Protein (Viridiplantae): 786990 [PGDBj - Ortholog DB

Lifescience Database Archive (English)

Full Text Available 79506 Solanum tuberosum MEETSTSSNNAKAKARVRVCITRKKTLKDKRAKLYIIRRCLYMLLCWKERAEFCNVGNRESTA ...3993 4070:3993 ... 424551:3993 ... 424574:3993 ... 4107:3993 ... 4113:2476 ... PREDICTED: uncharacterized protein LOC1025

Surveillance of Washington OSHA exposure data to identify uncharacterized or emerging occupational health hazards.

Science.gov (United States)

Lofgren, Don J; Reeb-Whitaker, Carolyn K; Adams, Darrin

2010-07-01

Chemical substance exposure data from the Washington State Occupational Safety and Health Administration (OSHA) program were reviewed to determine if inspections conducted as a result of a report of a hazard from a complainant or referent may alert the agency to uncharacterized or emerging health hazards. Exposure and other electronically stored data from 6890 health inspection reports conducted between April 2003 and August 2008 were extracted from agency records. A total of 515 (7%) inspections with one or more personal airborne chemical substance samples were identified for further study. Inspections by report of a hazard and by targeting were compared for the following: number of inspections, number and percentage of inspections with workers exposed to substances above an agency's permissible exposure limit, types of industries inspected, and number and type of chemical substances assessed. Report of a hazard inspections documented work sites with worker overexposure at the same rate as agency targeted inspections (approximately 35% of the time), suggesting that complainants and referents are a credible pool of observers capable of directing the agency to airborne chemical substance hazards. Report of a hazard inspections were associated with significantly broader distribution of industries as well as a greater variety of chemical substance exposures than were targeted inspections. Narrative text that described business type and processes inspected was more useful than NAICS codes alone and critical in identifying processes and industries that may be associated with new hazards. Finally, previously identified emerging hazards were found among the report of a hazard data. These findings indicate that surveillance of OSHA inspection data can be a valid tool to identify uncharacterized and emerging health hazards. Additional research is needed to develop criteria for objective review and prioritization of the data for intervention. Federal OSHA and other state

Protein (Viridiplantae): 850873 [PGDBj - Ortholog DB

Lifescience Database Archive (English)

Full Text Available OC100796589 Glycine max MEETSWEQRVQALTHILTSPTTTPSLHSQFFIATQIPCYLNWDYPPFLCSSNPQLLK...803:12853 ... 3814:12853 ... 163735:5410 ... 3846:8024 1462606:8024 3847:8024 ... PREDICTED: uncharacterized protein L

Protein (Viridiplantae): 128879 [PGDBj - Ortholog DB

Lifescience Database Archive (English)

Full Text Available 5:248 3803:248 ... 3814:248 ... 163722:3689 ... 3826:3689 ... 3827:3689 ... PREDICTED: uncharacterized protein LOC101515541 Cicer arietinum MNSSTI...CSLFLGLILISQSANAKGHGGGLVVTICKGATDRAACENILGSNSEISHAKSFSQL

The Starch Granule-Associated Protein EARLY STARVATION1 Is Required for the Control of Starch Degradation in Arabidopsis thaliana Leaves[OPEN

Science.gov (United States)

Feike, Doreen; Seung, David; Graf, Alexander; Bischof, Sylvain; Ellick, Tamaryn; Coiro, Mario; Soyk, Sebastian; Eicke, Simona; Mettler-Altmann, Tabea; Lu, Kuan Jen; Trick, Martin; Zeeman, Samuel C.

2016-01-01

To uncover components of the mechanism that adjusts the rate of leaf starch degradation to the length of the night, we devised a screen for mutant Arabidopsis thaliana plants in which starch reserves are prematurely exhausted. The mutation in one such mutant, named early starvation1 (esv1), eliminates a previously uncharacterized protein. Starch in mutant leaves is degraded rapidly and in a nonlinear fashion, so that reserves are exhausted 2 h prior to dawn. The ESV1 protein and a similar uncharacterized Arabidopsis protein (named Like ESV1 [LESV]) are located in the chloroplast stroma and are also bound into starch granules. The region of highest similarity between the two proteins contains a series of near-repeated motifs rich in tryptophan. Both proteins are conserved throughout starch-synthesizing organisms, from angiosperms and monocots to green algae. Analysis of transgenic plants lacking or overexpressing ESV1 or LESV, and of double mutants lacking ESV1 and another protein necessary for starch degradation, leads us to propose that these proteins function in the organization of the starch granule matrix. We argue that their misexpression affects starch degradation indirectly, by altering matrix organization and, thus, accessibility of starch polymers to starch-degrading enzymes. PMID:27207856

Binding Ligand Prediction for Proteins Using Partial Matching of Local Surface Patches

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Lee Sael

2010-12-01

Full Text Available Functional elucidation of uncharacterized protein structures is an important task in bioinformatics. We report our new approach for structure-based function prediction which captures local surface features of ligand binding pockets. Function of proteins, specifically, binding ligands of proteins, can be predicted by finding similar local surface regions of known proteins. To enable partial comparison of binding sites in proteins, a weighted bipartite matching algorithm is used to match pairs of surface patches. The surface patches are encoded with the 3D Zernike descriptors. Unlike the existing methods which compare global characteristics of the protein fold or the global pocket shape, the local surface patch method can find functional similarity between non-homologous proteins and binding pockets for flexible ligand molecules. The proposed method improves prediction results over global pocket shape-based method which was previously developed by our group.

Binding ligand prediction for proteins using partial matching of local surface patches.

Science.gov (United States)

Sael, Lee; Kihara, Daisuke

2010-01-01

Functional elucidation of uncharacterized protein structures is an important task in bioinformatics. We report our new approach for structure-based function prediction which captures local surface features of ligand binding pockets. Function of proteins, specifically, binding ligands of proteins, can be predicted by finding similar local surface regions of known proteins. To enable partial comparison of binding sites in proteins, a weighted bipartite matching algorithm is used to match pairs of surface patches. The surface patches are encoded with the 3D Zernike descriptors. Unlike the existing methods which compare global characteristics of the protein fold or the global pocket shape, the local surface patch method can find functional similarity between non-homologous proteins and binding pockets for flexible ligand molecules. The proposed method improves prediction results over global pocket shape-based method which was previously developed by our group.

An estimated 5% of new protein structures solved today represent a new Pfam family

International Nuclear Information System (INIS)

Mistry, Jaina; Kloppmann, Edda; Rost, Burkhard; Punta, Marco

2013-01-01

This study uses the Pfam database to show that the sequence redundancy of protein structures deposited in the PDB is increasing. The possible reasons behind this trend are discussed. High-resolution structural knowledge is key to understanding how proteins function at the molecular level. The number of entries in the Protein Data Bank (PDB), the repository of all publicly available protein structures, continues to increase, with more than 8000 structures released in 2012 alone. The authors of this article have studied how structural coverage of the protein-sequence space has changed over time by monitoring the number of Pfam families that acquired their first representative structure each year from 1976 to 2012. Twenty years ago, for every 100 new PDB entries released, an estimated 20 Pfam families acquired their first structure. By 2012, this decreased to only about five families per 100 structures. The reasons behind the slower pace at which previously uncharacterized families are being structurally covered were investigated. It was found that although more than 50% of current Pfam families are still without a structural representative, this set is enriched in families that are small, functionally uncharacterized or rich in problem features such as intrinsically disordered and transmembrane regions. While these are important constraints, the reasons why it may not yet be time to give up the pursuit of a targeted but more comprehensive structural coverage of the protein-sequence space are discussed

A statistical approach to the estimation of mechanical unfolding parameters from the unfolding patterns of protein heteropolymers

International Nuclear Information System (INIS)

Beddard, G S; Brockwell, D J

2010-01-01

A statistical calculation is described with which the saw-tooth-like unfolding patterns of concatenated heteropolymeric proteins can be used to estimate the forced unfolding parameters of a previously uncharacterized protein. The chance of observing the various sequences of unfolding events, such as ABAABBB or BBAAABB etc, for two proteins of types A and B is calculated using proteins with various ratios of A and B and at different values of effective unfolding rate constants. If the experimental rate constant for forced unfolding, k 0 , and distance to the transition state x u are known for one protein, then the calculation allows an estimation of values for the other. The predictions are compared with Monte Carlo simulations and experimental data. (communication)

A previously uncharacterized gene stm0551 plays a repressive role in the regulation of type 1 fimbriae in Salmonella enterica serotype Typhimurium

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Wang Ke-Chuan

2012-06-01

Full Text Available Abstract Background Salmonella enterica serotype Typhimurium produces surface-associated fimbriae that facilitate adherence of the bacteria to a variety of cells and tissues. Type 1 fimbriae with binding specificity to mannose residues are the most commonly found fimbrial type. In vitro, static-broth culture favors the growth of S. Typhimurium with type 1 fimbriae, whereas non-type 1 fimbriate bacteria are obtained by culture on solid-agar media. Previous studies demonstrated that the phenotypic expression of type 1 fimbriae is the result of the interaction and cooperation of the regulatory genes fimZ, fimY, fimW, and fimU within the fim gene cluster. Genome sequencing revealed a novel gene, stm0551, located between fimY and fimW that encodes an 11.4-kDa putative phosphodiesterase specific for the bacterial second messenger cyclic-diguanylate monophosphate (c-di-GMP. The role of stm0551 in the regulation of type 1 fimbriae in S. Typhimurium remains unclear. Results A stm0551-deleted stain constructed by allelic exchange constitutively produced type 1 fimbriae in both static-broth and solid-agar medium conditions. Quantative RT-PCR revealed that expression of the fimbrial major subunit gene, fimA, and one of the regulatory genes, fimZ, were comparably increased in the stm0551-deleted strain compared with those of the parental strain when grown on the solid-agar medium, a condition that normally inhibits expression of type 1 fimbriae. Following transformation with a plasmid possessing the coding sequence of stm0551, expression of fimA and fimZ decreased in the stm0551 mutant strain in both culture conditions, whereas transformation with the control vector pACYC184 relieved this repression. A purified STM0551 protein exhibited a phosphodiesterase activity in vitro while a point mutation in the putative EAL domain, substituting glutamic acid (E with alanine (A, of STM0551 or a FimY protein abolished this activity. Conclusions The finding that the

Cell signaling, post-translational protein modifications and NMR spectroscopy

International Nuclear Information System (INIS)

Theillet, Francois-Xavier; Smet-Nocca, Caroline; Liokatis, Stamatios; Thongwichian, Rossukon; Kosten, Jonas; Yoon, Mi-Kyung; Kriwacki, Richard W.; Landrieu, Isabelle; Lippens, Guy; Selenko, Philipp

2012-01-01

Post-translationally modified proteins make up the majority of the proteome and establish, to a large part, the impressive level of functional diversity in higher, multi-cellular organisms. Most eukaryotic post-translational protein modifications (PTMs) denote reversible, covalent additions of small chemical entities such as phosphate-, acyl-, alkyl- and glycosyl-groups onto selected subsets of modifiable amino acids. In turn, these modifications induce highly specific changes in the chemical environments of individual protein residues, which are readily detected by high-resolution NMR spectroscopy. In the following, we provide a concise compendium of NMR characteristics of the main types of eukaryotic PTMs: serine, threonine, tyrosine and histidine phosphorylation, lysine acetylation, lysine and arginine methylation, and serine, threonine O-glycosylation. We further delineate the previously uncharacterized NMR properties of lysine propionylation, butyrylation, succinylation, malonylation and crotonylation, which, altogether, define an initial reference frame for comprehensive PTM studies by high-resolution NMR spectroscopy.

Proteomic profiling of Plasmodium sporozoite maturation identifies new proteins essential for parasite development and infectivity

DEFF Research Database (Denmark)

Lasonder, Edwin; Janse, Chris J; van Gemert, Geert-Jan

2008-01-01

Plasmodium falciparum sporozoites that develop and mature inside an Anopheles mosquito initiate a malaria infection in humans. Here we report the first proteomic comparison of different parasite stages from the mosquito -- early and late oocysts containing midgut sporozoites, and the mature...... whose annotation suggest an involvement in sporozoite maturation, motility, infection of the human host and associated metabolic adjustments. Analyses of proteins identified in the P. falciparum sporozoite proteomes by orthologous gene disruption in the rodent malaria parasite, P. berghei, revealed...... three previously uncharacterized Plasmodium proteins that appear to be essential for sporozoite development at distinct points of maturation in the mosquito. This study sheds light on the development and maturation of the malaria parasite in an Anopheles mosquito and also identifies proteins that may...

Optimization of Saanen sperm genes amplification: evaluation of standardized protocols in genetically uncharacterized rural goats reared under a subtropical environment.

Science.gov (United States)

Barbour, Elie K; Saade, Maya F; Sleiman, Fawwak T; Hamadeh, Shady K; Mouneimne, Youssef; Kassaifi, Zeina; Kayali, Ghazi; Harakeh, Steve; Jaber, Lina S; Shaib, Houssam A

2012-10-01

The purpose of this research is to optimize quantitatively the amplification of specific sperm genes in reference genomically characterized Saanen goat and to evaluate the standardized protocols applicability on sperms of uncharacterized genome of rural goats reared under subtropical environment for inclusion in future selection programs. The optimization of the protocols in Saanen sperms included three production genes (growth hormone (GH) exons 2, 3, and 4, αS1-casein (CSN1S1), and α-lactalbumin) and two health genes (MHC class II DRB and prion (PrP)). The optimization was based on varying the primers concentrations and the inclusion of a PCR cosolvent (Triton X). The impact of the studied variables on statistically significant increase in the yield of amplicons was noticed in four out of five (80%) optimized protocols, namely in those related to GH, CSN1S1, α-lactalbumin, and PrP genes (P 0.05). The applicability of the optimized protocols of Saanen sperm genes on amplification of uncharacterized rural goat sperms revealed a 100% success in tested individuals for amplification of GH, CSN1S1, α-lactalbumin, and MHC class II DRB genes and a 75% success for the PrP gene. The significant success in applicability of the Saanen quantitatively optimized protocols to other uncharacterized genome of rural goats allows for their inclusion in future selection, targeting the sustainability of this farming system in a subtropical environment and the improvement of the farmers livelihood.

AVID: An integrative framework for discovering functional relationships among proteins

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Keating Amy E

2005-06-01

Full Text Available Abstract Background Determining the functions of uncharacterized proteins is one of the most pressing problems in the post-genomic era. Large scale protein-protein interaction assays, global mRNA expression analyses and systematic protein localization studies provide experimental information that can be used for this purpose. The data from such experiments contain many false positives and false negatives, but can be processed using computational methods to provide reliable information about protein-protein relationships and protein function. An outstanding and important goal is to predict detailed functional annotation for all uncharacterized proteins that is reliable enough to effectively guide experiments. Results We present AVID, a computational method that uses a multi-stage learning framework to integrate experimental results with sequence information, generating networks reflecting functional similarities among proteins. We illustrate use of the networks by making predictions of detailed Gene Ontology (GO annotations in three categories: molecular function, biological process, and cellular component. Applied to the yeast Saccharomyces cerevisiae, AVID provides 37,451 pair-wise functional linkages between 4,191 proteins. These relationships are ~65–78% accurate, as assessed by cross-validation testing. Assignments of highly detailed functional descriptors to proteins, based on the networks, are estimated to be ~67% accurate for GO categories describing molecular function and cellular component and ~52% accurate for terms describing biological process. The predictions cover 1,490 proteins with no previous annotation in GO and also assign more detailed functions to many proteins annotated only with less descriptive terms. Predictions made by AVID are largely distinct from those made by other methods. Out of 37,451 predicted pair-wise relationships, the greatest number shared in common with another method is 3,413. Conclusion AVID provides

Purification, crystallization and preliminary crystallographic analysis of SMU.1108c protein from Streptococcus mutans

International Nuclear Information System (INIS)

Feng, Ming-Jing; Fu, Tian-Min; Liu, Xiang; Li, Lan-Fen

2010-01-01

SMU.1108c, a putative uncharacterized protein from S. mutans, was crystallized and X-ray diffraction data were collected to a resolution of 2.2 Å. Streptococcus mutans SMU.1108c (KEGG database) encodes a functionally uncharacterized protein consisting of 270 amino-acid residues. This protein is predicted to have a haloacid dehalogenase hydrolase-like domain and is a homologue of haloacid dehalogenase phosphatases that catalyze phosphoryl-transfer reactions. In this work, SMU.1108c was cloned into the pET28a vector and overexpressed in Escherichia coli strain BL21 (DE3). The protein was purified to homogeneity and crystallized using the sitting-drop vapour-diffusion method. The best crystal diffracted to 2.0 Å resolution and belonged to space group C2, with unit-cell parameters a = 77.1, b = 80.2, c = 47.9 Å, β = 99.5°

Semi-supervised Learning Predicts Approximately One Third of the Alternative Splicing Isoforms as Functional Proteins

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Yanqi Hao

2015-07-01

Full Text Available Alternative splicing acts on transcripts from almost all human multi-exon genes. Notwithstanding its ubiquity, fundamental ramifications of splicing on protein expression remain unresolved. The number and identity of spliced transcripts that form stably folded proteins remain the sources of considerable debate, due largely to low coverage of experimental methods and the resulting absence of negative data. We circumvent this issue by developing a semi-supervised learning algorithm, positive unlabeled learning for splicing elucidation (PULSE; http://www.kimlab.org/software/pulse, which uses 48 features spanning various categories. We validated its accuracy on sets of bona fide protein isoforms and directly on mass spectrometry (MS spectra for an overall AU-ROC of 0.85. We predict that around 32% of “exon skipping” alternative splicing events produce stable proteins, suggesting that the process engenders a significant number of previously uncharacterized proteins. We also provide insights into the distribution of positive isoforms in various functional classes and into the structural effects of alternative splicing.

Implementation and assessment of a yeast orphan gene research project: involving undergraduates in authentic research experiences and progressing our understanding of uncharacterized open reading frames.

Science.gov (United States)

Bowling, Bethany V; Schultheis, Patrick J; Strome, Erin D

2016-02-01

Saccharomyces cerevisiae was the first eukaryotic organism to be sequenced; however, little progress has been made in recent years in furthering our understanding of all open reading frames (ORFs). From October 2012 to May 2015 the number of verified ORFs had only risen from 75.31% to 78%, while the number of uncharacterized ORFs had decreased from 12.8% to 11% (representing > 700 genes still left in this category; http://www.yeastgenome.org/genomesnapshot). Course-based research has been shown to increase student learning while providing experience with real scientific investigation; however, implementation in large, multi-section courses presents many challenges. This study sought to test the feasibility and effectiveness of incorporating authentic research into a core genetics course, with multiple instructors, to increase student learning and progress our understanding of uncharacterized ORFs. We generated a module-based annotation toolkit and utilized easily accessible bioinformatics tools to predict gene function for uncharacterized ORFs within the Saccharomyces Genome Database (SGD). Students were each assigned an uncharacterized ORF, which they annotated using contemporary comparative genomics methodologies, including multiple sequence alignment, conserved domain identification, signal peptide prediction and cellular localization algorithms. Student learning outcomes were measured by quizzes, project reports and presentations, as well as a post-project questionnaire. Our results indicate that the authentic research experience had positive impacts on students' perception of their learning and their confidence to conduct future research. Furthermore, we believe that creation of an online repository and adoption and/or adaptation of this project across multiple researchers and institutions could speed the process of gene function prediction. Copyright © 2015 John Wiley & Sons, Ltd.

Identification and characterization of proteins involved in nuclear organization using Drosophila GFP protein trap lines.

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Margaret Rohrbaugh

Full Text Available Strains from a collection of Drosophila GFP protein trap lines express GFP in the normal tissues where the endogenous protein is present. This collection can be used to screen for proteins distributed in the nucleus in a non-uniform pattern.We analyzed four lines that show peripheral or punctate nuclear staining. One of these lines affects an uncharacterized gene named CG11138. The CG11138 protein shows a punctate distribution in the nuclear periphery similar to that of Drosophila insulator proteins but does not co-localize with known insulators. Interestingly, mutations in Lamin proteins result in alterations in CG11138 localization, suggesting that this protein may be a novel component of the nuclear lamina. A second line affects the Decondensation factor 31 (Df31 gene, which encodes a protein with a unique nuclear distribution that appears to segment the nucleus into four different compartments. The X-chromosome of males is confined to one of these compartments. We also find that Drosophila Nucleoplasmin (dNlp is present in regions of active transcription. Heat shock leads to loss of dNlp from previously transcribed regions of polytene chromosome without redistribution to the heat shock genes. Analysis of Stonewall (Stwl, a protein previously found to be necessary for the maintenance of germline stem cells, shows that Stwl is present in a punctate pattern in the nucleus that partially overlaps with that of known insulator proteins. Finally we show that Stwl, dNlp, and Df31 form part of a highly interactive network. The properties of other components of this network may help understand the role of these proteins in nuclear biology.These results establish screening of GFP protein trap alleles as a strategy to identify factors with novel cellular functions. Information gained from the analysis of CG11138 Stwl, dNlp, and Df31 sets the stage for future studies of these proteins.

Protein Supplement Usage Among Male University Students: Comparisons Between Current and Previous Users.

Science.gov (United States)

Sung, Youngmo; Choi, Jinkyung

2018-02-01

Interest in specialized dietary supplements is leading market growth, and protein supplements are popular for increasing muscle mass among young males. Therefore, this study investigated the attitudes toward and satisfaction with protein supplements to identify detailed consumer behaviors related to the consumption of protein supplements. The study sample includes male university students in their 20s in South Korea. In total, 223 responses were entered for analysis. Questions related to attitudes, satisfaction, and future behavioral intentions were asked using 5-point Likert scales. The responses were divided into two groups, current and previous users, to identify significant differences in terms of attitudes, satisfaction, and future behavioral intentions. A descriptive analysis, analysis of variance (ANOVA), and multiple regression were run. The majority of respondents prefer the powdered form of supplements in bulk with a price range between 30,000 won and 60,000 won. Online shopping was preferred, while word of mouth and friends/family were considered credible information sources. The most common side effects experienced were problems with digestion and hives, although more than 78% did not experience side effects. In comparison between current and previous users in terms of attitudes and satisfaction, the following areas showed significances. Regarding attitudes, the importance of brand, preference for products from overseas, the search for nutritional facts, and reading carefully all product information were significant, while regarding satisfaction, price, effectiveness, and ease of consumption were significant. All significances showed that current users had more positive attitudes and greater satisfaction. Overall, consumers' satisfaction regarding ease of consumption influenced future behavioral intentions. The market for protein supplements has been growing, so measuring consumers' attitudes and satisfaction would help attract potential consumers. In

Monitoring protein phosphorylation by acrylamide pendant Phos-Tag™ in various plants

Directory of Open Access Journals (Sweden)

Slavka eBekesova

2015-05-01

Full Text Available The aim of the present study is to rationalize acrylamide pendant Phos-Tag™ in-gel discrimination of phosphorylated and non-phosphorylated plant protein species with standard immunoblot analysis, and optimize sample preparation, efficient electrophoretic separation and transfer. We tested variants of the method including extraction buffers suitable for preservation of phosphorylated protein species in crude extracts from plants and we addressed the importance of the cation (Mn2+ or Zn2+ used in the gel recipe for efficient transfer to PVDF membranes for further immunoblot analysis. We demonstrate the monitoring of Medicago sativa stress-induced mitogen activated protein kinase (SIMK in stress-treated wild type plants and transgenic SIMKK RNAi line. We further show the hyperosmotically-induced phosphorylation of the previously uncharacterized HvMPK4 of barley. The method is validated using inducible phosphorylation of barley and wheat α-tubulin and of Arabidopsis MPK6. Acrylamide pendant Phos-Tag™ offers a flexible tool for studying protein phosphorylation in crops and Arabidopsis circumventing radioactive labeling and the use of phosphorylation specific antibodies.

The Ser/Thr Protein Kinase Protein-Protein Interaction Map of M. tuberculosis.

Science.gov (United States)

Wu, Fan-Lin; Liu, Yin; Jiang, He-Wei; Luan, Yi-Zhao; Zhang, Hai-Nan; He, Xiang; Xu, Zhao-Wei; Hou, Jing-Li; Ji, Li-Yun; Xie, Zhi; Czajkowsky, Daniel M; Yan, Wei; Deng, Jiao-Yu; Bi, Li-Jun; Zhang, Xian-En; Tao, Sheng-Ce

2017-08-01

Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis, the leading cause of death among all infectious diseases. There are 11 eukaryotic-like serine/threonine protein kinases (STPKs) in Mtb, which are thought to play pivotal roles in cell growth, signal transduction and pathogenesis. However, their underlying mechanisms of action remain largely uncharacterized. In this study, using a Mtb proteome microarray, we have globally identified the binding proteins in Mtb for all of the STPKs, and constructed the first STPK protein interaction (KPI) map that includes 492 binding proteins and 1,027 interactions. Bioinformatics analysis showed that the interacting proteins reflect diverse functions, including roles in two-component system, transcription, protein degradation, and cell wall integrity. Functional investigations confirmed that PknG regulates cell wall integrity through key components of peptidoglycan (PG) biosynthesis, e.g. MurC. The global STPK-KPIs network constructed here is expected to serve as a rich resource for understanding the key signaling pathways in Mtb, thus facilitating drug development and effective control of Mtb. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Scoring protein relationships in functional interaction networks predicted from sequence data.

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Gaston K Mazandu

Full Text Available UNLABELLED: The abundance of diverse biological data from various sources constitutes a rich source of knowledge, which has the power to advance our understanding of organisms. This requires computational methods in order to integrate and exploit these data effectively and elucidate local and genome wide functional connections between protein pairs, thus enabling functional inferences for uncharacterized proteins. These biological data are primarily in the form of sequences, which determine functions, although functional properties of a protein can often be predicted from just the domains it contains. Thus, protein sequences and domains can be used to predict protein pair-wise functional relationships, and thus contribute to the function prediction process of uncharacterized proteins in order to ensure that knowledge is gained from sequencing efforts. In this work, we introduce information-theoretic based approaches to score protein-protein functional interaction pairs predicted from protein sequence similarity and conserved protein signature matches. The proposed schemes are effective for data-driven scoring of connections between protein pairs. We applied these schemes to the Mycobacterium tuberculosis proteome to produce a homology-based functional network of the organism with a high confidence and coverage. We use the network for predicting functions of uncharacterised proteins. AVAILABILITY: Protein pair-wise functional relationship scores for Mycobacterium tuberculosis strain CDC1551 sequence data and python scripts to compute these scores are available at http://web.cbio.uct.ac.za/~gmazandu/scoringschemes.

Comparative evaluation of low-molecular-mass proteins from Mycobacterium tuberculosis identifies members of the ESAT-6 family as immunodominant T-cell antigens

DEFF Research Database (Denmark)

Skjøt, R L; Oettinger, T; Rosenkrands, I

2000-01-01

Culture filtrate from Mycobacterium tuberculosis contains protective antigens of relevance for the generation of a new antituberculosis vaccine. We have identified two previously uncharacterized M. tuberculosis proteins (TB7.3 and TB10.4) from the highly active low-mass fraction of culture filtrate....... The molecules were characterized, mapped in a two-dimensional electrophoresis reference map of short-term culture filtrate, and compared with another recently identified low-mass protein, CFP10 (F. X. Berthet, P. B. Rasmussen, I. Rosenkrands, P. Andersen, and B. Gicquel. Microbiology 144:3195-3203, 1998......), and the well-described ESAT-6 antigen. Genetic analyses demonstrated that TB10.4 as well as CFP10 belongs to the ESAT-6 family of low-mass proteins, whereas TB7.3 is a low-molecular-mass protein outside this family. The proteins were expressed in Escherichia coli, and their immunogenicity was tested...

Maize sugary enhancer1 (se1) is a presence-absence variant of a previously uncharacterized gene and development of educational videos to raise the profile of plant breeding and improve curricula

Science.gov (United States)

Haro von Mogel, Karl J.

Carbohydrate metabolism is a biologically, economically, and culturally important process in crop plants. Humans have selected many crop species such as maize (Zea mays L.) in ways that have resulted in changes to carbohydrate metabolic pathways, and understanding the underlying genetics of this pathway is therefore exceedingly important. A previously uncharacterized starch metabolic pathway mutant, sugary enhancer1 (se1), is a recessive modifier of sugary1 (su1) sweet corn that increases the sugar content while maintaining an appealing creamy texture. This allele has been incorporated into many sweet corn varieties since its discovery in the 1970s, however, testing for the presence of this allele has been difficult. A genetic stock was developed that allowed the presence of se1 to be visually scored in segregating ears, which were used to genetically map se1 to the deletion of a single gene model located on the distal end of the long arm of chromosome 2. An analysis of homology found that this gene is specific to monocots, and the gene is expressed in the endosperm and developing leaf. The se1 allele increased water soluble polysaccharide (WSP) and decreased amylopectin in maize endosperm, but there was no overall effect on starch content in mature leaves due to se1. This discovery will lead to a greater understanding of starch metabolism, and the marker developed will assist in breeding. There is a present need for increased training for plant breeders to meet the growing needs of the human population. To raise the profile of plant breeding among young students, a series of videos called Fields of Study was developed. These feature interviews with plant breeders who talk about what they do as plant breeders and what they enjoy about their chosen profession. To help broaden the education of students in college biology courses, and assist with the training of plant breeders, a second video series, Pollination Methods was developed. Each video focuses on one or two

Outer membrane protein functions as integrator of protein import and DNA inheritance in mitochondria

Science.gov (United States)

Käser, Sandro; Oeljeklaus, Silke; Týč, Jiří; Vaughan, Sue; Warscheid, Bettina; Schneider, André

2016-01-01

Trypanosomatids are one of the earliest diverging eukaryotes that have fully functional mitochondria. pATOM36 is a trypanosomatid-specific essential mitochondrial outer membrane protein that has been implicated in protein import. Changes in the mitochondrial proteome induced by ablation of pATOM36 and in vitro assays show that pATOM36 is required for the assembly of the archaic translocase of the outer membrane (ATOM), the functional analog of the TOM complex in other organisms. Reciprocal pull-down experiments and immunofluorescence analyses demonstrate that a fraction of pATOM36 interacts and colocalizes with TAC65, a previously uncharacterized essential component of the tripartite attachment complex (TAC). The TAC links the single-unit mitochondrial genome to the basal body of the flagellum and mediates the segregation of the replicated mitochondrial genomes. RNAi experiments show that pATOM36, in line with its dual localization, is not only essential for ATOM complex assembly but also for segregation of the replicated mitochondrial genomes. However, the two functions are distinct, as a truncated version of pATOM36 lacking the 75 C-terminal amino acids can rescue kinetoplast DNA missegregation but not the lack of ATOM complex assembly. Thus, pATOM36 has a dual function and integrates mitochondrial protein import with mitochondrial DNA inheritance. PMID:27436903

Journal of Biosciences | Indian Academy of Sciences

Indian Academy of Sciences (India)

Amino acid sequence analysis corresponding to the PPE proteins in H37Rv and CDC1551 strains of the Mycobacterium tuberculosis genomes resulted in the identification of a previously uncharacterized 225 amino acidresidue common region in 22 proteins. The pairwise sequence identities were as low as 18%.

Identification of TOEFAZ1-interacting proteins reveals key regulators of Trypanosoma brucei cytokinesis.

Science.gov (United States)

Hilton, Nicholas A; Sladewski, Thomas E; Perry, Jenna A; Pataki, Zemplen; Sinclair-Davis, Amy N; Muniz, Richard S; Tran, Holly L; Wurster, Jenna I; Seo, Jiwon; de Graffenried, Christopher L

2018-05-21

The protist parasite Trypanosoma brucei is an obligate extracellular pathogen that retains its highly-polarized morphology during cell division and has evolved a novel cytokinetic process independent of non-muscle myosin II. The polo-like kinase homolog TbPLK is essential for transmission of cell polarity during division and for cytokinesis. We previously identified a putative TbPLK substrate named Tip of the Extending FAZ 1 (TOEFAZ1) as an essential kinetoplastid-specific component of the T. brucei cytokinetic machinery. We performed a proximity-dependent biotinylation (BioID) screen using TOEFAZ1 as a means to identify additional proteins that are involved in cytokinesis. Using quantitative proteomic methods, we identified nearly 500 TOEFAZ1-proximal proteins and characterized 59 in further detail. Among the candidates, we identified an essential putative phosphatase that regulates the expression level and localization of both TOEFAZ1 and TbPLK, a previously uncharacterized protein that is necessary for the assembly of a new cell posterior, and a microtubule plus-end directed orphan kinesin that is required for completing cleavage furrow ingression. The identification of these proteins provides new insight into T. brucei cytokinesis and establishes TOEFAZ1 as a key component of this essential and uniquely-configured process in kinetoplastids. This article is protected by copyright. All rights reserved. © 2018 John Wiley & Sons Ltd.

Mutation of the N-Terminal Region of Chikungunya Virus Capsid Protein: Implications for Vaccine Design.

Science.gov (United States)

Taylor, Adam; Liu, Xiang; Zaid, Ali; Goh, Lucas Y H; Hobson-Peters, Jody; Hall, Roy A; Merits, Andres; Mahalingam, Suresh

2017-02-21

Mosquito-transmitted chikungunya virus (CHIKV) is an arthritogenic alphavirus of the Togaviridae family responsible for frequent outbreaks of arthritic disease in humans. Capsid protein, a structural protein encoded by the CHIKV RNA genome, is able to translocate to the host cell nucleolus. In encephalitic alphaviruses, nuclear translocation induces host cell transcriptional shutoff; however, the role of capsid protein nucleolar localization in arthritogenic alphaviruses remains unclear. Using recombinant enhanced green fluorescent protein (EGFP)-tagged expression constructs and CHIKV infectious clones, we describe a nucleolar localization sequence (NoLS) in the N-terminal region of capsid protein, previously uncharacterized in CHIKV. Mutation of the NoLS by site-directed mutagenesis reduced efficiency of nuclear import of CHIKV capsid protein. In the virus, mutation of the capsid protein NoLS (CHIKV-NoLS) attenuated replication in mammalian and mosquito cells, producing a small-plaque phenotype. Attenuation of CHIKV-NoLS is likely due to disruption of the viral replication cycle downstream of viral RNA synthesis. In mice, CHIKV-NoLS infection caused no disease signs compared to wild-type CHIKV (CHIKV-WT)-infected mice; lack of disease signs correlated with significantly reduced viremia and decreased expression of proinflammatory factors. Mice immunized with CHIKV-NoLS, challenged with CHIKV-WT at 30 days postimmunization, develop no disease signs and no detectable viremia. Serum from CHIKV-NoLS-immunized mice is able to efficiently neutralize CHIKV infection in vitro Additionally, CHIKV-NoLS-immunized mice challenged with the related alphavirus Ross River virus showed reduced early and peak viremia postchallenge, indicating a cross-protective effect. The high degree of CHIKV-NoLS attenuation may improve CHIKV antiviral and rational vaccine design. IMPORTANCE CHIKV is a mosquito-borne pathogen capable of causing explosive epidemics of incapacitating joint pain

AcEST: BP919907 [AcEST

Lifescience Database Archive (English)

Full Text Available 5_9BACT Putative uncharacterized protein OS=Verrucomicrobiae bacterium DG1235 Align length 67 Score (bit) 35... Putative uncharacterized protein OS=Crypt... 33 9.2 >tr|B5JJK5|B5JJK5_9BACT Putative uncharacterized protein OS=Verrucomic

Effects of previous protein intake on rectal temperature, blood glucose, plasma thyroid hormone and minerals by laying hens during a forced molt

International Nuclear Information System (INIS)

Rodrigues, G.A.; Moraes, V.M.B.; Cherici, I; Furlan, R.L.; Macari, M.

1991-01-01

The effects of forced molting on blood glucose, rectal temperature, plasma T4, T3 and minerals were studied in hens previously fed rations with different protein contents (14, 17 and 20% crude protein). Blood samples were obtained from brachial veins for blood glucose, T4 and T3 were measured by radioimmunoassay, and plasma minerals were determined by atomic absorption spectroscopy. Blood glucose and rectal temperature were reduced during fasting regardless of previous protein intake. Pre molting T4 plasma level was higher in laying hens fed higher protein ration, but feed deprivation reduced T 4 and T 3 concentrations irrespective of protein intake, except T 4 level for 14% crude protein fed birds that increased during fasting. The data obtained in this experiment suggest that previous protein intake does not interfere with the metabolic changes during forced molt. (author). 19 refs, 1 fig, 4 tabs

Effects of previous protein intake on rectal temperature, blood glucose, plasma thyroid hormone and minerals by laying hens during a forced molt

Energy Technology Data Exchange (ETDEWEB)

Rodrigues, G A; Moraes, V M.B.; Cherici, I; Furlan, R L; Macari, M [UNESP, Jaboticabal, SP (Brazil). Faculdade de Ciencias Agrarias e Veterinarias

1991-12-01

The effects of forced molting on blood glucose, rectal temperature, plasma T4, T3 and minerals were studied in hens previously fed rations with different protein contents (14, 17 and 20% crude protein). Blood samples were obtained from brachial veins for blood glucose, T4 and T3 were measured by radioimmunoassay, and plasma minerals were determined by atomic absorption spectroscopy. Blood glucose and rectal temperature were reduced during fasting regardless of previous protein intake. Pre molting T4 plasma level was higher in laying hens fed higher protein ration, but feed deprivation reduced T{sub 4} and T{sub 3} concentrations irrespective of protein intake, except T{sub 4} level for 14% crude protein fed birds that increased during fasting. The data obtained in this experiment suggest that previous protein intake does not interfere with the metabolic changes during forced molt. (author). 19 refs, 1 fig, 4 tabs.

Atmospheric Detection of Perfluorotributyl Amine, an Uncharacterized Long-Lived Greenhouse Gas

Science.gov (United States)

Hong, A. C.; Young, C. J.; Mabury, S. A.

2012-12-01

Poly- and perfluoroalkyl substances (PFASs) are trace atmospheric constituents of radiative significance. In the atmosphere, PFASs may represent a class of potent long-lived greenhouse gases (LLGHGs) because they possess long lifetimes and exceptionally strong absorption bands in the infrared (IR) spectral region where other naturally occurring greenhouse gases (GHGs) do not absorb. The Intergovernmental Panel on Climate Change determined the radiative forcing (RF) of halocarbons to be +0.337 [± 0.03] W m-2, accounting for 13 % of the total RF attributed to LLGHGs. Although this value claims high certainty, it does not represent the actual perturbation from all environmentally relevant PFASs. Here we present the radiative efficiency (RE) and atmospheric concentration of a previously uncharacterized and unreported PFAS, perfluorotributyl amine (PFBAm). To assess the radiative properties of PFBAm, IR spectra were acquired by Fourier transform spectroscopy at 0.25 cm-1 resolution over the spectral range 0-2500 cm-1 at 296 K. The total integrated band strength, 7.08 x 10-16 cm2 molec-1 cm-1, was used to derive the cloudy-sky, instantaneous RE assuming a 0 to 1 ppbv change in concentration.The RE of PFBAm is calculated to be 0.86 W m-2 ppb-1, exceeding the RE of SF5CF3, the most effective GHG on a per molecule basis as reported in the literature to date. To evaluate the RF of PFBAm, a highly sensitive and selective method for detection was developed and validated. PFBAm was cryogenically extracted and pre-concentrated from bulk air samples for the offline detection by a custom-designed manifold coupled to a gas chromatograph-mass spectrometer. Quantitation was achieved by external calibration with a gravimetrically prepared, matrix-matched, authentic gaseous standard. Validation of the sampling method was performed by simultaneous measurement of several legacy chlorofluorocarbons and hydrochlorofluorocarbons. Preliminary results indicate that PFBAm is present in the

CUP-1 Is a Novel Protein Involved in Dietary Cholesterol Uptake in Caenorhabditis elegans

Science.gov (United States)

Valdes, Victor J.; Athie, Alejandro; Salinas, Laura S.; Navarro, Rosa E.; Vaca, Luis

2012-01-01

Sterols transport and distribution are essential processes in all multicellular organisms. Survival of the nematode Caenorhabditis elegans depends on dietary absorption of sterols present in the environment. However the general mechanisms associated to sterol uptake in nematodes are poorly understood. In the present work we provide evidence showing that a previously uncharacterized transmembrane protein, designated Cholesterol Uptake Protein-1 (CUP-1), is involved in dietary cholesterol uptake in C. elegans. Animals lacking CUP-1 showed hypersensitivity to cholesterol limitation and were unable to uptake cholesterol. A CUP-1-GFP fusion protein colocalized with cholesterol-rich vesicles, endosomes and lysosomes as well as the plasma membrane. Additionally, by FRET imaging, a direct interaction was found between the cholesterol analog DHE and the transmembrane “cholesterol recognition/interaction amino acid consensus” (CRAC) motif present in C. elegans CUP-1. In-silico analysis identified two mammalian homologues of CUP-1. Most interestingly, CRAC motifs are conserved in mammalian CUP-1 homologous. Our results suggest a role of CUP-1 in cholesterol uptake in C. elegans and open up the possibility for the existence of a new class of proteins involved in sterol absorption in mammals. PMID:22479487

The calcium-binding protein ALG-2 regulates protein secretion and trafficking via interactions with MISSL and MAP1B proteins.

Science.gov (United States)

Takahara, Terunao; Inoue, Kuniko; Arai, Yumika; Kuwata, Keiko; Shibata, Hideki; Maki, Masatoshi

2017-10-13

Mobilization of intracellular calcium is essential for a wide range of cellular processes, including signal transduction, apoptosis, and vesicular trafficking. Several lines of evidence have suggested that apoptosis-linked gene 2 (ALG-2, also known as PDCD6 ), a calcium-binding protein, acts as a calcium sensor linking calcium levels with efficient vesicular trafficking, especially at the endoplasmic reticulum (ER)-to-Golgi transport step. However, how ALG-2 regulates these processes remains largely unclear. Here, we report that M APK1- i nteracting and s pindle- s tabilizing (MISS)- l ike (MISSL), a previously uncharacterized protein, interacts with ALG-2 in a calcium-dependent manner. Live-cell imaging revealed that upon a rise in intracellular calcium levels, GFP-tagged MISSL (GFP-MISSL) dynamically relocalizes in a punctate pattern and colocalizes with ALG-2. MISSL knockdown caused disorganization of the components of the ER exit site, the ER-Golgi intermediate compartment, and Golgi. Importantly, knockdown of either MISSL or ALG-2 attenuated the secretion of se creted a lkaline p hosphatase (SEAP), a model secreted cargo protein, with similar reductions in secretion by single- and double-protein knockdowns, suggesting that MISSL and ALG-2 act in the same pathway to regulate the secretion process. Furthermore, ALG-2 or MISSL knockdown delayed ER-to-Golgi transport of procollagen type I. We also found that ALG-2 and MISSL interact with microtubule-associated protein 1B (MAP1B) and that MAP1B knockdown reverts the reduced secretion of SEAP caused by MISSL or ALG-2 depletion. These results suggest that a change in the intracellular calcium level plays a role in regulation of the secretory pathway via interaction of ALG-2 with MISSL and MAP1B. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Interactions among tobacco sieve element occlusion (SEO) proteins.

Science.gov (United States)

Jekat, Stephan B; Ernst, Antonia M; Zielonka, Sascia; Noll, Gundula A; Prüfer, Dirk

2012-12-01

Angiosperms transport their photoassimilates through sieve tubes, which comprise longitudinally-connected sieve elements. In dicots and also some monocots, the sieve elements contain parietal structural proteins known as phloem proteins or P-proteins. Following injury, P proteins disperse and accumulate as viscous plugs at the sieve plates to prevent the loss of valuable transport sugars. Tobacco (Nicotiana tabacum) P-proteins are multimeric complexes comprising subunits encoded by members of the SEO (sieve element occlusion) gene family. The existence of multiple subunits suggests that P-protein assembly involves interactions between SEO proteins, but this process is largely uncharacterized and it is unclear whether the different subunits perform unique roles or are redundant. We therefore extended our analysis of the tobacco P-proteins NtSEO1 and NtSEO2 to investigate potential interactions between them, and found that both proteins can form homomeric and heteromeric complexes in planta.

Lack of ADAM2, CALR3 and SAGE1 Cancer/Testis Antigen Expression in Lung and Breast Cancer

DEFF Research Database (Denmark)

Maheswaran, Emeaga; Pedersen, Christina B; Ditzel, Henrik J

2015-01-01

and antigenic properties, but the expression patterns of most of the more than 200 identified cancer/testis antigens in various cancers remain largely uncharacterized. In this study, we investigated the expression of the cancer/testis antigens ADAM2, CALR3 and SAGE1 in lung and breast cancer, the two most...... frequent human cancers, with the purpose of providing novel therapeutic targets for these diseases. We used a set of previously uncharacterized antibodies against the cancer/testis antigens ADAM2, CALR3 and SAGE1 to investigate their expression in a large panel of normal tissues as well as breast and lung...... cancers. Staining for the well-characterized MAGE-A proteins was included for comparison. Immunohistochemical staining confirmed previous mRNA analysis demonstrating that ADAM2, CALR3 and SAGE1 proteins are confined to testis in normal individuals. Negative tissues included plancenta, which express many...

Insulator function and topological domain border strength scale with architectural protein occupancy

Science.gov (United States)

2014-01-01

Background Chromosome conformation capture studies suggest that eukaryotic genomes are organized into structures called topologically associating domains. The borders of these domains are highly enriched for architectural proteins with characterized roles in insulator function. However, a majority of architectural protein binding sites localize within topological domains, suggesting sites associated with domain borders represent a functionally different subclass of these regulatory elements. How topologically associating domains are established and what differentiates border-associated from non-border architectural protein binding sites remain unanswered questions. Results By mapping the genome-wide target sites for several Drosophila architectural proteins, including previously uncharacterized profiles for TFIIIC and SMC-containing condensin complexes, we uncover an extensive pattern of colocalization in which architectural proteins establish dense clusters at the borders of topological domains. Reporter-based enhancer-blocking insulator activity as well as endogenous domain border strength scale with the occupancy level of architectural protein binding sites, suggesting co-binding by architectural proteins underlies the functional potential of these loci. Analyses in mouse and human stem cells suggest that clustering of architectural proteins is a general feature of genome organization, and conserved architectural protein binding sites may underlie the tissue-invariant nature of topologically associating domains observed in mammals. Conclusions We identify a spectrum of architectural protein occupancy that scales with the topological structure of chromosomes and the regulatory potential of these elements. Whereas high occupancy architectural protein binding sites associate with robust partitioning of topologically associating domains and robust insulator function, low occupancy sites appear reserved for gene-specific regulation within topological domains. PMID

NCBI nr-aa BLAST: CBRC-DMEL-04-0042 [SEVENS

Lifescience Database Archive (English)

Full Text Available ike protein [Geobacter uraniumreducens Rf4] gb|ABQ26219.1| Uncharacterized membrane-associated protein-like protein [Geobacter uraniumreducens Rf4] YP_001230792.1 4.3 28% ... ...CBRC-DMEL-04-0042 ref|YP_001230792.1| Uncharacterized membrane-associated protein-l

Rapid identification of sequences for orphan enzymes to power accurate protein annotation.

Directory of Open Access Journals (Sweden)

Kevin R Ramkissoon

Full Text Available The power of genome sequencing depends on the ability to understand what those genes and their proteins products actually do. The automated methods used to assign functions to putative proteins in newly sequenced organisms are limited by the size of our library of proteins with both known function and sequence. Unfortunately this library grows slowly, lagging well behind the rapid increase in novel protein sequences produced by modern genome sequencing methods. One potential source for rapidly expanding this functional library is the "back catalog" of enzymology--"orphan enzymes," those enzymes that have been characterized and yet lack any associated sequence. There are hundreds of orphan enzymes in the Enzyme Commission (EC database alone. In this study, we demonstrate how this orphan enzyme "back catalog" is a fertile source for rapidly advancing the state of protein annotation. Starting from three orphan enzyme samples, we applied mass-spectrometry based analysis and computational methods (including sequence similarity networks, sequence and structural alignments, and operon context analysis to rapidly identify the specific sequence for each orphan while avoiding the most time- and labor-intensive aspects of typical sequence identifications. We then used these three new sequences to more accurately predict the catalytic function of 385 previously uncharacterized or misannotated proteins. We expect that this kind of rapid sequence identification could be efficiently applied on a larger scale to make enzymology's "back catalog" another powerful tool to drive accurate genome annotation.

Rapid Identification of Sequences for Orphan Enzymes to Power Accurate Protein Annotation

Science.gov (United States)

Ojha, Sunil; Watson, Douglas S.; Bomar, Martha G.; Galande, Amit K.; Shearer, Alexander G.

2013-01-01

The power of genome sequencing depends on the ability to understand what those genes and their proteins products actually do. The automated methods used to assign functions to putative proteins in newly sequenced organisms are limited by the size of our library of proteins with both known function and sequence. Unfortunately this library grows slowly, lagging well behind the rapid increase in novel protein sequences produced by modern genome sequencing methods. One potential source for rapidly expanding this functional library is the “back catalog” of enzymology – “orphan enzymes,” those enzymes that have been characterized and yet lack any associated sequence. There are hundreds of orphan enzymes in the Enzyme Commission (EC) database alone. In this study, we demonstrate how this orphan enzyme “back catalog” is a fertile source for rapidly advancing the state of protein annotation. Starting from three orphan enzyme samples, we applied mass-spectrometry based analysis and computational methods (including sequence similarity networks, sequence and structural alignments, and operon context analysis) to rapidly identify the specific sequence for each orphan while avoiding the most time- and labor-intensive aspects of typical sequence identifications. We then used these three new sequences to more accurately predict the catalytic function of 385 previously uncharacterized or misannotated proteins. We expect that this kind of rapid sequence identification could be efficiently applied on a larger scale to make enzymology’s “back catalog” another powerful tool to drive accurate genome annotation. PMID:24386392

Inferring metabolic states in uncharacterized environments using gene-expression measurements.

Directory of Open Access Journals (Sweden)

Sergio Rossell

biotechnological processes that use complex, largely uncharacterized media, such as biofuel production.

Crystal structure of Homo sapiens protein LOC79017

Energy Technology Data Exchange (ETDEWEB)

Bae, Euiyoung; Bingman, Craig A.; Aceti, David J.; Phillips, Jr., George N. (UW)

2010-02-08

LOC79017 (MW 21.0 kDa, residues 1-188) was annotated as a hypothetical protein encoded by Homo sapiens chromosome 7 open reading frame 24. It was selected as a target by the Center for Eukaryotic Structural Genomics (CESG) because it did not share more than 30% sequence identity with any protein for which the three-dimensional structure is known. The biological function of the protein has not been established yet. Parts of LOC79017 were identified as members of uncharacterized Pfam families (residues 1-95 as PB006073 and residues 104-180 as PB031696). BLAST searches revealed homologues of LOC79017 in many eukaryotes, but none of them have been functionally characterized. Here, we report the crystal structure of H. sapiens protein LOC79017 (UniGene code Hs.530024, UniProt code O75223, CESG target number go.35223).

Comprehensive Protein Interactome Analysis of a Key RNA Helicase: Detection of Novel Stress Granule Proteins

Directory of Open Access Journals (Sweden)

Rebecca Bish

2015-07-01

Full Text Available DDX6 (p54/RCK is a human RNA helicase with central roles in mRNA decay and translation repression. To help our understanding of how DDX6 performs these multiple functions, we conducted the first unbiased, large-scale study to map the DDX6-centric protein-protein interactome using immunoprecipitation and mass spectrometry. Using DDX6 as bait, we identify a high-confidence and high-quality set of protein interaction partners which are enriched for functions in RNA metabolism and ribosomal proteins. The screen is highly specific, maximizing the number of true positives, as demonstrated by the validation of 81% (47/58 of the RNA-independent interactors through known functions and interactions. Importantly, we minimize the number of indirect interaction partners through use of a nuclease-based digestion to eliminate RNA. We describe eleven new interactors, including proteins involved in splicing which is an as-yet unknown role for DDX6. We validated and characterized in more detail the interaction of DDX6 with Nuclear fragile X mental retardation-interacting protein 2 (NUFIP2 and with two previously uncharacterized proteins, FAM195A and FAM195B (here referred to as granulin-1 and granulin-2, or GRAN1 and GRAN2. We show that NUFIP2, GRAN1, and GRAN2 are not P-body components, but re-localize to stress granules upon exposure to stress, suggesting a function in translation repression in the cellular stress response. Using a complementary analysis that resolved DDX6’s multiple complex memberships, we further validated these interaction partners and the presence of splicing factors. As DDX6 also interacts with the E3 SUMO ligase TIF1β, we tested for and observed a significant enrichment of sumoylation amongst DDX6’s interaction partners. Our results represent the most comprehensive screen for direct interaction partners of a key regulator of RNA life cycle and localization, highlighting new stress granule components and possible DDX6 functions�

A CRM domain protein functions dually in group I and group II intron splicing in land plant chloroplasts.

Science.gov (United States)

Asakura, Yukari; Barkan, Alice

2007-12-01

The CRM domain is a recently recognized RNA binding domain found in three group II intron splicing factors in chloroplasts, in a bacterial protein that associates with ribosome precursors, and in a family of uncharacterized proteins in plants. To elucidate the functional repertoire of proteins with CRM domains, we studied CFM2 (for CRM Family Member 2), which harbors four CRM domains. RNA coimmunoprecipitation assays showed that CFM2 in maize (Zea mays) chloroplasts is associated with the group I intron in pre-trnL-UAA and group II introns in the ndhA and ycf3 pre-mRNAs. T-DNA insertions in the Arabidopsis thaliana ortholog condition a defective-seed phenotype (strong allele) or chlorophyll-deficient seedlings with impaired splicing of the trnL group I intron and the ndhA, ycf3-int1, and clpP-int2 group II introns (weak alleles). CFM2 and two previously described CRM proteins are bound simultaneously to the ndhA and ycf3-int1 introns and act in a nonredundant fashion to promote their splicing. With these findings, CRM domain proteins are implicated in the activities of three classes of catalytic RNA: group I introns, group II introns, and 23S rRNA.

ATP-binding Cassette (ABC) Transport System Solute-binding Protein-guided Identification of Novel d-Altritol and Galactitol Catabolic Pathways in Agrobacterium tumefaciens C58*

Science.gov (United States)

Wichelecki, Daniel J.; Vetting, Matthew W.; Chou, Liyushang; Al-Obaidi, Nawar; Bouvier, Jason T.; Almo, Steven C.; Gerlt, John A.

2015-01-01

Innovations in the discovery of the functions of uncharacterized proteins/enzymes have become increasingly important as advances in sequencing technology flood protein databases with an exponentially growing number of open reading frames. This study documents one such innovation developed by the Enzyme Function Initiative (EFI; U54GM093342), the use of solute-binding proteins for transport systems to identify novel metabolic pathways. In a previous study, this strategy was applied to the tripartite ATP-independent periplasmic transporters. Here, we apply this strategy to the ATP-binding cassette transporters and report the discovery of novel catabolic pathways for d-altritol and galactitol in Agrobacterium tumefaciens C58. These efforts resulted in the description of three novel enzymatic reactions as follows: 1) oxidation of d-altritol to d-tagatose via a dehydrogenase in Pfam family PF00107, a previously unknown reaction; 2) phosphorylation of d-tagatose to d-tagatose 6-phosphate via a kinase in Pfam family PF00294, a previously orphan EC number; and 3) epimerization of d-tagatose 6-phosphate C-4 to d-fructose 6-phosphate via a member of Pfam family PF08013, another previously unknown reaction. The epimerization reaction catalyzed by a member of PF08013 is especially noteworthy, because the functions of members of PF08013 have been unknown. These discoveries were assisted by the following two synergistic bioinformatics web tools made available by the Enzyme Function Initiative: the EFI-Enzyme Similarity Tool and the EFI-Genome Neighborhood Tool. PMID:26472925

A comprehensive software suite for protein family construction and functional site prediction.

Directory of Open Access Journals (Sweden)

David Renfrew Haft

Full Text Available In functionally diverse protein families, conservation in short signature regions may outperform full-length sequence comparisons for identifying proteins that belong to a subgroup within which one specific aspect of their function is conserved. The SIMBAL workflow (Sites Inferred by Metabolic Background Assertion Labeling is a data-mining procedure for finding such signature regions. It begins by using clues from genomic context, such as co-occurrence or conserved gene neighborhoods, to build a useful training set from a large number of uncharacterized but mutually homologous proteins. When training set construction is successful, the YES partition is enriched in proteins that share function with the user's query sequence, while the NO partition is depleted. A selected query sequence is then mined for short signature regions whose closest matches overwhelmingly favor proteins from the YES partition. High-scoring signature regions typically contain key residues critical to functional specificity, so proteins with the highest sequence similarity across these regions tend to share the same function. The SIMBAL algorithm was described previously, but significant manual effort, expertise, and a supporting software infrastructure were required to prepare the requisite training sets. Here, we describe a new, distributable software suite that speeds up and simplifies the process for using SIMBAL, most notably by providing tools that automate training set construction. These tools have broad utility for comparative genomics, allowing for flexible collection of proteins or protein domains based on genomic context as well as homology, a capability that can greatly assist in protein family construction. Armed with this new software suite, SIMBAL can serve as a fast and powerful in silico alternative to direct experimentation for characterizing proteins and their functional interactions.

Sub-terahertz spectroscopy reveals that proteins influence the properties of water at greater distances than previously detected

Energy Technology Data Exchange (ETDEWEB)

Sushko, Oleksandr; Dubrovka, Rostyslav; Donnan, Robert S., E-mail: r.donnan@qmul.ac.uk [School of Electronic Engineering and Computer Science, Queen Mary University of London, Mile End Road, London E1 4NS (United Kingdom)

2015-02-07

The initial purpose of the study is to systematically investigate the solvation properties of different proteins in water solution by terahertz (THz) radiation absorption. Transmission measurements of protein water solutions have been performed using a vector network analyser-driven quasi-optical bench covering the WR-3 waveguide band (0.220–0.325 THz). The following proteins, ranging from low to high molecular weight, were chosen for this study: lysozyme, myoglobin, and bovine serum albumin (BSA). Absorption properties of solutions were studied at different concentrations of proteins ranging from 2 to 100 mg/ml. The concentration-dependent absorption of protein molecules was determined by treating the solution as a two-component model first; then, based on protein absorptivity, the extent of the hydration shell is estimated. Protein molecules are shown to possess a concentration-dependent absorptivity in water solutions. Absorption curves of all three proteins sharply peak towards a dilution-limit that is attributed to the enhanced flexibility of protein and amino acid side chains. An alternative approach to the determination of hydration shell thickness is thereby suggested, based on protein absorptivity. The proposed approach is independent of the absorption of the hydration shell. The derived estimate of hydration shell thickness for each protein supports previous findings that protein-water interaction dynamics extends beyond 2-3 water solvation-layers as predicted by molecular dynamics simulations and other techniques such as NMR, X-ray scattering, and neutron scattering. According to our estimations, the radius of the dynamic hydration shell is 16, 19, and 25 Å, respectively, for lysozyme, myoglobin, and BSA proteins and correlates with the dipole moment of the protein. It is also seen that THz radiation can serve as an initial estimate of the protein hydrophobicity.

Sub-terahertz spectroscopy reveals that proteins influence the properties of water at greater distances than previously detected

International Nuclear Information System (INIS)

Sushko, Oleksandr; Dubrovka, Rostyslav; Donnan, Robert S.

2015-01-01

The initial purpose of the study is to systematically investigate the solvation properties of different proteins in water solution by terahertz (THz) radiation absorption. Transmission measurements of protein water solutions have been performed using a vector network analyser-driven quasi-optical bench covering the WR-3 waveguide band (0.220–0.325 THz). The following proteins, ranging from low to high molecular weight, were chosen for this study: lysozyme, myoglobin, and bovine serum albumin (BSA). Absorption properties of solutions were studied at different concentrations of proteins ranging from 2 to 100 mg/ml. The concentration-dependent absorption of protein molecules was determined by treating the solution as a two-component model first; then, based on protein absorptivity, the extent of the hydration shell is estimated. Protein molecules are shown to possess a concentration-dependent absorptivity in water solutions. Absorption curves of all three proteins sharply peak towards a dilution-limit that is attributed to the enhanced flexibility of protein and amino acid side chains. An alternative approach to the determination of hydration shell thickness is thereby suggested, based on protein absorptivity. The proposed approach is independent of the absorption of the hydration shell. The derived estimate of hydration shell thickness for each protein supports previous findings that protein-water interaction dynamics extends beyond 2-3 water solvation-layers as predicted by molecular dynamics simulations and other techniques such as NMR, X-ray scattering, and neutron scattering. According to our estimations, the radius of the dynamic hydration shell is 16, 19, and 25 Å, respectively, for lysozyme, myoglobin, and BSA proteins and correlates with the dipole moment of the protein. It is also seen that THz radiation can serve as an initial estimate of the protein hydrophobicity

Previously Unidentified Single Nucleotide Polymorphisms in HIV/AIDS Cases Associate with Clinical Parameters and Disease Progression

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Vladimir V. Anokhin

2016-01-01

Full Text Available The genetic background of an individual plays an important role in the progression of HIV infection to AIDS. Identifying previously unknown or uncharacterized single nucleotide polymorphisms (SNPs that associate with disease progression may reveal important therapeutic targets and provide a greater understanding of disease pathogenesis. In the present study, we employed ultra-high multiplex PCR on an Ion Torrent next-generation sequencing platform to sequence 23 innate immune genes from 94 individuals with HIV/AIDS. This data was used to identify potential associations of SNPs with clinical parameters and disease progression. SNPs that associated with an increased viral load were identified in the genes for the interleukin 15 receptor (IL15RA, toll-like receptor 7 (TLR7, tripartite motif-containing protein 5 (TRIM5, and two killer-cell immunoglobulin-like receptors (KIR2DL1 and KIR2DL3. Additionally, SNPs that associated with progression from HIV infection to AIDS were identified in two 2′-5′-oligoadenylate synthetase genes (OAS2 and OAS3. In contrast, other SNPs identified in OAS2 and OAS3 genes, as well as in the TRIM5 and KIR2DS4 genes, were associated with a slower progression of disease. Taken together, our data demonstrates the utility of ultra-high multiplex PCR in identifying polymorphisms of potential clinical significance and further,identifies SNPs that may play a role in HIV pathogenesis.

HD-GYP domain proteins regulate biofilm formation and virulence in Pseudomonas aeruginosa

DEFF Research Database (Denmark)

Ryan, Robert P.; Lucey, Jean; O'Donovan, Karen

2009-01-01

residues (YN-GYP). Here we have investigated the role of these proteins in biofilm formation, virulence factor synthesis and virulence of P. aeruginosa. Mutation of PA4108 and PA4781 led to an increase in the level of cyclic-di-GMP in P. aeruginosa, consistent with the predicted activity of the encoded......2572 had a negative influence on swarming that was cryptic and was revealed only after removal of an uncharacterized C-terminal domain. Mutation of PA4108, PA4781 and PA2572 had distinct effects on biofilm formation and architecture of P. aeruginosa. All three proteins contributed to virulence of P...

The uncharacterized gene 1700093K21Rik and flanking regions are correlated with reproductive isolation in the house mouse, Mus musculus.

Science.gov (United States)

Kass, David H; Janoušek, Václav; Wang, Liuyang; Tucker, Priscilla K

2014-06-01

Reproductive barriers exist between the house mouse subspecies, Mus musculus musculus and M. m. domesticus, members of the Mus musculus species complex, primarily as a result of hybrid male infertility, and a hybrid zone exists where their ranges intersect in Europe. Using single nucleotide polymorphisms (SNPs) diagnostic for the two taxa, the extent of introgression across the genome was previously compared in these hybrid populations. Sixty-nine of 1316 autosomal SNPs exhibited reduced introgression in two hybrid zone transects suggesting maladaptive interactions among certain loci. One of these markers is within a region on chromosome 11 that, in other studies, has been associated with hybrid male sterility of these subspecies. We assessed sequence variation in a 20 Mb region on chromosome 11 flanking this marker, and observed its inclusion within a roughly 150 kb stretch of DNA showing elevated sequence differentiation between the two subspecies. Four genes are associated with this genomic subregion, with two entirely encompassed. One of the two genes, the uncharacterized 1700093K21Rik gene, displays distinguishing features consistent with a potential role in reproductive isolation between these subspecies. Along with its expression specifically within spermatogenic cells, we present various sequence analyses that demonstrate a high rate of molecular evolution of this gene, as well as identify a subspecies amino acid variant resulting in a structural difference. Taken together, the data suggest a role for this gene in reproductive isolation.

