactions?

    NASA Astrophysics Data System (ADS)

    Eyrisch, Susanne; Helms, Volkhard

    2009-02-01

    We previously showed for the proteins BCL-XL, IL-2, and MDM2 that transient pockets at their protein-protein binding interfaces can be identified by applying the PASS algorithm to molecular dynamics (MD) snapshots. We now investigated which aspects of the natural conformational dynamics of proteins induce the formation of such pockets. The pocket detection protocol was applied to three different conformational ensembles for the same proteins that were extracted from MD simulations of the inhibitor bound crystal conformation in water and the free crystal/NMR structure in water and in methanol. Additional MD simulations studied the impact of backbone mobility. The more efficient CONCOORD or normal mode analysis (NMA) techniques gave significantly smaller pockets than MD simulations, whereas tCONCOORD generated pockets comparable to those observed in MD simulations for two of the three systems. Our findings emphasize the influence of solvent polarity and backbone rearrangements on the formation of pockets on protein surfaces and should be helpful in future generation of transient pockets as putative ligand binding sites at protein-protein interfaces.

  18. 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

  19. 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.

  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. 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. Kelch-repeat proteins interacting with the Gα protein Gpa2 bypass adenylate cyclase for direct regulation of protein kinase A in yeast

    PubMed Central

    Peeters, Tom; Louwet, Wendy; Geladé, Ruud; Nauwelaers, David; Thevelein, Johan M.; Versele, Matthias

    2006-01-01

    The cAMP–PKA pathway consists of an extracellular ligand-sensitive G protein-coupled receptor, a G protein signal transmitter, and the effector, adenylate cyclase, of which the product, cAMP, acts as an intracellular second messenger. cAMP activates PKA by dissociating the regulatory subunit from the catalytic subunit. Yeast cells (Saccharomyces cerevisiae) contain a glucose/sucrose-sensitive seven-transmembrane domain receptor, Gpr1, that was proposed to activate adenylate cyclase through the Gα protein Gpa2. Consistently, we show here that adenylate cyclase binds only to active, GTP-bound Gpa2. Two related kelch-repeat proteins, Krh1/Gpb2 and Krh2/Gpb1, are associated with Gpa2 and were suggested to act as Gβ mimics for Gpa2, based on their predicted seven-bladed β-propeller structure. However, we find that although Krh1 associates with both GDP and GTP-bound Gpa2, it displays a preference for GTP-Gpa2. The strong down-regulation of PKA targets by Krh1 and Krh2 does not require Gpa2 but is strictly dependent on both the catalytic and the regulatory subunits of PKA. Krh1 directly interacts with PKA by means of the catalytic subunits, and Krh1/2 stimulate the association between the catalytic and regulatory subunits in vivo. Indeed, both a constitutively active GPA2 allele and deletion of KRH1/2 lower the cAMP requirement of PKA for growth. We propose that active Gpa2 relieves the inhibition imposed by the kelch-repeat proteins on PKA, thereby bypassing adenylate cyclase for direct regulation of PKA. Importantly, we show that Krh1/2 also enhance the association between mouse R and C subunits, suggesting that Krh control of PKA has been evolutionarily conserved. PMID:16924114

  3. The Toll immune-regulated Drosophila protein Fondue is involved in hemolymph clotting and puparium formation.

    PubMed

    Scherfer, Christoph; Qazi, Mousumi R; Takahashi, Kuniaki; Ueda, Ryu; Dushay, Mitchell S; Theopold, Ulrich; Lemaitre, Bruno

    2006-07-01

    Clotting is critical in limiting hemolymph loss and initiating wound healing in insects as in vertebrates. It is also an important immune defense, quickly forming a secondary barrier to infection, immobilizing bacteria and thereby promoting their killing. However, hemolymph clotting is one of the least understood immune responses in insects. Here, we characterize fondue (fon; CG15825), an immune-responsive gene of Drosophila melanogaster that encodes an abundant hemolymph protein containing multiple repeat blocks. After knockdown of fon by RNAi, bead aggregation activity of larval hemolymph is strongly reduced, and wound closure is affected. fon is thus the second Drosophila gene after hemolectin (hml), for which a knockdown causes a clotting phenotype. In contrast to hml-RNAi larvae, clot fibers are still observed in samples from fon-RNAi larvae. However, clot fibers from fon-RNAi larvae are more ductile and longer than in wt hemolymph samples, indicating that Fondue might be involved in cross-linking of fiber proteins. In addition, fon-RNAi larvae exhibit melanotic tumors and constitutive expression of the antifungal peptide gene Drosomycin (Drs), while fon-RNAi pupae display an aberrant pupal phenotype. Altogether, our studies indicate that Fondue is a major hemolymph protein required for efficient clotting in Drosophila.

  4. Functional consequences of B-repeat sequence variation in the staphylococcal biofilm protein Aap: deciphering the assembly code.

    PubMed

    Shelton, Catherine L; Conrady, Deborah G; Herr, Andrew B

    2017-02-01

    Staphylococcus epidermidis is an opportunistic pathogen that can form robust biofilms that render the bacteria resistant to antibiotic action and immune responses. Intercellular adhesion in S. epidermidis biofilms is mediated by the cell wall-associated accumulation-associated protein (Aap), via zinc-mediated self-assembly of its B-repeat region. This region contains up to 17 nearly identical sequence repeats, with each repeat assumed to be functionally equivalent. However, Aap B-repeats exist as two subtypes, defined by a cluster of consensus or variant amino acids. These variable residues are positioned near the zinc-binding (and dimerization) site and the stability determinant for the B-repeat fold. We have characterized four B-repeat constructs to assess the functional relevance of the two Aap B-repeat subtypes. Analytical ultracentrifugation experiments demonstrated that constructs with the variant sequence show reduced or absent Zn(2+)-induced dimerization. Likewise, circular dichroism thermal denaturation experiments showed that the variant sequence could significantly stabilize the fold, depending on its location within the construct. Crystal structures of three of the constructs revealed that the side chains from the variant sequence form an extensive bonding network that can stabilize the fold. Furthermore, altered distribution of charged residues between consensus and variant sequences changes the electrostatic potential in the vicinity of the Zn(2+)-binding site, providing a mechanistic explanation for the loss of zinc-induced dimerization in the variant constructs. These data suggest an assembly code that defines preferred oligomerization modes of the B-repeat region of Aap and a slip-grip model for initial contact followed by firm intercellular adhesion during biofilm formation. © 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.

