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Sample records for cellular protein database

  1. ComPPI: a cellular compartment-specific database for protein–protein interaction network analysis

    PubMed Central

    Veres, Daniel V.; Gyurkó, Dávid M.; Thaler, Benedek; Szalay, Kristóf Z.; Fazekas, Dávid; Korcsmáros, Tamás; Csermely, Peter

    2015-01-01

    Here we present ComPPI, a cellular compartment-specific database of proteins and their interactions enabling an extensive, compartmentalized protein–protein interaction network analysis (URL: http://ComPPI.LinkGroup.hu). ComPPI enables the user to filter biologically unlikely interactions, where the two interacting proteins have no common subcellular localizations and to predict novel properties, such as compartment-specific biological functions. ComPPI is an integrated database covering four species (S. cerevisiae, C. elegans, D. melanogaster and H. sapiens). The compilation of nine protein–protein interaction and eight subcellular localization data sets had four curation steps including a manually built, comprehensive hierarchical structure of >1600 subcellular localizations. ComPPI provides confidence scores for protein subcellular localizations and protein–protein interactions. ComPPI has user-friendly search options for individual proteins giving their subcellular localization, their interactions and the likelihood of their interactions considering the subcellular localization of their interacting partners. Download options of search results, whole-proteomes, organelle-specific interactomes and subcellular localization data are available on its website. Due to its novel features, ComPPI is useful for the analysis of experimental results in biochemistry and molecular biology, as well as for proteome-wide studies in bioinformatics and network science helping cellular biology, medicine and drug design. PMID:25348397

  2. Human Mitochondrial Protein Database

    National Institute of Standards and Technology Data Gateway

    SRD 131 Human Mitochondrial Protein Database (Web, free access)   The Human Mitochondrial Protein Database (HMPDb) provides comprehensive data on mitochondrial and human nuclear encoded proteins involved in mitochondrial biogenesis and function. This database consolidates information from SwissProt, LocusLink, Protein Data Bank (PDB), GenBank, Genome Database (GDB), Online Mendelian Inheritance in Man (OMIM), Human Mitochondrial Genome Database (mtDB), MITOMAP, Neuromuscular Disease Center and Human 2-D PAGE Databases. This database is intended as a tool not only to aid in studying the mitochondrion but in studying the associated diseases.

  3. The Halophile protein database.

    PubMed

    Sharma, Naveen; Farooqi, Mohammad Samir; Chaturvedi, Krishna Kumar; Lal, Shashi Bhushan; Grover, Monendra; Rai, Anil; Pandey, Pankaj

    2014-01-01

    Halophilic archaea/bacteria adapt to different salt concentration, namely extreme, moderate and low. These type of adaptations may occur as a result of modification of protein structure and other changes in different cell organelles. Thus proteins may play an important role in the adaptation of halophilic archaea/bacteria to saline conditions. The Halophile protein database (HProtDB) is a systematic attempt to document the biochemical and biophysical properties of proteins from halophilic archaea/bacteria which may be involved in adaptation of these organisms to saline conditions. In this database, various physicochemical properties such as molecular weight, theoretical pI, amino acid composition, atomic composition, estimated half-life, instability index, aliphatic index and grand average of hydropathicity (Gravy) have been listed. These physicochemical properties play an important role in identifying the protein structure, bonding pattern and function of the specific proteins. This database is comprehensive, manually curated, non-redundant catalogue of proteins. The database currently contains 59 897 proteins properties extracted from 21 different strains of halophilic archaea/bacteria. The database can be accessed through link. Database URL: http://webapp.cabgrid.res.in/protein/

  4. Protein Structure Databases.

    PubMed

    Laskowski, Roman A

    2016-01-01

    Web-based protein structure databases come in a wide variety of types and levels of information content. Those having the most general interest are the various atlases that describe each experimentally determined protein structure and provide useful links, analyses, and schematic diagrams relating to its 3D structure and biological function. Also of great interest are the databases that classify 3D structures by their folds as these can reveal evolutionary relationships which may be hard to detect from sequence comparison alone. Related to these are the numerous servers that compare folds-particularly useful for newly solved structures, and especially those of unknown function. Beyond these are a vast number of databases for the more specialized user, dealing with specific families, diseases, structural features, and so on. PMID:27115626

  5. The Protein Ensemble Database.

    PubMed

    Varadi, Mihaly; Tompa, Peter

    2015-01-01

    The scientific community's major conceptual notion of structural biology has recently shifted in emphasis from the classical structure-function paradigm due to the emergence of intrinsically disordered proteins (IDPs). As opposed to their folded cousins, these proteins are defined by the lack of a stable 3D fold and a high degree of inherent structural heterogeneity that is closely tied to their function. Due to their flexible nature, solution techniques such as small-angle X-ray scattering (SAXS), nuclear magnetic resonance (NMR) spectroscopy and fluorescence resonance energy transfer (FRET) are particularly well-suited for characterizing their biophysical properties. Computationally derived structural ensembles based on such experimental measurements provide models of the conformational sampling displayed by these proteins, and they may offer valuable insights into the functional consequences of inherent flexibility. The Protein Ensemble Database (http://pedb.vib.be) is the first openly accessible, manually curated online resource storing the ensemble models, protocols used during the calculation procedure, and underlying primary experimental data derived from SAXS and/or NMR measurements. By making this previously inaccessible data freely available to researchers, this novel resource is expected to promote the development of more advanced modelling methodologies, facilitate the design of standardized calculation protocols, and consequently lead to a better understanding of how function arises from the disordered state.

  6. DIP: The Database of Interacting Proteins

    DOE Data Explorer

    The DIP Database catalogs experimentally determined interactions between proteins. It combines information from a variety of sources to create a single, consistent set of protein-protein interactions. By interaction, the DIP Database creators mean that two amino acid chains were experimentally identified to bind to each other. The database lists such pairs to aid those studying a particular protein-protein interaction but also those investigating entire regulatory and signaling pathways as well as those studying the organisation and complexity of the protein interaction network at the cellular level. The data stored within the DIP database were curated, both, manually by expert curators and also automatically using computational approaches that utilize the knowledge about the protein-protein interaction networks extracted from the most reliable, core subset of the DIP data. It is a relational database that can be searched by protein, sequence, motif, article information, and pathBLAST. The website also serves as an access point to a number of projects related to DIP, such as LiveDIP, The Database of Ligand-Receptor Partners (DLRP) and JDIP. Users have free and open access to DIP after login. [Taken from the DIP Guide and the DIP website] (Specialized Interface) (Registration Required)

  7. A comparative cellular and molecular biology of longevity database.

    PubMed

    Stuart, Jeffrey A; Liang, Ping; Luo, Xuemei; Page, Melissa M; Gallagher, Emily J; Christoff, Casey A; Robb, Ellen L

    2013-10-01

    Discovering key cellular and molecular traits that promote longevity is a major goal of aging and longevity research. One experimental strategy is to determine which traits have been selected during the evolution of longevity in naturally long-lived animal species. This comparative approach has been applied to lifespan research for nearly four decades, yielding hundreds of datasets describing aspects of cell and molecular biology hypothesized to relate to animal longevity. Here, we introduce a Comparative Cellular and Molecular Biology of Longevity Database, available at ( http://genomics.brocku.ca/ccmbl/ ), as a compendium of comparative cell and molecular data presented in the context of longevity. This open access database will facilitate the meta-analysis of amalgamated datasets using standardized maximum lifespan (MLSP) data (from AnAge). The first edition contains over 800 data records describing experimental measurements of cellular stress resistance, reactive oxygen species metabolism, membrane composition, protein homeostasis, and genome homeostasis as they relate to vertebrate species MLSP. The purpose of this review is to introduce the database and briefly demonstrate its use in the meta-analysis of combined datasets.

  8. Protein accounting in the cellular economy.

    PubMed

    Vázquez-Laslop, Nora; Mankin, Alexander S

    2014-04-24

    Knowing the copy number of cellular proteins is critical for understanding cell physiology. By being able to measure the absolute synthesis rates of the majority of cellular proteins, Li et al. gain insights into key aspects of translation regulation and fundamental principles of cellular strategies to adjust protein synthesis according to the functional needs.

  9. Pfam: the protein families database

    PubMed Central

    Finn, Robert D.; Bateman, Alex; Clements, Jody; Coggill, Penelope; Eberhardt, Ruth Y.; Eddy, Sean R.; Heger, Andreas; Hetherington, Kirstie; Holm, Liisa; Mistry, Jaina; Sonnhammer, Erik L. L.; Tate, John; Punta, Marco

    2014-01-01

    Pfam, available via servers in the UK (http://pfam.sanger.ac.uk/) and the USA (http://pfam.janelia.org/), is a widely used database of protein families, containing 14 831 manually curated entries in the current release, version 27.0. Since the last update article 2 years ago, we have generated 1182 new families and maintained sequence coverage of the UniProt Knowledgebase (UniProtKB) at nearly 80%, despite a 50% increase in the size of the underlying sequence database. Since our 2012 article describing Pfam, we have also undertaken a comprehensive review of the features that are provided by Pfam over and above the basic family data. For each feature, we determined the relevance, computational burden, usage statistics and the functionality of the feature in a website context. As a consequence of this review, we have removed some features, enhanced others and developed new ones to meet the changing demands of computational biology. Here, we describe the changes to Pfam content. Notably, we now provide family alignments based on four different representative proteome sequence data sets and a new interactive DNA search interface. We also discuss the mapping between Pfam and known 3D structures. PMID:24288371

  10. The Pfam protein families database

    PubMed Central

    Finn, Robert D.; Mistry, Jaina; Tate, John; Coggill, Penny; Heger, Andreas; Pollington, Joanne E.; Gavin, O. Luke; Gunasekaran, Prasad; Ceric, Goran; Forslund, Kristoffer; Holm, Liisa; Sonnhammer, Erik L. L.; Eddy, Sean R.; Bateman, Alex

    2010-01-01

    Pfam is a widely used database of protein families and domains. This article describes a set of major updates that we have implemented in the latest release (version 24.0). The most important change is that we now use HMMER3, the latest version of the popular profile hidden Markov model package. This software is ∼100 times faster than HMMER2 and is more sensitive due to the routine use of the forward algorithm. The move to HMMER3 has necessitated numerous changes to Pfam that are described in detail. Pfam release 24.0 contains 11 912 families, of which a large number have been significantly updated during the past two years. Pfam is available via servers in the UK (http://pfam.sanger.ac.uk/), the USA (http://pfam.janelia.org/) and Sweden (http://pfam.sbc.su.se/). PMID:19920124

  11. Protein-protein interaction databases: keeping up with growing interactomes

    PubMed Central

    2009-01-01

    Over the past few years, the number of known protein-protein interactions has increased substantially. To make this information more readily available, a number of publicly available databases have set out to collect and store protein-protein interaction data. Protein-protein interactions have been retrieved from six major databases, integrated and the results compared. The six databases (the Biological General Repository for Interaction Datasets [BioGRID], the Molecular INTeraction database [MINT], the Biomolecular Interaction Network Database [BIND], the Database of Interacting Proteins [DIP], the IntAct molecular interaction database [IntAct] and the Human Protein Reference Database [HPRD]) differ in scope and content; integration of all datasets is non-trivial owing to differences in data annotation. With respect to human protein-protein interaction data, HPRD seems to be the most comprehensive. To obtain a complete dataset, however, interactions from all six databases have to be combined. To overcome this limitation, meta-databases such as the Agile Protein Interaction Database (APID) offer access to integrated protein-protein interaction datasets, although these also currently have certain restrictions. PMID:19403463

  12. The PIR-International Protein Sequence Database.

    PubMed

    Barker, W C; Garavelli, J S; McGarvey, P B; Marzec, C R; Orcutt, B C; Srinivasarao, G Y; Yeh, L S; Ledley, R S; Mewes, H W; Pfeiffer, F; Tsugita, A; Wu, C

    1999-01-01

    The Protein Information Resource (PIR; http://www-nbrf.georgetown. edu/pir/) supports research on molecular evolution, functional genomics, and computational biology by maintaining a comprehensive, non-redundant, well-organized and freely available protein sequence database. Since 1988 the database has been maintained collaboratively by PIR-International, an international association of data collection centers cooperating to develop this resource during a period of explosive growth in new sequence data and new computer technologies. The PIR Protein Sequence Database entries are classified into superfamilies, families and homology domains, for which sequence alignments are available. Full-scale family classification supports comparative genomics research, aids sequence annotation, assists database organization and improves database integrity. The PIR WWW server supports direct on-line sequence similarity searches, information retrieval, and knowledge discovery by providing the Protein Sequence Database and other supplementary databases. Sequence entries are extensively cross-referenced and hypertext-linked to major nucleic acid, literature, genome, structure, sequence alignment and family databases. The weekly release of the Protein Sequence Database can be accessed through the PIR Web site. The quarterly release of the database is freely available from our anonymous FTP server and is also available on CD-ROM with the accompanying ATLAS database search program.