NCBI nr-aa BLAST: CBRC-ACAR-01-0087 [SEVENS

Lifescience Database Archive (English)

Full Text Available CBRC-ACAR-01-0087 ref|YP_677558.1| CHU large protein; uncharacterized [Cytophaga hutchinson...ii ATCC 33406] gb|ABG58218.1| CHU large protein; uncharacterized [Cytophaga hutchinsonii ATCC 33406] YP_677558.1 3.8 22% ...

Bayesian modeling of the yeast SH3 domain interactome predicts spatiotemporal dynamics of endocytosis proteins.

Directory of Open Access Journals (Sweden)

Raffi Tonikian

2009-10-01

Full Text Available SH3 domains are peptide recognition modules that mediate the assembly of diverse biological complexes. We scanned billions of phage-displayed peptides to map the binding specificities of the SH3 domain family in the budding yeast, Saccharomyces cerevisiae. Although most of the SH3 domains fall into the canonical classes I and II, each domain utilizes distinct features of its cognate ligands to achieve binding selectivity. Furthermore, we uncovered several SH3 domains with specificity profiles that clearly deviate from the two canonical classes. In conjunction with phage display, we used yeast two-hybrid and peptide array screening to independently identify SH3 domain binding partners. The results from the three complementary techniques were integrated using a Bayesian algorithm to generate a high-confidence yeast SH3 domain interaction map. The interaction map was enriched for proteins involved in endocytosis, revealing a set of SH3-mediated interactions that underlie formation of protein complexes essential to this biological pathway. We used the SH3 domain interaction network to predict the dynamic localization of several previously uncharacterized endocytic proteins, and our analysis suggests a novel role for the SH3 domains of Lsb3p and Lsb4p as hubs that recruit and assemble several endocytic complexes.

AcEST: BP915397 [AcEST

Lifescience Database Archive (English)

Full Text Available UPTR Putative uncharacterized protein OS=Cupriavidus taiwanensis (strain R1 / LMG...Cupri... 33 9.9 >tr|B3R9M4|B3R9M4_CUPTR Putative uncharacterized protein OS=Cupriavidus taiwanensis (strain

Structural basis for plant plasma membrane protein dynamics and organization into functional nanodomains.

Science.gov (United States)

Gronnier, Julien; Crowet, Jean-Marc; Habenstein, Birgit; Nasir, Mehmet Nail; Bayle, Vincent; Hosy, Eric; Platre, Matthieu Pierre; Gouguet, Paul; Raffaele, Sylvain; Martinez, Denis; Grelard, Axelle; Loquet, Antoine; Simon-Plas, Françoise; Gerbeau-Pissot, Patricia; Der, Christophe; Bayer, Emmanuelle M; Jaillais, Yvon; Deleu, Magali; Germain, Véronique; Lins, Laurence; Mongrand, Sébastien

2017-07-31

Plasma Membrane is the primary structure for adjusting to ever changing conditions. PM sub-compartmentalization in domains is thought to orchestrate signaling. Yet, mechanisms governing membrane organization are mostly uncharacterized. The plant-specific REMORINs are proteins regulating hormonal crosstalk and host invasion. REMs are the best-characterized nanodomain markers via an uncharacterized moiety called REMORIN C-terminal Anchor. By coupling biophysical methods, super-resolution microscopy and physiology, we decipher an original mechanism regulating the dynamic and organization of nanodomains. We showed that targeting of REMORIN is independent of the COP-II-dependent secretory pathway and mediated by PI4P and sterol. REM-CA is an unconventional lipid-binding motif that confers nanodomain organization. Analyses of REM-CA mutants by single particle tracking demonstrate that mobility and supramolecular organization are critical for immunity. This study provides a unique mechanistic insight into how the tight control of spatial segregation is critical in the definition of PM domain necessary to support biological function.

EST2Prot: Mapping EST sequences to proteins

Directory of Open Access Journals (Sweden)

Lin David M

2006-03-01

Full Text Available Abstract Background EST libraries are used in various biological studies, from microarray experiments to proteomic and genetic screens. These libraries usually contain many uncharacterized ESTs that are typically ignored since they cannot be mapped to known genes. Consequently, new discoveries are possibly overlooked. Results We describe a system (EST2Prot that uses multiple elements to map EST sequences to their corresponding protein products. EST2Prot uses UniGene clusters, substring analysis, information about protein coding regions in existing DNA sequences and protein database searches to detect protein products related to a query EST sequence. Gene Ontology terms, Swiss-Prot keywords, and protein similarity data are used to map the ESTs to functional descriptors. Conclusion EST2Prot extends and significantly enriches the popular UniGene mapping by utilizing multiple relations between known biological entities. It produces a mapping between ESTs and proteins in real-time through a simple web-interface. The system is part of the Biozon database and is accessible at http://biozon.org/tools/est/.

Effect of Irradiation on the Mixture of Egg White Proteins Responsible for Foaming Property

International Nuclear Information System (INIS)

Xian De Liu; Rong Xiu Han; Dong-Il Jin; Soo-Kee Lee; Cheo-Run Jo

2009-01-01

Irradiation of egg white increased foaming ability significantly. To investigate the protein modification by irradiation responsible for the increase of foaming ability, 3 major egg white proteins were purchased and mixed (7.7 g/L ovalbumin, 1.8 g/L ovotransferrin, 0.5 g/L lysozyme) as a model system and irradiated at 0, 2.5, and 5 kGy. The different protein expressions were evaluated using 2-D electrophoresis and it was found that ovotransferrin was cleaved by irradiation and molecular weight and isoelectric point were changed. In addition, many uncharacterized proteins were found and it indicated that irradiation modified proteins randomly but mainly fragmentation was observed. Therefore, it can be concluded that protein fragmentation of 3 major egg white proteins responsible for foaming ability may be the main reason for the improvement of foaming ability

Bioinformatic and Comparative Localization of Rab Proteins Reveals Functional Insights into the Uncharacterized GTPases Ypt10p and Ypt11p†

OpenAIRE

Buvelot Frei, Stéphanie; Rahl, Peter B.; Nussbaum, Maria; Briggs, Benjamin J.; Calero, Monica; Janeczko, Stephanie; Regan, Andrew D.; Chen, Catherine Z.; Barral, Yves; Whittaker, Gary R.; Collins, Ruth N.

2006-01-01

A striking characteristic of a Rab protein is its steady-state localization to the cytosolic surface of a particular subcellular membrane. In this study, we have undertaken a combined bioinformatic and experimental approach to examine the evolutionary conservation of Rab protein localization. A comprehensive primary sequence classification shows that 10 out of the 11 Rab proteins identified in the yeast (Saccharomyces cerevisiae) genome can be grouped within a major subclass, each comprising ...

Proteins in Soy Might Have a Higher Role in Cancer Prevention than Previously Expected: Soybean Protein Fractions Are More Effective MMP-9 Inhibitors Than Non-Protein Fractions, Even in Cooked Seeds

Directory of Open Access Journals (Sweden)

Ana Lima

2017-02-01

Full Text Available The search for anticancer MMP-9 inhibitors (MMPIs in food products has become a major goal for research. MMPIs in soy have been related only to saponins and isoflavones, but recently, low specific protein fractions in soybeans were shown to reduce MMP-9 activity as well. The present work aimed at comparing the MMPI potential of protein fractions (P and non-protein fractions (NP isolated from soybean seeds, before and after soaking and cooking, mimicking dietary exposures. Reverse and substrate zymography, as well as a fluoregenic DQ gelatin assay were used to evaluate MMP-9 activities. Colon cancer cell migration and proliferation was also tested in HT29 cells. Regarding MMP-9 inhibition, proteins in soy presented IC50 values 100 times lower than non-protein extracts, and remained active after cooking, suggesting that proteins may be more effective MMP-9 inhibitors than non-protein compounds. Using the determined IC50 concentrations, NP fractions were able to induce higher inhibitions of HT29 cell migration and proliferation, but not through MMP-9 inhibition, whilst protein fractions were shown to specifically inhibit MMP-9 activity. Overall, our results show that protein fractions in soybeans might have a higher role in soy-related cancer prevention as MMPIs than previously expected. Being nontoxic and active at lower concentrations, the discovery of these heat-resistant specific MMPI proteins in soy can be of significant importance for cancer preventive diets, particularly considering the increasing use of soy proteins in food products and the controversy around isoflavones amongst consumers.

Cul8/Rtt101 Forms a Variety of Protein Complexes That Regulate DNA Damage Response and Transcriptional Silencing*

OpenAIRE

Mimura, Satoru; Yamaguchi, Tsuyoshi; Ishii, Satoru; Noro, Emiko; Katsura, Tomoya; Obuse, Chikashi; Kamura, Takumi

2010-01-01

The budding yeast, Saccharomyces cerevisiae, has three cullin proteins, which act as platforms for Cullin-based E3 ubiquitin ligases. Genetic evidence indicates that Cul8, together with Mms1, Mms22, and Esc4, is involved in the repair of DNA damage that can occur during DNA replication. Cul8 is thought to form a complex with these proteins, but the composition and the function of Cul8-based E3 ubiquitin ligases remain largely uncharacterized. Herein, we report a comprehensive biochemical anal...

Identification of a mitochondrial target of thiazolidinedione insulin sensitizers (mTOT--relationship to newly identified mitochondrial pyruvate carrier proteins.

Directory of Open Access Journals (Sweden)

Jerry R Colca

Full Text Available Thiazolidinedione (TZD insulin sensitizers have the potential to effectively treat a number of human diseases, however the currently available agents have dose-limiting side effects that are mediated via activation of the transcription factor PPARγ. We have recently shown PPARγ-independent actions of TZD insulin sensitizers, but the molecular target of these molecules remained to be identified. Here we use a photo-catalyzable drug analog probe and mass spectrometry-based proteomics to identify a previously uncharacterized mitochondrial complex that specifically recognizes TZDs. These studies identify two well-conserved proteins previously known as brain protein 44 (BRP44 and BRP44 Like (BRP44L, which recently have been renamed Mpc2 and Mpc1 to signify their function as a mitochondrial pyruvate carrier complex. Knockdown of Mpc1 or Mpc2 in Drosophila melanogaster or pre-incubation with UK5099, an inhibitor of pyruvate transport, blocks the crosslinking of mitochondrial membranes by the TZD probe. Knockdown of these proteins in Drosophila also led to increased hemolymph glucose and blocked drug action. In isolated brown adipose tissue (BAT cells, MSDC-0602, a PPARγ-sparing TZD, altered the incorporation of (13C-labeled carbon from glucose into acetyl CoA. These results identify Mpc1 and Mpc2 as components of the mitochondrial target of TZDs (mTOT and suggest that understanding the modulation of this complex, which appears to regulate pyruvate entry into the mitochondria, may provide a viable target for insulin sensitizing pharmacology.

AcEST: BP920733 [AcEST

Lifescience Database Archive (English)

Full Text Available TrEMBL tr_hit_id B8CKP7 Definition tr|B8CKP7|B8CKP7_9GAMM Putative uncharacterized protein OS=Shewanella piezo...B8CKP7|B8CKP7_9GAMM Putative uncharacterized protein OS=Shewanella piezotolerans

Molecular and functional analyses of a maize autoactive NB-LRR protein identify precise structural requirements for activity.

Directory of Open Access Journals (Sweden)

Guan-Feng Wang

2015-02-01

Full Text Available Plant disease resistance is often mediated by nucleotide binding-leucine rich repeat (NLR proteins which remain auto-inhibited until recognition of specific pathogen-derived molecules causes their activation, triggering a rapid, localized cell death called a hypersensitive response (HR. Three domains are recognized in one of the major classes of NLR proteins: a coiled-coil (CC, a nucleotide binding (NB-ARC and a leucine rich repeat (LRR domains. The maize NLR gene Rp1-D21 derives from an intergenic recombination event between two NLR genes, Rp1-D and Rp1-dp2 and confers an autoactive HR. We report systematic structural and functional analyses of Rp1 proteins in maize and N. benthamiana to characterize the molecular mechanism of NLR activation/auto-inhibition. We derive a model comprising the following three main features: Rp1 proteins appear to self-associate to become competent for activity. The CC domain is signaling-competent and is sufficient to induce HR. This can be suppressed by the NB-ARC domain through direct interaction. In autoactive proteins, the interaction of the LRR domain with the NB-ARC domain causes de-repression and thus disrupts the inhibition of HR. Further, we identify specific amino acids and combinations thereof that are important for the auto-inhibition/activity of Rp1 proteins. We also provide evidence for the function of MHD2, a previously uncharacterized, though widely conserved NLR motif. This work reports several novel insights into the precise structural requirement for NLR function and informs efforts towards utilizing these proteins for engineering disease resistance.

Molecular and functional analyses of a maize autoactive NB-LRR protein identify precise structural requirements for activity.

Science.gov (United States)

Wang, Guan-Feng; Ji, Jiabing; El-Kasmi, Farid; Dangl, Jeffery L; Johal, Guri; Balint-Kurti, Peter J

2015-02-01

Plant disease resistance is often mediated by nucleotide binding-leucine rich repeat (NLR) proteins which remain auto-inhibited until recognition of specific pathogen-derived molecules causes their activation, triggering a rapid, localized cell death called a hypersensitive response (HR). Three domains are recognized in one of the major classes of NLR proteins: a coiled-coil (CC), a nucleotide binding (NB-ARC) and a leucine rich repeat (LRR) domains. The maize NLR gene Rp1-D21 derives from an intergenic recombination event between two NLR genes, Rp1-D and Rp1-dp2 and confers an autoactive HR. We report systematic structural and functional analyses of Rp1 proteins in maize and N. benthamiana to characterize the molecular mechanism of NLR activation/auto-inhibition. We derive a model comprising the following three main features: Rp1 proteins appear to self-associate to become competent for activity. The CC domain is signaling-competent and is sufficient to induce HR. This can be suppressed by the NB-ARC domain through direct interaction. In autoactive proteins, the interaction of the LRR domain with the NB-ARC domain causes de-repression and thus disrupts the inhibition of HR. Further, we identify specific amino acids and combinations thereof that are important for the auto-inhibition/activity of Rp1 proteins. We also provide evidence for the function of MHD2, a previously uncharacterized, though widely conserved NLR motif. This work reports several novel insights into the precise structural requirement for NLR function and informs efforts towards utilizing these proteins for engineering disease resistance.

Characterization of the Raf kinase inhibitory protein (RKIP) binding pocket: NMR-based screening identifies small-molecule ligands.

Science.gov (United States)

Shemon, Anne N; Heil, Gary L; Granovsky, Alexey E; Clark, Mathew M; McElheny, Dan; Chimon, Alexander; Rosner, Marsha R; Koide, Shohei

2010-05-05

Raf kinase inhibitory protein (RKIP), also known as phoshaptidylethanolamine binding protein (PEBP), has been shown to inhibit Raf and thereby negatively regulate growth factor signaling by the Raf/MAP kinase pathway. RKIP has also been shown to suppress metastasis. We have previously demonstrated that RKIP/Raf interaction is regulated by two mechanisms: phosphorylation of RKIP at Ser-153, and occupation of RKIP's conserved ligand binding domain with a phospholipid (2-dihexanoyl-sn-glycero-3-phosphoethanolamine; DHPE). In addition to phospholipids, other ligands have been reported to bind this domain; however their binding properties remain uncharacterized. In this study, we used high-resolution heteronuclear NMR spectroscopy to screen a chemical library and assay a number of potential RKIP ligands for binding to the protein. Surprisingly, many compounds previously postulated as RKIP ligands showed no detectable binding in near-physiological solution conditions even at millimolar concentrations. In contrast, we found three novel ligands for RKIP that specifically bind to the RKIP pocket. Interestingly, unlike the phospholipid, DHPE, these newly identified ligands did not affect RKIP binding to Raf-1 or RKIP phosphorylation. One out of the three ligands displayed off target biological effects, impairing EGF-induced MAPK and metabolic activity. This work defines the binding properties of RKIP ligands under near physiological conditions, establishing RKIP's affinity for hydrophobic ligands and the importance of bulky aliphatic chains for inhibiting its function. The common structural elements of these compounds defines a minimal requirement for RKIP binding and thus they can be used as lead compounds for future design of RKIP ligands with therapeutic potential.

Characterization of the Raf kinase inhibitory protein (RKIP binding pocket: NMR-based screening identifies small-molecule ligands.

Directory of Open Access Journals (Sweden)

Anne N Shemon

2010-05-01

Full Text Available Raf kinase inhibitory protein (RKIP, also known as phoshaptidylethanolamine binding protein (PEBP, has been shown to inhibit Raf and thereby negatively regulate growth factor signaling by the Raf/MAP kinase pathway. RKIP has also been shown to suppress metastasis. We have previously demonstrated that RKIP/Raf interaction is regulated by two mechanisms: phosphorylation of RKIP at Ser-153, and occupation of RKIP's conserved ligand binding domain with a phospholipid (2-dihexanoyl-sn-glycero-3-phosphoethanolamine; DHPE. In addition to phospholipids, other ligands have been reported to bind this domain; however their binding properties remain uncharacterized.In this study, we used high-resolution heteronuclear NMR spectroscopy to screen a chemical library and assay a number of potential RKIP ligands for binding to the protein. Surprisingly, many compounds previously postulated as RKIP ligands showed no detectable binding in near-physiological solution conditions even at millimolar concentrations. In contrast, we found three novel ligands for RKIP that specifically bind to the RKIP pocket. Interestingly, unlike the phospholipid, DHPE, these newly identified ligands did not affect RKIP binding to Raf-1 or RKIP phosphorylation. One out of the three ligands displayed off target biological effects, impairing EGF-induced MAPK and metabolic activity.This work defines the binding properties of RKIP ligands under near physiological conditions, establishing RKIP's affinity for hydrophobic ligands and the importance of bulky aliphatic chains for inhibiting its function. The common structural elements of these compounds defines a minimal requirement for RKIP binding and thus they can be used as lead compounds for future design of RKIP ligands with therapeutic potential.

Impact of protein and ligand impurities on ITC-derived protein-ligand thermodynamics.

Science.gov (United States)

Grüner, Stefan; Neeb, Manuel; Barandun, Luzi Jakob; Sielaff, Frank; Hohn, Christoph; Kojima, Shun; Steinmetzer, Torsten; Diederich, François; Klebe, Gerhard

2014-09-01

The thermodynamic characterization of protein-ligand interactions by isothermal titration calorimetry (ITC) is a powerful tool in drug design, giving valuable insight into the interaction driving forces. ITC is thought to require protein and ligand solutions of high quality, meaning both the absence of contaminants as well as accurately determined concentrations. Ligands synthesized to deviating purity and protein of different pureness were titrated by ITC. Data curation was attempted also considering information from analytical techniques to correct stoichiometry. We used trypsin and tRNA-guanine transglycosylase (TGT), together with high affinity ligands to investigate the effect of errors in protein concentration as well as the impact of ligand impurities on the apparent thermodynamics. We found that errors in protein concentration did not change the thermodynamic properties obtained significantly. However, most ligand impurities led to pronounced changes in binding enthalpy. If protein binding of the respective impurity is not expected, the actual ligand concentration was corrected for and the thus revised data compared to thermodynamic properties obtained with the respective pure ligand. Even in these cases, we observed differences in binding enthalpy of about 4kJ⋅mol(-1), which is considered significant. Our results indicate that ligand purity is the critical parameter to monitor if accurate thermodynamic data of a protein-ligand complex are to be recorded. Furthermore, artificially changing fitting parameters to obtain a sound interaction stoichiometry in the presence of uncharacterized ligand impurities may lead to thermodynamic parameters significantly deviating from the accurate thermodynamic signature. Copyright © 2014 Elsevier B.V. All rights reserved.

Identification of herpesvirus proteins that contribute to G1/S arrest.

Science.gov (United States)

Paladino, Patrick; Marcon, Edyta; Greenblatt, Jack; Frappier, Lori

2014-04-01

Lytic infection by herpesviruses induces cell cycle arrest at the G1/S transition. This appears to be a function of multiple herpesvirus proteins, but only a minority of herpesvirus proteins have been examined for cell cycle effects. To gain a more comprehensive understanding of the viral proteins that contribute to G1/S arrest, we screened a library of over 200 proteins from herpes simplex virus type 1, human cytomegalovirus, and Epstein-Barr virus (EBV) for effects on the G1/S interface, using HeLa fluorescent, ubiquitination-based cell cycle indicator (Fucci) cells in which G1/S can be detected colorimetrically. Proteins from each virus were identified that induce accumulation of G1/S cells, predominantly tegument, early, and capsid proteins. The identification of several capsid proteins in this screen suggests that incoming viral capsids may function to modulate cellular processes. The cell cycle effects of selected EBV proteins were further verified and examined for effects on p53 and p21 as regulators of the G1/S transition. Two EBV replication proteins (BORF2 and BMRF1) were found to induce p53 but not p21, while a previously uncharacterized tegument protein (BGLF2) was found to induce p21 protein levels in a p53-independent manner. Proteomic analyses of BGLF2-interacting proteins identified interactions with the NIMA-related protein kinase (NEK9) and GEM-interacting protein (GMIP). Silencing of either NEK9 or GMIP induced p21 without affecting p53 and abrogated the ability of BGLF2 to further induce p21. Collectively, these results suggest multiple viral proteins contribute to G1/S arrest, including BGLF2, which induces p21 levels likely by interfering with the functions of NEK9 and GMIP. Most people are infected with multiple herpesviruses, whose proteins alter the infected cells in several ways. During lytic infection, the viral proteins block cell proliferation just before the cellular DNA replicates. We used a novel screening method to identify proteins

Limonene Arrests Parasite Development and Inhibits Isoprenylation of Proteins in Plasmodium falciparum

Science.gov (United States)

Moura, Ivan Cruz; Wunderlich, Gerhard; Uhrig, Maria L.; Couto, Alicia S.; Peres, Valnice J.; Katzin, Alejandro M.; Kimura, Emília A.

2001-01-01

Isoprenylation is an essential protein modification in eukaryotic cells. Herein, we report that in Plasmodium falciparum, a number of proteins were labeled upon incubation of intraerythrocytic forms with either [3H]farnesyl pyrophosphate or [3H]geranylgeranyl pyrophosphate. By thin-layer chromatography, we showed that attached isoprenoids are partially modified to dolichol and other, uncharacterized, residues, confirming active isoprenoid metabolism in this parasite. Incubation of blood-stage P. falciparum treated with the isoprenylation inhibitor limonene significantly decreased the parasites' progression from the ring stage to the trophozoite stage and at 1.22 mM, 50% of the parasites died after the first cycle. Using Ras- and Rap-specific monoclonal antibodies, putative Rap and Ras proteins of P. falciparum were immunoprecipitated. Upon treatment with 0.5 mM limonene, isoprenylation of these proteins was significantly decreased, possibly explaining the observed arrest of parasite development. PMID:11502528

Development of aptamers against unpurified proteins.

Science.gov (United States)

Goto, Shinichi; Tsukakoshi, Kaori; Ikebukuro, Kazunori

2017-12-01

SELEX (Systematic Evolution of Ligands by EXponential enrichment) has been widely used for the generation of aptamers against target proteins. However, its requirement for pure target proteins remains a major problem in aptamer selection, as procedures for protein purification from crude bio-samples are not only complicated but also time and labor consuming. This is because native proteins can be found in a large number of diverse forms because of posttranslational modifications and their complicated molecular conformations. Moreover, several proteins are difficult to purify owing to their chemical fragility and/or rarity in native samples. An alternative route is the use of recombinant proteins for aptamer selection, because they are homogenous and easily purified. However, aptamers generated against recombinant proteins produced in prokaryotic cells may not interact with the same proteins expressed in eukaryotic cells because of posttranslational modifications. Moreover, to date recombinant proteins have been constructed for only a fraction of proteins expressed in the human body. Therefore, the demand for advanced SELEX methods not relying on complicated purification processes from native samples or recombinant proteins is growing. This review article describes several such techniques that allow researchers to directly develop an aptamer from various unpurified samples, such as whole cells, tissues, serum, and cell lysates. The key advantages of advanced SELEX are that it does not require a purification process from a crude bio-sample, maintains the functional states of target proteins, and facilitates the development of aptamers against unidentified and uncharacterized proteins in unpurified biological samples. © 2017 Wiley Periodicals, Inc.

AcEST: DK957741 [AcEST

Lifescience Database Archive (English)

Full Text Available yza sativa subs... 148 3e-34 tr|A2Y2K9|A2Y2K9_ORYSI Putative uncharacterized protein OS=Oryza... 148 3e-34 t...70 SALLKIDVPLMFIGDDASPGL 190 >tr|A2Y2K9|A2Y2K9_ORYSI Putative uncharacterized protein OS=Oryza sativa subsp.

Activation of the ATR kinase by the RPA-binding protein ETAA1

DEFF Research Database (Denmark)

Haahr, Peter; Hoffmann, Saskia; Tollenaere, Maxim A X

2016-01-01

Activation of the ATR kinase following perturbations to DNA replication relies on a complex mechanism involving ATR recruitment to RPA-coated single-stranded DNA via its binding partner ATRIP and stimulation of ATR kinase activity by TopBP1. Here, we discovered an independent ATR activation pathway...... in vertebrates, mediated by the uncharacterized protein ETAA1 (Ewing's tumour-associated antigen 1). Human ETAA1 accumulates at DNA damage sites via dual RPA-binding motifs and promotes replication fork progression and integrity, ATR signalling and cell survival after genotoxic insults. Mechanistically...

A systematic analysis of the early transcribed membrane protein family throughout the life cycle of Plasmodium yoelii.

Science.gov (United States)

MacKellar, Drew C; Vaughan, Ashley M; Aly, Ahmed S I; DeLeon, Sasha; Kappe, Stefan H I

2011-11-01

The early transcribed membrane proteins (ETRAMPs) are a family of small, highly charged transmembrane proteins unique to malaria parasites. Some members of the ETRAMP family have been localized to the parasitophorous vacuole membrane that separates the intracellular parasite from the host cell and thus presumably have a role in host-parasite interactions. Although it was previously shown that two ETRAMPs are critical for rodent malaria parasite liver-stage development, the importance of most ETRAMPs during the parasite life cycle remains unknown. Here, we comprehensively identify nine new etramps in the genome of the rodent malaria parasite Plasmodium yoelii, and elucidate their conservation in other malaria parasites. etramp expression profiles are diverse throughout the parasite life cycle as measured by RT-PCR. Epitope tagging of two ETRAMPs demonstrates protein expression in blood and liver stages, and reveals differences in both their timing of expression and their subcellular localization. Gene targeting studies of each of the nine uncharacterized etramps show that two are refractory to deletion and thus likely essential for blood-stage replication. Seven etramps are not essential for any life cycle stage. Systematic characterization of the members of the ETRAMP family reveals the diversity in importance of each family member at the interface between host and parasite throughout the developmental cycle of the malaria parasite. © 2011 Blackwell Publishing Ltd.

Structural basis of O-GlcNAc recognition by mammalian 14-3-3 proteins.

Science.gov (United States)

Toleman, Clifford A; Schumacher, Maria A; Yu, Seok-Ho; Zeng, Wenjie; Cox, Nathan J; Smith, Timothy J; Soderblom, Erik J; Wands, Amberlyn M; Kohler, Jennifer J; Boyce, Michael

2018-05-21

O-GlcNAc is an intracellular posttranslational modification that governs myriad cell biological processes and is dysregulated in human diseases. Despite this broad pathophysiological significance, the biochemical effects of most O-GlcNAcylation events remain uncharacterized. One prevalent hypothesis is that O-GlcNAc moieties may be recognized by "reader" proteins to effect downstream signaling. However, no general O-GlcNAc readers have been identified, leaving a considerable gap in the field. To elucidate O-GlcNAc signaling mechanisms, we devised a biochemical screen for candidate O-GlcNAc reader proteins. We identified several human proteins, including 14-3-3 isoforms, that bind O-GlcNAc directly and selectively. We demonstrate that 14-3-3 proteins bind O-GlcNAc moieties in human cells, and we present the structures of 14-3-3β/α and γ bound to glycopeptides, providing biophysical insights into O-GlcNAc-mediated protein-protein interactions. Because 14-3-3 proteins also bind to phospho-serine and phospho-threonine, they may integrate information from O-GlcNAc and O-phosphate signaling pathways to regulate numerous physiological functions.

Differential Protein Expressions in Virus-Infected and Uninfected Trichomonas vaginalis.

Science.gov (United States)

He, Ding; Pengtao, Gong; Ju, Yang; Jianhua, Li; He, Li; Guocai, Zhang; Xichen, Zhang

2017-04-01

Protozoan viruses may influence the function and pathogenicity of the protozoa. Trichomonas vaginalis is a parasitic protozoan that could contain a double stranded RNA (dsRNA) virus, T. vaginalis virus (TVV). However, there are few reports on the properties of the virus. To further determine variations in protein expression of T. vaginalis , we detected 2 strains of T. vaginalis ; the virus-infected (V + ) and uninfected (V - ) isolates to examine differentially expressed proteins upon TVV infection. Using a stable isotope N-terminal labeling strategy (iTRAQ) on soluble fractions to analyze proteomes, we identified 293 proteins, of which 50 were altered in V + compared with V - isolates. The results showed that the expression of 29 proteins was increased, and 21 proteins decreased in V + isolates. These differentially expressed proteins can be classified into 4 categories: ribosomal proteins, metabolic enzymes, heat shock proteins, and putative uncharacterized proteins. Quantitative PCR was used to detect 4 metabolic processes proteins: glycogen phosphorylase, malate dehydrogenase, triosephosphate isomerase, and glucose-6-phosphate isomerase, which were differentially expressed in V + and V - isolates. Our findings suggest that mRNA levels of these genes were consistent with protein expression levels. This study was the first which analyzed protein expression variations upon TVV infection. These observations will provide a basis for future studies concerning the possible roles of these proteins in host-parasite interactions.

3D-SURFER 2.0: web platform for real-time search and characterization of protein surfaces.

Science.gov (United States)

Xiong, Yi; Esquivel-Rodriguez, Juan; Sael, Lee; Kihara, Daisuke

2014-01-01

The increasing number of uncharacterized protein structures necessitates the development of computational approaches for function annotation using the protein tertiary structures. Protein structure database search is the basis of any structure-based functional elucidation of proteins. 3D-SURFER is a web platform for real-time protein surface comparison of a given protein structure against the entire PDB using 3D Zernike descriptors. It can smoothly navigate the protein structure space in real-time from one query structure to another. A major new feature of Release 2.0 is the ability to compare the protein surface of a single chain, a single domain, or a single complex against databases of protein chains, domains, complexes, or a combination of all three in the latest PDB. Additionally, two types of protein structures can now be compared: all-atom-surface and backbone-atom-surface. The server can also accept a batch job for a large number of database searches. Pockets in protein surfaces can be identified by VisGrid and LIGSITE (csc) . The server is available at http://kiharalab.org/3d-surfer/.

eggNOG 4.5: a hierarchical orthology framework with improved functional annotations for eukaryotic, prokaryotic and viral sequences

OpenAIRE

Huerta-Cepas, J.; Szklarczyk, D.; Forslund, K.; Cook, H.; Heller, D.; Walter, M.C.; Rattei, T.; Mende, D.R.; Sunagawa, S.; Kuhn, M.; Jensen, L.J.; von Mering, C.; Bork, P.

2016-01-01

eggNOG is a public resource that provides Orthologous Groups (OGs) of proteins at different taxonomic levels, each with integrated and summarized functional annotations. Developments since the latest public release include changes to the algorithm for creating OGs across taxonomic levels, making nested groups hierarchically consistent. This allows for a better propagation of functional terms across nested OGs and led to the novel annotation of 95 890 previously uncharacterized OGs, increasing...

The L1TD1 Protein Interactome Reveals the Importance of Post-transcriptional Regulation in Human Pluripotency

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Maheswara Reddy Emani

2015-03-01

Full Text Available The RNA-binding protein L1TD1 is one of the most specific and abundant proteins in pluripotent stem cells and is essential for the maintenance of pluripotency in human cells. Here, we identify the protein interaction network of L1TD1 in human embryonic stem cells (hESCs and provide insights into the interactome network constructed in human pluripotent cells. Our data reveal that L1TD1 has an important role in RNA splicing, translation, protein traffic, and degradation. L1TD1 interacts with multiple stem-cell-specific proteins, many of which are still uncharacterized in the context of development. Further, we show that L1TD1 is a part of the pluripotency interactome network of OCT4, SOX2, and NANOG, bridging nuclear and cytoplasmic regulation and highlighting the importance of RNA biology in pluripotency.

A guild of 45 CRISPR-associated (Cas protein families and multiple CRISPR/Cas subtypes exist in prokaryotic genomes.

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Daniel H Haft

2005-11-01

Full Text Available Clustered regularly interspaced short palindromic repeats (CRISPRs are a family of DNA direct repeats found in many prokaryotic genomes. Repeats of 21-37 bp typically show weak dyad symmetry and are separated by regularly sized, nonrepetitive spacer sequences. Four CRISPR-associated (Cas protein families, designated Cas1 to Cas4, are strictly associated with CRISPR elements and always occur near a repeat cluster. Some spacers originate from mobile genetic elements and are thought to confer "immunity" against the elements that harbor these sequences. In the present study, we have systematically investigated uncharacterized proteins encoded in the vicinity of these CRISPRs and found many additional protein families that are strictly associated with CRISPR loci across multiple prokaryotic species. Multiple sequence alignments and hidden Markov models have been built for 45 Cas protein families. These models identify family members with high sensitivity and selectivity and classify key regulators of development, DevR and DevS, in Myxococcus xanthus as Cas proteins. These identifications show that CRISPR/cas gene regions can be quite large, with up to 20 different, tandem-arranged cas genes next to a repeat cluster or filling the region between two repeat clusters. Distinctive subsets of the collection of Cas proteins recur in phylogenetically distant species and correlate with characteristic repeat periodicity. The analyses presented here support initial proposals of mobility of these units, along with the likelihood that loci of different subtypes interact with one another as well as with host cell defensive, replicative, and regulatory systems. It is evident from this analysis that CRISPR/cas loci are larger, more complex, and more heterogeneous than previously appreciated.

Structures of a putative ζ-class glutathione S-transferase from the pathogenic fungus Coccidioides immitis

International Nuclear Information System (INIS)

Edwards, Thomas E.; Bryan, Cassie M.; Leibly, David J.; Dieterich, Shellie H.; Abendroth, Jan; Sankaran, Banumathi; Sivam, Dhileep; Staker, Bart L.; Van Voorhis, Wesley C.; Myler, Peter J.; Stewart, Lance J.

2011-01-01

The pathogenic fungus C. immitis causes coccidioidomycosis, a potentially fatal disease. Here, apo and glutathione-bound crystal structures of a previously uncharacterized protein from C. immitis that appears to be a ζ-class glutathione S-transferase are presented. Coccidioides immitis is a pathogenic fungus populating the southwestern United States and is a causative agent of coccidioidomycosis, sometimes referred to as Valley Fever. Although the genome of this fungus has been sequenced, many operons are not properly annotated. Crystal structures are presented for a putative uncharacterized protein that shares sequence similarity with ζ-class glutathione S-transferases (GSTs) in both apo and glutathione-bound forms. The apo structure reveals a nonsymmetric homodimer with each protomer comprising two subdomains: a C-terminal helical domain and an N-terminal thioredoxin-like domain that is common to all GSTs. Half-site binding is observed in the glutathione-bound form. Considerable movement of some components of the active site relative to the glutathione-free form was observed, indicating an induced-fit mechanism for cofactor binding. The sequence homology, structure and half-site occupancy imply that the protein is a ζ-class glutathione S-transferase, a maleylacetoacetate isomerase (MAAI)

Preparation, crystallization and preliminary X-ray analysis of YjcG protein from Bacillus subtilis

International Nuclear Information System (INIS)

Li, Dan; Chan, Chiomui; Liang, Yu-He; Zheng, Xiaofeng; Li, Lanfen; Su, Xiao-Dong

2005-01-01

B. subtilis YjcG protein was expressed, purified and crystallized. A complete diffraction data set was collected at BSRF beamline 3W1A and processed to 2.3 Å resolution. Bacillus subtilis YjcG is a functionally uncharacterized protein with 171 residues that has no structural homologue in the Protein Data Bank. However, it shows sequence homology to bacterial and archaeal 2′–5′ RNA ligases. In order to identify its exact function via structural studies, the yjcG gene was amplified from B. subtilis genomic DNA and cloned into the expression vector pET21-DEST. The protein was expressed in a soluble form in Escherichia coli and was purified to homogeneity. Crystals suitable for X-ray analysis were obtained that diffracted to 2.3 Å and belonged to space group C2, with unit-cell parameters a = 99.66, b = 73.93, c = 61.77 Å, β = 113.56°

Unification of Cas protein families and a simple scenario for the origin and evolution of CRISPR-Cas systems

Directory of Open Access Journals (Sweden)

Wolf Yuri I

2011-07-01

Full Text Available Abstract Background The CRISPR-Cas adaptive immunity systems that are present in most Archaea and many Bacteria function by incorporating fragments of alien genomes into specific genomic loci, transcribing the inserts and using the transcripts as guide RNAs to destroy the genome of the cognate virus or plasmid. This RNA interference-like immune response is mediated by numerous, diverse and rapidly evolving Cas (CRISPR-associated proteins, several of which form the Cascade complex involved in the processing of CRISPR transcripts and cleavage of the target DNA. Comparative analysis of the Cas protein sequences and structures led to the classification of the CRISPR-Cas systems into three Types (I, II and III. Results A detailed comparison of the available sequences and structures of Cas proteins revealed several unnoticed homologous relationships. The Repeat-Associated Mysterious Proteins (RAMPs containing a distinct form of the RNA Recognition Motif (RRM domain, which are major components of the CRISPR-Cas systems, were classified into three large groups, Cas5, Cas6 and Cas7. Each of these groups includes many previously uncharacterized proteins now shown to adopt the RAMP structure. Evidence is presented that large subunits contained in most of the CRISPR-Cas systems could be homologous to Cas10 proteins which contain a polymerase-like Palm domain and are predicted to be enzymatically active in Type III CRISPR-Cas systems but inactivated in Type I systems. These findings, the fact that the CRISPR polymerases, RAMPs and Cas2 all contain core RRM domains, and distinct gene arrangements in the three types of CRISPR-Cas systems together provide for a simple scenario for origin and evolution of the CRISPR-Cas machinery. Under this scenario, the CRISPR-Cas system originated in thermophilic Archaea and subsequently spread horizontally among prokaryotes. Conclusions Because of the extreme diversity of CRISPR-Cas systems, in-depth sequence and structure

The Escherichia coli BolA Protein IbaG Forms a Histidine-Ligated [2Fe-2S]-Bridged Complex with Grx4.

Science.gov (United States)

Dlouhy, Adrienne C; Li, Haoran; Albetel, Angela-Nadia; Zhang, Bo; Mapolelo, Daphne T; Randeniya, Sajini; Holland, Ashley A; Johnson, Michael K; Outten, Caryn E

2016-12-13

Two ubiquitous protein families have emerged as key players in iron metabolism, the CGFS-type monothiol glutaredoxins (Grxs) and the BolA proteins. Monothiol Grxs and BolA proteins form heterocomplexes that have been implicated in Fe-S cluster assembly and trafficking. The Escherichia coli genome encodes members of both of these proteins families, namely, the monothiol glutaredoxin Grx4 and two BolA family proteins, BolA and IbaG. Previous work has demonstrated that E. coli Grx4 and BolA interact as both apo and [2Fe-2S]-bridged heterodimers that are spectroscopically distinct from [2Fe-2S]-bridged Grx4 homodimers. However, the physical and functional interactions between Grx4 and IbaG are uncharacterized. Here we show that co-expression of Grx4 with IbaG yields a [2Fe-2S]-bridged Grx4-IbaG heterodimer. In vitro interaction studies indicate that IbaG binds the [2Fe-2S] Grx4 homodimer to form apo Grx4-IbaG heterodimer as well as the [2Fe-2S] Grx4-IbaG heterodimer, altering the cluster stability and coordination environment. Additionally, spectroscopic and mutagenesis studies provide evidence that IbaG ligates the Fe-S cluster via the conserved histidine that is present in all BolA proteins and by a second conserved histidine that is present in the H/C loop of two of the four classes of BolA proteins. These results suggest that IbaG may function in Fe-S cluster assembly and trafficking in E. coli as demonstrated for other BolA homologues that interact with monothiol Grxs.

Gypsy transposition correlates with the production of a retroviral envelope-like protein under the tissue-specific control of the Drosophila flamenco gene.

OpenAIRE

Pélisson, A; Song, S U; Prud'homme, N; Smith, P A; Bucheton, A; Corces, V G

1994-01-01

Gypsy displays striking similarities to vertebrate retroviruses, including the presence of a yet uncharacterized additional open reading frame (ORF3) and the recent evidence for infectivity. It is mobilized with high frequency in the germline of the progeny of females homozygous for the flamenco permissive mutation. We report the characterization of a gypsy subgenomic ORF3 RNA encoding typical retroviral envelope proteins. In females, env expression is strongly repressed by one copy of the no...

Development of a high-throughput method for the systematic identification of human proteins nuclear translocation potential

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Kawai Jun

2009-09-01

Full Text Available Abstract Background Important clues to the function of novel and uncharacterized proteins can be obtained by identifying their ability to translocate in the nucleus. In addition, a comprehensive definition of the nuclear proteome undoubtedly represents a key step toward a better understanding of the biology of this organelle. Although several high-throughput experimental methods have been developed to explore the sub-cellular localization of proteins, these methods tend to focus on the predominant localizations of gene products and may fail to provide a complete catalog of proteins that are able to transiently locate into the nucleus. Results We have developed a method for examining the nuclear localization potential of human gene products at the proteome scale by adapting a mammalian two-hybrid system we have previously developed. Our system is composed of three constructs co-transfected into a mammalian cell line. First, it contains a PCR construct encoding a fusion protein composed of a tested protein, the PDZ-protein TIP-1, and the transactivation domain of TNNC2 (referred to as ACT construct. Second, our system contains a PCR construct encoding a fusion protein composed of the DNA binding domain of GAL4 and the PDZ binding domain of rhotekin (referred to as the BIND construct. Third, a GAL4-responsive luciferase reporter is used to detect the reconstitution of a transcriptionally active BIND-ACT complex through the interaction of TIP-1 and rhotekin, which indicates the ability of the tested protein to translocate into the nucleus. We validated our method in a small-scale feasibility study by comparing it to green fluorescent protein (GFP fusion-based sub-cellular localization assays, sequence-based computational prediction of protein sub-cellular localization, and current sub-cellular localization data available from the literature for 22 gene products. Conclusion Our reporter-based system can rapidly screen gene products for their ability

Novel E3 ubiquitin ligases that regulate histone protein levels in the budding yeast Saccharomyces cerevisiae.

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Rakesh Kumar Singh

Full Text Available Core histone proteins are essential for packaging the genomic DNA into chromatin in all eukaryotes. Since multiple genes encode these histone proteins, there is potential for generating more histones than what is required for chromatin assembly. The positively charged histones have a very high affinity for negatively charged molecules such as DNA, and any excess of histone proteins results in deleterious effects on genomic stability and cell viability. Hence, histone levels are known to be tightly regulated via transcriptional, posttranscriptional and posttranslational mechanisms. We have previously elucidated the posttranslational regulation of histone protein levels by the ubiquitin-proteasome pathway involving the E2 ubiquitin conjugating enzymes Ubc4/5 and the HECT (Homologous to E6-AP C-Terminus domain containing E3 ligase Tom1 in the budding yeast. Here we report the identification of four additional E3 ligases containing the RING (Really Interesting New Gene finger domains that are involved in the ubiquitylation and subsequent degradation of excess histones in yeast. These E3 ligases are Pep5, Snt2 as well as two previously uncharacterized Open Reading Frames (ORFs YKR017C and YDR266C that we have named Hel1 and Hel2 (for Histone E3 Ligases respectively. Mutants lacking these E3 ligases are sensitive to histone overexpression as they fail to degrade excess histones and accumulate high levels of endogenous histones on histone chaperones. Co-immunoprecipitation assays showed that these E3 ligases interact with the major E2 enzyme Ubc4 that is involved in the degradation related ubiquitylation of histones. Using mutagenesis we further demonstrate that the RING domains of Hel1, Hel2 and Snt2 are required for histone regulation. Lastly, mutants corresponding to Hel1, Hel2 and Pep5 are sensitive to replication inhibitors. Overall, our results highlight the importance of posttranslational histone regulatory mechanisms that employ multiple E3

PFP: Automated prediction of gene ontology functional annotations with confidence scores using protein sequence data.

Science.gov (United States)

Hawkins, Troy; Chitale, Meghana; Luban, Stanislav; Kihara, Daisuke

2009-02-15

Protein function prediction is a central problem in bioinformatics, increasing in importance recently due to the rapid accumulation of biological data awaiting interpretation. Sequence data represents the bulk of this new stock and is the obvious target for consideration as input, as newly sequenced organisms often lack any other type of biological characterization. We have previously introduced PFP (Protein Function Prediction) as our sequence-based predictor of Gene Ontology (GO) functional terms. PFP interprets the results of a PSI-BLAST search by extracting and scoring individual functional attributes, searching a wide range of E-value sequence matches, and utilizing conventional data mining techniques to fill in missing information. We have shown it to be effective in predicting both specific and low-resolution functional attributes when sufficient data is unavailable. Here we describe (1) significant improvements to the PFP infrastructure, including the addition of prediction significance and confidence scores, (2) a thorough benchmark of performance and comparisons to other related prediction methods, and (3) applications of PFP predictions to genome-scale data. We applied PFP predictions to uncharacterized protein sequences from 15 organisms. Among these sequences, 60-90% could be annotated with a GO molecular function term at high confidence (>or=80%). We also applied our predictions to the protein-protein interaction network of the Malaria plasmodium (Plasmodium falciparum). High confidence GO biological process predictions (>or=90%) from PFP increased the number of fully enriched interactions in this dataset from 23% of interactions to 94%. Our benchmark comparison shows significant performance improvement of PFP relative to GOtcha, InterProScan, and PSI-BLAST predictions. This is consistent with the performance of PFP as the overall best predictor in both the AFP-SIG '05 and CASP7 function (FN) assessments. PFP is available as a web service at http

Predictive and comparative analysis of Ebolavirus proteins

Science.gov (United States)

Cong, Qian; Pei, Jimin; Grishin, Nick V

2015-01-01

Ebolavirus is the pathogen for Ebola Hemorrhagic Fever (EHF). This disease exhibits a high fatality rate and has recently reached a historically epidemic proportion in West Africa. Out of the 5 known Ebolavirus species, only Reston ebolavirus has lost human pathogenicity, while retaining the ability to cause EHF in long-tailed macaque. Significant efforts have been spent to determine the three-dimensional (3D) structures of Ebolavirus proteins, to study their interaction with host proteins, and to identify the functional motifs in these viral proteins. Here, in light of these experimental results, we apply computational analysis to predict the 3D structures and functional sites for Ebolavirus protein domains with unknown structure, including a zinc-finger domain of VP30, the RNA-dependent RNA polymerase catalytic domain and a methyltransferase domain of protein L. In addition, we compare sequences of proteins that interact with Ebolavirus proteins from RESTV-resistant primates with those from RESTV-susceptible monkeys. The host proteins that interact with GP and VP35 show an elevated level of sequence divergence between the RESTV-resistant and RESTV-susceptible species, suggesting that they may be responsible for host specificity. Meanwhile, we detect variable positions in protein sequences that are likely associated with the loss of human pathogenicity in RESTV, map them onto the 3D structures and compare their positions to known functional sites. VP35 and VP30 are significantly enriched in these potential pathogenicity determinants and the clustering of such positions on the surfaces of VP35 and GP suggests possible uncharacterized interaction sites with host proteins that contribute to the virulence of Ebolavirus. PMID:26158395

Predictive and comparative analysis of Ebolavirus proteins.

Science.gov (United States)

Cong, Qian; Pei, Jimin; Grishin, Nick V

2015-01-01

Ebolavirus is the pathogen for Ebola Hemorrhagic Fever (EHF). This disease exhibits a high fatality rate and has recently reached a historically epidemic proportion in West Africa. Out of the 5 known Ebolavirus species, only Reston ebolavirus has lost human pathogenicity, while retaining the ability to cause EHF in long-tailed macaque. Significant efforts have been spent to determine the three-dimensional (3D) structures of Ebolavirus proteins, to study their interaction with host proteins, and to identify the functional motifs in these viral proteins. Here, in light of these experimental results, we apply computational analysis to predict the 3D structures and functional sites for Ebolavirus protein domains with unknown structure, including a zinc-finger domain of VP30, the RNA-dependent RNA polymerase catalytic domain and a methyltransferase domain of protein L. In addition, we compare sequences of proteins that interact with Ebolavirus proteins from RESTV-resistant primates with those from RESTV-susceptible monkeys. The host proteins that interact with GP and VP35 show an elevated level of sequence divergence between the RESTV-resistant and RESTV-susceptible species, suggesting that they may be responsible for host specificity. Meanwhile, we detect variable positions in protein sequences that are likely associated with the loss of human pathogenicity in RESTV, map them onto the 3D structures and compare their positions to known functional sites. VP35 and VP30 are significantly enriched in these potential pathogenicity determinants and the clustering of such positions on the surfaces of VP35 and GP suggests possible uncharacterized interaction sites with host proteins that contribute to the virulence of Ebolavirus.

Proteolysis of complement factors iC3b and C5 by the serine protease prostate-specific antigen in prostatic fluid and seminal plasma.

Science.gov (United States)

Manning, Michael L; Williams, Simon A; Jelinek, Christine A; Kostova, Maya B; Denmeade, Samuel R

2013-03-15

Prostate-specific Ag (PSA) is a serine protease that is expressed exclusively by normal and malignant prostate epithelial cells. The continued high-level expression of PSA by the majority of men with both high- and low-grade prostate cancer throughout the course of disease progression, even in the androgen-ablated state, suggests that PSA has a role in the pathogenesis of disease. Current experimental and clinical evidence suggests that chronic inflammation, regardless of the cause, may predispose men to prostate cancer. The responsibility of the immune system in immune surveillance and eventually tumor progression is well appreciated but not completely understood. In this study, we used a mass spectrometry-based evaluation of prostatic fluid obtained from diseased prostates after removal by radical prostatectomy to identify potential immunoregulatory proteins. This analysis revealed the presence of Igs and the complement system proteins C3, factor B, and clusterin. Verification of these findings by Western blot confirmed the high-level expression of C3 in the prostatic fluid and the presence of a previously uncharacterized C-terminal C3 cleavage product. Biochemical analysis of this C3 cleavage fragment revealed a putative PSA cleavage site after tyrosine-1348. Purified PSA was able to cleave iC3b and the related complement protein C5. These results suggest a previously uncharacterized function of PSA as an immunoregulatory protease that could help to create an environment hospitable to malignancy through proteolysis of the complement system.

The tumor necrosis factor-alpha-induced protein 8 family in immune homeostasis and inflammatory cancer diseases.

Science.gov (United States)

Luan, Y Y; Yao, Y M; Sheng, Z Y

2013-01-01

Within the immune system homeostasis is maintained by a myriad of mechanisms that include the regulation of immune cell activation and programmed cell death. The breakdown of immune homeostasis may lead to fatal inflammatory diseases. We set out to identify genes of tumor necrosis factor-alpha-induced protein 8 (TNFAIP8) family that has a functional role in the process of immune homeostasis. Tumor necrosis factor-alpha-induced protein 8 (TNFAIP8), which functions as an oncogenic molecule, is also associated with enhanced cell survival and inhibition of apoptosis. Tumor necrosis factor-alpha-induced protein 8-like 2 (TIPE2) governs immune homeostasis in both the innate and adaptive immune system and prevents hyper-responsiveness by negatively regulating signaling via T cell receptors and Toll-like receptors (TLRs). There also exist two highly homologous but uncharacterized proteins, TIPE1 and TIPE3. This review is an attempt to provide a summary of TNFAIP8 family associated with immune homeostasis and inflammatory cancer diseases.

Long-term outcome on renal replacement therapy in patients who previously received a keto acid-supplemented very-low-protein diet.

Science.gov (United States)

Chauveau, Philippe; Couzi, Lionel; Vendrely, Benoit; de Précigout, Valérie; Combe, Christian; Fouque, Denis; Aparicio, Michel

2009-10-01

The consequences of a supplemented very-low-protein diet remain a matter of debate with regard to patient outcome before or after the onset of renal replacement therapy. We evaluated the long-term clinical outcome during maintenance dialysis and/or transplantation in patients who previously received a supplemented very-low-protein diet. We assessed the outcome of 203 patients who received a supplemented very-low-protein diet for >3 mo (inclusion period: 1985-2000) and started dialysis after a mean diet duration of 33.1 mo (4-230 mo). The survival rate in the whole cohort was 79% and 63% at 5 and 10 y, respectively. One hundred two patients continued with chronic dialysis during the entire follow-up, and 101 patients were grafted at least once. Patient outcomes were similar to those of the French Dialysis Registry patients for the dialysis group and similar to the 865 patients who were transplanted in Bordeaux during the same period for the transplant group. There was no correlation between death rate and duration of diet. The lack of correlation between death rate and duration of diet and the moderate mortality rate observed during the first 10 y of renal replacement therapy confirm that a supplemented very-low-protein diet has no detrimental effect on the outcome of patients with chronic kidney disease who receive renal replacement therapy.

Phospho-dependent binding of the clathrin AP2 adaptor complex to GABAA receptors regulates the efficacy of inhibitory synaptic transmission

OpenAIRE

Kittler, Josef T.; Chen, Guojun; Honing, Stephan; Bogdanov, Yury; McAinsh, Kristina; Arancibia-Carcamo, I. Lorena; Jovanovic, Jasmina N.; Pangalos, Menelas N.; Haucke, Volker; Yan, Zhen; Moss, Stephen J.

2005-01-01

The efficacy of synaptic inhibition depends on the number of γ-aminobutyric acid type A receptors (GABAARs) expressed on the cell surface of neurons. The clathrin adaptor protein 2 (AP2) complex is a critical regulator of GABAAR endocytosis and, hence, surface receptor number. Here, we identify a previously uncharacterized atypical AP2 binding motif conserved within the intracellular domains of all GABAAR β subunit isoforms. This AP2 binding motif (KTHLRRRSSQLK in the β3 subunit) incorporates...

The coiled-coil domain containing protein CCDC40 is essential for motile cilia function and left-right axis formation

DEFF Research Database (Denmark)

Becker-Heck, Anita; Zohn, Irene E; Okabe, Noriko

2011-01-01

Primary ciliary dyskinesia (PCD) is a genetically heterogeneous autosomal recessive disorder characterized by recurrent infections of the respiratory tract associated with the abnormal function of motile cilia. Approximately half of individuals with PCD also have alterations in the left-right...... organization of their internal organ positioning, including situs inversus and situs ambiguous (Kartagener's syndrome). Here, we identify an uncharacterized coiled-coil domain containing a protein, CCDC40, essential for correct left-right patterning in mouse, zebrafish and human. In mouse and zebrafish, Ccdc40...

Yeast Surface Display of Two Proteins Previously Shown to Be Protective Against White Spot Syndrome Virus (WSSV) in Shrimp.

Science.gov (United States)

Ananphongmanee, Vorawit; Srisala, Jiraporn; Sritunyalucksana, Kallaya; Boonchird, Chuenchit

2015-01-01

Cell surface display using the yeasts Saccharomyces cerevisiae and Pichia pastoris has been extensively developed for application in bioindustrial processes. Due to the rigid structure of their cell walls, a number of proteins have been successfully displayed on their cell surfaces. It was previously reported that the viral binding protein Rab7 from the giant tiger shrimp Penaeus monodon (PmRab7) and its binding partner envelope protein VP28 of white spot syndrome virus (WSSV) could independently protect shrimp against WSSV infection. Thus, we aimed to display these two proteins independently on the cell surfaces of 2 yeast clones with the ultimate goal of using a mixture of the two clones as an orally deliverable, antiviral agent to protect shrimp against WSSV infection. PmRab7 and VP28 were modified by N-terminal tagging to the C-terminal half of S. cerevisiae α-agglutinin. DNA fragments, harboring fused-gene expression cassettes under control of an alcohol oxidase I (AOX1) promoter were constructed and used to transform the yeast cells. Immunofluorescence microscopy with antibodies specific to both proteins demonstrated that mutated PmRab7 (mPmRab7) and partial VP28 (pVP28) were localized on the cell surfaces of the respective clones, and fluorescence intensity for each was significantly higher than that of control cells by flow cytometry. Enzyme-linked immunosorbant assay (ELISA) using cells displaying mPmRab7 or pVP28 revealed that the binding of specific antibodies for each was dose-dependent, and could be saturated. In addition, the binding of mPmRab7-expressing cells with free VP28, and vice versa was dose dependent. Binding between the two surface-expressed proteins was confirmed by an assay showing agglutination between cells expressing complementary mPmRab7 and pVP28. In summary, our genetically engineered P. pastoris can display biologically active mPmRab7 and pVP28 and is now ready for evaluation of efficacy in protecting shrimp against WSSV by oral

Yeast Surface Display of Two Proteins Previously Shown to Be Protective Against White Spot Syndrome Virus (WSSV in Shrimp.

Directory of Open Access Journals (Sweden)

Vorawit Ananphongmanee

Full Text Available Cell surface display using the yeasts Saccharomyces cerevisiae and Pichia pastoris has been extensively developed for application in bioindustrial processes. Due to the rigid structure of their cell walls, a number of proteins have been successfully displayed on their cell surfaces. It was previously reported that the viral binding protein Rab7 from the giant tiger shrimp Penaeus monodon (PmRab7 and its binding partner envelope protein VP28 of white spot syndrome virus (WSSV could independently protect shrimp against WSSV infection. Thus, we aimed to display these two proteins independently on the cell surfaces of 2 yeast clones with the ultimate goal of using a mixture of the two clones as an orally deliverable, antiviral agent to protect shrimp against WSSV infection. PmRab7 and VP28 were modified by N-terminal tagging to the C-terminal half of S. cerevisiae α-agglutinin. DNA fragments, harboring fused-gene expression cassettes under control of an alcohol oxidase I (AOX1 promoter were constructed and used to transform the yeast cells. Immunofluorescence microscopy with antibodies specific to both proteins demonstrated that mutated PmRab7 (mPmRab7 and partial VP28 (pVP28 were localized on the cell surfaces of the respective clones, and fluorescence intensity for each was significantly higher than that of control cells by flow cytometry. Enzyme-linked immunosorbant assay (ELISA using cells displaying mPmRab7 or pVP28 revealed that the binding of specific antibodies for each was dose-dependent, and could be saturated. In addition, the binding of mPmRab7-expressing cells with free VP28, and vice versa was dose dependent. Binding between the two surface-expressed proteins was confirmed by an assay showing agglutination between cells expressing complementary mPmRab7 and pVP28. In summary, our genetically engineered P. pastoris can display biologically active mPmRab7 and pVP28 and is now ready for evaluation of efficacy in protecting shrimp against

Exploring and Expanding the Fatty-Acid-Binding Protein Superfamily in Fasciola Species.

Science.gov (United States)

Morphew, Russell M; Wilkinson, Toby J; Mackintosh, Neil; Jahndel, Veronika; Paterson, Steve; McVeigh, Paul; Abbas Abidi, Syed M; Saifullah, Khalid; Raman, Muthusamy; Ravikumar, Gopalakrishnan; LaCourse, James; Maule, Aaron; Brophy, Peter M

2016-09-02

The liver flukes Fasciola hepatica and F. gigantica infect livestock worldwide and threaten food security with climate change and problematic control measures spreading disease. Fascioliasis is also a foodborne disease with up to 17 million humans infected. In the absence of vaccines, treatment depends on triclabendazole (TCBZ), and overuse has led to widespread resistance, compromising future TCBZ control. Reductionist biology from many laboratories has predicted new therapeutic targets. To this end, the fatty-acid-binding protein (FABP) superfamily has proposed multifunctional roles, including functions intersecting vaccine and drug therapy, such as immune modulation and anthelmintic sequestration. Research is hindered by a lack of understanding of the full FABP superfamily complement. Although discovery studies predicted FABPs as promising vaccine candidates, it is unclear if uncharacterized FABPs are more relevant for vaccine formulations. We have coupled genome, transcriptome, and EST data mining with proteomics and phylogenetics to reveal a liver fluke FABP superfamily of seven clades: previously identified clades I-III and newly identified clades IV-VII. All new clade FABPs were analyzed using bioinformatics and cloned from both liver flukes. The extended FABP data set will provide new study tools to research the role of FABPs in parasite biology and as therapy targets.

CYP2D6 and CYP2C19 in Papua New Guinea: High frequency of previously uncharacterized CYP2D6 alleles and heterozygote excess.

Science.gov (United States)

von Ahsen, Nicolas; Tzvetkov, Mladen; Karunajeewa, Harin A; Gomorrai, Servina; Ura, Alice; Brockmöller, Jürgen; Davis, Timothy M E; Mueller, Ivo; Ilett, Kenneth F; Oellerich, Michael

2010-08-18

A high frequency of previously unknown CYP2D6 alleles have been reported in Oceania populations. Genetic and functional properties of these alleles remain unknown. We performed analyses of the genetic variability of CYP2D6 and CYP2C19 genes using AmpliChip genotyping in cohorts from two distinct Papua New Guinea (PNG) populations (Kunjingini, n=88; Alexishafen, n=84) focussing on the genetic characterisation of PNG-specific alleles by re-sequencing. Previously unknown CYP2D6 alleles have population frequencies of 24% (Kunjingini) and 12% (Alexishafen). An allele similar to CYP2D6*1, but carrying the 1661G>C substitution, was the second most frequent CYP2D6 allele (20% Kunjingini and 10% Alexishafen population frequency). Sequencing suggests the CYP2D6* 1661G>C allele originated from a cross-over between CYP2D6*1 and *2 and thus is predicted to confer fully active CYP2D6 enzyme. Two additional predicted full activity alleles [1661G>C;4180G>C] and 31G>A were found in the Kunjingini cohort (frequencies 3 c/c and 1%, respectively) and a novel predicted reduced activity allele [100C>T;1039C>T] was found in the Alexishafen cohort (frequency 2%). A high frequency of ultra-rapid (15%) and notably low frequencies of intermediate and poor CYP2D6 metabolizers (exogamy and recent introduction of alleles by migration that are yet to reach HWE in relatively isolated populations. The CYP2D6*1661 allele common in Oceania may be regarded as functionally equivalent to the full activity CYP2D6*1 allele.