  5. Protein and hypervariable tandem repeat diversity in eight African-derived South American populations: inferred relationships do not coincide.

    PubMed

    Bortolini, M C; da Silva-Júnior, W A; Weimer, T de A; Zago, M A; de Guerra, D C; Schneider, M P; Layrisse, Z; Castellano, H M; Salzano, F M

    1998-06-01

    We compared data from individuals living in 4 African Venezuelan and 4 African Brazilian communities for 11 protein loci (551 subjects) and 8 hypervariable tandem repeat polymorphisms (252 subjects). There is heterogeneity in diversity within and between the two sets of loci. On the other hand, African-derived Brazilians and Venezuelans do not present marked variability differences between themselves. Although the hypervariable loci show gene diversities that are about four times higher than those obtained from the protein data, they are not more discriminative at the interpopulation level (averages 6% and 4%, respectively). Interpopulation differences do not strictly parallel the geographic distances between the groups, and population relationships obtained from the protein data are not the same as those indicated by hypervariable tandem repeat polymorphisms. Caution is needed in establishing relationships considering just one level of the biological hierarchy.

  6. Hexanucleotide Repeats in ALS/FTD Form Length-Dependent RNA Foci, Sequester RNA Binding Proteins, and Are Neurotoxic

    PubMed Central

    Lee, Youn-Bok; Chen, Han-Jou; Peres, João N.; Gomez-Deza, Jorge; Attig, Jan; Štalekar, 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.

    2013-01-01

    Summary 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. PMID:24290757

  7. 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.

  8. Involvement of cysteinyl leukotrienes in airway smooth muscle cell DNA synthesis after repeated allergen exposure in sensitized Brown Norway rats

    PubMed Central

    Salmon, Michael; Walsh, David A; Huang, Tung-Jung; Barnes, Peter J; Leonard, Thomas B; Hay, Douglas W P; Chung, K Fan

    1999-01-01

    Airway smooth muscle thickening is a characteristic feature of airway wall remodelling in chronic asthma. We have investigated the role of the leukotrienes in airway smooth muscle (ASM) and epithelial cell DNA synthesis and ASM thickening following repeated allergen exposure in Brown Norway rats sensitized to ovalbumin. There was a 3 fold increase in ASM cell DNA synthesis, as measured by percentage bromodeoxyuridine (BrdU) incorporation, in repeatedly ovalbumin-exposed (4.1%, 3.6–4.6; mean, 95% c.i.) compared to chronically saline-exposed rats (1.3%, 0.6–2.1; P<0.001). Treatment with a 5-lipoxygenase enzyme inhibitor (SB 210661, 10 mg kg−1, p.o.) and a specific cysteinyl leukotriene (CysLT1) receptor antagonist, pranlukast (SB 205312, 30 mg kg−1, p.o.), both attenuated ASM cell DNA synthesis. Treatment with a specific leukotriene B4 (BLT) receptor antagonist (SB 201146, 15 mg kg−1, p.o.) had no effect. There was also a significant, 2 fold increase in the number of epithelial cells incorporating BrdU per unit length of basement membrane after repeated allergen exposure. This response was not inhibited by treatment with SB 210661, pranlukast or SB 201146. A significant increase in ASM thickness was identified following repeated allergen exposure and this response was attenuated significantly by SB 210661, pranlukast and SB 201146. Rats exposed to chronic allergen exhibited bronchial hyperresponsiveness to acetylcholine and had significant eosinophil recruitment into the lungs. Treatment with SB 210661, pranlukast or SB 201146 significantly attenuated eosinophil recruitment into the lungs, whilst having no significant effect on airway hyperresponsiveness. These data indicate that the cysteinyl leukotrienes are important mediators in allergen-induced ASM cell DNA synthesis in rats, while both LTB4 and cysteinyl leukotrienes contribute to ASM thickening and eosinophil recruitment following repeated allergen exposure. PMID:10455261

  9. 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.

  10. 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

  11. A highly parallel method for synthesizing DNA repeats enables the discovery of 'smart' protein polymers.

    PubMed

    Amiram, Miriam; Quiroz, Felipe Garcia; Callahan, Daniel J; Chilkoti, Ashutosh

    2011-02-01

    Robust high-throughput synthesis methods are needed to expand the repertoire of repetitive protein-polymers for different applications. To address this need, we developed a new method, overlap extension rolling circle amplification (OERCA), for the highly parallel synthesis of genes encoding repetitive protein-polymers. OERCA involves a single PCR-type reaction for the rolling circle amplification of a circular DNA template and simultaneous overlap extension by thermal cycling. We characterized the variables that control OERCA and demonstrated its superiority over existing methods, its robustness, high-throughput and versatility by synthesizing variants of elastin-like polypeptides (ELPs) and protease-responsive polymers of glucagon-like peptide-1 analogues. Despite the GC-rich, highly repetitive sequences of ELPs, we synthesized remarkably large genes without recursive ligation. OERCA also enabled us to discover 'smart' biopolymers that exhibit fully reversible thermally responsive behaviour. This powerful strategy generates libraries of repetitive genes over a wide and tunable range of molecular weights in a 'one-pot' parallel format.