  13. Cellular phenotype database: a repository for systems microscopy data

    PubMed Central

    Kirsanova, Catherine; Brazma, Alvis; Rustici, Gabriella; Sarkans, Ugis

    2015-01-01

    Motivation: The Cellular Phenotype Database (CPD) is a repository for data derived from high-throughput systems microscopy studies. The aims of this resource are: (i) to provide easy access to cellular phenotype and molecular localization data for the broader research community; (ii) to facilitate integration of independent phenotypic studies by means of data aggregation techniques, including use of an ontology and (iii) to facilitate development of analytical methods in this field. Results: In this article we present CPD, its data structure and user interface, propose a minimal set of information describing RNA interference experiments, and suggest a generic schema for management and aggregation of outputs from phenotypic or molecular localization experiments. The database has a flexible structure for management of data from heterogeneous sources of systems microscopy experimental outputs generated by a variety of protocols and technologies and can be queried by gene, reagent, gene attribute, study keywords, phenotype or ontology terms. Availability and implementation: CPD is developed as part of the Systems Microscopy Network of Excellence and is accessible at http://www.ebi.ac.uk/fg/sym. Contact: jes@ebi.ac.uk or ugis@ebi.ac.uk Supplementary information: Supplementary data are available at Bioinformatics online. PMID:25861964

  14. Protein databases and software on BIONET.

    PubMed

    Maulik, S

    1989-02-01

    BIONET provides databases, software, and networking/communications tools to over 2500 molecular biologists worldwide. Software for the analysis of nucleic acid and protein sequence data is provided by both IntelliGenetics and academic contributors. BIONET is currently implementing dedicated high speed servers for searching protein databases, as well as providing more flexible tools for protein structure recognition and prediction. In this review, protein databases and analysis software available on the BIONET resource are described, and progress in providing new tools for structure prediction, comparative sequence analysis, and pattern recognition using Artificial Intelligence (AI) techniques are summarized. PMID:2652143

  15. Cellular regulation by protein phosphorylation.

    PubMed

    Fischer, Edmond H

    2013-01-11

    A historical account of the discovery of reversible protein phosphorylation is presented. This process was uncovered in the mid 1950s in a study undertaken with Edwin G. Krebs to elucidate the complex hormonal regulation of skeletal muscle glycogen phosphorylase. Contrary to the known activation of this enzyme by AMP which serves as an allosteric effector, its hormonal regulation results from a phosphorylation of the protein by phosphorylase kinase following the activation of the latter by Ca(2+) and ATP. The study led to the establishment of the first hormonal cascade of successive enzymatic reactions, kinases acting on kinases, initiated by cAMP discovered by Earl Sutherland. It also showed how two different physiological processes, carbohydrate metabolism and muscle contraction, could be regulated in concert.

  16. SENTRA, a database of signal transduction proteins.

    SciTech Connect

    D'Souza, M.; Romine, M. F.; Maltsev, N.; Mathematics and Computer Science; PNNL

    2000-01-01

    SENTRA, available via URL http://wit.mcs.anl.gov/WIT2/Sentra/, is a database of proteins associated with microbial signal transduction. The database currently includes the classical two-component signal transduction pathway proteins and methyl-accepting chemotaxis proteins, but will be expanded to also include other classes of signal transduction systems that are modulated by phosphorylation or methylation reactions. Although the majority of database entries are from prokaryotic systems, eukaroytic proteins with bacterial-like signal transduction domains are also included. Currently SENTRA contains signal transduction proteins in 34 complete and almost completely sequenced prokaryotic genomes, as well as sequences from 243 organisms available in public databases (SWISS-PROT and EMBL). The analysis was carried out within the framework of the WIT2 system, which is designed and implemented to support genetic sequence analysis and comparative analysis of sequenced genomes.

  17. Dynameomics: A comprehensive database of protein dynamics

    PubMed Central

    van der Kamp, Marc W.; Schaeffer, Richard D.; Jonsson, Amanda L.; Scouras, Alexander D.; Simms, Andrew; Toofanny, Rudesh D.; Benson, Noah C.; Anderson, Peter C.; Merkley, Eric D.; Rysavy, Steve; Bromley, Denny; Beck, David A. C.; Daggett, Valerie

    2010-01-01

    Summary The dynamic behavior of proteins is important for an understanding of their function and folding. We have performed molecular dynamics simulations of the native state and unfolding pathways of over 1000 proteins, representing the majority of folds in globular proteins. These data are stored and organized using an innovative database approach, which can be mined to obtain both general and specific information about the dynamics and folding/unfolding of proteins, relevant subsets thereof, and individual proteins. Here we describe the project in general terms and the type of information contained in the database. Then we provide examples of mining the database for information relevant to protein folding, structure building, the effect of single-nucleotide polymorphisms, and drug design. The native state simulation data and corresponding analyses for the 100 most populated metafolds, together with related resources, are publicly accessible through www.dynameomics.org. PMID:20399180

  18. CPLM: a database of protein lysine modifications

    PubMed Central

    Liu, Zexian; Wang, Yongbo; Gao, Tianshun; Pan, Zhicheng; Cheng, Han; Yang, Qing; Cheng, Zhongyi; Guo, Anyuan; Ren, Jian; Xue, Yu

    2014-01-01

    We reported an integrated database of Compendium of Protein Lysine Modifications (CPLM; http://cplm.biocuckoo.org) for protein lysine modifications (PLMs), which occur at active ε-amino groups of specific lysine residues in proteins and are critical for orchestrating various biological processes. The CPLM database was updated from our previously developed database of Compendium of Protein Lysine Acetylation (CPLA), which contained 7151 lysine acetylation sites in 3311 proteins. Here, we manually collected experimentally identified substrates and sites for 12 types of PLMs, including acetylation, ubiquitination, sumoylation, methylation, butyrylation, crotonylation, glycation, malonylation, phosphoglycerylation, propionylation, succinylation and pupylation. In total, the CPLM database contained 203 972 modification events on 189 919 modified lysines in 45 748 proteins for 122 species. With the dataset, we totally identified 76 types of co-occurrences of various PLMs on the same lysine residues, and the most abundant PLM crosstalk is between acetylation and ubiquitination. Up to 53.5% of acetylation and 33.1% of ubiquitination events co-occur at 10 746 lysine sites. Thus, the various PLM crosstalks suggested that a considerable proportion of lysines were competitively and dynamically regulated in a complicated manner. Taken together, the CPLM database can serve as a useful resource for further research of PLMs. PMID:24214993

  19. VirusMINT: a viral protein interaction database

    PubMed Central

    Chatr-aryamontri, Andrew; Ceol, Arnaud; Peluso, Daniele; Nardozza, Aurelio; Panni, Simona; Sacco, Francesca; Tinti, Michele; Smolyar, Alex; Castagnoli, Luisa; Vidal, Marc; Cusick, Michael E.; Cesareni, Gianni

    2009-01-01

    Understanding the consequences on host physiology induced by viral infection requires complete understanding of the perturbations caused by virus proteins on the cellular protein interaction network. The VirusMINT database (http://mint.bio.uniroma2.it/virusmint/) aims at collecting all protein interactions between viral and human proteins reported in the literature. VirusMINT currently stores over 5000 interactions involving more than 490 unique viral proteins from more than 110 different viral strains. The whole data set can be easily queried through the search pages and the results can be displayed with a graphical viewer. The curation effort has focused on manuscripts reporting interactions between human proteins and proteins encoded by some of the most medically relevant viruses: papilloma viruses, human immunodeficiency virus 1, Epstein–Barr virus, hepatitis B virus, hepatitis C virus, herpes viruses and Simian virus 40. PMID:18974184

  20. Proteomics: Protein Identification Using Online Databases

    ERIC Educational Resources Information Center

    Eurich, Chris; Fields, Peter A.; Rice, Elizabeth

    2012-01-01

    Proteomics is an emerging area of systems biology that allows simultaneous study of thousands of proteins expressed in cells, tissues, or whole organisms. We have developed this activity to enable high school or college students to explore proteomic databases using mass spectrometry data files generated from yeast proteins in a college laboratory…

  1. Regulation of cellular transport by klotho protein.

    PubMed

    Sopjani, Mentor; Rinnerthaler, Mark; Almilaji, Ahmad; Ahmeti, Salih; Dermaku-Sopjani, Miribane

    2014-01-01

    The antiaging protein of Klotho is a transmembrane protein mainly expressed in the kidney, parathyroid glands and choroid plexus of the brain. The Klotho protein exists in two forms, a full-length membrane form and a soluble secreted form. The extracellular domain of Klotho can be enzymatically cleaved off and released into the systemic circulation where it acts as β-glucuronidase and a hormone. Soluble Klotho can be found in the blood, cerebrospinal fluid, and the urine of mammals. Klotho deficiency results in early appearance of multiple age-related disorders and premature death, whereas overexpression of Klotho exerts the opposite effect. Klotho may influence cellular transport processes across the cell membrane by inhibiting calcitriol (1,25(OH) (2)D(3)), formation or by directly affecting transporter proteins, including ion channels, carriers and pumps. Accordingly, Klotho protein is a powerful regulator of transport mechanisms across the cell membrane. Klotho regulates diverse calcium and potassium ion channels, as well as several carriers including the Na(+)-coupled excitatory amino acid transporters EAAT3 and EAAT4, the Na(+)-coupled phosphate cotransporters, NaPi-IIa and NaPi-IIb, and a Na(+)/K(+)-ATPase. All those cellular transport regulations contribute in the aging suppressor role of Klotho. Future studies will help to determine if the Klotho protein regulates cell-surface expression of other transport proteins and is affecting underlying mechanisms.

  2. HypoxiaDB: a database of hypoxia-regulated proteins.

    PubMed

    Khurana, Pankaj; Sugadev, Ragumani; Jain, Jaspreet; Singh, Shashi Bala

    2013-01-01

    There has been intense interest in the cellular response to hypoxia, and a large number of differentially expressed proteins have been identified through various high-throughput experiments. These valuable data are scattered, and there have been no systematic attempts to document the various proteins regulated by hypoxia. Compilation, curation and annotation of these data are important in deciphering their role in hypoxia and hypoxia-related disorders. Therefore, we have compiled HypoxiaDB, a database of hypoxia-regulated proteins. It is a comprehensive, manually-curated, non-redundant catalog of proteins whose expressions are shown experimentally to be altered at different levels and durations of hypoxia. The database currently contains 72 000 manually curated entries taken on 3500 proteins extracted from 73 peer-reviewed publications selected from PubMed. HypoxiaDB is distinctive from other generalized databases: (i) it compiles tissue-specific protein expression changes under different levels and duration of hypoxia. Also, it provides manually curated literature references to support the inclusion of the protein in the database and establish its association with hypoxia. (ii) For each protein, HypoxiaDB integrates data on gene ontology, KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway, protein-protein interactions, protein family (Pfam), OMIM (Online Mendelian Inheritance in Man), PDB (Protein Data Bank) structures and homology to other sequenced genomes. (iii) It also provides pre-compiled information on hypoxia-proteins, which otherwise requires tedious computational analysis. This includes information like chromosomal location, identifiers like Entrez, HGNC, Unigene, Uniprot, Ensembl, Vega, GI numbers and Genbank accession numbers associated with the protein. These are further cross-linked to respective public databases augmenting HypoxiaDB to the external repositories. (iv) In addition, HypoxiaDB provides an online sequence-similarity search tool for

  3. The Pfam protein families database.

    PubMed

    Bateman, Alex; Birney, Ewan; Cerruti, Lorenzo; Durbin, Richard; Etwiller, Laurence; Eddy, Sean R; Griffiths-Jones, Sam; Howe, Kevin L; Marshall, Mhairi; Sonnhammer, Erik L L

    2002-01-01

    Pfam is a large collection of protein multiple sequence alignments and profile hidden Markov models. Pfam is available on the World Wide Web in the UK at http://www.sanger.ac.uk/Software/Pfam/, in Sweden at http://www.cgb.ki.se/Pfam/, in France at http://pfam.jouy.inra.fr/ and in the US at http://pfam.wustl.edu/. The latest version (6.6) of Pfam contains 3071 families, which match 69% of proteins in SWISS-PROT 39 and TrEMBL 14. Structural data, where available, have been utilised to ensure that Pfam families correspond with structural domains, and to improve domain-based annotation. Predictions of non-domain regions are now also included. In addition to secondary structure, Pfam multiple sequence alignments now contain active site residue mark-up. New search tools, including taxonomy search and domain query, greatly add to the functionality and usability of the Pfam resource.