Thioredoxin Selectivity for Thiol-based Redox Regulation of Target Proteins in Chloroplasts*

Science.gov (United States)

Yoshida, Keisuke; Hara, Satoshi; Hisabori, Toru

2015-01-01

Redox regulation based on the thioredoxin (Trx) system is believed to ensure light-responsive control of various functions in chloroplasts. Five Trx subtypes have been reported to reside in chloroplasts, but their functional diversity in the redox regulation of Trx target proteins remains poorly clarified. To directly address this issue, we studied the Trx-dependent redox shifts of several chloroplast thiol-modulated enzymes in vitro and in vivo. In vitro assays using a series of Arabidopsis recombinant proteins provided new insights into Trx selectivity for the redox regulation as well as the underpinning for previous suggestions. Most notably, by combining the discrimination of thiol status with mass spectrometry and activity measurement, we identified an uncharacterized aspect of the reductive activation of NADP-malate dehydrogenase; two redox-active Cys pairs harbored in this enzyme were reduced via distinct utilization of Trxs even within a single polypeptide. In our in vitro assays, Trx-f was effective in reducing all thiol-modulated enzymes analyzed here. We then investigated the in vivo physiological relevance of these in vitro findings, using Arabidopsis wild-type and Trx-f-deficient plants. Photoreduction of fructose-1,6-bisphosphatase was partially impaired in Trx-f-deficient plants, but the global impact of Trx-f deficiency on the redox behaviors of thiol-modulated enzymes was not as striking as expected from the in vitro data. Our results provide support for the in vivo functionality of the Trx system and also highlight the complexity and plasticity of the chloroplast redox network. PMID:25878252

A Clostridium difficile-Specific, Gel-Forming Protein Required for Optimal Spore Germination

Directory of Open Access Journals (Sweden)

M. Lauren Donnelly

2017-01-01

Full Text Available Clostridium difficile is a Gram-positive spore-forming obligate anaerobe that is a leading cause of antibiotic-associated diarrhea worldwide. In order for C. difficile to initiate infection, its aerotolerant spore form must germinate in the gut of mammalian hosts. While almost all spore-forming organisms use transmembrane germinant receptors to trigger germination, C. difficile uses the pseudoprotease CspC to sense bile salt germinants. CspC activates the related subtilisin-like protease CspB, which then proteolytically activates the cortex hydrolase SleC. Activated SleC degrades the protective spore cortex layer, a step that is essential for germination to proceed. Since CspC incorporation into spores also depends on CspA, a related pseudoprotease domain, Csp family proteins play a critical role in germination. However, how Csps are incorporated into spores remains unknown. In this study, we demonstrate that incorporation of the CspC, CspB, and CspA germination regulators into spores depends on CD0311 (renamed GerG, a previously uncharacterized hypothetical protein. The reduced levels of Csps in gerG spores correlate with reduced responsiveness to bile salt germinants and increased germination heterogeneity in single-spore germination assays. Interestingly, asparagine-rich repeat sequences in GerG’s central region facilitate spontaneous gel formation in vitro even though they are dispensable for GerG-mediated control of germination. Since GerG is found exclusively in C. difficile, our results suggest that exploiting GerG function could represent a promising avenue for developing C. difficile-specific anti-infective therapies.

MadR1, a Mycobacterium tuberculosis cell cycle stress response protein that is a member of a widely conserved protein class of prokaryotic, eukaryotic and archeal origin.

Science.gov (United States)

Crew, Rebecca; Ramirez, Melissa V; England, Kathleen; Slayden, Richard A

2015-05-01

Stress-induced molecular programs designed to stall division progression are nearly ubiquitous in bacteria, with one well-known example being the participation of the SulA septum inhibiting protein in the SOS DNA damage repair response. Mycobacteria similarly demonstrate stress-altered growth kinetics, however no such regulators have been found in these organisms. We therefore set out to identify SulA-like regulatory proteins in Mycobacterium tuberculosis. A bioinformatics modeling-based approach led to the identification of rv2216 as encoding for a protein with weak similarity to SulA, further analysis distinguished this protein as belonging to a group of uncharacterized growth promoting proteins. We have named the mycobacterial protein encoded by rv2216 morphology altering division regulator protein 1, MadR1. Overexpression of madR1 modulated cell length while maintaining growth kinetics similar to wild-type, and increased the proportion of bent or V-form cells in the population. The presence of MadR1-GFP at regions of cellular elongation (poles) and morphological differentiation (V-form) suggests MadR1 involvement in phenotypic heterogeneity and longitudinal cellular growth. Global transcriptional analysis indicated that MadR1 functionality is linked to lipid editing programs required for growth and persistence. This is the first report to differentiate the larger class of these conserved proteins from SulA proteins and characterizes MadR1 effects on the mycobacterial cell. Copyright © 2015 Elsevier Ltd. All rights reserved.

Fine-tuning of protein domain boundary by minimizing potential coiled coil regions

International Nuclear Information System (INIS)

Iwaya, Naoko; Goda, Natsuko; Unzai, Satoru; Fujiwara, Kenichiro; Tanaka, Toshiki; Tomii, Kentaro; Tochio, Hidehito; Shirakawa, Masahiro; Hiroaki, Hidekazu

2007-01-01

Structural determination of individual protein domains isolated from multidomain proteins is a common approach in the post-genomic era. Novel and thus uncharacterized domains liberated from intact proteins often self-associate due to incorrectly defined domain boundaries. Self-association results in missing signals, poor signal dispersion and a low signal-to-noise ratio in 1 H- 15 N HSQC spectra. We have found that a putative, non-canonical coiled coil region close to a domain boundary can cause transient hydrophobic self-association and monomer-dimer equilibrium in solution. Here we propose a rational method to predict putative coiled coil regions adjacent to the globular core domain using the program COILS. Except for the amino acid sequence, no preexisting knowledge concerning the domain is required. A small number of mutant proteins with a minimized coiled coil region have been rationally designed and tested. The engineered domains exhibit decreased self-association as assessed by 1 H- 15 N HSQC spectra with improved peak dispersion and sharper cross peaks. Two successful examples of isolating novel N-terminal domains from AAA-ATPases are demonstrated. Our method is useful for the experimental determination of domain boundaries suited for structural genomics studies

Fine-tuning of protein domain boundary by minimizing potential coiled coil regions.

Science.gov (United States)

Iwaya, Naoko; Goda, Natsuko; Unzai, Satoru; Fujiwara, Kenichiro; Tanaka, Toshiki; Tomii, Kentaro; Tochio, Hidehito; Shirakawa, Masahiro; Hiroaki, Hidekazu

2007-01-01

Structural determination of individual protein domains isolated from multidomain proteins is a common approach in the post-genomic era. Novel and thus uncharacterized domains liberated from intact proteins often self-associate due to incorrectly defined domain boundaries. Self-association results in missing signals, poor signal dispersion and a low signal-to-noise ratio in (1)H-(15)N HSQC spectra. We have found that a putative, non-canonical coiled coil region close to a domain boundary can cause transient hydrophobic self-association and monomer-dimer equilibrium in solution. Here we propose a rational method to predict putative coiled coil regions adjacent to the globular core domain using the program COILS. Except for the amino acid sequence, no preexisting knowledge concerning the domain is required. A small number of mutant proteins with a minimized coiled coil region have been rationally designed and tested. The engineered domains exhibit decreased self-association as assessed by (1)H-(15)N HSQC spectra with improved peak dispersion and sharper cross peaks. Two successful examples of isolating novel N-terminal domains from AAA-ATPases are demonstrated. Our method is useful for the experimental determination of domain boundaries suited for structural genomics studies.

Characterization of Helicobacter pylori dapE and construction of a conditionally lethal dapE mutant.

OpenAIRE

Karita, M; Etterbeek, M L; Forsyth, M H; Tummuru, M K; Blaser, M J

1997-01-01

Helicobacter pylori colonizes the human gastric mucosa and causes gastritis, ulceration, or gastric cancer. A previously uncharacterized region of the H. pylori genome was identified and sequenced. This region includes a putative operon containing three open reading frames termed gidA (1,866 bp), dapE (1,167 bp), and orf2 (753 bp); the gidA and dapE products are highly homologous to other bacterial proteins. In E. coli, dapE encodes N-succinyl-L-diaminopimelic acid desuccinylase, which cataly...

Proteomic Mapping of Dental Enamel Matrix from Inbred Mouse Strains: Unraveling Potential New Players in Enamel.

Science.gov (United States)

Lima Leite, Aline; Silva Fernandes, Mileni; Charone, Senda; Whitford, Gary Milton; Everett, Eric T; Buzalaf, Marília Afonso Rabelo

2018-01-01

Enamel formation is a complex 2-step process by which proteins are secreted to form an extracellular matrix, followed by massive protein degradation and subsequent mineralization. Excessive systemic exposure to fluoride can disrupt this process and lead to a condition known as dental fluorosis. The genetic background influences the responses of mineralized tissues to fluoride, such as dental fluorosis, observed in A/J and 129P3/J mice. The aim of the present study was to map the protein profile of enamel matrix from A/J and 129P3/J strains. Enamel matrix samples were obtained from A/J and 129P3/J mice and analyzed by 2-dimensional electrophoresis and liquid chromatography coupled with mass spectrometry. A total of 120 proteins were identified, and 7 of them were classified as putative uncharacterized proteins and analyzed in silico for structural and functional characterization. An interesting finding was the possibility of the uncharacterized sequence Q8BIS2 being an enzyme involved in the degradation of matrix proteins. Thus, the results provide a comprehensive view of the structure and function for putative uncharacterized proteins found in the enamel matrix that could help to elucidate the mechanisms involved in enamel biomineralization and genetic susceptibility to dental fluorosis. © 2018 S. Karger AG, Basel.

Ligands in PSI structures

International Nuclear Information System (INIS)

Kumar, Abhinav; Chiu, Hsiu-Ju; Axelrod, Herbert L.; Morse, Andrew; Elsliger, Marc-André; Wilson, Ian A.; Deacon, Ashley

2010-01-01

A survey of the types and frequency of ligands that are bound to PSI structures is analyzed as well as their utility in functional annotation of previously uncharacterized proteins. Approximately 65% of PSI structures report some type of ligand(s) that is bound in the crystal structure. Here, a description is given of how such ligands are handled and analyzed at the JCSG and a survey of the types, variety and frequency of ligands that are observed in the PSI structures is also compiled and analyzed, including illustrations of how these bound ligands have provided functional clues for annotation of proteins with little or no previous experimental characterization. Furthermore, a web server was developed as a tool to mine and analyze the PSI structures for bound ligands and other identifying features

Evidence-based annotation of the malaria parasite's genome using comparative expression profiling.

Directory of Open Access Journals (Sweden)

Yingyao Zhou

2008-02-01

Full Text Available A fundamental problem in systems biology and whole genome sequence analysis is how to infer functions for the many uncharacterized proteins that are identified, whether they are conserved across organisms of different phyla or are phylum-specific. This problem is especially acute in pathogens, such as malaria parasites, where genetic and biochemical investigations are likely to be more difficult. Here we perform comparative expression analysis on Plasmodium parasite life cycle data derived from P. falciparum blood, sporozoite, zygote and ookinete stages, and P. yoelii mosquito oocyst and salivary gland sporozoites, blood and liver stages and show that type II fatty acid biosynthesis genes are upregulated in liver and insect stages relative to asexual blood stages. We also show that some universally uncharacterized genes with orthologs in Plasmodium species, Saccharomyces cerevisiae and humans show coordinated transcription patterns in large collections of human and yeast expression data and that the function of the uncharacterized genes can sometimes be predicted based on the expression patterns across these diverse organisms. We also use a comprehensive and unbiased literature mining method to predict which uncharacterized parasite-specific genes are likely to have roles in processes such as gliding motility, host-cell interactions, sporozoite stage, or rhoptry function. These analyses, together with protein-protein interaction data, provide probabilistic models that predict the function of 926 uncharacterized malaria genes and also suggest that malaria parasites may provide a simple model system for the study of some human processes. These data also provide a foundation for further studies of transcriptional regulation in malaria parasites.

ZUFSP Deubiquitylates K63-Linked Polyubiquitin Chains to Promote Genome Stability

DEFF Research Database (Denmark)

Haahr, Peter; Borgermann, Nikoline; Guo, Xiaohu

2018-01-01

Deubiquitylating enzymes (DUBs) enhance the dynamics of the versatile ubiquitin (Ub) code by reversing and regulating cellular ubiquitylation processes at multiple levels. Here we discovered that the uncharacterized human protein ZUFSP (zinc finger with UFM1-specific peptidase domain protein/C6orf......113/ZUP1), which has been annotated as a potentially inactive UFM1 protease, and its fission yeast homolog Mug105 define a previously unrecognized class of evolutionarily conserved cysteine protease DUBs. Human ZUFSP selectively interacts with and cleaves long K63-linked poly-Ub chains by means...... establish ZUFSP as a new type of linkage-selective cysteine peptidase DUB with a role in genome maintenance pathways....

SOLO: a meiotic protein required for centromere cohesion, coorientation, and SMC1 localization in Drosophila melanogaster.

Science.gov (United States)

Yan, Rihui; Thomas, Sharon E; Tsai, Jui-He; Yamada, Yukihiro; McKee, Bruce D

2010-02-08

Sister chromatid cohesion is essential to maintain stable connections between homologues and sister chromatids during meiosis and to establish correct centromere orientation patterns on the meiosis I and II spindles. However, the meiotic cohesion apparatus in Drosophila melanogaster remains largely uncharacterized. We describe a novel protein, sisters on the loose (SOLO), which is essential for meiotic cohesion in Drosophila. In solo mutants, sister centromeres separate before prometaphase I, disrupting meiosis I centromere orientation and causing nondisjunction of both homologous and sister chromatids. Centromeric foci of the cohesin protein SMC1 are absent in solo mutants at all meiotic stages. SOLO and SMC1 colocalize to meiotic centromeres from early prophase I until anaphase II in wild-type males, but both proteins disappear prematurely at anaphase I in mutants for mei-S332, which encodes the Drosophila homologue of the cohesin protector protein shugoshin. The solo mutant phenotypes and the localization patterns of SOLO and SMC1 indicate that they function together to maintain sister chromatid cohesion in Drosophila meiosis.

The human-bacterial pathogen protein interaction networks of Bacillus anthracis, Francisella tularensis, and Yersinia pestis.

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Matthew D Dyer

2010-08-01

Full Text Available Bacillus anthracis, Francisella tularensis, and Yersinia pestis are bacterial pathogens that can cause anthrax, lethal acute pneumonic disease, and bubonic plague, respectively, and are listed as NIAID Category A priority pathogens for possible use as biological weapons. However, the interactions between human proteins and proteins in these bacteria remain poorly characterized leading to an incomplete understanding of their pathogenesis and mechanisms of immune evasion.In this study, we used a high-throughput yeast two-hybrid assay to identify physical interactions between human proteins and proteins from each of these three pathogens. From more than 250,000 screens performed, we identified 3,073 human-B. anthracis, 1,383 human-F. tularensis, and 4,059 human-Y. pestis protein-protein interactions including interactions involving 304 B. anthracis, 52 F. tularensis, and 330 Y. pestis proteins that are uncharacterized. Computational analysis revealed that pathogen proteins preferentially interact with human proteins that are hubs and bottlenecks in the human PPI network. In addition, we computed modules of human-pathogen PPIs that are conserved amongst the three networks. Functionally, such conserved modules reveal commonalities between how the different pathogens interact with crucial host pathways involved in inflammation and immunity.These data constitute the first extensive protein interaction networks constructed for bacterial pathogens and their human hosts. This study provides novel insights into host-pathogen interactions.

LIFEGUARD proteins support plant colonization by biotrophic powdery mildew fungi.

Science.gov (United States)

Weis, Corina; Hückelhoven, Ralph; Eichmann, Ruth

2013-09-01

Pathogenic microbes manipulate eukaryotic cells during invasion and target plant proteins to achieve host susceptibility. BAX INHIBITOR-1 (BI-1) is an endoplasmic reticulum-resident cell death suppressor in plants and animals and is required for full susceptibility of barley to the barley powdery mildew fungus Blumeria graminis f.sp. hordei. LIFEGUARD (LFG) proteins resemble BI-1 proteins in terms of predicted membrane topology and cell-death-inhibiting function in metazoans, but display clear sequence-specific distinctions. This work shows that barley (Hordeum vulgare L.) and Arabidopsis thaliana genomes harbour five LFG genes, HvLFGa-HvLFGe and AtLFG1-AtLFG5, whose functions are largely uncharacterized. As observed for HvBI-1, single-cell overexpression of HvLFGa supports penetration success of B. graminis f.sp. hordei into barley epidermal cells, while transient-induced gene silencing restricts it. In penetrated barley epidermal cells, a green fluorescent protein-tagged HvLFGa protein accumulates at the site of fungal entry, around fungal haustoria and in endosomal or vacuolar membranes. The data further suggest a role of LFG proteins in plant-powdery mildew interactions in both monocot and dicot plants, because stable overexpression or knockdown of AtLFG1 or AtLFG2 also support or delay development of the powdery mildew fungus Erysiphe cruciferarum on the respective Arabidopsis mutants. Together, this work has identified new modulators of plant-powdery mildew interactions, and the data further support functional similarities between BI-1 and LFG proteins beyond cell death regulation.

Structural and Function Prediction of Musa acuminata subsp. Malaccensis Protein

Directory of Open Access Journals (Sweden)

Anum Munir

2016-03-01

Full Text Available Hypothetical proteins (HPs are the proteins whose presence has been anticipated, yet in vivo function has not been built up. Illustrating the structural and functional privileged insights of these HPs might likewise prompt a superior comprehension of the protein-protein associations or networks in diverse types of life. Bananas (Musa acuminata spp., including sweet and cooking types, are giant perennial monocotyledonous herbs of the order Zingiberales, a sister grouped to the all-around considered Poales, which incorporate oats. Bananas are crucial for nourishment security in numerous tropical and subtropical nations and the most prominent organic product in industrialized nations. In the present study, the hypothetical protein of M. acuminata (Banana was chosen for analysis and modeling by distinctive bioinformatics apparatuses and databases. As indicated by primary and secondary structure analysis, XP_009393594.1 is a stable hydrophobic protein containing a noteworthy extent of α-helices; Homology modeling was done utilizing SWISS-MODEL server where the templates identity with XP_009393594.1 protein was less which demonstrated novelty of our protein. Ab initio strategy was conducted to produce its 3D structure. A few evaluations of quality assessment and validation parameters determined the generated protein model as stable with genuinely great quality. Functional analysis was completed by ProtFun 2.2, and KEGG (KAAS, recommended that the hypothetical protein is a transcription factor with cytoplasmic domain as zinc finger. The protein was observed to be vital for translation process, involved in metabolism, signaling and cellular processes, genetic information processing and Zinc ion binding. It is suggested that further test approval would help to anticipate the structures and functions of other uncharacterized proteins of different plants and living being.

Cell-free protein synthesis for structure determination by X-ray crystallography.

Science.gov (United States)

Watanabe, Miki; Miyazono, Ken-ichi; Tanokura, Masaru; Sawasaki, Tatsuya; Endo, Yaeta; Kobayashi, Ichizo

2010-01-01

Structure determination has been difficult for those proteins that are toxic to the cells and cannot be prepared in a large amount in vivo. These proteins, even when biologically very interesting, tend to be left uncharacterized in the structural genomics projects. Their cell-free synthesis can bypass the toxicity problem. Among the various cell-free systems, the wheat-germ-based system is of special interest due to the following points: (1) Because the gene is placed under a plant translational signal, its toxic expression in a bacterial host is reduced. (2) It has only little codon preference and, especially, little discrimination between methionine and selenomethionine (SeMet), which allows easy preparation of selenomethionylated proteins for crystal structure determination by SAD and MAD methods. (3) Translation is uncoupled from transcription, so that the toxicity of the translation product on DNA and its transcription, if any, can be bypassed. We have shown that the wheat-germ-based cell-free protein synthesis is useful for X-ray crystallography of one of the 4-bp cutter restriction enzymes, which are expected to be very toxic to all forms of cells retaining the genome. Our report on its structure represents the first report of structure determination by X-ray crystallography using protein overexpressed with the wheat-germ-based cell-free protein expression system. This will be a method of choice for cytotoxic proteins when its cost is not a problem. Its use will become popular when the crystal structure determination technology has evolved to require only a tiny amount of protein.

Identification of a novel Leucine-rich repeat protein and candidate PP1 regulatory subunit expressed in developing spermatids

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Sperry Ann O

2008-01-01

Full Text Available Abstract Background Spermatogenesis is comprised of a series of highly regulated developmental changes that transform the precursor germ cell into a highly specialized spermatozoon. The last phase of spermatogenesis, termed spermiogenesis, involves dramatic morphological change including formation of the acrosome, elongation and condensation of the nucleus, formation of the flagella, and disposal of unnecessary cytoplasm. A prominent cytoskeletal component of the developing spermatid is the manchette, a unique microtubular structure that surrounds the nucleus of the developing spermatid and is thought to assist in both the reshaping of the nucleus and redistribution of spermatid cytoplasm. Although the molecular motor KIFC1 has been shown to associate with the manchette, its precise role in function of the manchette and the identity of its testis specific protein partners are unknown. The purpose of this study was to identify proteins in the testis that interact with KIFC1 using a yeast 2 hybrid screen of a testis cDNA library. Results Thirty percent of the interacting clones identified in our screen contain an identical cDNA encoding a 40 kD protein. This interacting protein has 4 leucine-rich repeats in its amino terminal half and is expressed primarily in the testis; therefore we have named this protein testis leucine-rich repeat protein or TLRR. TLRR was also found to associate tightly with the KIFC1 targeting domain using affinity chromatography. In addition to the leucine-rich repeats, TLRR contains a consensus-binding site for protein phosphatase-1 (PP1. Immunocytochemistry using a TLRR specific antibody demonstrates that this protein is found near the manchette of developing spermatids. Conclusion We have identified a previously uncharacterized leucine-rich repeat protein that is expressed abundantly in the testis and associates with the manchette of developing spermatids, possibly through its interaction with the KIFC1 molecular motor

Red fluorescent protein responsible for pigmentation in trematode-infected Porites compressa tissues.

Science.gov (United States)

Palmer, Caroline V; Roth, Melissa S; Gates, Ruth D

2009-02-01

Reports of coral disease have increased dramatically over the last decade; however, the biological mechanisms that corals utilize to limit infection and resist disease remain poorly understood. Compromised coral tissues often display non-normal pigmentation that potentially represents an inflammation-like response, although these pigments remain uncharacterized. Using spectral emission analysis and cryo-histological and electrophoretic techniques, we investigated the pink pigmentation associated with trematodiasis, infection with Podocotyloides stenometre larval trematode, in Porites compressa. Spectral emission analysis reveals that macroscopic areas of pink pigmentation fluoresce under blue light excitation (450 nm) and produce a broad emission peak at 590 nm (+/-6) with a 60-nm full width at half maximum. Electrophoretic protein separation of pigmented tissue extract confirms the red fluorescence to be a protein rather than a low-molecular-weight compound. Histological sections demonstrate green fluorescence in healthy coral tissue and red fluorescence in the trematodiasis-compromised tissue. The red fluorescent protein (FP) is limited to the epidermis, is not associated with cells or granules, and appears unstructured. These data collectively suggest that the red FP is produced and localized in tissue infected by larval trematodes and plays a role in the immune response in corals.

Shotgun proteomics of Aspergillus niger microsomes upon D-xylose induction

NARCIS (Netherlands)

Ferreira de Oliveira, J.M.P.; Passel, van M.W.J.; Schaap, P.J.; Graaff, de L.H.

2010-01-01

Protein secretion plays an eminent role in cell maintenance and adaptation to the extracellular environment of microorganisms. Although protein secretion is an extremely efficient process in filamentous fungi, the mechanisms underlying protein secretion have remained largely uncharacterized in these

Bacterial motility complexes require the actin-like protein, MreB and the Ras homologue, MglA.

Science.gov (United States)

Mauriello, Emilia M F; Mouhamar, Fabrice; Nan, Beiyan; Ducret, Adrien; Dai, David; Zusman, David R; Mignot, Tâm

2010-01-20

Gliding motility in the bacterium Myxococcus xanthus uses two motility engines: S-motility powered by type-IV pili and A-motility powered by uncharacterized motor proteins and focal adhesion complexes. In this paper, we identified MreB, an actin-like protein, and MglA, a small GTPase of the Ras superfamily, as essential for both motility systems. A22, an inhibitor of MreB cytoskeleton assembly, reversibly inhibited S- and A-motility, causing rapid dispersal of S- and A-motility protein clusters, FrzS and AglZ. This suggests that the MreB cytoskeleton is involved in directing the positioning of these proteins. We also found that a DeltamglA motility mutant showed defective localization of AglZ and FrzS clusters. Interestingly, MglA-YFP localization mimicked both FrzS and AglZ patterns and was perturbed by A22 treatment, consistent with results indicating that both MglA and MreB bind to motility complexes. We propose that MglA and the MreB cytoskeleton act together in a pathway to localize motility proteins such as AglZ and FrzS to assemble the A-motility machineries. Interestingly, M. xanthus motility systems, like eukaryotic systems, use an actin-like protein and a small GTPase spatial regulator.

Predicting highly-connected hubs in protein interaction networks by QSAR and biological data descriptors

Science.gov (United States)

Hsing, Michael; Byler, Kendall; Cherkasov, Artem

2009-01-01

Hub proteins (those engaged in most physical interactions in a protein interaction network (PIN) have recently gained much research interest due to their essential role in mediating cellular processes and their potential therapeutic value. It is straightforward to identify hubs if the underlying PIN is experimentally determined; however, theoretical hub prediction remains a very challenging task, as physicochemical properties that differentiate hubs from less connected proteins remain mostly uncharacterized. To adequately distinguish hubs from non-hub proteins we have utilized over 1300 protein descriptors, some of which represent QSAR (quantitative structure-activity relationship) parameters, and some reflect sequence-derived characteristics of proteins including domain composition and functional annotations. Those protein descriptors, together with available protein interaction data have been processed by a machine learning method (boosting trees) and resulted in the development of hub classifiers that are capable of predicting highly interacting proteins for four model organisms: Escherichia coli, Saccharomyces cerevisiae, Drosophila melanogaster and Homo sapiens. More importantly, through the analyses of the most relevant protein descriptors, we are able to demonstrate that hub proteins not only share certain common physicochemical and structural characteristics that make them different from non-hub counterparts, but they also exhibit species-specific characteristics that should be taken into account when analyzing different PINs. The developed prediction models can be used for determining highly interacting proteins in the four studied species to assist future proteomics experiments and PIN analyses. Availability The source code and executable program of the hub classifier are available for download at: http://www.cnbi2.ca/hub-analysis/ PMID:20198194

A Member of the p38 Mitogen-Activated Protein Kinase Family Is Responsible for Transcriptional Induction of Dopa decarboxylase in the Epidermis of Drosophila melanogaster during the Innate Immune Response▿ †

Science.gov (United States)

Davis, Monica M.; Primrose, David A.; Hodgetts, Ross B.

2008-01-01

Drosophila innate immunity is controlled primarily by the activation of IMD (immune deficiency) or Toll signaling leading to the production of antimicrobial peptides (AMPs). IMD signaling also activates the JUN N-terminal kinase (JNK) cascade, which is responsible for immune induction of non-antimicrobial peptide immune gene transcription though the transcription factor AP-1. Transcription of the Dopa decarboxylase (Ddc) gene is induced in response to gram-negative and gram-positive septic injury, but not aseptic wounding. Transcription is induced throughout the epidermis and not specifically at the site of infection. Ddc transcripts are detectible within 2 h and remain high for several hours following infection with either gram-negative or gram-positive bacteria. Using Ddc-green fluorescent protein (GFP) reporter gene constructs, we show that a conserved consensus AP-1 binding site upstream of the Ddc transcription start site is required for induction. However, neither the Toll, IMD, nor JNK pathway is involved. Rather, Ddc transcription depends on a previously uncharacterized member of the p38 mitogen-activated protein kinase family, p38c. We propose that the involvement of DDC in a new pathway involved in Drosophila immunity increases the levels of dopamine, which is metabolized to produce reactive quinones that exert an antimicrobial effect on invading bacteria. PMID:18519585

ProClusEnsem: Predicting membrane protein types by fusing different modes of pseudo amino acid composition

KAUST Repository

Wang, Jim Jing-Yan; Li, Yongping; Wang, Quanquan; You, Xinge; Man, Jiaju; Wang, Chao; Gao, Xin

2012-01-01

Knowing the type of an uncharacterized membrane protein often provides a useful clue in both basic research and drug discovery. With the explosion of protein sequences generated in the post genomic era, determination of membrane protein types by experimental methods is expensive and time consuming. It therefore becomes important to develop an automated method to find the possible types of membrane proteins. In view of this, various computational membrane protein prediction methods have been proposed. They extract protein feature vectors, such as PseAAC (pseudo amino acid composition) and PsePSSM (pseudo position-specific scoring matrix) for representation of protein sequence, and then learn a distance metric for the KNN (K nearest neighbor) or NN (nearest neighbor) classifier to predicate the final type. Most of the metrics are learned using linear dimensionality reduction algorithms like Principle Components Analysis (PCA) and Linear Discriminant Analysis (LDA). Such metrics are common to all the proteins in the dataset. In fact, they assume that the proteins lie on a uniform distribution, which can be captured by the linear dimensionality reduction algorithm. We doubt this assumption, and learn local metrics which are optimized for local subset of the whole proteins. The learning procedure is iterated with the protein clustering. Then a novel ensemble distance metric is given by combining the local metrics through Tikhonov regularization. The experimental results on a benchmark dataset demonstrate the feasibility and effectiveness of the proposed algorithm named ProClusEnsem. © 2012 Elsevier Ltd.

ProClusEnsem: Predicting membrane protein types by fusing different modes of pseudo amino acid composition

KAUST Repository

Wang, Jim Jing-Yan

2012-05-01

Knowing the type of an uncharacterized membrane protein often provides a useful clue in both basic research and drug discovery. With the explosion of protein sequences generated in the post genomic era, determination of membrane protein types by experimental methods is expensive and time consuming. It therefore becomes important to develop an automated method to find the possible types of membrane proteins. In view of this, various computational membrane protein prediction methods have been proposed. They extract protein feature vectors, such as PseAAC (pseudo amino acid composition) and PsePSSM (pseudo position-specific scoring matrix) for representation of protein sequence, and then learn a distance metric for the KNN (K nearest neighbor) or NN (nearest neighbor) classifier to predicate the final type. Most of the metrics are learned using linear dimensionality reduction algorithms like Principle Components Analysis (PCA) and Linear Discriminant Analysis (LDA). Such metrics are common to all the proteins in the dataset. In fact, they assume that the proteins lie on a uniform distribution, which can be captured by the linear dimensionality reduction algorithm. We doubt this assumption, and learn local metrics which are optimized for local subset of the whole proteins. The learning procedure is iterated with the protein clustering. Then a novel ensemble distance metric is given by combining the local metrics through Tikhonov regularization. The experimental results on a benchmark dataset demonstrate the feasibility and effectiveness of the proposed algorithm named ProClusEnsem. © 2012 Elsevier Ltd.

Quantitative and functional characterization of the hyper-conserved protein of Prochlorococcus and marine Synechococcus.

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Caroline E Whidden

Full Text Available A large fraction of any bacterial genome consists of hypothetical protein-coding open reading frames (ORFs. While most of these ORFs are present only in one or a few sequenced genomes, a few are conserved, often across large phylogenetic distances. Such conservation provides clues to likely uncharacterized cellular functions that need to be elucidated. Marine cyanobacteria from the Prochlorococcus/marine Synechococcus clade are dominant bacteria in oceanic waters and are significant contributors to global primary production. A Hyper Conserved Protein (PSHCP of unknown function is 100% conserved at the amino acid level in genomes of Prochlorococcus/marine Synechococcus, but lacks homologs outside of this clade. In this study we investigated Prochlorococcus marinus strains MED4 and MIT 9313 and Synechococcus sp. strain WH 8102 for the transcription of the PSHCP gene using RT-Q-PCR, for the presence of the protein product through quantitative immunoblotting, and for the protein's binding partners in a pull down assay. Significant transcription of the gene was detected in all strains. The PSHCP protein content varied between 8±1 fmol and 26±9 fmol per ug total protein, depending on the strain. The 50 S ribosomal protein L2, the Photosystem I protein PsaD and the Ycf48-like protein were found associated with the PSHCP protein in all strains and not appreciably or at all in control experiments. We hypothesize that PSHCP is a protein associated with the ribosome, and is possibly involved in photosystem assembly.

NHS-Esters As Versatile Reactivity-Based Probes for Mapping Proteome-Wide Ligandable Hotspots.

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Ward, Carl C; Kleinman, Jordan I; Nomura, Daniel K

2017-06-16

Most of the proteome is considered undruggable, oftentimes hindering translational efforts for drug discovery. Identifying previously unknown druggable hotspots in proteins would enable strategies for pharmacologically interrogating these sites with small molecules. Activity-based protein profiling (ABPP) has arisen as a powerful chemoproteomic strategy that uses reactivity-based chemical probes to map reactive, functional, and ligandable hotspots in complex proteomes, which has enabled inhibitor discovery against various therapeutic protein targets. Here, we report an alkyne-functionalized N-hydroxysuccinimide-ester (NHS-ester) as a versatile reactivity-based probe for mapping the reactivity of a wide range of nucleophilic ligandable hotspots, including lysines, serines, threonines, and tyrosines, encompassing active sites, allosteric sites, post-translational modification sites, protein interaction sites, and previously uncharacterized potential binding sites. Surprisingly, we also show that fragment-based NHS-ester ligands can be made to confer selectivity for specific lysine hotspots on specific targets including Dpyd, Aldh2, and Gstt1. We thus put forth NHS-esters as promising reactivity-based probes and chemical scaffolds for covalent ligand discovery.

Expanded microbial genome coverage and improved protein family annotation in the COG database.

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Galperin, Michael Y; Makarova, Kira S; Wolf, Yuri I; Koonin, Eugene V

2015-01-01

Microbial genome sequencing projects produce numerous sequences of deduced proteins, only a small fraction of which have been or will ever be studied experimentally. This leaves sequence analysis as the only feasible way to annotate these proteins and assign to them tentative functions. The Clusters of Orthologous Groups of proteins (COGs) database (http://www.ncbi.nlm.nih.gov/COG/), first created in 1997, has been a popular tool for functional annotation. Its success was largely based on (i) its reliance on complete microbial genomes, which allowed reliable assignment of orthologs and paralogs for most genes; (ii) orthology-based approach, which used the function(s) of the characterized member(s) of the protein family (COG) to assign function(s) to the entire set of carefully identified orthologs and describe the range of potential functions when there were more than one; and (iii) careful manual curation of the annotation of the COGs, aimed at detailed prediction of the biological function(s) for each COG while avoiding annotation errors and overprediction. Here we present an update of the COGs, the first since 2003, and a comprehensive revision of the COG annotations and expansion of the genome coverage to include representative complete genomes from all bacterial and archaeal lineages down to the genus level. This re-analysis of the COGs shows that the original COG assignments had an error rate below 0.5% and allows an assessment of the progress in functional genomics in the past 12 years. During this time, functions of many previously uncharacterized COGs have been elucidated and tentative functional assignments of many COGs have been validated, either by targeted experiments or through the use of high-throughput methods. A particularly important development is the assignment of functions to several widespread, conserved proteins many of which turned out to participate in translation, in particular rRNA maturation and tRNA modification. The new version of the

Purification of recombinant Chlamydia trachomatis histone H1-like protein Hc2, and comparative functional analysis of Hc2 and Hc1

DEFF Research Database (Denmark)

Pedersen, LB; Birkelund, Svend; Christiansen, Gunna

1996-01-01

The metabolically inactive developmental form of Chlamydia trachomatis, the elementary body, contains two very basic DNA-binding proteins with homology to eukaryotic histone H1. One of these, Hc1, is relatively well characterized and induces DNA condensation in vitro, whereas the other, Hc2......, is functionally virtually uncharacterized. In this study we describe the purification of Hc2, and a detailed comparative functional analysis of Hc2 and Hc1 is presented. By gel shift assays and electron microscopy, marked differences in the nucleic acid-binding properties of Hc2 and Hc1 were observed. Furthermore...

FmvB: A Francisella tularensis Magnesium-Responsive Outer Membrane Protein that Plays a Role in Virulence.

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Xiaojun Wu

Full Text Available Francisella tularensis is the causative agent of the lethal disease tularemia. Despite decades of research, little is understood about why F. tularensis is so virulent. Bacterial outer membrane proteins (OMPs are involved in various virulence processes, including protein secretion, host cell attachment, and intracellular survival. Many pathogenic bacteria require metals for intracellular survival and OMPs often play important roles in metal uptake. Previous studies identified three F. tularensis OMPs that play roles in iron acquisition. In this study, we examined two previously uncharacterized proteins, FTT0267 (named fmvA, for Francisella metal and virulence and FTT0602c (fmvB, which are homologs of the previously studied F. tularensis iron acquisition genes and are predicted OMPs. To study the potential roles of FmvA and FmvB in metal acquisition and virulence, we first examined fmvA and fmvB expression following pulmonary infection of mice, finding that fmvB was upregulated up to 5-fold during F. tularensis infection of mice. Despite sequence homology to previously-characterized iron-acquisition genes, FmvA and FmvB do not appear to be involved iron uptake, as neither fmvA nor fmvB were upregulated in iron-limiting media and neither ΔfmvA nor ΔfmvB exhibited growth defects in iron limitation. However, when other metals were examined in this study, magnesium-limitation significantly induced fmvB expression, ΔfmvB was found to express significantly higher levels of lipopolysaccharide (LPS in magnesium-limiting medium, and increased numbers of surface protrusions were observed on ΔfmvB in magnesium-limiting medium, compared to wild-type F. tularensis grown in magnesium-limiting medium. RNA sequencing analysis of ΔfmvB revealed the potential mechanism for increased LPS expression, as LPS synthesis genes kdtA and wbtA were significantly upregulated in ΔfmvB, compared with wild-type F. tularensis. To provide further evidence for the potential

Mua (HP0868) Is a Nickel-Binding Protein That Modulates Urease Activity in Helicobacter pylori

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Benoit, Stéphane L.; Maier, Robert J.

2011-01-01

A novel mechanism aimed at controlling urease expression in Helicobacter pylori in the presence of ample nickel is described. Higher urease activities were observed in an hp0868 mutant (than in the wild type) in cells supplemented with nickel, suggesting that the HP0868 protein (herein named Mua for modulator of urease activity) represses urease activity when nickel concentrations are ample. The increase in urease activity in the Δmua mutant was linked to an increase in urease transcription and synthesis, as shown by quantitative real-time PCR, SDS-PAGE, and immunoblotting against UreAB. Increased urease synthesis was also detected in a Δmua ΔnikR double mutant strain. The Δmua mutant was more sensitive to nickel toxicity but more resistant to acid challenge than was the wild-type strain. Pure Mua protein binds 2 moles of Ni2+ per mole of dimer. Electrophoretic mobility shift assays did not reveal any binding of Mua to the ureA promoter or other selected promoters (nikR, arsRS, 5′ ureB-sRNAp). Previous yeast two-hybrid studies indicated that Mua and RpoD may interact; however, only a weak interaction was detected via cross-linking with pure components and this could not be verified by another approach. There was no significant difference in the intracellular nickel level between wild-type and mua mutant cells. Taken together, our results suggest the HP0868 gene product represses urease transcription when nickel levels are high through an as-yet-uncharacterized mechanism, thus counterbalancing the well-described NikR-mediated activation. PMID:21505055

Analysis of triglyceride synthesis unveils a green algal soluble diacylglycerol acyltransferase and provides clues to potential enzymatic components of the chloroplast pathway.

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Bagnato, Carolina; Prados, María B; Franchini, Gisela R; Scaglia, Natalia; Miranda, Silvia E; Beligni, María V

2017-03-09

Microalgal triglyceride (TAG) synthesis has attracted considerable attention. Particular emphasis has been put towards characterizing the algal homologs of the canonical rate-limiting enzymes, diacylglycerol acyltransferase (DGAT) and phospholipid:diacylglycerol acyltransferase (PDAT). Less work has been done to analyze homologs from a phylogenetic perspective. In this work, we used HMMER iterative profiling and phylogenetic and functional analyses to determine the number and sequence characteristics of algal DGAT and PDAT, as well as related sequences that constitute their corresponding superfamilies. We included most algae with available genomes, as well as representative eukaryotic and prokaryotic species. Amongst our main findings, we identified a novel clade of DGAT1-like proteins exclusive to red algae and glaucophyta and a previously uncharacterized subclade of DGAT2 proteins with an unusual number of transmembrane segments. Our analysis also revealed the existence of a novel DGAT exclusive to green algae with moderate similarity to plant soluble DGAT3. The DGAT3 clade shares a most recent ancestor with a group of uncharacterized proteins from cyanobacteria. Subcellular targeting prediction suggests that most green algal DGAT3 proteins are imported to the chloroplast, evidencing that the green algal chloroplast might have a soluble pathway for the de novo synthesis of TAGs. Heterologous expression of C. reinhardtii DGAT3 produces an increase in the accumulation of TAG, as evidenced by thin layer chromatography. Our analysis contributes to advance in the knowledge of complex superfamilies involved in lipid metabolism and provides clues to possible enzymatic players of chloroplast TAG synthesis.

Improving protein function prediction methods with integrated literature data

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Gabow Aaron P

2008-04-01

Full Text Available Abstract Background Determining the function of uncharacterized proteins is a major challenge in the post-genomic era due to the problem's complexity and scale. Identifying a protein's function contributes to an understanding of its role in the involved pathways, its suitability as a drug target, and its potential for protein modifications. Several graph-theoretic approaches predict unidentified functions of proteins by using the functional annotations of better-characterized proteins in protein-protein interaction networks. We systematically consider the use of literature co-occurrence data, introduce a new method for quantifying the reliability of co-occurrence and test how performance differs across species. We also quantify changes in performance as the prediction algorithms annotate with increased specificity. Results We find that including information on the co-occurrence of proteins within an abstract greatly boosts performance in the Functional Flow graph-theoretic function prediction algorithm in yeast, fly and worm. This increase in performance is not simply due to the presence of additional edges since supplementing protein-protein interactions with co-occurrence data outperforms supplementing with a comparably-sized genetic interaction dataset. Through the combination of protein-protein interactions and co-occurrence data, the neighborhood around unknown proteins is quickly connected to well-characterized nodes which global prediction algorithms can exploit. Our method for quantifying co-occurrence reliability shows superior performance to the other methods, particularly at threshold values around 10% which yield the best trade off between coverage and accuracy. In contrast, the traditional way of asserting co-occurrence when at least one abstract mentions both proteins proves to be the worst method for generating co-occurrence data, introducing too many false positives. Annotating the functions with greater specificity is harder

The fungus Ustilago maydis and humans share disease-related proteins that are not found in Saccharomyces cerevisiae

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Steinberg Gero

2007-12-01

Full Text Available Abstract Background The corn smut fungus Ustilago maydis is a well-established model system for molecular phytopathology. In addition, it recently became evident that U. maydis and humans share proteins and cellular processes that are not found in the standard fungal model Saccharomyces cerevisiae. This prompted us to do a comparative analysis of the predicted proteome of U. maydis, S. cerevisiae and humans. Results At a cut off at 20% identity over protein length, all three organisms share 1738 proteins, whereas both fungi share only 541 conserved proteins. Despite the evolutionary distance between U. maydis and humans, 777 proteins were shared. When applying a more stringent criterion (≥ 20% identity with a homologue in one organism over at least 50 amino acids and ≥ 10% less in the other organism, we found 681 proteins for the comparison of U. maydis and humans, whereas the both fungi share only 622 fungal specific proteins. Finally, we found that S. cerevisiae and humans shared 312 proteins. In the U. maydis to H. sapiens homology set 454 proteins are functionally classified and 42 proteins are related to serious human diseases. However, a large portion of 222 proteins are of unknown function. Conclusion The fungus U. maydis has a long history of being a model system for understanding DNA recombination and repair, as well as molecular plant pathology. The identification of functionally un-characterized genes that are conserved in humans and U. maydis opens the door for experimental work, which promises new insight in the cell biology of the mammalian cell.

Regulator of G protein signaling 5 (RGS5) inhibits sonic hedgehog function in mouse cortical neurons.

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Liu, Chuanliang; Hu, Qiongqiong; Jing, Jia; Zhang, Yun; Jin, Jing; Zhang, Liulei; Mu, Lili; Liu, Yumei; Sun, Bo; Zhang, Tongshuai; Kong, Qingfei; Wang, Guangyou; Wang, Dandan; Zhang, Yao; Liu, Xijun; Zhao, Wei; Wang, Jinghua; Feng, Tao; Li, Hulun

2017-09-01

Regulator of G protein signaling 5 (RGS5) acts as a GTPase-activating protein (GAP) for the Gαi subunit and negatively regulates G protein-coupled receptor signaling. However, its presence and function in postmitotic differentiated primary neurons remains largely uncharacterized. During neural development, sonic hedgehog (Shh) signaling is involved in cell signaling pathways via Gαi activity. In particular, Shh signaling is essential for embryonic neural tube patterning, which has been implicated in neuronal polarization involving neurite outgrowth. Here, we examined whether RGS5 regulates Shh signaling in neurons. RGS5 transcripts were found to be expressed in cortical neurons and their expression gradually declined in a time-dependent manner in culture system. When an adenovirus expressing RGS5 was introduced into an in vitro cell culture model of cortical neurons, RGS5 overexpression significantly reduced neurite outgrowth and FM4-64 uptake, while cAMP-PKA signaling was also affected. These findings suggest that RGS5 inhibits Shh function during neurite outgrowth and the presynaptic terminals of primary cortical neurons mature via modulation of cAMP. Copyright © 2017 Elsevier Inc. All rights reserved.

ErpC, a member of the complement regulator-acquiring family of surface proteins from Borrelia burgdorferi, possesses an architecture previously unseen in this protein family

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Caesar, Joseph J. E.; Johnson, Steven; Kraiczy, Peter; Lea, Susan M.

2013-01-01

The structure of ErpC, a member of the complement regulator-acquiring surface protein family from B. burgdorferi, has been solved, providing insights into the strategies of complement evasion by this zoonotic bacterium and suggesting a common architecture for other members of this protein family. Borrelia burgdorferi is a spirochete responsible for Lyme disease, the most commonly occurring vector-borne disease in Europe and North America. The bacterium utilizes a set of proteins, termed complement regulator-acquiring surface proteins (CRASPs), to aid evasion of the human complement system by recruiting and presenting complement regulator factor H on its surface in a manner that mimics host cells. Presented here is the atomic resolution structure of a member of this protein family, ErpC. The structure provides new insights into the mechanism of recruitment of factor H and other factor H-related proteins by acting as a molecular mimic of host glycosaminoglycans. It also describes the architecture of other CRASP proteins belonging to the OspE/F-related paralogous protein family and suggests that they have evolved to bind specific complement proteins, aiding survival of the bacterium in different hosts

Dissecting protein loops with a statistical scalpel suggests a functional implication of some structural motifs.

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Regad, Leslie; Martin, Juliette; Camproux, Anne-Claude

2011-06-20

One of the strategies for protein function annotation is to search particular structural motifs that are known to be shared by proteins with a given function. Here, we present a systematic extraction of structural motifs of seven residues from protein loops and we explore their correspondence with functional sites. Our approach is based on the structural alphabet HMM-SA (Hidden Markov Model - Structural Alphabet), which allows simplification of protein structures into uni-dimensional sequences, and advanced pattern statistics adapted to short sequences. Structural motifs of interest are selected by looking for structural motifs significantly over-represented in SCOP superfamilies in protein loops. We discovered two types of structural motifs significantly over-represented in SCOP superfamilies: (i) ubiquitous motifs, shared by several superfamilies and (ii) superfamily-specific motifs, over-represented in few superfamilies. A comparison of ubiquitous words with known small structural motifs shows that they contain well-described motifs as turn, niche or nest motifs. A comparison between superfamily-specific motifs and biological annotations of Swiss-Prot reveals that some of them actually correspond to functional sites involved in the binding sites of small ligands, such as ATP/GTP, NAD(P) and SAH/SAM. Our findings show that statistical over-representation in SCOP superfamilies is linked to functional features. The detection of over-represented motifs within structures simplified by HMM-SA is therefore a promising approach for prediction of functional sites and annotation of uncharacterized proteins.

Dissecting protein loops with a statistical scalpel suggests a functional implication of some structural motifs

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Martin Juliette

2011-06-01

Full Text Available Abstract Background One of the strategies for protein function annotation is to search particular structural motifs that are known to be shared by proteins with a given function. Results Here, we present a systematic extraction of structural motifs of seven residues from protein loops and we explore their correspondence with functional sites. Our approach is based on the structural alphabet HMM-SA (Hidden Markov Model - Structural Alphabet, which allows simplification of protein structures into uni-dimensional sequences, and advanced pattern statistics adapted to short sequences. Structural motifs of interest are selected by looking for structural motifs significantly over-represented in SCOP superfamilies in protein loops. We discovered two types of structural motifs significantly over-represented in SCOP superfamilies: (i ubiquitous motifs, shared by several superfamilies and (ii superfamily-specific motifs, over-represented in few superfamilies. A comparison of ubiquitous words with known small structural motifs shows that they contain well-described motifs as turn, niche or nest motifs. A comparison between superfamily-specific motifs and biological annotations of Swiss-Prot reveals that some of them actually correspond to functional sites involved in the binding sites of small ligands, such as ATP/GTP, NAD(P and SAH/SAM. Conclusions Our findings show that statistical over-representation in SCOP superfamilies is linked to functional features. The detection of over-represented motifs within structures simplified by HMM-SA is therefore a promising approach for prediction of functional sites and annotation of uncharacterized proteins.

Genome-wide analysis of gene expression and protein secretion of Babesia canis during virulent infection identifies potential pathogenicity factors.

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Eichenberger, Ramon M; Ramakrishnan, Chandra; Russo, Giancarlo; Deplazes, Peter; Hehl, Adrian B

2017-06-13

Infections of dogs with virulent strains of Babesia canis are characterized by rapid onset and high mortality, comparable to complicated human malaria. As in other apicomplexan parasites, most Babesia virulence factors responsible for survival and pathogenicity are secreted to the host cell surface and beyond where they remodel and biochemically modify the infected cell interacting with host proteins in a very specific manner. Here, we investigated factors secreted by B. canis during acute infections in dogs and report on in silico predictions and experimental analysis of the parasite's exportome. As a backdrop, we generated a fully annotated B. canis genome sequence of a virulent Hungarian field isolate (strain BcH-CHIPZ) underpinned by extensive genome-wide RNA-seq analysis. We find evidence for conserved factors in apicomplexan hemoparasites involved in immune-evasion (e.g. VESA-protein family), proteins secreted across the iRBC membrane into the host bloodstream (e.g. SA- and Bc28 protein families), potential moonlighting proteins (e.g. profilin and histones), and uncharacterized antigens present during acute crisis in dogs. The combined data provides a first predicted and partially validated set of potential virulence factors exported during fatal infections, which can be exploited for urgently needed innovative intervention strategies aimed at facilitating diagnosis and management of canine babesiosis.

Computational mining for hypothetical patterns of amino acid side chains in protein data bank (PDB)

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Ghani, Nur Syatila Ab; Firdaus-Raih, Mohd

2018-04-01

The three-dimensional structure of a protein can provide insights regarding its function. Functional relationship between proteins can be inferred from fold and sequence similarities. In certain cases, sequence or fold comparison fails to conclude homology between proteins with similar mechanism. Since the structure is more conserved than the sequence, a constellation of functional residues can be similarly arranged among proteins of similar mechanism. Local structural similarity searches are able to detect such constellation of amino acids among distinct proteins, which can be useful to annotate proteins of unknown function. Detection of such patterns of amino acids on a large scale can increase the repertoire of important 3D motifs since available known 3D motifs currently, could not compensate the ever-increasing numbers of uncharacterized proteins to be annotated. Here, a computational platform for an automated detection of 3D motifs is described. A fuzzy-pattern searching algorithm derived from IMagine an Amino Acid 3D Arrangement search EnGINE (IMAAAGINE) was implemented to develop an automated method for searching of hypothetical patterns of amino acid side chains in Protein Data Bank (PDB), without the need for prior knowledge on related sequence or structure of pattern of interest. We present an example of the searches, which is the detection of a hypothetical pattern derived from known structural motif of C2H2 structural pattern from zinc fingers. The conservation of particular patterns of amino acid side chains in unrelated proteins is highlighted. This approach can act as a complementary method for available structure- and sequence-based platforms and may contribute in improving functional association between proteins.

Exoproteome Analysis of the Seaweed Pathogen Nautella italica R11 Reveals Temperature-Dependent Regulation of RTX-Like Proteins

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Melissa Gardiner

2017-06-01

Full Text Available Climate fluctuations have been linked to an increased prevalence of disease in seaweeds, including the red alga Delisea pulchra, which is susceptible to a bleaching disease caused by the bacterium Nautella italica R11 under elevated seawater temperatures. To further investigate the role of temperature in the induction of disease by N. italica R11, we assessed the effect of temperature on the expression of the extracellular proteome (exoproteome in this bacterium. Label-free quantitative mass spectrometry was used to identify 207 proteins secreted into supernatant fraction, which is equivalent to 5% of the protein coding genes in the N. italica R11 genome. Comparative analysis demonstrated that expression of over 30% of the N. italica R11 exoproteome is affected by temperature. The temperature-dependent proteins include traits that could facilitate the ATP-dependent transport of amino acid and carbohydrate, as well as several uncharacterized proteins. Further, potential virulence determinants, including two RTX-like proteins, exhibited significantly higher expression in the exoproteome at the disease inducing temperature of 24°C relative to non-inducing temperature (16°C. This is the first study to demonstrate that temperature has an influence exoproteome expression in a macroalgal pathogen. The results have revealed several temperature regulated candidate virulence factors that may have a role in macroalgal colonization and invasion at elevated sea-surface temperatures, including novel RTX-like proteins.

PVP-SVM: Sequence-Based Prediction of Phage Virion Proteins Using a Support Vector Machine.

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Manavalan, Balachandran; Shin, Tae H; Lee, Gwang

2018-01-01

Accurately identifying bacteriophage virion proteins from uncharacterized sequences is important to understand interactions between the phage and its host bacteria in order to develop new antibacterial drugs. However, identification of such proteins using experimental techniques is expensive and often time consuming; hence, development of an efficient computational algorithm for the prediction of phage virion proteins (PVPs) prior to in vitro experimentation is needed. Here, we describe a support vector machine (SVM)-based PVP predictor, called PVP-SVM, which was trained with 136 optimal features. A feature selection protocol was employed to identify the optimal features from a large set that included amino acid composition, dipeptide composition, atomic composition, physicochemical properties, and chain-transition-distribution. PVP-SVM achieved an accuracy of 0.870 during leave-one-out cross-validation, which was 6% higher than control SVM predictors trained with all features, indicating the efficiency of the feature selection method. Furthermore, PVP-SVM displayed superior performance compared to the currently available method, PVPred, and two other machine-learning methods developed in this study when objectively evaluated with an independent dataset. For the convenience of the scientific community, a user-friendly and publicly accessible web server has been established at www.thegleelab.org/PVP-SVM/PVP-SVM.html.

PVP-SVM: Sequence-Based Prediction of Phage Virion Proteins Using a Support Vector Machine

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Balachandran Manavalan

2018-03-01

Full Text Available Accurately identifying bacteriophage virion proteins from uncharacterized sequences is important to understand interactions between the phage and its host bacteria in order to develop new antibacterial drugs. However, identification of such proteins using experimental techniques is expensive and often time consuming; hence, development of an efficient computational algorithm for the prediction of phage virion proteins (PVPs prior to in vitro experimentation is needed. Here, we describe a support vector machine (SVM-based PVP predictor, called PVP-SVM, which was trained with 136 optimal features. A feature selection protocol was employed to identify the optimal features from a large set that included amino acid composition, dipeptide composition, atomic composition, physicochemical properties, and chain-transition-distribution. PVP-SVM achieved an accuracy of 0.870 during leave-one-out cross-validation, which was 6% higher than control SVM predictors trained with all features, indicating the efficiency of the feature selection method. Furthermore, PVP-SVM displayed superior performance compared to the currently available method, PVPred, and two other machine-learning methods developed in this study when objectively evaluated with an independent dataset. For the convenience of the scientific community, a user-friendly and publicly accessible web server has been established at www.thegleelab.org/PVP-SVM/PVP-SVM.html.

Identification of Protein-Protein Interactions with Glutathione-S-Transferase (GST) Fusion Proteins.

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Einarson, Margret B; Pugacheva, Elena N; Orlinick, Jason R

2007-08-01

INTRODUCTIONGlutathione-S-transferase (GST) fusion proteins have had a wide range of applications since their introduction as tools for synthesis of recombinant proteins in bacteria. GST was originally selected as a fusion moiety because of several desirable properties. First and foremost, when expressed in bacteria alone, or as a fusion, GST is not sequestered in inclusion bodies (in contrast to previous fusion protein systems). Second, GST can be affinity-purified without denaturation because it binds to immobilized glutathione, which provides the basis for simple purification. Consequently, GST fusion proteins are routinely used for antibody generation and purification, protein-protein interaction studies, and biochemical analysis. This article describes the use of GST fusion proteins as probes for the identification of protein-protein interactions.

Identification of a High Affinity Nucleocapsid Protein Binding Element from The Bovine Leukemia Virus Genome

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Yildiz, F. Zehra; Babalola, Kathleen; Summers, Michael F.

2012-01-01

Retroviral genome recognition is mediated by interactions between the nucleocapsid (NC) domain of the virally encoded Gag polyprotein and cognate RNA packaging elements that, for most retroviruses, appear to reside primarily within the 5′-untranslated region (5′-UTR) of the genome. Recent studies suggest that a major packaging determinant of Bovine Leukemia Virus (BLV), a member of the human T-cell leukemia virus (HTLV)/BLV family and a non-primate animal model for HTLV-induced leukemogenesis, resides within the gag open reading frame. We have prepared and purified the recombinant BLV NC protein and conducted electrophoretic mobility shift and isothermal titration calorimetry studies with RNA fragments corresponding to these proposed packaging elements. The gag-derived RNAs did not exhibit significant affinity for NC, suggesting an alternate role in packaging. However, an 83-nucleotide fragment of the 5′-UTR that resides just upstream of the gag start codon binds NC stoichiometrically and with high affinity (Kd = 136 ± 21 nM). These nucleotides were predicted to form tandem hairpin structures, and studies with smaller fragments indicate that the NC binding site resides exclusively within the distal hairpin (residues G369- U399, Kd = 67 ± 8 nM at physiological ionic strength). Unlike all other structurally characterized retroviral NC binding RNAs, this fragment is not expected to contain exposed guanosines, suggesting that RNA binding may be mediated by a previously uncharacterized mechanism. PMID:22846919

In silico pattern-based analysis of the human cytomegalovirus genome.

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Rigoutsos, Isidore; Novotny, Jiri; Huynh, Tien; Chin-Bow, Stephen T; Parida, Laxmi; Platt, Daniel; Coleman, David; Shenk, Thomas

2003-04-01

More than 200 open reading frames (ORFs) from the human cytomegalovirus genome have been reported as potentially coding for proteins. We have used two pattern-based in silico approaches to analyze this set of putative viral genes. With the help of an objective annotation method that is based on the Bio-Dictionary, a comprehensive collection of amino acid patterns that describes the currently known natural sequence space of proteins, we have reannotated all of the previously reported putative genes of the human cytomegalovirus. Also, with the help of MUSCA, a pattern-based multiple sequence alignment algorithm, we have reexamined the original human cytomegalovirus gene family definitions. Our analysis of the genome shows that many of the coded proteins comprise amino acid combinations that are unique to either the human cytomegalovirus or the larger group of herpesviruses. We have confirmed that a surprisingly large portion of the analyzed ORFs encode membrane proteins, and we have discovered a significant number of previously uncharacterized proteins that are predicted to be G-protein-coupled receptor homologues. The analysis also indicates that many of the encoded proteins undergo posttranslational modifications such as hydroxylation, phosphorylation, and glycosylation. ORFs encoding proteins with similar functional behavior appear in neighboring regions of the human cytomegalovirus genome. All of the results of the present study can be found and interactively explored online (http://cbcsrv.watson.ibm.com/virus/).

Expression of Haemophilus ducreyi collagen binding outer membrane protein NcaA is required for virulence in swine and human challenge models of chancroid.

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Fulcher, Robert A; Cole, Leah E; Janowicz, Diane M; Toffer, Kristen L; Fortney, Kate R; Katz, Barry P; Orndorff, Paul E; Spinola, Stanley M; Kawula, Thomas H

2006-05-01

Haemophilus ducreyi, the etiologic agent of the sexually transmitted genital ulcer disease chancroid, has been shown to associate with dermal collagen fibers within infected skin lesions. Here we describe NcaA, a previously uncharacterized outer membrane protein that is important for H. ducreyi collagen binding and host colonization. An H. ducreyi strain lacking the ncaA gene was impaired in adherence to type I collagen but not fibronectin (plasma or cellular form) or heparin. The mutation had no effect on serum resistance or binding to HaCaT keratinocytes or human foreskin fibroblasts in vitro. Escherichia coli expressing H. ducreyi NcaA bound to type I collagen, demonstrating that NcaA is sufficient to confer collagen attachment. The importance of NcaA in H. ducreyi pathogenesis was assessed using both swine and human experimental models of chancroid. In the swine model, 20% of lesions from sites inoculated with the ncaA mutant were culture positive for H. ducreyi 7 days after inoculation, compared to 73% of wild-type-inoculated sites. The average number of CFU recovered from mutant-inoculated lesions was also significantly reduced compared to that recovered from wild-type-inoculated sites at both 2 and 7 days after inoculation. In the human challenge model, 8 of 30 sites inoculated with wild-type H. ducreyi progressed to the pustular stage, compared to 0 of 30 sites inoculated with the ncaA mutant. Together these results demonstrate that the collagen binding protein NcaA is required for H. ducreyi infection.

Identification of the PDI-family member ERp90 as an interaction partner of ERFAD.

Directory of Open Access Journals (Sweden)

Jan Riemer

Full Text Available In the endoplasmic reticulum (ER, members of the protein disulfide isomerase (PDI family perform critical functions during protein maturation. Herein, we identify the previously uncharacterized PDI-family member ERp90. In cultured human cells, we find ERp90 to be a soluble ER-luminal glycoprotein that comprises five potential thioredoxin (Trx-like domains. Mature ERp90 contains 10 cysteine residues, of which at least some form intramolecular disulfides. While none of the Trx domains contain a canonical Cys-Xaa-Xaa-Cys active-site motif, other conserved cysteines could endow the protein with redox activity. Importantly, we show that ERp90 co-immunoprecipitates with ERFAD, a flavoprotein involved in ER-associated degradation (ERAD, through what is most likely a direct interaction. We propose that the function of ERp90 is related to substrate recruitment or delivery to the ERAD retrotranslocation machinery by ERFAD.

A Machine Learning Approach for Hot-Spot Detection at Protein-Protein Interfaces

NARCIS (Netherlands)

Melo, Rita; Fieldhouse, Robert; Melo, André; Correia, João D G; Cordeiro, Maria Natália D S; Gümüş, Zeynep H; Costa, Joaquim; Bonvin, Alexandre M J J; de Sousa Moreira, Irina

2016-01-01

Understanding protein-protein interactions is a key challenge in biochemistry. In this work, we describe a more accurate methodology to predict Hot-Spots (HS) in protein-protein interfaces from their native complex structure compared to previous published Machine Learning (ML) techniques. Our model

Stereochemical criteria for prediction of the effects of proline mutations on protein stability.

Directory of Open Access Journals (Sweden)

Kanika Bajaj

2007-12-01

Full Text Available When incorporated into a polypeptide chain, proline (Pro differs from all other naturally occurring amino acid residues in two important respects. The phi dihedral angle of Pro is constrained to values close to -65 degrees and Pro lacks an amide hydrogen. Consequently, mutations which result in introduction of Pro can significantly affect protein stability. In the present work, we describe a procedure to accurately predict the effect of Pro introduction on protein thermodynamic stability. Seventy-seven of the 97 non-Pro amino acid residues in the model protein, CcdB, were individually mutated to Pro, and the in vivo activity of each mutant was characterized. A decision tree to classify the mutation as perturbing or nonperturbing was created by correlating stereochemical properties of mutants to activity data. The stereochemical properties including main chain dihedral angle phi and main chain amide H-bonds (hydrogen bonds were determined from 3D models of the mutant proteins built using MODELLER. We assessed the performance of the decision tree on a large dataset of 163 single-site Pro mutations of T4 lysozyme, 74 nsSNPs, and 52 other Pro substitutions from the literature. The overall accuracy of this algorithm was found to be 81% in the case of CcdB, 77% in the case of lysozyme, 76% in the case of nsSNPs, and 71% in the case of other Pro substitution data. The accuracy of Pro scanning mutagenesis for secondary structure assignment was also assessed and found to be at best 69%. Our prediction procedure will be useful in annotating uncharacterized nsSNPs of disease-associated proteins and for protein engineering and design.