  12. Highly Parallel Method for Synthesis of DNA Repeats Enables Discovery of “Smart” Protein Polymers

    PubMed Central

    Amiram, Miriam; Quiroz, Felipe Garcia; Callahan, Daniel J.; Chilkoti, Ashutosh

    2010-01-01

    Robust high-throughput synthesis methods are needed to expand the repertoire of repetitive protein-polymers for different applications. To address this need, we developed a new method, overlap-extension rolling circle amplification (OERCA), for the highly parallel synthesis of genes encoding repetitive protein-polymers. OERCA involves a single PCR-type reaction for the rolling circle amplification of a circular DNA template and simultaneous overlap extension by thermal cycling. We characterized the variables that control OERCA and demonstrated its superiority over existing methods, its robustness, throughput and versatility by synthesizing variants of elastin-like polypeptides (ELPs) and protease-responsive polymers of a glucagon-like peptide-1 analog. Despite the GC-rich, highly repetitive sequences of ELPs, we synthesized remarkably large genes without recursive ligation. OERCA also enabled us to discover “smart” biopolymers that exhibit fully reversible thermally responsive behavior. This powerful strategy generates libraries of repetitive genes over a wide and tunable range of molecular weights in a “one-pot” parallel format. PMID:21258353

  13. Host membrane proteins involved in the replication of tobamovirus RNA.

    PubMed

    Ishibashi, Kazuhiro; Miyashita, Shuhei; Katoh, Etsuko; Ishikawa, Masayuki

    2012-12-01

    Eukaryotic positive-strand RNA viruses replicate their genomes in membrane-bound replication complexes composed of viral replication proteins and negative-strand RNA templates. These replication proteins are programmed to exhibit RNA polymerase and other replication-related activities only in replication complexes to avoid inducing double-stranded RNA-mediated host defenses. Host membrane components (e.g. proteins and lipids) should play important roles in the activation of replication proteins. Two host membrane proteins are components of the replication complex and activate the replication proteins of tobamoviruses. Interaction analyses using deletion mutants constructed based on structural information suggest a conformational change in replication proteins during the formation of a protein complex with RNA 5'-capping activity.

  14. 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.

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

    PubMed

    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.

  16. 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

  17. 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.

  18. Selection of Specific Protein Binders for Pre-Defined Targets from an Optimized Library of Artificial Helicoidal Repeat Proteins (alphaRep)

    PubMed Central

    Chevrel, Anne; Graille, Marc; Fourati-Kammoun, Zaineb; Desmadril, Michel; van Tilbeurgh, Herman; Minard, Philippe

    2013-01-01

    We previously designed a new family of artificial proteins named αRep based on a subgroup of thermostable helicoidal HEAT-like repeats. We have now assembled a large optimized αRep library. In this library, the side chains at each variable position are not fully randomized but instead encoded by a distribution of codons based on the natural frequency of side chains of the natural repeats family. The library construction is based on a polymerization of micro-genes and therefore results in a distribution of proteins with a variable number of repeats. We improved the library construction process using a “filtration” procedure to retain only fully coding modules that were recombined to recreate sequence diversity. The final library named Lib2.1 contains 1.7×109 independent clones. Here, we used phage display to select, from the previously described library or from the new library, new specific αRep proteins binding to four different non-related predefined protein targets. Specific binders were selected in each case. The results show that binders with various sizes are selected including relatively long sequences, with up to 7 repeats. ITC-measured affinities vary with Kd values ranging from micromolar to nanomolar ranges. The formation of complexes is associated with a significant thermal stabilization of the bound target protein. The crystal structures of two complexes between αRep and their cognate targets were solved and show that the new interfaces are established by the variable surfaces of the repeated modules, as well by the variable N-cap residues. These results suggest that αRep library is a new and versatile source of tight and specific binding proteins with favorable biophysical properties. PMID:24014183

  19. A diverse host thrombospondin-type-1 repeat protein repertoire promotes symbiont colonization during establishment of cnidarian-dinoflagellate symbiosis.

    PubMed

    Neubauer, Emilie-Fleur; Poole, Angela Z; Neubauer, Philipp; Detournay, Olivier; Tan, Kenneth; Davy, Simon K; Weis, Virginia M

    2017-05-08

    The mutualistic endosymbiosis between cnidarians and dinoflagellates is mediated by complex inter-partner signaling events, where the host cnidarian innate immune system plays a crucial role in recognition and regulation of symbionts. To date, little is known about the diversity of thrombospondin-type-1 repeat (TSR) domain proteins in basal metazoans or their potential role in regulation of cnidarian-dinoflagellate mutualisms. We reveal a large and diverse repertoire of TSR proteins in seven anthozoan species, and show that in the model sea anemone Aiptasia pallida the TSR domain promotes colonization of the host by the symbiotic dinoflagellate Symbiodinium minutum. Blocking TSR domains led to decreased colonization success, while adding exogenous TSRs resulted in a 'super colonization'. Furthermore, gene expression of TSR proteins was highest at early time-points during symbiosis establishment. Our work characterizes the diversity of cnidarian TSR proteins and provides evidence that these proteins play an important role in the establishment of cnidarian-dinoflagellate symbiosis.

  20. A low protein diet alters bone material level properties and the response to in vitro repeated mechanical loading.

    PubMed

    Dubois-Ferrière, Victor; Rizzoli, René; Ammann, Patrick

    2014-01-01

    Low protein intake is associated with an alteration of bone microstructure and material level properties. However, it remains unknown whether these alterations of bone tissue could influence the response to repeated mechanical loading. The authors investigated the in vitro effect of repeated loading on bone strength in humeri collected from 20 6-month-old female rats pair-fed with a control (15% casein) or an isocaloric low protein (2.5% casein) diet for 10 weeks. Bone specimens were cyclically loaded in three-point bending under load control for 2000 cycles. Humeri were then monotonically loaded to failure. The load-displacement curve of the in vitro cyclically loaded humerus was compared to the contralateral noncyclically loaded humerus and the influence of both protein diets. Material level properties were also evaluated through a nanoindentation test. Cyclic loading decreased postyield load and plastic deflection in rats fed a low protein diet, but not in those on a regular diet. Bone material level properties were altered in rats fed a low protein diet. This suggests that bone biomechanical alterations consequent to cyclic loading are more likely to occur in rats fed a low protein diet than in control animals subjected to the same in vitro cyclic loading regimen.