  4. Molecular and cellular constraints on proteins

    NASA Astrophysics Data System (ADS)

    Kortemme, Tanja

    Engineering proteins with new sequences, structures and functions has many exciting practical applications, and provides new ways to dissect design principles for function. Recent successes in computational protein design provide a cause for optimism. Yet many functions are currently too complex to engineer predictively, and successful design of new biological activities also requires an understanding of the functional pressures acting on proteins in the context of cells and organisms. I will present two vignettes describing our progress with dissecting both molecular and cellular constraints on protein function. In the first, we characterized the cost and benefit of protein production upon sequence perturbations in a classic system for gene regulation, the lac operon. Our results were unexpected in light of the common assumption that the dominant fitness costs are due to protein expression. Instead, we discovered a direct linear relationship between cost and lacpermease activity, not protein or mRNA production. The magnitude of the cost of permease activity, relative to protein production, has consequences for regulation. Our model predicts an advantage of direct regulation of protein activity (not just expression), providing a new explanation for the long-known mechanism of ``inducer exclusion'' that inhibits transport through the permease. Similar pressures and cost/benefit tradeoffs may be key to engineering synthetic systems with improved fitness. In the second vignette, I will describe our recent efforts to develop computational approaches that predict protein sequences consistent with multiple functional conformations. We expect such ``multi-constraint'' models to improve predictions of functional sequences determined by deep mutational scanning in bacteria, to provide insights into how the balance between functional conformations shapes sequence space, and to highlight molecular and cellular constraints that cannot be captured by the model.

  5. SCOP: a structural classification of proteins database.

    PubMed

    Hubbard, T J; Murzin, A G; Brenner, S E; Chothia, C

    1997-01-01

    The Structural Classification of Proteins (SCOP) database provides a detailed and comprehensive description of the relationships of all known proteins structures. The classification is on hierarchical levels: the first two levels, family and superfamily, describe near and far evolutionary relationships; the third, fold, describes geometrical relationships. The distinction between evolutionary relationships and those that arise from the physics and chemistry of proteins is a feature that is unique to this database, so far. SCOP also provides for each structure links to atomic co-ordinates, images of the structures, interactive viewers, sequence data, data on any conformational changes related to function and literature references. The database is freely accessible on the World Wide Web (WWW) with an entry point at URL http://scop.mrc-lmb.cam.ac.uk/scop/

  6. WD40 proteins propel cellular networks.

    PubMed

    Stirnimann, Christian U; Petsalaki, Evangelia; Russell, Robert B; Müller, Christoph W

    2010-10-01

    Recent findings indicate that WD40 domains play central roles in biological processes by acting as hubs in cellular networks; however, they have been studied less intensely than other common domains, such as the kinase, PDZ or SH3 domains. As suggested by various interactome studies, they are among the most promiscuous interactors. Structural studies suggest that this property stems from their ability, as scaffolds, to interact with diverse proteins, peptides or nucleic acids using multiple surfaces or modes of interaction. A general scaffolding role is supported by the fact that no WD40 domain has been found with intrinsic enzymatic activity despite often being part of large molecular machines. We discuss the WD40 domain distributions in protein networks and structures of WD40-containing assemblies to demonstrate their versatility in mediating critical cellular functions.

  7. Sentra, a database of signal transduction proteins.

    SciTech Connect

    Maltsev, N.; Marland, E.; Yu, G. X.; Bhatnagar, S.; Lusk, R.; Mathematics and Computer Science

    2002-01-01

    Sentra (http://www-wit.mcs.anl.gov/sentra) is a database of signal transduction proteins with the emphasis on microbial signal transduction. The database was updated to include classes of signal transduction systems modulated by either phosphorylation or methylation reactions such as PAS proteins and serine/threonine kinases, as well as the classical two-component histidine kinases and methyl-accepting chemotaxis proteins. Currently, Sentra contains signal transduction proteins from 43 completely sequenced prokaryotic genomes as well as sequences from SWISS-PROT and TrEMBL. Signal transduction proteins are annotated with information describing conserved domains, paralogous and orthologous sequences, and conserved chromosomal gene clusters. The newly developed user interface supports flexible search capabilities and extensive visualization of the data.

  8. TransportDB: a relational database of cellular membrane transport systems.

    PubMed

    Ren, Qinghu; Kang, Katherine H; Paulsen, Ian T

    2004-01-01

    TransportDB (http://www.membranetransport.org) is a relational database designed for describing the predicted cellular membrane transport proteins in organisms whose complete genome sequences are available. For each organism, the complete set of membrane transport systems was identified and classified into different types and families according to putative membrane topology, protein family, bioenergetics and substrate specificities. Web pages were created to provide user-friendly interfaces to easily access, query and download the data. Additional features, such as a BLAST search tool against known transporter protein sequences, comparison of transport systems from different organisms and phylogenetic trees of individual transporter families are also provided. TransportDB will be regularly updated with data obtained from newly sequenced genomes.

  9. Cellular Mechanisms of Membrane Protein Folding

    PubMed Central

    Skach, William R.

    2010-01-01

    The membrane protein folding problem can be articulated by two central questions. How is protein topology established by selective peptide transport to opposite sides of the cellular membrane? And how are transmembrane segments inserted, integrated and folded within the lipid bilayer? In eukaryotes, this process usually takes place in the endoplasmic reticulum (ER) coincident with protein synthesis, and is facilitated by the translating Ribosome and the Sec61 Translocon Complex (RTC). At its core, the RTC forms a dynamic pathway through which the elongating nascent polypeptide moves as it is delivered into cytosolic, lumenal and lipid compartments. This perspective will focus on emerging evidence that the RTC functions as a protein folding machine that restricts conformational space by establishing transmembrane topology and yet provides a permissive environment that enables nascent transmembrane domains to efficiently progress down their folding energy landscape. PMID:19491932

  10. Cellular Prion Protein: From Physiology to Pathology

    PubMed Central

    Yusa, Sei-ichi; Oliveira-Martins, José B.; Sugita-Konishi, Yoshiko; Kikuchi, Yutaka

    2012-01-01

    The human cellular prion protein (PrPC) is a glycosylphosphatidylinositol (GPI) anchored membrane glycoprotein with two N-glycosylation sites at residues 181 and 197. This protein migrates in several bands by Western blot analysis (WB). Interestingly, PNGase F treatment of human brain homogenates prior to the WB, which is known to remove the N-glycosylations, unexpectedly gives rise to two dominant bands, which are now known as C-terminal (C1) and N-terminal (N1) fragments. This resembles the β-amyloid precursor protein (APP) in Alzheimer disease (AD), which can be physiologically processed by α-, β-, and γ-secretases. The processing of APP has been extensively studied, while the identity of the cellular proteases involved in the proteolysis of PrPC and their possible role in prion biology has remained limited and controversial. Nevertheless, there is a strong correlation between the neurotoxicity caused by prion proteins and the blockade of their normal proteolysis. For example, expression of non-cleavable PrPC mutants in transgenic mice generates neurotoxicity, even in the absence of infectious prions, suggesting that PrPC proteolysis is physiologically and pathologically important. As many mouse models of prion diseases have recently been developed and the knowledge about the proteases responsible for the PrPC proteolysis is accumulating, we examine the historical experimental evidence and highlight recent studies that shed new light on this issue. PMID:23202518

  11. The PIR-International Protein Sequence Database.

    PubMed

    Barker, W C; Garavelli, J S; Haft, D H; Hunt, L T; Marzec, C R; Orcutt, B C; Srinivasarao, G Y; Yeh, L S; Ledley, R S; Mewes, H W; Pfeiffer, F; Tsugita, A

    1998-01-01

    From its origin the Protein Information Resource (http://www-nbrf. georgetown.edu/pir/) has supported research on evolution and computational biology by designing and compiling a comprehensive, quality controlled, and well-organized protein sequence database. The database has been produced and updated on a regular schedule since 1984. Since 1988 it has been maintained collaboratively by the PIR-International, an association of data collection centers engaged in international cooperation for the development of this research resource during a period of explosive acquisition of new data. As of June 1997, essentially all sequence entries have been classified into families, allowing the efficient application of methods to propagate and standardize annotation among related sequences. The databases are available through the Internet by the World-Wide Web and FTP, or on CD-ROM and magnetic media.

  12. Chromophore assisted laser inactivation of cellular proteins

    NASA Astrophysics Data System (ADS)

    Jay, Daniel G.; Wang, F. S.; Chang, H. Y.; Sydor, A. M.; Liao, J. C.

    1997-05-01

    A molecular understanding of biology requires that we establish the in situ functions of the proteins in cellular processes. To address this, we developed chromophore- assisted laser inactivation (CALI) for probing the in vivo function of proteins. CALI inactivates specific proteins in living cells by using non-blocking antibodies conjugated with malachite green (MG) dye. MG absorbs 620 nm laser light (which is not absorbed by cells) to generate short lived free radicals with limited range of oxidative damage (15 angstroms) around the dye. This inactivates the bound protein without significantly affecting its neighbors. CALI has been applied to 40 proteins and achieved specific inactivation in almost all those tested. We have developed micro-CALI which uses a focused laser beam (10 micrometers ) to acutely inactivate specific proteins within cells. We have used this to address the molecular mechanisms of neuronal growth cone motility and has implicated a diversity of proteins (e.g. molecular motors, cytoskeletal, and signaling molecules) in discrete steps of growth cone motility. We hope that micro-CALI will be a useful research tool for addressing dynamic processes in biology and medicine.

  13. Total Cellular RNA Modulates Protein Activity.

    PubMed

    Majumder, Subhabrata; DeMott, Christopher M; Reverdatto, Sergey; Burz, David S; Shekhtman, Alexander

    2016-08-16

    RNA constitutes up to 20% of a cell's dry weight, corresponding to ∼20 mg/mL. This high concentration of RNA facilitates low-affinity protein-RNA quinary interactions, which may play an important role in facilitating and regulating biological processes. In the yeast Pichia pastoris, the level of ubiquitin-RNA colocalization increases when cells are grown in the presence of dextrose and methanol instead of methanol as the sole carbon source. Total RNA isolated from cells grown in methanol increases β-galactosidase activity relative to that seen with RNA isolated from cells grown in the presence of dextrose and methanol. Because the total cellular RNA content changes with growth medium, protein-RNA quinary interactions can alter in-cell protein biochemistry and may play an important role in cell adaptation, critical to many physiological and pathological states. PMID:27456029

  14. AntiJen: a quantitative immunology database integrating functional, thermodynamic, kinetic, biophysical, and cellular data.

    PubMed

    Toseland, Christopher P; Clayton, Debra J; McSparron, Helen; Hemsley, Shelley L; Blythe, Martin J; Paine, Kelly; Doytchinova, Irini A; Guan, Pingping; Hattotuwagama, Channa K; Flower, Darren R

    2005-10-01

    AntiJen is a database system focused on the integration of kinetic, thermodynamic, functional, and cellular data within the context of immunology and vaccinology. Compared to its progenitor JenPep, the interface has been completely rewritten and redesigned and now offers a wider variety of search methods, including a nucleotide and a peptide BLAST search. In terms of data archived, AntiJen has a richer and more complete breadth, depth, and scope, and this has seen the database increase to over 31,000 entries. AntiJen provides the most complete and up-to-date dataset of its kind. While AntiJen v2.0 retains a focus on both T cell and B cell epitopes, its greatest novelty is the archiving of continuous quantitative data on a variety of immunological molecular interactions. This includes thermodynamic and kinetic measures of peptide binding to TAP and the Major Histocompatibility Complex (MHC), peptide-MHC complexes binding to T cell receptors, antibodies binding to protein antigens and general immunological protein-protein interactions. The database also contains quantitative specificity data from position-specific peptide libraries and biophysical data, in the form of diffusion co-efficients and cell surface copy numbers, on MHCs and other immunological molecules. The uses of AntiJen include the design of vaccines and diagnostics, such as tetramers, and other laboratory reagents, as well as helping parameterize the bioinformatic or mathematical in silico modeling of the immune system. The database is accessible from the URL: http://www.jenner.ac.uk/antijen. PMID:16305757

  15. The SYSTERS Protein Family Database in 2005.

    PubMed

    Meinel, Thomas; Krause, Antje; Luz, Hannes; Vingron, Martin; Staub, Eike

    2005-01-01

    The SYSTERS project aims to provide a meaningful partitioning of the whole protein sequence space by a fully automatic procedure. A refined two-step algorithm assigns each protein to a family and a superfamily. The sequence data underlying SYSTERS release 4 now comprise several protein sequence databases derived from completely sequenced genomes (ENSEMBL, TAIR, SGD and GeneDB), in addition to the comprehensive Swiss-Prot/TrEMBL databases. The SYSTERS web server (http://systers.molgen.mpg.de) provides access to 158 153 SYSTERS protein families. To augment the automatically derived results, information from external databases like Pfam and Gene Ontology are added to the web server. Furthermore, users can retrieve pre-processed analyses of families like multiple alignments and phylogenetic trees. New query options comprise a batch retrieval tool for functional inference about families based on automatic keyword extraction from sequence annotations. A new access point, PhyloMatrix, allows the retrieval of phylogenetic profiles of SYSTERS families across organisms with completely sequenced genomes.