Stereochemical criteria for prediction of the effects of proline mutations on protein stability.

Science.gov (United States)

Bajaj, Kanika; Madhusudhan, M S; Adkar, Bharat V; Chakrabarti, Purbani; Ramakrishnan, C; Sali, Andrej; Varadarajan, Raghavan

2007-12-01

When incorporated into a polypeptide chain, proline (Pro) differs from all other naturally occurring amino acid residues in two important respects. The phi dihedral angle of Pro is constrained to values close to -65 degrees and Pro lacks an amide hydrogen. Consequently, mutations which result in introduction of Pro can significantly affect protein stability. In the present work, we describe a procedure to accurately predict the effect of Pro introduction on protein thermodynamic stability. Seventy-seven of the 97 non-Pro amino acid residues in the model protein, CcdB, were individually mutated to Pro, and the in vivo activity of each mutant was characterized. A decision tree to classify the mutation as perturbing or nonperturbing was created by correlating stereochemical properties of mutants to activity data. The stereochemical properties including main chain dihedral angle phi and main chain amide H-bonds (hydrogen bonds) were determined from 3D models of the mutant proteins built using MODELLER. We assessed the performance of the decision tree on a large dataset of 163 single-site Pro mutations of T4 lysozyme, 74 nsSNPs, and 52 other Pro substitutions from the literature. The overall accuracy of this algorithm was found to be 81% in the case of CcdB, 77% in the case of lysozyme, 76% in the case of nsSNPs, and 71% in the case of other Pro substitution data. The accuracy of Pro scanning mutagenesis for secondary structure assignment was also assessed and found to be at best 69%. Our prediction procedure will be useful in annotating uncharacterized nsSNPs of disease-associated proteins and for protein engineering and design.

Epidemic spreading model to characterize misfolded proteins propagation in aging and associated neurodegenerative disorders.

Science.gov (United States)

Iturria-Medina, Yasser; Sotero, Roberto C; Toussaint, Paule J; Evans, Alan C

2014-11-01

Misfolded proteins (MP) are a key component in aging and associated neurodegenerative disorders. For example, misfolded Amyloid-ß (Aß) and tau proteins are two neuropathogenic hallmarks of Alzheimer's disease. Mechanisms underlying intra-brain MP propagation/deposition remain essentially uncharacterized. Here, is introduced an epidemic spreading model (ESM) for MP dynamics that considers propagation-like interactions between MP agents and the brain's clearance response across the structural connectome. The ESM reproduces advanced Aß deposition patterns in the human brain (explaining 46∼56% of the variance in regional Aß loads, in 733 subjects from the ADNI database). Furthermore, this model strongly supports a) the leading role of Aß clearance deficiency and early Aß onset age during Alzheimer's disease progression, b) that effective anatomical distance from Aß outbreak region explains regional Aß arrival time and Aß deposition likelihood, c) the multi-factorial impact of APOE e4 genotype, gender and educational level on lifetime intra-brain Aß propagation, and d) the modulatory impact of Aß propagation history on tau proteins concentrations, supporting the hypothesis of an interrelated pathway between Aß pathophysiology and tauopathy. To our knowledge, the ESM is the first computational model highlighting the direct link between structural brain networks, production/clearance of pathogenic proteins and associated intercellular transfer mechanisms, individual genetic/demographic properties and clinical states in health and disease. In sum, the proposed ESM constitutes a promising framework to clarify intra-brain region to region transference mechanisms associated with aging and neurodegenerative disorders.

Epidemic spreading model to characterize misfolded proteins propagation in aging and associated neurodegenerative disorders.

Directory of Open Access Journals (Sweden)

Yasser Iturria-Medina

2014-11-01

Full Text Available Misfolded proteins (MP are a key component in aging and associated neurodegenerative disorders. For example, misfolded Amyloid-ß (Aß and tau proteins are two neuropathogenic hallmarks of Alzheimer's disease. Mechanisms underlying intra-brain MP propagation/deposition remain essentially uncharacterized. Here, is introduced an epidemic spreading model (ESM for MP dynamics that considers propagation-like interactions between MP agents and the brain's clearance response across the structural connectome. The ESM reproduces advanced Aß deposition patterns in the human brain (explaining 46∼56% of the variance in regional Aß loads, in 733 subjects from the ADNI database. Furthermore, this model strongly supports a the leading role of Aß clearance deficiency and early Aß onset age during Alzheimer's disease progression, b that effective anatomical distance from Aß outbreak region explains regional Aß arrival time and Aß deposition likelihood, c the multi-factorial impact of APOE e4 genotype, gender and educational level on lifetime intra-brain Aß propagation, and d the modulatory impact of Aß propagation history on tau proteins concentrations, supporting the hypothesis of an interrelated pathway between Aß pathophysiology and tauopathy. To our knowledge, the ESM is the first computational model highlighting the direct link between structural brain networks, production/clearance of pathogenic proteins and associated intercellular transfer mechanisms, individual genetic/demographic properties and clinical states in health and disease. In sum, the proposed ESM constitutes a promising framework to clarify intra-brain region to region transference mechanisms associated with aging and neurodegenerative disorders.

AcEST: BP918418 [AcEST

Lifescience Database Archive (English)

Full Text Available 9GAMM GGDEF domain protein OS=Shewanella piezot... 33 8.1 >tr|Q5CY37|Q5CY37_CRYPV Putative uncharacterized p...KTLAKLLPVEAKVAKNKKK 296 >tr|B8CPU2|B8CPU2_9GAMM GGDEF domain protein OS=Shewanella piezotolerans WP3 GN=swp_

ATGL and CGI-58 are lipid droplet proteins of the hepatic stellate cell line HSC-T6.

Science.gov (United States)

Eichmann, Thomas O; Grumet, Lukas; Taschler, Ulrike; Hartler, Jürgen; Heier, Christoph; Woblistin, Aaron; Pajed, Laura; Kollroser, Manfred; Rechberger, Gerald; Thallinger, Gerhard G; Zechner, Rudolf; Haemmerle, Günter; Zimmermann, Robert; Lass, Achim

2015-10-01

Lipid droplets (LDs) of hepatic stellate cells (HSCs) contain large amounts of vitamin A [in the form of retinyl esters (REs)] as well as other neutral lipids such as TGs. During times of insufficient vitamin A availability, RE stores are mobilized to ensure a constant supply to the body. To date, little is known about the enzymes responsible for the hydrolysis of neutral lipid esters, in particular of REs, in HSCs. In this study, we aimed to identify LD-associated neutral lipid hydrolases by a proteomic approach using the rat stellate cell line HSC-T6. First, we loaded cells with retinol and FAs to promote lipid synthesis and deposition within LDs. Then, LDs were isolated and lipid composition and the LD proteome were analyzed. Among other proteins, we found perilipin 2, adipose TG lipase (ATGL), and comparative gene identification-58 (CGI-58), known and established LD proteins. Bioinformatic search of the LD proteome for α/β-hydrolase fold-containing proteins revealed no yet uncharacterized neutral lipid hydrolases. In in vitro activity assays, we show that rat (r)ATGL, coactivated by rat (r)CGI-58, efficiently hydrolyzes TGs and REs. These findings suggest that rATGL and rCGI-58 are LD-resident proteins in HSCs and participate in the mobilization of both REs and TGs. Copyright © 2015 by the American Society for Biochemistry and Molecular Biology, Inc.

Identification and quantification of protein S-nitrosation by nitrite in the mouse heart during ischemia.

Science.gov (United States)

Chouchani, Edward T; James, Andrew M; Methner, Carmen; Pell, Victoria R; Prime, Tracy A; Erickson, Brian K; Forkink, Marleen; Lau, Gigi Y; Bright, Thomas P; Menger, Katja E; Fearnley, Ian M; Krieg, Thomas; Murphy, Michael P

2017-09-01

Nitrate (NO 3 - ) and nitrite (NO 2 - ) are known to be cardioprotective and to alter energy metabolism in vivo NO 3 - action results from its conversion to NO 2 - by salivary bacteria, but the mechanism(s) by which NO 2 - affects metabolism remains obscure. NO 2 - may act by S -nitrosating protein thiols, thereby altering protein activity. But how this occurs, and the functional importance of S -nitrosation sites across the mammalian proteome, remain largely uncharacterized. Here we analyzed protein thiols within mouse hearts in vivo using quantitative proteomics to determine S -nitrosation site occupancy. We extended the thiol-redox proteomic technique, isotope-coded affinity tag labeling, to quantify the extent of NO 2 - -dependent S -nitrosation of proteins thiols in vivo Using this approach, called SNOxICAT ( S -nitrosothiol redox isotope-coded affinity tag), we found that exposure to NO 2 - under normoxic conditions or exposure to ischemia alone results in minimal S -nitrosation of protein thiols. However, exposure to NO 2 - in conjunction with ischemia led to extensive S -nitrosation of protein thiols across all cellular compartments. Several mitochondrial protein thiols exposed to the mitochondrial matrix were selectively S -nitrosated under these conditions, potentially contributing to the beneficial effects of NO 2 - on mitochondrial metabolism. The permeability of the mitochondrial inner membrane to HNO 2 , but not to NO 2 - , combined with the lack of S -nitrosation during anoxia alone or by NO 2 - during normoxia places constraints on how S -nitrosation occurs in vivo and on its mechanisms of cardioprotection and modulation of energy metabolism. Quantifying S -nitrosated protein thiols now allows determination of modified cysteines across the proteome and identification of those most likely responsible for the functional consequences of NO 2 - exposure. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

AcEST: BP912743 [AcEST

Lifescience Database Archive (English)

Full Text Available 6_TRIAD Putative uncharacterized protein OS=Trich... 37 0.65 tr|Q44443|Q44443_9RHIZ Nopal...DKLAIMENYLGKKSPTNLAQTNNQLTGLSITEEMNQNHHHHHPITSN 3868 >tr|Q44443|Q44443_9RHIZ Nopaline synthase OS=Agrobacter

AcEST: DK959286 [AcEST

Lifescience Database Archive (English)

Full Text Available 80_9HYPO Coatamer subunit protein OS=Epichloe festucae Align length 57 Score (bit...J80|B2CJ80_9HYPO Coatamer subunit protein OS=Epichloe fest... 34 3.2 tr|B3SAW3|B3...SAW3_TRIAD Putative uncharacterized protein OS=Trich... 33 7.2 >tr|B2CJ80|B2CJ80_9HYPO Coatamer subunit protein OS=Epichloe

pLoc-mPlant: predict subcellular localization of multi-location plant proteins by incorporating the optimal GO information into general PseAAC.

Science.gov (United States)

Cheng, Xiang; Xiao, Xuan; Chou, Kuo-Chen

2017-08-22

One of the fundamental goals in cellular biochemistry is to identify the functions of proteins in the context of compartments that organize them in the cellular environment. To realize this, it is indispensable to develop an automated method for fast and accurate identification of the subcellular locations of uncharacterized proteins. The current study is focused on plant protein subcellular location prediction based on the sequence information alone. Although considerable efforts have been made in this regard, the problem is far from being solved yet. Most of the existing methods can be used to deal with single-location proteins only. Actually, proteins with multi-locations may have some special biological functions. This kind of multiplex protein is particularly important for both basic research and drug design. Using the multi-label theory, we present a new predictor called "pLoc-mPlant" by extracting the optimal GO (Gene Ontology) information into the Chou's general PseAAC (Pseudo Amino Acid Composition). Rigorous cross-validation on the same stringent benchmark dataset indicated that the proposed pLoc-mPlant predictor is remarkably superior to iLoc-Plant, the state-of-the-art method for predicting plant protein subcellular localization. To maximize the convenience of most experimental scientists, a user-friendly web-server for the new predictor has been established at , by which users can easily get their desired results without the need to go through the complicated mathematics involved.

Prolyl hydroxylation regulates protein degradation, synthesis, and splicing in human induced pluripotent stem cell-derived cardiomyocytes.

Science.gov (United States)

Stoehr, Andrea; Yang, Yanqin; Patel, Sajni; Evangelista, Alicia M; Aponte, Angel; Wang, Guanghui; Liu, Poching; Boylston, Jennifer; Kloner, Philip H; Lin, Yongshun; Gucek, Marjan; Zhu, Jun; Murphy, Elizabeth

2016-06-01

Protein hydroxylases are oxygen- and α-ketoglutarate-dependent enzymes that catalyse hydroxylation of amino acids such as proline, thus linking oxygen and metabolism to enzymatic activity. Prolyl hydroxylation is a dynamic post-translational modification that regulates protein stability and protein-protein interactions; however, the extent of this modification is largely uncharacterized. The goals of this study are to investigate the biological consequences of prolyl hydroxylation and to identify new targets that undergo prolyl hydroxylation in human cardiomyocytes. We used human induced pluripotent stem cell-derived cardiomyocytes in combination with pulse-chase amino acid labelling and proteomics to analyse the effects of prolyl hydroxylation on protein degradation and synthesis. We identified 167 proteins that exhibit differences in degradation with inhibition of prolyl hydroxylation by dimethyloxalylglycine (DMOG); 164 were stabilized. Proteins involved in RNA splicing such as serine/arginine-rich splicing factor 2 (SRSF2) and splicing factor and proline- and glutamine-rich (SFPQ) were stabilized with DMOG. DMOG also decreased protein translation of cytoskeletal and sarcomeric proteins such as α-cardiac actin. We searched the mass spectrometry data for proline hydroxylation and identified 134 high confidence peptides mapping to 78 unique proteins. We identified SRSF2, SFPQ, α-cardiac actin, and cardiac titin as prolyl hydroxylated. We identified 29 prolyl hydroxylated proteins that showed a significant difference in either protein degradation or synthesis. Additionally, we performed next-generation RNA sequencing and showed that the observed decrease in protein synthesis was not due to changes in mRNA levels. Because RNA splicing factors were prolyl hydroxylated, we investigated splicing ± inhibition of prolyl hydroxylation and detected 369 alternative splicing events, with a preponderance of exon skipping. This study provides the first extensive

Expressional and Biochemical Characterization of Rice Disease Resistance Gene Xa3/Xa26 Family

Institute of Scientific and Technical Information of China (English)

Songjie Xu; Yinglong Cao; Xianghua Li; Shiping Wang

2007-01-01

The rice (Oryza sativa L.) Xa3/Xa26 gene, conferring race-specific resistance to bacterial blight disease and encoding a leucine-rich repeat (LRR) receptor kinase-like protein, belongs to a multigene family consisting of tandem clustered homologous genes, colocalizing with several uncharacterized genes for resistance to bacterial blight or fungal blast. To provide more information on the expressional and biochemical characteristics of the Xa3/Xa26 family, we analyzed the family members. Four Xa3/Xa26 family members in the indica rice variety Teqing, which carries a bacterial blight resistance gene with a chromosomal location tightly linked to Xa3/Xa26, and five Xa3/Xa26 family members in the japonica rice variety Nipponbare, which carries at least one uncharacterized blast resistance gene, were constitutively expressed in leaf tissue. The result suggests that some of the family members may be candidates of these uncharacterized resistance genes. At least five putative N-glycosylation sites in the LRR domain of XA3/XA26 protein are not glycosylated. The XA3/XA26 and its family members MRKa and MRKc all possess the consensus sequences of paired cysteines, which putatively function in dimerization of the receptor proteins for signal transduction, immediately before the first LRR and immediately after the last LRR. However, no homo-dimer between the XA3/XA26 molecules or hetero-dimer between XA3/XA26 and MRKa or MRKc were formed, indicating that XA3/XA26 protein might function either as a monomer or a hetero-dimer formed with other protein outside of the XA3/XA26 family. These results provide valuable information for further extensive investigation into this multiple protein family.

AcEST: DK949530 [AcEST

Lifescience Database Archive (English)

Full Text Available L + R+ID G Sbjct: 451 PRTRFGAFARGAKVVLYTKKSGAHLRIIDGG 481 >sp|Q9NQ79|CRAC1_HUMAN Cartilage acidic protein 1 OS=Homo sapiens GN=CRTAC1...ovel protein similar to H.sapiens CRTAC1... 146 1e-33 tr|B6MKC0|B6MKC0_BRAFL Putative uncharacterized protei

Genome-Wide Sensitivity Analysis of the Microsymbiont Sinorhizobium meliloti to Symbiotically Important, Defensin-Like Host Peptides

Directory of Open Access Journals (Sweden)

Markus F. F. Arnold

2017-08-01

Full Text Available The model legume species Medicago truncatula expresses more than 700 nodule-specific cysteine-rich (NCR signaling peptides that mediate the differentiation of Sinorhizobium meliloti bacteria into nitrogen-fixing bacteroids. NCR peptides are essential for a successful symbiosis in legume plants of the inverted-repeat-lacking clade (IRLC and show similarity to mammalian defensins. In addition to signaling functions, many NCR peptides exhibit antimicrobial activity in vitro and in vivo. Bacterial resistance to these antimicrobial activities is likely to be important for symbiosis. However, the mechanisms used by S. meliloti to resist antimicrobial activity of plant peptides are poorly understood. To address this, we applied a global genetic approach using transposon mutagenesis followed by high-throughput sequencing (Tn-seq to identify S. meliloti genes and pathways that increase or decrease bacterial competitiveness during exposure to the well-studied cationic NCR247 peptide and also to the unrelated model antimicrobial peptide polymyxin B. We identified 78 genes and several diverse pathways whose interruption alters S. meliloti resistance to NCR247. These genes encode the following: (i cell envelope polysaccharide biosynthesis and modification proteins, (ii inner and outer membrane proteins, (iii peptidoglycan (PG effector proteins, and (iv non-membrane-associated factors such as transcriptional regulators and ribosome-associated factors. We describe a previously uncharacterized yet highly conserved peptidase, which protects S. meliloti from NCR247 and increases competitiveness during symbiosis. Additionally, we highlight a considerable number of uncharacterized genes that provide the basis for future studies to investigate the molecular basis of symbiotic development as well as chronic pathogenic interactions.

Tools and strategies for discovering novel enzymes and metabolic pathways

Directory of Open Access Journals (Sweden)

John A. Gerlt

2016-12-01

Full Text Available The number of entries in the sequence databases continues to increase exponentially – the UniProt database is increasing with a doubling time of ∼4 years (2% increase/month. Approximately 50% of the entries have uncertain, unknown, or incorrect function annotations because these are made by automated methods based on sequence homology. If the potential in complete genome sequences is to be realized, strategies and tools must be developed to facilitate experimental assignment of functions to uncharacterized proteins discovered in genome projects. The Enzyme Function Initiative (EFI; previously supported by U54GM093342 from the National Institutes of Health, now supported by P01GM118303 developed web tools for visualizing and analyzing (1 sequence and function space in protein families (EFI-EST and (2 genome neighbourhoods in microbial and fungal genomes (EFI-GNT to assist the design of experimental strategies for discovering the in vitro activities and in vivo metabolic functions of uncharacterized enzymes. The EFI developed an experimental platform for large-scale production of the solute binding proteins (SBPs for ABC, TRAP, and TCT transport systems and their screening with a physical ligand library to identify the identities of the ligands for these transport systems. Because the genes that encode transport systems are often co-located with the genes that encode the catabolic pathways for the transported solutes, the identity of the SBP ligand together with the EFI-EST and EFI-GNT web tools can be used to discover new enzyme functions and new metabolic pathways. This approach is demonstrated with the characterization of a novel pathway for ethanolamine catabolism.

The Ras GTPase-activating protein Rasal3 supports survival of naive T cells.

Directory of Open Access Journals (Sweden)

Ryunosuke Muro

Full Text Available The Ras-mitogen-activated protein kinase (MAPK pathway is crucial for T cell receptor (TCR signaling in the development and function of T cells. The significance of various modulators of the Ras-MAPK pathway in T cells, however, remains to be fully understood. Ras-activating protein-like 3 (Rasal3 is an uncharacterized member of the SynGAP family that contains a conserved Ras GTPase-activating protein (GAP domain, and is predominantly expressed in the T cell lineage. In the current study, we investigated the function and physiological roles of Rasal3. Our results showed that Rasal3 possesses RasGAP activity, but not Rap1GAP activity, and represses TCR-stimulated ERK phosphorylation in a T cell line. In systemic Rasal3-deficient mice, T cell development in the thymus including positive selection, negative selection, and β-selection was unaffected. However, the number of naive, but not effector memory CD4 and CD8 T cell in the periphery was significantly reduced in Rasal3-deficient mice, and associated with a marked increase in apoptosis of these cells. Indeed, survival of Rasal3 deficient naive CD4 T cells in vivo by adoptive transfer was significantly impaired, whereas IL-7-dependent survival of naive CD4 T cells in vitro was unaltered. Collectively, Rasal3 is required for in vivo survival of peripheral naive T cells, contributing to the maintenance of optimal T cell numbers.

Neutrophils alter the inflammatory milieu by signal-dependent translation of constitutive messenger RNAs

Science.gov (United States)

Lindemann, Stephan W.; Yost, Christian C.; Denis, Melvin M.; McIntyre, Thomas M.; Weyrich, Andrew S.; Zimmerman, Guy A.

2004-05-01

The mechanisms by which neutrophils, key effector cells of the innate immune system, express new gene products in inflammation are largely uncharacterized. We found that they rapidly translate constitutive mRNAs when activated, a previously unrecognized response. One of the proteins synthesized without a requirement for transcription is the soluble IL-6 receptor , which translocates to endothelial cells and induces a temporal switch to mononuclear leukocyte recruitment. Its synthesis is regulated by a specialized translational control pathway that is inhibited by rapamycin, a bacterial macrolide with therapeutic efficacy in transplantation, inflammatory syndromes, and neoplasia. Signal-dependent translation in activated neutrophils may be a critical mechanism for alteration of the inflammatory milieu and a therapeutic target.

A Computational Framework for Proteome-Wide Pursuit and Prediction of Metalloproteins using ICP-MS and MS/MS Data

Directory of Open Access Journals (Sweden)

Trauger Sunia A

2011-02-01

Full Text Available Abstract Background Metal-containing proteins comprise a diverse and sizable category within the proteomes of organisms, ranging from proteins that use metals to catalyze reactions to proteins in which metals play key structural roles. Unfortunately, reliably predicting that a protein will contain a specific metal from its amino acid sequence is not currently possible. We recently developed a generally-applicable experimental technique for finding metalloproteins on a genome-wide scale. Applying this metal-directed protein purification approach (ICP-MS and MS/MS based to the prototypical microbe Pyrococcus furiosus conclusively demonstrated the extent and diversity of the uncharacterized portion of microbial metalloproteomes since a majority of the observed metal peaks could not be assigned to known or predicted metalloproteins. However, even using this technique, it is not technically feasible to purify to homogeneity all metalloproteins in an organism. In order to address these limitations and complement the metal-directed protein purification, we developed a computational infrastructure and statistical methodology to aid in the pursuit and identification of novel metalloproteins. Results We demonstrate that our methodology enables predictions of metal-protein interactions using an experimental data set derived from a chromatography fractionation experiment in which 870 proteins and 10 metals were measured over 2,589 fractions. For each of the 10 metals, cobalt, iron, manganese, molybdenum, nickel, lead, tungsten, uranium, vanadium, and zinc, clusters of proteins frequently occurring in metal peaks (of a specific metal within the fractionation space were defined. This resulted in predictions that there are from 5 undiscovered vanadium- to 13 undiscovered cobalt-containing proteins in Pyrococcus furiosus. Molybdenum and nickel were chosen for additional assessment producing lists of genes predicted to encode metalloproteins or metalloprotein

AcEST: DK963038 [AcEST

Lifescience Database Archive (English)

Full Text Available tr_hit_id A5WW09 Definition tr|A5WW09|A5WW09_DANRE Novel protein similar to autism susceptibility candidate ... Novel protein similar to autism susceptib... 42 0.028 tr|A9H6U3|A9H6U3_GLUDA Put...Putative uncharacterized protein OS=Planc... 33 9.9 >tr|A5WW09|A5WW09_DANRE Novel protein similar to autism

AcEST: DK962662 [AcEST

Lifescience Database Archive (English)

Full Text Available T0P7_JANMA Uncharacterized conserved protein OS=Janthinobacterium sp. (strain Marseille) (Minibacterium massili...n Marseille) (Minibacterium massiliensis) GN=mma_2404 PE=4 SV=1 Length = 322 Score = 33.9 bits (76), Expect

LncRNA NEAT1 promotes autophagy in MPTP-induced Parkinson's disease through stabilizing PINK1 protein.

Science.gov (United States)

Yan, Wang; Chen, Zhao-Ying; Chen, Jia-Qi; Chen, Hui-Min

2018-02-19

Long non-coding RNA nuclear paraspeckle assembly transcript 1 (lncRNA NEAT1) was found to be closely related to the pathological changes in brain and nervous system. However, the role of NEAT1 and its potential mechanism in Parkinson's disease (PD) largely remain uncharacterized. In this study, PD mouse model was established by intraperitoneal injection of MPTP. The numbers of TH + neurons, NEAT1 expression and the level of PINK1, LC3-II, LC3-I protein were assessed in PD mice. SH-SY5Y cells were treated with MPP + as PD cell model. RNA pull-down assay was used to identify the interaction between NEAT1 and PINK1 in vitro. The endogenous expression of NEAT1 was modified by lentiviral vector carrying interference sequence for NEAT1 in vivo. The numbers of TH + neurons significantly decreased in PD mice compared with the control. The expressions of NEAT1, PINK1 protein and LC3-II/LC3-I level were increased by MPTP in vitro and in vivo. Moreover, NEAT1 positively regulated the protein level of PINK1 through inhibition of PINK1 protein degradation. And NEAT1 mediated the effects of MPP + on SH-SY5Y cells through stabilization of PINK1 protein. The results of in vivo experiments revealed that NEAT1 knockdown could effectively suppress MPTP-induced autophagy in vivo that alleviated dopaminergic neuronal injury. LncRNA NEAT1 promoted the MPTP-induced autophagy in PD through stabilization of PINK1 protein. Copyright © 2017 Elsevier Inc. All rights reserved.

AcEST: BP917290 [AcEST

Lifescience Database Archive (English)

Full Text Available 7DTG3|SRTD4_MOUSE SERTA domain-containing protein 4 OS=Mus m... 31 3.7 sp|O41174|POLG_PEV9U Genome polyprotein OS=Porcine enterovirus...1DP68_NEOFI Putative uncharacterized protein OS=Neosa... 33 5.7 tr|Q68T42|Q68T42_9ENTO Polyprotein OS=Human enterovirus

Roles of the cytoskeleton and of Protein Phosphorylation Events in the Osmotic Stress Response in EEL Intestinal Epithelium

DEFF Research Database (Denmark)

Lionetto, Maria G; Pedersen, Stine F; Hoffmann, Else K

2002-01-01

The eel intestinal epithelium responds to an acute hypertonic challenge by a biphasic increase of the rate of Cl(-) absorption (measured as short circuit current, Isc, and creating a negative transepithelial potential, V(te), at the basolateral side of the epithelium). While the first, transient...... phase is bumetanide-insensitive, the second, sustained phase is bumetanide-sensitive, reflecting activation of the apically located Na(+)-K(+)-2Cl(-) (NKCC) cotransporter, which correlates with the cellular RVI response. Here, we investigated the involvement of the cytoskeleton and of serine....../threonine phosphorylation events in the osmotic stress-induced ion transport in the eel intestinal epithelium, focusing on the sustained RVI phase, as well as on the previously uncharacterized response to hypotonic stress. The study was carried out using confocal laser scanning microscopy, a quantitative F-actin assay...

pSuc-Lys: Predict lysine succinylation sites in proteins with PseAAC and ensemble random forest approach.

Science.gov (United States)

Jia, Jianhua; Liu, Zi; Xiao, Xuan; Liu, Bingxiang; Chou, Kuo-Chen

2016-04-07

Being one type of post-translational modifications (PTMs), protein lysine succinylation is important in regulating varieties of biological processes. It is also involved with some diseases, however. Consequently, from the angles of both basic research and drug development, we are facing a challenging problem: for an uncharacterized protein sequence having many Lys residues therein, which ones can be succinylated, and which ones cannot? To address this problem, we have developed a predictor called pSuc-Lys through (1) incorporating the sequence-coupled information into the general pseudo amino acid composition, (2) balancing out skewed training dataset by random sampling, and (3) constructing an ensemble predictor by fusing a series of individual random forest classifiers. Rigorous cross-validations indicated that it remarkably outperformed the existing methods. A user-friendly web-server for pSuc-Lys has been established at http://www.jci-bioinfo.cn/pSuc-Lys, by which users can easily obtain their desired results without the need to go through the complicated mathematical equations involved. It has not escaped our notice that the formulation and approach presented here can also be used to analyze many other problems in computational proteomics. Copyright © 2016 Elsevier Ltd. All rights reserved.

A Human XPC Protein Interactome—A Resource

Directory of Open Access Journals (Sweden)

Abigail Lubin

2013-12-01

Full Text Available Global genome nucleotide excision repair (GG-NER is responsible for identifying and removing bulky adducts from non-transcribed DNA that result from damaging agents such as UV radiation and cisplatin. Xeroderma pigmentosum complementation group C (XPC is one of the essential damage recognition proteins of the GG-NER pathway and its dysfunction results in xeroderma pigmentosum (XP, a disorder involving photosensitivity and a predisposition to cancer. To better understand the identification of DNA damage by XPC in the context of chromatin and the role of XPC in the pathogenesis of XP, we characterized the interactome of XPC using a high throughput yeast two-hybrid screening. Our screening showed 49 novel interactors of XPC involved in DNA repair and replication, proteolysis and post-translational modifications, transcription regulation, signal transduction, and metabolism. Importantly, we validated the XPC-OTUD4 interaction by co-IP and provided evidence that OTUD4 knockdown in human cells indeed affects the levels of ubiquitinated XPC, supporting a hypothesis that the OTUD4 deubiquitinase is involved in XPC recycling by cleaving the ubiquitin moiety. This high-throughput characterization of the XPC interactome provides a resource for future exploration and suggests that XPC may have many uncharacterized cellular functions.

Protein function prediction using neighbor relativity in protein-protein interaction network.

Science.gov (United States)

Moosavi, Sobhan; Rahgozar, Masoud; Rahimi, Amir

2013-04-01

There is a large gap between the number of discovered proteins and the number of functionally annotated ones. Due to the high cost of determining protein function by wet-lab research, function prediction has become a major task for computational biology and bioinformatics. Some researches utilize the proteins interaction information to predict function for un-annotated proteins. In this paper, we propose a novel approach called "Neighbor Relativity Coefficient" (NRC) based on interaction network topology which estimates the functional similarity between two proteins. NRC is calculated for each pair of proteins based on their graph-based features including distance, common neighbors and the number of paths between them. In order to ascribe function to an un-annotated protein, NRC estimates a weight for each neighbor to transfer its annotation to the unknown protein. Finally, the unknown protein will be annotated by the top score transferred functions. We also investigate the effect of using different coefficients for various types of functions. The proposed method has been evaluated on Saccharomyces cerevisiae and Homo sapiens interaction networks. The performance analysis demonstrates that NRC yields better results in comparison with previous protein function prediction approaches that utilize interaction network. Copyright © 2012 Elsevier Ltd. All rights reserved.

Predicting membrane protein types using various decision tree classifiers based on various modes of general PseAAC for imbalanced datasets.

Science.gov (United States)

Sankari, E Siva; Manimegalai, D

2017-12-21

Predicting membrane protein types is an important and challenging research area in bioinformatics and proteomics. Traditional biophysical methods are used to classify membrane protein types. Due to large exploration of uncharacterized protein sequences in databases, traditional methods are very time consuming, expensive and susceptible to errors. Hence, it is highly desirable to develop a robust, reliable, and efficient method to predict membrane protein types. Imbalanced datasets and large datasets are often handled well by decision tree classifiers. Since imbalanced datasets are taken, the performance of various decision tree classifiers such as Decision Tree (DT), Classification And Regression Tree (CART), C4.5, Random tree, REP (Reduced Error Pruning) tree, ensemble methods such as Adaboost, RUS (Random Under Sampling) boost, Rotation forest and Random forest are analysed. Among the various decision tree classifiers Random forest performs well in less time with good accuracy of 96.35%. Another inference is RUS boost decision tree classifier is able to classify one or two samples in the class with very less samples while the other classifiers such as DT, Adaboost, Rotation forest and Random forest are not sensitive for the classes with fewer samples. Also the performance of decision tree classifiers is compared with SVM (Support Vector Machine) and Naive Bayes classifier. Copyright © 2017 Elsevier Ltd. All rights reserved.

Mitochondrial nucleoid interacting proteins support mitochondrial protein synthesis.

Science.gov (United States)

He, J; Cooper, H M; Reyes, A; Di Re, M; Sembongi, H; Litwin, T R; Gao, J; Neuman, K C; Fearnley, I M; Spinazzola, A; Walker, J E; Holt, I J

2012-07-01

Mitochondrial ribosomes and translation factors co-purify with mitochondrial nucleoids of human cells, based on affinity protein purification of tagged mitochondrial DNA binding proteins. Among the most frequently identified proteins were ATAD3 and prohibitin, which have been identified previously as nucleoid components, using a variety of methods. Both proteins are demonstrated to be required for mitochondrial protein synthesis in human cultured cells, and the major binding partner of ATAD3 is the mitochondrial ribosome. Altered ATAD3 expression also perturbs mtDNA maintenance and replication. These findings suggest an intimate association between nucleoids and the machinery of protein synthesis in mitochondria. ATAD3 and prohibitin are tightly associated with the mitochondrial membranes and so we propose that they support nucleic acid complexes at the inner membrane of the mitochondrion.

Identification of putative drug targets in Vancomycin-resistant Staphylococcus aureus (VRSA) using computer aided protein data analysis.

Science.gov (United States)

Hasan, Md Anayet; Khan, Md Arif; Sharmin, Tahmina; Hasan Mazumder, Md Habibul; Chowdhury, Afrin Sultana

2016-01-01

Vancomycin-resistant Staphylococcus aureus (VRSA) is a Gram-positive, facultative aerobic bacterium which is evolved from the extensive exposure of Vancomycin to Methicillin resistant S. aureus (MRSA) that had become the most common cause of hospital and community-acquired infections. Due to the emergence of different antibiotic resistance strains, there is an exigency to develop novel drug targets to address the provocation of multidrug-resistant bacteria. In this study, in-silico genome subtraction methodology was used to design potential and pathogen specific drug targets against VRSA. Our study divulged 1987 proteins from the proteome of 34,549 proteins, which have no homologues in human genome after sequential analysis through CD-HIT and BLASTp. The high stringency analysis of the remaining proteins against database of essential genes (DEG) resulted in 169 proteins which are essential for S. aureus. Metabolic pathway analysis of human host and pathogen by KAAS at the KEGG server sorted out 19 proteins involved in unique metabolic pathways. 26 human non-homologous membrane-bound essential proteins including 4 which were also involved in unique metabolic pathway were deduced through PSORTb, CELLO v.2.5, ngLOC. Functional classification of uncharacterized proteins through SVMprot derived 7 human non-homologous membrane-bound hypothetical essential proteins. Study of potential drug target against Drug Bank revealed pbpA-penicillin-binding protein 1 and hypothetical protein MQW_01796 as the best drug target candidate. 2D structure was predicted by PRED-TMBB, 3D structure and functional analysis was also performed. Protein-protein interaction network of potential drug target proteins was analyzed by using STRING. The identified drug targets are expected to have great potential for designing novel drugs against VRSA infections and further screening of the compounds against these new targets may result in the discovery of novel therapeutic compounds that can be

Crystallization and preliminary X-ray diffraction analysis of the C-terminal domain of the human spliceosomal DExD/H-box protein hPrp22

International Nuclear Information System (INIS)

Kudlinzki, Denis; Nagel, Christian; Ficner, Ralf

2009-01-01

The cloning, purification and crystallization of the C-terminal domain of human hPrp22 are reported. This communication also contains data for the preliminary X-ray diffraction analysis. The Homo sapiens DExD/H-box protein hPrp22 is a crucial component of the eukaryotic pre-mRNA splicing machinery. Within the splicing cycle, it is involved in the ligation of exons and generation of the lariat and it additionally catalyzes the release of mature mRNA from the spliceosomal U5 snRNP. The yeast homologue of this protein, yPrp22, shows ATP-dependent RNA-helicase activity and is capable of unwinding RNA/RNA duplex molecules. A truncated construct coding for residues 950–1183 of human Prp22, comprising the structurally and functionally uncharacterized C-terminal domain, was cloned into an Escherichia coli expression vector. The protein was subsequently overproduced, purified and crystallized. The crystals obtained diffracted to 2.1 Å resolution, belonged to the tetragonal space group P4 1 2 1 2 or P4 3 2 1 2, with unit-cell parameters a = b = 78.2, c = 88.4 Å, and contained one molecule in the asymmetric unit

A previously unidentified deletion in G protein-coupled receptor 143 causing X-linked congenital nystagmus in a Chinese family

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Jing Liu

2016-01-01

Full Text Available Background: Congenital nystagmus (CN is characterized by conjugated, spontaneous, and involuntary ocular oscillations. It is an inherited disease and the most common inheritance pattern is X-linked CN. In this study, our aim is to identify the disease-causing mutation in a large sixth-generation Chinese family with X-linked CN. Methods: It has been reported that mutations in four-point-one, ezrin, radixin, moesin domain-containing 7 gene (FRMD7 and G protein-coupled receptor 143 gene (GPR143 account for the majority patients of X-linked nystagmus. We collected 8 ml blood samples from members of a large sixth-generation pedigree with X-linked CN and 100 normal controls. FRMD7 and GPR143 were scanned by polymerase chain reaction (PCR-based DNA sequencing assays, and multiplex PCR assays were applied to detect deletions. Results: We identified a previously unreported deletion covering 7 exons in GPR143 in a Chinese family. The heterozygous deletion from exon 3 to exon 9 of GPR143 was detected in all affected males in the family, while it was not detected in other unaffected relatives or 100 normal controls. Conclusions: This is the first report of molecular characterization in GPR143 gene in the CN family. Our results expand the spectrum of GPR143 mutations causing CN and further confirm the role of GPR143 in the pathogenesis of CN.

An ice-binding and tandem beta-sandwich domain-containing protein in Shewanella frigidimarina is a potential new type of ice adhesin.

Science.gov (United States)

Vance, Tyler D R; Graham, Laurie A; Davies, Peter L

2018-04-01

Out of the dozen different ice-binding protein (IBP) structures known, the DUF3494 domain is the most widespread, having been passed many times between prokaryotic and eukaryotic microorganisms by horizontal gene transfer. This ~25-kDa β-solenoid domain with an adjacent parallel α-helix is most commonly associated with an N-terminal secretory signal peptide. However, examples of the DUF3494 domain preceded by tandem Bacterial Immunoglobulin-like (BIg) domains are sometimes found, though uncharacterized. Here, we present one such protein (SfIBP_1) from the Antarctic bacterium Shewanella frigidimarina. We have confirmed and characterized the ice-binding activity of its ice-binding domain using thermal hysteresis measurements, fluorescent ice plane affinity analysis, and ice recrystallization inhibition assays. X-ray crystallography was used to solve the structure of the SfIBP_1 ice-binding domain, to further characterize its ice-binding surface and unique method of stabilizing or 'capping' the ends of the solenoid structure. The latter is formed from the interaction of two loops mediated by a combination of tandem prolines and electrostatic interactions. Furthermore, given their domain architecture and membrane association, we propose that these BIg-containing DUF3494 IBPs serve as ice-binding adhesion proteins that are capable of adsorbing their host bacterium onto ice. Submitted new structure to the Protein Data Bank (PDB: 6BG8). © 2018 Federation of European Biochemical Societies.

The Golgi localized bifunctional UDP-rhamnose/UDP-galactose transporter family of Arabidopsis

DEFF Research Database (Denmark)

Rautengarten, Carsten; Ebert, Berit; Moreno, Ignacio

2014-01-01

Delivery of nucleotide sugar substrates into the Golgi apparatus and endoplasmic reticulum for processes such as cell wall biosynthesis and protein glycosylation is critical for plant growth and development. Plant genomes encode large families of uncharacterized nucleotide sugar transporters that...

Genomic Enzymology: Web Tools for Leveraging Protein Family Sequence-Function Space and Genome Context to Discover Novel Functions.

Science.gov (United States)

Gerlt, John A

2017-08-22

The exponentially increasing number of protein and nucleic acid sequences provides opportunities to discover novel enzymes, metabolic pathways, and metabolites/natural products, thereby adding to our knowledge of biochemistry and biology. The challenge has evolved from generating sequence information to mining the databases to integrating and leveraging the available information, i.e., the availability of "genomic enzymology" web tools. Web tools that allow identification of biosynthetic gene clusters are widely used by the natural products/synthetic biology community, thereby facilitating the discovery of novel natural products and the enzymes responsible for their biosynthesis. However, many novel enzymes with interesting mechanisms participate in uncharacterized small-molecule metabolic pathways; their discovery and functional characterization also can be accomplished by leveraging information in protein and nucleic acid databases. This Perspective focuses on two genomic enzymology web tools that assist the discovery novel metabolic pathways: (1) Enzyme Function Initiative-Enzyme Similarity Tool (EFI-EST) for generating sequence similarity networks to visualize and analyze sequence-function space in protein families and (2) Enzyme Function Initiative-Genome Neighborhood Tool (EFI-GNT) for generating genome neighborhood networks to visualize and analyze the genome context in microbial and fungal genomes. Both tools have been adapted to other applications to facilitate target selection for enzyme discovery and functional characterization. As the natural products community has demonstrated, the enzymology community needs to embrace the essential role of web tools that allow the protein and genome sequence databases to be leveraged for novel insights into enzymological problems.

The Development of Protein Microarrays and Their Applications in DNA-Protein and Protein-Protein Interaction Analyses of Arabidopsis Transcription Factors

Science.gov (United States)

Gong, Wei; He, Kun; Covington, Mike; Dinesh-Kumar, S. P.; Snyder, Michael; Harmer, Stacey L.; Zhu, Yu-Xian; Deng, Xing Wang

2009-01-01

We used our collection of Arabidopsis transcription factor (TF) ORFeome clones to construct protein microarrays containing as many as 802 TF proteins. These protein microarrays were used for both protein-DNA and protein-protein interaction analyses. For protein-DNA interaction studies, we examined AP2/ERF family TFs and their cognate cis-elements. By careful comparison of the DNA-binding specificity of 13 TFs on the protein microarray with previous non-microarray data, we showed that protein microarrays provide an efficient and high throughput tool for genome-wide analysis of TF-DNA interactions. This microarray protein-DNA interaction analysis allowed us to derive a comprehensive view of DNA-binding profiles of AP2/ERF family proteins in Arabidopsis. It also revealed four TFs that bound the EE (evening element) and had the expected phased gene expression under clock-regulation, thus providing a basis for further functional analysis of their roles in clock regulation of gene expression. We also developed procedures for detecting protein interactions using this TF protein microarray and discovered four novel partners that interact with HY5, which can be validated by yeast two-hybrid assays. Thus, plant TF protein microarrays offer an attractive high-throughput alternative to traditional techniques for TF functional characterization on a global scale. PMID:19802365

Structures of the Mycobacterium tuberculosis GlpX protein (class II fructose-1,6-bisphosphatase): implications for the active oligomeric state, catalytic mechanism and citrate inhibition.

Science.gov (United States)

Wolf, Nina M; Gutka, Hiten J; Movahedzadeh, Farahnaz; Abad-Zapatero, Celerino

2018-04-01

The crystal structures of native class II fructose-1,6-bisphosphatase (FBPaseII) from Mycobacterium tuberculosis at 2.6 Å resolution and two active-site protein variants are presented. The variants were complexed with the reaction product fructose 6-phosphate (F6P). The Thr84Ala mutant is inactive, while the Thr84Ser mutant has a lower catalytic activity. The structures reveal the presence of a 222 tetramer, similar to those described for fructose-1,6/sedoheptulose-1,7-bisphosphatase from Synechocystis (strain 6803) as well as the equivalent enzyme from Thermosynechococcus elongatus. This homotetramer corresponds to a homologous oligomer that is present but not described in the crystal structure of FBPaseII from Escherichia coli and is probably conserved in all FBPaseIIs. The constellation of amino-acid residues in the active site of FBPaseII from M. tuberculosis (MtFBPaseII) is conserved and is analogous to that described previously for the E. coli enzyme. Moreover, the structure of the active site of the partially active (Thr84Ser) variant and the analysis of the kinetics are consistent with the previously proposed catalytic mechanism. The presence of metabolites in the crystallization medium (for example citrate and malonate) and in the corresponding crystal structures of MtFBPaseII, combined with their observed inhibitory effect, could suggest the existence of an uncharacterized inhibition of this class of enzymes besides the allosteric inhibition by adenosine monophosphate observed for the Synechocystis enzyme. The structural and functional insights derived from the structure of MtFBPaseII will provide critical information for the design of lead inhibitors, which will be used to validate this target for future chemical intervention.

Prediction of novel archaeal enzymes from sequence-derived features

DEFF Research Database (Denmark)

Jensen, Lars Juhl; Skovgaard, Marie; Brunak, Søren

2002-01-01

The completely sequenced archaeal genomes potentially encode, among their many functionally uncharacterized genes, novel enzymes of biotechnological interest. We have developed a prediction method for detection and classification of enzymes from sequence alone (available at http://www.cbs.dtu.dk/......The completely sequenced archaeal genomes potentially encode, among their many functionally uncharacterized genes, novel enzymes of biotechnological interest. We have developed a prediction method for detection and classification of enzymes from sequence alone (available at http......://www.cbs.dtu.dk/services/ArchaeaFun/). The method does not make use of sequence similarity; rather, it relies on predicted protein features like cotranslational and posttranslational modifications, secondary structure, and simple physical/chemical properties....

Specificity and affinity quantification of protein-protein interactions.

Science.gov (United States)

Yan, Zhiqiang; Guo, Liyong; Hu, Liang; Wang, Jin

2013-05-01

Most biological processes are mediated by the protein-protein interactions. Determination of the protein-protein structures and insight into their interactions are vital to understand the mechanisms of protein functions. Currently, compared with the isolated protein structures, only a small fraction of protein-protein structures are experimentally solved. Therefore, the computational docking methods play an increasing role in predicting the structures and interactions of protein-protein complexes. The scoring function of protein-protein interactions is the key responsible for the accuracy of the computational docking. Previous scoring functions were mostly developed by optimizing the binding affinity which determines the stability of the protein-protein complex, but they are often lack of the consideration of specificity which determines the discrimination of native protein-protein complex against competitive ones. We developed a scoring function (named as SPA-PP, specificity and affinity of the protein-protein interactions) by incorporating both the specificity and affinity into the optimization strategy. The testing results and comparisons with other scoring functions show that SPA-PP performs remarkably on both predictions of binding pose and binding affinity. Thus, SPA-PP is a promising quantification of protein-protein interactions, which can be implemented into the protein docking tools and applied for the predictions of protein-protein structure and affinity. The algorithm is implemented in C language, and the code can be downloaded from http://dl.dropbox.com/u/1865642/Optimization.cpp.

Immunoprotective efficacy of Acinetobacter baumannii outer membrane protein, FilF, predicted in silico as a potential vaccine candidate

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Ravinder eSingh

2016-02-01

Full Text Available Acinetobacter baumannii is emerging as a serious nosocomial pathogen with multidrug resistance that has made it difficult to cure and development of efficacious treatment against this pathogen is direly needed. This has led to investigate vaccine approach to prevent and treat A. baumannii infections. In this work, an outer membrane putative pilus assembly protein, FilF, was predicted as vaccine candidate by in silico analysis of A. baumannii proteome and was found to be conserved among the A. baumannii strains. It was cloned and expressed in E. coli BL21(DE3 and purified by Ni-NTA chromatography. Immunization with FilF generated high antibody titer (>64000 and provided 50% protection against a standardized lethal dose (10*8 CFU of A. baumannii in murine pneumonia model. FilF immunization reduced the bacterial load in lungs by 2 and 4 log cycles, 12 and 24 h post infection as compared to adjuvant control; reduced the levels of pro-inflammatory cytokines TNF-α, IL-6, IL-33, IFN-γ and IL-1β significantly and histology of lung tissue supported the data by showing considerably reduced damage and infiltration of neutrophils in lungs. These results demonstrate the in vivo validation of immunoprotective efficacy of a protein predicted as a vaccine candidate by in silico proteomic analysis and open the possibilities for exploration of a large array of uncharacterized proteins.

Suppression of NF-κB signal pathway by NLRC3-like protein in stony coral Acropora aculeus under heat stress.

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Zhou, Zhi; Wu, Yibo; Zhang, Chengkai; Li, Can; Chen, Guangmei; Yu, Xiaopeng; Shi, Xiaowei; Xu, Yanlai; Wang, Lingui; Huang, Bo

2017-08-01

Heat stress is the most common factor for coral bleaching, which has increased both in frequency and severity due to global warming. In the present study, the stony coral Acropora aculeus was subjected to acute heat stress and entire transcriptomes were sequenced via the next generation sequencing platform. Four paired-end libraries were constructed and sequenced in two groups, including a control and a heat stress group. A total of 120,319,751 paired-end reads with lengths of 2 × 100 bp were assembled and 55,021 coral-derived genes were obtained. After read mapping and abundance estimation, 9110 differentially expressed genes were obtained in the comparison between the control and heat stress group, including 4465 significantly upregulated and 4645 significantly downregulated genes. Twenty-three GO terms in the Biological Process category were overrepresented for significantly upregulated genes, and divided into six groups according to their relationship. These three groups were related to the NF-κB signal pathway, and the remaining three groups were relevant for pathogen response, immunocyte activation and protein ubiquitination. Forty-three common genes were found in four GO terms, which were directly related to the NF-κB signal pathway. These included 2 NACHT, LRR, PYD domains-containing protein, 5 nucleotide-binding oligomerization domain-containing protein, 29 NLRC3-like protein, 4 NLRC5-like protein, and 3 uncharacterized protein. For significantly downregulated genes, 27 overrepresented GO terms were found in the Biological Process category, which were relevant to protein ubiquitination and ATP metabolism. Our results indicate that heat stress suppressed the immune response level via the NLRC3-like protein, the fine-tuning of protein turnover activity, and ATP metabolism. This might disrupt the balance of coral-zooxanthellae symbiosis and result in the bleaching of the coral A. aculeus. Copyright © 2017 Elsevier Ltd. All rights reserved.

Internalization of the human CRF receptor 1 is independent of classical phosphorylation sites and of beta-arrestin 1 recruitment

DEFF Research Database (Denmark)

Rasmussen, Trine N; Novak, Ivana; Nielsen, Søren M

2004-01-01

The corticotropin releasing factor receptor 1 (CRFR1) belongs to the superfamily of G-protein coupled receptors. Though CRF is involved in the aetiology of several stress-related disorders, including depression and anxiety, details of CRFR1 regulation such as internalization remain uncharacterized...

Protein-protein interactions: an application of Tus-Ter mediated protein microarray system.

Science.gov (United States)

Sitaraman, Kalavathy; Chatterjee, Deb K

2011-01-01

In this chapter, we present a novel, cost-effective microarray strategy that utilizes expression-ready plasmid DNAs to generate protein arrays on-demand and its use to validate protein-protein interactions. These expression plasmids were constructed in such a way so as to serve a dual purpose of synthesizing the protein of interest as well as capturing the synthesized protein. The microarray system is based on the high affinity binding of Escherichia coli "Tus" protein to "Ter," a 20 bp DNA sequence involved in the regulation of DNA replication. The protein expression is carried out in a cell-free protein synthesis system, with rabbit reticulocyte lysates, and the target proteins are detected either by labeled incorporated tag specific or by gene-specific antibodies. This microarray system has been successfully used for the detection of protein-protein interaction because both the target protein and the query protein can be transcribed and translated simultaneously in the microarray slides. The utility of this system for detecting protein-protein interaction is demonstrated by a few well-known examples: Jun/Fos, FRB/FKBP12, p53/MDM2, and CDK4/p16. In all these cases, the presence of protein complexes resulted in the localization of fluorophores at the specific sites of the immobilized target plasmids. Interestingly, during our interactions studies we also detected a previously unknown interaction between CDK2 and p16. Thus, this Tus-Ter based system of protein microarray can be used for the validation of known protein interactions as well as for identifying new protein-protein interactions. In addition, it can be used to examine and identify targets of nucleic acid-protein, ligand-receptor, enzyme-substrate, and drug-protein interactions.

Identification and Isolation of Protein Markers Associated with Somatic Embryogenesis in Oil Palm

Institute of Scientific and Technical Information of China (English)

Chin Chiew Foan; Nguyen Thi Thuy Van

2012-01-01

uncharacterized protein Sb07g019140 (C5YKJ0_SORBI).These proteins could be related to the amenability of the explants in tissue culture as they were also expressed abundantly only in leaves with high proliferation rate.

Characterizing the Syphilis-Causing Treponema pallidum ssp. pallidum Proteome Using Complementary Mass Spectrometry.

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Kara K Osbak

2016-09-01

Full Text Available The spirochete bacterium Treponema pallidum ssp. pallidum is the etiological agent of syphilis, a chronic multistage disease. Little is known about the global T. pallidum proteome, therefore mass spectrometry studies are needed to bring insights into pathogenicity and protein expression profiles during infection.To better understand the T. pallidum proteome profile during infection, we studied T. pallidum ssp. pallidum DAL-1 strain bacteria isolated from rabbits using complementary mass spectrometry techniques, including multidimensional peptide separation and protein identification via matrix-assisted laser desorption ionization-time of flight (MALDI-TOF/TOF and electrospray ionization (ESI-LTQ-Orbitrap tandem mass spectrometry. A total of 6033 peptides were detected, corresponding to 557 unique T. pallidum proteins at a high level of confidence, representing 54% of the predicted proteome. A previous gel-based T. pallidum MS proteome study detected 58 of these proteins. One hundred fourteen of the detected proteins were previously annotated as hypothetical or uncharacterized proteins; this is the first account of 106 of these proteins at the protein level. Detected proteins were characterized according to their predicted biological function and localization; half were allocated into a wide range of functional categories. Proteins annotated as potential membrane proteins and proteins with unclear functional annotations were subjected to an additional bioinformatics pipeline analysis to facilitate further characterization. A total of 116 potential membrane proteins were identified, of which 16 have evidence supporting outer membrane localization. We found 8/12 proteins related to the paralogous tpr gene family: TprB, TprC/D, TprE, TprG, TprH, TprI and TprJ. Protein abundance was semi-quantified using label-free spectral counting methods. A low correlation (r = 0.26 was found between previous microarray signal data and protein abundance.This is

Nanosilver pathophysiology in earthworms: Transcriptional profiling of secretory proteins and the implication for the protein corona

DEFF Research Database (Denmark)

Hayashi, Yuya; Miclaus, Teodora; Engelmann, Péter

2016-01-01

Previously we have identified lysenin as a key protein constituent of the secretome from Eisenia fetida coelomocytes and revealed its critical importance in priming interactions between the cells and the protein corona around nanosilver. As alterations of the protein environment can directly affe...

Isolation of the Binding Protein of Periplocoside E from BBMVs in Midgut of the Oriental Amyworm Mythimna separata Walker (Lepidoptera: Noctuidae through Affinity Chromatography

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Mingxing Feng

2016-05-01

Full Text Available Periplocosides, which are insecticidal compounds isolated from the root bark of Periploca sepium Bunge, can affect the digestive system of insects. However, the mechanism though which periplocosides induces a series of symptoms remains unknown. In this study, affinity chromatography was conducted by coupling periplocoside E-semi-succinic acid ester with epoxy amino hexyl (EAH sepharose 4B. Sodium dodecyl sulfonate-polyacrylamide gelelectrophoresis (SDS-PAGE was performed to analyze the fraction eluted by periplocoside E. Eight binding proteins (luciferin 4-monooxygenase, aminopeptidase N, aminopeptidase N3, nicotinamide adenine dinucleotide health (NADH dehydrogenase subunit 5, phosphatidylinositol 3-phosphate 3-phosphatase myotubularin, actin, uncharacterized family 31 glucosidase KIAA1161, and 2OG-Fe(2 oxygenase superfamily protein were obtained and identified through liquid chromatography/quadrupole-time of flight-mass spectrometry (LC/Q-TOF-MS analysis of the midgut epithelium cells of Mythimna separata larvae. Aminopeptidase N and N3 are potential putative targets of periplocosides. This study establishes the foundation for further research on the mechanism of action and target localization of periplocosides in agricultural pests.

The Chlamydomonas Genome Reveals the Evolution of Key Animal and Plant Functions

Energy Technology Data Exchange (ETDEWEB)

Merchant, Sabeeha S

2007-04-09

Chlamydomonas reinhardtii is a unicellular green alga whose lineage diverged from land plants over 1 billion years ago. It is a model system for studying chloroplast-based photosynthesis, as well as the structure, assembly, and function of eukaryotic flagella (cilia), which were inherited from the common ancestor of plants and animals, but lost in land plants. We sequenced the 120-megabase nuclear genome of Chlamydomonas and performed comparative phylogenomic analyses, identifying genes encoding uncharacterized proteins that are likely associated with the function and biogenesis of chloroplasts or eukaryotic flagella. Analyses of the Chlamydomonas genome advance our understanding of the ancestral eukaryotic cell, reveal previously unknown genes associated with photosynthetic and flagellar functions, and establish links between ciliopathy and the composition and function of flagella.

Characterization of Danio rerio Mn2+-dependent ADP-ribose/CDP-alcohol diphosphatase, the structural prototype of the ADPRibase-Mn-like protein family.

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Joaquim Rui Rodrigues

Full Text Available The ADPRibase-Mn-like protein family, that belongs to the metallo-dependent phosphatase superfamily, has different functional and structural prototypes. The functional one is the Mn(2+-dependent ADP-ribose/CDP-alcohol diphosphatase from Rattus norvegicus, which is essentially inactive with Mg(2+ and active with low micromolar Mn(2+ in the hydrolysis of the phosphoanhydride linkages of ADP-ribose, CDP-alcohols and cyclic ADP-ribose (cADPR in order of decreasing efficiency. The structural prototype of the family is a Danio rerio protein with a known crystallographic structure but functionally uncharacterized. To estimate the structure-function correlation with the same protein, the activities of zebrafish ADPRibase-Mn were studied. Differences between zebrafish and rat enzymes are highlighted. The former showed a complex activity dependence on Mn(2+, significant (≈25% Mg(2+-dependent activity, but was almost inactive on cADPR (150-fold less efficient than the rat counterpart. The low cADPR hydrolase activity agreed with the zebrafish genome lacking genes coding for proteins with significant homology with cADPR-forming enzymes. Substrate-docking to zebrafish wild-type protein, and characterization of the ADPRibase-Mn H97A mutant pointed to a role of His-97 in catalysis by orientation, and to a bidentate water bridging the dinuclear metal center as the potential nucleophile. Finally, three structural elements that delimit the active site entrance in the zebrafish protein were identified as unique to the ADPRibase-Mn-like family within the metallo-dependent phosphatase superfamily.

Chlamydia co-opts the rod shape-determining proteins MreB and Pbp2 for cell division.

Science.gov (United States)

Ouellette, Scot P; Karimova, Gouzel; Subtil, Agathe; Ladant, Daniel

2012-07-01

Chlamydiae are obligate intracellular bacterial pathogens that have extensively reduced their genome in adapting to the intracellular environment. The chlamydial genome contains only three annotated cell division genes and lacks ftsZ. How this obligate intracellular pathogen divides is uncharacterized. Chlamydiae contain two high-molecular-weight (HMW) penicillin binding proteins (Pbp) implicated in peptidoglycan synthesis, Pbp2 and Pbp3/FtsI. We show here, using HMW Pbp-specific penicillin derivatives, that both Pbp2 and Pbp3 are essential for chlamydial cell division. Ultrastructural analyses of antibiotic-treated cultures revealed distinct phenotypes: Pbp2 inhibition induced internal cell bodies within a single outer membrane whereas Pbp3 inhibition induced elongated phenotypes with little internal division. Each HMW Pbp interacts with the Chlamydia cell division protein FtsK. Chlamydiae are coccoid yet contain MreB, a rod shape-determining protein linked to Pbp2 in bacilli. Using MreB-specific antibiotics, we show that MreB is essential for chlamydial growth and division. Importantly, co-treatment with MreB-specific and Pbp-specific antibiotics resulted in the MreB-inhibited phenotype, placing MreB upstream of Pbp function in chlamydial cell division. Finally, we showed that MreB also interacts with FtsK. We propose that, in Chlamydia, MreB acts as a central co-ordinator at the division site to substitute for the lack of FtsZ in this bacterium. © 2012 Blackwell Publishing Ltd.

Molecular characterization of a Penicillium chrysogenum exo-rhamnogalacturonan lyase that is structurally distinct from other polysaccharide lyase family proteins.

Science.gov (United States)

Iwai, Marin; Kawakami, Takuya; Ikemoto, Takeshi; Fujiwara, Daisuke; Takenaka, Shigeo; Nakazawa, Masami; Ueda, Mitsuhiro; Sakamoto, Tatsuji

2015-10-01

We previously described an endo-acting rhamnogalacturonan (RG) lyase, termed PcRGL4A, of Penicillium chrysogenum 31B. Here, we describe a second RG lyase, called PcRGLX. We determined the cDNA sequence of the Pcrglx gene, which encodes PcRGLX. Based on analyses using a BLAST search and a conserved domain search, PcRGLX was found to be structurally distinct from known RG lyases and might belong to a new polysaccharide lyase family together with uncharacterized fungal proteins of Nectria haematococca, Aspergillus oryzae, and Fusarium oxysporum. The Pcrglx cDNA gene product (rPcRGLX) expressed in Escherichia coli demonstrated specific activity against RG but not against homogalacturonan. Divalent cations were not essential for the enzymatic activity of rPcRGLX. rPcRGLX mainly released unsaturated galacturonosyl rhamnose (ΔGR) from RG backbones used as the substrate from the initial stage of the reaction, indicating that the enzyme can be classified as an exo-acting RG lyase (EC 4.2.2.24). This is the first report of an RG lyase with this mode of action in Eukaryota. rPcRGLX acted synergistically with PcRGL4A to degrade soybean RG and released ΔGR. This ΔGR was partially decorated with galactose (Gal) residues, indicating that rPcRGLX preferred oligomeric RGs to polymeric RGs, that the enzyme did not require Gal decoration of RG backbones for degradation, and that the enzyme bypassed the Gal side chains of RG backbones. These characteristics of rPcRGLX might be useful in the determination of complex structures of pectins.

iPhos-PseEvo: Identifying Human Phosphorylated Proteins by Incorporating Evolutionary Information into General PseAAC via Grey System Theory.