  1. 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

  2. Intrinsic disorder in proteins involved in amyotrophic lateral sclerosis.

    PubMed

    Santamaria, Nikolas; Alhothali, Marwa; Alfonso, Maria Harreguy; Breydo, Leonid; Uversky, Vladimir N

    2017-04-01

    Five structurally and functionally different proteins, an enzyme superoxide dismutase 1 (SOD1), a TAR-DNA binding protein-43 (TDP-43), an RNA-binding protein FUS, a cofilin-binding protein C9orf72, and polypeptides generated as a result of its intronic hexanucleotide expansions, and to lesser degree actin-binding profilin-1 (PFN1), are considered to be the major drivers of amyotrophic lateral sclerosis. One of the features common to these proteins is the presence of significant levels of intrinsic disorder. The goal of this study is to consider these neurodegeneration-related proteins from the intrinsic disorder perspective. To this end, we employed a broad set of computational tools for intrinsic disorder analysis and conducted intensive literature search to gain information on the structural peculiarities of SOD1, TDP-43, FUS, C9orf72, and PFN1 and their intrinsic disorder predispositions, and the roles of intrinsic disorder in their normal and pathological functions.

  3. DUF581 Is Plant Specific FCS-Like Zinc Finger Involved in Protein-Protein Interaction

    PubMed Central

    K, Muhammed Jamsheer; Laxmi, Ashverya

    2014-01-01

    Zinc fingers are a ubiquitous class of protein domain with considerable variation in structure and function. Zf-FCS is a highly diverged group of C2-C2 zinc finger which is present in animals, prokaryotes and viruses, but not in plants. In this study we identified that a plant specific domain of unknown function, DUF581 is a zf-FCS type zinc finger. Based on HMM-HMM comparison and signature motif similarity we named this domain as FCS-Like Zinc finger (FLZ) domain. A genome wide survey identified that FLZ domain containing genes are bryophytic in origin and this gene family is expanded in spermatophytes. Expression analysis of selected FLZ gene family members of A. thaliana identified an overlapping expression pattern suggesting a possible redundancy in their function. Unlike the zf-FCS domain, the FLZ domain found to be highly conserved in sequence and structure. Using a combination of bioinformatic and protein-protein interaction tools, we identified that FLZ domain is involved in protein-protein interaction. PMID:24901469

  4. Repeat instability: mechanisms of dynamic mutations.

    PubMed

    Pearson, Christopher E; Nichol Edamura, Kerrie; Cleary, John D

    2005-10-01

    Disease-causing repeat instability is an important and unique form of mutation that is linked to more than 40 neurological, neurodegenerative and neuromuscular disorders. DNA repeat expansion mutations are dynamic and ongoing within tissues and across generations. The patterns of inherited and tissue-specific instability are determined by both gene-specific cis-elements and trans-acting DNA metabolic proteins. Repeat instability probably involves the formation of unusual DNA structures during DNA replication, repair and recombination. Experimental advances towards explaining the mechanisms of repeat instability have broadened our understanding of this mutational process. They have revealed surprising ways in which metabolic pathways can drive or protect from repeat instability.

  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. 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.

  7. 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.

  8. Characterization of a novel spore wall protein NbSWP16 with proline-rich tandem repeats from Nosema bombycis (microsporidia).

    PubMed

    Wang, Ying; Dang, Xiaoqun; Ma, Qiang; Liu, Fangyan; Pan, Guoqing; Li, Tian; Zhou, Zeyang

    2015-04-01

    Nosema bombycis, a pathogen of silkworm pebrine, is an obligate unicellular eukaryotic parasite. It is reported that the spore wall proteins have essential functions in the adherence and infection process of microsporidia. To date, the information related to spore wall proteins from microsporidia is still limited. Here, a 44 kDa spore wall protein NbSWP16 was characterized in N. bombycis. In NbSWP16, a 25 amino acids signal peptide and 3 heparin binding motifs were predicted. Interestingly, a region that contains 3 proline-rich tandem repeats lacking homology to any known protein was also present in this protein. The immunofluorescence analysis (IFA) demonstrated that distinct fluorescent signals were detected both on the surface of mature spores and the germinated spore coats. Immunolocation by electron microscopy revealed that NbSWP16 localized on the exospore regions. Finally, spore adherence analysis indicated that spore adherence to host cell was decreased more than 20% by anti-NbSWP16 blocking compared with the negative control in vitro. In contrast with anti-NbSWP16, no remarkable decrement inhibition was detected when antibodies of NbSWP16 and NbSWP5 were used simultaneously. Collectively, these results suggest that NbSWP16 is a new exospore protein and probably be involved in spore adherence of N. bombycis.

  9. 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.

  10. Taxonomic distribution, repeats, and functions of the S1 domain-containing proteins as members of the OB-fold family.

    PubMed

    Deryusheva, Evgeniia I; Machulin, Andrey V; Selivanova, Olga M; Galzitskaya, Oxana V

    2017-04-01

    Proteins of the nucleic acid-binding proteins superfamily perform such functions as processing, transport, storage, stretching, translation, and degradation of RNA. It is one of the 16 superfamilies containing the OB-fold in protein structures. Here, we have analyzed the superfamily of nucleic acid-binding proteins (the number of sequences exceeds 200,000) and obtained that this superfamily prevalently consists of proteins containing the cold shock DNA-binding domain (ca. 131,000 protein sequences). Proteins containing the S1 domain compose 57% from the cold shock DNA-binding domain family. Furthermore, we have found that the S1 domain was identified mainly in the bacterial proteins (ca. 83%) compared to the eukaryotic and archaeal proteins, which are available in the UniProt database. We have found that the number of multiple repeats of S1 domain in the S1 domain-containing proteins depends on the taxonomic affiliation. All archaeal proteins contain one copy of the S1 domain, while the number of repeats in the eukaryotic proteins varies between 1 and 15 and correlates with the protein size. In the bacterial proteins, the number of repeats is no more than 6, regardless of the protein size. The large variation of the repeat number of S1 domain as one of the structural variants of the OB-fold is a distinctive feature of S1 domain-containing proteins. Proteins from the other families and superfamilies have either one OB-fold or change slightly the repeat numbers. On the whole, it can be supposed that the repeat number is a vital for multifunctional activity of the S1 domain-containing proteins. Proteins 2017; 85:602-613. © 2016 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  11. Specific Binding of Tetratricopeptide Repeat Proteins to Heat Shock Protein 70 (Hsp70) and Heat Shock Protein 90 (Hsp90) Is Regulated by Affinity and Phosphorylation.