  16. DAPS: Database of Aligned Protein Structures

    DOE Data Explorer

    Mallick, Parag; Rice, Danny; Eisenberg, David

    DAPS is based on the FSSP, DSSP, PDB and CATH databases. There also exists a subset of DAPS known as DDAPS (also pronounced DAPS) - Database of Distant Aligned Protein Structures. It is a database of structures that have low sequence similarity but share a similar fold. There are a number of filters used to make the DDAPS list more useful. The algorithm requires that an FSSP file exists for one of the members of a pair and that the other member is listed in that FSSP file. It requires that each member of the pair be within the CATH database and share a common CAT classification. It also requires that the secondary structure can be determined by DSSP. How is DAPS constructed? We begin with the set of all chains from the current release of the PDB. An all on all search is done on the list to find pairs that have the same fold acoording to both the FSSP and CATH databases and clustered into groups by a representative structure (representative structures have less than 25% sequence identity to each other). For each protein pair, regions aligned by the DALI program are extracted from the corresponding FSSP file, or recomputed using DALI-lite. In domain DAPS, only regions that are called "domains" by CATH are included in the alignment. The amino acid type, secondary structure type, and solvent accessibility are extracted from the DSSP file and written pairwise into the database. DAPS is updated with updates of CATH.[Taken from http://nihserver.mbi.ucla.edu/DAPS/daps_help.html

  17. MOPED: Model Organism Protein Expression Database.

    PubMed

    Kolker, Eugene; Higdon, Roger; Haynes, Winston; Welch, Dean; Broomall, William; Lancet, Doron; Stanberry, Larissa; Kolker, Natali

    2012-01-01

    Large numbers of mass spectrometry proteomics studies are being conducted to understand all types of biological processes. The size and complexity of proteomics data hinders efforts to easily share, integrate, query and compare the studies. The Model Organism Protein Expression Database (MOPED, htttp://moped.proteinspire.org) is a new and expanding proteomics resource that enables rapid browsing of protein expression information from publicly available studies on humans and model organisms. MOPED is designed to simplify the comparison and sharing of proteomics data for the greater research community. MOPED uniquely provides protein level expression data, meta-analysis capabilities and quantitative data from standardized analysis. Data can be queried for specific proteins, browsed based on organism, tissue, localization and condition and sorted by false discovery rate and expression. MOPED empowers users to visualize their own expression data and compare it with existing studies. Further, MOPED links to various protein and pathway databases, including GeneCards, Entrez, UniProt, KEGG and Reactome. The current version of MOPED contains over 43,000 proteins with at least one spectral match and more than 11 million high certainty spectra.

  18. Database for protein adsorption: update on developments

    NASA Astrophysics Data System (ADS)

    Paszek, Ewa; Vasina, Elena N.; Nicolau, Dan V.

    2008-12-01

    Protein adsorption at solid-liquid interfaces is critical to many applications, including biomaterials, protein microarrays and lab-on-a-chip devices. Despite this general interest, and a large amount of research in the last half a century, protein adsorption cannot be predicted with an engineering level, design-orientated accuracy. Here we describe a Biomolecular Adsorption Database (BAD), freely available online, which archives the published protein adsorption data. Piecewise linear regression with breakpoint applied to the data in the BAD suggests that the input variables to protein adsorption, i.e., protein concentration in solution; protein descriptors derived from primary structure (number of residues, protein hydrophobicity and spread of amino acid hydrophobicity, isoelectric point); surface descriptors (contact angle); and fluid environment descriptors (pH, ionic strength), correlate well with the output variable - the protein concentration on the surface. Furthermore, neural network analysis revealed that the size of the BAD makes it sufficiently representative, with a neural network-based predictive error of 5% or less. Interestingly, a consistently better fit is obtained if the BAD is divided into two separate subsets representing protein adsorption on hydrophilic and hydrophobic surfaces. Based on these findings, selected entries from the BAD have been used to construct neural network-based estimation routines, which predict the amount of adsorbed protein, the thickness of the absorbed layer and the surface tension of the proteincovered surface. While the BAD is of general interest, the prediction of the thickness and the surface tension of the protein-covered layers are of particular relevance to the design of microfluidics devices.

  19. Celecoxib transiently inhibits cellular protein synthesis.

    PubMed

    Pyrko, Peter; Kardosh, Adel; Schönthal, Axel H

    2008-01-15

    To uncover the full spectrum of its pharmacological activities, the selective COX-2 inhibitor celecoxib is routinely being used at concentrations of up to 100 microM in cell culture. At these elevated concentrations, several COX-2-independent effects were identified, although many details of these events have remained unclear. Here, we report a COX-2-independent effect of celecoxib that might have profound consequences for the interpretation of previous results obtained at elevated concentrations of this drug in vitro. We found that celecoxib rapidly inhibits general protein translation at concentrations as low as 30 microM. This appears to be a consequence of endoplasmic reticulum (ER) stress and entails the phosphorylation and inactivation of eukaryotic translation initiation factor 2 alpha (eIF2alpha). These effects were not achieved by other coxibs (rofecoxib, valdecoxib) or traditional NSAIDs (indomethacin, flurbiprofen), but were mimicked by the COX-2-inactive celecoxib analog, 2,5-dimethyl-celecoxib (DMC), indicating COX-2 independence. Considering the obvious impact of blocked translation on cellular function, we provide evidence that this severe inhibition of protein synthesis might suffice to explain some of the previously reported COX-2-independent effects of celecoxib, such as the down-regulation of the essential cell cycle regulatory protein cyclin D, which is a short-lived protein that rapidly disappears in response to the inhibition of protein synthesis. Taken together, our findings establish ER stress-induced inhibition of general translation as a critical outcome of celecoxib treatment in vitro, and suggest that this effect needs to be considered when interpreting observations from the use of this drug in cell culture. PMID:17920040

  20. Cellular proteomes have broad distributions of protein stability.

    PubMed

    Ghosh, Kingshuk; Dill, Ken

    2010-12-15

    Biological cells are extremely sensitive to temperature. What is the mechanism? We compute the thermal stabilities of the whole proteomes of Escherichia coli, yeast, and Caenorhabditis elegans using an analytical model and an extensive database of stabilities of individual proteins. Our results support the hypothesis that a cell's thermal sensitivities arise from the collective instability of its proteins. This model shows a denaturation catastrophe at temperatures of 49-55°C, roughly the thermal death point of mesophiles. Cells live on the edge of a proteostasis catastrophe. According to the model, it is not that the average protein is problematic; it is the tail of the distribution. About 650 of E. coli's 4300 proteins are less than 4 kcal mol(-1) stable to denaturation. And upshifting by only 4° from 37° to 41°C is estimated to destabilize an average protein by nearly 20%. This model also treats effects of denaturants, osmolytes, and other physical stressors. In addition, it predicts the dependence of cellular growth rates on temperature. This approach may be useful for studying physical forces in biological evolution and the role of climate change on biology.

  1. The Protein Naming Utility: a rules database for protein nomenclature.

    PubMed

    Goll, Johannes; Montgomery, Robert; Brinkac, Lauren M; Schobel, Seth; Harkins, Derek M; Sebastian, Yinong; Shrivastava, Susmita; Durkin, Scott; Sutton, Granger

    2010-01-01

    Generation of syntactically correct and unambiguous names for proteins is a challenging, yet vital task for functional annotation processes. Proteins are often named based on homology to known proteins, many of which have problematic names. To address the need to generate high-quality protein names, and capture our significant experience correcting protein names manually, we have developed the Protein Naming Utility (PNU, http://www.jcvi.org/pn-utility). The PNU is a web-based database for storing and applying naming rules to identify and correct syntactically incorrect protein names, or to replace synonyms with their preferred name. The PNU allows users to generate and manage collections of naming rules, optionally building upon the growing body of rules generated at the J. Craig Venter Institute (JCVI). Since communities often enforce disparate conventions for naming proteins, the PNU supports grouping rules into user-managed collections. Users can check their protein names against a selected PNU rule collection, generating both statistics and corrected names. The PNU can also be used to correct GenBank table files prior to submission to GenBank. Currently, the database features 3080 manual rules that have been entered by JCVI Bioinformatics Analysts as well as 7458 automatically imported names.

  2. MTBreg: The Database of Conditionally Regulated Proteins in Mycobacterium Tuberculosis

    DOE Data Explorer

    Kaufman, Markus; Pal, Debnath; Eisenberg, David

    Proteins up- and down- regulated in Mycobacterium tuberculosis grown under conditions mimicking infection are included in this database. It also includes information on proteins that are regulated by selected transcription factors or other regulatory proteins. The literature data provided here is complimentary to the databases provided by Michael Strong that include recent TB computational functional linkages and the Prolinks Database by Peter Bowers. The experimental condition, the experimental dataset and a literature reference will be displayed, including links to the computationally linked proteins in the Prolinks Database and the entry in the Mycobacterium tuberculosis Structural Genomics Database.[Copied from information at http://www.doe-mbi.ucla.edu/Services/MTBreg/

  3. Prion protein scrapie and the normal cellular prion protein.

    PubMed

    Atkinson, Caroline J; Zhang, Kai; Munn, Alan L; Wiegmans, Adrian; Wei, Ming Q

    2016-01-01

    Prions are infectious proteins and over the past few decades, some prions have become renowned for their causative role in several neurodegenerative diseases in animals and humans. Since their discovery, the mechanisms and mode of transmission and molecular structure of prions have begun to be established. There is, however, still much to be elucidated about prion diseases, including the development of potential therapeutic strategies for treatment. The significance of prion disease is discussed here, including the categories of human and animal prion diseases, disease transmission, disease progression and the development of symptoms and potential future strategies for treatment. Furthermore, the structure and function of the normal cellular prion protein (PrP(C)) and its importance in not only in prion disease development, but also in diseases such as cancer and Alzheimer's disease will also be discussed. PMID:26645475

  4. [Cellular immunity in protein-caloric malnutrition].

    PubMed

    Khadraoui, S; Lopez, V; Hamza, B; Smith, N J

    1977-02-01

    In order to have a clearer idea on the relationship between infectious diseases and malnutrition, cellular immunity is studied in 3 to 18 months old malnourished and healthy infants. Nutritional status is evaluated by the food intake the anthropologic measurements and some biological parameters like transferrin. Cellular immunity is investigated by skin tests to tuberculin, monilia, P.H.A. and DNCB. Absolute number of peripheral lymphocytes and percentage of T cells are studied too. The results show that there is a cellular immunity deficiency: frequent non-response to antigens, weak sensitization to DNCB. Some patients have a low percentage of spontaneous rosettes. The transferrin titering is useful to appreciate the nutritional status and the iron therapy opportunity, the prognostic and the immunitary possibilities.

  5. Increased coverage of protein families with the blocks database servers.

    PubMed

    Henikoff, J G; Greene, E A; Pietrokovski, S; Henikoff, S

    2000-01-01

    The Blocks Database WWW (http://blocks.fhcrc.org ) and Email (blocks@blocks.fhcrc.org ) servers provide tools to search DNA and protein queries against the Blocks+ Database of multiple alignments, which represent conserved protein regions. Blocks+ nearly doubles the number of protein families included in the database by adding families from the Pfam-A, ProDom and Domo databases to those from PROSITE and PRINTS. Other new features include improved Block Searcher statistics, searching with NCBI's IMPALA program and 3D display of blocks on PDB structures.

  6. Proteomic profiling of cellular proteins interacting with the hepatitis C virus core protein.

    PubMed

    Kang, Su-Min; Shin, Min-Jung; Kim, Jung-Hee; Oh, Jong-Won

    2005-05-01

    Hepatitis C virus (HCV) is a causative agent of chronic hepatitis and hepatocellular carcinoma. The core protein of HCV packages the viral RNA genome to form a nucleocapsid. In addition to its function as a structural protein, core protein is involved in regulation of cellular transcription, virus-induced transformation, and pathogenesis. To gain insights into cellular functions of the core protein by identification of cellular proteins interacting with the core protein, we employed a proteomic approach. Hepatocytes soluble cytoplasmic proteins were applied to the core proteins immobilized on Ni-nitrilotriacetic resin and total bound cellular proteins were resolved by 2-DE. Analyses of interacting proteins by matrix-assisted laser desorption/ionization-time of flight mass spectrometry allowed identification of 14 cellular proteins binding to the core protein. These proteins include DEAD-box polypeptide 5, similar in function to a known protein identified previously by yeast two-hybrid screening and 13 newly identified cellular proteins. Interestingly, nine protein spots were identified as intermediate microfilament proteins, including cytokeratins (five spots for cytokeratin 8, two for cytokeratin 19, and one for cytokeratin 18) and vimentin. Cytokeratin 8 and vimentin, which were previously shown to be involved in the infection processes of other viruses, were further analyzed to confirm their in vivo interactions with the core protein by immunoblotting and immunofluorescence microscopy. We discuss the functional implications of the interactions of the core protein with newly identified cellular proteins in HCV infection and pathogenesis.