Science.gov (United States)

Qiu, Wang-Ren; Sun, Bi-Qian; Xiao, Xuan; Xu, Dong; Chou, Kuo-Chen

2017-05-01

Protein phosphorylation plays a critical role in human body by altering the structural conformation of a protein, causing it to become activated/deactivated, or functional modification. Given an uncharacterized protein sequence, can we predict whether it may be phosphorylated or may not? This is no doubt a very meaningful problem for both basic research and drug development. Unfortunately, to our best knowledge, so far no high throughput bioinformatics tool whatsoever has been developed to address such a very basic but important problem due to its extremely complexity and lacking sufficient training data. Here we proposed a predictor called iPhos-PseEvo by (1) incorporating the protein sequence evolutionary information into the general pseudo amino acid composition (PseAAC) via the grey system theory, (2) balancing out the skewed training datasets by the asymmetric bootstrap approach, and (3) constructing an ensemble predictor by fusing an array of individual random forest classifiers thru a voting system. Rigorous jackknife tests have indicated that very promising success rates have been achieved by iPhos-PseEvo even for such a difficult problem. A user-friendly web-server for iPhos-PseEvo has been established at http://www.jci-bioinfo.cn/iPhos-PseEvo, by which users can easily obtain their desired results without the need to go through the complicated mathematical equations involved. It has not escaped our notice that the formulation and approach presented here can be used to analyze many other problems in protein science as well. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

AcEST: BP918427 [AcEST

Lifescience Database Archive (English)

Full Text Available tative uncharacterized protein OS=Vitis... 116 7e-25 tr|Q8RZ67|Q8RZ67_ORYSJ Putative rice retrotransposon retrofit... + Sbjct: 384 ATVRIILSLAVTSGLRLHKLDVKNAFLHGFLNEEVYMEQPPGYTDPY 430 >tr|Q8RZ67|Q8RZ67_ORYSJ Putative rice retrotransposon retrofit

AcEST: BP918938 [AcEST

Lifescience Database Archive (English)

Full Text Available 46_RAT Uncharacterized protein C6orf146 homolog OS=... 30 4.5 sp|O93988|ACL1_SORMA ATP-citrate synthase subunit 1 OS=Sordaria...ESPAEQLVLQLTVSEHAHSRSQNNNQGVFQLW 89 >sp|O93988|ACL1_SORMA ATP-citrate synthase subunit 1 OS=Sordaria macrosp

AcEST: DK960956 [AcEST

Lifescience Database Archive (English)

Full Text Available GR ++ EV S A Sbjct: 357 RLLTGLESITRGKPFIGDVRGRGLMLGLEVVSAEAA 392 >tr|B5JMW8|B5JMW8_9BACT Putative uncharacterized protein OS=Verrucom...icrobiae bacterium DG1235 GN=VDG1235_2840 PE=4 SV=1 Length = 963 Score = 35.4 bits

Protein tyrosine nitration in the cell cycle

International Nuclear Information System (INIS)

Jia, Min; Mateoiu, Claudia; Souchelnytskyi, Serhiy

2011-01-01

Highlights: → Enrichment of 3-nitrotyrosine containing proteins from cells synchronized in different phases of the cell cycle. → Identification of 76 tyrosine nitrated proteins that change expression during the cell cycle. → Nineteen identified proteins were previously described as regulators of cell proliferation. -- Abstract: Nitration of tyrosine residues in proteins is associated with cell response to oxidative/nitrosative stress. Tyrosine nitration is relatively low abundant post-translational modification that may affect protein functions. Little is known about the extent of protein tyrosine nitration in cells during progression through the cell cycle. Here we report identification of proteins enriched for tyrosine nitration in cells synchronized in G0/G1, S or G2/M phases of the cell cycle. We identified 27 proteins in cells synchronized in G0/G1 phase, 37 proteins in S phase synchronized cells, and 12 proteins related to G2/M phase. Nineteen of the identified proteins were previously described as regulators of cell proliferation. Thus, our data indicate which tyrosine nitrated proteins may affect regulation of the cell cycle.

An early cytoplasmic step of peptidoglycan synthesis is associated to MreB in Bacillus subtilis.

Science.gov (United States)

Rueff, Anne-Stéphanie; Chastanet, Arnaud; Domínguez-Escobar, Julia; Yao, Zhizhong; Yates, James; Prejean, Maria-Victoria; Delumeau, Olivier; Noirot, Philippe; Wedlich-Söldner, Roland; Filipe, Sergio R; Carballido-López, Rut

2014-01-01

MreB proteins play a major role during morphogenesis of rod-shaped bacteria by organizing biosynthesis of the peptidoglycan cell wall. However, the mechanisms underlying this process are not well understood. In Bacillus subtilis, membrane-associated MreB polymers have been shown to be associated to elongation-specific complexes containing transmembrane morphogenetic factors and extracellular cell wall assembly proteins. We have now found that an early intracellular step of cell wall synthesis is also associated to MreB. We show that the previously uncharacterized protein YkuR (renamed DapI) is required for synthesis of meso-diaminopimelate (m-DAP), an essential constituent of the peptidoglycan precursor, and that it physically interacts with MreB. Highly inclined laminated optical sheet microscopy revealed that YkuR forms uniformly distributed foci that exhibit fast motion in the cytoplasm, and are not detected in cells lacking MreB. We propose a model in which soluble MreB organizes intracellular steps of peptidoglycan synthesis in the cytoplasm to feed the membrane-associated cell wall synthesizing machineries. © 2013 John Wiley & Sons Ltd.

ISG15 counteracts Listeria monocytogenes infection

Science.gov (United States)

Radoshevich, Lilliana; Impens, Francis; Ribet, David; Quereda, Juan J; Nam Tham, To; Nahori, Marie-Anne; Bierne, Hélène; Dussurget, Olivier; Pizarro-Cerdá, Javier; Knobeloch, Klaus-Peter; Cossart, Pascale

2015-01-01

ISG15 is an interferon-stimulated, linear di-ubiquitin-like protein, with anti-viral activity. The role of ISG15 during bacterial infection remains elusive. We show that ISG15 expression in nonphagocytic cells is dramatically induced upon Listeria infection. Surprisingly this induction can be type I interferon independent and depends on the cytosolic surveillance pathway, which senses bacterial DNA and signals through STING, TBK1, IRF3 and IRF7. Most importantly, we observed that ISG15 expression restricts Listeria infection in vitro and in vivo. We made use of stable isotope labeling in tissue culture (SILAC) to identify ISGylated proteins that could be responsible for the protective effect. Strikingly, infection or overexpression of ISG15 leads to ISGylation of ER and Golgi proteins, which correlates with increased secretion of cytokines known to counteract infection. Together, our data reveal a previously uncharacterized ISG15-dependent restriction of Listeria infection, reinforcing the view that ISG15 is a key component of the innate immune response. DOI: http://dx.doi.org/10.7554/eLife.06848.001 PMID:26259872

Interaction between plate make and protein in protein crystallisation screening.

Directory of Open Access Journals (Sweden)

Gordon J King

Full Text Available BACKGROUND: Protein crystallisation screening involves the parallel testing of large numbers of candidate conditions with the aim of identifying conditions suitable as a starting point for the production of diffraction quality crystals. Generally, condition screening is performed in 96-well plates. While previous studies have examined the effects of protein construct, protein purity, or crystallisation condition ingredients on protein crystallisation, few have examined the effect of the crystallisation plate. METHODOLOGY/PRINCIPAL FINDINGS: We performed a statistically rigorous examination of protein crystallisation, and evaluated interactions between crystallisation success and plate row/column, different plates of same make, different plate makes and different proteins. From our analysis of protein crystallisation, we found a significant interaction between plate make and the specific protein being crystallised. CONCLUSIONS/SIGNIFICANCE: Protein crystal structure determination is the principal method for determining protein structure but is limited by the need to produce crystals of the protein under study. Many important proteins are difficult to crystallize, so that identification of factors that assist crystallisation could open up the structure determination of these more challenging targets. Our findings suggest that protein crystallisation success may be improved by matching a protein with its optimal plate make.

Characterization of novel and uncharacterized p53 SNPs in the Chinese population--intron 2 SNP co-segregates with the common codon 72 polymorphism.

Directory of Open Access Journals (Sweden)

Beng Hooi Phang

Full Text Available Multiple single nucleotide polymorphisms (SNPs have been identified in the tumor suppressor gene p53, though the relevance of many of them is unclear. Some of them are also differentially distributed in various ethnic populations, suggesting selective functionality. We have therefore sequenced all exons and flanking regions of p53 from the Singaporean Chinese population and report here the characterization of some novel and uncharacterized SNPs - four in intron 1 (nucleotide positions 8759/10361/10506/11130, three in intron 3 (11968/11969/11974 and two in the 3'UTR (19168/19514. Allelic frequencies were determined for all these and some known SNPs, and were compared in a limited scale to leukemia and lung cancer patient samples. Intron 2 (11827 and 7 (14181/14201 SNPs were found to have a high minor allele frequency of between 26-47%, in contrast to the lower frequencies found in the US population, but similar in trend to the codon 72 polymorphism (SNP12139 that shows a distribution pattern correlative with latitude. Several of the SNPs were linked, such as those in introns 1, 3 and 7. Most interestingly, we noticed the co-segregation of the intron 2 and the codon 72 SNPs, the latter which has been shown to be expressed in an allele-specific manner, suggesting possible regulatory cross-talk. Association analysis indicated that the T/G alleles in both the co-segregating intron 7 SNPs and a 4tagSNP haplotype was strongly associated increased susceptibility to lung cancer in non-smoker females [OR: 1.97 (1.32, 3.394]. These data together demonstrate high SNP diversity in p53 gene between different populations, highlighting ethnicity-based differences, and their association with cancer risk.

2018-04-28T19:44:25Z https://www.ajol.info/index.php/all/oai oai:ojs ...

African Journals Online (AJOL)

Mechanically transmissible viruses were isolated from Amaranthus hybridus, Celosia argentea, Cucurbita moschata, Telfairia occidentalis and Brassica oleracea, while virus-like symptoms (causal agents not sap-transmissible), were observed on Solanum macrocarpon. Two previously uncharacterized viruses, one each ...

Niakha virus: a novel member of the family Rhabdoviridae isolated from phlebotomine sandflies in Senegal.

Science.gov (United States)

Vasilakis, Nikos; Widen, Steven; Mayer, Sandra V; Seymour, Robert; Wood, Thomas G; Popov, Vsevolov; Guzman, Hilda; Travassos da Rosa, Amelia P A; Ghedin, Elodie; Holmes, Edward C; Walker, Peter J; Tesh, Robert B

2013-09-01

Members of the family Rhabdoviridae have been assigned to eight genera but many remain unassigned. Rhabdoviruses have a remarkably diverse host range that includes terrestrial and marine animals, invertebrates and plants. Transmission of some rhabdoviruses often requires an arthropod vector, such as mosquitoes, midges, sandflies, ticks, aphids and leafhoppers, in which they replicate. Herein we characterize Niakha virus (NIAV), a previously uncharacterized rhabdovirus isolated from phebotomine sandflies in Senegal. Analysis of the 11,124 nt genome sequence indicates that it encodes the five common rhabdovirus proteins with alternative ORFs in the M, G and L genes. Phylogenetic analysis of the L protein indicate that NIAV's closest relative is Oak Vale rhabdovirus, although in this analysis NIAV is still so phylogenetically distinct that it might be classified as distinct from the eight currently recognized Rhabdoviridae genera. This observation highlights the vast, and yet not fully recognized diversity, of this family. Copyright © 2013 Elsevier Inc. All rights reserved.

Niakha virus: A novel member of the family Rhabdoviridae isolated from phlebotomine sandflies in Senegal

Science.gov (United States)

Vasilakis, Nikos; Widen, Steven; Mayer, Sandra V.; Seymour, Robert; Wood, Thomas G.; Popov, Vsevolov; Guzman, Hilda; da Rosa, Amelia P.A. Travassos; Ghedin, Elodie; Holmes, Edward C.; Walker, Peter J.; Tesh, Robert B.

2013-01-01

Members of the family Rhabdoviridae have been assigned to eight genera but many remain unassigned. Rhabdoviruses have a remarkably diverse host range that includes terrestrial and marine animals, invertebrates and plants. Transmission of some rhabdoviruses often requires an arthropod vector, such as mosquitoes, midges, sandflies, ticks, aphids and leafhoppers, in which they replicate. Herein we characterize Niakha virus (NIAV), a previously uncharacterized rhabdovirus isolated from phebotomine sandflies in Senegal. Analysis of the 11,124 nt genome sequence indicates that it encodes the five common rhabdovirus proteins with alternative ORFs in the M, G and L genes. Phylogenetic analysis of the L protein indicate that NIAV’s closest relative is Oak Vale rhabdovirus, although in this analysis NIAV is still so phylogenetically distinct that it might be classified as distinct from the eight currently recognized Rhabdoviridae genera. This observation highlights the vast, and yet not fully recognized diversity, of this family. PMID:23773405

Identification and functional verification of archaeal-type phosphoenolpyruvate carboxylase, a missing link in archaeal central carbohydrate metabolism.

Science.gov (United States)

Ettema, Thijs J G; Makarova, Kira S; Jellema, Gera L; Gierman, Hinco J; Koonin, Eugene V; Huynen, Martijn A; de Vos, Willem M; van der Oost, John

2004-11-01

Despite the fact that phosphoenolpyruvate carboxylase (PEPC) activity has been measured and in some cases even purified from some Archaea, the gene responsible for this activity has not been elucidated. Using sensitive sequence comparison methods, we detected a highly conserved, uncharacterized archaeal gene family that is distantly related to the catalytic core of the canonical PEPC. To verify the predicted function of this archaeal gene family, we cloned a representative from the hyperthermophilic acidophile Sulfolobus solfataricus and functionally produced the corresponding enzyme as a fusion with the Escherichia coli maltose-binding protein. The purified fusion protein indeed displayed highly thermostable PEPC activity. The structural and biochemical properties of the characterized archaeal-type PEPC (atPEPC) from S. solfataricus are in good agreement with previously reported biochemical analyses of other archaeal PEPC enzymes. The newly identified atPEPC, with its distinct properties, constitutes yet another example of the versatility of the enzymes of the central carbon metabolic pathways in the archaeal domain.

AcEST: DK962031 [AcEST

Lifescience Database Archive (English)

Full Text Available VSFLTGLGCPNCIECFTSQGLQ 392 >tr|B1AX90|B1AX90_MOUSE Transformation related protein 73 OS=Mus musculus GN=Trp7...tr|B1AX90|B1AX90_MOUSE Transformation related protein 73 OS=Mus ... 37 1.5 tr|B7Q4E7|B7Q4E7_IXOSC Putative u...rized protein OS=Mus m... 37 1.5 tr|Q9CU77|Q9CU77_MOUSE Putative uncharacterized protein (Fragmen... 37 1.5

The intake of total protein, natural protein and protein substitute and growth of height and head circumference in Dutch infants with phenylketonuria

NARCIS (Netherlands)

Hoeksma, M.; van Rijn, M. [=Margreet; Verkerk, P. H.; Bosch, A. M.; Mulder, M. F.; de Klerk, J. B. C.; de Koning, T. J.; Rubio-Gozalbo, E.; de Vries, M.; Sauer, P. J. J.; van Spronsen, F. J.

2005-01-01

In a previous study, Dutch children with phenylketonuria (PKU) were found to be slightly shorter than their healthy counterparts. In the literature, it has been hypothesized that a higher protein intake is necessary to optimize growth in PKU patients. The study aimed to investigate whether protein

Impact of protein uptake and degradation on recombinant protein secretion in yeast

DEFF Research Database (Denmark)

Tyo, Keith E. J.; Liu, Zihe; Magnusson, Ylva

2014-01-01

Protein titers, a key bioprocessing metric, depend both on the synthesis of protein and the degradation of protein. Secreted recombinant protein production in Saccharomyces cerevisiae is an attractive platform as minimal media can be used for cultivation, thus reducing fermentation costs...... and transcriptomics, we identify metabolic and regulatory markers that are consistent with uptake of whole proteins by endocytosis, followed by intracellular degradation and catabolism of substituent amino acids. Uptake and degradation of recombinant protein products may be common in S. cerevisiae protein secretion...... and simplifying downstream purification, compared to other systems that require complex media. As such, engineering S. cerevisiae to improve titers has been then the subject of significant attention, but the majority of previous efforts have been focused on improving protein synthesis. Here, we characterize...

The nematode homologue of Mediator complex subunit 28, F28F8.5, is a critical regulator of C. elegans development.

Science.gov (United States)

Kostrouchová, Markéta; Kostrouch, David; Chughtai, Ahmed A; Kaššák, Filip; Novotný, Jan P; Kostrouchová, Veronika; Benda, Aleš; Krause, Michael W; Saudek, Vladimír; Kostrouchová, Marta; Kostrouch, Zdeněk

2017-01-01

The evolutionarily conserved Mediator complex is a critical player in regulating transcription. Comprised of approximately two dozen proteins, the Mediator integrates diverse regulatory signals through direct protein-protein interactions that, in turn, modulate the influence of Mediator on RNA Polymerase II activity. One Mediator subunit, MED28, is known to interact with cytoplasmic structural proteins, providing a potential direct link between cytoplasmic dynamics and the control of gene transcription. Although identified in many animals and plants, MED28 is not present in yeast; no bona fide MED28 has been described previously in Caenorhabditis elegans. Here, we identify bioinformatically F28F8.5, an uncharacterized predicted protein, as the nematode homologue of MED28. As in other Metazoa, F28F8.5 has dual nuclear and cytoplasmic localization and plays critical roles in the regulation of development. F28F8.5 is a vital gene and its null mutants have severely malformed gonads and do not reproduce. F28F8.5 interacts on the protein level with the Mediator subunits MDT-6 and MDT-30. Our results indicate that F28F8.5 is an orthologue of MED28 and suggest that the potential to link cytoplasmic and nuclear events is conserved between MED28 vertebrate and nematode orthologues.

Disruption of SMIM1 causes the Vel− blood type

Science.gov (United States)

Ballif, Bryan A; Helias, Virginie; Peyrard, Thierry; Menanteau, Cécile; Saison, Carole; Lucien, Nicole; Bourgouin, Sébastien; Le Gall, Maude; Cartron, Jean-Pierre; Arnaud, Lionel

2013-01-01

Here, we report the biochemical and genetic basis of the Vel blood group antigen, which has been a vexing mystery for decades, especially as anti-Vel regularly causes severe haemolytic transfusion reactions. The protein carrying the Vel blood group antigen was biochemically purified from red blood cell membranes. Mass spectrometry-based de novo peptide sequencing identified this protein to be small integral membrane protein 1 (SMIM1), a previously uncharacterized single-pass membrane protein. Expression of SMIM1 cDNA in Vel− cultured cells generated anti-Vel cell surface reactivity, confirming that SMIM1 encoded the Vel blood group antigen. A cohort of 70 Vel− individuals was found to be uniformly homozygous for a 17 nucleotide deletion in the coding sequence of SMIM1. The genetic homogeneity of the Vel− blood type, likely having a common origin, facilitated the development of two highly specific DNA-based tests for rapid Vel genotyping, which can be easily integrated into blood group genotyping platforms. These results answer a 60-year-old riddle and provide tools of immediate assistance to all clinicians involved in the care of Vel− patients. PMID:23505126

Bioinformatic evidence for a widely distributed, ribosomally produced electron carrier precursor, its maturation proteins, and its nicotinoprotein redox partners

Directory of Open Access Journals (Sweden)

Haft Daniel H

2011-01-01

Full Text Available Abstract Background Enzymes in the radical SAM (rSAM domain family serve in a wide variety of biological processes, including RNA modification, enzyme activation, bacteriocin core peptide maturation, and cofactor biosynthesis. Evolutionary pressures and relationships to other cellular constituents impose recognizable grammars on each class of rSAM-containing system, shaping patterns in results obtained through various comparative genomics analyses. Results An uncharacterized gene cluster found in many Actinobacteria and sporadically in Firmicutes, Chloroflexi, Deltaproteobacteria, and one Archaeal plasmid contains a PqqE-like rSAM protein family that includes Rv0693 from Mycobacterium tuberculosis. Members occur clustered with a strikingly well-conserved small polypeptide we designate "mycofactocin," similar in size to bacteriocins and PqqA, precursor of pyrroloquinoline quinone (PQQ. Partial Phylogenetic Profiling (PPP based on the distribution of these markers identifies the mycofactocin cluster, but also a second tier of high-scoring proteins. This tier, strikingly, is filled with up to thirty-one members per genome from three variant subfamilies that occur, one each, in three unrelated classes of nicotinoproteins. The pattern suggests these variant enzymes require not only NAD(P, but also the novel gene cluster. Further study was conducted using SIMBAL, a PPP-like tool, to search these nicotinoproteins for subsequences best correlated across multiple genomes to the presence of mycofactocin. For both the short chain dehydrogenase/reductase (SDR and iron-containing dehydrogenase families, aligning SIMBAL's top-scoring sequences to homologous solved crystal structures shows signals centered over NAD(P-binding sites rather than over substrate-binding or active site residues. Previous studies on some of these proteins have revealed a non-exchangeable NAD cofactor, such that enzymatic activity in vitro requires an artificial electron acceptor such

Mating in the Closest Living Relatives of Animals Is Induced by a Bacterial Chondroitinase.

Science.gov (United States)

Woznica, Arielle; Gerdt, Joseph P; Hulett, Ryan E; Clardy, Jon; King, Nicole

2017-09-07

We serendipitously discovered that the marine bacterium Vibrio fischeri induces sexual reproduction in one of the closest living relatives of animals, the choanoflagellate Salpingoeca rosetta. Although bacteria influence everything from nutrition and metabolism to cell biology and development in eukaryotes, bacterial regulation of eukaryotic mating was unexpected. Here, we show that a single V. fischeri protein, the previously uncharacterized EroS, fully recapitulates the aphrodisiac-like activity of live V. fischeri. EroS is a chondroitin lyase; although its substrate, chondroitin sulfate, was previously thought to be an animal synapomorphy, we demonstrate that S. rosetta produces chondroitin sulfate and thus extend the ancestry of this important glycosaminoglycan to the premetazoan era. Finally, we show that V. fischeri, purified EroS, and other bacterial chondroitin lyases induce S. rosetta mating at environmentally relevant concentrations, suggesting that bacteria likely regulate choanoflagellate mating in nature. Copyright © 2017 Elsevier Inc. All rights reserved.

On the performance of de novo pathway enrichment

DEFF Research Database (Denmark)

Batra, Richa; Alcaraz, Nicolas; Gitzhofer, Kevin

2017-01-01

De novo pathway enrichment is a powerful approach to discover previously uncharacterized molecular mechanisms in addition to already known pathways. To achieve this, condition-specific functional modules are extracted from large interaction networks. Here, we give an overview of the state...

Differential binding of calmodulin-related proteins to their targets revealed through high-density Arabidopsis protein microarrays

Science.gov (United States)

Popescu, Sorina C.; Popescu, George V.; Bachan, Shawn; Zhang, Zimei; Seay, Montrell; Gerstein, Mark; Snyder, Michael; Dinesh-Kumar, S. P.

2007-01-01

Calmodulins (CaMs) are the most ubiquitous calcium sensors in eukaryotes. A number of CaM-binding proteins have been identified through classical methods, and many proteins have been predicted to bind CaMs based on their structural homology with known targets. However, multicellular organisms typically contain many CaM-like (CML) proteins, and a global identification of their targets and specificity of interaction is lacking. In an effort to develop a platform for large-scale analysis of proteins in plants we have developed a protein microarray and used it to study the global analysis of CaM/CML interactions. An Arabidopsis thaliana expression collection containing 1,133 ORFs was generated and used to produce proteins with an optimized medium-throughput plant-based expression system. Protein microarrays were prepared and screened with several CaMs/CMLs. A large number of previously known and novel CaM/CML targets were identified, including transcription factors, receptor and intracellular protein kinases, F-box proteins, RNA-binding proteins, and proteins of unknown function. Multiple CaM/CML proteins bound many binding partners, but the majority of targets were specific to one or a few CaMs/CMLs indicating that different CaM family members function through different targets. Based on our analyses, the emergent CaM/CML interactome is more extensive than previously predicted. Our results suggest that calcium functions through distinct CaM/CML proteins to regulate a wide range of targets and cellular activities. PMID:17360592

Annotating the protein-RNA interaction sites in proteins using evolutionary information and protein backbone structure.

Science.gov (United States)

Li, Tao; Li, Qian-Zhong

2012-11-07

RNA-protein interactions play important roles in various biological processes. The precise detection of RNA-protein interaction sites is very important for understanding essential biological processes and annotating the function of the proteins. In this study, based on various features from amino acid sequence and structure, including evolutionary information, solvent accessible surface area and torsion angles (φ, ψ) in the backbone structure of the polypeptide chain, a computational method for predicting RNA-binding sites in proteins is proposed. When the method is applied to predict RNA-binding sites in three datasets: RBP86 containing 86 protein chains, RBP107 containing 107 proteins chains and RBP109 containing 109 proteins chains, better sensitivities and specificities are obtained compared to previously published methods in five-fold cross-validation tests. In order to make further examination for the efficiency of our method, the RBP107 dataset is used as training set, RBP86 and RBP109 datasets are used as the independent test sets. In addition, as examples of our prediction, RNA-binding sites in a few proteins are presented. The annotated results are consistent with the PDB annotation. These results show that our method is useful for annotating RNA binding sites of novel proteins.

Model of OSBP-Mediated Cholesterol Supply to Aichi Virus RNA Replication Sites Involving Protein-Protein Interactions among Viral Proteins, ACBD3, OSBP, VAP-A/B, and SAC1.

Science.gov (United States)

Ishikawa-Sasaki, Kumiko; Nagashima, Shigeo; Taniguchi, Koki; Sasaki, Jun

2018-04-15

Positive-strand RNA viruses, including picornaviruses, utilize cellular machinery for genome replication. Previously, we reported that each of the 2B, 2BC, 2C, 3A, and 3AB proteins of Aichi virus (AiV), a picornavirus, forms a complex with the Golgi apparatus protein ACBD3 and phosphatidylinositol 4-kinase IIIβ (PI4KB) at viral RNA replication sites (replication organelles [ROs]), enhancing PI4KB-dependent phosphatidylinositol 4-phosphate (PI4P) production. Here, we demonstrate AiV hijacking of the cellular cholesterol transport system involving oxysterol-binding protein (OSBP), a PI4P-binding cholesterol transfer protein. AiV RNA replication was inhibited by silencing cellular proteins known to be components of this pathway, OSBP, the ER membrane proteins VAPA and VAPB (VAP-A/B), the PI4P-phosphatase SAC1, and PI-transfer protein β. OSBP, VAP-A/B, and SAC1 were present at RNA replication sites. We also found various previously unknown interactions among the AiV proteins (2B, 2BC, 2C, 3A, and 3AB), ACBD3, OSBP, VAP-A/B, and SAC1, and the interactions were suggested to be involved in recruiting the component proteins to AiV ROs. Importantly, the OSBP-2B interaction enabled PI4P-independent recruitment of OSBP to AiV ROs, indicating preferential recruitment of OSBP among PI4P-binding proteins. Protein-protein interaction-based OSBP recruitment has not been reported for other picornaviruses. Cholesterol was accumulated at AiV ROs, and inhibition of OSBP-mediated cholesterol transfer impaired cholesterol accumulation and AiV RNA replication. Electron microscopy showed that AiV-induced vesicle-like structures were close to ER membranes. Altogether, we conclude that AiV directly recruits the cholesterol transport machinery through protein-protein interactions, resulting in formation of membrane contact sites between the ER and AiV ROs and cholesterol supply to the ROs. IMPORTANCE Positive-strand RNA viruses utilize host pathways to modulate the lipid composition of

The Major Outer Membrane Protein MopB Is Required for Twitching Movement and Affects Biofilm Formation and Virulence in Two Xylella fastidiosa strains.

Science.gov (United States)

Chen, Hongyu; Kandel, Prem P; Cruz, Luisa F; Cobine, Paul A; De La Fuente, Leonardo

2017-11-01

MopB is a major outer membrane protein (OMP) in Xylella fastidiosa, a bacterial plant pathogen that causes losses on many economically important crops. Based on in silico analysis, the uncharacterized MopB protein of X. fastidiosa contains a β-barrel structure with an OmpA-like domain and a predicted calcium-binding motif. Here, MopB function was studied by mutational analysis taking advantage of the natural competence of X. fastidiosa. Mutants of mopB were constructed in two different X. fastidiosa strains, the type strain Temecula and the more virulent WM1-1. Deletion of the mopB gene impaired cell-to-cell aggregation, surface attachment, and biofilm formation in both strains. Interestingly, mopB deletion completely abolished twitching motility. Electron microscopy of the bacterial cell surface revealed that mopB deletion eliminated type IV and type I pili formation, potentially caused by destabilization of the outer membrane. Both mopB mutants showed reduced virulence using tobacco (Nicotiana tabacum) as a host under greenhouse conditions. These results suggest that MopB has pleiotropic functions in biofilm formation and twitching motility and is important for virulence of X. fastidiosa.

Two Chimeric Regulators of G-protein Signaling (RGS) Proteins Differentially Modulate Soybean Heterotrimeric G-protein Cycle*

Science.gov (United States)

Roy Choudhury, Swarup; Westfall, Corey S.; Laborde, John P.; Bisht, Naveen C.; Jez, Joseph M.; Pandey, Sona

2012-01-01

Heterotrimeric G-proteins and the regulator of G-protein signaling (RGS) proteins, which accelerate the inherent GTPase activity of Gα proteins, are common in animals and encoded by large gene families; however, in plants G-protein signaling is thought to be more limited in scope. For example, Arabidopsis thaliana contains one Gα, one Gβ, three Gγ, and one RGS protein. Recent examination of the Glycine max (soybean) genome reveals a larger set of G-protein-related genes and raises the possibility of more intricate G-protein networks than previously observed in plants. Stopped-flow analysis of GTP-binding and GDP/GTP exchange for the four soybean Gα proteins (GmGα1–4) reveals differences in their kinetic properties. The soybean genome encodes two chimeric RGS proteins with an N-terminal seven transmembrane domain and a C-terminal RGS box. Both GmRGS interact with each of the four GmGα and regulate their GTPase activity. The GTPase-accelerating activities of GmRGS1 and -2 differ for each GmGα, suggesting more than one possible rate of the G-protein cycle initiated by each of the Gα proteins. The differential effects of GmRGS1 and GmRGS2 on GmGα1–4 result from a single valine versus alanine difference. The emerging picture suggests complex regulation of the G-protein cycle in soybean and in other plants with expanded G-protein networks. PMID:22474294

Perturbation metatranscriptomics for studying complex microbial communities

DEFF Research Database (Denmark)

Williams, Rohan B.H.; Kirkegaard, Rasmus Hansen; Arumugam, Krithika

Studying the functional state of natural or engineered microbial communities presents substantial challenges due to both the complexities of field sampling, and, in the laboratory context, the inability of culture or reactor systems to maintain community composition ex situ over long periods. Here...... correlation between orthologous genes (Pearson r=0.4). We also used these data to annotate uncharacterized genes in the Ca. nitrospira defluvii genome: finding clear evidence for several previously unrecognized denitrification related genes, using a combination of expression profiles and protein domain data...... are associated with the transition from anoxic to aerobic conditions, and are observable at a whole community level and 3) these data provide a means of identifying unannotated genes in reference genomes that are likely to be associated with specific functional processes. More broadly, our approach permits...

Optimised 'on demand' protein arraying from DNA by cell free expression with the 'DNA to Protein Array' (DAPA) technology.

Science.gov (United States)

Schmidt, Ronny; Cook, Elizabeth A; Kastelic, Damjana; Taussig, Michael J; Stoevesandt, Oda

2013-08-02

We have previously described a protein arraying process based on cell free expression from DNA template arrays (DNA Array to Protein Array, DAPA). Here, we have investigated the influence of different array support coatings (Ni-NTA, Epoxy, 3D-Epoxy and Polyethylene glycol methacrylate (PEGMA)). Their optimal combination yields an increased amount of detected protein and an optimised spot morphology on the resulting protein array compared to the previously published protocol. The specificity of protein capture was improved using a tag-specific capture antibody on a protein repellent surface coating. The conditions for protein expression were optimised to yield the maximum amount of protein or the best detection results using specific monoclonal antibodies or a scaffold binder against the expressed targets. The optimised DAPA system was able to increase by threefold the expression of a representative model protein while conserving recognition by a specific antibody. The amount of expressed protein in DAPA was comparable to those of classically spotted protein arrays. Reaction conditions can be tailored to suit the application of interest. DAPA represents a cost effective, easy and convenient way of producing protein arrays on demand. The reported work is expected to facilitate the application of DAPA for personalized medicine and screening purposes. Copyright © 2013 Elsevier B.V. All rights reserved.

AcEST: DK957284 [AcEST

Lifescience Database Archive (English)

Full Text Available tr|Q092J0|Q092J0_STIAU Putative uncharacterized protein OS=Stigm... 40 0.094 tr|Q69340|Q69340_9ALPH Pseudorabies...PP--------VPLPRPVPGCGE 263 >tr|Q69340|Q69340_9ALPH Pseudorabies virus ORF1, ORF2, and ORF3 OS=Suid herpesvir

AcEST: BP917903 [AcEST

Lifescience Database Archive (English)

Full Text Available Uncharacterized protein KIAA2012 homolog O... 40 0.005 sp|Q7M3Y3|TNNI_CHLNI Troponin I OS=Chlamys nipponensis akazara...RQWQEEEERRQ 610 >sp|Q7M3Y3|TNNI_CHLNI Troponin I OS=Chlamys nipponensis akazara PE=1 SV=2 Length = 292 Score

Preparation of GST Fusion Proteins.

Science.gov (United States)

Einarson, Margret B; Pugacheva, Elena N; Orlinick, Jason R

2007-04-01

INTRODUCTIONThis protocol describes the preparation of glutathione-S-transferase (GST) fusion proteins, which have had a wide range of applications since their introduction as tools for synthesis of recombinant proteins in bacteria. GST was originally selected as a fusion moiety because of several desirable properties. First and foremost, when expressed in bacteria alone, or as a fusion, GST is not sequestered in inclusion bodies (in contrast to previous fusion protein systems). Second, GST can be affinity-purified without denaturation because it binds to immobilized glutathione, which provides the basis for simple purification. Consequently, GST fusion proteins are routinely used for antibody generation and purification, protein-protein interaction studies, and biochemical analysis.

Protein Correlation Profiles Identify Lipid Droplet Proteins with High Confidence*

Science.gov (United States)

Krahmer, Natalie; Hilger, Maximiliane; Kory, Nora; Wilfling, Florian; Stoehr, Gabriele; Mann, Matthias; Farese, Robert V.; Walther, Tobias C.

2013-01-01

Lipid droplets (LDs) are important organelles in energy metabolism and lipid storage. Their cores are composed of neutral lipids that form a hydrophobic phase and are surrounded by a phospholipid monolayer that harbors specific proteins. Most well-established LD proteins perform important functions, particularly in cellular lipid metabolism. Morphological studies show LDs in close proximity to and interacting with membrane-bound cellular organelles, including the endoplasmic reticulum, mitochondria, peroxisomes, and endosomes. Because of these close associations, it is difficult to purify LDs to homogeneity. Consequently, the confident identification of bona fide LD proteins via proteomics has been challenging. Here, we report a methodology for LD protein identification based on mass spectrometry and protein correlation profiles. Using LD purification and quantitative, high-resolution mass spectrometry, we identified LD proteins by correlating their purification profiles to those of known LD proteins. Application of the protein correlation profile strategy to LDs isolated from Drosophila S2 cells led to the identification of 111 LD proteins in a cellular LD fraction in which 1481 proteins were detected. LD localization was confirmed in a subset of identified proteins via microscopy of the expressed proteins, thereby validating the approach. Among the identified LD proteins were both well-characterized LD proteins and proteins not previously known to be localized to LDs. Our method provides a high-confidence LD proteome of Drosophila cells and a novel approach that can be applied to identify LD proteins of other cell types and tissues. PMID:23319140

Computational Studies of Snake Venom Toxins

OpenAIRE

Paola G. Ojeda; David Ramírez; Jans Alzate-Morales; Julio Caballero; Quentin Kaas; Wendy González

2017-01-01

Most snake venom toxins are proteins, and participate to envenomation through a diverse array of bioactivities, such as bleeding, inflammation, and pain, cytotoxic, cardiotoxic or neurotoxic effects. The venom of a single snake species contains hundreds of toxins, and the venoms of the 725 species of venomous snakes represent a large pool of potentially bioactive proteins. Despite considerable discovery efforts, most of the snake venom toxins are still uncharacterized. Modern bioinformatics t...

Generation of Adducts of 4-Hydroxy-2-nonenal with Heat Shock 60 kDa Protein 1 in Human Promyelocytic HL-60 and Monocytic THP-1 Cell Lines

Directory of Open Access Journals (Sweden)

Alessia Arcaro

2015-01-01

Full Text Available Heat shock 60 kDa protein 1 (HSP60 is a chaperone and stress response protein responsible for protein folding and delivery of endogenous peptides to antigen-presenting cells and also a target of autoimmunity implicated in the pathogenesis of atherosclerosis. By two-dimensional electrophoresis and mass spectrometry, we found that exposure of human promyelocytic HL-60 cells to a nontoxic concentration (10 μM of 4-hydroxy-2-nonenal (HNE yielded a HSP60 modified with HNE. We also detected adducts of HNE with putative uncharacterized protein CXorf49, the product of an open reading frame identified in various cell and tissue proteomes. Moreover, exposure of human monocytic THP-1 cells differentiated with phorbol 12-myristate 13-acetate to 10 μM HNE, and to light density lipoprotein modified with HNE (HNE-LDL or by copper-catalyzed oxidation (oxLDL, but not to native LDL, stimulated the formation of HNE adducts with HSP60, as detected by immunoprecipitation and western blot, well over basal levels. The identification of HNE-HSP60 adducts outlines a framework of mutually reinforcing interactions between endothelial cell stressors, like oxLDL and HSP60, whose possible outcomes, such as the amplification of endothelial dysfunction, the spreading of lipoxidative damage to other proteins, such as CXorf49, the activation of antigen-presenting cells, and the breaking of tolerance to HSP60 are discussed.

Sequence analysis reveals how G protein-coupled receptors transduce the signal to the G protein.

NARCIS (Netherlands)

Oliveira, L.; Paiva, P.B.; Paiva, A.C.; Vriend, G.

2003-01-01

Sequence entropy-variability plots based on alignments of very large numbers of sequences-can indicate the location in proteins of the main active site and modulator sites. In the previous article in this issue, we applied this observation to a series of well-studied proteins and concluded that it

Consumption of Milk Protein or Whey Protein Results in a Similar Increase in Muscle Protein Synthesis in Middle Aged Men.

Science.gov (United States)

Mitchell, Cameron J; McGregor, Robin A; D'Souza, Randall F; Thorstensen, Eric B; Markworth, James F; Fanning, Aaron C; Poppitt, Sally D; Cameron-Smith, David

2015-10-21

The differential ability of various milk protein fractions to stimulate muscle protein synthesis (MPS) has been previously described, with whey protein generally considered to be superior to other fractions. However, the relative ability of a whole milk protein to stimulate MPS has not been compared to whey. Sixteen healthy middle-aged males ingested either 20 g of milk protein (n = 8) or whey protein (n = 8) while undergoing a primed constant infusion of ring (13)C₆ phenylalanine. Muscle biopsies were obtained 120 min prior to consumption of the protein and 90 and 210 min afterwards. Resting myofibrillar fractional synthetic rates (FSR) were 0.019% ± 0.009% and 0.021% ± 0.018% h(-1) in the milk and whey groups respectively. For the first 90 min after protein ingestion the FSR increased (p whey groups respectively with no difference between groups (p = 0.810). FSR returned to baseline in both groups between 90 and 210 min after protein ingestion. Despite evidence of increased rate of digestion and leucine availability following the ingestion of whey protein, there was similar activation of MPS in middle-aged men with either 20 g of milk protein or whey protein.

iHyd-PseAAC: Predicting Hydroxyproline and Hydroxylysine in Proteins by Incorporating Dipeptide Position-Specific Propensity into Pseudo Amino Acid Composition

Directory of Open Access Journals (Sweden)

Yan Xu

2014-05-01

Full Text Available Post-translational modifications (PTMs play crucial roles in various cell functions and biological processes. Protein hydroxylation is one type of PTM that usually occurs at the sites of proline and lysine. Given an uncharacterized protein sequence, which site of its Pro (or Lys can be hydroxylated and which site cannot? This is a challenging problem, not only for in-depth understanding of the hydroxylation mechanism, but also for drug development, because protein hydroxylation is closely relevant to major diseases, such as stomach and lung cancers. With the avalanche of protein sequences generated in the post-genomic age, it is highly desired to develop computational methods to address this problem. In view of this, a new predictor called “iHyd-PseAAC” (identify hydroxylation by pseudo amino acid composition was proposed by incorporating the dipeptide position-specific propensity into the general form of pseudo amino acid composition. It was demonstrated by rigorous cross-validation tests on stringent benchmark datasets that the new predictor is quite promising and may become a useful high throughput tool in this area. A user-friendly web-server for iHyd-PseAAC is accessible at http://app.aporc.org/iHyd-PseAAC/. Furthermore, for the convenience of the majority of experimental scientists, a step-by-step guide on how to use the web-server is given. Users can easily obtain their desired results by following these steps without the need of understanding the complicated mathematical equations presented in this paper just for its integrity.

mCSF1, a nucleus-encoded CRM protein required for the processing of many mitochondrial introns, is involved in the biogenesis of respiratory complexes I and IV in Arabidopsis.

Science.gov (United States)

Zmudjak, Michal; Colas des Francs-Small, Catherine; Keren, Ido; Shaya, Felix; Belausov, Eduard; Small, Ian; Ostersetzer-Biran, Oren

2013-07-01

The coding regions of many mitochondrial genes in plants are interrupted by intervening sequences that are classified as group II introns. Their splicing is essential for the expression of the genes they interrupt and hence for respiratory function, and is facilitated by various protein cofactors. Despite the importance of these cofactors, only a few of them have been characterized. CRS1-YhbY domain (CRM) is a recently recognized RNA-binding domain that is present in several characterized splicing factors in plant chloroplasts. The Arabidopsis genome encodes 16 CRM proteins, but these are largely uncharacterized. Here, we analyzed the intracellular location of one of these hypothetical proteins in Arabidopsis, mitochondrial CAF-like splicing factor 1 (mCSF1; At4 g31010), and analyzed the growth phenotypes and organellar activities associated with mcsf1 mutants in plants. Our data indicated that mCSF1 resides within mitochondria and its functions are essential during embryogenesis. Mutant plants with reduced mCSF1 displayed inhibited germination and retarded growth phenotypes that were tightly associated with reduced complex I and IV activities. Analogously to the functions of plastid-localized CRM proteins, analysis of the RNA profiles in wildtype and mcsf1 plants showed that mCSF1 acts in the splicing of many of the group II intron RNAs in Arabidopsis mitochondria. © 2013 The Authors. New Phytologist © 2013 New Phytologist Trust.

Txl1 and Txc1 are co-factors of the 26S proteasome in fission yeast

DEFF Research Database (Denmark)

Andersen, Katrine M; Jensen, Camilla; Kriegenburg, Franziska

2011-01-01

and thereby equips proteasomes with redox capabilities. Here, we characterize the fission yeast orthologue of Txnl1, called Txl1. Txl1 associates with the 26S proteasome via its C-terminal domain. This domain is also found in the uncharacterized protein, Txc1, which was also found to interact with 26S...

AcEST: DK948427 [AcEST

Lifescience Database Archive (English)

Full Text Available 0.025 tr|A6BIV0|A6BIV0_9FIRM Putative uncharacterized protein OS=Dorea... 39 0.36 tr|B6K130|B6K130_SCHJP Shuttle craft...SEENSGETEDNSEEEDKAEDLSSGE 721 >tr|B6K130|B6K130_SCHJP Shuttle craft like transcri

A new protein-protein interaction sensor based on tripartite split-GFP association.

Science.gov (United States)

Cabantous, Stéphanie; Nguyen, Hau B; Pedelacq, Jean-Denis; Koraïchi, Faten; Chaudhary, Anu; Ganguly, Kumkum; Lockard, Meghan A; Favre, Gilles; Terwilliger, Thomas C; Waldo, Geoffrey S

2013-10-04

Monitoring protein-protein interactions in living cells is key to unraveling their roles in numerous cellular processes and various diseases. Previously described split-GFP based sensors suffer from poor folding and/or self-assembly background fluorescence. Here, we have engineered a micro-tagging system to monitor protein-protein interactions in vivo and in vitro. The assay is based on tripartite association between two twenty amino-acids long GFP tags, GFP10 and GFP11, fused to interacting protein partners, and the complementary GFP1-9 detector. When proteins interact, GFP10 and GFP11 self-associate with GFP1-9 to reconstitute a functional GFP. Using coiled-coils and FRB/FKBP12 model systems we characterize the sensor in vitro and in Escherichia coli. We extend the studies to mammalian cells and examine the FK-506 inhibition of the rapamycin-induced association of FRB/FKBP12. The small size of these tags and their minimal effect on fusion protein behavior and solubility should enable new experiments for monitoring protein-protein association by fluorescence.

Essential multimeric enzymes in kinetoplastid parasites: A host of potentially druggable protein-protein interactions.

Science.gov (United States)

Wachsmuth, Leah M; Johnson, Meredith G; Gavenonis, Jason

2017-06-01

Parasitic diseases caused by kinetoplastid parasites of the genera Trypanosoma and Leishmania are an urgent public health crisis in the developing world. These closely related species possess a number of multimeric enzymes in highly conserved pathways involved in vital functions, such as redox homeostasis and nucleotide synthesis. Computational alanine scanning of these protein-protein interfaces has revealed a host of potentially ligandable sites on several established and emerging anti-parasitic drug targets. Analysis of interfaces with multiple clustered hotspots has suggested several potentially inhibitable protein-protein interactions that may have been overlooked by previous large-scale analyses focusing solely on secondary structure. These protein-protein interactions provide a promising lead for the development of new peptide and macrocycle inhibitors of these enzymes.

Secretion of intact proteins and peptide fragments by lysosomal pathways of protein degradation

International Nuclear Information System (INIS)

Isenman, L.D.; Dice, J.F.

1989-01-01

We report that degradation of proteins microinjected into human fibroblasts is accompanied by release into the culture medium of peptide fragments and intact proteins as well as single amino acids. For the nine proteins and polypeptides microinjected, acid-precipitable radioactivity, i.e. peptide fragments and/or intact proteins, ranged from 10 to 67% of the total released radioactivity. Peptide fragments and/or intact protein accounted for 60% of the radioactivity released into the medium by cells microinjected with ribonuclease A. Two major radiolabeled peptide fragments were found, and one was of an appropriate size to function as an antigen in antigen-presenting cells. The peptides released from microinjected ribonuclease A were derived from lysosomal pathways of proteolysis based on several lines of evidence. Previous studies have shown that microinjected ribonuclease A is degraded to single amino acids entirely within lysosomes. We show that release of free amino acids and peptide fragments and/or intact protein was equivalently stimulated by serum deprivation and equivalently inhibited by NH4Cl. We also show that lysosomal degradation of endocytosed [3H]ribonuclease A was accompanied by the release of two peptide fragments similar in size and charge to those from microinjected [ 3 H]ribonuclease A. These findings demonstrate that degradation within lysosomes occurs in a manner that spares specific peptides; they also suggest a previously unsuspected pathway by which cells can secrete cytosol-derived polypeptides

Eight previously unidentified mutations found in the OA1 ocular albinism gene

Directory of Open Access Journals (Sweden)

Dufier Jean-Louis

2006-04-01

Full Text Available Abstract Background Ocular albinism type 1 (OA1 is an X-linked ocular disorder characterized by a severe reduction in visual acuity, nystagmus, hypopigmentation of the retinal pigmented epithelium, foveal hypoplasia, macromelanosomes in pigmented skin and eye cells, and misrouting of the optical tracts. This disease is primarily caused by mutations in the OA1 gene. Methods The ophthalmologic phenotype of the patients and their family members was characterized. We screened for mutations in the OA1 gene by direct sequencing of the nine PCR-amplified exons, and for genomic deletions by PCR-amplification of large DNA fragments. Results We sequenced the nine exons of the OA1 gene in 72 individuals and found ten different mutations in seven unrelated families and three sporadic cases. The ten mutations include an amino acid substitution and a premature stop codon previously reported by our team, and eight previously unidentified mutations: three amino acid substitutions, a duplication, a deletion, an insertion and two splice-site mutations. The use of a novel Taq polymerase enabled us to amplify large genomic fragments covering the OA1 gene. and to detect very likely six distinct large deletions. Furthermore, we were able to confirm that there was no deletion in twenty one patients where no mutation had been found. Conclusion The identified mutations affect highly conserved amino acids, cause frameshifts or alternative splicing, thus affecting folding of the OA1 G protein coupled receptor, interactions of OA1 with its G protein and/or binding with its ligand.

Macrophage replication screen identifies a novel Francisella hydroperoxide resistance protein involved in virulence.

Directory of Open Access Journals (Sweden)

Anna C Llewellyn

Full Text Available Francisella tularensis is a gram-negative facultative intracellular pathogen and the causative agent of tularemia. Recently, genome-wide screens have identified Francisella genes required for virulence in mice. However, the mechanisms by which most of the corresponding proteins contribute to pathogenesis are still largely unknown. To further elucidate the roles of these virulence determinants in Francisella pathogenesis, we tested whether each gene was required for replication of the model pathogen F. novicida within macrophages, an important virulence trait. Fifty-three of the 224 genes tested were involved in intracellular replication, including many of those within the Francisella pathogenicity island (FPI, validating our results. Interestingly, over one third of the genes identified are annotated as hypothetical, indicating that F. novicida likely utilizes novel virulence factors for intracellular replication. To further characterize these virulence determinants, we selected two hypothetical genes to study in more detail. As predicted by our screen, deletion mutants of FTN_0096 and FTN_1133 were attenuated for replication in macrophages. The mutants displayed differing levels of attenuation in vivo, with the FTN_1133 mutant being the most attenuated. FTN_1133 has sequence similarity to the organic hydroperoxide resistance protein Ohr, an enzyme involved in the bacterial response to oxidative stress. We show that FTN_1133 is required for F. novicida resistance to, and degradation of, organic hydroperoxides as well as resistance to the action of the NADPH oxidase both in macrophages and mice. Furthermore, we demonstrate that F. holarctica LVS, a strain derived from a highly virulent human pathogenic species of Francisella, also requires this protein for organic hydroperoxide resistance as well as replication in macrophages and mice. This study expands our knowledge of Francisella's largely uncharacterized intracellular lifecycle and

Complete Genome Sequence of a Porcine Polyomavirus from Nasal Swabs of Pigs with Respiratory Disease.

Science.gov (United States)

Hause, Ben M; Smith, Catherine; Bishop, Brian; Stewart, Chelsea; Simonson, Randy

2018-04-26

Metagenomic sequencing of pooled nasal swabs from pigs with unexplained respiratory disease identified a large number of reads mapping to a previously uncharacterized porcine polyomavirus. Sus scrofa polyomavirus 2 was most closely related to betapolyomaviruses frequently detected in mammalian respiratory samples. Copyright © 2018 Hause et al.

Growth and physiological responses of isohydric and anisohydric poplars to drought

Science.gov (United States)

Ziv Attia; Jean-Christophe Domec; Ram Oren; Danielle A. Way; Menachem Moshelion

2015-01-01

Understanding how different plants prioritize carbon gain and drought vulnerability under a variable water supply is important for predicting which trees will maximize woody biomass production under different environmental conditions. Here, Populus balsamifera (BS, isohydric genotype), P. simonii (SI, previously uncharacterized stomatal behaviour), and their cross, P....

Type VI Secretion is a Major Virulence Determinant in Burkholderia Mallei

National Research Council Canada - National Science Library

Schell, Mark A; Ulrich, Ricky L; Ribot, Wilson J; Brueggemann, Ernst E; Hines, Harry B; Chen, Dan; Lipscomb, Lyla; Kim, H. S; Mrazek, Jan; Nierman, William C; DeShazer, David

2007-01-01

... > 2.5-fold, but capsule was still produced in the absence of virAG. Actin tail formation required virAG as well as bimB, bimC and bimE, three previously uncharacterized genes that were activated four- to 15-fold when VirAG was overproduced...

Prokaryotic homologs of Argonaute proteins are predicted to function as key components of a novel system of defense against mobile genetic elements

Directory of Open Access Journals (Sweden)

van der Oost John

2009-08-01

Full Text Available Abstract Background In eukaryotes, RNA interference (RNAi is a major mechanism of defense against viruses and transposable elements as well of regulating translation of endogenous mRNAs. The RNAi systems recognize the target RNA molecules via small guide RNAs that are completely or partially complementary to a region of the target. Key components of the RNAi systems are proteins of the Argonaute-PIWI family some of which function as slicers, the nucleases that cleave the target RNA that is base-paired to a guide RNA. Numerous prokaryotes possess the CRISPR-associated system (CASS of defense against phages and plasmids that is, in part, mechanistically analogous but not homologous to eukaryotic RNAi systems. Many prokaryotes also encode homologs of Argonaute-PIWI proteins but their functions remain unknown. Results We present a detailed analysis of Argonaute-PIWI protein sequences and the genomic neighborhoods of the respective genes in prokaryotes. Whereas eukaryotic Ago/PIWI proteins always contain PAZ (oligonucleotide binding and PIWI (active or inactivated nuclease domains, the prokaryotic Argonaute homologs (pAgos fall into two major groups in which the PAZ domain is either present or absent. The monophyly of each group is supported by a phylogenetic analysis of the conserved PIWI-domains. Almost all pAgos that lack a PAZ domain appear to be inactivated, and the respective genes are associated with a variety of predicted nucleases in putative operons. An additional, uncharacterized domain that is fused to various nucleases appears to be a unique signature of operons encoding the short (lacking PAZ pAgo form. By contrast, almost all PAZ-domain containing pAgos are predicted to be active nucleases. Some proteins of this group (e.g., that from Aquifex aeolicus have been experimentally shown to possess nuclease activity, and are not typically associated with genes for other (putative nucleases. Given these observations, the apparent extensive

In silico modeling of the yeast protein and protein family interaction network

Science.gov (United States)

Goh, K.-I.; Kahng, B.; Kim, D.

2004-03-01

Understanding of how protein interaction networks of living organisms have evolved or are organized can be the first stepping stone in unveiling how life works on a fundamental ground. Here we introduce an in silico ``coevolutionary'' model for the protein interaction network and the protein family network. The essential ingredient of the model includes the protein family identity and its robustness under evolution, as well as the three previously proposed: gene duplication, divergence, and mutation. This model produces a prototypical feature of complex networks in a wide range of parameter space, following the generalized Pareto distribution in connectivity. Moreover, we investigate other structural properties of our model in detail with some specific values of parameters relevant to the yeast Saccharomyces cerevisiae, showing excellent agreement with the empirical data. Our model indicates that the physical constraints encoded via the domain structure of proteins play a crucial role in protein interactions.

Ulysses - an application for the projection of molecular interactions across species.

Science.gov (United States)

Kemmer, Danielle; Huang, Yong; Shah, Sohrab P; Lim, Jonathan; Brumm, Jochen; Yuen, Macaire M S; Ling, John; Xu, Tao; Wasserman, Wyeth W; Ouellette, B F Francis

2005-01-01

We developed Ulysses as a user-oriented system that uses a process called Interolog Analysis for the parallel analysis and display of protein interactions detected in various species. Ulysses was designed to perform such Interolog Analysis by the projection of model organism interaction data onto homologous human proteins, and thus serves as an accelerator for the analysis of uncharacterized human proteins. The relevance of projections was assessed and validated against published reference collections. All source code is freely available, and the Ulysses system can be accessed via a web interface http://www.cisreg.ca/ulysses.

Of monkeys and men: immunomic profiling of sera from humans and non-human primates resistant to schistosomiasis reveals novel potential vaccine candidates.

Science.gov (United States)

Pearson, Mark S; Becker, Luke; Driguez, Patrick; Young, Neil D; Gaze, Soraya; Mendes, Tiago; Li, Xiao-Hong; Doolan, Denise L; Midzi, Nicholas; Mduluza, Takafira; McManus, Donald P; Wilson, R Alan; Bethony, Jeffrey M; Nausch, Norman; Mutapi, Francisca; Felgner, Philip L; Loukas, Alex

2015-01-01

Schistosoma haematobium affects more than 100 million people throughout Africa and is the causative agent of urogenital schistosomiasis. The parasite is strongly associated with urothelial cancer in infected individuals and as such is designated a group I carcinogen by the International Agency for Research on Cancer. Using a protein microarray containing schistosome proteins, we sought to identify antigens that were the targets of protective IgG1 immune responses in S. haematobium-exposed individuals that acquire drug-induced resistance (DIR) to schistosomiasis after praziquantel treatment. Numerous antigens with known vaccine potential were identified, including calpain (Smp80), tetraspanins, glutathione-S-transferases, and glucose transporters (SGTP1), as well as previously uncharacterized proteins. Reactive IgG1 responses were not elevated in exposed individuals who did not acquire DIR. To complement our human subjects study, we screened for antigen targets of rhesus macaques rendered resistant to S. japonicum by experimental infection followed by self-cure, and discovered a number of new and known vaccine targets, including major targets recognized by our human subjects. This study has further validated the immunomics-based approach to schistosomiasis vaccine antigen discovery and identified numerous novel potential vaccine antigens.

Small RNA-based feedforward loop with AND-gate logic regulates extrachromosomal DNA transfer in Salmonella.

Science.gov (United States)

Papenfort, Kai; Espinosa, Elena; Casadesús, Josep; Vogel, Jörg

2015-08-25

Horizontal gene transfer via plasmid conjugation is a major driving force in microbial evolution but constitutes a complex process that requires synchronization with the physiological state of the host bacteria. Although several host transcription factors are known to regulate plasmid-borne transfer genes, RNA-based regulatory circuits for host-plasmid communication remain unknown. We describe a posttranscriptional mechanism whereby the Hfq-dependent small RNA, RprA, inhibits transfer of pSLT, the virulence plasmid of Salmonella enterica. RprA employs two separate seed-pairing domains to activate the mRNAs of both the sigma-factor σ(S) and the RicI protein, a previously uncharacterized membrane protein here shown to inhibit conjugation. Transcription of ricI requires σ(S) and, together, RprA and σ(S) orchestrate a coherent feedforward loop with AND-gate logic to tightly control the activation of RicI synthesis. RicI interacts with the conjugation apparatus protein TraV and limits plasmid transfer under membrane-damaging conditions. To our knowledge, this study reports the first small RNA-controlled feedforward loop relying on posttranscriptional activation of two independent targets and an unexpected role of the conserved RprA small RNA in controlling extrachromosomal DNA transfer.

Of monkeys and men: immunomic profiling of sera from humans and non-human primates resistant to schistosomiasis reveals novel potential vaccine candidates

Directory of Open Access Journals (Sweden)

Mark ePearson

2015-05-01

Full Text Available Schistosoma haematobium affects more than 100 million people throughout Africa and is the causative agent of urogenital schistosomiasis. The parasite is strongly associated with urothelial cancer in infected individuals and as such is designated a group I carcinogen by the International Agency for Research on Cancer. Using a protein microarray containing schistosome proteins, we sought to identify antigens that were the targets of protective IgG1 immune responses in S. haematobium-exposed individuals that acquire drug-induced resistance (DIR to schistosomiasis after praziquantel treatment. Numerous antigens with known vaccine potential were identified, including calpain (Smp80, tetraspanins, glutathione-S-transferases and glucose transporters (SGTP1, as well as previously uncharacterized proteins. Reactive IgG1 responses were not elevated in exposed individuals who did not acquire DIR. To complement our human subjects study, we screened for antigen targets of rhesus macaques rendered resistant to Schistosoma japonicum by experimental infection followed by self-cure, and discovered a number of new and known vaccine targets, including major targets recognised by our human subjects. This study has further validated the immunomics-based approach to schistosomiasis vaccine antigen discovery and identified numerous novel potential vaccine antigens.

Identification of factors promoting ex vivo maintenance of mouse hematopoietic stem cells by long-term single-cell quantification.

Science.gov (United States)

Kokkaliaris, Konstantinos D; Drew, Erin; Endele, Max; Loeffler, Dirk; Hoppe, Philipp S; Hilsenbeck, Oliver; Schauberger, Bernhard; Hinzen, Christoph; Skylaki, Stavroula; Theodorou, Marina; Kieslinger, Matthias; Lemischka, Ihor; Moore, Kateri; Schroeder, Timm

2016-09-01

The maintenance of hematopoietic stem cells (HSCs) during ex vivo culture is an important prerequisite for their therapeutic manipulation. However, despite intense research, culture conditions for robust maintenance of HSCs are still missing. Cultured HSCs are quickly lost, preventing their improved analysis and manipulation. Identification of novel factors supporting HSC ex vivo maintenance is therefore necessary. Coculture with the AFT024 stroma cell line is capable of maintaining HSCs ex vivo long-term, but the responsible molecular players remain unknown. Here, we use continuous long-term single-cell observation to identify the HSC behavioral signature under supportive or nonsupportive stroma cocultures. We report early HSC survival as a major characteristic of HSC-maintaining conditions. Behavioral screening after manipulation of candidate molecules revealed that the extracellular matrix protein dermatopontin (Dpt) is involved in HSC maintenance. DPT knockdown in supportive stroma impaired HSC survival, whereas ectopic expression of the Dpt gene or protein in nonsupportive conditions restored HSC survival. Supplementing defined stroma- and serum-free culture conditions with recombinant DPT protein improved HSC clonogenicity. These findings illustrate a previously uncharacterized role of Dpt in maintaining HSCs ex vivo. © 2016 by The American Society of Hematology.

Arabidopsis EMB1990 Encoding a Plastid-Targeted YlmG Protein Is Required for Chloroplast Biogenesis and Embryo Development

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Hongyu Chen

2018-02-01

Full Text Available In higher plants, embryo development originated from fertilized egg cell is the first step of the life cycle. The chloroplast participates in many essential metabolic pathways, and its function is highly associated with embryo development. However, the mechanisms and relevant genetic components by which the chloroplast functions in embryogenesis are largely uncharacterized. In this paper, we describe the Arabidopsis EMB1990 gene, encoding a plastid-targeted YlmG protein which is required for chloroplast biogenesis and embryo development. Loss of the EMB1990/YLMG1-1 resulted in albino seeds containing abortive embryos, and the morphological development of homozygous emb1990 embryos was disrupted after the globular stage. Our results showed that EMB1990/YLMG1-1 was expressed in the primordia and adaxial region of cotyledon during embryogenesis, and the encoded protein was targeted to the chloroplast. TEM observation of cellular ultrastructure showed that chloroplast biogenesis was impaired in emb1990 embryo cells. Expression of certain plastid genes was also affected in the loss-of-function mutants, including genes encoding core protein complex subunits located in the thylakoid membrane. Moreover, the tissue-specific genes of embryo development were misexpressed in emb1990 mutant, including genes known to delineate cell fate decisions in the SAM (shoot apical meristem, cotyledon and hypophysis. Taken together, we propose that the nuclear-encoded YLMG1-1 is targeted to the chloroplast and required for normal plastid gene expression. Hence, YLMG1-1 plays a critical role in Arabidopsis embryogenesis through participating in chloroplast biogenesis.

Completion of autobuilt protein models using a database of protein fragments

International Nuclear Information System (INIS)

Cowtan, Kevin

2012-01-01

Two developments in the process of automated protein model building in the Buccaneer software are described: the use of a database of protein fragments in improving the model completeness and the assembly of disconnected chain fragments into complete molecules. Two developments in the process of automated protein model building in the Buccaneer software are presented. A general-purpose library for protein fragments of arbitrary size is described, with a highly optimized search method allowing the use of a larger database than in previous work. The problem of assembling an autobuilt model into complete chains is discussed. This involves the assembly of disconnected chain fragments into complete molecules and the use of the database of protein fragments in improving the model completeness. Assembly of fragments into molecules is a standard step in existing model-building software, but the methods have not received detailed discussion in the literature

AcEST: DK959903 [AcEST

Lifescience Database Archive (English)

Full Text Available 5|Q8K2E5_MOUSE Budding uninhibited by benzimidazoles 1 h... 102 3e-20 tr|Q3UZD6|Q...3UZD6_MOUSE Putative uncharacterized protein OS=Mus m... 102 3e-20 tr|A2ARS1|A2ARS1_MOUSE Budding uninhibited by benzimidazoles

The nematode homologue of Mediator complex subunit 28, F28F8.5, is a critical regulator of C. elegans development

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Markéta Kostrouchová

2017-06-01

Full Text Available The evolutionarily conserved Mediator complex is a critical player in regulating transcription. Comprised of approximately two dozen proteins, the Mediator integrates diverse regulatory signals through direct protein-protein interactions that, in turn, modulate the influence of Mediator on RNA Polymerase II activity. One Mediator subunit, MED28, is known to interact with cytoplasmic structural proteins, providing a potential direct link between cytoplasmic dynamics and the control of gene transcription. Although identified in many animals and plants, MED28 is not present in yeast; no bona fide MED28 has been described previously in Caenorhabditis elegans. Here, we identify bioinformatically F28F8.5, an uncharacterized predicted protein, as the nematode homologue of MED28. As in other Metazoa, F28F8.5 has dual nuclear and cytoplasmic localization and plays critical roles in the regulation of development. F28F8.5 is a vital gene and its null mutants have severely malformed gonads and do not reproduce. F28F8.5 interacts on the protein level with the Mediator subunits MDT-6 and MDT-30. Our results indicate that F28F8.5 is an orthologue of MED28 and suggest that the potential to link cytoplasmic and nuclear events is conserved between MED28 vertebrate and nematode orthologues.