    PubMed

    Assimon, Victoria A; Southworth, Daniel R; Gestwicki, Jason E

    2015-12-08

    Heat shock protein 70 (Hsp70) and heat shock protein 90 (Hsp90) require the help of tetratricopeptide repeat (TPR) domain-containing cochaperones for many of their functions. Each monomer of Hsp70 or Hsp90 can interact with only a single TPR cochaperone at a time, and each member of the TPR cochaperone family brings distinct functions to the complex. Thus, competition for TPR binding sites on Hsp70 and Hsp90 appears to shape chaperone activity. Recent structural and biophysical efforts have improved our understanding of chaperone-TPR contacts, focusing on the C-terminal EEVD motif that is present in both chaperones. To better understand these important protein-protein interactions on a wider scale, we measured the affinity of five TPR cochaperones, CHIP, Hop, DnaJC7, FKBP51, and FKBP52, for the C-termini of four members of the chaperone family, Hsc70, Hsp72, Hsp90α, and Hsp90β, in vitro. These studies identified some surprising selectivity among the chaperone-TPR pairs, including the selective binding of FKBP51/52 to Hsp90α/β. These results also revealed that other TPR cochaperones are only able to weakly discriminate between the chaperones or between their paralogs. We also explored whether mimicking phosphorylation of serine and threonine residues near the EEVD motif might impact affinity and found that pseudophosphorylation had selective effects on binding to CHIP but not other cochaperones. Together, these findings suggest that both intrinsic affinity and post-translational modifications tune the interactions between the Hsp70 and Hsp90 proteins and the TPR cochaperones.

  12. Involvement of the Transmembrane Protein p23 in Biosynthetic Protein Transport

    PubMed Central

    Rojo, Manuel; Pepperkok, Rainer; Emery, Gregory; Kellner, Roland; Stang, Espen; Parton, Robert G.; Gruenberg, Jean

    1997-01-01

    Here, we report the localization and characterization of BHKp23, a member of the p24 family of transmembrane proteins, in mammalian cells. We find that p23 is a major component of tubulovesicular membranes at the cis side of the Golgi complex (estimated density: 12,500 copies/μm2 membrane surface area, or ≈30% of the total protein). Our data indicate that BHKp23-containing membranes are part of the cis-Golgi network/intermediate compartment . Using the G protein of vesicular stomatitis virus as a transmembrane cargo molecule, we find that p23 membranes are an obligatory station in forward biosynthetic membrane transport, but that p23 itself is absent from transport vesicles that carry the G protein to and beyond the Golgi complex. Our data show that p23 is not present to any significant extent in coat protein (COP) I-coated vesicles generated in vitro and does not colocalize with COP I buds and vesicles. Moreover, we find that p23 cytoplasmic domain is not involved in COP I membrane recruitment. Our data demonstrate that microinjected antibodies against the cytoplasmic tail of p23 inhibit G protein transport from the cis-Golgi network/ intermediate compartment to the cell surface, suggesting that p23 function is required for the transport of transmembrane cargo molecules. These observations together with the fact that p23 is a highly abundant component in the intermediate compartment, lead us to propose that p23 contributes to membrane structure, and that this contribution is necessary for efficient segregation and transport. PMID:9382861

  13. The VHL short variant involves in protein quality control.

    PubMed

    Liu, Yanbin; Yang, Haixia; Zuo, Feifei; Chen, Liang

    2016-09-01

    The von Hippel-Lindau (VHL) is the most important and frequently mutated gene in human clear cell renal cell carcinoma (ccRCC). In contrast to its long counterpart, the internal translational variant of VHL protein (VHLs) is evolutionarily conserved. Herein we present evidence that VHLs associates with ribosome complex via interaction with the large subunit 6 (RPL6). Manipulation of VHLs expression significantly alters protein synthesis, cell size and mitochondrial mass. VHLs deficiency leads to remarkable sensitivity to drug treatments eliciting nascent protein mis-folding and translational errors. The ubiquitination of nascent peptides are dramatically increased upon the ectopic over-expression of VHLs, which simultaneously co-localizes with proteasome and thus may facilitate the ubiquitin-proteasome mediated degradation. In summary, VHLs contributes to protein quality control in addition to its canonical function in maintaining homeostasis of hypoxia-induced factors alpha subunit (HIFα) in response to environmental oxygen supply.

  14. Sex Hormones Regulate Cytoskeletal Proteins Involved in Brain Plasticity

    PubMed Central

    Hansberg-Pastor, Valeria; González-Arenas, Aliesha; Piña-Medina, Ana Gabriela; Camacho-Arroyo, Ignacio

    2015-01-01

    In the brain of female mammals, including humans, a number of physiological and behavioral changes occur as a result of sex hormone exposure. Estradiol and progesterone regulate several brain functions, including learning and memory. Sex hormones contribute to shape the central nervous system by modulating the formation and turnover of the interconnections between neurons as well as controlling the function of glial cells. The dynamics of neuron and glial cells morphology depends on the cytoskeleton and its associated proteins. Cytoskeletal proteins are necessary to form neuronal dendrites and dendritic spines, as well as to regulate the diverse functions in astrocytes. The expression pattern of proteins, such as actin, microtubule-associated protein 2, Tau, and glial fibrillary acidic protein, changes in a tissue-specific manner in the brain, particularly when variations in sex hormone levels occur during the estrous or menstrual cycles or pregnancy. Here, we review the changes in structure and organization of neurons and glial cells that require the participation of cytoskeletal proteins whose expression and activity are regulated by estradiol and progesterone. PMID:26635640