  7. Designed Proteins To Modulate Cellular Networks

    PubMed Central

    Cortajarena, Aitziber L.; Liu, Tina Y.; Hochstrasser, Mark; Regan, Lynne

    2012-01-01

    A major challenge of protein design is to create useful new proteins that interact specifically with biological targets in living cells. Such binding modules have many potential applications, including the targeted perturbation of protein networks. As a general approach to create such modules, we designed a library with approximately 109 different binding specificities based on a small 3-tetratricopeptide repeat (TPR) motif framework. We employed a novel strategy, based on split GFP reassembly, to screen the library for modules with the desired binding specificity. Using this approach, we identified modules that bind tightly and specifically to Dss1, a small human protein that interacts with the tumor suppressor protein BRCA2. We showed that these modules also bind the yeast homologue of Dss1, Sem1. Furthermore, we demonstrated that these modules inhibit Sem1 activity in yeast. This strategy will be generally applicable to make novel genetically encoded tools for systems/synthetic biology applications. PMID:20020775

  8. Piezo proteins: regulators of mechanosensation and other cellular processes.

    PubMed

    Bagriantsev, Sviatoslav N; Gracheva, Elena O; Gallagher, Patrick G

    2014-11-14

    Piezo proteins have recently been identified as ion channels mediating mechanosensory transduction in mammalian cells. Characterization of these channels has yielded important insights into mechanisms of somatosensation, as well as other mechano-associated biologic processes such as sensing of shear stress, particularly in the vasculature, and regulation of urine flow and bladder distention. Other roles for Piezo proteins have emerged, some unexpected, including participation in cellular development, volume regulation, cellular migration, proliferation, and elongation. Mutations in human Piezo proteins have been associated with a variety of disorders including hereditary xerocytosis and several syndromes with muscular contracture as a prominent feature.

  9. Degradation of cellular and viral Fos proteins.

    PubMed

    Acquaviva, C; Ferrara, P; Bossis, G; Brockly, F; Salvat, C; Jariel-Encontre, I; Piechaczyk, M

    2001-01-01

    c-Fos proto-oncoprotein is a short-lived transcription factor with oncogenic potential. We have shown that it is massively degraded by the proteasome in vivo under various experimental conditions. Other proteolytic systems including lysosomes and calpains, might, however, also marginally operate on it. Although there is evidence that c-Fos can be ubiquitinylated in vitro, the unambiguous demonstration that ubiquitinylation is necessary for its addressing to the proteasome in vivo is still lacking. c-Jun, one of the main dimerization partners of c-Fos within the AP-1 transcription complex, is also an unstable protein. Its degradation is clearly proteasome- and ubiquitin-dependent in vivo. Interestingly, several lines of evidence indicate that the addressing of c-Fos and c-Jun to the proteasome is, at least in part, governed by different mechanisms. c-Fos has been transduced by two murine osteosarcomatogenic retroviruses under mutated forms which are more stable and more oncogenic. The stabilization is not simply accounted for by simple deletion of c-Fos main destabilizer but, rather, by a complex balance between opposing destabilizing and stabilizing mutations. Though mutations in viral Fos proteins confer full resistance to proteasomal degradation, stabilization is limited because mutations also entail sensitivity to an unidentified proteolytic system. This observation is consistent with the idea that Fos-expressing viruses have evolved to ensure control protein levels to avoid high protein accumulation-linked apoptosis. In conclusion, the unveiling of the complex mechanism network responsible for the degradation of AP-1 family members is still at its beginning and a number of issues regarding the regulation of this process and the addressing to the proteasome are still unresolved.

  10. Cellular strategies for regulating functional and nonfunctional protein aggregation.

    PubMed

    Gsponer, Jörg; Babu, M Madan

    2012-11-29

    Growing evidence suggests that aggregation-prone proteins are both harmful and functional for a cell. How do cellular systems balance the detrimental and beneficial effect of protein aggregation? We reveal that aggregation-prone proteins are subject to differential transcriptional, translational, and degradation control compared to nonaggregation-prone proteins, which leads to their decreased synthesis, low abundance, and high turnover. Genetic modulators that enhance the aggregation phenotype are enriched in genes that influence expression homeostasis. Moreover, genes encoding aggregation-prone proteins are more likely to be harmful when overexpressed. The trends are evolutionarily conserved and suggest a strategy whereby cellular mechanisms specifically modulate the availability of aggregation-prone proteins to (1) keep concentrations below the critical ones required for aggregation and (2) shift the equilibrium between the monomeric and oligomeric/aggregate form, as explained by Le Chatelier's principle. This strategy may prevent formation of undesirable aggregates and keep functional assemblies/aggregates under control. PMID:23168257

  11. Cellular proteostasis: degradation of misfolded proteins by lysosomes

    PubMed Central

    Jackson, Matthew P.

    2016-01-01

    Proteostasis refers to the regulation of the cellular concentration, folding, interactions and localization of each of the proteins that comprise the proteome. One essential element of proteostasis is the disposal of misfolded proteins by the cellular pathways of protein degradation. Lysosomes are an important site for the degradation of misfolded proteins, which are trafficked to this organelle by the pathways of macroautophagy, chaperone-mediated autophagy and endocytosis. Conversely, amyloid diseases represent a failure in proteostasis, in which proteins misfold, forming amyloid deposits that are not degraded effectively by cells. Amyloid may then exacerbate this failure by disrupting autophagy and lysosomal proteolysis. However, targeting the pathways that regulate autophagy and the biogenesis of lysosomes may present approaches that can rescue cells from the deleterious effects of amyloidogenic proteins. PMID:27744333

  12. Cooperative protein transport in cellular organelles

    NASA Astrophysics Data System (ADS)

    Dmitrieff, S.; Sens, P.

    2011-04-01

    Compartmentalization into biochemically distinct organelles constantly exchanging material is one of the hallmarks of eukaryotic cells. In the most naive picture of interorganelle transport driven by concentration gradients, concentration differences between organelles should relax. We determine the conditions under which cooperative transport, i.e., based on molecular recognition, allows for the existence and maintenance of distinct organelle identities. Cooperative transport is also shown to control the flux of material transiting through a compartmentalized system, dramatically increasing the transit time under high incoming flux. By including chemical processing of the transported species, we show that this property provides a strong functional advantage to a system responsible for protein maturation and sorting.

  13. ApoCanD: Database of human apoptotic proteins in the context of cancer

    PubMed Central

    Kumar, Rahul; Raghava, Gajendra P. S.

    2016-01-01

    In the past decade, apoptosis pathway has gained a serious consideration being a critical cellular process in determining the cancer progression. Inverse relationship between cancer progression and apoptosis rate has been well established in the literature. It causes apoptosis proteins under the investigative scanner for developing anticancer therapies, which certainly got a success in the case of few apoptosis proteins as drug targets. In the present study, we have developed a dedicated database of 82 apoptosis proteins called ApoCanD. This database comprises of crucial information of apoptosis proteins in the context of cancer. Genomic status of proteins in the form of mutation, copy number variation and expression in thousands of tumour samples and cancer cell lines are the major bricks of this database. In analysis, we have found that TP53 and MYD88 are the two most frequently mutated proteins in cancer. Availability of other information e.g. gene essentiality data, tertiary structure, sequence alignments, sequences profiles, post-translational modifications makes it even more useful for the researchers. A user-friendly web interface is provided to ameliorate the use of ApoCanD. We anticipate that, this database will facilitate the research community working in the field of apoptosis and cancer. The database can be accessed at: http://crdd.osdd.net/raghava/apocand. PMID:26861916

  14. ATtRACT-a database of RNA-binding proteins and associated motifs.

    PubMed

    Giudice, Girolamo; Sánchez-Cabo, Fátima; Torroja, Carlos; Lara-Pezzi, Enrique

    2016-01-01

    RNA-binding proteins (RBPs) play a crucial role in key cellular processes, including RNA transport, splicing, polyadenylation and stability. Understanding the interaction between RBPs and RNA is key to improve our knowledge of RNA processing, localization and regulation in a global manner. Despite advances in recent years, a unified non-redundant resource that includes information on experimentally validated motifs, RBPs and integrated tools to exploit this information is lacking. Here, we developed a database named ATtRACT (available athttp://attract.cnic.es) that compiles information on 370 RBPs and 1583 RBP consensus binding motifs, 192 of which are not present in any other database. To populate ATtRACT we (i) extracted and hand-curated experimentally validated data from CISBP-RNA, SpliceAid-F, RBPDB databases, (ii) integrated and updated the unavailable ASD database and (iii) extracted information from Protein-RNA complexes present in Protein Data Bank database through computational analyses. ATtRACT provides also efficient algorithms to search a specific motif and scan one or more RNA sequences at a time. It also allows discoveringde novomotifs enriched in a set of related sequences and compare them with the motifs included in the database.Database URL:http:// attract. cnic. es. PMID:27055826

  15. ATtRACT-a database of RNA-binding proteins and associated motifs.

    PubMed

    Giudice, Girolamo; Sánchez-Cabo, Fátima; Torroja, Carlos; Lara-Pezzi, Enrique

    2016-01-01

    RNA-binding proteins (RBPs) play a crucial role in key cellular processes, including RNA transport, splicing, polyadenylation and stability. Understanding the interaction between RBPs and RNA is key to improve our knowledge of RNA processing, localization and regulation in a global manner. Despite advances in recent years, a unified non-redundant resource that includes information on experimentally validated motifs, RBPs and integrated tools to exploit this information is lacking. Here, we developed a database named ATtRACT (available athttp://attract.cnic.es) that compiles information on 370 RBPs and 1583 RBP consensus binding motifs, 192 of which are not present in any other database. To populate ATtRACT we (i) extracted and hand-curated experimentally validated data from CISBP-RNA, SpliceAid-F, RBPDB databases, (ii) integrated and updated the unavailable ASD database and (iii) extracted information from Protein-RNA complexes present in Protein Data Bank database through computational analyses. ATtRACT provides also efficient algorithms to search a specific motif and scan one or more RNA sequences at a time. It also allows discoveringde novomotifs enriched in a set of related sequences and compare them with the motifs included in the database.Database URL:http:// attract. cnic. es.

  16. ATtRACT—a database of RNA-binding proteins and associated motifs

    PubMed Central

    Giudice, Girolamo; Sánchez-Cabo, Fátima; Torroja, Carlos; Lara-Pezzi, Enrique

    2016-01-01

    RNA-binding proteins (RBPs) play a crucial role in key cellular processes, including RNA transport, splicing, polyadenylation and stability. Understanding the interaction between RBPs and RNA is key to improve our knowledge of RNA processing, localization and regulation in a global manner. Despite advances in recent years, a unified non-redundant resource that includes information on experimentally validated motifs, RBPs and integrated tools to exploit this information is lacking. Here, we developed a database named ATtRACT (available at http://attract.cnic.es) that compiles information on 370 RBPs and 1583 RBP consensus binding motifs, 192 of which are not present in any other database. To populate ATtRACT we (i) extracted and hand-curated experimentally validated data from CISBP-RNA, SpliceAid–F, RBPDB databases, (ii) integrated and updated the unavailable ASD database and (iii) extracted information from Protein-RNA complexes present in Protein Data Bank database through computational analyses. ATtRACT provides also efficient algorithms to search a specific motif and scan one or more RNA sequences at a time. It also allows discovering de novo motifs enriched in a set of related sequences and compare them with the motifs included in the database. Database URL: http:// attract. cnic. es PMID:27055826

  17. Nipah virus matrix protein: expert hacker of cellular machines.

    PubMed

    Watkinson, Ruth E; Lee, Benhur

    2016-08-01

    Nipah virus (NiV, Henipavirus) is a highly lethal emergent zoonotic paramyxovirus responsible for repeated human outbreaks of encephalitis in South East Asia. There are no approved vaccines or treatments, thus improved understanding of NiV biology is imperative. NiV matrix protein recruits a plethora of cellular machinery to scaffold and coordinate virion budding. Intriguingly, matrix also hijacks cellular trafficking and ubiquitination pathways to facilitate transient nuclear localization. While the biological significance of matrix nuclear localization for an otherwise cytoplasmic virus remains enigmatic, the molecular details have begun to be characterized, and are conserved among matrix proteins from divergent paramyxoviruses. Matrix protein appropriation of cellular machinery will be discussed in terms of its early nuclear targeting and later role in virion assembly. PMID:27350027

  18. Nipah virus matrix protein: expert hacker of cellular machines.

    PubMed

    Watkinson, Ruth E; Lee, Benhur

    2016-08-01

    Nipah virus (NiV, Henipavirus) is a highly lethal emergent zoonotic paramyxovirus responsible for repeated human outbreaks of encephalitis in South East Asia. There are no approved vaccines or treatments, thus improved understanding of NiV biology is imperative. NiV matrix protein recruits a plethora of cellular machinery to scaffold and coordinate virion budding. Intriguingly, matrix also hijacks cellular trafficking and ubiquitination pathways to facilitate transient nuclear localization. While the biological significance of matrix nuclear localization for an otherwise cytoplasmic virus remains enigmatic, the molecular details have begun to be characterized, and are conserved among matrix proteins from divergent paramyxoviruses. Matrix protein appropriation of cellular machinery will be discussed in terms of its early nuclear targeting and later role in virion assembly.