Comparative Membrane Proteomics Reveals a Nonannotated E. coli Heat Shock Protein.

Science.gov (United States)

Yuan, Peijia; D'Lima, Nadia G; Slavoff, Sarah A

2018-01-09

Recent advances in proteomics and genomics have enabled discovery of thousands of previously nonannotated small open reading frames (smORFs) in genomes across evolutionary space. Furthermore, quantitative mass spectrometry has recently been applied to analysis of regulated smORF expression. However, bottom-up proteomics has remained relatively insensitive to membrane proteins, suggesting they may have been underdetected in previous studies. In this report, we add biochemical membrane protein enrichment to our previously developed label-free quantitative proteomics protocol, revealing a never-before-identified heat shock protein in Escherichia coli K12. This putative smORF-encoded heat shock protein, GndA, is likely to be ∼36-55 amino acids in length and contains a predicted transmembrane helix. We validate heat shock-regulated expression of the gndA smORF and demonstrate that a GndA-GFP fusion protein cofractionates with the cell membrane. Quantitative membrane proteomics therefore has the ability to reveal nonannotated small proteins that may play roles in bacterial stress responses.

Interaction of a non-histone chromatin protein (high-mobility group protein 2) with DNA

International Nuclear Information System (INIS)

Goodwin, G.H.; Shooter, K.V.; Johns, E.W.

1975-01-01

The interaction with DNA of the calf thymus chromatin non-histone protein termed the high-mobility group protein 2 has been studied by sedimentation analysis in the ultracentrifuge and by measuring the binding of the 125 I-labelled protein to DNA. The results have been compared with those obtained previously by us [Eur. J. Biochem. (1974) 47, 263-270] for the interaction of high-mobility group protein 1 with DNA. Although the binding parameters are similar for these two proteins, high-mobility group protein 2 differs from high-mobility group protein 1 in that the former appears to change the shape of the DNA to a more compact form. The molecular weight of high-mobility group protein 2 has been determined by equilibrium sedimentation and a mean value of 26,000 was obtained. A low level of nuclease activity detected in one preparation of high-mobility group protein 2 has been investigated. (orig.) [de

Dietary Proteins and Angiogenesis

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Miguel Ángel Medina

2014-01-01

Full Text Available Both defective and persistent angiogenesis are linked to pathological situations in the adult. Compounds able to modulate angiogenesis have a potential value for the treatment of such pathologies. Several small molecules present in the diet have been shown to have modulatory effects on angiogenesis. This review presents the current state of knowledge on the potential modulatory roles of dietary proteins on angiogenesis. There is currently limited available information on the topic. Milk contains at least three proteins for which modulatory effects on angiogenesis have been previously demonstrated. On the other hand, there is some scarce information on the potential of dietary lectins, edible plant proteins and high protein diets to modulate angiogenesis.

SACE_5599, a putative regulatory protein, is involved in morphological differentiation and erythromycin production in Saccharopolyspora erythraea.

Science.gov (United States)

Kirm, Benjamin; Magdevska, Vasilka; Tome, Miha; Horvat, Marinka; Karničar, Katarina; Petek, Marko; Vidmar, Robert; Baebler, Spela; Jamnik, Polona; Fujs, Štefan; Horvat, Jaka; Fonovič, Marko; Turk, Boris; Gruden, Kristina; Petković, Hrvoje; Kosec, Gregor

2013-12-17

Erythromycin is a medically important antibiotic, biosynthesized by the actinomycete Saccharopolyspora erythraea. Genes encoding erythromycin biosynthesis are organized in a gene cluster, spanning over 60 kbp of DNA. Most often, gene clusters encoding biosynthesis of secondary metabolites contain regulatory genes. In contrast, the erythromycin gene cluster does not contain regulatory genes and regulation of its biosynthesis has therefore remained poorly understood, which has for a long time limited genetic engineering approaches for erythromycin yield improvement. We used a comparative proteomic approach to screen for potential regulatory proteins involved in erythromycin biosynthesis. We have identified a putative regulatory protein SACE_5599 which shows significantly higher levels of expression in an erythromycin high-producing strain, compared to the wild type S. erythraea strain. SACE_5599 is a member of an uncharacterized family of putative regulatory genes, located in several actinomycete biosynthetic gene clusters. Importantly, increased expression of SACE_5599 was observed in the complex fermentation medium and at controlled bioprocess conditions, simulating a high-yield industrial fermentation process in the bioreactor. Inactivation of SACE_5599 in the high-producing strain significantly reduced erythromycin yield, in addition to drastically decreasing sporulation intensity of the SACE_5599-inactivated strains when cultivated on ABSM4 agar medium. In contrast, constitutive overexpression of SACE_5599 in the wild type NRRL23338 strain resulted in an increase of erythromycin yield by 32%. Similar yield increase was also observed when we overexpressed the bldD gene, a previously identified regulator of erythromycin biosynthesis, thereby for the first time revealing its potential for improving erythromycin biosynthesis. SACE_5599 is the second putative regulatory gene to be identified in S. erythraea which has positive influence on erythromycin yield. Like bld

Membrane Stored Curvature Elastic Stress Modulates Recruitment of Maintenance Proteins PspA and Vipp1.

Science.gov (United States)

McDonald, Christopher; Jovanovic, Goran; Ces, Oscar; Buck, Martin

2015-09-01

Phage shock protein A (PspA), which is responsible for maintaining inner membrane integrity under stress in enterobacteria, and vesicle-inducting protein in plastids 1 (Vipp1), which functions for membrane maintenance and thylakoid biogenesis in cyanobacteria and plants, are similar peripheral membrane-binding proteins. Their homologous N-terminal amphipathic helices are required for membrane binding; however, the membrane features recognized and required for expressing their functionalities have remained largely uncharacterized. Rigorously controlled, in vitro methodologies with lipid vesicles and purified proteins were used in this study and provided the first biochemical and biophysical characterizations of membrane binding by PspA and Vipp1. Both proteins are found to sense stored curvature elastic (SCE) stress and anionic lipids within the membrane. PspA has an enhanced sensitivity for SCE stress and a higher affinity for the membrane than Vipp1. These variations in binding may be crucial for some of the proteins' differing roles in vivo. Assays probing the transcriptional regulatory function of PspA in the presence of vesicles showed that a relief of transcription inhibition occurs in an SCE stress-specific manner. This in vitro recapitulation of membrane stress-dependent transcription control suggests that the Psp response may be mounted in vivo when a cell's inner membrane experiences increased SCE stress. All cell types maintain the integrity of their membrane systems. One widely distributed membrane stress response system in bacteria is the phage shock protein (Psp) system. The central component, peripheral membrane protein PspA, which mitigates inner membrane stress in bacteria, has a counterpart, Vipp1, which functions for membrane maintenance and thylakoid biogenesis in plants and photosynthetic bacteria. Membrane association of both these proteins is accepted as playing a pivotal role in their functions. Here we show that direct membrane binding by

The Use of Contact Mode Atomic Force Microscopy in Aqueous Medium for Structural Analysis of Spinach Photosynthetic Complexes

Energy Technology Data Exchange (ETDEWEB)

Phuthong, Witchukorn; Huang, Zubin; Wittkopp, Tyler M.; Sznee, Kinga; Heinnickel, Mark L.; Dekker, Jan P.; Frese, Raoul N.; Prinz, Fritz B.; Grossman, Arthur R.

2015-07-28

To investigate the dynamics of photosynthetic pigment-protein complexes in vascular plants at high resolution in an aqueous environment, membrane-protruding oxygen-evolving complexes (OECs) associated with photosystem II (PSII) on spinach (Spinacia oleracea) grana membranes were examined using contact mode atomic force microscopy. This study represents, to our knowledge, the first use of atomic force microscopy to distinguish the putative large extrinsic loop of Photosystem II CP47 reaction center protein (CP47) from the putative oxygen-evolving enhancer proteins 1, 2, and 3 (PsbO, PsbP, and PsbQ) and large extrinsic loop of Photosystem II CP43 reaction center protein (CP43) in the PSII-OEC extrinsic domains of grana membranes under conditions resulting in the disordered arrangement of PSII-OEC particles. Moreover, we observed uncharacterized membrane particles that, based on their physical characteristics and electrophoretic analysis of the polypeptides associated with the grana samples, are hypothesized to be a domain of photosystem I that protrudes from the stromal face of single thylakoid bilayers. Our results are interpreted in the context of the results of others that were obtained using cryo-electron microscopy (and single particle analysis), negative staining and freeze-fracture electron microscopy, as well as previous atomic force microscopy studies.

Loss of PRDM11 promotes MYC-driven lymphomagenesis

DEFF Research Database (Denmark)

Fog-Tonnesen, Cathrine Kolster; Asmar, Fazila; Côme, Christophe Roger Michel

2015-01-01

of the previously uncharacterized PR-domain family member Prdm11 and overexpression of MYC. Overexpression of PRDM11 inhibits proliferation and induces apoptosis. Prdm11 knockout mice are viable, and loss of Prdm11 accelerates MYC-driven lymphomagenesis in the Eµ-Myc mouse model. Moreover, we show that patients...

Salvage of Failed Protein Targets by Reductive Alkylation

Science.gov (United States)

Tan, Kemin; Kim, Youngchang; Hatzos-Skintges, Catherine; Chang, Changsoo; Cuff, Marianne; Chhor, Gekleng; Osipiuk, Jerzy; Michalska, Karolina; Nocek, Boguslaw; An, Hao; Babnigg, Gyorgy; Bigelow, Lance; Joachimiak, Grazyna; Li, Hui; Mack, Jamey; Makowska-Grzyska, Magdalena; Maltseva, Natalia; Mulligan, Rory; Tesar, Christine; Zhou, Min; Joachimiak, Andrzej

2014-01-01

The growth of diffraction-quality single crystals is of primary importance in protein X-ray crystallography. Chemical modification of proteins can alter their surface properties and crystallization behavior. The Midwest Center for Structural Genomics (MCSG) has previously reported how reductive methylation of lysine residues in proteins can improve crystallization of unique proteins that initially failed to produce diffraction-quality crystals. Recently, this approach has been expanded to include ethylation and isopropylation in the MCSG protein crystallization pipeline. Applying standard methods, 180 unique proteins were alkylated and screened using standard crystallization procedures. Crystal structures of 12 new proteins were determined, including the first ethylated and the first isopropylated protein structures. In a few cases, the structures of native and methylated or ethylated states were obtained and the impact of reductive alkylation of lysine residues was assessed. Reductive methylation tends to be more efficient and produces the most alkylated protein structures. Structures of methylated proteins typically have higher resolution limits. A number of well-ordered alkylated lysine residues have been identified, which make both intermolecular and intramolecular contacts. The previous report is updated and complemented with the following new data; a description of a detailed alkylation protocol with results, structural features, and roles of alkylated lysine residues in protein crystals. These contribute to improved crystallization properties of some proteins. PMID:24590719

Salvage of failed protein targets by reductive alkylation.

Science.gov (United States)

Tan, Kemin; Kim, Youngchang; Hatzos-Skintges, Catherine; Chang, Changsoo; Cuff, Marianne; Chhor, Gekleng; Osipiuk, Jerzy; Michalska, Karolina; Nocek, Boguslaw; An, Hao; Babnigg, Gyorgy; Bigelow, Lance; Joachimiak, Grazyna; Li, Hui; Mack, Jamey; Makowska-Grzyska, Magdalena; Maltseva, Natalia; Mulligan, Rory; Tesar, Christine; Zhou, Min; Joachimiak, Andrzej

2014-01-01

The growth of diffraction-quality single crystals is of primary importance in protein X-ray crystallography. Chemical modification of proteins can alter their surface properties and crystallization behavior. The Midwest Center for Structural Genomics (MCSG) has previously reported how reductive methylation of lysine residues in proteins can improve crystallization of unique proteins that initially failed to produce diffraction-quality crystals. Recently, this approach has been expanded to include ethylation and isopropylation in the MCSG protein crystallization pipeline. Applying standard methods, 180 unique proteins were alkylated and screened using standard crystallization procedures. Crystal structures of 12 new proteins were determined, including the first ethylated and the first isopropylated protein structures. In a few cases, the structures of native and methylated or ethylated states were obtained and the impact of reductive alkylation of lysine residues was assessed. Reductive methylation tends to be more efficient and produces the most alkylated protein structures. Structures of methylated proteins typically have higher resolution limits. A number of well-ordered alkylated lysine residues have been identified, which make both intermolecular and intramolecular contacts. The previous report is updated and complemented with the following new data; a description of a detailed alkylation protocol with results, structural features, and roles of alkylated lysine residues in protein crystals. These contribute to improved crystallization properties of some proteins.

Protein function prediction involved on radio-resistant bacteria

International Nuclear Information System (INIS)

Mezhoud, Karim; Mankai, Houda; Sghaier, Haitham; Barkallah, Insaf

2009-01-01

Previously, we identified 58 proteins under positive selection in ionizing-radiation-resistant bacteria (IRRB) but absent in all ionizing-radiation-sensitive bacteria (IRSB). These are good reasons to believe these 58 proteins with their interactions with other proteins (interactomes) are a part of the answer to the question as to how IRRB resist to radiation, because our knowledge of interactomes of positively selected orphan proteins in IRRB might allow us to define cellular pathways important to ionizing-radiation resistance. Using the Database of Interacting Proteins and the PSIbase, we have predicted interactions of orthologs of the 58 proteins under positive selection in IRRB but absent in all IRSB. We used integrate experimental data sets with molecular interaction networks and protein structure prediction from databases. Among these, 18 proteins with their interactomes were identified in Deinococcus radiodurans R1. DNA checkpoint and repair, kinases pathways, energetic and nucleotide metabolisms were the important biological process that found. We predicted the interactomes of 58 proteins under positive selection in IRRB. It is hoped our data will provide new clues as to the cellular pathways that are important for ionizing-radiation resistance. We have identified news proteins involved on DNA management which were not previously mentioned. It is an important input in addition to protein that studied. It does still work to deepen our study on these new proteins

ProteinHistorian: tools for the comparative analysis of eukaryote protein origin.

Directory of Open Access Journals (Sweden)

John A Capra

Full Text Available The evolutionary history of a protein reflects the functional history of its ancestors. Recent phylogenetic studies identified distinct evolutionary signatures that characterize proteins involved in cancer, Mendelian disease, and different ontogenic stages. Despite the potential to yield insight into the cellular functions and interactions of proteins, such comparative phylogenetic analyses are rarely performed, because they require custom algorithms. We developed ProteinHistorian to make tools for performing analyses of protein origins widely available. Given a list of proteins of interest, ProteinHistorian estimates the phylogenetic age of each protein, quantifies enrichment for proteins of specific ages, and compares variation in protein age with other protein attributes. ProteinHistorian allows flexibility in the definition of protein age by including several algorithms for estimating ages from different databases of evolutionary relationships. We illustrate the use of ProteinHistorian with three example analyses. First, we demonstrate that proteins with high expression in human, compared to chimpanzee and rhesus macaque, are significantly younger than those with human-specific low expression. Next, we show that human proteins with annotated regulatory functions are significantly younger than proteins with catalytic functions. Finally, we compare protein length and age in many eukaryotic species and, as expected from previous studies, find a positive, though often weak, correlation between protein age and length. ProteinHistorian is available through a web server with an intuitive interface and as a set of command line tools; this allows biologists and bioinformaticians alike to integrate these approaches into their analysis pipelines. ProteinHistorian's modular, extensible design facilitates the integration of new datasets and algorithms. The ProteinHistorian web server, source code, and pre-computed ages for 32 eukaryotic genomes are

Semantic role labeling for protein transport predicates

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Martin James H

2008-06-01

Full Text Available Abstract Background Automatic semantic role labeling (SRL is a natural language processing (NLP technique that maps sentences to semantic representations. This technique has been widely studied in the recent years, but mostly with data in newswire domains. Here, we report on a SRL model for identifying the semantic roles of biomedical predicates describing protein transport in GeneRIFs – manually curated sentences focusing on gene functions. To avoid the computational cost of syntactic parsing, and because the boundaries of our protein transport roles often did not match up with syntactic phrase boundaries, we approached this problem with a word-chunking paradigm and trained support vector machine classifiers to classify words as being at the beginning, inside or outside of a protein transport role. Results We collected a set of 837 GeneRIFs describing movements of proteins between cellular components, whose predicates were annotated for the semantic roles AGENT, PATIENT, ORIGIN and DESTINATION. We trained these models with the features of previous word-chunking models, features adapted from phrase-chunking models, and features derived from an analysis of our data. Our models were able to label protein transport semantic roles with 87.6% precision and 79.0% recall when using manually annotated protein boundaries, and 87.0% precision and 74.5% recall when using automatically identified ones. Conclusion We successfully adapted the word-chunking classification paradigm to semantic role labeling, applying it to a new domain with predicates completely absent from any previous studies. By combining the traditional word and phrasal role labeling features with biomedical features like protein boundaries and MEDPOST part of speech tags, we were able to address the challenges posed by the new domain data and subsequently build robust models that achieved F-measures as high as 83.1. This system for extracting protein transport information from Gene

AcEST: DK953909 [AcEST

Lifescience Database Archive (English)

Full Text Available A3B212_ORYSJ Putative uncharacterized protein OS=Oryza... 49 1e-04 tr|A2Y2K5|A2Y2K5_ORYSI Putative uncharact...+ ++DP I +SKSA + Y + + Sbjct: 76 VEEENDTPSATKSIRDPPVITVSKSATYALYALTY 110 >tr|A2Y2K5|A2Y2K5_ORYSI Putative un

AcEST: BP921549 [AcEST

Lifescience Database Archive (English)

Full Text Available 62... 34 4.1 tr|B3LCU9|B3LCU9_PLAKH Putative uncharacterized protein OS=Plasm... 34 4.1 tr|Q87HL0|Q87HL0_VIBPA Putative biofilm...-associated surface prote... 33 5.4 tr|A6AZS7|A6AZS7_VIBPA Putative biofilm-associated su

Identifying Hierarchical and Overlapping Protein Complexes Based on Essential Protein-Protein Interactions and “Seed-Expanding” Method

Directory of Open Access Journals (Sweden)

Jun Ren

2014-01-01

Full Text Available Many evidences have demonstrated that protein complexes are overlapping and hierarchically organized in PPI networks. Meanwhile, the large size of PPI network wants complex detection methods have low time complexity. Up to now, few methods can identify overlapping and hierarchical protein complexes in a PPI network quickly. In this paper, a novel method, called MCSE, is proposed based on λ-module and “seed-expanding.” First, it chooses seeds as essential PPIs or edges with high edge clustering values. Then, it identifies protein complexes by expanding each seed to a λ-module. MCSE is suitable for large PPI networks because of its low time complexity. MCSE can identify overlapping protein complexes naturally because a protein can be visited by different seeds. MCSE uses the parameter λ_th to control the range of seed expanding and can detect a hierarchical organization of protein complexes by tuning the value of λ_th. Experimental results of S. cerevisiae show that this hierarchical organization is similar to that of known complexes in MIPS database. The experimental results also show that MCSE outperforms other previous competing algorithms, such as CPM, CMC, Core-Attachment, Dpclus, HC-PIN, MCL, and NFC, in terms of the functional enrichment and matching with known protein complexes.

Characterization of Prochlorococcus clades from iron-depleted oceanic regions.

Science.gov (United States)

Rusch, Douglas B; Martiny, Adam C; Dupont, Christopher L; Halpern, Aaron L; Venter, J Craig

2010-09-14

Prochlorococcus describes a diverse and abundant genus of marine photosynthetic microbes. It is primarily found in oligotrophic waters across the globe and plays a crucial role in energy and nutrient cycling in the ocean ecosystem. The abundance, global distribution, and availability of isolates make Prochlorococcus a model system for understanding marine microbial diversity and biogeochemical cycling. Analysis of 73 metagenomic samples from the Global Ocean Sampling expedition acquired in the Atlantic, Pacific, and Indian Oceans revealed the presence of two uncharacterized Prochlorococcus clades. A phylogenetic analysis using six different genetic markers places the clades close to known lineages adapted to high-light environments. The two uncharacterized clades consistently cooccur and dominate the surface waters of high-temperature, macronutrient-replete, and low-iron regions of the Eastern Equatorial Pacific upwelling and the tropical Indian Ocean. They are genetically distinct from each other and other high-light Prochlorococcus isolates and likely define a previously unrecognized ecotype. Our detailed genomic analysis indicates that these clades comprise organisms that are adapted to iron-depleted environments by reducing their iron quota through the loss of several iron-containing proteins that likely function as electron sinks in the photosynthetic pathway in other Prochlorococcus clades from high-light environments. The presence and inferred physiology of these clades may explain why Prochlorococcus populations from iron-depleted regions do not respond to iron fertilization experiments and further expand our understanding of how phytoplankton adapt to variations in nutrient availability in the ocean.

A SNARE-Like Superfamily Protein SbSLSP from the Halophyte Salicornia brachiata Confers Salt and Drought Tolerance by Maintaining Membrane Stability, K(+)/Na(+) Ratio, and Antioxidant Machinery.

Science.gov (United States)

Singh, Dinkar; Yadav, Narendra Singh; Tiwari, Vivekanand; Agarwal, Pradeep K; Jha, Bhavanath

2016-01-01

About 1000 salt-responsive ESTs were identified from an extreme halophyte Salicornia brachiata. Among these, a novel salt-inducible gene SbSLSP (Salicornia brachiata SNARE-like superfamily protein), showed up-regulation upon salinity and dehydration stress. The presence of cis-regulatory motifs related to abiotic stress in the putative promoter region supports our finding that SbSLSP gene is inducible by abiotic stress. The SbSLSP protein showed a high sequence identity to hypothetical/uncharacterized proteins from Beta vulgaris, Spinacia oleracea, Eucalyptus grandis, and Prunus persica and with SNARE-like superfamily proteins from Zostera marina and Arabidopsis thaliana. Bioinformatics analysis predicted a clathrin adaptor complex small-chain domain and N-myristoylation site in the SbSLSP protein. Subcellular localization studies indicated that the SbSLSP protein is mainly localized in the plasma membrane. Using transgenic tobacco lines, we establish that overexpression of SbSLSP resulted in elevated tolerance to salt and drought stress. The improved tolerance was confirmed by alterations in a range of physiological parameters, including high germination and survival rate, higher leaf chlorophyll contents, and reduced accumulation of Na(+) ion and reactive oxygen species (ROS). Furthermore, overexpressing lines also showed lower water loss, higher cell membrane stability, and increased accumulation of proline and ROS-scavenging enzymes. Overexpression of SbSLSP also enhanced the transcript levels of ROS-scavenging and signaling enzyme genes. This study is the first investigation of the function of the SbSLSP gene as a novel determinant of salinity/drought tolerance. The results suggest that SbSLSP could be a potential candidate to increase salinity and drought tolerance in crop plants for sustainable agriculture in semi-arid saline soil.

Rapid acquisition and model-based analysis of cell-free transcription–translation reactions from nonmodel bacteria

Science.gov (United States)

Wienecke, Sarah; Ishwarbhai, Alka; Tsipa, Argyro; Aw, Rochelle; Kylilis, Nicolas; Bell, David J.; McClymont, David W.; Jensen, Kirsten; Biedendieck, Rebekka

2018-01-01

Native cell-free transcription–translation systems offer a rapid route to characterize the regulatory elements (promoters, transcription factors) for gene expression from nonmodel microbial hosts, which can be difficult to assess through traditional in vivo approaches. One such host, Bacillus megaterium, is a giant Gram-positive bacterium with potential biotechnology applications, although many of its regulatory elements remain uncharacterized. Here, we have developed a rapid automated platform for measuring and modeling in vitro cell-free reactions and have applied this to B. megaterium to quantify a range of ribosome binding site variants and previously uncharacterized endogenous constitutive and inducible promoters. To provide quantitative models for cell-free systems, we have also applied a Bayesian approach to infer ordinary differential equation model parameters by simultaneously using time-course data from multiple experimental conditions. Using this modeling framework, we were able to infer previously unknown transcription factor binding affinities and quantify the sharing of cell-free transcription–translation resources (energy, ribosomes, RNA polymerases, nucleotides, and amino acids) using a promoter competition experiment. This allows insights into resource limiting-factors in batch cell-free synthesis mode. Our combined automated and modeling platform allows for the rapid acquisition and model-based analysis of cell-free transcription–translation data from uncharacterized microbial cell hosts, as well as resource competition within cell-free systems, which potentially can be applied to a range of cell-free synthetic biology and biotechnology applications. PMID:29666238

Validation of protein carbonyl measurement

DEFF Research Database (Denmark)

Augustyniak, Edyta; Adam, Aisha; Wojdyla, Katarzyna

2015-01-01

Protein carbonyls are widely analysed as a measure of protein oxidation. Several different methods exist for their determination. A previous study had described orders of magnitude variance that existed when protein carbonyls were analysed in a single laboratory by ELISA using different commercial...... protein carbonyl analysis across Europe. ELISA and Western blotting techniques detected an increase in protein carbonyl formation between 0 and 5min of UV irradiation irrespective of method used. After irradiation for 15min, less oxidation was detected by half of the laboratories than after 5min...... irradiation. Three of the four ELISA carbonyl results fell within 95% confidence intervals. Likely errors in calculating absolute carbonyl values may be attributed to differences in standardisation. Out of up to 88 proteins identified as containing carbonyl groups after tryptic cleavage of irradiated...

ProteinAC: a frequency domain technique for analyzing protein dynamics

Science.gov (United States)

Bozkurt Varolgunes, Yasemin; Demir, Alper

2018-03-01

It is widely believed that the interactions of proteins with ligands and other proteins are determined by their dynamic characteristics as opposed to only static, time-invariant processes. We propose a novel computational technique, called ProteinAC (PAC), that can be used to analyze small scale functional protein motions as well as interactions with ligands directly in the frequency domain. PAC was inspired by a frequency domain analysis technique that is widely used in electronic circuit design, and can be applied to both coarse-grained and all-atom models. It can be considered as a generalization of previously proposed static perturbation-response methods, where the frequency of the perturbation becomes the key. We discuss the precise relationship of PAC to static perturbation-response schemes. We show that the frequency of the perturbation may be an important factor in protein dynamics. Perturbations at different frequencies may result in completely different response behavior while magnitude and direction are kept constant. Furthermore, we introduce several novel frequency dependent metrics that can be computed via PAC in order to characterize response behavior. We present results for the ferric binding protein that demonstrate the potential utility of the proposed techniques.

MEGADOCK-Web: an integrated database of high-throughput structure-based protein-protein interaction predictions.

Science.gov (United States)

Hayashi, Takanori; Matsuzaki, Yuri; Yanagisawa, Keisuke; Ohue, Masahito; Akiyama, Yutaka

2018-05-08

Protein-protein interactions (PPIs) play several roles in living cells, and computational PPI prediction is a major focus of many researchers. The three-dimensional (3D) structure and binding surface are important for the design of PPI inhibitors. Therefore, rigid body protein-protein docking calculations for two protein structures are expected to allow elucidation of PPIs different from known complexes in terms of 3D structures because known PPI information is not explicitly required. We have developed rapid PPI prediction software based on protein-protein docking, called MEGADOCK. In order to fully utilize the benefits of computational PPI predictions, it is necessary to construct a comprehensive database to gather prediction results and their predicted 3D complex structures and to make them easily accessible. Although several databases exist that provide predicted PPIs, the previous databases do not contain a sufficient number of entries for the purpose of discovering novel PPIs. In this study, we constructed an integrated database of MEGADOCK PPI predictions, named MEGADOCK-Web. MEGADOCK-Web provides more than 10 times the number of PPI predictions than previous databases and enables users to conduct PPI predictions that cannot be found in conventional PPI prediction databases. In MEGADOCK-Web, there are 7528 protein chains and 28,331,628 predicted PPIs from all possible combinations of those proteins. Each protein structure is annotated with PDB ID, chain ID, UniProt AC, related KEGG pathway IDs, and known PPI pairs. Additionally, MEGADOCK-Web provides four powerful functions: 1) searching precalculated PPI predictions, 2) providing annotations for each predicted protein pair with an experimentally known PPI, 3) visualizing candidates that may interact with the query protein on biochemical pathways, and 4) visualizing predicted complex structures through a 3D molecular viewer. MEGADOCK-Web provides a huge amount of comprehensive PPI predictions based on

Feature generation and representations for protein-protein interaction classification.

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Lan, Man; Tan, Chew Lim; Su, Jian

2009-10-01

Automatic detecting protein-protein interaction (PPI) relevant articles is a crucial step for large-scale biological database curation. The previous work adopted POS tagging, shallow parsing and sentence splitting techniques, but they achieved worse performance than the simple bag-of-words representation. In this paper, we generated and investigated multiple types of feature representations in order to further improve the performance of PPI text classification task. Besides the traditional domain-independent bag-of-words approach and the term weighting methods, we also explored other domain-dependent features, i.e. protein-protein interaction trigger keywords, protein named entities and the advanced ways of incorporating Natural Language Processing (NLP) output. The integration of these multiple features has been evaluated on the BioCreAtIvE II corpus. The experimental results showed that both the advanced way of using NLP output and the integration of bag-of-words and NLP output improved the performance of text classification. Specifically, in comparison with the best performance achieved in the BioCreAtIvE II IAS, the feature-level and classifier-level integration of multiple features improved the performance of classification 2.71% and 3.95%, respectively.

Prediction of protein subcellular localization using support vector machine with the choice of proper kernel

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Al Mehedi Hasan

2017-07-01

Full Text Available The prediction of subcellular locations of proteins can provide useful hints for revealing their functions as well as for understanding the mechanisms of some diseases and, finally, for developing novel drugs. As the number of newly discovered proteins has been growing exponentially, laboratory-based experiments to determine the location of an uncharacterized protein in a living cell have become both expensive and time-consuming. Consequently, to tackle these challenges, computational methods are being developed as an alternative to help biologists in selecting target proteins and designing related experiments. However, the success of protein subcellular localization prediction is still a complicated and challenging problem, particularly when query proteins may have multi-label characteristics, i.e. their simultaneous existence in more than one subcellular location, or if they move between two or more different subcellular locations as well. At this point, to get rid of this problem, several types of subcellular localization prediction methods with different levels of accuracy have been proposed. The support vector machine (SVM has been employed to provide potential solutions for problems connected with the prediction of protein subcellular localization. However, the practicability of SVM is affected by difficulties in selecting its appropriate kernel as well as in selecting the parameters of that selected kernel. The literature survey has shown that most researchers apply the radial basis function (RBF kernel to build a SVM based subcellular localization prediction system. Surprisingly, there are still many other kernel functions which have not yet been applied in the prediction of protein subcellular localization. However, the nature of this classification problem requires the application of different kernels for SVM to ensure an optimal result. From this viewpoint, this paper presents the work to apply different kernels for SVM in protein

Laparoscopy After Previous Laparotomy

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Zulfo Godinjak

2006-11-01

Full Text Available Following the abdominal surgery, extensive adhesions often occur and they can cause difficulties during laparoscopic operations. However, previous laparotomy is not considered to be a contraindication for laparoscopy. The aim of this study is to present that an insertion of Veres needle in the region of umbilicus is a safe method for creating a pneumoperitoneum for laparoscopic operations after previous laparotomy. In the last three years, we have performed 144 laparoscopic operations in patients that previously underwent one or two laparotomies. Pathology of digestive system, genital organs, Cesarean Section or abdominal war injuries were the most common causes of previouslaparotomy. During those operations or during entering into abdominal cavity we have not experienced any complications, while in 7 patients we performed conversion to laparotomy following the diagnostic laparoscopy. In all patients an insertion of Veres needle and trocar insertion in the umbilical region was performed, namely a technique of closed laparoscopy. Not even in one patient adhesions in the region of umbilicus were found, and no abdominal organs were injured.

Influence of Bleaching on Flavor of 34% Whey Protein Concentrate and Residual Benzoic Acid Concentration in Dried Whey Proteins

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Previous studies have shown that bleaching negatively affects the flavor of 70% whey protein concentrate (WPC70), but bleaching effects on lower-protein products have not been established. Benzoyl peroxide (BP), a whey bleaching agent, degrades to benzoic acid (BA) and may elevate BA concentrations...

Assisted suicide of a selfish gene.

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Thomson, M S; Beeman, R W

1999-01-01

Medea (M) factors and the hybrid incompatibility factor (H) are involved in two incompatibility systems in flour beetles that were previously thought to be independent. M factors are a novel class of selfish genes that act by maternal lethality to nonself. The H factor causes the death of hybrids with a paternally derived H gene and previously uncharacterized maternal cofactors. We now find that M factors exhibit their selfish behavior only in the absence of the H factor. Furthermore, we show that the previously uncharacterized maternal cofactors required for H-associated hybrid inviability are identical to M factors. We propose that incompatibility between H strains and M strains is due to suppression by the H factor of the self-rescuing activity of the lethal M genes. This interaction has the effect of converting M elements from selfish into self-destructive or "suicidal" genes. M factors are globally widespread, but are conspicuously absent from India, the only country where the H factor is known to occur. Such a mechanism could prevent the spread of selfish M elements by establishing an absolute barrier to hybridization in the boundary between M and non-M zones.

Molecular evolution of a novel family of putative calcium transporters.

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Didier Demaegd

Full Text Available The UPF0016 family is a group of uncharacterized membrane proteins, well conserved through evolution and defined by the presence of one or two copies of an E-Φ-G-D-(KR-(ST consensus motif. Our previous results have shown that two members of this family, the human TMEM165 and the budding yeast Gdt1p, are functionally related and might form a new group of cation/Ca2+ exchangers. Most members of the family are made of two homologous clusters of three transmembrane spans, separated by a central loop and assembled with an opposite orientation in the membrane. However, some bacterial members of the family have only one cluster of transmembrane domains. Among these 'single-domain membrane proteins' some cyanobacterial members were found as pairs of adjacent genes within the genome, but each gene was slightly different. We performed a bioinformatic analysis to propose the molecular evolution of the UPF0016 family and the emergence of the antiparallel topology. Our hypotheses were confirmed experimentally using functional complementation in yeast. This suggests an important and conserved function for UPF0016 proteins in a fundamental cellular process. We also show that members of the UPF0016 family share striking similarities, but no primary sequence homology, with members of the cation/Ca2+ exchangers (CaCA superfamily. Such similarities could be an example of convergent evolution, supporting the previous hypothesis that members of the UPF0016 family are cation/Ca2+ exchangers.

A new essential protein discovery method based on the integration of protein-protein interaction and gene expression data

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Li Min

2012-03-01

Full Text Available Abstract Background Identification of essential proteins is always a challenging task since it requires experimental approaches that are time-consuming and laborious. With the advances in high throughput technologies, a large number of protein-protein interactions are available, which have produced unprecedented opportunities for detecting proteins' essentialities from the network level. There have been a series of computational approaches proposed for predicting essential proteins based on network topologies. However, the network topology-based centrality measures are very sensitive to the robustness of network. Therefore, a new robust essential protein discovery method would be of great value. Results In this paper, we propose a new centrality measure, named PeC, based on the integration of protein-protein interaction and gene expression data. The performance of PeC is validated based on the protein-protein interaction network of Saccharomyces cerevisiae. The experimental results show that the predicted precision of PeC clearly exceeds that of the other fifteen previously proposed centrality measures: Degree Centrality (DC, Betweenness Centrality (BC, Closeness Centrality (CC, Subgraph Centrality (SC, Eigenvector Centrality (EC, Information Centrality (IC, Bottle Neck (BN, Density of Maximum Neighborhood Component (DMNC, Local Average Connectivity-based method (LAC, Sum of ECC (SoECC, Range-Limited Centrality (RL, L-index (LI, Leader Rank (LR, Normalized α-Centrality (NC, and Moduland-Centrality (MC. Especially, the improvement of PeC over the classic centrality measures (BC, CC, SC, EC, and BN is more than 50% when predicting no more than 500 proteins. Conclusions We demonstrate that the integration of protein-protein interaction network and gene expression data can help improve the precision of predicting essential proteins. The new centrality measure, PeC, is an effective essential protein discovery method.

Searching the protein structure database for ligand-binding site similarities using CPASS v.2

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Caprez Adam

2011-01-01

Full Text Available Abstract Background A recent analysis of protein sequences deposited in the NCBI RefSeq database indicates that ~8.5 million protein sequences are encoded in prokaryotic and eukaryotic genomes, where ~30% are explicitly annotated as "hypothetical" or "uncharacterized" protein. Our Comparison of Protein Active-Site Structures (CPASS v.2 database and software compares the sequence and structural characteristics of experimentally determined ligand binding sites to infer a functional relationship in the absence of global sequence or structure similarity. CPASS is an important component of our Functional Annotation Screening Technology by NMR (FAST-NMR protocol and has been successfully applied to aid the annotation of a number of proteins of unknown function. Findings We report a major upgrade to our CPASS software and database that significantly improves its broad utility. CPASS v.2 is designed with a layered architecture to increase flexibility and portability that also enables job distribution over the Open Science Grid (OSG to increase speed. Similarly, the CPASS interface was enhanced to provide more user flexibility in submitting a CPASS query. CPASS v.2 now allows for both automatic and manual definition of ligand-binding sites and permits pair-wise, one versus all, one versus list, or list versus list comparisons. Solvent accessible surface area, ligand root-mean square difference, and Cβ distances have been incorporated into the CPASS similarity function to improve the quality of the results. The CPASS database has also been updated. Conclusions CPASS v.2 is more than an order of magnitude faster than the original implementation, and allows for multiple simultaneous job submissions. Similarly, the CPASS database of ligand-defined binding sites has increased in size by ~ 38%, dramatically increasing the likelihood of a positive search result. The modification to the CPASS similarity function is effective in reducing CPASS similarity scores

Illuminating structural proteins in viral "dark matter" with metaproteomics.

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Brum, Jennifer R; Ignacio-Espinoza, J Cesar; Kim, Eun-Hae; Trubl, Gareth; Jones, Robert M; Roux, Simon; VerBerkmoes, Nathan C; Rich, Virginia I; Sullivan, Matthew B

2016-03-01

Viruses are ecologically important, yet environmental virology is limited by dominance of unannotated genomic sequences representing taxonomic and functional "viral dark matter." Although recent analytical advances are rapidly improving taxonomic annotations, identifying functional dark matter remains problematic. Here, we apply paired metaproteomics and dsDNA-targeted metagenomics to identify 1,875 virion-associated proteins from the ocean. Over one-half of these proteins were newly functionally annotated and represent abundant and widespread viral metagenome-derived protein clusters (PCs). One primarily unannotated PC dominated the dataset, but structural modeling and genomic context identified this PC as a previously unidentified capsid protein from multiple uncultivated tailed virus families. Furthermore, four of the five most abundant PCs in the metaproteome represent capsid proteins containing the HK97-like protein fold previously found in many viruses that infect all three domains of life. The dominance of these proteins within our dataset, as well as their global distribution throughout the world's oceans and seas, supports prior hypotheses that this HK97-like protein fold is the most abundant biological structure on Earth. Together, these culture-independent analyses improve virion-associated protein annotations, facilitate the investigation of proteins within natural viral communities, and offer a high-throughput means of illuminating functional viral dark matter.

Identification of structural protein-protein interactions of herpes simplex virus type 1.

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Lee, Jin H; Vittone, Valerio; Diefenbach, Eve; Cunningham, Anthony L; Diefenbach, Russell J

2008-09-01

In this study we have defined protein-protein interactions between the structural proteins of herpes simplex virus type 1 (HSV-1) using a LexA yeast two-hybrid system. The majority of the capsid, tegument and envelope proteins of HSV-1 were screened in a matrix approach. A total of 40 binary interactions were detected including 9 out of 10 previously identified tegument-tegument interactions (Vittone, V., Diefenbach, E., Triffett, D., Douglas, M.W., Cunningham, A.L., and Diefenbach, R.J., 2005. Determination of interactions between tegument proteins of herpes simplex virus type 1. J. Virol. 79, 9566-9571). A total of 12 interactions involving the capsid protein pUL35 (VP26) and 11 interactions involving the tegument protein pUL46 (VP11/12) were identified. The most significant novel interactions detected in this study, which are likely to play a role in viral assembly, include pUL35-pUL37 (capsid-tegument), pUL46-pUL37 (tegument-tegument) and pUL49 (VP22)-pUS9 (tegument-envelope). This information will provide further insights into the pathways of HSV-1 assembly and the identified interactions are potential targets for new antiviral drugs.

Dimerization in the Grb7 Protein

OpenAIRE

Peterson, Tabitha A.; Benallie, Renee L.; Bradford, Andrew M.; Pias, Sally C.; Yazzie, Jaron.; Lor, Siamee N.; Haulsee, Zachary M.; Park, Chad K.; Johnson, Dennis L.; Rohrschneider, Larry R.; Spuches, Anne.; Lyons, Barbara A.

2012-01-01

In previous studies, we showed that the tyrosine phosphorylation state of growth factor receptor–bound protein 7 (Grb7) affects its ability to bind to the transcription regulator FHL2 and the cortactin-interacting protein, human HS-1-associated protein-1. Here, we present results describing the importance of dimerization in the Grb7–Src homology 2 (SH2) domain in terms of its structural integrity and the ability to bind phosphorylated tyrosine peptide ligands. A tyrosine phosphorylation-mimic...

Preoperative screening: value of previous tests.

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Macpherson, D S; Snow, R; Lofgren, R P

1990-12-15

To determine the frequency of tests done in the year before elective surgery that might substitute for preoperative screening tests and to determine the frequency of test results that change from a normal value to a value likely to alter perioperative management. Retrospective cohort analysis of computerized laboratory data (complete blood count, sodium, potassium, and creatinine levels, prothrombin time, and partial thromboplastin time). Urban tertiary care Veterans Affairs Hospital. Consecutive sample of 1109 patients who had elective surgery in 1988. At admission, 7549 preoperative tests were done, 47% of which duplicated tests performed in the previous year. Of 3096 previous results that were normal as defined by hospital reference range and done closest to the time of but before admission (median interval, 2 months), 13 (0.4%; 95% CI, 0.2% to 0.7%), repeat values were outside a range considered acceptable for surgery. Most of the abnormalities were predictable from the patient's history, and most were not noted in the medical record. Of 461 previous tests that were abnormal, 78 (17%; CI, 13% to 20%) repeat values at admission were outside a range considered acceptable for surgery (P less than 0.001, frequency of clinically important abnormalities of patients with normal previous results with those with abnormal previous results). Physicians evaluating patients preoperatively could safely substitute the previous test results analyzed in this study for preoperative screening tests if the previous tests are normal and no obvious indication for retesting is present.

Global Analysis of Photosynthesis Transcriptional Regulatory Networks

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Imam, Saheed; Noguera, Daniel R.; Donohue, Timothy J.

2014-01-01

Photosynthesis is a crucial biological process that depends on the interplay of many components. This work analyzed the gene targets for 4 transcription factors: FnrL, PrrA, CrpK and MppG (RSP_2888), which are known or predicted to control photosynthesis in Rhodobacter sphaeroides. Chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) identified 52 operons under direct control of FnrL, illustrating its regulatory role in photosynthesis, iron homeostasis, nitrogen metabolism and regulation of sRNA synthesis. Using global gene expression analysis combined with ChIP-seq, we mapped the regulons of PrrA, CrpK and MppG. PrrA regulates ∼34 operons encoding mainly photosynthesis and electron transport functions, while CrpK, a previously uncharacterized Crp-family protein, regulates genes involved in photosynthesis and maintenance of iron homeostasis. Furthermore, CrpK and FnrL share similar DNA binding determinants, possibly explaining our observation of the ability of CrpK to partially compensate for the growth defects of a ΔFnrL mutant. We show that the Rrf2 family protein, MppG, plays an important role in photopigment biosynthesis, as part of an incoherent feed-forward loop with PrrA. Our results reveal a previously unrealized, high degree of combinatorial regulation of photosynthetic genes and significant cross-talk between their transcriptional regulators, while illustrating previously unidentified links between photosynthesis and the maintenance of iron homeostasis. PMID:25503406

Global analysis of photosynthesis transcriptional regulatory networks.

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Saheed Imam

2014-12-01

Full Text Available Photosynthesis is a crucial biological process that depends on the interplay of many components. This work analyzed the gene targets for 4 transcription factors: FnrL, PrrA, CrpK and MppG (RSP_2888, which are known or predicted to control photosynthesis in Rhodobacter sphaeroides. Chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq identified 52 operons under direct control of FnrL, illustrating its regulatory role in photosynthesis, iron homeostasis, nitrogen metabolism and regulation of sRNA synthesis. Using global gene expression analysis combined with ChIP-seq, we mapped the regulons of PrrA, CrpK and MppG. PrrA regulates ∼34 operons encoding mainly photosynthesis and electron transport functions, while CrpK, a previously uncharacterized Crp-family protein, regulates genes involved in photosynthesis and maintenance of iron homeostasis. Furthermore, CrpK and FnrL share similar DNA binding determinants, possibly explaining our observation of the ability of CrpK to partially compensate for the growth defects of a ΔFnrL mutant. We show that the Rrf2 family protein, MppG, plays an important role in photopigment biosynthesis, as part of an incoherent feed-forward loop with PrrA. Our results reveal a previously unrealized, high degree of combinatorial regulation of photosynthetic genes and significant cross-talk between their transcriptional regulators, while illustrating previously unidentified links between photosynthesis and the maintenance of iron homeostasis.

MIR144* inhibits antimicrobial responses against Mycobacterium tuberculosis in human monocytes and macrophages by targeting the autophagy protein DRAM2.

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Kim, Jin Kyung; Lee, Hye-Mi; Park, Ki-Sun; Shin, Dong-Min; Kim, Tae Sung; Kim, Yi Sak; Suh, Hyun-Woo; Kim, Soo Yeon; Kim, In Soo; Kim, Jin-Man; Son, Ji-Woong; Sohn, Kyung Mok; Jung, Sung Soo; Chung, Chaeuk; Han, Sang-Bae; Yang, Chul-Su; Jo, Eun-Kyeong

2017-02-01

Autophagy is an important antimicrobial effector process that defends against Mycobacterium tuberculosis (Mtb), the human pathogen causing tuberculosis (TB). MicroRNAs (miRNAs), endogenous noncoding RNAs, are involved in various biological functions and act as post-transcriptional regulators to target mRNAs. The process by which miRNAs affect antibacterial autophagy and host defense mechanisms against Mtb infections in human monocytes and macrophages is largely uncharacterized. In this study, we show that Mtb significantly induces the expression of MIR144*/hsa-miR-144-5p, which targets the 3'-untranslated region of DRAM2 (DNA damage regulated autophagy modulator 2) in human monocytes and macrophages. Mtb infection downregulated, whereas the autophagy activators upregulated, DRAM2 expression in human monocytes and macrophages by activating AMP-activated protein kinase. In addition, overexpression of MIR144* decreased DRAM2 expression and formation of autophagosomes in human monocytes, whereas inhibition of MIR144* had the opposite effect. Moreover, the levels of MIR144* were elevated, whereas DRAM2 levels were reduced, in human peripheral blood cells and tissues in TB patients, indicating the clinical significance of MIR144* and DRAM2 in human TB. Notably, DRAM2 interacted with BECN1 and UVRAG, essential components of the autophagic machinery, leading to displacement of RUBCN from the BECN1 complex and enhancement of Ptdlns3K activity. Furthermore, MIR144* and DRAM2 were critically involved in phagosomal maturation and enhanced antimicrobial effects against Mtb. Our findings identify a previously unrecognized role of human MIR144* in the inhibition of antibacterial autophagy and the innate host immune response to Mtb. Additionally, these data reveal that DRAM2 is a key coordinator of autophagy activation that enhances antimicrobial activity against Mtb.

In Women with Previous Pregnancy Hypertension, Levels of Cardiovascular Risk Biomarkers May Be Modulated by Haptoglobin Polymorphism

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Andreia Matos

2014-01-01

Full Text Available Preeclampsia (PE may affect the risk for future cardiovascular disease. Haptoglobin (Hp, an acute phase protein with functional genetic polymorphism, synthesized in the hepatocyte and in many peripheral tissues secondary of oxidative stress of PE, may modulate that risk through the antioxidant, angiogenic, and anti-inflammatory differential effects of their genotypes. We performed a prospective study in 352 women aged 35±5.48 years, which 165 had previous PE, 2 to 16 years ago. We studied demographic, anthropometric, and haemodynamic biomarkers such as C-reactive protein (CRP, myeloperoxidase (MPO, and nitric oxide metabolites (total and nitrites, and others associated with liver function (AST and ALT and lipid profile (total LDL and cholesterol HDL, non-HDL, and apolipoproteins A and B. Finally, we study the influence of Hp genetic polymorphism on all these biomarkers and as a predisposing factor for PE and its remote cardiovascular disease prognosis. Previously preeclamptic women either hypertensive or normotensive presented significant differences in those risk biomarkers (MPO, nitrites, and ALT, whose variation may be modulated by Hp 1/2 functional genetic polymorphism. The history of PE may be relevant, in association with these biomarkers to the cardiovascular risk in premenopausal women.

Identification of Ser/Thr kinase and Forkhead Associated Domains in Mycobacterium ulcerans: Characterization of Novel Association between Protein Kinase Q and MupFHA

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Singhal, Anshika; Joshi, Jayadev; Virmani, Richa; Gupta, Meetu; Verma, Nupur; Maji, Abhijit; Misra, Richa; Baronian, Grégory; Pandey, Amit K.; Molle, Virginie; Singh, Yogendra

2014-01-01

Background Mycobacterium ulcerans, the causative agent of Buruli ulcer in humans, is unique among the members of Mycobacterium genus due to the presence of the virulence determinant megaplasmid pMUM001. This plasmid encodes multiple virulence-associated genes, including mup011, which is an uncharacterized Ser/Thr protein kinase (STPK) PknQ. Methodology/Principal Findings In this study, we have characterized PknQ and explored its interaction with MupFHA (Mup018c), a FHA domain containing protein also encoded by pMUM001. MupFHA was found to interact with PknQ and suppress its autophosphorylation. Subsequent protein-protein docking and molecular dynamic simulation analyses showed that this interaction involves the FHA domain of MupFHA and PknQ activation loop residues Ser170 and Thr174. FHA domains are known to recognize phosphothreonine residues, and therefore, MupFHA may be acting as one of the few unusual FHA-domain having overlapping specificity. Additionally, we elucidated the PknQ-dependent regulation of MupDivIVA (Mup012c), which is a DivIVA domain containing protein encoded by pMUM001. MupDivIVA interacts with MupFHA and this interaction may also involve phospho-threonine/serine residues of MupDivIVA. Conclusions/Significance Together, these results describe novel signaling mechanisms in M. ulcerans and show a three-way regulation of PknQ, MupFHA, and MupDivIVA. FHA domains have been considered to be only pThr specific and our results indicate a novel mechanism of pSer as well as pThr interaction exhibited by MupFHA. These results signify the need of further re-evaluating the FHA domain –pThr/pSer interaction model. MupFHA may serve as the ideal candidate for structural studies on this unique class of modular enzymes. PMID:25412098

Identification of Ser/Thr kinase and forkhead associated domains in Mycobacterium ulcerans: characterization of novel association between protein kinase Q and MupFHA.

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Gunjan Arora

2014-11-01

Full Text Available Mycobacterium ulcerans, the causative agent of Buruli ulcer in humans, is unique among the members of Mycobacterium genus due to the presence of the virulence determinant megaplasmid pMUM001. This plasmid encodes multiple virulence-associated genes, including mup011, which is an uncharacterized Ser/Thr protein kinase (STPK PknQ.In this study, we have characterized PknQ and explored its interaction with MupFHA (Mup018c, a FHA domain containing protein also encoded by pMUM001. MupFHA was found to interact with PknQ and suppress its autophosphorylation. Subsequent protein-protein docking and molecular dynamic simulation analyses showed that this interaction involves the FHA domain of MupFHA and PknQ activation loop residues Ser170 and Thr174. FHA domains are known to recognize phosphothreonine residues, and therefore, MupFHA may be acting as one of the few unusual FHA-domain having overlapping specificity. Additionally, we elucidated the PknQ-dependent regulation of MupDivIVA (Mup012c, which is a DivIVA domain containing protein encoded by pMUM001. MupDivIVA interacts with MupFHA and this interaction may also involve phospho-threonine/serine residues of MupDivIVA.Together, these results describe novel signaling mechanisms in M. ulcerans and show a three-way regulation of PknQ, MupFHA, and MupDivIVA. FHA domains have been considered to be only pThr specific and our results indicate a novel mechanism of pSer as well as pThr interaction exhibited by MupFHA. These results signify the need of further re-evaluating the FHA domain -pThr/pSer interaction model. MupFHA may serve as the ideal candidate for structural studies on this unique class of modular enzymes.

Cdk1-Cyclin B1-mediated Phosphorylation of Tumor-associated Microtubule-associated Protein/Cytoskeleton-associated Protein 2 in Mitosis*

OpenAIRE

Uk Hong, Kyung; Kim, Hyun-Jun; Kim, Hyo-Sil; Seong, Yeon-Sun; Hong, Kyeong-Man; Bae, Chang-Dae; Park, Joobae

2009-01-01

During mitosis, establishment of structurally and functionally sound bipolar spindles is necessary for maintaining the fidelity of chromosome segregation. Tumor-associated microtubule-associated protein (TMAP), also known as cytoskeleton-associated protein 2 (CKAP2), is a mitotic spindle-associated protein whose level is frequently up-regulated in various malignancies. Previous reports have suggested that TMAP is a potential regulator of mitotic spindle assembly and dynamics and that it is re...

Short Toxin-like Proteins Abound in Cnidaria Genomes

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Michal Linial

2012-11-01

Full Text Available Cnidaria is a rich phylum that includes thousands of marine species. In this study, we focused on Anthozoa and Hydrozoa that are represented by the Nematostella vectensis (Sea anemone and Hydra magnipapillata genomes. We present a method for ranking the toxin-like candidates from complete proteomes of Cnidaria. Toxin-like functions were revealed using ClanTox, a statistical machine-learning predictor trained on ion channel inhibitors from venomous animals. Fundamental features that were emphasized in training ClanTox include cysteines and their spacing along the sequences. Among the 83,000 proteins derived from Cnidaria representatives, we found 170 candidates that fulfill the properties of toxin-like-proteins, the vast majority of which were previously unrecognized as toxins. An additional 394 short proteins exhibit characteristics of toxin-like proteins at a moderate degree of confidence. Remarkably, only 11% of the predicted toxin-like proteins were previously classified as toxins. Based on our prediction methodology and manual annotation, we inferred functions for over 400 of these proteins. Such functions include protease inhibitors, membrane pore formation, ion channel blockers and metal binding proteins. Many of the proteins belong to small families of paralogs. We conclude that the evolutionary expansion of toxin-like proteins in Cnidaria contributes to their fitness in the complex environment of the aquatic ecosystem.

Candida albicans Hom6 is a homoserine dehydrogenase involved in protein synthesis and cell adhesion

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Pei-Wen Tsai

2017-12-01

Full Text Available Background/Purpose: Candida albicans is a common fungal pathogen in humans. In healthy individuals, C. albicans represents a harmless commensal organism, but infections can be life threatening in immunocompromised patients. The complete genome sequence of C. albicans is extremely useful for identifying genes that may be potential drug targets and important for pathogenic virulence. However, there are still many uncharacterized genes in the Candida genome database. In this study, we investigated C. albicans Hom6, the functions of which remain undetermined experimentally. Methods: HOM6-deleted and HOM6-reintegrated mutant strains were constructed. The mutant strains were compared with wild-type in their growth in various media and enzyme activity. Effects of HOM6 deletion on translation were further investigated by cell susceptibility to hygromycin B or cycloheximide, as well as by polysome profiling, and cell adhesion to polystyrene was also determined. Results: C. albicans Hom6 exhibits homoserine dehydrogenase activity and is involved in the biosynthesis of methionine and threonine. HOM6 deletion caused translational arrest in cells grown under amino acid starvation conditions. Additionally, Hom6 protein was found in both cytosolic and cell-wall fractions of cultured cells. Furthermore, HOM6 deletion reduced C. albicans cell adhesion to polystyrene, which is a common plastic used in many medical devices. Conclusion: Given that there is no Hom6 homologue in mammalian cells, our results provided an important foundation for future development of new antifungal drugs. Keywords: Candida albicans, cell adhesion, Hom6, homoserine dehydrogenase, protein synthesis

Phosphorylation of mitogen-activated protein kinase (MAPK) is required for cytokinesis and progression of cell cycle in tobacco BY-2 cells.

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Ma, Zhaowu; Yu, Guanghui

2010-02-15

The role of mitogen-activated protein kinase (MAPK) in plant cytokinesis remains largely uncharacterized. To elucidate its role, tobacco Bright Yellow-2 (BY-2) cells have been synchronized using a two-step procedure, and the different phases of the cell cycle identified by Histone 4 gene expression and the mitotic index. MAPK expression was analyzed by semi-quantitative (SQ) RT-PCR and protein gel blot analysis for phosphorylated MAPK during cell cycle progression. The SQ RT-PCR analysis indicated that MAPK expression is lower in mitosis than in interphase (G1, G2 and S). However, the amount of phosphorylated MAPK remained stable throughout the cell cycle, indicating that MAPK activity is predominantly regulated at the post-translational level and that phosphorylation of MAPK plays an important role in mitosis. Application of the specific MAPK phosphorylation inhibitor U0126 revealed that while U0126 treatment decreases the phosphorylation of MAPK and the progression from telophase to early cytokinesis is significantly inhibited. The formation of the phragmoplast is also negatively affected at this stage. These results demonstrate that MAPK phosphorylation is involved in the formation of the cell plate within the phragmoplast during cytokinesis and that MAPK predominantly functions during the cytokinesis stage of the cell cycle in tobacco BY-2 cells. Copyright 2009 Elsevier GmbH. All rights reserved.

INTS3 controls the hSSB1-mediated DNA damage response

OpenAIRE

Skaar, Jeffrey R.; Richard, Derek J.; Saraf, Anita; Toschi, Alfredo; Bolderson, Emma; Florens, Laurence; Washburn, Michael P.; Khanna, Kum Kum; Pagano, Michele

2009-01-01

Human SSB1 (single-stranded binding protein 1 [hSSB1]) was recently identified as a part of the ataxia telangiectasia mutated (ATM) signaling pathway. To investigate hSSB1 function, we performed tandem affinity purifications of hSSB1 mutants mimicking the unphosphorylated and ATM-phosphorylated states. Both hSSB1 mutants copurified a subset of Integrator complex subunits and the uncharacterized protein LOC58493/c9orf80 (henceforth minute INTS3/hSSB-associated element [MISE]). The INTS3?MISE?h...

Protein-protein interactions within late pre-40S ribosomes.

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Melody G Campbell

2011-01-01

Full Text Available Ribosome assembly in eukaryotic organisms requires more than 200 assembly factors to facilitate and coordinate rRNA transcription, processing, and folding with the binding of the ribosomal proteins. Many of these assembly factors bind and dissociate at defined times giving rise to discrete assembly intermediates, some of which have been partially characterized with regards to their protein and RNA composition. Here, we have analyzed the protein-protein interactions between the seven assembly factors bound to late cytoplasmic pre-40S ribosomes using recombinant proteins in binding assays. Our data show that these factors form two modules: one comprising Enp1 and the export adaptor Ltv1 near the beak structure, and the second comprising the kinase Rio2, the nuclease Nob1, and a regulatory RNA binding protein Dim2/Pno1 on the front of the head. The GTPase-like Tsr1 and the universally conserved methylase Dim1 are also peripherally connected to this second module. Additionally, in an effort to further define the locations for these essential proteins, we have analyzed the interactions between these assembly factors and six ribosomal proteins: Rps0, Rps3, Rps5, Rps14, Rps15 and Rps29. Together, these results and previous RNA-protein crosslinking data allow us to propose a model for the binding sites of these seven assembly factors. Furthermore, our data show that the essential kinase Rio2 is located at the center of the pre-ribosomal particle and interacts, directly or indirectly, with every other assembly factor, as well as three ribosomal proteins required for cytoplasmic 40S maturation. These data suggest that Rio2 could play a central role in regulating cytoplasmic maturation steps.

Proteins analysed as virtual knots

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Alexander, Keith; Taylor, Alexander J.; Dennis, Mark R.

2017-02-01

Long, flexible physical filaments are naturally tangled and knotted, from macroscopic string down to long-chain molecules. The existence of knotting in a filament naturally affects its configuration and properties, and may be very stable or disappear rapidly under manipulation and interaction. Knotting has been previously identified in protein backbone chains, for which these mechanical constraints are of fundamental importance to their molecular functionality, despite their being open curves in which the knots are not mathematically well defined; knotting can only be identified by closing the termini of the chain somehow. We introduce a new method for resolving knotting in open curves using virtual knots, which are a wider class of topological objects that do not require a classical closure and so naturally capture the topological ambiguity inherent in open curves. We describe the results of analysing proteins in the Protein Data Bank by this new scheme, recovering and extending previous knotting results, and identifying topological interest in some new cases. The statistics of virtual knots in protein chains are compared with those of open random walks and Hamiltonian subchains on cubic lattices, identifying a regime of open curves in which the virtual knotting description is likely to be important.

Measuring Dynamic and Kinetic Information in the Previously Inaccessible Supra-tc Window of Nanoseconds to Microseconds by Solution NMR Spectroscopy

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Donghan Lee

2013-09-01

Full Text Available Nuclear Magnetic Resonance (NMR spectroscopy is a powerful tool that has enabled experimentalists to characterize molecular dynamics and kinetics spanning a wide range of time-scales from picoseconds to days. This review focuses on addressing the previously inaccessible supra-τc window (defined as τc < supra-τc < 40 μs; in which τc is the overall tumbling time of a molecule from the perspective of local inter-nuclear vector dynamics extracted from residual dipolar couplings (RDCs and from the perspective of conformational exchange captured by relaxation dispersion measurements (RD. The goal of the first section is to present a detailed analysis of how to extract protein dynamics encoded in RDCs and how to relate this information to protein functionality within the previously inaccessible supra-τc window. In the second section, the current state of the art for RD is analyzed, as well as the considerable progress toward pushing the sensitivity of RD further into the supra-τc scale by up to a factor of two (motion up to 25 ms. From the data obtained with these techniques and methodology, the importance of the supra-τ c scale for protein function and molecular recognition is becoming increasingly clearer as the connection between motion on the supra-τc scale and protein functionality from the experimental side is further strengthened with results from molecular dynamics simulations.

The Protein Kinase RSK Family - Roles in Prostate Cancer

National Research Council Canada - National Science Library

Lannigan, Deborah

2006-01-01

The Ser/Thr protein kinase p90-kDa ribosomal S6 kinase (RSK) is an important downstream effector of mitogen-activated protein kinase but its roles in prostate cancer have not been previously examined...