  15. The essential WD40 protein Cia1 is involved in a late step of cytosolic and nuclear iron-sulfur protein assembly.

    PubMed

    Balk, Janneke; Aguilar Netz, Daili J; Tepper, Katharina; Pierik, Antonio J; Lill, Roland

    2005-12-01

    The assembly of cytosolic and nuclear iron-sulfur (Fe/S) proteins in yeast is dependent on the iron-sulfur cluster assembly and export machineries in mitochondria and three recently identified extramitochondrial proteins, the P-loop NTPases Cfd1 and Nbp35 and the hydrogenase-like Nar1. However, the molecular mechanism of Fe/S protein assembly in the cytosol is far from being understood, and more components are anticipated to take part in this process. Here, we have identified and functionally characterized a novel WD40 repeat protein, designated Cia1, as an essential component required for Fe/S cluster assembly in vivo on cytosolic and nuclear, but not mitochondrial, Fe/S proteins. Surprisingly, Nbp35 and Nar1, themselves Fe/S proteins, could assemble their Fe/S clusters in the absence of Cia1, demonstrating that these components act before Cia1. Consequently, Cia1 is involved in a late step of Fe/S cluster incorporation into target proteins. Coimmunoprecipitation assays demonstrated a specific interaction between Cia1 and Nar1. In contrast to the mostly cytosolic Nar1, Cia1 is preferentially localized to the nucleus, suggesting an additional function of Cia1. Taken together, our results indicate that Cia1 is a new member of the cytosolic Fe/S protein assembly (CIA) machinery participating in a step after Nbp35 and Nar1.

  16. The Essential WD40 Protein Cia1 Is Involved in a Late Step of Cytosolic and Nuclear Iron-Sulfur Protein Assembly

    PubMed Central

    Balk, Janneke; Aguilar Netz, Daili J.; Tepper, Katharina; Pierik, Antonio J.; Lill, Roland

    2005-01-01

    The assembly of cytosolic and nuclear iron-sulfur (Fe/S) proteins in yeast is dependent on the iron-sulfur cluster assembly and export machineries in mitochondria and three recently identified extramitochondrial proteins, the P-loop NTPases Cfd1 and Nbp35 and the hydrogenase-like Nar1. However, the molecular mechanism of Fe/S protein assembly in the cytosol is far from being understood, and more components are anticipated to take part in this process. Here, we have identified and functionally characterized a novel WD40 repeat protein, designated Cia1, as an essential component required for Fe/S cluster assembly in vivo on cytosolic and nuclear, but not mitochondrial, Fe/S proteins. Surprisingly, Nbp35 and Nar1, themselves Fe/S proteins, could assemble their Fe/S clusters in the absence of Cia1, demonstrating that these components act before Cia1. Consequently, Cia1 is involved in a late step of Fe/S cluster incorporation into target proteins. Coimmunoprecipitation assays demonstrated a specific interaction between Cia1 and Nar1. In contrast to the mostly cytosolic Nar1, Cia1 is preferentially localized to the nucleus, suggesting an additional function of Cia1. Taken together, our results indicate that Cia1 is a new member of the cytosolic Fe/S protein assembly (CIA) machinery participating in a step after Nbp35 and Nar1. PMID:16314508

  17. Autologous adipose tissue-derived mesenchymal stem cells are involved in rat liver regeneration following repeat partial hepatectomy

    PubMed Central

    LIU, TAO; MU, HONG; SHEN, ZHONGYANG; SONG, ZHUOLUN; CHEN, XIAOBO; WANG, YULIANG

    2016-01-01

    Adipose tissue-derived mesenchymal stem cells (ADSCs) have been considered to be attractive and readily available adult mesenchymal stem cells, and they are becoming increasingly popular for use in regenerative cell therapy, as they are readily accessible through minimally invasive techniques. The present study investigated whether autologous ADSC transplantation promoted liver regeneration following a repeat partial hepatectomy in rats. The rats were divided into three groups as follows: 70% partial hepatectomy (PH) group; repeat PH (R-PH) group and R-PH/ADSC group, subjected to R-PH and treated with autologous ADSCs via portal vein injection. In each group, the rats were sacrificed at different time points postoperatively in order to evaluate the changes in liver function and to estimate the liver regenerative response. The expression of proliferating cell nuclear antigen (PCNA) labeling index in the liver was measured using immunohistochemistry. The expression levels of hepatocyte growth factor (HGF) mRNA were measured using reverse transcription polymerase chain reaction. The results showed that regeneration of the remaining liver following R-PH was significantly promoted by ADSC transplantation, as shown by a significant increase in liver to body weight ratio and the PCNA labeling index at 24 h post-hepatectomy. Additionally, ADSC transplantation markedly inhibited the elevation of serum levels of alanine aminotransferase, aspartate aminotransferase and total bilirubin, increased HGF content and also attenuated hepatic vacuolar degeneration 24 h postoperatively. Furthermore, the liver was found to almost fully recover from hepatocellular damage due to hepatectomy among the three groups at 168 h postoperatively. These results indicated that autologous ADSC transplantation enhanced the regenerative capacity of the remnant liver tissues in the early phase following R-PH. PMID:26783183

  18. Differentiation of HL60 cells: involvement of protein phosphorylation

    SciTech Connect

    Spearman, T.N.; Fontana, J.A.; Butcher, F.R.; Durham, J.P.

    1986-05-01

    The addition of retinoic acid (RA) to the human promyelocytic leukemic cell line HL60 in culture results in the cessation of growth and the acquisition of a more mature phenotype. Previous work in these laboratories has demonstrated a concomitant increase in the activity of calcium-dependent, phospholipid-sensitive protein kinase (PK-C). HL60 cells were incubated with /sup 32/P-P/sub i/ in the absence and presence of RA, homogenized, and aliquots subjected to two-dimensional electrophoresis. A comparison of autoradiograms made from these gels revealed several phosphoproteins whose radiolabeling was affected by RA. The radiolabeling of one particular phosphoprotein (49kd, pI 4.8) was found to be increased prior to phenotypic evidence of differentiation. It was demonstrated via incubating HL60 cytosol with /sup 32/P -ATP and Ca/sup 2 +/ in the absence and presence of phosphatidylserine and resolving the labeled proteins as above that this protein is phosphorylated by PK-C. The labeling of this protein was also increased by RA in other leukemic cell lines which showed phenotypic evidence of differentiation while no effect was seen in HL60 sublines resistant to RA or in mature neutrophils (the end product of myeloid differentiation). These results suggest that this protein may be an important intermediate in myeloid differentiation.