  19. Searching the MINT database for protein interaction information.

    PubMed

    Cesareni, Gianni; Chatr-aryamontri, Andrew; Licata, Luana; Ceol, Arnaud

    2008-06-01

    The Molecular Interactions Database (MINT) is a relational database designed to store information about protein interactions. Expert curators extract the relevant information from the scientific literature and deposit it in a computer readable form. Currently (April 2008), MINT contains information on almost 29,000 proteins and more than 100,000 interactions from more than 30 model organisms. This unit provides protocols for searching MINT over the Internet, using the MINT Viewer.

  20. How well are protein structures annotated in secondary databases?

    PubMed

    Rother, Kristian; Michalsky, Elke; Leser, Ulf

    2005-09-01

    We investigated to what extent Protein Data Bank (PDB) entries are annotated with second-party information based on existing cross-references between PDB and 15 other databases. We report 2 interesting findings. First, there is a clear "annotation gap" for structures less than 7 years old for secondary databases that are manually curated. Second, the examined databases overlap with each other quite well, dividing the PDB into 2 well-annotated thirds and one poorly annotated third. Both observations should be taken into account in any study depending on the selection of protein structures by their annotation.

  1. HOMSTRAD: a database of protein structure alignments for homologous families.

    PubMed

    Mizuguchi, K; Deane, C M; Blundell, T L; Overington, J P

    1998-11-01

    We describe a database of protein structure alignments for homologous families. The database HOMSTRAD presently contains 130 protein families and 590 aligned structures, which have been selected on the basis of quality of the X-ray analysis and accuracy of the structure. For each family, the database provides a structure-based alignment derived using COMPARER and annotated with JOY in a special format that represents the local structural environment of each amino acid residue. HOMSTRAD also provides a set of superposed atomic coordinates obtained using MNYFIT, which can be viewed with a graphical user interface or used for comparative modeling studies. The database is freely available on the World Wide Web at: http://www-cryst.bioc.cam. ac.uk/-homstrad/, with search facilities and links to other databases.

  2. PROFESS: a PROtein function, evolution, structure and sequence database.

    PubMed

    Triplet, Thomas; Shortridge, Matthew D; Griep, Mark A; Stark, Jaime L; Powers, Robert; Revesz, Peter

    2010-07-06

    The proliferation of biological databases and the easy access enabled by the Internet is having a beneficial impact on biological sciences and transforming the way research is conducted. There are approximately 1100 molecular biology databases dispersed throughout the Internet. To assist in the functional, structural and evolutionary analysis of the abundant number of novel proteins continually identified from whole-genome sequencing, we introduce the PROFESS (PROtein Function, Evolution, Structure and Sequence) database. Our database is designed to be versatile and expandable and will not confine analysis to a pre-existing set of data relationships. A fundamental component of this approach is the development of an intuitive query system that incorporates a variety of similarity functions capable of generating data relationships not conceived during the creation of the database. The utility of PROFESS is demonstrated by the analysis of the structural drift of homologous proteins and the identification of potential pancreatic cancer therapeutic targets based on the observation of protein-protein interaction networks. Database URL: http://cse.unl.edu/~profess/

  3. Small G proteins and their regulators in cellular signalling.

    PubMed

    Csépányi-Kömi, Roland; Lévay, Magdolna; Ligeti, Erzsébet

    2012-04-28

    Small molecular weight GTPases (small G proteins) are essential in the transduction of signals from different plasma membrane receptors. Due to their endogenous GTP-hydrolyzing activity, these proteins function as time-dependent biological switches controlling diverse cellular functions including cell shape and migration, cell proliferation, gene transcription, vesicular transport and membrane-trafficking. This review focuses on endocrine diseases linked to small G proteins. We provide examples for the regulation of the activity of small G proteins by various mechanisms such as posttranslational modifications, guanine nucleotide exchange factors (GEFs), GTPase activating proteins (GAPs) or guanine nucleotide dissociation inhibitors (GDIs). Finally we summarize endocrine diseases where small G proteins or their regulatory proteins have been revealed as the cause.

  4. SwissPalm: Protein Palmitoylation database

    PubMed Central

    Abrami, Laurence; Migliozzi, Daniel; Armand, Florence; Bürgi, Jérôme; van der Goot, Françoise Gisou

    2015-01-01

    Protein S-palmitoylation is a reversible post-translational modification that regulates many key biological processes, although the full extent and functions of protein S-palmitoylation remain largely unexplored. Recent developments of new chemical methods have allowed the establishment of palmitoyl-proteomes of a variety of cell lines and tissues from different species.  As the amount of information generated by these high-throughput studies is increasing, the field requires centralization and comparison of this information. Here we present SwissPalm ( http://swisspalm.epfl.ch), our open, comprehensive, manually curated resource to study protein S-palmitoylation. It currently encompasses more than 5000 S-palmitoylated protein hits from seven species, and contains more than 500 specific sites of S-palmitoylation. SwissPalm also provides curated information and filters that increase the confidence in true positive hits, and integrates predictions of S-palmitoylated cysteine scores, orthologs and isoform multiple alignments. Systems analysis of the palmitoyl-proteome screens indicate that 10% or more of the human proteome is susceptible to S-palmitoylation. Moreover, ontology and pathway analyses of the human palmitoyl-proteome reveal that key biological functions involve this reversible lipid modification. Comparative analysis finally shows a strong crosstalk between S-palmitoylation and other post-translational modifications. Through the compilation of data and continuous updates, SwissPalm will provide a powerful tool to unravel the global importance of protein S-palmitoylation. PMID:26339475

  5. MAPU: Max-Planck Unified database of organellar, cellular, tissue and body fluid proteomes.

    PubMed

    Zhang, Yanling; Zhang, Yong; Adachi, Jun; Olsen, Jesper V; Shi, Rong; de Souza, Gustavo; Pasini, Erica; Foster, Leonard J; Macek, Boris; Zougman, Alexandre; Kumar, Chanchal; Wisniewski, Jacek R; Jun, Wang; Mann, Matthias

    2007-01-01

    Mass spectrometry (MS)-based proteomics has become a powerful technology to map the protein composition of organelles, cell types and tissues. In our department, a large-scale effort to map these proteomes is complemented by the Max-Planck Unified (MAPU) proteome database. MAPU contains several body fluid proteomes; including plasma, urine, and cerebrospinal fluid. Cell lines have been mapped to a depth of several thousand proteins and the red blood cell proteome has also been analyzed in depth. The liver proteome is represented with 3200 proteins. By employing high resolution MS and stringent validation criteria, false positive identification rates in MAPU are lower than 1:1000. Thus MAPU datasets can serve as reference proteomes in biomarker discovery. MAPU contains the peptides identifying each protein, measured masses, scores and intensities and is freely available at http://www.mapuproteome.com using a clickable interface of cell or body parts. Proteome data can be queried across proteomes by protein name, accession number, sequence similarity, peptide sequence and annotation information. More than 4500 mouse and 2500 human proteins have already been identified in at least one proteome. Basic annotation information and links to other public databases are provided in MAPU and we plan to add further analysis tools.

  6. MAPU: Max-Planck Unified database of organellar, cellular, tissue and body fluid proteomes

    PubMed Central

    Zhang, Yanling; Zhang, Yong; Adachi, Jun; Olsen, Jesper V.; Shi, Rong; de Souza, Gustavo; Pasini, Erica; Foster, Leonard J.; Macek, Boris; Zougman, Alexandre; Kumar, Chanchal; Wiśniewski, Jacek R.; Jun, Wang; Mann, Matthias

    2007-01-01

    Mass spectrometry (MS)-based proteomics has become a powerful technology to map the protein composition of organelles, cell types and tissues. In our department, a large-scale effort to map these proteomes is complemented by the Max-Planck Unified (MAPU) proteome database. MAPU contains several body fluid proteomes; including plasma, urine, and cerebrospinal fluid. Cell lines have been mapped to a depth of several thousand proteins and the red blood cell proteome has also been analyzed in depth. The liver proteome is represented with 3200 proteins. By employing high resolution MS and stringent validation criteria, false positive identification rates in MAPU are lower than 1:1000. Thus MAPU datasets can serve as reference proteomes in biomarker discovery. MAPU contains the peptides identifying each protein, measured masses, scores and intensities and is freely available at using a clickable interface of cell or body parts. Proteome data can be queried across proteomes by protein name, accession number, sequence similarity, peptide sequence and annotation information. More than 4500 mouse and 2500 human proteins have already been identified in at least one proteome. Basic annotation information and links to other public databases are provided in MAPU and we plan to add further analysis tools. PMID:17090601

  7. Human protein reference database as a discovery resource for proteomics

    PubMed Central

    Peri, Suraj; Navarro, J. Daniel; Kristiansen, Troels Z.; Amanchy, Ramars; Surendranath, Vineeth; Muthusamy, Babylakshmi; Gandhi, T. K. B.; Chandrika, K. N.; Deshpande, Nandan; Suresh, Shubha; Rashmi, B. P.; Shanker, K.; Padma, N.; Niranjan, Vidya; Harsha, H. C.; Talreja, Naveen; Vrushabendra, B. M.; Ramya, M. A.; Yatish, A. J.; Joy, Mary; Shivashankar, H. N.; Kavitha, M. P.; Menezes, Minal; Choudhury, Dipanwita Roy; Ghosh, Neelanjana; Saravana, R.; Chandran, Sreenath; Mohan, Sujatha; Jonnalagadda, Chandra Kiran; Prasad, C. K.; Kumar-Sinha, Chandan; Deshpande, Krishna S.; Pandey, Akhilesh

    2004-01-01

    The rapid pace at which genomic and proteomic data is being generated necessitates the development of tools and resources for managing data that allow integration of information from disparate sources. The Human Protein Reference Database (http://www.hprd.org) is a web-based resource based on open source technologies for protein information about several aspects of human proteins including protein–protein interactions, post-translational modifications, enzyme–substrate relationships and disease associations. This information was derived manually by a critical reading of the published literature by expert biologists and through bioinformatics analyses of the protein sequence. This database will assist in biomedical discoveries by serving as a resource of genomic and proteomic information and providing an integrated view of sequence, structure, function and protein networks in health and disease. PMID:14681466

  8. A palette of fluorescent proteins optimized for diverse cellular environments

    PubMed Central

    Costantini, Lindsey M.; Baloban, Mikhail; Markwardt, Michele L.; Rizzo, Mark; Guo, Feng; Verkhusha, Vladislav V.; Snapp, Erik L.