RNA-Binding Proteins in Trichomonas vaginalis: Atypical Multifunctional Proteins

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Elisa E. Figueroa-Angulo

2015-11-01

Full Text Available Iron homeostasis is highly regulated in vertebrates through a regulatory system mediated by RNA-protein interactions between the iron regulatory proteins (IRPs that interact with an iron responsive element (IRE located in certain mRNAs, dubbed the IRE-IRP regulatory system. Trichomonas vaginalis, the causal agent of trichomoniasis, presents high iron dependency to regulate its growth, metabolism, and virulence properties. Although T. vaginalis lacks IRPs or proteins with aconitase activity, possesses gene expression mechanisms of iron regulation at the transcriptional and posttranscriptional levels. However, only one gene with iron regulation at the transcriptional level has been described. Recently, our research group described an iron posttranscriptional regulatory mechanism in the T. vaginalis tvcp4 and tvcp12 cysteine proteinase mRNAs. The tvcp4 and tvcp12 mRNAs have a stem-loop structure in the 5'-coding region or in the 3'-UTR, respectively that interacts with T. vaginalis multifunctional proteins HSP70, α-Actinin, and Actin under iron starvation condition, causing translation inhibition or mRNA stabilization similar to the previously characterized IRE-IRP system in eukaryotes. Herein, we summarize recent progress and shed some light on atypical RNA-binding proteins that may participate in the iron posttranscriptional regulation in T. vaginalis.

RNA-Binding Proteins in Trichomonas vaginalis: Atypical Multifunctional Proteins.

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Figueroa-Angulo, Elisa E; Calla-Choque, Jaeson S; Mancilla-Olea, Maria Inocente; Arroyo, Rossana

2015-11-26

Iron homeostasis is highly regulated in vertebrates through a regulatory system mediated by RNA-protein interactions between the iron regulatory proteins (IRPs) that interact with an iron responsive element (IRE) located in certain mRNAs, dubbed the IRE-IRP regulatory system. Trichomonas vaginalis, the causal agent of trichomoniasis, presents high iron dependency to regulate its growth, metabolism, and virulence properties. Although T. vaginalis lacks IRPs or proteins with aconitase activity, possesses gene expression mechanisms of iron regulation at the transcriptional and posttranscriptional levels. However, only one gene with iron regulation at the transcriptional level has been described. Recently, our research group described an iron posttranscriptional regulatory mechanism in the T. vaginalis tvcp4 and tvcp12 cysteine proteinase mRNAs. The tvcp4 and tvcp12 mRNAs have a stem-loop structure in the 5'-coding region or in the 3'-UTR, respectively that interacts with T. vaginalis multifunctional proteins HSP70, α-Actinin, and Actin under iron starvation condition, causing translation inhibition or mRNA stabilization similar to the previously characterized IRE-IRP system in eukaryotes. Herein, we summarize recent progress and shed some light on atypical RNA-binding proteins that may participate in the iron posttranscriptional regulation in T. vaginalis.

Pulmonary heat shock protein expression after exposure to a metabolically activated Clara cell toxicant: relationship to protein adduct formation

International Nuclear Information System (INIS)

Williams, Kurt J.; Cruikshank, Michael K.; Plopper, Charles G.

2003-01-01

Heat shock proteins/stress proteins (Hsps) participate in regulation of protein synthesis and degradation and serve as general cytoprotectants, yet their role in lethal Clara cell injury is not clear. To define the pattern of Hsp expression in acute lethal Clara cell injury, mice were treated with the Clara cell-specific toxicant naphthalene (NA), and patterns of expression compared to electrophilic protein adduction and previously established organellar degradation and gluathione (GSH) depletion. In sites of lethal injury (distal bronchiole), prior to organellar degradation (1 h post-NA), protein adduction is detectable and ubiquitin, Hsp 25, Hsp 72, and heme-oxygenase 1 (HO-1) are increased. Maximal Hsp expression, protein adduction, and GSH depletion occur simultaneous (by 2-3 h) with early organelle disruption. Hsp expression is higher later (6-24 h), only in exfoliating cells. In airway sites (proximal bronchiole) with nonlethal Clara cell injury elevation of Hsp 25, 72, and HO-1 expression follows significant GSH depletion (greater than 50% 2 h post-NA). This data build upon our previous studies and we conclude that (1) in lethal (terminal bronchiole) and nonlethal (proximal bronchiole) Clara cell injury, Hsp induction is associated with the loss of GSH and increased protein adduction, and (2) in these same sites, organelle disruption is not a prerequisite for Hsp induction

Integrated genomics and proteomics of the Torpedo californica electric organ: concordance with the mammalian neuromuscular junction

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Mate Suzanne E

2011-05-01

Full Text Available Abstract Background During development, the branchial mesoderm of Torpedo californica transdifferentiates into an electric organ capable of generating high voltage discharges to stun fish. The organ contains a high density of cholinergic synapses and has served as a biochemical model for the membrane specialization of myofibers, the neuromuscular junction (NMJ. We studied the genome and proteome of the electric organ to gain insight into its composition, to determine if there is concordance with skeletal muscle and the NMJ, and to identify novel synaptic proteins. Results Of 435 proteins identified, 300 mapped to Torpedo cDNA sequences with ≥2 peptides. We identified 14 uncharacterized proteins in the electric organ that are known to play a role in acetylcholine receptor clustering or signal transduction. In addition, two human open reading frames, C1orf123 and C6orf130, showed high sequence similarity to electric organ proteins. Our profile lists several proteins that are highly expressed in skeletal muscle or are muscle specific. Synaptic proteins such as acetylcholinesterase, acetylcholine receptor subunits, and rapsyn were present in the electric organ proteome but absent in the skeletal muscle proteome. Conclusions Our integrated genomic and proteomic analysis supports research describing a muscle-like profile of the organ. We show that it is a repository of NMJ proteins but we present limitations on its use as a comprehensive model of the NMJ. Finally, we identified several proteins that may become candidates for signaling proteins not previously characterized as components of the NMJ.

Markers of protein oxidation

DEFF Research Database (Denmark)

Headlam, Henrietta A; Davies, Michael Jonathan

2004-01-01

Exposure of proteins to radicals in the presence of O2 gives both side-chain oxidation and backbone fragmentation. These processes can be interrelated, with initial side-chain oxidation giving rise to backbone damage via transfer reactions. We have shown previously that alkoxyl radicals formed...... of this process depends on the extent of oxidation at C-3 compared with other sites. HO*, generated by gamma radiolysis, gave the highest total carbonyl yield, with protein-bound carbonyls predominating over released. In contrast, metal ion/H2O2 systems, gave more released than bound carbonyls, with this ratio...... modulated by EDTA. This is ascribed to metal ion-protein interactions affecting the sites of initial oxidation. Hypochlorous acid gave low concentrations of released carbonyls, but high yields of protein-bound material. The peroxyl radical generator 2,2'-azobis(2-amidinopropane) hydrochloride...

Identification of proteins in the postsynaptic density fraction by mass spectrometry

DEFF Research Database (Denmark)

Walikonis, R S; Jensen, Ole Nørregaard; Mann, M

2000-01-01

Our understanding of the organization of postsynaptic signaling systems at excitatory synapses has been aided by the identification of proteins in the postsynaptic density (PSD) fraction, a subcellular fraction enriched in structures with the morphology of PSDs. In this study, we have completed...... not previously known to be constituents of the PSD fraction and 24 that had previously been associated with the PSD by other methods. The newly identified proteins include the heavy chain of myosin-Va (dilute myosin), a motor protein thought to be involved in vesicle trafficking, and the mammalian homolog...

Evolution, diversification and expression of KNOX proteins in plants

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Jie eGao

2015-10-01

Full Text Available The KNOX (KNOTTED1-like homeobox transcription factors play a pivotal role in leaf and meristem development. The majority of these proteins are characterized by the KNOX1, KNOX2, ELK and homeobox domains whereas the proteins of the KNATM family contain only the KNOX domains. We carried out an extensive inventory of these proteins and here report on a total of 394 KNOX proteins from 48 species. The land plant proteins fall into two classes (I and II as previously shown where the class I family seems to be most closely related to the green algae homologs. The KNATM proteins are restricted to Eudicots and some species have multiple paralogs of this protein. Certain plants are characterized by a significant increase in the number of KNOX paralogs; one example is Glycine max. Through the analysis of public gene expression data we show that the class II proteins of this plant have a relatively broad expression specificity as compared to class I proteins, consistent with previous studies of other plants. In G. max, class I protein are mainly distributed in axis tissues and KNATM paralogs are overall poorly expressed; highest expression is in the early plumular axis. Overall, analysis of gene expression in G. max demonstrates clearly that the expansion in gene number is associated with functional diversification.

Triglycerides as an early pathophysiological marker of endothelial dysfunction in nondiabetic women with a previous history of gestational diabetes.

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Sokup, Alina; Góralczyk, Barbara; Góralczyk, Krzysztof; Rość, Danuta

2012-02-01

To investigate whether baseline triglyceride levels are associated with early glucose dysregulation and/or cardiovascular risk in women with a previous history of gestational diabetes. Prospective postpregnancy cohort study. Polish university hospitals. Participants included 125 women with previous gestational diabetes and 40 women with normal glucose regulation during pregnancy. All women were studied 2-24 months (mean 12 ± 10 months) after the index pregnancy. Women with previous gestational diabetes were divided into tertiles in accordance with baseline triglyceride levels. We assessed glucose regulation (oral glucose tolerance test), insulin resistance (homeostasis model assessment), markers of endothelial dysfunction (soluble: intercellular adhesion molecule-1, vascular cell adhesion molecule-1, E-selectin, tissue plasminogen activator antigen, von Willebrand factor antigen), fibrinolysis (plasminogen activator inhibitor antigen), inflammation (high-sensitivity C-reactive protein) and lipid levels. Women with previous gestational diabetes (78% normal glucose regulation, 22% impaired glucose tolerance) had a high cardiometabolic risk profile compared with control women (100% normal glucose regulation). Baseline triglycerides >0.83 mmol/l were associated with a higher prevalence of impaired glucose tolerance, higher high-sensitivity C-reactive protein and triglyceride/high-density lipoprotein-cholesterol ratio. Triglycerides >1.22 mmol/l were associated with higher body fat indexes, higher insulin resistance, higher levels of endothelial dysfunction biomarkers, higher plasminogen activator inhibitor antigen and dyslipidemia. Only E-selectin was independently associated with triglyceride levels. Baseline triglyceride levels are a cardiovascular risk marker as well as a pathophysiological parameter independently associated with endothelial dysfunction in nondiabetic women with previous gestational diabetes at 2-24 months after an index pregnancy. Normalization of

Nonlinear deterministic structures and the randomness of protein sequences

CERN Document Server

Huang Yan Zhao

2003-01-01

To clarify the randomness of protein sequences, we make a detailed analysis of a set of typical protein sequences representing each structural classes by using nonlinear prediction method. No deterministic structures are found in these protein sequences and this implies that they behave as random sequences. We also give an explanation to the controversial results obtained in previous investigations.

Myofibrillar protein synthesis following ingestion of soy protein isolate at rest and after resistance exercise in elderly men

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Yang Yifan

2012-06-01

Full Text Available Abstract Background Increased amino acid availability stimulates muscle protein synthesis, however, aged muscle appears less responsive to the anabolic effects of amino acids when compared to the young. We aimed to compare changes in myofibrillar protein synthesis (MPS in elderly men at rest and after resistance exercise following ingestion of different doses of soy protein and compare the responses to those we previously observed with ingestion of whey protein isolate. Methods Thirty elderly men (age 71 ± 5 y completed a bout of unilateral knee-extensor resistance exercise prior to ingesting no protein (0 g, or either 20 g or 40 g of soy protein isolate (0, S20, and S40 respectively. We compared these responses to previous responses from similar aged men who had ingested 20 g and 40 g of whey protein isolate (W20 and W40. A primed constant infusion of L-[1-13 C]leucine and L-[ring-13 C6]phenylalanine and skeletal muscle biopsies were used to measure whole-body leucine oxidation and MPS over 4 h post-protein consumption in both exercised and non-exercised legs. Results Whole-body leucine oxidation increased with protein ingestion and was significantly greater for S20 vs. W20 (P = 0.003. Rates of MPS for S20 were less than W20 (P = 0.02 and not different from 0 g (P = 0.41 in both exercised and non-exercised leg muscles. For S40, MPS was also reduced compared with W40 under both rested and post-exercise conditions (both P P = 0.04. Conclusions The relationship between protein intake and MPS is both dose and protein source-dependent, with isolated soy showing a reduced ability, as compared to isolated whey protein, to stimulate MPS under both rested and post-exercise conditions. These differences may relate to the lower postprandial leucinemia and greater rates of amino acid oxidation following ingestion of soy versus whey protein.

Solving Classification Problems for Large Sets of Protein Sequences with the Example of Hox and ParaHox Proteins

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Stefanie D. Hueber

2016-02-01

Full Text Available Phylogenetic methods are key to providing models for how a given protein family evolved. However, these methods run into difficulties when sequence divergence is either too low or too high. Here, we provide a case study of Hox and ParaHox proteins so that additional insights can be gained using a new computational approach to help solve old classification problems. For two (Gsx and Cdx out of three ParaHox proteins the assignments differ between the currently most established view and four alternative scenarios. We use a non-phylogenetic, pairwise-sequence-similarity-based method to assess which of the previous predictions, if any, are best supported by the sequence-similarity relationships between Hox and ParaHox proteins. The overall sequence-similarities show Gsx to be most similar to Hox2–3, and Cdx to be most similar to Hox4–8. The results indicate that a purely pairwise-sequence-similarity-based approach can provide additional information not only when phylogenetic inference methods have insufficient information to provide reliable classifications (as was shown previously for central Hox proteins, but also when the sequence variation is so high that the resulting phylogenetic reconstructions are likely plagued by long-branch-attraction artifacts.

Brain calbindin-D28k and an Mr 29,000 calcium binding protein in cerebellum are different but related proteins: Evidence obtained from sequence analysis by tandem mass spectroscopy

International Nuclear Information System (INIS)

Gabrielides, C.; Christakos, S.; McCormack, A.L.; Hunt, D.F.

1991-01-01

A calcium binding protein of M r 29,000 which cross-reacts with antibodies raised against chick calbindin-D 28k was previously reported to be present in rat cerebellum. It was suggested that the M r 29,000 protein represents another form of calbindin-D 28k . In the authors laboratory they were able to identify M r 28,000 and 29,000 proteins in rat, human, and chick cerebellum by their ability to bind 45 Ca in a 45 Ca blot assay. Two calcium binding proteins of M r 27,680 and 29,450 were isolated from rat cerebelli by the use of gel permeation chromatography and preparative gel electrophoresis. After reverse-phase high-performance liquid chromatography (HPLC) the proteins were sequenced. Sequence analysis by tandem mass spectrometry indicated only 52% identity between the rat cerebellar M r 28,000 and 29,000 proteins. Thus they are not different forms of the same protein, as previously suggested. Eighty-nine percent identity was observed between the rate cerebellar M r 29,000 protein and chick calretinin. The difference in identity between the rat cerebellar M r 29,000 protein and chick calretinin may be due to species differences, and thus this protein is most likely rat calretinin. These results suggest either posttranscriptional regulation of calretinin in cerebellum or species differences. The study also suggests that previous immunocytochemical mapping for calbindin using antisera which cross-reacted with both proteins detected brain regions that expressed not only calbindin but also calretinin or a calretinin-like protein

Recovering protein-protein and domain-domain interactions from aggregation of IP-MS proteomics of coregulator complexes.

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Amin R Mazloom

2011-12-01

Full Text Available Coregulator proteins (CoRegs are part of multi-protein complexes that transiently assemble with transcription factors and chromatin modifiers to regulate gene expression. In this study we analyzed data from 3,290 immuno-precipitations (IP followed by mass spectrometry (MS applied to human cell lines aimed at identifying CoRegs complexes. Using the semi-quantitative spectral counts, we scored binary protein-protein and domain-domain associations with several equations. Unlike previous applications, our methods scored prey-prey protein-protein interactions regardless of the baits used. We also predicted domain-domain interactions underlying predicted protein-protein interactions. The quality of predicted protein-protein and domain-domain interactions was evaluated using known binary interactions from the literature, whereas one protein-protein interaction, between STRN and CTTNBP2NL, was validated experimentally; and one domain-domain interaction, between the HEAT domain of PPP2R1A and the Pkinase domain of STK25, was validated using molecular docking simulations. The scoring schemes presented here recovered known, and predicted many new, complexes, protein-protein, and domain-domain interactions. The networks that resulted from the predictions are provided as a web-based interactive application at http://maayanlab.net/HT-IP-MS-2-PPI-DDI/.

Arabidopsis KHZ1 and KHZ2, two novel non-tandem CCCH zinc-finger and K-homolog domain proteins, have redundant roles in the regulation of flowering and senescence.

Science.gov (United States)

Yan, Zongyun; Jia, Jianheng; Yan, Xiaoyuan; Shi, Huiying; Han, Yuzhen

2017-12-01

The two novel CCCH zinc-finger and K-homolog (KH) proteins, KHZ1 and KHZ2, play important roles in regulating flowering and senescence redundantly in Arabidopsis. The CCCH zinc-finger proteins and K-homolog (KH) proteins play important roles in plant development and stress responses. However, the biological functions of many CCCH zinc-finger proteins and KH proteins remain uncharacterized. In Arabidopsis, KHZ1 and KHZ2 are characterized as two novel CCCH zinc-finger and KH domain proteins which belong to subfamily VII in CCCH family. We obtained khz1, khz2 mutants and khz1 khz2 double mutants, as well as overexpression (OE) lines of KHZ1 and KHZ2. Compared with the wild type (WT), the khz2 mutants displayed no defects in growth and development, and the khz1 mutants were slightly late flowering, whereas the khz1 khz2 double mutants showed a pronounced late flowering phenotype. In contrast, artificially overexpressing KHZ1 and KHZ2 led to the early flowering. Consistent with the late flowering phenotype, the expression of flowering repressor gene FLC was up-regulated, while the expression of flowering integrator and floral meristem identity (FMI) genes were down-regulated significantly in khz1 khz2. In addition, we also observed that the OE plants of KHZ1 and KHZ2 showed early leaf senescence significantly, whereas the khz1 khz2 double mutants showed delayed senescence of leaf and the whole plant. Both KHZ1 and KHZ2 were ubiquitously expressed throughout the tissues of Arabidopsis. KHZ1 and KHZ2 were localized to the nucleus, and possessed both transactivation activities and RNA-binding abilities. Taken together, we conclude that KHZ1 and KHZ2 have redundant roles in the regulation of flowering and senescence in Arabidopsis.

Identification of proteins from tuberculin purified protein derivative (PPD) by LC-MS/MS.

Science.gov (United States)

Borsuk, Sibele; Newcombe, Jane; Mendum, Tom A; Dellagostin, Odir A; McFadden, Johnjoe

2009-11-01

The tuberculin purified protein derivative (PPD) is a widely used diagnostic antigen for tuberculosis, however it is poorly defined. Most mycobacterial proteins are extensively denatured by the procedure employed in its preparation, which explains previous difficulties in identifying constituents from PPD to characterize their behaviour in B- and T-cell reactions. We here described a proteomics-based characterization of PPD from several different sources by LC-MS/MS, which combines the solute separation power of HPLC, with the detection power of a mass spectrometer. The technique is able to identify proteins from complex mixtures of peptide fragments. A total of 171 different proteins were identified among the four PPD samples (two bovine PPD and two avium PPD) from Brazil and UK. The majority of the proteins were cytoplasmic (77.9%) and involved in intermediary metabolism and respiration (24.25%) but there was a preponderance of proteins involved in lipid metabolism. We identified a group of 21 proteins that are present in both bovine PPD but were not detected in avium PPD preparation. In addition, four proteins found in bovine PPD are absent in Mycobacterium bovis BCG vaccine strain. This study provides a better understanding of the tuberculin PPD components leading to the identification of additional antigens useful as reagents for specific diagnosis of tuberculosis.

Positive Selection and Centrality in the Yeast and Fly Protein-Protein Interaction Networks

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Sandip Chakraborty

2016-01-01

Full Text Available Proteins within a molecular network are expected to be subject to different selective pressures depending on their relative hierarchical positions. However, it is not obvious what genes within a network should be more likely to evolve under positive selection. On one hand, only mutations at genes with a relatively high degree of control over adaptive phenotypes (such as those encoding highly connected proteins are expected to be “seen” by natural selection. On the other hand, a high degree of pleiotropy at these genes is expected to hinder adaptation. Previous analyses of the human protein-protein interaction network have shown that genes under long-term, recurrent positive selection (as inferred from interspecific comparisons tend to act at the periphery of the network. It is unknown, however, whether these trends apply to other organisms. Here, we show that long-term positive selection has preferentially targeted the periphery of the yeast interactome. Conversely, in flies, genes under positive selection encode significantly more connected and central proteins. These observations are not due to covariation of genes’ adaptability and centrality with confounding factors. Therefore, the distribution of proteins encoded by genes under recurrent positive selection across protein-protein interaction networks varies from one species to another.

Periodontitis and type 2 diabetes among women with previous gestational diabetes: epidemiological and immunological aspects in a follow-up of three years.

Science.gov (United States)

Esteves Lima, Rafael Paschoal; Cota, Luis Otávio Miranda; Silva, Tarcília Aparecida; Cortelli, Sheila Cavalca; Cortelli, José Roberto; Costa, Fernando Oliveira

2017-01-01

The aim of this study was to verify the incidence on the development of type 2 diabetes in women with previous gestational diabetes with and without periodontitis after a three-year time interval. Initial sample of this follow-up study consisted of 90 women diagnosed with gestational diabetes who underwent periodontal examination. After three years, 49 women were subjected to new periodontal examination and biological, behavioral, and social data of interest were collected. Additionally, the quantification of the C-reactive protein in blood samples was performed. Fasting glucose and glycated hemoglobin levels were requested. Saliva samples were collected for quantification of interleukin 6 and 10, tumor necrosis factor α, matrix metalloproteinase 2 and 9. The incidence of type 2 diabetes mellitus was 18.4% and of periodontitis was 10.2%. There was no significant difference in the incidence of type 2 diabetes mellitus among women with and without periodontitis. It was observed impact of C-reactive protein in the development of type 2 diabetes mellitus. However, it was not observed impact of periodontitis on the development of type 2 diabetes mellitus among women with previous gestational diabetes. It was not observed impact of periodontitis on the development of type 2 diabetes among women with previous gestational diabetes. The impact of C-reactive protein in the development of type 2 diabetes mellitus highlights the importance of an inflammatory process in the diabetes pathogenesis.

TMEM45A Is Dispensable for Epidermal Morphogenesis, Keratinization and Barrier Formation.

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Aurélie Hayez

Full Text Available TMEM45A gene encodes an initially uncharacterized predicted transmembrane protein. We previously showed that this gene is highly expressed in keratinocytes where its expression correlates with keratinization, suggesting a role in normal epidermal physiology. To test this hypothesis, we generated TMEM45A knockout mice and found that these mice develop without any evident phenotype. The morphology of the epidermis assessed by histology and by labelling differentiation markers in immunofluorescence was not altered. Toluidine blue permeability assay showed that the epidermal barrier develops normally during embryonic development. We also showed that depletion of TMEM45A in human keratinocytes does not alter their potential to form in vitro 3D-reconstructed epidermis. Indeed, epidermis with normal morphogenesis were generated from TMEM45A-silenced keratinocytes. Their expression of differentiation markers quantified by RT-qPCR and evidenced by immunofluorescence labelling as well as their barrier function estimated by Lucifer yellow permeability were similar to the control epidermis. In summary, TMEM45A gene expression is dispensable for epidermal morphogenesis, keratinization and barrier formation. If this protein plays a role in the epidermis, its experimental depletion can possibly be compensated by other proteins in the two experimental models analyzed in this study.

Deletion of the Vaccinia Virus I2 Protein Interrupts Virion Morphogenesis, Leading to Retention of the Scaffold Protein and Mislocalization of Membrane-Associated Entry Proteins.

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Hyun, Seong-In; Weisberg, Andrea; Moss, Bernard

2017-08-01

The I2L open reading frame of vaccinia virus (VACV) encodes a conserved 72-amino-acid protein with a putative C-terminal transmembrane domain. Previous studies with a tetracycline-inducible mutant demonstrated that I2-deficient virions are defective in cell entry. The purpose of the present study was to determine the step of replication or entry that is affected by loss of the I2 protein. Fluorescence microscopy experiments showed that I2 colocalized with a major membrane protein of immature and mature virions. We generated a cell line that constitutively expressed I2 and allowed construction of the VACV I2L deletion mutant vΔI2. As anticipated, vΔI2 was unable to replicate in cells that did not express I2. Unexpectedly, morphogenesis was interrupted at a stage after immature virion formation, resulting in the accumulation of dense spherical particles instead of brick-shaped mature virions with well-defined core structures. The abnormal particles retained the D13 scaffold protein of immature virions, were severely deficient in the transmembrane proteins that comprise the entry fusion complex (EFC), and had increased amounts of unprocessed membrane and core proteins. Total lysates of cells infected with vΔI2 also had diminished EFC proteins due to instability attributed to their hydrophobicity and failure to be inserted into viral membranes. A similar instability of EFC proteins had previously been found with unrelated mutants blocked earlier in morphogenesis that also accumulated viral membranes retaining the D13 scaffold. We concluded that I2 is required for virion morphogenesis, release of the D13 scaffold, and the association of EFC proteins with viral membranes. IMPORTANCE Poxviruses comprise a large family that infect vertebrates and invertebrates, cause disease in both in humans and in wild and domesticated animals, and are being engineered as vectors for vaccines and cancer therapy. In addition, investigations of poxviruses have provided insights into

Coupled Transcriptome and Proteome Analysis of Human Lymphotropic Tumor Viruses: Insights on the Detection and Discovery of Viral Genes

Energy Technology Data Exchange (ETDEWEB)

Dresang, Lindsay R.; Teuton, Jeremy R.; Feng, Huichen; Jacobs, Jon M.; Camp, David G.; Purvine, Samuel O.; Gritsenko, Marina A.; Li, Zhihua; Smith, Richard D.; Sugden, Bill; Moore, Patrick S.; Chang, Yuan

2011-12-20

Kaposi's sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV) are related human tumor viruses that cause primary effusion lymphomas (PEL) and Burkitt's lymphomas (BL), respectively. Viral genes expressed in naturally-infected cancer cells contribute to disease pathogenesis; knowing which viral genes are expressed is critical in understanding how these viruses cause cancer. To evaluate the expression of viral genes, we used high-resolution separation and mass spectrometry coupled with custom tiling arrays to align the viral proteomes and transcriptomes of three PEL and two BL cell lines under latent and lytic culture conditions. Results The majority of viral genes were efficiently detected at the transcript and/or protein level on manipulating the viral life cycle. Overall the correlation of expressed viral proteins and transcripts was highly complementary in both validating and providing orthogonal data with latent/lytic viral gene expression. Our approach also identified novel viral genes in both KSHV and EBV, and extends viral genome annotation. Several previously uncharacterized genes were validated at both transcript and protein levels. Conclusions This systems biology approach coupling proteome and transcriptome measurements provides a comprehensive view of viral gene expression that could not have been attained using each methodology independently. Detection of viral proteins in combination with viral transcripts is a potentially powerful method for establishing virus-disease relationships.

The defense-responsive genes showing enhanced and repressed expression after pathogen infection in rice (Oryza sativa L.)

Institute of Scientific and Technical Information of China (English)

ZHOU; Bin(周斌); PENG; Kaiman(彭开蔓); CHU; Zhaohui(储昭晖); WANG; Shiping(王石平); ZHANG; Qifa(张启发)

2002-01-01

Despite large numbers of studies about defense response, processes involved in the resistance of plants to incompatible pathogens are still largely uncharacterized. The objective of this study was to identify genes involved in defense response by cDNA array analysis and to gain knowledge about the functions of the genes involved in defense response. Approximately 20000 rice cDNA clones were arrayed on nylon filters. RNA samples isolated from different rice lines after infection with incompatible strains or isolates of Xanthomonas oryzae pv. oryzae or Pyricularia grisea, respectively, were used to synthesize cDNA as probes for screening the cDNA arrays. A total of 100 differentially expressed unique sequences were identified from 5 pathogen-host combinations. Fifty-three sequences were detected as showing enhanced expression and 47 sequences were detected as showing repressed expression after pathogen infection. Sequence analysis revealed that most of the 100 sequences had various degrees of homology with genes in databases which encode or putatively encode transcription regulating proteins, translation regulating proteins, transport proteins, kinases, metabolic enzymes, and proteins involved in other functions. Most of the genes have not been previously reported as being involved in the disease resistance response in rice. The results from cDNA arrays, reverse transcription-polymerase chain reaction, and RNA gel blot analysis suggest that activation or repression of most of these genes might occur commonly in the defense response.

Evidence for close side-chain packing in an early protein folding intermediate previously assumed to be a molten globule.

Science.gov (United States)

Rosen, Laura E; Connell, Katelyn B; Marqusee, Susan

2014-10-14

The molten globule, a conformational ensemble with significant secondary structure but only loosely packed tertiary structure, has been suggested to be a ubiquitous intermediate in protein folding. However, it is difficult to assess the tertiary packing of transiently populated species to evaluate this hypothesis. Escherichia coli RNase H is known to populate an intermediate before the rate-limiting barrier to folding that has long been thought to be a molten globule. We investigated this hypothesis by making mimics of the intermediate that are the ground-state conformation at equilibrium, using two approaches: a truncation to generate a fragment mimic of the intermediate, and selective destabilization of the native state using point mutations. Spectroscopic characterization and the response of the mimics to further mutation are consistent with studies on the transient kinetic intermediate, indicating that they model the early intermediate. Both mimics fold cooperatively and exhibit NMR spectra indicative of a closely packed conformation, in contrast to the hypothesis of molten tertiary packing. This result is important for understanding the nature of the subsequent rate-limiting barrier to folding and has implications for the assumption that many other proteins populate molten globule folding intermediates.

From nonspecific DNA-protein encounter complexes to the prediction of DNA-protein interactions.

Directory of Open Access Journals (Sweden)

Mu Gao

2009-03-01

Full Text Available DNA-protein interactions are involved in many essential biological activities. Because there is no simple mapping code between DNA base pairs and protein amino acids, the prediction of DNA-protein interactions is a challenging problem. Here, we present a novel computational approach for predicting DNA-binding protein residues and DNA-protein interaction modes without knowing its specific DNA target sequence. Given the structure of a DNA-binding protein, the method first generates an ensemble of complex structures obtained by rigid-body docking with a nonspecific canonical B-DNA. Representative models are subsequently selected through clustering and ranking by their DNA-protein interfacial energy. Analysis of these encounter complex models suggests that the recognition sites for specific DNA binding are usually favorable interaction sites for the nonspecific DNA probe and that nonspecific DNA-protein interaction modes exhibit some similarity to specific DNA-protein binding modes. Although the method requires as input the knowledge that the protein binds DNA, in benchmark tests, it achieves better performance in identifying DNA-binding sites than three previously established methods, which are based on sophisticated machine-learning techniques. We further apply our method to protein structures predicted through modeling and demonstrate that our method performs satisfactorily on protein models whose root-mean-square Calpha deviation from native is up to 5 A from their native structures. This study provides valuable structural insights into how a specific DNA-binding protein interacts with a nonspecific DNA sequence. The similarity between the specific DNA-protein interaction mode and nonspecific interaction modes may reflect an important sampling step in search of its specific DNA targets by a DNA-binding protein.

TorsinA and the torsinA-interacting protein printor have no impact on endoplasmic reticulum stress or protein trafficking in yeast.

Directory of Open Access Journals (Sweden)

Julie S Valastyan

Full Text Available Early-onset torsion dystonia is a severe, life-long disease that leads to loss of motor control and involuntary muscle contractions. While the molecular etiology of the disease is not fully understood, a mutation in an AAA+ ATPase, torsinA, has been linked to disease onset. Previous work on torsinA has shown that it localizes to the endoplasmic reticulum, where there is evidence that it plays roles in protein trafficking, and potentially also protein folding. Given the high level of evolutionary conservation among proteins involved in these processes, the ability of human such proteins to function effectively in yeast, as well as the previous successes achieved in examining other proteins involved in complex human diseases in yeast, we hypothesized that Saccharomyces cerevisiae might represent a useful model system for studying torsinA function and the effects of its mutants. Since torsinA is proposed to function in protein homeostasis, we tested cells for their ability to respond to various stressors, using a fluorescent reporter to measure the unfolded protein response, as well as their rate of protein secretion. TorsinA did not impact these processes, even after co-expression of its recently identified interacting partner, printor. In light of these findings, we propose that yeast may lack an additional cofactor necessary for torsinA function or proteins required for essential post-translational modifications of torsinA. Alternatively, torsinA may not function in endoplasmic reticulum protein homeostasis. The strains and assays we describe may provide useful tools for identifying and investigating these possibilities and are freely available.

Indirect labeling of proteins with radioiodine

International Nuclear Information System (INIS)

Araujo, Elaine Bortoleti de; Lavinas, Tatiana; Muramoto, Emiko; Pereira, Nilda P.S. de; Silva, Constancia P.G.; Tavares, Leoberto C.

2000-01-01

A procedure is described for the radioiodination of proteins using an iodinated derivative of N succinimidyl 3-(tri-n-butylstannyl)benzoate (ATE), previously described by Zalutsky. ATE was obtained in a high pure form and the iodination has been performed with 131-Iodine in 70-80% yield. Protein labeling studies performed with human IgG indicate that the ATE intermediate is an important alternative to conventional labeling methods. (author)

Bioreducible Lipid-like Nanoparticles for Intracellular Protein Delivery

Science.gov (United States)

Arellano, Carlos Luis

Protein-based therapy is one of the most direct ways to manipulate cell function and treat human disease. Although protein therapeutics has made its way to clinical practice, with five of the top fifteen global pharmaceuticals being peptide or protein-based drugs, one common limitation is that the effects of protein therapy are only achieved through the targeting of cell surface receptors and intracellular domains. Due to the impermeability of the cell membrane to most foreign materials, entire classes of potentially therapeutic proteins cannot thoroughly be studied without a safe and efficient method of transporting proteins into the cytosol. We report the use of a combinatorially-designed bioreducible lipid-like material (termed "lipidoid") - based protein delivery platform for the transfection of human cancer cell lines. Lipidoid nanoparticles are synthesized through a thin film dispersion method. The degradation of the bioreducible nanoparticles was observed when exposed to glutathione, a highly reductive compound present in the cytosol. We demonstrate that the nanoparticles are capable of transfecting a dose-dependent concentration of our model protein, beta-galactosidase into HeLa cells. Furthermore, formulations of the lipidoid containing the cytotoxic proteins saporin and RNase-A are both capable of inhibiting tumor cell proliferation as observed in in vitro treatment of different human cancer cell lines. There was no observed loss in protein activity after lyophilization and long--term storage, indicating the potential of pre-clinical applications. Overall, we demonstrate an effective approach to protein formulation and intracellular delivery. We believe that our formulations will lead to the study of a whole class of previously untapped therapeutics that may generate new solutions for previously untreatable diseases.

Electrophoretic transfer protein zymography.

Science.gov (United States)

Pan, Daniel; Hill, Adam P; Kashou, Anthony; Wilson, Karl A; Tan-Wilson, Anna

2011-04-15

Zymography detects and characterizes proteolytic enzymes by electrophoresis of protease-containing samples into a nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel containing a copolymerized protein substrate. The usefulness of zymography for molecular weight determination and proteomic analysis is hampered by the fact that some proteases exhibit slower migration through a gel that contains substrate protein. This article introduces electrophoretic transfer protein zymography as one solution to this problem. In this technique, samples containing proteolytic enzymes are first resolved in nonreducing SDS-PAGE on a gel without protein substrate. The proteins in the resolving gel are then electrophoretically transferred to a receiving gel previously prepared with a copolymerized protein substrate. The receiving gel is then developed as a zymogram to visualize clear or lightly stained bands in a dark background. Band intensities are linearly related to the amount of protease, extending the usefulness of the technique so long as conditions for transfer and development of the zymogram are kept constant. Conditions of transfer, such as the pore sizes of resolving and receiving gels and the transfer time relative to the molecular weight of the protease, are explored. Copyright © 2011 Elsevier Inc. All rights reserved.

Non-uniform tube representation of proteins

DEFF Research Database (Denmark)

Hansen, Mikael Sonne

Treating the full protein structure is often neither computationally nor physically possible. Instead one is forced to consider various reduced models capturing the properties of interest. Previous work have used tubular neighborhoods of the C-alpha backbone. However, assigning a unique radius...... might not correctly capture volume exclusion - of crucial importance when trying to understand a proteins $3$d-structure. We propose a new reduced model treating the protein as a non-uniform tube with a radius reflecting the positions of atoms. The tube representation is well suited considering X......-ray crystallographic resolution ~ 3Å while a varying radius accounts for the different sizes of side chains. Such a non-uniform tube better capture the protein geometry and has numerous applications in structural/computational biology from the classification of protein structures to sequence-structure prediction....

Prediction of Protein Configurational Entropy (Popcoen).

Science.gov (United States)

Goethe, Martin; Gleixner, Jan; Fita, Ignacio; Rubi, J Miguel

2018-03-13

A knowledge-based method for configurational entropy prediction of proteins is presented; this methodology is extremely fast, compared to previous approaches, because it does not involve any type of configurational sampling. Instead, the configurational entropy of a query fold is estimated by evaluating an artificial neural network, which was trained on molecular-dynamics simulations of ∼1000 proteins. The predicted entropy can be incorporated into a large class of protein software based on cost-function minimization/evaluation, in which configurational entropy is currently neglected for performance reasons. Software of this type is used for all major protein tasks such as structure predictions, proteins design, NMR and X-ray refinement, docking, and mutation effect predictions. Integrating the predicted entropy can yield a significant accuracy increase as we show exemplarily for native-state identification with the prominent protein software FoldX. The method has been termed Popcoen for Prediction of Protein Configurational Entropy. An implementation is freely available at http://fmc.ub.edu/popcoen/ .

New insights into potential functions for the protein 4.1superfamily of proteins in kidney epithelium

Energy Technology Data Exchange (ETDEWEB)

Calinisan, Venice; Gravem, Dana; Chen, Ray Ping-Hsu; Brittin,Sachi; Mohandas, Narla; Lecomte, Marie-Christine; Gascard, Philippe

2005-06-17

Members of the protein 4.1 family of adapter proteins are expressed in a broad panel of tissues including various epithelia where they likely play an important role in maintenance of cell architecture and polarity and in control of cell proliferation. We have recently characterized the structure and distribution of three members of the protein 4.1 family, 4.1B, 4.1R and 4.1N, in mouse kidney. We describe here binding partners for renal 4.1 proteins, identified through the screening of a rat kidney yeast two-hybrid system cDNA library. The identification of putative protein 4.1-based complexes enables us to envision potential functions for 4.1 proteins in kidney: organization of signaling complexes, response to osmotic stress, protein trafficking, and control of cell proliferation. We discuss the relevance of these protein 4.1-based interactions in kidney physio-pathology in the context of their previously identified functions in other cells and tissues. Specifically, we will focus on renal 4.1 protein interactions with beta amyloid precursor protein (beta-APP), 14-3-3 proteins, and the cell swelling-activated chloride channel pICln. We also discuss the functional relevance of another member of the protein 4.1 superfamily, ezrin, in kidney physiopathology.

A dual small-molecule rheostat for precise control of protein concentration in Mammalian cells.

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Lin, Yu Hsuan; Pratt, Matthew R

2014-04-14

One of the most successful strategies for controlling protein concentrations in living cells relies on protein destabilization domains (DD). Under normal conditions, a DD will be rapidly degraded by the proteasome. However, the same DD can be stabilized or "shielded" in a stoichiometric complex with a small molecule, enabling dose-dependent control of its concentration. This process has been exploited by several labs to post-translationally control the expression levels of proteins in vitro as well as in vivo, although the previous technologies resulted in permanent fusion of the protein of interest to the DD, which can affect biological activity and complicate results. We previously reported a complementary strategy, termed traceless shielding (TShld), in which the protein of interest is released in its native form. Here, we describe an optimized protein concentration control system, TTShld, which retains the traceless features of TShld but utilizes two tiers of small molecule control to set protein concentrations in living cells. These experiments provide the first protein concentration control system that results in both a wide range of protein concentrations and proteins free from engineered fusion constructs. The TTShld system has a greatly improved dynamic range compared to our previously reported system, and the traceless feature is attractive for elucidation of the consequences of protein concentration in cell biology. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Tritium Suicide Selection Identifies Proteins Involved in the Uptake and Intracellular Transport of Sterols in Saccharomyces cerevisiae▿

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Sullivan, David P.; Georgiev, Alexander; Menon, Anant K.

2009-01-01

Sterol transport between the plasma membrane (PM) and the endoplasmic reticulum (ER) occurs by a nonvesicular mechanism that is poorly understood. To identify proteins required for this process, we isolated Saccharomyces cerevisiae mutants with defects in sterol transport. We used Upc2-1 cells that have the ability to take up sterols under aerobic conditions and exploited the observation that intracellular accumulation of exogenously supplied [3H]cholesterol in the form of [3H]cholesteryl ester requires an intact PM-ER sterol transport pathway. Upc2-1 cells were mutagenized using a transposon library, incubated with [3H]cholesterol, and subjected to tritium suicide selection to isolate mutants with a decreased ability to accumulate [3H]cholesterol. Many of the mutants had defects in the expression and trafficking of Aus1 and Pdr11, PM-localized ABC transporters that are required for sterol uptake. Through characterization of one of the mutants, a new role was uncovered for the transcription factor Mot3 in controlling expression of Aus1 and Pdr11. A number of mutants had transposon insertions in the uncharacterized Ydr051c gene, which we now refer to as DET1 (decreased ergosterol transport). These mutants expressed Aus1 and Pdr11 normally but were severely defective in the ability to accumulate exogenously supplied cholesterol. The transport of newly synthesized sterols from the ER to the PM was also defective in det1Δ cells. These data indicate that the cytoplasmic protein encoded by DET1 is involved in intracellular sterol transport. PMID:19060182

Characterization of dry globular proteins and protein fibrils by synchrotron radiation vacuum UV circular dichroism

DEFF Research Database (Denmark)

Nesgaard, Lise W.; Hoffmann, Søren Vrønning; Andersen, Christian Beyschau

2008-01-01

Circular dichroism using synchrotron radiation (SRCD) can extend the spectral range down to approximately 130 nm for dry proteins, potentially providing new structural information. Using a selection of dried model proteins, including alpha-helical, beta-sheet, and mixed-structure proteins, we...... with previously published theoretical calculations related to pi-orbital transitions. We also show that drying does not lead to large changes in the secondary structure and does not induce orientational artifacts. In combination with principal component analysis, our SRCD data allow us to distinguish between two...... different types of protein fibrils, highlighting that bona fide fibrils formed by lysozyme are structurally more similar to the nonclassical fibrillar aggregates formed by the SerADan peptide than with the amyloid formed by alpha-synuclein. Thus, despite the lack of direct structural conclusions...

Identification and characterization of secreted proteins in Eimeria tenella

Science.gov (United States)

Ramlee, Intan Azlinda; Firdaus-Raih, Mohd; Wan, Kiew-Lian

2015-09-01

Eimeria tenella is a protozoan parasite that causes coccidiosis, an economically important disease in the poultry industry. The characterization of proteins that are secreted by parasites have been shown to play important roles in parasite invasion and are considered to be potential control agents. In this study, 775 proteins potentially secreted by E. tenella were identified. These proteins were further filtered to remove mitochondrial proteins. Out of 763 putative secreted proteins, 259 proteins possess transmembrane domains while another 150 proteins have GPI (Glycosylphosphatidylinositol) anchors. Homology search revealed that 315 and 448 proteins have matches with known and hypothetical proteins in the database, respectively. Within this data set, previously characterized secretory proteins such as micronemes, rhoptry kinases and dense granules were detected.

A Library of Plasmodium vivax Recombinant Merozoite Proteins Reveals New Vaccine Candidates and Protein-Protein Interactions

Science.gov (United States)

Hostetler, Jessica B.; Sharma, Sumana; Bartholdson, S. Josefin; Wright, Gavin J.; Fairhurst, Rick M.; Rayner, Julian C.

2015-01-01

Background A vaccine targeting Plasmodium vivax will be an essential component of any comprehensive malaria elimination program, but major gaps in our understanding of P. vivax biology, including the protein-protein interactions that mediate merozoite invasion of reticulocytes, hinder the search for candidate antigens. Only one ligand-receptor interaction has been identified, that between P. vivax Duffy Binding Protein (PvDBP) and the erythrocyte Duffy Antigen Receptor for Chemokines (DARC), and strain-specific immune responses to PvDBP make it a complex vaccine target. To broaden the repertoire of potential P. vivax merozoite-stage vaccine targets, we exploited a recent breakthrough in expressing full-length ectodomains of Plasmodium proteins in a functionally-active form in mammalian cells and initiated a large-scale study of P. vivax merozoite proteins that are potentially involved in reticulocyte binding and invasion. Methodology/Principal Findings We selected 39 P. vivax proteins that are predicted to localize to the merozoite surface or invasive secretory organelles, some of which show homology to P. falciparum vaccine candidates. Of these, we were able to express 37 full-length protein ectodomains in a mammalian expression system, which has been previously used to express P. falciparum invasion ligands such as PfRH5. To establish whether the expressed proteins were correctly folded, we assessed whether they were recognized by antibodies from Cambodian patients with acute vivax malaria. IgG from these samples showed at least a two-fold change in reactivity over naïve controls in 27 of 34 antigens tested, and the majority showed heat-labile IgG immunoreactivity, suggesting the presence of conformation-sensitive epitopes and native tertiary protein structures. Using a method specifically designed to detect low-affinity, extracellular protein-protein interactions, we confirmed a predicted interaction between P. vivax 6-cysteine proteins P12 and P41, further

A membrane protein / signaling protein interaction network for Arabidopsis version AMPv2

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Sylvie Lalonde

2010-09-01

Full Text Available Interactions between membrane proteins and the soluble fraction are essential for signal transduction and for regulating nutrient transport. To gain insights into the membrane-based interactome, 3,852 open reading frames (ORFs out of a target list of 8,383 representing membrane and signaling proteins from Arabidopsis thaliana were cloned into a Gateway compatible vector. The mating-based split-ubiquitin system was used to screen for potential protein-protein interactions (pPPIs among 490 Arabidopsis ORFs. A binary robotic screen between 142 receptor-like kinases, 72 transporters, 57 soluble protein kinases and phosphatases, 40 glycosyltransferases, 95 proteins of various functions and 89 proteins with unknown function detected 387 out of 90,370 possible PPIs. A secondary screen confirmed 343 (of 387 pPPIs between 179 proteins, yielding a scale-free network (r2=0.863. Eighty of 142 transmembrane receptor-like kinases (RLK tested positive, identifying three homomers, 63 heteromers and 80 pPPIs with other proteins. Thirty-one out of 142 RLK interactors (including RLKs had previously been found to be phosphorylated; thus interactors may be substrates for respective RLKs. None of the pPPIs described here had been reported in the major interactome databases, including potential interactors of G protein-coupled receptors, phospholipase C, and AMT ammonium transporters. Two RLKs found as putative interactors of AMT1;1 were independently confirmed using a split luciferase assay in Arabidopsis protoplasts. These RLKs may be involved in ammonium-dependent phosphorylation of the C-terminus and regulation of ammonium uptake activity. The robotic screening method established here will enable a systematic analysis of membrane protein interactions in fungi, plants and metazoa.

Twelve previously unknown phage genera are ubiquitous in global oceans.

Science.gov (United States)

Holmfeldt, Karin; Solonenko, Natalie; Shah, Manesh; Corrier, Kristen; Riemann, Lasse; Verberkmoes, Nathan C; Sullivan, Matthew B

2013-07-30

Viruses are fundamental to ecosystems ranging from oceans to humans, yet our ability to study them is bottlenecked by the lack of ecologically relevant isolates, resulting in "unknowns" dominating culture-independent surveys. Here we present genomes from 31 phages infecting multiple strains of the aquatic bacterium Cellulophaga baltica (Bacteroidetes) to provide data for an underrepresented and environmentally abundant bacterial lineage. Comparative genomics delineated 12 phage groups that (i) each represent a new genus, and (ii) represent one novel and four well-known viral families. This diversity contrasts the few well-studied marine phage systems, but parallels the diversity of phages infecting human-associated bacteria. Although all 12 Cellulophaga phages represent new genera, the podoviruses and icosahedral, nontailed ssDNA phages were exceptional, with genomes up to twice as large as those previously observed for each phage type. Structural novelty was also substantial, requiring experimental phage proteomics to identify 83% of the structural proteins. The presence of uncommon nucleotide metabolism genes in four genera likely underscores the importance of scavenging nutrient-rich molecules as previously seen for phages in marine environments. Metagenomic recruitment analyses suggest that these particular Cellulophaga phages are rare and may represent a first glimpse into the phage side of the rare biosphere. However, these analyses also revealed that these phage genera are widespread, occurring in 94% of 137 investigated metagenomes. Together, this diverse and novel collection of phages identifies a small but ubiquitous fraction of unknown marine viral diversity and provides numerous environmentally relevant phage-host systems for experimental hypothesis testing.

3D profile-based approach to proteome-wide discovery of novel human chemokines.

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Aurelie Tomczak

Full Text Available Chemokines are small secreted proteins with important roles in immune responses. They consist of a conserved three-dimensional (3D structure, so-called IL8-like chemokine fold, which is supported by disulfide bridges characteristic of this protein family. Sequence- and profile-based computational methods have been proficient in discovering novel chemokines by making use of their sequence-conserved cysteine patterns. However, it has been recently shown that some chemokines escaped annotation by these methods due to low sequence similarity to known chemokines and to different arrangement of cysteines in sequence and in 3D. Innovative methods overcoming the limitations of current techniques may allow the discovery of new remote homologs in the still functionally uncharacterized fraction of the human genome. We report a novel computational approach for proteome-wide identification of remote homologs of the chemokine family that uses fold recognition techniques in combination with a scaffold-based automatic mapping of disulfide bonds to define a 3D profile of the chemokine protein family. By applying our methodology to all currently uncharacterized human protein sequences, we have discovered two novel proteins that, without having significant sequence similarity to known chemokines or characteristic cysteine patterns, show strong structural resemblance to known anti-HIV chemokines. Detailed computational analysis and experimental structural investigations based on mass spectrometry and circular dichroism support our structural predictions and highlight several other chemokine-like features. The results obtained support their functional annotation as putative novel chemokines and encourage further experimental characterization. The identification of remote homologs of human chemokines may provide new insights into the molecular mechanisms causing pathologies such as cancer or AIDS, and may contribute to the development of novel treatments. Besides

Allergenicity assessment strategy for novel food proteins and protein sources.

Science.gov (United States)

Verhoeckx, Kitty; Broekman, Henrike; Knulst, André; Houben, Geert

2016-08-01

To solve the future food insecurity problem, alternative and sustainable protein sources (e.g. insects, rapeseed, fava bean and algae) are now being explored for the production of food and feed. To approve these novel protein sources for future food a comprehensive risk assessment is needed according to the European food legislation. Allergenicity risk assessment might pose some major difficulties, since detailed guidance on how to assess the allergenic potential of novel foods is not available. At present, the approach relies mostly on the guidance of allergenicity assessment for genetically modified (GM) plant foods. The most recent one was proposed by EFSA (2010 and 2011); "weight-of-evidence approach". However this guidance is difficult to interpret, not completely applicable or validated for novel foods and therefore needs some adjustments. In this paper we propose a conceptual strategy which is based on the "weight-of-evidence approach" for food derived from GM plants and other strategies that were previously published in the literature. This strategy will give more guidance on how to assess the allergenicity of novel food proteins and protein sources. Copyright © 2016 Elsevier Inc. All rights reserved.

Prediction of thermodynamic instabilities of protein solutions from simple protein–protein interactions

International Nuclear Information System (INIS)

D’Agostino, Tommaso; Solana, José Ramón; Emanuele, Antonio

2013-01-01

Highlights: ► We propose a model of effective protein–protein interaction embedding solvent effects. ► A previous square-well model is enhanced by giving to the interaction a free energy character. ► The temperature dependence of the interaction is due to entropic effects of the solvent. ► The validity of the original SW model is extended to entropy driven phase transitions. ► We get good fits for lysozyme and haemoglobin spinodal data taken from literature. - Abstract: Statistical thermodynamics of protein solutions is often studied in terms of simple, microscopic models of particles interacting via pairwise potentials. Such modelling can reproduce the short range structure of protein solutions at equilibrium and predict thermodynamics instabilities of these systems. We introduce a square well model of effective protein–protein interaction that embeds the solvent’s action. We modify an existing model [45] by considering a well depth having an explicit dependence on temperature, i.e. an explicit free energy character, thus encompassing the statistically relevant configurations of solvent molecules around proteins. We choose protein solutions exhibiting demixing upon temperature decrease (lysozyme, enthalpy driven) and upon temperature increase (haemoglobin, entropy driven). We obtain satisfactory fits of spinodal curves for both the two proteins without adding any mean field term, thus extending the validity of the original model. Our results underline the solvent role in modulating or stretching the interaction potential

The hybrid-cluster protein ('prismane protein') from Escherichia coli. Characterization of the hybrid-cluster protein, redox properties of the [2Fe-2S] and [4Fe-2S-2O] clusters and identification of an associated NADH oxidoreductase containing FAD and[2Fe-2S

NARCIS (Netherlands)

Berg, van den W.A.M.; Hagen, W.R.; Dongen, van W.M.A.M.

2000-01-01

Hybrid-cluster proteins ('prismane proteins') have previously been isolated and characterized from strictly anaerobic sulfate-reducing bacteria. These proteins contain two types of Fe/S clusters unique in biological systems: a [4Fe-4S] cubane cluster with spin-admixed S = 3/2 ground-state

Rv0004 is a new essential member of the mycobacterial DNA replication machinery.

Science.gov (United States)

Mann, Katherine M; Huang, Deborah L; Hooppaw, Anna J; Logsdon, Michelle M; Richardson, Kirill; Lee, Hark Joon; Kimmey, Jacqueline M; Aldridge, Bree B; Stallings, Christina L

2017-11-01

DNA replication is fundamental for life, yet a detailed understanding of bacterial DNA replication is limited outside the organisms Escherichia coli and Bacillus subtilis. Many bacteria, including mycobacteria, encode no identified homologs of helicase loaders or regulators of the initiator protein DnaA, despite these factors being essential for DNA replication in E. coli and B. subtilis. In this study we discover that a previously uncharacterized protein, Rv0004, from the human pathogen Mycobacterium tuberculosis is essential for bacterial viability and that depletion of Rv0004 leads to a block in cell cycle progression. Using a combination of genetic and biochemical approaches, we found that Rv0004 has a role in DNA replication, interacts with DNA and the replicative helicase DnaB, and affects DnaB-DnaA complex formation. We also identify a conserved domain in Rv0004 that is predicted to structurally resemble the N-terminal protein-protein interaction domain of DnaA. Mutation of a single conserved tryptophan within Rv0004's DnaA N-terminal-like domain leads to phenotypes similar to those observed upon Rv0004 depletion and can affect the association of Rv0004 with DnaB. In addition, using live cell imaging during depletion of Rv0004, we have uncovered a previously unappreciated role for DNA replication in coordinating mycobacterial cell division and cell size. Together, our data support that Rv0004 encodes a homolog of the recently identified DciA family of proteins found in most bacteria that lack the DnaC-DnaI helicase loaders in E. coli and B. subtilis. Therefore, the mechanisms of Rv0004 elucidated here likely apply to other DciA homologs and reveal insight into the diversity of bacterial strategies in even the most conserved biological processes.

Effects of ubiquilin 1 on the unfolded protein response.

Science.gov (United States)

Lu, Alice; Hiltunen, Mikko; Romano, Donna M; Soininen, Hilkka; Hyman, Bradley T; Bertram, Lars; Tanzi, Rudolph E

2009-05-01

Previous studies have implicated the unfolded protein response (UPR) in the pathogenesis of Alzheimer's disease (AD). We previously reported that DNA variants in the ubiquilin 1 (UBQLN1) gene increase the risk for AD. Since UBQLN1 has been shown to play a role in the UPR, we assessed the effects of overexpression and downregulation of UBQLN1 splice variants during tunicamycin-induced ER stress. In addition to previously described transcript variants, TV1 and TV2, we identified two novel transcript variants of UBQLN1 in brain: TV3 (lacking exons 2-4) and TV4 (lacking exon 4). Overexpression of TV1-3, but not TV4 significantly decreased the mRNA induction of UPR-inducible genes, C/EBP homologous protein (CHOP), BiP/GRP78, and protein disulfide isomerase (PDI) during the UPR. Stable overexpression of TV1-3, but not TV4, also significantly decreased the induction of CHOP protein and increased cell viability during the UPR. In contrast, downregulation of UBQLN1 did not affect CHOP mRNA induction, but instead increased PDI mRNA levels. These findings suggest that overexpression UBQLN1 transcript variants TV1-3, but not TV4, exert a protective effect during the UPR by attenuating CHOP induction and potentially increasing cell viability.

Identification of stress-induced genes from the drought-tolerant plant Prosopis juliflora (Swartz) DC. through analysis of expressed sequence tags.

Science.gov (United States)

George, Suja; Venkataraman, Gayatri; Parida, Ajay

2007-05-01

Abiotic stresses such as cold, salinity, drought, wounding, and heavy metal contamination adversely affect crop productivity throughout the world. Prosopis juliflora is a phreatophyte that can tolerate severe adverse environmental conditions such as drought, salinity, and heavy metal contamination. As a first step towards the characterization of genes that contribute to combating abiotic stress, construction and analysis of a cDNA library of P. juliflora genes is reported here. Random expressed sequence tag (EST) sequencing of 1750 clones produced 1467 high-quality reads. These clones were classified into functional categories, and BLAST comparisons revealed that 114 clones were homologous to genes implicated in stress response(s) and included heat shock proteins, metallothioneins, lipid transfer proteins, and late embryogenesis abundant proteins. Of the ESTs analyzed, 26% showed homology to previously uncharacterized genes in the databases. Fifty-two clones from this category were selected for reverse Northern analysis: 21 were shown to be upregulated and 16 downregulated. The results obtained by reverse Northern analysis were confirmed by Northern analysis. Clustering of the 1467 ESTs produced a total of 295 contigs encompassing 790 ESTs, resulting in a 54.2% redundancy. Two of the abundant genes coding for a nonspecific lipid transfer protein and late embryogenesis abundant protein were sequenced completely. Northern analysis (after polyethylene glycol stress) of the 2 genes was carried out. The implications of the analyzed genes in abiotic stress tolerance are also discussed.

GSHSite: exploiting an iteratively statistical method to identify s-glutathionylation sites with substrate specificity.

Directory of Open Access Journals (Sweden)

Yi-Ju Chen

Full Text Available S-glutathionylation, the covalent attachment of a glutathione (GSH to the sulfur atom of cysteine, is a selective and reversible protein post-translational modification (PTM that regulates protein activity, localization, and stability. Despite its implication in the regulation of protein functions and cell signaling, the substrate specificity of cysteine S-glutathionylation remains unknown. Based on a total of 1783 experimentally identified S-glutathionylation sites from mouse macrophages, this work presents an informatics investigation on S-glutathionylation sites including structural factors such as the flanking amino acids composition and the accessible surface area (ASA. TwoSampleLogo presents that positively charged amino acids flanking the S-glutathionylated cysteine may influence the formation of S-glutathionylation in closed three-dimensional environment. A statistical method is further applied to iteratively detect the conserved substrate motifs with statistical significance. Support vector machine (SVM is then applied to generate predictive model considering the substrate motifs. According to five-fold cross-validation, the SVMs trained with substrate motifs could achieve an enhanced sensitivity, specificity, and accuracy, and provides a promising performance in an independent test set. The effectiveness of the proposed method is demonstrated by the correct identification of previously reported S-glutathionylation sites of mouse thioredoxin (TXN and human protein tyrosine phosphatase 1b (PTP1B. Finally, the constructed models are adopted to implement an effective web-based tool, named GSHSite (http://csb.cse.yzu.edu.tw/GSHSite/, for identifying uncharacterized GSH substrate sites on the protein sequences.

Cellular responses to modified Plasmodium falciparum MSP119 antigens in individuals previously exposed to natural malaria infection

Directory of Open Access Journals (Sweden)

Awobode Henrietta O

2009-11-01

Full Text Available Abstract Background MSP1 processing-inhibitory antibodies bind to epitopes on the 19 kDa C-terminal region of the Plasmodium falciparum merozoite surface protein 1 (MSP119, inhibiting erythrocyte invasion. Blocking antibodies also bind to this antigen but prevent inhibitory antibodies binding, allowing invasion to proceed. Recombinant MSP119 had been modified previously to allow inhibitory but not blocking antibodies to continue to bind. Immunization with these modified proteins, therefore, has the potential to induce more effective protective antibodies. However, it was unclear whether the modification of MSP119 would affect critical T-cell responses to epitopes in this antigen. Methods The cellular responses to wild-type MSP119 and a panel of modified MSP119 antigens were measured using an in-vitro assay for two groups of individuals: the first were malaria-naïve and the second had been naturally exposed to Plasmodium falciparum infection. The cellular responses to the modified proteins were examined using cells from malaria-exposed infants and adults. Results Interestingly, stimulation indices (SI for responses induced by some of the modified proteins were at least two-fold higher than those elicited by the wild-type MSP119. A protein with four amino acid substitutions (Glu27→Tyr, Leu31→Arg, Tyr34→Ser and Glu43→Leu had the highest stimulation index (SI up to 360 and induced large responses in 64% of the samples that had significant cellular responses to the modified proteins. Conclusion This study suggests that specific MSP119 variants that have been engineered to improve their antigenicity for inhibitory antibodies, retain T-cell epitopes and the ability to induce cellular responses. These proteins are candidates for the development of MSP1-based malaria vaccines.

Species specificity for HBsAg binding protein endonexin II

NARCIS (Netherlands)

deBruin, WCC; Leenders, WPJ; Moshage, H; vanHaelst, UJGM

Background/Aims: Hepatitis B virus displays a distinct species and tissue tropism, Previously we have demonstrated that a human liver plasma membrane protein,vith a molecular weight of approximately 34 kiloDalton specifically binds to HBsAg. This protein was identified as endonexin II, a Ca2+

Studies on new antifreeze protein from the psychrophilic diatom ...

African Journals Online (AJOL)

Administrator

2011-09-12

Sep 12, 2011 ... 27-kDa protein modified with His-tag. According to bioinformatics data, a comparison ... for plant biotechnology application on tomato. MATERIALS AND METHODS. The ESTs library was ... specific expression in tomato as previously described (Deng, 2003). Sequence analysis and prediction of protein ...

HIV-1 Tat protein induces glial cell autophagy through enhancement of BAG3 protein levels.

Science.gov (United States)

Bruno, Anna Paola; De Simone, Francesca Isabella; Iorio, Vittoria; De Marco, Margot; Khalili, Kamel; Sariyer, Ilker Kudret; Capunzo, Mario; Nori, Stefania Lucia; Rosati, Alessandra

2014-01-01

BAG3 protein has been described as an anti-apoptotic and pro-autophagic factor in several neoplastic and normal cells. We previously demonstrated that BAG3 expression is elevated upon HIV-1 infection of glial and T lymphocyte cells. Among HIV-1 proteins, Tat is highly involved in regulating host cell response to viral infection. Therefore, we investigated the possible role of Tat protein in modulating BAG3 protein levels and the autophagic process itself. In this report, we show that transfection with Tat raises BAG3 levels in glioblastoma cells. Moreover, BAG3 silencing results in highly reducing Tat- induced levels of LC3-II and increasing the appearance of sub G0/G1 apoptotic cells, in keeping with the reported role of BAG3 in modulating the autophagy/apoptosis balance. These results demonstrate for the first time that Tat protein is able to stimulate autophagy through increasing BAG3 levels in human glial cells.