  19. Villidin, a Novel WD-repeat and Villin-related Protein from Dictyostelium, Is Associated with Membranes and the Cytoskeleton

    PubMed Central

    Gloss, Annika; Rivero, Francisco; Khaire, Nandkumar; Müller, Rolf; Loomis, William F.; Schleicher, Michael; Noegel, Angelika A.

    2003-01-01

    Villidin is a novel multidomain protein (190 kDa) from Dictyostelium amoebae containing WD repeats at its N-terminus, three PH domains in the middle of the molecule, and five gelsolin-like segments at the C-terminus, followed by a villin-like headpiece. Villidin mRNA and protein are present in low amounts during growth and early aggregation, but increase during development and reach their highest levels at the tipped mound stage. The protein is present in the cytosol as well as in the cytoskeletal and membrane fractions. GFP-tagged full-length villidin exhibits a similar distribution as native villidin, including a distinct colocalization with Golgi structures. Interestingly, GFP fusions with the gelsolin/villin-like region are uniformly dispersed in the cytoplasm, whereas GFP fusions of the N-terminal WD repeats codistribute with F-actin and are associated with the Triton-insoluble cytoskeleton. Strains lacking villidin because of targeted deletion of its gene grow normally and can develop into fruiting bodies. However, cell motility is reduced during aggregation and phototaxis is impaired in the mutant strains. We conclude that villidin harbors a major F-actin binding site in the N-terminal domain and not in the villin-like region as expected; association of villidin with vesicular membranes suggests that the protein functions as a linker between membranes and the actin cytoskeleton. PMID:12857859

  20. Villidin, a novel WD-repeat and villin-related protein from Dictyostelium, is associated with membranes and the cytoskeleton.

    PubMed

    Gloss, Annika; Rivero, Francisco; Khaire, Nandkumar; Müller, Rolf; Loomis, William F; Schleicher, Michael; Noegel, Angelika A

    2003-07-01

    Villidin is a novel multidomain protein (190 kDa) from Dictyostelium amoebae containing WD repeats at its N-terminus, three PH domains in the middle of the molecule, and five gelsolin-like segments at the C-terminus, followed by a villin-like headpiece. Villidin mRNA and protein are present in low amounts during growth and early aggregation, but increase during development and reach their highest levels at the tipped mound stage. The protein is present in the cytosol as well as in the cytoskeletal and membrane fractions. GFP-tagged full-length villidin exhibits a similar distribution as native villidin, including a distinct colocalization with Golgi structures. Interestingly, GFP fusions with the gelsolin/villin-like region are uniformly dispersed in the cytoplasm, whereas GFP fusions of the N-terminal WD repeats codistribute with F-actin and are associated with the Triton-insoluble cytoskeleton. Strains lacking villidin because of targeted deletion of its gene grow normally and can develop into fruiting bodies. However, cell motility is reduced during aggregation and phototaxis is impaired in the mutant strains. We conclude that villidin harbors a major F-actin binding site in the N-terminal domain and not in the villin-like region as expected; association of villidin with vesicular membranes suggests that the protein functions as a linker between membranes and the actin cytoskeleton.

  1. 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.

  2. The Rice Pentatricopeptide Repeat Protein RF5 Restores Fertility in Hong-Lian Cytoplasmic Male-Sterile Lines via a Complex with the Glycine-Rich Protein GRP162[C][W

    PubMed Central

    Hu, Jun; Wang, Kun; Huang, Wenchao; Liu, Gai; Gao, Ya; Wang, Jianming; Huang, Qi; Ji, Yanxiao; Qin, Xiaojian; Wan, Lei; Zhu, Renshan; Li, Shaoqing; Yang, Daichang; Zhu, Yingguo

    2012-01-01

    The cytoplasmic male sterility (CMS) phenotype in plants can be reversed by the action of nuclear-encoded fertility restorer (Rf) genes. The molecular mechanism involved in Rf gene–mediated processing of CMS-associated transcripts is unclear, as are the identities of other proteins that may be involved in the CMS–Rf interaction. In this study, we cloned the restorer gene Rf5 for Hong-Lian CMS in rice and studied its fertility restoration mechanism with respect to the processing of the CMS-associated transcript atp6-orfH79. RF5, a pentatricopeptide repeat (PPR) protein, was unable to bind to this CMS-associated transcript; however, a partner protein of RF5 (GRP162, a Gly-rich protein encoding 162 amino acids) was identified to bind to atp6-orfH79. GRP162 was found to physically interact with RF5 and to bind to atp6-orfH79 via an RNA recognition motif. Furthermore, we found that RF5 and GRP162 are both components of a restoration of fertility complex (RFC) that is 400 to 500 kD in size and can cleave CMS-associated transcripts in vitro. Evidence that a PPR protein interacts directly with a Gly-rich protein to form a subunit of the RFC provides a new perspective on the molecular mechanisms underlying fertility restoration. PMID:22247252

  3. Association between levels of C-reactive protein and leukocytes and cancer: Three repeated measurements in the Swedish AMORIS study

    PubMed Central

    Van Hemelrijck, Mieke; Holmberg, Lars; Garmo, Hans; Hammar, Niklas; Walldius, Göran; Binda, Elisa; Lambe, Mats; Jungner, Ingmar