    2015-01-01

    To perform quantitative live cell imaging, investigators require fluorescent reporters that accurately report protein localization and levels, while minimally perturbing the cell. Yet, within the biochemically distinct environments of cellular organelles, popular fluorescent proteins (FPs), including EGFP, can be unreliable for quantitative imaging, resulting in underestimation of protein levels and incorrect localization. Specifically, within the secretory pathway, significant populations of FPs misfold and fail to fluoresce due to non-native disulphide bond formation. Furthermore, transmembrane FP fusion constructs can disrupt organelle architecture due to oligomerizing tendencies of numerous common FPs. Here, we describe a powerful set of bright and inert FPs optimized for use in multiple cellular compartments, especially oxidizing environments and biological membranes. Also, we provide new insights into use of red FPs in the secretory pathway. Our monomeric "oxFPs" finally resolve long standing, underappreciated, and important problems of cell biology and should be useful for a number of applications. PMID:26158227

  9. AMYPdb: A database dedicated to amyloid precursor proteins

    PubMed Central

    Pawlicki, Sandrine; Le Béchec, Antony; Delamarche, Christian

    2008-01-01

    Background Misfolding and aggregation of proteins into ordered fibrillar structures is associated with a number of severe pathologies, including Alzheimer's disease, prion diseases, and type II diabetes. The rapid accumulation of knowledge about the sequences and structures of these proteins allows using of in silico methods to investigate the molecular mechanisms of their abnormal conformational changes and assembly. However, such an approach requires the collection of accurate data, which are inconveniently dispersed among several generalist databases. Results We therefore created a free online knowledge database (AMYPdb) dedicated to amyloid precursor proteins and we have performed large scale sequence analysis of the included data. Currently, AMYPdb integrates data on 31 families, including 1,705 proteins from nearly 600 organisms. It displays links to more than 2,300 bibliographic references and 1,200 3D-structures. A Wiki system is available to insert data into the database, providing a sharing and collaboration environment. We generated and analyzed 3,621 amino acid sequence patterns, reporting highly specific patterns for each amyloid family, along with patterns likely to be involved in protein misfolding and aggregation. Conclusion AMYPdb is a comprehensive online database aiming at the centralization of bioinformatic data regarding all amyloid proteins and their precursors. Our sequence pattern discovery and analysis approach unveiled protein regions of significant interest. AMYPdb is freely accessible [1]. PMID:18544157

  10. Atlas of protein expression: image capture, analysis, and design of terabyte image database

    NASA Astrophysics Data System (ADS)

    Wu, Jiahua; Maslen, Gareth; Warford, Anthony; Griffin, Gareth; Xie, Jane; Crowther, Sandra; McCafferty, John

    2006-03-01

    The activity of genes in health and disease are manifested through the proteins which they encode. Ultimately, proteins drive functional processes in cells and tissues and so by measuring individual protein levels, studying modifications and discovering their sites of action we will understand better their function. It is possible to visualize the location of proteins of interest in tissue sections using labeled antibodies which bind to the target protein. This procedure, known as immunohistochemistry (IHC), provides valuable information on the cellular and sub-cellular distribution of proteins in tissue. The project, atlas of protein expression, aims to create a quality, information rich database of protein expression profiles, which is accessible to the world-wide research community. For the long term archival value of the data, the accompanying validated antibody and protein clones will potentially have great research, diagnostic and possibly therapeutic potential. To achieve this we had introduced a number of novel technologies, e.g. express recombinant proteins, select antibodies, stain proteins present in tissue section, and tissue microarray (TMA) image analysis. These are currently being optimized, automated and integrated into a multi-disciplinary production process. We had also created infrastructure for multi-terabyte scale image capture, established an image analysis capability for initial screening and quantization.

  11. Cellular reprogramming through mitogen-activated protein kinases

    PubMed Central

    Lee, Justin; Eschen-Lippold, Lennart; Lassowskat, Ines; Böttcher, Christoph; Scheel, Dierk

    2015-01-01

    Mitogen-activated protein kinase (MAPK) cascades are conserved eukaryote signaling modules where MAPKs, as the final kinases in the cascade, phosphorylate protein substrates to regulate cellular processes. While some progress in the identification of MAPK substrates has been made in plants, the knowledge on the spectrum of substrates and their mechanistic action is still fragmentary. In this focused review, we discuss the biological implications of the data in our original paper (Sustained mitogen-activated protein kinase activation reprograms defense metabolism and phosphoprotein profile in Arabidopsis thaliana; Frontiers in Plant Science 5: 554) in the context of related research. In our work, we mimicked in vivo activation of two stress-activated MAPKs, MPK3 and MPK6, through transgenic manipulation of Arabidopsis thaliana and used phosphoproteomics analysis to identify potential novel MAPK substrates. Here, we plotted the identified putative MAPK substrates (and downstream phosphoproteins) as a global protein clustering network. Based on a highly stringent selection confidence level, the core networks highlighted a MAPK-induced cellular reprogramming at multiple levels of gene and protein expression—including transcriptional, post-transcriptional, translational, post-translational (such as protein modification, folding, and degradation) steps, and also protein re-compartmentalization. Additionally, the increase in putative substrates/phosphoproteins of energy metabolism and various secondary metabolite biosynthesis pathways coincides with the observed accumulation of defense antimicrobial substances as detected by metabolome analysis. Furthermore, detection of protein networks in phospholipid or redox elements suggests activation of downstream signaling events. Taken in context with other studies, MAPKs are key regulators that reprogram cellular events to orchestrate defense signaling in eukaryotes. PMID:26579181

  12. TIGRFAMS: The TIGRFAMs database of protein families

    DOE Data Explorer

    TIGRFAMs are protein families based on Hidden Markov Models or HMMs. Use this page to see the curated seed alignmet for each TIGRFam, the full alignment of all family members and the cutoff scores for inclusion in each of the TIGRFAMs. Also use this page to search through the TIGRFAMs and HMMs for text in the TIGRFAMs Text Search or search for specific sequences in the TIGRFAMs Sequence Search.[Copied from the Overview at http://www.jcvi.org/cms/research/projects/tigrfams/overview/] See also TIGRFAMs ordered by the roles they play at http://cmr.jcvi.org/tigr-scripts/CMR/shared/EvidenceList.cgi?ev_type=TIGRFAM&order_type=role.

  13. TOPDOM: database of conservatively located domains and motifs in proteins

    PubMed Central

    Varga, Julia; Dobson, László; Tusnády, Gábor E.

    2016-01-01

    Summary: The TOPDOM database—originally created as a collection of domains and motifs located consistently on the same side of the membranes in α-helical transmembrane proteins—has been updated and extended by taking into consideration consistently localized domains and motifs in globular proteins, too. By taking advantage of the recently developed CCTOP algorithm to determine the type of a protein and predict topology in case of transmembrane proteins, and by applying a thorough search for domains and motifs as well as utilizing the most up-to-date version of all source databases, we managed to reach a 6-fold increase in the size of the whole database and a 2-fold increase in the number of transmembrane proteins. Availability and implementation: TOPDOM database is available at http://topdom.enzim.hu. The webpage utilizes the common Apache, PHP5 and MySQL software to provide the user interface for accessing and searching the database. The database itself is generated on a high performance computer. Contact: tusnady.gabor@ttk.mta.hu. Supplementary information: Supplementary data are available at Bioinformatics online. PMID:27153630

  14. Cellular Strategies for Regulating Functional and Nonfunctional Protein Aggregation

    PubMed Central

    Gsponer, Jörg; Babu, M. Madan

    2012-01-01

    Summary Growing evidence suggests that aggregation-prone proteins are both harmful and functional for a cell. How do cellular systems balance the detrimental and beneficial effect of protein aggregation? We reveal that aggregation-prone proteins are subject to differential transcriptional, translational, and degradation control compared to nonaggregation-prone proteins, which leads to their decreased synthesis, low abundance, and high turnover. Genetic modulators that enhance the aggregation phenotype are enriched in genes that influence expression homeostasis. Moreover, genes encoding aggregation-prone proteins are more likely to be harmful when overexpressed. The trends are evolutionarily conserved and suggest a strategy whereby cellular mechanisms specifically modulate the availability of aggregation-prone proteins to (1) keep concentrations below the critical ones required for aggregation and (2) shift the equilibrium between the monomeric and oligomeric/aggregate form, as explained by Le Chatelier’s principle. This strategy may prevent formation of undesirable aggregates and keep functional assemblies/aggregates under control. PMID:23168257

  15. PEPPI: a peptidomic database of human protein isoforms for proteomics experiments

    PubMed Central

    2010-01-01

    Abstract Background Protein isoform generation, which may derive from alternative splicing, genetic polymorphism, and posttranslational modification, is an essential source of achieving molecular diversity by eukaryotic cells. Previous studies have shown that protein isoforms play critical roles in disease diagnosis, risk assessment, sub-typing, prognosis, and treatment outcome predictions. Understanding the types, presence, and abundance of different protein isoforms in different cellular and physiological conditions is a major task in functional proteomics, and may pave ways to molecular biomarker discovery of human diseases. In tandem mass spectrometry (MS/MS) based proteomics analysis, peptide peaks with exact matches to protein sequence records in the proteomics database may be identified with mass spectrometry (MS) search software. However, due to limited annotation and poor coverage of protein isoforms in proteomics databases, high throughput protein isoform identifications, particularly those arising from alternative splicing and genetic polymorphism, have not been possible. Results Therefore, we present the PEPtidomics Protein Isoform Database (PEPPI, http://bio.informatics.iupui.edu/peppi), a comprehensive database of computationally-synthesized human peptides that can identify protein isoforms derived from either alternatively spliced mRNA transcripts or SNP variations. We collected genome, pre-mRNA alternative splicing and SNP information from Ensembl. We synthesized in silico isoform transcripts that cover all exons and theoretically possible junctions of exons and introns, as well as all their variations derived from known SNPs. With three case studies, we further demonstrated that the database can help researchers discover and characterize new protein isoform biomarkers from experimental proteomics data. Conclusions We developed a new tool for the proteomics community to characterize protein isoforms from MS-based proteomics experiments. By

  16. Cellular Recycling of Proteins in Seed Dormancy Alleviation and Germination

    PubMed Central

    Oracz, Krystyna; Stawska, Marlena

    2016-01-01

    Each step of the seed-to-seed cycle of plant development including seed germination is characterized by a specific set of proteins. The continual renewal and/or replacement of these biomolecules are crucial for optimal plant adaptation. As proteins are the main effectors inside the cells, their levels need to be tightly regulated. This is partially achieved by specific proteolytic pathways via multicatalytic protease complexes defined as 20S and 26S proteasomes. In plants, the 20S proteasome is responsible for degradation of carbonylated proteins, while the 26S being a part of ubiquitin-proteasome pathway is known to be involved in proteolysis of phytohormone signaling regulators. On the other hand, the role of translational control of plant development is also well-documented, especially in the context of pollen tube growth and light signaling. Despite the current progress that has been made in seed biology, the sequence of cellular events that determine if the seed can germinate or not are still far from complete understanding. The role and mechanisms of regulation of proteome composition during processes occurring in the plant’s photosynthetic tissues have been well-characterized since many years, but in non-photosynthetic seeds it has emerged as a tempting research task only since the last decade. This review discusses the recent discoveries providing insights into the role of protein turnover in seed dormancy alleviation, and germination, with a focus on the control of translation and proteasomal proteolysis. The presented novel data of translatome profiling in seeds highlighted that post-transcriptional regulation of germination results from a timely regulated initiation of translation. In addition, the importance of 26S proteasome in the degradation of regulatory elements of cellular signaling and that of the 20S complex in proteolysis of specific carbonylated proteins in hormonal- and light-dependent processes occurring in seeds is discussed. Based on the

  17. Cellular Recycling of Proteins in Seed Dormancy Alleviation and Germination.

    PubMed

    Oracz, Krystyna; Stawska, Marlena

    2016-01-01

    Each step of the seed-to-seed cycle of plant development including seed germination is characterized by a specific set of proteins. The continual renewal and/or replacement of these biomolecules are crucial for optimal plant adaptation. As proteins are the main effectors inside the cells, their levels need to be tightly regulated. This is partially achieved by specific proteolytic pathways via multicatalytic protease complexes defined as 20S and 26S proteasomes. In plants, the 20S proteasome is responsible for degradation of carbonylated proteins, while the 26S being a part of ubiquitin-proteasome pathway is known to be involved in proteolysis of phytohormone signaling regulators. On the other hand, the role of translational control of plant development is also well-documented, especially in the context of pollen tube growth and light signaling. Despite the current progress that has been made in seed biology, the sequence of cellular events that determine if the seed can germinate or not are still far from complete understanding. The role and mechanisms of regulation of proteome composition during processes occurring in the plant's photosynthetic tissues have been well-characterized since many years, but in non-photosynthetic seeds it has emerged as a tempting research task only since the last decade. This review discusses the recent discoveries providing insights into the role of protein turnover in seed dormancy alleviation, and germination, with a focus on the control of translation and proteasomal proteolysis. The presented novel data of translatome profiling in seeds highlighted that post-transcriptional regulation of germination results from a timely regulated initiation of translation. In addition, the importance of 26S proteasome in the degradation of regulatory elements of cellular signaling and that of the 20S complex in proteolysis of specific carbonylated proteins in hormonal- and light-dependent processes occurring in seeds is discussed. Based on the