Wongabel Rhabdovirus Accessory Protein U3 Targets the SWI/SNF Chromatin Remodeling Complex

Science.gov (United States)

Joubert, D. Albert; Rodriguez-Andres, Julio; Monaghan, Paul; Cummins, Michelle; McKinstry, William J.; Paradkar, Prasad N.; Moseley, Gregory W.

2014-01-01

ABSTRACT Wongabel virus (WONV) is an arthropod-borne rhabdovirus that infects birds. It is one of the growing array of rhabdoviruses with complex genomes that encode multiple accessory proteins of unknown function. In addition to the five canonical rhabdovirus structural protein genes (N, P, M, G, and L), the 13.2-kb negative-sense single-stranded RNA (ssRNA) WONV genome contains five uncharacterized accessory genes, one overlapping the N gene (Nx or U4), three located between the P and M genes (U1 to U3), and a fifth one overlapping the G gene (Gx or U5). Here we show that WONV U3 is expressed during infection in insect and mammalian cells and is required for efficient viral replication. A yeast two-hybrid screen against a mosquito cell cDNA library identified that WONV U3 interacts with the 83-amino-acid (aa) C-terminal domain of SNF5, a component of the SWI/SNF chromatin remodeling complex. The interaction was confirmed by affinity chromatography, and nuclear colocalization was established by confocal microscopy. Gene expression studies showed that SNF5 transcripts are upregulated during infection of mosquito cells with WONV, as well as West Nile virus (Flaviviridae) and bovine ephemeral fever virus (Rhabdoviridae), and that SNF5 knockdown results in increased WONV replication. WONV U3 also inhibits SNF5-regulated expression of the cytokine gene CSF1. The data suggest that WONV U3 targets the SWI/SNF complex to block the host response to infection. IMPORTANCE The rhabdoviruses comprise a large family of RNA viruses infecting plants, vertebrates, and invertebrates. In addition to the major structural proteins (N, P, M, G, and L), many rhabdoviruses encode a diverse array of accessory proteins of largely unknown function. Understanding the role of these proteins may reveal much about host-pathogen interactions in infected cells. Here we examine accessory protein U3 of Wongabel virus, an arthropod-borne rhabdovirus that infects birds. We show that U3 enters the

[Meningitis and white matter lesions due to Streptococcus mitis in a previously healthy child].

Science.gov (United States)

Yiş, Reyhan; Yüksel, Ciğdem Nükhet; Derundere, Umit; Yiş, Uluç

2011-10-01

Streptococcus mitis, an important member of viridans streptococci, is found in the normal flora of the oropharynx, gastrointestinal tract, female genital tract and skin. Although it is of low pathogenicity and virulence, it may cause serious infections in immunocompromised patients. Meningitis caused by S.mitis has been described in patients with previous spinal anesthesia, neurosurgical procedure, malignancy, bacterial endocarditis with neurological complications and alcoholics, but it is rare in patients who are previously healthy. In this report, a rare case of meningoencephalitis caused by S.mitis developed in a previously healthy child has been presented. A previously healthy eight-year-old girl who presented with fever, altered state of consciousness, and headache was hospitalized in intensive care unit with the diagnosis of meningitis. Past history revealed that she was treated with amoxicillin-clavulanate for acute sinusitis ten days before her admission. Whole blood count revealed the followings: hemoglobin 13 g/dl, white blood cell count 18.6 x 109/L (90% neutrophils), platelet count 200 x 109/L and 150 leucocytes were detected on cerebrospinal fluid (CSF) examination. Protein and glucose levels of CSF were 80 mg/dl and 40 mg/dl (concomitant blood glucose 100 mg/dl), respectively. Brain magnetic resonance imaging (MRI) revealed widespread white matter lesions, and alpha-hemolytic streptococci were grown in CSF culture. The isolate was identified as S.mitis with conventional methods, and also confirmed by VITEK2 (bioMerieux, France) and API 20 STREP (bioMerieux, France) systems. Isolate was found susceptible to penicillin, erythromycin, clindamycin, tetracycline, cefotaxime, vancomycin and chloramphenicol. Regarding the etiology, echocardiography revealed no vegetation nor valve pathology, and peripheral blood smear showed no abnormality. Immunoglobulin and complement levels were within normal limits. Ongoing inflammation in maxillary sinuses detected in

Mpn1, Mutated in Poikiloderma with Neutropenia Protein 1, Is a Conserved 3′-to-5′ RNA Exonuclease Processing U6 Small Nuclear RNA

Directory of Open Access Journals (Sweden)

Vadim Shchepachev

2012-10-01

Full Text Available Clericuzio-type poikiloderma with neutropenia (PN is a rare genodermatosis associated with mutations in the C16orf57 gene, which codes for the uncharacterized protein hMpn1. We show here that, in both fission yeasts and humans, Mpn1 processes the spliceosomal U6 small nuclear RNA (snRNA posttranscriptionally. In Mpn1-deficient cells, U6 molecules carry 3′ end polyuridine tails that are longer than those in normal cells and lack a terminal 2′,3′ cyclic phosphate group. In mpn1Δ yeast cells, U6 snRNA and U4/U6 di-small nuclear RNA protein complex levels are diminished, leading to precursor messenger RNA splicing defects, which are reverted by expression of either yeast or human Mpn1 and by overexpression of U6. Recombinant hMpn1 is a 3′-to-5′ RNA exonuclease that removes uridines from U6 3′ ends, generating terminal 2′,3′ cyclic phosphates in vitro. Finally, U6 degradation rates increase in mpn1Δ yeasts and in lymphoblasts established from individuals affected by PN. Our data indicate that Mpn1 promotes U6 stability through 3′ end posttranscriptional processing and implicate altered U6 metabolism as a potential mechanism for PN pathogenesis.

Human salivary agglutinin binds to lung surfactant protein-D and is identical with scavenger receptor protein gp-340

DEFF Research Database (Denmark)

Ligtenberg, T J; Bikker, F J; Groenink, J

2001-01-01

bound in a similar way to Streptococcus mutans and surfactant protein-D. Histochemically, the distribution of gp-340 in the submandibular salivary glands was identical with the agglutinin distribution, as shown in a previous paper [Takano, Bogert, Malamud, Lally and Hand (1991) Anat. Rec. 230, 307......-318]. We conclude that agglutinin is identical with gp-340, and that this molecule interacts with S. mutans and surfactant protein-D....

Measurement of glutathione-protein mixed disulfides

International Nuclear Information System (INIS)

Livesey, J.C.; Reed, D.J.

1984-01-01

The development of a sensitive and highly specific assay for the presence of mixed disulfides between protein thiol groups and endogenous thiols has been undertaken. Previous investigations on the concentrations of glutathione (GSH), glutathione disulfide (GSSG) and protein glutathione mixed disulfides (ProSSG) have been of limited usefulness because of the poor specificity of the assays used. Our assay for these forms of glutathione is based on high performance liquid chromatography (HPLC) and is an extension of an earlier method. After perchloric acid precipitation, the protein sample is washed with an organic solvent to fully denature the protein. Up to a 10-fold increase in GSH released from fetal bovine serum (FBS) protein has been found when the protein precipitate is washed with ethanol rather than ether, as earlier suggested. Similar effects have been observed with an as yet unidentified thiol which elutes in the chromatography system with a retention volume similar to cysteine

Vaccinia-based influenza vaccine overcomes previously induced immunodominance hierarchy for heterosubtypic protection.

Science.gov (United States)

Kwon, Ji-Sun; Yoon, Jungsoon; Kim, Yeon-Jung; Kang, Kyuho; Woo, Sunje; Jung, Dea-Im; Song, Man Ki; Kim, Eun-Ha; Kwon, Hyeok-Il; Choi, Young Ki; Kim, Jihye; Lee, Jeewon; Yoon, Yeup; Shin, Eui-Cheol; Youn, Jin-Won

2014-08-01

Growing concerns about unpredictable influenza pandemics require a broadly protective vaccine against diverse influenza strains. One of the promising approaches was a T cell-based vaccine, but the narrow breadth of T-cell immunity due to the immunodominance hierarchy established by previous influenza infection and efficacy against only mild challenge condition are important hurdles to overcome. To model T-cell immunodominance hierarchy in humans in an experimental setting, influenza-primed C57BL/6 mice were chosen and boosted with a mixture of vaccinia recombinants, individually expressing consensus sequences from avian, swine, and human isolates of influenza internal proteins. As determined by IFN-γ ELISPOT and polyfunctional cytokine secretion, the vaccinia recombinants of influenza expanded the breadth of T-cell responses to include subdominant and even minor epitopes. Vaccine groups were successfully protected against 100 LD50 challenges with PR/8/34 and highly pathogenic avian influenza H5N1, which contained the identical dominant NP366 epitope. Interestingly, in challenge with pandemic A/Cal/04/2009 containing mutations in the dominant epitope, only the group vaccinated with rVV-NP + PA showed improved protection. Taken together, a vaccinia-based influenza vaccine expressing conserved internal proteins improved the breadth of influenza-specific T-cell immunity and provided heterosubtypic protection against immunologically close as well as distant influenza strains. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

AcEST: BP916224 [AcEST

Lifescience Database Archive (English)

Full Text Available 70 5e-11 tr|A3C6S4|A3C6S4_ORYSJ Putative uncharacterized protein OS=Oryza... 70 5e-11 tr|B6U1W6|B6U1W6_MAIZE Angus...ent) OS=Arab... 65 2e-09 tr|Q84JM5|Q84JM5_IPONI Angustifolia OS=Ipomoea nil GN=IA

AcEST: BP915041 [AcEST

Lifescience Database Archive (English)

Full Text Available . 172 1e-41 tr|B6U1W6|B6U1W6_MAIZE Angustifolia OS=Zea mays PE=2 SV=1 170 6e-41 tr|Q948X7|Q948X7_ARATH C-ter...YQ6_ARATH Putative uncharacterized protein (Putativ... 166 1e-39 tr|Q84JM5|Q84JM5_IPONI Angustifolia OS=Ipom

AcEST: BP914074 [AcEST

Lifescience Database Archive (English)

Full Text Available d P36357 Definition sp|P36357|VS09_ROTA1 Glycoprotein VP7 OS=Rotavirus A (strain ...VS09_ROTA1 Glycoprotein VP7 OS=Rotavirus A (strain Tur... 30 4.2 sp|P59051|CU087_HUMAN Uncharacterized prote...-like protein 1 OS=Homo ... 29 9.3 >sp|P36357|VS09_ROTA1 Glycoprotein VP7 OS=Rotavirus A (strain Turkey/Irel

Photoswitchable method for the ordered attachment of proteins to surfaces

Science.gov (United States)

Camarero, Julio A.; De Yoreo, James J.; Kwon, Youngeun

2010-04-20

Described herein is a method for the attachment of proteins to any solid support with control over the orientation of the attachment. The method is extremely efficient, not requiring the previous purification of the protein to be attached, and can be activated by UV-light. Spatially addressable arrays of multiple protein components can be generated by using standard photolithographic techniques.

Photoswitchable method for the ordered attachment of proteins to surfaces

Science.gov (United States)

Camarero, Julio A [Livermore, CA; DeYoreo, James J [Clayton, CA; Kwon, Youngeun [Livermore, CA

2011-07-05

Described herein is a method for the attachment of proteins to any solid support with control over the orientation of the attachment. The method is extremely efficient, not requiring the previous purification of the protein to be attached, and can be activated by UV-light. Spatially addressable arrays of multiple protein components can be generated by using standard photolithographic techniques.

Protein gels and emulsions from mixtures of Cape hake and pea proteins.

Science.gov (United States)

Tomé, Ana Sofia; Pires, Carla; Batista, Irineu; Sousa, Isabel; Raymundo, Anabela

2015-01-01

Portioning of frozen fish generates by-products such as fish 'sawdust' and cut-offs which can be further processed into protein concentrates and isolates. The objective of the present work was to produce gels and emulsions using recovered Cape hake protein powder (HPP). In previous works, the structures of the gels produced by HPP were found to be strong, with a high rubbery character. In this work, the addition of commercial pea proteins (PPC) to HPP gels and emulsions was studied. Physical properties of gels and emulsions prepared with different proportions of mixtures of PPC and HPP were evaluated. In general, gels and emulsions showed high values for whiteness and, as expected, the higher content of HPP in the protein mixtures led to higher firmness values of the gels. The gel network was rapidly formed upon heating due to the fish protein macromolecules and further reinforced by the pea protein macromolecules when cooled to 5 °C. Both visco-elastic parameters, storage and loss moduli, of the produced gels increased with the HPP proportion in the protein mixtures, corresponding to more structured systems. For the emulsions, two different pH environments were studied: 3.8 and 7.0. At neutral pH a synergy was found between the vegetable and fish protein, which is not so strong when pH is lowered to 3.8, near the isoelectric point of pea proteins (pI = 4.5). This evidence was supported by the results from the texture measurements, viscosity and visco-elastic parameters. Gels made from Cape hake proteins showed a softer texture and were less rubbery with the addition of pea proteins. Emulsions stabilised by these mixtures showed slightly different behaviour when produced at pH 7.0 or pH 3.8. © 2014 Society of Chemical Industry.

Two Nucleolar Proteins, GDP1 and OLI2, Function As Ribosome Biogenesis Factors and Are Preferentially Involved in Promotion of Leaf Cell Proliferation without Strongly Affecting Leaf Adaxial–Abaxial Patterning in Arabidopsis thaliana

Directory of Open Access Journals (Sweden)

Koji Kojima

2018-01-01

Full Text Available Leaf abaxial–adaxial patterning is dependent on the mutual repression of leaf polarity genes expressed either adaxially or abaxially. In Arabidopsis thaliana, this process is strongly affected by mutations in ribosomal protein genes and in ribosome biogenesis genes in a sensitized genetic background, such as asymmetric leaves2 (as2. Most ribosome-related mutants by themselves do not show leaf abaxialization, and one of their typical phenotypes is the formation of pointed rather than rounded leaves. In this study, we characterized two ribosome-related mutants to understand how ribosome biogenesis is linked to several aspects of leaf development. Previously, we isolated oligocellula2 (oli2 which exhibits the pointed-leaf phenotype and has a cell proliferation defect. OLI2 encodes a homolog of Nop2 in Saccharomyces cerevisiae, a ribosome biogenesis factor involved in pre-60S subunit maturation. In this study, we found another pointed-leaf mutant that carries a mutation in a gene encoding an uncharacterized protein with a G-patch domain. Similar to oli2, this mutant, named g-patch domain protein1 (gdp1, has a reduced number of leaf cells. In addition, gdp1 oli2 double mutants showed a strong genetic interaction such that they synergistically impaired cell proliferation in leaves and produced markedly larger cells. On the other hand, they showed additive phenotypes when combined with several known ribosomal protein mutants. Furthermore, these mutants have a defect in pre-rRNA processing. GDP1 and OLI2 are strongly expressed in tissues with high cell proliferation activity, and GDP1-GFP and GFP-OLI2 are localized in the nucleolus. These results suggest that OLI2 and GDP1 are involved in ribosome biogenesis. We then examined the effects of gdp1 and oli2 on adaxial–abaxial patterning by crossing them with as2. Interestingly, neither gdp1 nor oli2 strongly enhanced the leaf polarity defect of as2. Similar results were obtained with as2 gdp1 oli2

Diversity and evolution of ABC proteins in basidiomycetes.

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Kovalchuk, Andriy; Lee, Yong-Hwan; Asiegbu, Fred O

2013-01-01

ABC proteins constitute one of the largest families of proteins. They are implicated in wide variety of cellular processes ranging from ribosome biogenesis to multidrug resistance. With the advance of fungal genomics, the number of known fungal ABC proteins increases rapidly but the information on their biological functions remains scarce. In this work we extended the previous analysis of fungal ABC proteins to include recently sequenced species of basidiomycetes. We performed an identification and initial cataloging of ABC proteins from 23 fungal species representing 10 orders within class Agaricomycotina. We identified more than 1000 genes coding for ABC proteins. Comparison of sets of ABC proteins present in basidiomycetes and ascomycetes revealed the existence of two groups of ABC proteins specific for basidiomycetes. Results of survey should contribute to the better understanding of evolution of ABC proteins in fungi and support further experimental work on their characterization.

Merging in-silico and in vitro salivary protein complex partners using the STRING database: A tutorial.

Science.gov (United States)

Crosara, Karla Tonelli Bicalho; Moffa, Eduardo Buozi; Xiao, Yizhi; Siqueira, Walter Luiz

2018-01-16

Protein-protein interaction is a common physiological mechanism for protection and actions of proteins in an organism. The identification and characterization of protein-protein interactions in different organisms is necessary to better understand their physiology and to determine their efficacy. In a previous in vitro study using mass spectrometry, we identified 43 proteins that interact with histatin 1. Six previously documented interactors were confirmed and 37 novel partners were identified. In this tutorial, we aimed to demonstrate the usefulness of the STRING database for studying protein-protein interactions. We used an in-silico approach along with the STRING database (http://string-db.org/) and successfully performed a fast simulation of a novel constructed histatin 1 protein-protein network, including both the previously known and the predicted interactors, along with our newly identified interactors. Our study highlights the advantages and importance of applying bioinformatics tools to merge in-silico tactics with experimental in vitro findings for rapid advancement of our knowledge about protein-protein interactions. Our findings also indicate that bioinformatics tools such as the STRING protein network database can help predict potential interactions between proteins and thus serve as a guide for future steps in our exploration of the Human Interactome. Our study highlights the usefulness of the STRING protein database for studying protein-protein interactions. The STRING database can collect and integrate data about known and predicted protein-protein associations from many organisms, including both direct (physical) and indirect (functional) interactions, in an easy-to-use interface. Copyright © 2017 Elsevier B.V. All rights reserved.

High-throughput assessment of context-dependent effects of chromatin proteins

NARCIS (Netherlands)

Brueckner, L. (Laura); Van Arensbergen, J. (Joris); Akhtar, W. (Waseem); L. Pagie (Ludo); B. van Steensel (Bas)

2016-01-01

textabstractBackground: Chromatin proteins control gene activity in a concerted manner. We developed a high-throughput assay to study the effects of the local chromatin environment on the regulatory activity of a protein of interest. The assay combines a previously reported multiplexing strategy

Association of connexin43 with a receptor protein tyrosine phosphatase

NARCIS (Netherlands)

Giepmans, Ben N G; Feiken, Elles; Gebbink, Martijn F B G; Moolenaar, Wouter H

2003-01-01

Connexin-43(Cx43)-based gap junctional communication is transiently inhibited by certain G protein-coupled receptor agonists, including lysophosphatidic acid, endothelin and thrombin. Our previous studies have implicated the c-Src protein tyrosine kinase in mediating closure of Cx43 based gap

The apoptotic effect and the plausible mechanism of microwave radiation on rat myocardial cells.

Science.gov (United States)

Zhu, Wenhe; Cui, Yan; Feng, Xianmin; Li, Yan; Zhang, Wei; Xu, Junjie; Wang, Huiyan; Lv, Shijie

2016-08-01

Microwaves may exert adverse biological effects on the cardiovascular system at the integrated system and cellular levels. However, the mechanism underlying such effects remains poorly understood. Here, we report a previously uncharacterized mechanism through which microwaves damage myocardial cells. Rats were treated with 2450 MHz microwave radiation at 50, 100, 150, or 200 mW/cm(2) for 6 min. Microwave treatment significantly enhanced the levels of various enzymes in serum. In addition, it increased the malondialdehyde content while decreasing the levels of antioxidative stress enzymes, activities of enzyme complexes I-IV, and ATP in myocardial tissues. Notably, irradiated myocardial cells exhibited structural damage and underwent apoptosis. Furthermore, Western blot analysis revealed significant changes in expression levels of proteins involved in oxidative stress regulation and apoptotic signaling pathways, indicating that microwave irradiation could induce myocardial cell apoptosis by interfering with oxidative stress and cardiac energy metabolism. Our findings provide useful insights into the mechanism of microwave-induced damage to the cardiovascular system.

Heat-induced whey protein isolate fibrils: Conversion, hydrolysis, and disulphide bond formation

NARCIS (Netherlands)

Bolder, S.G.; Vasbinder, A.; Sagis, L.M.C.; Linden, van der E.

2007-01-01

Fibril formation of individual pure whey proteins and whey protein isolate (WPI) was studied. The heat-induced conversion of WPI monomers into fibrils at pH 2 and low ionic strength increased with heating time and protein concentration. Previous studies, using a precipitation method, size-exclusion

Quantifying the mechanisms of domain gain in animal proteins.

Science.gov (United States)

Buljan, Marija; Frankish, Adam; Bateman, Alex

2010-01-01

Protein domains are protein regions that are shared among different proteins and are frequently functionally and structurally independent from the rest of the protein. Novel domain combinations have a major role in evolutionary innovation. However, the relative contributions of the different molecular mechanisms that underlie domain gains in animals are still unknown. By using animal gene phylogenies we were able to identify a set of high confidence domain gain events and by looking at their coding DNA investigate the causative mechanisms. Here we show that the major mechanism for gains of new domains in metazoan proteins is likely to be gene fusion through joining of exons from adjacent genes, possibly mediated by non-allelic homologous recombination. Retroposition and insertion of exons into ancestral introns through intronic recombination are, in contrast to previous expectations, only minor contributors to domain gains and have accounted for less than 1% and 10% of high confidence domain gain events, respectively. Additionally, exonization of previously non-coding regions appears to be an important mechanism for addition of disordered segments to proteins. We observe that gene duplication has preceded domain gain in at least 80% of the gain events. The interplay of gene duplication and domain gain demonstrates an important mechanism for fast neofunctionalization of genes.

Binding free energy analysis of protein-protein docking model structures by evERdock.

Science.gov (United States)

Takemura, Kazuhiro; Matubayasi, Nobuyuki; Kitao, Akio

2018-03-14

To aid the evaluation of protein-protein complex model structures generated by protein docking prediction (decoys), we previously developed a method to calculate the binding free energies for complexes. The method combines a short (2 ns) all-atom molecular dynamics simulation with explicit solvent and solution theory in the energy representation (ER). We showed that this method successfully selected structures similar to the native complex structure (near-native decoys) as the lowest binding free energy structures. In our current work, we applied this method (evERdock) to 100 or 300 model structures of four protein-protein complexes. The crystal structures and the near-native decoys showed the lowest binding free energy of all the examined structures, indicating that evERdock can successfully evaluate decoys. Several decoys that show low interface root-mean-square distance but relatively high binding free energy were also identified. Analysis of the fraction of native contacts, hydrogen bonds, and salt bridges at the protein-protein interface indicated that these decoys were insufficiently optimized at the interface. After optimizing the interactions around the interface by including interfacial water molecules, the binding free energies of these decoys were improved. We also investigated the effect of solute entropy on binding free energy and found that consideration of the entropy term does not necessarily improve the evaluations of decoys using the normal model analysis for entropy calculation.

Phosphoproteome analysis of streptomyces development reveals extensive protein phosphorylation accompanying bacterial differentiation

DEFF Research Database (Denmark)

Manteca, Angel; Ye, Juanying; Sánchez, Jesús

2011-01-01

Streptomycetes are bacterial species that undergo a complex developmental cycle that includes programmed cell death (PCD) events and sporulation. They are widely used in biotechnology because they produce most clinically relevant secondary metabolites. Although Streptomyces coelicolor is one...... events were detected during the presporulation and sporulation stages (80%). Most of these phosphorylations were not reported before in Streptomyces, and included sporulation factors, transcriptional regulators, protein kinases and other regulatory proteins. Several of the identified phosphorylated...... proteins, FtsZ, DivIVA, and FtsH2, were previously demonstrated to be involved in the sporulation process. We thus established for the first time the widespread occurrence and dynamic features of Ser/Thr/Tyr protein phosphorylation in a bacteria species and also revealed a previously unrecognized...

Pleiotropic Regulation of Virulence Genes in Streptococcus mutans by the Conserved Small Protein SprV.

Science.gov (United States)

Shankar, Manoharan; Hossain, Mohammad S; Biswas, Indranil

2017-04-15

Streptococcus mutans , an oral pathogen associated with dental caries, colonizes tooth surfaces as polymicrobial biofilms known as dental plaque. S. mutans expresses several virulence factors that allow the organism to tolerate environmental fluctuations and compete with other microorganisms. We recently identified a small hypothetical protein (90 amino acids) essential for the normal growth of the bacterium. Inactivation of the gene, SMU.2137, encoding this protein caused a significant growth defect and loss of various virulence-associated functions. An S. mutans strain lacking this gene was more sensitive to acid, temperature, osmotic, oxidative, and DNA damage-inducing stresses. In addition, we observed an altered protein profile and defects in biofilm formation, bacteriocin production, and natural competence development, possibly due to the fitness defect associated with SMU.2137 deletion. Transcriptome sequencing revealed that nearly 20% of the S. mutans genes were differentially expressed upon SMU.2137 deletion, thereby suggesting a pleiotropic effect. Therefore, we have renamed this hitherto uncharacterized gene as sprV ( s treptococcal p leiotropic r egulator of v irulence). The transcript levels of several relevant genes in the sprV mutant corroborated the phenotypes observed upon sprV deletion. Owing to its highly conserved nature, inactivation of the sprV ortholog in Streptococcus gordonii also resulted in poor growth and defective UV tolerance and competence development as in the case of S. mutans Our experiments suggest that SprV is functionally distinct from its homologs identified by structure and sequence homology. Nonetheless, our current work is aimed at understanding the importance of SprV in the S. mutans biology. IMPORTANCE Streptococcus mutans employs several virulence factors and stress resistance mechanisms to colonize tooth surfaces and cause dental caries. Bacterial pathogenesis is generally controlled by regulators of fitness that are

Biochemical characterization of the small hydrophobic protein of avian metapneumovirus.

Science.gov (United States)

Deng, Qiji; Song, Minxun; Demers, Andrew; Weng, Yuejin; Lu, Wuxun; Wang, Dan; Kaushik, Radhey S; Yu, Qingzhong; Li, Feng

2012-08-01

Avian metapneumovirus (AMPV) is a paramyxovirus that has three membrane proteins (G, F, and SH). Among them, the SH protein is a small type II integral membrane protein that is incorporated into virions and is only present in certain paramyxoviruses. In the present study, we show that the AMPV SH protein is modified by N-linked glycans and can be released into the extracellular environment. Furthermore, we demonstrate that glycosylated AMPV SH proteins form homodimers through cysteine-mediated disulfide bonds, which has not been reported previously for SH proteins of paramyxoviruses. Copyright © 2012 Elsevier B.V. All rights reserved.

Fragment-Based Protein-Protein Interaction Antagonists of a Viral Dimeric Protease.

Science.gov (United States)

Gable, Jonathan E; Lee, Gregory M; Acker, Timothy M; Hulce, Kaitlin R; Gonzalez, Eric R; Schweigler, Patrick; Melkko, Samu; Farady, Christopher J; Craik, Charles S

2016-04-19

Fragment-based drug discovery has shown promise as an approach for challenging targets such as protein-protein interfaces. We developed and applied an activity-based fragment screen against dimeric Kaposi's sarcoma-associated herpesvirus protease (KSHV Pr) using an optimized fluorogenic substrate. Dose-response determination was performed as a confirmation screen, and NMR spectroscopy was used to map fragment inhibitor binding to KSHV Pr. Kinetic assays demonstrated that several initial hits also inhibit human cytomegalovirus protease (HCMV Pr). Binding of these hits to HCMV Pr was also confirmed by NMR spectroscopy. Despite the use of a target-agnostic fragment library, more than 80 % of confirmed hits disrupted dimerization and bound to a previously reported pocket at the dimer interface of KSHV Pr, not to the active site. One class of fragments, an aminothiazole scaffold, was further explored using commercially available analogues. These compounds demonstrated greater than 100-fold improvement of inhibition. This study illustrates the power of fragment-based screening for these challenging enzymatic targets and provides an example of the potential druggability of pockets at protein-protein interfaces. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Reducing dietary protein in pond production of hybrid striped bass - study shows a significant reduction is possible in digestible protein level in commercial diets

Science.gov (United States)

In previous work, we demonstrated that diets containing 40% digestible protein (DP) (45% crude protein) and 18 %lipid supplemented with Met and Lys resulted in superior performance and nutrient retentions in hybrid striped bass compared to less energy-dense diets when rearing hybrid striped bass at ...

Interplay between human high mobility group protein 1 and replication protein A on psoralen-cross-linked DNA

DEFF Research Database (Denmark)

Reddy, Madhava C; Christensen, Jesper; Vasquez, Karen M

2005-01-01

-DNA interstrand cross-link (ICL) to a specific site to determine the effect of HMGB proteins on recognition of these lesions. Our results reveal that human HMGB1 (but not HMGB2) binds with high affinity and specificity to psoralen ICLs, and interacts with the essential NER protein, replication protein A (RPA......), at these lesions. RPA, shown previously to bind tightly to these lesions, also binds in the presence of HMGB1, without displacing HMGB1. A discrete ternary complex is formed, containing HMGB1, RPA, and psoralen-damaged DNA. Thus, HMGB1 has the ability to recognize ICLs, can cooperate with RPA in doing so...

Chlamydia trachomatis Mip-like protein

DEFF Research Database (Denmark)

Lundemose, AG; Rousch, DA; Birkelund, Svend

1992-01-01

A 27 kDa Chlamydia trachomatis Mip-like protein with homology of a 175-amino-acid C-terminal fragment to the surface-exposed Legionella pneumophila mip-gene product has previously been described. In this paper the entire chlamydia Mip-like sequence of C. trachomatis serovar L2 (lymphogranuloma...... venereum (LGV) biovar) is presented. The sequence shows high similarity to the legionella Mip protein and its C-terminal region, like that of the legionella Mip, has high amino acid similarity to eukaryotic and prokaryotic FK506-binding proteins. The chlamydial mip-like gene was detected by polymerase...... chain reaction (PCR) in other C. trachomatis serovars and by sequencing of the mip-like genes of serovars B and E (trachoma biovar) was shown to be highly conserved within the two major biovars of C. trachomatis. Monoclonal and polyclonal antibodies raised against the recombinant Mip-like protein failed...

Tools for controlling protein interactions with light

Science.gov (United States)

Tucker, Chandra L.; Vrana, Justin D.; Kennedy, Matthew J.

2014-01-01

Genetically-encoded actuators that allow control of protein-protein interactions with light, termed ‘optical dimerizers’, are emerging as new tools for experimental biology. In recent years, numerous new and versatile dimerizer systems have been developed. Here we discuss the design of optical dimerizer experiments, including choice of a dimerizer system, photoexcitation sources, and coordinate use of imaging reporters. We provide detailed protocols for experiments using two dimerization systems we previously developed, CRY2/CIB and UVR8/UVR8, for use controlling transcription, protein localization, and protein secretion with light. Additionally, we provide instructions and software for constructing a pulse-controlled LED light device for use in experiments requiring extended light treatments. PMID:25181301

Gene Prioritization by Integrated Analysis of Protein Structural and Network Topological Properties for the Protein-Protein Interaction Network of Neurological Disorders

Directory of Open Access Journals (Sweden)

Yashna Paul

2016-01-01

Full Text Available Neurological disorders are known to show similar phenotypic manifestations like anxiety, depression, and cognitive impairment. There is a need to identify shared genetic markers and molecular pathways in these diseases, which lead to such comorbid conditions. Our study aims to prioritize novel genetic markers that might increase the susceptibility of patients affected with one neurological disorder to other diseases with similar manifestations. Identification of pathways involving common candidate markers will help in the development of improved diagnosis and treatments strategies for patients affected with neurological disorders. This systems biology study for the first time integratively uses 3D-structural protein interface descriptors and network topological properties that characterize proteins in a neurological protein interaction network, to aid the identification of genes that are previously not known to be shared between these diseases. Results of protein prioritization by machine learning have identified known as well as new genetic markers which might have direct or indirect involvement in several neurological disorders. Important gene hubs have also been identified that provide an evidence for shared molecular pathways in the neurological disease network.

Data quality objectives for sampling of sludge from the K West and K East Basin floor and from other Basin areas

International Nuclear Information System (INIS)

MAKENAS, B.J.

1998-01-01

This document addresses the characterization strategy for those types of sludge not previously characterized or discussed in previous DQO documents. It seeks to ascertain those characteristics of uncharacterized Sludge which are unique with respect to the properties already determined for canister and K East Basin floor Sludge. Also recent decisions have resulted in the need for treatment of the Sludge prior to its currently identified disposal path to the Hanford waste tanks. This has resulted in a need for process development testing for the treatment system development

A tensegrity model for hydrogen bond networks in proteins

OpenAIRE

Bywater, Robert P.

2017-01-01

Hydrogen-bonding networks in proteins considered as structural tensile elements are in balance separately from any other stabilising interactions that may be in operation. The hydrogen bond arrangement in the network is reminiscent of tensegrity structures in architecture and sculpture. Tensegrity has been discussed before in cells and tissues and in proteins. In contrast to previous work only hydrogen bonds are studied here. The other interactions within proteins are either much stronger − c...

Local anesthetic inhibition of G protein-coupled receptor signaling by interference with Galpha(q) protein function

NARCIS (Netherlands)

Hollmann, M. W.; Wieczorek, K. S.; Berger, A.; Durieux, M. E.

2001-01-01

Although local anesthetics are considered primarily Na(+) channel blockers, previous studies suggest a common intracellular site of action on different G protein-coupled receptors. In the present study, we characterized this site for the LPA, m1 muscarinic, and trypsin receptor. Xenopus laevis

Isomeric Detergent Comparison for Membrane Protein Stability

DEFF Research Database (Denmark)

Cho, Kyung Ho; Hariharan, Parameswaran; Mortensen, Jonas S.

2016-01-01

and utility, particularly for eukaryotic membrane proteins and membrane protein complexes. Thus, a number of new agents have been devised; some have made significant contributions to membrane protein structural studies. However, few detergent design principles are available. In this study, we prepared meta...... and ortho isomers of the previously reported para-substituted xylene-linked maltoside amphiphiles (XMAs), along with alkyl chain-length variation. The isomeric XMAs were assessed with three membrane proteins, and the meta isomer with a C12 alkyl chain was most effective at maintaining solubility....../stability of the membrane proteins. We propose that interplay between the hydrophile–lipophile balance (HLB) and alkyl chain length is of central importance for high detergent efficacy. In addition, differences in inter-alkyl-chain distance between the isomers influence the ability of the detergents to stabilise membrane...

Virus variants with differences in the P1 protein coexist in a Plum pox virus population and display particular host-dependent pathogenicity features.

Science.gov (United States)

Maliogka, Varvara I; Salvador, Beatriz; Carbonell, Alberto; Sáenz, Pilar; León, David San; Oliveros, Juan Carlos; Delgadillo, Ma Otilia; García, Juan Antonio; Simón-Mateo, Carmen

2012-10-01

Subisolates segregated from an M-type Plum pox virus (PPV) isolate, PPV-PS, differ widely in pathogenicity despite their high degree of sequence similarity. A single amino acid substitution, K109E, in the helper component proteinase (HCPro) protein of PPV caused a significant enhancement of symptom severity in herbaceous hosts, and notably modified virus infectivity in peach seedlings. The presence of this substitution in certain subisolates that induced mild symptoms in herbaceous hosts and did not infect peach seedlings suggested the existence of uncharacterized attenuating factors in these subisolates. In this study, we show that two amino acid changes in the P1 protein are specifically associated with the mild pathogenicity exhibited by some PS subisolates. Site-directed mutagenesis studies demonstrated that both substitutions, W29R and V139E, but especially W29R, resulted in lower levels of virus accumulation and symptom severity in a woody host, Prunus persica. Furthermore, when W29R and V139E mutations were expressed concomitantly, PPV infectivity was completely abolished in this host. In contrast, the V139E substitution, but not W29R, was found to be responsible for symptom attenuation in herbaceous hosts. Deep sequencing analysis demonstrated that the W29R and V139E heterogeneities already existed in the original PPV-PS isolate before its segregation in different subisolates by local lesion cloning. These results highlight the potential complexity of potyviral populations and the relevance of the P1 protein of potyviruses in pathogenesis and viral adaptation to the host. © 2012 THE AUTHORS. MOLECULAR PLANT PATHOLOGY © 2012 BSPP AND BLACKWELL PUBLISHING LTD.

The novel BTB/POZ and zinc finger factor Zbtb45 is essential for proper glial differentiation of neural and oligodendrocyte progenitor cells

DEFF Research Database (Denmark)

Södersten, Erik; Lilja, Tobias; Hermanson, Ola

2010-01-01

Understanding the regulatory mechanisms controlling the fate decisions of neural stem cells (NSCs) is a crucial issue to shed new light on mammalian central nervous system (CNS) development in health and disease. We have investigated a possible role for the previously uncharacterized BTB/POZ-doma......Understanding the regulatory mechanisms controlling the fate decisions of neural stem cells (NSCs) is a crucial issue to shed new light on mammalian central nervous system (CNS) development in health and disease. We have investigated a possible role for the previously uncharacterized BTB....../POZ-domain containing zinc finger factor Zbtb45 in the differentiation of NSCs and postnatal oligodendrocyte precursors. In situ hybridization histochemistry and RT-qPCR analysis revealed that Zbtb45 mRNA was ubiquitously expressed in the developing CNS in mouse embryos at embryonic day (E) 12.5 and 14.5. Zbtb45 m......RNA knockdown in embryonic forebrain NSCs by siRNA resulted in a rapid decrease in the expression of oligodendrocyte-characteristic genes after mitogen (FGF2) withdrawal, whereas the expression of astrocyte-associated genes such as CD44 and GFAP increased compared to control. Accordingly, the number...

Computational design of binding proteins to EGFR domain II.

Directory of Open Access Journals (Sweden)

Yoon Sup Choi

Full Text Available We developed a process to produce novel interactions between two previously unrelated proteins. This process selects protein scaffolds and designs protein interfaces that bind to a surface patch of interest on a target protein. Scaffolds with shapes complementary to the target surface patch were screened using an exhaustive computational search of the human proteome and optimized by directed evolution using phage display. This method was applied to successfully design scaffolds that bind to epidermal growth factor receptor (EGFR domain II, the interface of EGFR dimerization, with high reactivity toward the target surface patch of EGFR domain II. One potential application of these tailor-made protein interactions is the development of therapeutic agents against specific protein targets.

Biospecific protein immobilization for rapid analysis of weak protein interactions using self-interaction nanoparticle spectroscopy.

Science.gov (United States)

Bengali, Aditya N; Tessier, Peter M

2009-10-01

"Reversible" protein interactions govern diverse biological behavior ranging from intracellular transport and toxic protein aggregation to protein crystallization and inactivation of protein therapeutics. Much less is known about weak protein interactions than their stronger counterparts since they are difficult to characterize, especially in a parallel format (in contrast to a sequential format) necessary for high-throughput screening. We have recently introduced a highly efficient approach of characterizing protein self-association, namely self-interaction nanoparticle spectroscopy (SINS; Tessier et al., 2008; J Am Chem Soc 130:3106-3112). This approach exploits the separation-dependent optical properties of gold nanoparticles to detect weak self-interactions between proteins immobilized on nanoparticles. A limitation of our previous work is that differences in the sequence and structure of proteins can lead to significant differences in their affinity to adsorb to nanoparticle surfaces, which complicates analysis of the corresponding protein self-association behavior. In this work we demonstrate a highly specific approach for coating nanoparticles with proteins using biotin-avidin interactions to generate protein-nanoparticle conjugates that report protein self-interactions through changes in their optical properties. Using lysozyme as a model protein that is refractory to characterization by conventional SINS, we demonstrate that surface Plasmon wavelengths for gold-avidin-lysozyme conjugates over a range of solution conditions (i.e., pH and ionic strength) are well correlated with lysozyme osmotic second virial coefficient measurements. Since SINS requires orders of magnitude less protein and time than conventional methods (e.g., static light scattering), we envision this approach will find application in large screens of protein self-association aimed at either preventing (e.g., protein aggregation) or promoting (e.g., protein crystallization) these

AcEST: BP921212 [AcEST

Lifescience Database Archive (English)

Full Text Available YMU001_000147_A09 361 Adiantum capillus-veneris mRNA. clone: YMU001_000147_A09. BP921212 - Show BP921212...is mRNA. clone: YMU001_000147_A09. Accession BP921212 Tissue type prothallium Developmental stage - Contig I...protein database search programs, Nucleic Acids Res. 25:3389-3402. Query= BP921212|Adiantum capillus-veneris... mRNA, clone: YMU001_000147_A09. (361 letters) Database: uniprot_sprot.fasta 412,...itol-4,5-bisphosphate 3-ki... 30 2.9 sp|O14338|YB33_SCHPO Uncharacterized serine-rich protein C2F12.0... 29

Dietary protein to maximize resistance training: a review and examination of protein spread and change theories.

Science.gov (United States)

Bosse, John D; Dixon, Brian M

2012-09-08

An appreciable volume of human clinical data supports increased dietary protein for greater gains from resistance training, but not all findings are in agreement. We recently proposed "protein spread theory" and "protein change theory" in an effort to explain discrepancies in the response to increased dietary protein in weight management interventions. The present review aimed to extend "protein spread theory" and "protein change theory" to studies examining the effects of protein on resistance training induced muscle and strength gains. Protein spread theory proposed that there must have been a sufficient spread or % difference in g/kg/day protein intake between groups during a protein intervention to see muscle and strength differences. Protein change theory postulated that for the higher protein group, there must be a sufficient change from baseline g/kg/day protein intake to during study g/kg/day protein intake to see muscle and strength benefits. Seventeen studies met inclusion criteria. In studies where a higher protein intervention was deemed successful there was, on average, a 66.1% g/kg/day between group intake spread versus a 10.2% g/kg/day spread in studies where a higher protein diet was no more effective than control. The average change in habitual protein intake in studies showing higher protein to be more effective than control was +59.5% compared to +6.5% when additional protein was no more effective than control. The magnitudes of difference between the mean spreads and changes of the present review are similar to our previous review on these theories in a weight management context. Providing sufficient deviation from habitual intake appears to be an important factor in determining the success of additional protein in enhancing muscle and strength gains from resistance training. An increase in dietary protein favorably effects muscle and strength during resistance training.

A Global Survey of ATPase Activity in Plasmodium falciparum Asexual Blood Stages and Gametocytes

Energy Technology Data Exchange (ETDEWEB)

Ortega, Corrie; Frando, Andrew; Webb-Robertson, Bobbie-Jo; Anderson, Lindsey N.; Fleck, Neil; Flannery, Erika L.; Fishbaugher, Matthew; Murphree, Taylor A.; Hansen, Joshua R.; Smith, Richard D.; Kappe, Stefan H. I.; Wright, Aaron T.; Grundner, Christoph

2017-10-27

Effective malaria control and elimination in hyperendemic areas of the world will require treatment of disease-causing Plasmodium falciparum (Pf) blood stage infection but also blocking parasite transmission from humans to mosquito to prevent disease spread. Numerous antimalarial drugs have become ineffective due to parasite drug resistance and many currently used therapies do not kill gametocytes, highly specialized sexual parasite stages with distinct physiology that are necessary for transmission from the human host to the mosquito vector. Further confounding next generation drug development against Pf is the lack of known biochemical activity for most parasite gene products as well as the unknown metabolic needs of non-replicating gametocyte. Here, we take a systematic activity-based proteomics approach to survey the large and druggable ATPase family that is associated with replicating blood stage asexual parasites and transmissible gametocytes. We experimentally confirm existing annotation and predict ATPase function for 38 uncharacterized proteins. ATPase activity broadly changes during the transition from asexual schizonts to gametocytes, indicating altered metabolism and regulatory roles of ATPases specific for each lifecycle stage. By mapping the activity of ATPases associated with gametocytogenesis, we assign biochemical activity to a large number of uncharacterized proteins and identify new candidate transmission blocking targets.

Milk proteins interact with goat Binder of SPerm (BSP) proteins and decrease their binding to sperm.

Science.gov (United States)

de Menezes, Erika Bezerra; van Tilburg, Mauricio; Plante, Geneviève; de Oliveira, Rodrigo V; Moura, Arlindo A; Manjunath, Puttaswamy

2016-11-01

Seminal plasma Binder of SPerm (BSP) proteins bind to sperm at ejaculation and promote capacitation. When in excess, however, BSP proteins damage the sperm membrane. It has been suggested that milk components of semen extenders associate with BSP proteins, potentially protecting sperm. Thus, this study was conducted to investigate if milk proteins interact with BSP proteins and reduce BSP binding to goat sperm. Using gel filtration chromatography, milk was incubated with goat seminal plasma proteins and loaded onto columns with and without calcium. Milk was also fractionated into parts containing mostly whey proteins or mostly caseins, incubated with seminal plasma proteins and subjected to gel filtration. Eluted fractions were evaluated by immunoblot using anti-goat BSP antibodies, confirming milk protein-BSP protein interactions. As determined by ELISA, milk proteins coated on polystyrene wells bound to increasing of goat BSP proteins. Far-western dot blots confirmed that BSP proteins bound to caseins and β-lactoglobulin in a concentration-dependent manner. Then, cauda epididymal sperm from five goats was incubated with seminal plasma; seminal plasma followed by milk; and milk followed by seminal plasma. Sperm membrane proteins were extracted and evaluated by immunoblotting. The pattern of BSP binding to sperm membrane proteins was reduced by 59.3 % when epididymal sperm were incubated with seminal plasma and then with skimmed milk (p  0.05). In conclusion, goat BSP proteins have an affinity for caseins and whey proteins. Milk reduces BSP binding to goat sperm, depending whether or not sperm had been previously exposed to seminal plasma. Such events may explain the protective effect of milk during goat sperm preservation.

Improved detection of calcium-binding proteins in polyacrylamide gels

International Nuclear Information System (INIS)

Anthony, F.A.; Babitch, J.A.

1984-01-01

The authors refined the method of Schibeci and Martonosi (1980) to enhance detection of calcium-binding proteins in polyacrylamide gels using 45 Ca 2+ . Their efforts have produced a method which is shorter, has 40-fold greater sensitivity over the previous method, and will detect 'EF hand'-containing calcium-binding proteins in polyacrylamide gels below the 0.5 μg level. In addition this method will detect at least one example from every described class of calcium-binding protein, including lectins and γ-carboxyglutamic acid containing calcium-binding proteins. The method should be useful for detecting calcium-binding proteins which may trigger neurotransmitter release. (Auth.)

AcEST: DK958010 [AcEST

Lifescience Database Archive (English)

Full Text Available 3. 5' end sequence. DK958010 CL175Contig1 Show DK958010 Clone id TST39A01NGRL0029_O03 Library TST39 Length 6...65 Definition Adiantum capillus-veneris mRNA. clone: TST39A01NGRL0029_O03. 5' end sequence. Accession DK95801...of protein database search programs, Nucleic Acids Res. 25:3389-3402. Query= DK95801...c Acids Res. 25:3389-3402. Query= DK958010|Adiantum capillus-veneris mRNA, clone: TST39A01NGRL0029_O03, 5' (...tive uncharacterized protein OS=Picea... 115 3e-24 tr|A8Y801|A8Y801_ZANAE Rieske iron-sulphur protein OS=Zan

Do vulnerable populations consume adequate amounts of dietary protein?

Science.gov (United States)

In the previous year there has been a renewed interest in the adequacy of protein intake to maintain optimal health and to promote normal growth and development (1, 2). In this issue of the Journal there is an excellent report on protein consumption among children aged 6–36 mo from low-income countr...

Open reading frames associated with cancer in the dark matter of the human genome.

Science.gov (United States)

Delgado, Ana Paula; Brandao, Pamela; Chapado, Maria Julia; Hamid, Sheilin; Narayanan, Ramaswamy

2014-01-01

The uncharacterized proteins (open reading frames, ORFs) in the human genome offer an opportunity to discover novel targets for cancer. A systematic analysis of the dark matter of the human proteome for druggability and biomarker discovery is crucial to mining the genome. Numerous data mining tools are available to mine these ORFs to develop a comprehensive knowledge base for future target discovery and validation. Using the Genetic Association Database, the ORFs of the human dark matter proteome were screened for evidence of association with neoplasms. The Phenome-Genome Integrator tool was used to establish phenotypic association with disease traits including cancer. Batch analysis of the tools for protein expression analysis, gene ontology and motifs and domains was used to characterize the ORFs. Sixty-two ORFs were identified for neoplasm association. The expression Quantitative Trait Loci (eQTL) analysis identified thirteen ORFs related to cancer traits. Protein expression, motifs and domain analysis and genome-wide association studies verified the relevance of these OncoORFs in diverse tumors. The OncoORFs are also associated with a wide variety of human diseases and disorders. Our results link the OncoORFs to diverse diseases and disorders. This suggests a complex landscape of the uncharacterized proteome in human diseases. These results open the dark matter of the proteome to novel cancer target research. Copyright© 2014, International Institute of Anticancer Research (Dr. John G. Delinasios), All rights reserved.

Phylogenomic analysis of the GIY-YIG nuclease superfamily

Directory of Open Access Journals (Sweden)

Bujnicki Janusz M

2006-04-01

Full Text Available Abstract Background The GIY-YIG domain was initially identified in homing endonucleases and later in other selfish mobile genetic elements (including restriction enzymes and non-LTR retrotransposons and in enzymes involved in DNA repair and recombination. However, to date no systematic search for novel members of the GIY-YIG superfamily or comparative analysis of these enzymes has been reported. Results We carried out database searches to identify all members of known GIY-YIG nuclease families. Multiple sequence alignments together with predicted secondary structures of identified families were represented as Hidden Markov Models (HMM and compared by the HHsearch method to the uncharacterized protein families gathered in the COG, KOG, and PFAM databases. This analysis allowed for extending the GIY-YIG superfamily to include members of COG3680 and a number of proteins not classified in COGs and to predict that these proteins may function as nucleases, potentially involved in DNA recombination and/or repair. Finally, all old and new members of the GIY-YIG superfamily were compared and analyzed to infer the phylogenetic tree. Conclusion An evolutionary classification of the GIY-YIG superfamily is presented for the very first time, along with the structural annotation of all (subfamilies. It provides a comprehensive picture of sequence-structure-function relationships in this superfamily of nucleases, which will help to design experiments to study the mechanism of action of known members (especially the uncharacterized ones and will facilitate the prediction of function for the newly discovered ones.

Discrimination of thermophilic and mesophilic proteins

Directory of Open Access Journals (Sweden)

Vaisman Iosif I

2010-05-01

Full Text Available Abstract Background There is a considerable literature on the source of the thermostability of proteins from thermophilic organisms. Understanding the mechanisms for this thermostability would provide insights into proteins generally and permit the design of synthetic hyperstable biocatalysts. Results We have systematically tested a large number of sequence and structure derived quantities for their ability to discriminate thermostable proteins from their non-thermostable orthologs using sets of mesophile-thermophile ortholog pairs. Most of the quantities tested correspond to properties previously reported to be associated with thermostability. Many of the structure related properties were derived from the Delaunay tessellation of protein structures. Conclusions Carefully selected sequence based indices discriminate better than purely structure based indices. Combined sequence and structure based indices improve performance somewhat further. Based on our analysis, the strongest contributors to thermostability are an increase in ion pairs on the protein surface and a more strongly hydrophobic interior.

Sequence-based prediction of protein protein interaction using a deep-learning algorithm.

Science.gov (United States)

Sun, Tanlin; Zhou, Bo; Lai, Luhua; Pei, Jianfeng

2017-05-25

Protein-protein interactions (PPIs) are critical for many biological processes. It is therefore important to develop accurate high-throughput methods for identifying PPI to better understand protein function, disease occurrence, and therapy design. Though various computational methods for predicting PPI have been developed, their robustness for prediction with external datasets is unknown. Deep-learning algorithms have achieved successful results in diverse areas, but their effectiveness for PPI prediction has not been tested. We used a stacked autoencoder, a type of deep-learning algorithm, to study the sequence-based PPI prediction. The best model achieved an average accuracy of 97.19% with 10-fold cross-validation. The prediction accuracies for various external datasets ranged from 87.99% to 99.21%, which are superior to those achieved with previous methods. To our knowledge, this research is the first to apply a deep-learning algorithm to sequence-based PPI prediction, and the results demonstrate its potential in this field.

Sequence alignment reveals possible MAPK docking motifs on HIV proteins.

Directory of Open Access Journals (Sweden)

Perry Evans

Full Text Available Over the course of HIV infection, virus replication is facilitated by the phosphorylation of HIV proteins by human ERK1 and ERK2 mitogen-activated protein kinases (MAPKs. MAPKs are known to phosphorylate their substrates by first binding with them at a docking site. Docking site interactions could be viable drug targets because the sequences guiding them are more specific than phosphorylation consensus sites. In this study we use multiple bioinformatics tools to discover candidate MAPK docking site motifs on HIV proteins known to be phosphorylated by MAPKs, and we discuss the possibility of targeting docking sites with drugs. Using sequence alignments of HIV proteins of different subtypes, we show that MAPK docking patterns previously described for human proteins appear on the HIV matrix, Tat, and Vif proteins in a strain dependent manner, but are absent from HIV Rev and appear on all HIV Nef strains. We revise the regular expressions of previously annotated MAPK docking patterns in order to provide a subtype independent motif that annotates all HIV proteins. One revision is based on a documented human variant of one of the substrate docking motifs, and the other reduces the number of required basic amino acids in the standard docking motifs from two to one. The proposed patterns are shown to be consistent with in silico docking between ERK1 and the HIV matrix protein. The motif usage on HIV proteins is sufficiently different from human proteins in amino acid sequence similarity to allow for HIV specific targeting using small-molecule drugs.

A dual role of lipasin (betatrophin) in lipid metabolism and glucose homeostasis: consensus and controversy

OpenAIRE

Zhang, Ren; Abou-Samra, Abdul B

2014-01-01

Metabolic syndrome includes glucose intolerance and dyslipidemia, both of which are strong risk factors for developing diabetes and atherosclerotic cardiovascular diseases. Recently, multiple groups independently studied a previously uncharacterized gene, officially named C19orf80 (human) and Gm6484 (mouse), but more commonly known as RIFL, Angptl8, betatrophin and lipasin. Both exciting and conflicting results have been obtained, and significant controversy is ongoing. Accumulating evidence ...

Niakha virus: A novel member of the family Rhabdoviridae isolated from phlebotomine sandflies in Senegal

OpenAIRE

Vasilakis, Nikos; Widen, Steven; Mayer, Sandra V.; Seymour, Robert; Wood, Thomas G.; Popov, Vsevolov; Guzman, Hilda; Travassos da Rosa, Amelia P.A.; Ghedin, Elodie; Holmes, Edward C.; Walker, Peter J.; Tesh, Robert B.

2013-01-01

Members of the family Rhabdoviridae have been assigned to eight genera but many remain unassigned. Rhabdoviruses have a remarkably diverse host range that includes terrestrial and marine animals, invertebrates and plants. Transmission of some rhabdoviruses often requires an arthropod vector, such as mosquitoes, midges, sandflies, ticks, aphids and leafhoppers, in which they replicate. Herein we characterize Niakha virus (NIAV), a previously uncharacterized rhabdovirus isolated from phebotomin...

Engineering and Functional Characterization of Fusion Genes Identifies Novel Oncogenic Drivers of Cancer. | Office of Cancer Genomics

Science.gov (United States)

Oncogenic gene fusions drive many human cancers, but tools to more quickly unravel their functional contributions are needed. Here we describe methodology permitting fusion gene construction for functional evaluation. Using this strategy, we engineered the known fusion oncogenes, BCR-ABL1, EML4-ALK, and ETV6-NTRK3, as well as 20 previously uncharacterized fusion genes identified in TCGA datasets.

High glucose suppresses embryonic stem cell differentiation into neural lineage cells

OpenAIRE

Yang, Penghua; Shen, Wei-bin; Reece, E. Albert; Chen, Xi; Yang, Peixin

2016-01-01

Abnormal neurogenesis occurs during embryonic development in human diabetic pregnancies and in animal models of diabetic embryopathy. Our previous studies in a mouse model of diabetic embryopathy have implicated that high glucose of maternal diabetes delays neurogenesis in the developing neuroepithelium leading to neural tube defects. However, the underlying process in high glucose-impaired neurogenesis is uncharacterized. Neurogenesis from embryonic stem (ES) cells provides a valuable model ...

49 CFR 173.23 - Previously authorized packaging.

Science.gov (United States)

2010-10-01

... 49 Transportation 2 2010-10-01 2010-10-01 false Previously authorized packaging. 173.23 Section... REQUIREMENTS FOR SHIPMENTS AND PACKAGINGS Preparation of Hazardous Materials for Transportation § 173.23 Previously authorized packaging. (a) When the regulations specify a packaging with a specification marking...

Characterization of pathogenic germline mutations in human Protein Kinases

Directory of Open Access Journals (Sweden)

Orengo Christine A

2011-07-01

Full Text Available Abstract Background Protein Kinases are a superfamily of proteins involved in crucial cellular processes such as cell cycle regulation and signal transduction. Accordingly, they play an important role in cancer biology. To contribute to the study of the relation between kinases and disease we compared pathogenic mutations to neutral mutations as an extension to our previous analysis of cancer somatic mutations. First, we analyzed native and mutant proteins in terms of amino acid composition. Secondly, mutations were characterized according to their potential structural effects and finally, we assessed the location of the different classes of polymorphisms with respect to kinase-relevant positions in terms of subfamily specificity, conservation, accessibility and functional sites. Results Pathogenic Protein Kinase mutations perturb essential aspects of protein function, including disruption of substrate binding and/or effector recognition at family-specific positions. Interestingly these mutations in Protein Kinases display a tendency to avoid structurally relevant positions, what represents a significant difference with respect to the average distribution of pathogenic mutations in other protein families. Conclusions Disease-associated mutations display sound differences with respect to neutral mutations: several amino acids are specific of each mutation type, different structural properties characterize each class and the distribution of pathogenic mutations within the consensus structure of the Protein Kinase domain is substantially different to that for non-pathogenic mutations. This preferential distribution confirms previous observations about the functional and structural distribution of the controversial cancer driver and passenger somatic mutations and their use as a proxy for the study of the involvement of somatic mutations in cancer development.

Systematic analysis of protein turnover in primary cells.

Science.gov (United States)

Mathieson, Toby; Franken, Holger; Kosinski, Jan; Kurzawa, Nils; Zinn, Nico; Sweetman, Gavain; Poeckel, Daniel; Ratnu, Vikram S; Schramm, Maike; Becher, Isabelle; Steidel, Michael; Noh, Kyung-Min; Bergamini, Giovanna; Beck, Martin; Bantscheff, Marcus; Savitski, Mikhail M

2018-02-15

A better understanding of proteostasis in health and disease requires robust methods to determine protein half-lives. Here we improve the precision and accuracy of peptide ion intensity-based quantification, enabling more accurate protein turnover determination in non-dividing cells by dynamic SILAC-based proteomics. This approach allows exact determination of protein half-lives ranging from 10 to >1000 h. We identified 4000-6000 proteins in several non-dividing cell types, corresponding to 9699 unique protein identifications over the entire data set. We observed similar protein half-lives in B-cells, natural killer cells and monocytes, whereas hepatocytes and mouse embryonic neurons show substantial differences. Our data set extends and statistically validates the previous observation that subunits of protein complexes tend to have coherent turnover. Moreover, analysis of different proteasome and nuclear pore complex assemblies suggests that their turnover rate is architecture dependent. These results illustrate that our approach allows investigating protein turnover and its implications in various cell types.

AcEST: DK959619 [AcEST

Lifescience Database Archive (English)

Full Text Available cotiana bentha... 149 1e-34 tr|Q5W6B7|Q5W6B7_ORYSJ Os05g0270000 protein OS=Oryza sativa subs... 139 2e-31 tr|A2Y2K9|A2Y2K...KIRVNVPLVF 613 A ++++VPL+F Sbjct: 170 SALLKIDVPLMF 181 >tr|A2Y2K9|A2Y2K9_ORYSI Putative uncharacterized prot

AcEST: BP911764 [AcEST

Lifescience Database Archive (English)

Full Text Available SESSSSCSSIHSMDTNSSGMSSLIN 609 Query: 275 NLSNPVASN 301 LS+P + N Sbjct: 610 PLSSPPSCN 618 >sp|Q4G0P3|HYDIN_HUMAN Hydrocephalus...erase OS=Pseudom... 30 6.1 sp|Q9NZL6|RGL1_HUMAN Ral guanine nucleotide dissociation stimula... 30 8.0 sp|Q4G0P3|HYDIN_HUMAN Hydroceph...alus-inducing protein homolog OS=... 30 8.0 >sp|P36495|YCF78_CHLRE Uncharacterized

A Machine Learning Approach for Hot-Spot Detection at Protein-Protein Interfaces

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Rita Melo

2016-07-01

Full Text Available Understanding protein-protein interactions is a key challenge in biochemistry. In this work, we describe a more accurate methodology to predict Hot-Spots (HS in protein-protein interfaces from their native complex structure compared to previous published Machine Learning (ML techniques. Our model is trained on a large number of complexes and on a significantly larger number of different structural- and evolutionary sequence-based features. In particular, we added interface size, type of interaction between residues at the interface of the complex, number of different types of residues at the interface and the Position-Specific Scoring Matrix (PSSM, for a total of 79 features. We used twenty-seven algorithms from a simple linear-based function to support-vector machine models with different cost functions. The best model was achieved by the use of the conditional inference random forest (c-forest algorithm with a dataset pre-processed by the normalization of features and with up-sampling of the minor class. The method has an overall accuracy of 0.80, an F1-score of 0.73, a sensitivity of 0.76 and a specificity of 0.82 for the independent test set.

Effect of the level of dietary protein on the utilization of alpha-ketoisocaproate for protein synthesis

Energy Technology Data Exchange (ETDEWEB)

Kang, C.W.; Tungsanga, K.; Walser, M.

1986-04-01

The efficiency of alpha-ketoisocaproate (KIC) as a dietary substitute for leucine in rats on varying protein intake was estimated by an isotopic method, previously shown to yield the same results as comparative growth experiments. /sup 14/C-KIC and /sup 3/H-leucine are injected orally. Six hours later the ratio, R, of /sup 14/C//sup 3/H in isolated proteins, divided by the same ratio in the injectate is measured. This ratio has been shown to be approximately equal to nutritional efficiency of KIC relative to leucine. As dietary protein increased from 6.3% to 48.3%, whole body protein R decreased from 0.515 +/- 0.045 to 0.299 +/- 0.016. Variations with protein intake were noted in R of protein isolated from individual organs. The magnitude of R in these organs varied two-fold, in the following sequence: brain greater than heart greater than or equal to skeletal muscle greater than or equal to salivary gland greater than or equal to kidney greater than liver. Whole body protein R could be confidently predicted (r2 = 0.992) from R in the protein of kidney and muscle. Thus, the nutritional efficiency of KIC as a dietary substitute for leucine in individual organs as well as in the whole animal is strongly dependent on the level of protein intake.

Effect of the level of dietary protein on the utilization of alpha-ketoisocaproate for protein synthesis

International Nuclear Information System (INIS)

Kang, C.W.; Tungsanga, K.; Walser, M.

1986-01-01

The efficiency of alpha-ketoisocaproate (KIC) as a dietary substitute for leucine in rats on varying protein intake was estimated by an isotopic method, previously shown to yield the same results as comparative growth experiments. 14 C-KIC and 3 H-leucine are injected orally. Six hours later the ratio, R, of 14 C/ 3 H in isolated proteins, divided by the same ratio in the injectate is measured. This ratio has been shown to be approximately equal to nutritional efficiency of KIC relative to leucine. As dietary protein increased from 6.3% to 48.3%, whole body protein R decreased from 0.515 +/- 0.045 to 0.299 +/- 0.016. Variations with protein intake were noted in R of protein isolated from individual organs. The magnitude of R in these organs varied two-fold, in the following sequence: brain greater than heart greater than or equal to skeletal muscle greater than or equal to salivary gland greater than or equal to kidney greater than liver. Whole body protein R could be confidently predicted (r2 = 0.992) from R in the protein of kidney and muscle. Thus, the nutritional efficiency of KIC as a dietary substitute for leucine in individual organs as well as in the whole animal is strongly dependent on the level of protein intake