    2011-01-01

    Objective To study levels of C-reactive protein (CRP) and leukocytes, as inflammatory markers, in the context of cancer risk. Methods From the Apolipoprotein MOrtality RISk (AMORIS) study, we selected 102,749 persons with one measurement and 9,273 persons with three repeated measurements of CRP and leukocytes. Multivariate Cox proportional hazards regression was applied to categories of CRP (<10, 10-15, 15-25, 25-50, >50 g/L) and quartiles of leukocytes. An Inflammation-based Predictive Score (IPS) indicated whether someone had CRP levels >10mg/L combined with leukocytes >10×109/L. Reverse causality was assessed by excluding those with <3, 5, or 7 years of follow-up. To analyze repeated measurements of CRP and leukocytes the repeated IPS (IPSr) was calculated by adding the IPS of each measurement. Results In the cohort with one measurement, there was a positive trend between CRP and cancer, with the lowest category being the reference: 0.99 (0.92-1.06), 1.28 (1.11-1.47), 1.27 (1.09-1.49), 1.22 (1.01-1.48) for the 2nd to 5th categories, respectively. This association disappeared when excluding those with follow-up <3, 5 or 7 years. The association between leukocytes and cancer was slightly stronger. In the cohort with repeated measurements the IPSr was strongly associated with cancer risk: 1.87 (1.33-2.63), 1.51 (0.56-4.06), 4.46 (1.43-13.87) for IPSr =1, 2, and 3, compared to IPSr =0. The association remained after excluding those with follow-up <1 year. Conclusions and impact Our large prospective cohort study adds evidence for a link between inflammatory markers and cancer risk by using repeated measurements and ascertaining reverse causality. PMID:21297038

  4. 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

  5. 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

  6. Enzymatic transformations involved in the biosynthesis of microbial exo-polysaccharides based on the assembly of repeat units.

    PubMed

    Schmid, Jochen; Sieber, Volker

    2015-05-26

    Microbial exo-polysaccharides can serve as valuable biopolymers in medicine, food and the feed industry as well as in various technical applications as substitutes of petro-based polymers or with unusual performance. Due to their different natural functions, they have vastly diverse structures, which lead to a very different properties. This structural diversity is brought about by complex biosyntheses based on enzymes whose genes are mostly encoded in clusters within the genomes of the different microbial species. The organisation of the genes and the chemical structures of the corresponding polysaccharides are closely related. Here, we will mainly focus on the genetics and biosynthesis of some major bacterial hetero-polysaccharides that are based on repeat unit assembly and will present specific examples of enzymatic transformation steps. Finally, a short outlook will be given on how in vivo modifications based on enzymatic transformations could be used to engineer these polymers. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  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. Specific binding of tetratricopeptide repeat (TPR) proteins to heat shock protein 70 (Hsp70) and heat shock protein 90 (Hsp90) is regulated by affinity and phosphorylation

    PubMed Central

    Assimon, Victoria A.; Southworth, Daniel R.; Gestwicki, Jason E.

    2016-01-01

    The heat shock proteins Hsp70 and Hsp90 require the help of tetratricopeptide repeat (TPR) domain-containing co-chaperones for many of their functions. Each monomer of Hsp70 or Hsp90 can only interact with a single TPR co-chaperone at a time and each member of the TPR co-chaperone family brings distinct functions into the complex. Thus, competition for TPR binding sites on Hsp70 and Hsp90 appears to shape chaperone activity. Recent structural and biophysical efforts have improved our understanding of chaperone-TPR contacts, focusing on the C-terminal EEVD motif that is present in both chaperones. To better understand these important protein-protein interactions on a wider scale, we measured the affinity of five TPR co-chaperones, CHIP, Hop, DnaJC7, FKBP51, and FKBP52, for the C-termini of four members of the chaperone family, Hsc70, Hsp72, Hsp90α, and Hsp90β, in vitro. These studies identified some surprising selectivity amongst the chaperone-TPR pairs, including the selective binding of FKBP51/52 to Hsp90α/β. These results also revealed that other TPR co-chaperones are only able to weakly discriminate between the chaperones or between their paralogs. We also explored whether mimicking phosphorylation of serine and threonine residues near the EEVD motif might impact affinity and found that pseudophosphorylation had selective effects on binding to CHIP but not other co-chaperones. Together, these findings suggest that both intrinsic affinity and post-translational modifications tune the interactions between Hsp70/90 and the TPR co-chaperones. PMID:26565746

  9. Different combinations of the heat-shock cognate protein 70 (hsc70) C-terminal functional groups are utilized to interact with distinct tetratricopeptide repeat-containing proteins.

    PubMed

    Wu, S J; Liu, F H; Hu, S M; Wang, C

    2001-10-15

    A group of tetratricopeptide repeat (TPR)-containing proteins has been shown to interact with the C-terminal domain of the 70 kDa heat-shock cognate protein (hsc70). In the present study, the effect of the TPR-containing proteins, including the C-terminus of hsc70-interacting protein (CHIP), TPR1 and human glutamine-rich TPR-containing protein (hSGT), on refolding of luciferase by DnaJ and hsc70 was investigated. These proteins inhibited the restoration of luciferase activity by the chaperones. The inhibitory effect exerted by TPR1 and hSGT depended upon their binding to hsc70. However, the interaction with hsc70 did not appear to be required for the inhibition of luciferase refolding by CHIP. We also demonstrate that the peptide, GPTIEEVD, corresponding to the C-terminal end of hsc70, abolished the association of [(3)H]hsc70 with CHIP, TPR1 and hSGT. This implied that the GPTIEEVD motif of hsc70 was responsible for interacting with these TPR-containing proteins. However, the GGXP-repeats (where X is any aliphatic residue), another C-terminal conserved motif of vertebrate hsc70s, were not essential for interacting with the TPR-containing proteins. On the basis of mutagenesis studies, it was clear that a unique combination of the functional groups in the GPTIEEVD motif were utilized to interact with each TPR-containing protein, suggesting that inhibitors can be designed and used to elucidate the functional role of these interactions.

  10. 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

  11. 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.

  12. 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.

  13. 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.

  14. 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.

  15. 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.

  16. 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.

  17. 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-linke