  18. Cellular Recycling of Proteins in Seed Dormancy Alleviation and Germination.

    PubMed

    Oracz, Krystyna; Stawska, Marlena

    2016-01-01

    Each step of the seed-to-seed cycle of plant development including seed germination is characterized by a specific set of proteins. The continual renewal and/or replacement of these biomolecules are crucial for optimal plant adaptation. As proteins are the main effectors inside the cells, their levels need to be tightly regulated. This is partially achieved by specific proteolytic pathways via multicatalytic protease complexes defined as 20S and 26S proteasomes. In plants, the 20S proteasome is responsible for degradation of carbonylated proteins, while the 26S being a part of ubiquitin-proteasome pathway is known to be involved in proteolysis of phytohormone signaling regulators. On the other hand, the role of translational control of plant development is also well-documented, especially in the context of pollen tube growth and light signaling. Despite the current progress that has been made in seed biology, the sequence of cellular events that determine if the seed can germinate or not are still far from complete understanding. The role and mechanisms of regulation of proteome composition during processes occurring in the plant's photosynthetic tissues have been well-characterized since many years, but in non-photosynthetic seeds it has emerged as a tempting research task only since the last decade. This review discusses the recent discoveries providing insights into the role of protein turnover in seed dormancy alleviation, and germination, with a focus on the control of translation and proteasomal proteolysis. The presented novel data of translatome profiling in seeds highlighted that post-transcriptional regulation of germination results from a timely regulated initiation of translation. In addition, the importance of 26S proteasome in the degradation of regulatory elements of cellular signaling and that of the 20S complex in proteolysis of specific carbonylated proteins in hormonal- and light-dependent processes occurring in seeds is discussed. Based on the

  19. MultitaskProtDB: a database of multitasking proteins.

    PubMed

    Hernández, Sergio; Ferragut, Gabriela; Amela, Isaac; Perez-Pons, JosepAntoni; Piñol, Jaume; Mozo-Villarias, Angel; Cedano, Juan; Querol, Enrique

    2014-01-01

    We have compiled MultitaskProtDB, available online at http://wallace.uab.es/multitask, to provide a repository where the many multitasking proteins found in the literature can be stored. Multitasking or moonlighting is the capability of some proteins to execute two or more biological functions. Usually, multitasking proteins are experimentally revealed by serendipity. This ability of proteins to perform multitasking functions helps us to understand one of the ways used by cells to perform many complex functions with a limited number of genes. Even so, the study of this phenomenon is complex because, among other things, there is no database of moonlighting proteins. The existence of such a tool facilitates the collection and dissemination of these important data. This work reports the database, MultitaskProtDB, which is designed as a friendly user web page containing >288 multitasking proteins with their NCBI and UniProt accession numbers, canonical and additional biological functions, monomeric/oligomeric states, PDB codes when available and bibliographic references. This database also serves to gain insight into some characteristics of multitasking proteins such as frequencies of the different pairs of functions, phylogenetic conservation and so forth.

  20. MultitaskProtDB: a database of multitasking proteins.

    PubMed

    Hernández, Sergio; Ferragut, Gabriela; Amela, Isaac; Perez-Pons, JosepAntoni; Piñol, Jaume; Mozo-Villarias, Angel; Cedano, Juan; Querol, Enrique

    2014-01-01

    We have compiled MultitaskProtDB, available online at http://wallace.uab.es/multitask, to provide a repository where the many multitasking proteins found in the literature can be stored. Multitasking or moonlighting is the capability of some proteins to execute two or more biological functions. Usually, multitasking proteins are experimentally revealed by serendipity. This ability of proteins to perform multitasking functions helps us to understand one of the ways used by cells to perform many complex functions with a limited number of genes. Even so, the study of this phenomenon is complex because, among other things, there is no database of moonlighting proteins. The existence of such a tool facilitates the collection and dissemination of these important data. This work reports the database, MultitaskProtDB, which is designed as a friendly user web page containing >288 multitasking proteins with their NCBI and UniProt accession numbers, canonical and additional biological functions, monomeric/oligomeric states, PDB codes when available and bibliographic references. This database also serves to gain insight into some characteristics of multitasking proteins such as frequencies of the different pairs of functions, phylogenetic conservation and so forth. PMID:24253302

  1. YPL.db2: the Yeast Protein Localization database, version 2.0.

    PubMed

    Kals, Mathias; Natter, Klaus; Thallinger, Gerhard G; Trajanoski, Zlatko; Kohlwein, Sepp D

    2005-02-01

    The Yeast Protein Localization database (YPL.db(2)) is an archive of microscopic image data of protein localization patterns in the yeast Saccharomyces cerevisiae. The current version of YPL.db(2) harbours 500 sets of image data derived from high-resolution microscopic analyses of proteins tagged with the green fluorescent protein (GFP). Major functional improvements in YPL.db(2) over a previous release are a web-based experiment and image submission interface, facilitating standardized data entry by remote users through the Internet. The image display page provides image gallery and image scrolling features. In addition, fluorescence and transmission images can be superimposed, allowing image fading for precise correlation of the protein's localization in the cellular context. The reference structure database displaying 'prototypic' localization patterns was extended, and a feature to display and manipulate 3D-image datasets, using a freely available VRML plug-in, was included. Access to the Yeast Protein Localization database version 2.0 (YPL.db(2)) is available through http://YPL.uni-graz.at.

  2. Cellular Disulfide Bond Formation in Bioactive Peptides and Proteins

    PubMed Central

    Patil, Nitin A.; Tailhades, Julien; Hughes, Richard Anthony; Separovic, Frances; Wade, John D.; Hossain, Mohammed Akhter

    2015-01-01

    Bioactive peptides play important roles in metabolic regulation and modulation and many are used as therapeutics. These peptides often possess disulfide bonds, which are important for their structure, function and stability. A systematic network of enzymes—a disulfide bond generating enzyme, a disulfide bond donor enzyme and a redox cofactor—that function inside the cell dictates the formation and maintenance of disulfide bonds. The main pathways that catalyze disulfide bond formation in peptides and proteins in prokaryotes and eukaryotes are remarkably similar and share several mechanistic features. This review summarizes the formation of disulfide bonds in peptides and proteins by cellular and recombinant machinery. PMID:25594871

  3. Direct Cellular Lysis/Protein Extraction Protocol for Soil Metaproteomics

    SciTech Connect

    Chourey, Karuna; Jansson, Janet; Verberkmoes, Nathan C; Shah, Manesh B; Chavarria, Krystle L.; Tom, Lauren M; Brodie, Eoin L.; Hettich, Robert {Bob} L

    2010-01-01

    We present a novel direct protocol for deep proteome characterization of microorganisms in soil. The method employs thermally assisted detergent-based cellular lysis (SDS) of soil samples, followed by TCA precipitation for proteome extraction/cleanup prior to liquid chromatography-mass spectrometric characterization. This approach was developed and optimized using different soils inoculated with genome-sequenced bacteria (Gram-negative Pseudomonas putida or Gram-positive Arthrobacter chlorophenolicus). Direct soil protein extraction was compared to protein extraction from cells isolated from the soil matrix prior to lysis (indirect method). Each approach resulted in identification of greater than 500 unique proteins, with a wide range in molecular mass and functional categories. To our knowledge, this SDS-TCA approach enables the deepest proteome characterizations of microbes in soil to date, without significant biases in protein size, localization, or functional category compared to pure cultures. This protocol should provide a powerful tool for ecological studies of soil microbial communities.

  4. PIMADb: A Database of Protein–Protein Interactions in Huge Macromolecular Assemblies

    PubMed Central

    Mathew, Oommen K.; Sowdhamini, Ramanathan

    2016-01-01

    Protein–protein interactions play a very important role in the process of cellular functionality. Intricate details about the interactions between the proteins in a macromolecular assembly are important to understand the function and significance of protein complexes. We are reporting about a database of protein–protein interactions in huge macromolecular assemblies (PIMADb) that records the intrinsic details of 189,532 interchain interactions in 40,049 complexes from the Protein Data Bank. These details include the results of the quantification and analysis of all the interactions in the complex. The availability of interprotomer interaction networks can enable the design of point mutation experiments. PIMADb can be accessed from the URL: http://caps.ncbs.res.in/pimadb PMID:27478368

  5. DDRprot: a database of DNA damage response-related proteins

    PubMed Central

    Andrés-León, Eduardo; Cases, Ildefonso; Arcas, Aida; Rojas, Ana M.

    2016-01-01

    The DNA Damage Response (DDR) signalling network is an essential system that protects the genome’s integrity. The DDRprot database presented here is a resource that integrates manually curated information on the human DDR network and its sub-pathways. For each particular DDR protein, we present detailed information about its function. If involved in post-translational modifications (PTMs) with each other, we depict the position of the modified residue/s in the three-dimensional structures, when resolved structures are available for the proteins. All this information is linked to the original publication from where it was obtained. Phylogenetic information is also shown, including time of emergence and conservation across 47 selected species, family trees and sequence alignments of homologues. The DDRprot database can be queried by different criteria: pathways, species, evolutionary age or involvement in (PTM). Sequence searches using hidden Markov models can be also used. Database URL: http://ddr.cbbio.es. PMID:27577567

  6. DDRprot: a database of DNA damage response-related proteins.

    PubMed

    Andrés-León, Eduardo; Cases, Ildefonso; Arcas, Aida; Rojas, Ana M

    2016-01-01

    The DNA Damage Response (DDR) signalling network is an essential system that protects the genome's integrity. The DDRprot database presented here is a resource that integrates manually curated information on the human DDR network and its sub-pathways. For each particular DDR protein, we present detailed information about its function. If involved in post-translational modifications (PTMs) with each other, we depict the position of the modified residue/s in the three-dimensional structures, when resolved structures are available for the proteins. All this information is linked to the original publication from where it was obtained. Phylogenetic information is also shown, including time of emergence and conservation across 47 selected species, family trees and sequence alignments of homologues. The DDRprot database can be queried by different criteria: pathways, species, evolutionary age or involvement in (PTM). Sequence searches using hidden Markov models can be also used.Database URL: http://ddr.cbbio.es. PMID:27577567

  7. Protein carbonylation and metal-catalyzed protein oxidation in a cellular perspective.

    PubMed

    Møller, Ian M; Rogowska-Wrzesinska, Adelina; Rao, R S P

    2011-10-19

    Proteins can become oxidatively modified in many different ways, either by direct oxidation of amino acid side chains and protein backbone or indirectly by conjugation with oxidation products of polyunsaturated fatty acids and carbohydrates. While reversible oxidative modifications are thought to be relevant in physiological processes, irreversible oxidative modifications are known to contribute to cellular damage and disease. The most well-studied irreversible protein oxidation is carbonylation. In this work we first examine how protein carbonylation occurs via metal-catalyzed oxidation (MCO) in vivo and in vitro with an emphasis on cellular metal ion homeostasis and metal binding. We then review proteomic methods currently used for identifying carbonylated proteins and their sites of modification. Finally, we discuss the identified carbonylated proteins and the pattern of carbonylation sites in relation to cellular metabolism using the mitochondrion as a case story.

  8. Kinomer v. 1.0: a database of systematically classified eukaryotic protein kinases.

    PubMed

    Martin, David M A; Miranda-Saavedra, Diego; Barton, Geoffrey J

    2009-01-01

    The regulation of protein function through reversible phosphorylation by protein kinases and phosphatases is a general mechanism controlling virtually every cellular activity. Eukaryotic protein kinases can be classified into distinct, well-characterized groups based on amino acid sequence similarity and function. We recently reported a highly sensitive and accurate hidden Markov model-based method for the automatic detection and classification of protein kinases into these specific groups. The Kinomer v. 1.0 database presented here contains annotated classifications for the protein kinase complements of 43 eukaryotic genomes. These span the taxonomic range and include fungi (16 species), plants (6), diatoms (1), amoebas (2), protists (1) and animals (17). The kinomes are stored in a relational database and are accessible through a web interface on the basis of species, kinase group or a combination of both. In addition, the Kinomer v. 1.0 HMM library is made available for users to perform classification on arbitrary sequences. The Kinomer v. 1.0 database is a continually updated resource where direct comparison of kinase sequences across kinase groups and across species can give insights into kinase function and evolution. Kinomer v. 1.0 is available at http://www.compbio.dundee.ac.uk/kinomer/.

  9. Functional and Structural Mimicry of Cellular Protein Kinase A Anchoring Proteins by a Viral Oncoprotein

    PubMed Central

    King, Cason R.; Cohen, Michael J.; Fonseca, Gregory J.; Dirk, Brennan S.; Dikeakos, Jimmy D.; Mymryk, Joe S.

    2016-01-01

    The oncoproteins of the small DNA tumor viruses interact with a plethora of cellular regulators to commandeer control of the infected cell. During infection, adenovirus E1A deregulates cAMP signalling and repurposes it for activation of viral gene expression. We show that E1A structurally and functionally mimics a cellular A-kinase anchoring protein (AKAP). E1A interacts with and relocalizes protein kinase A (PKA) to the nucleus, likely to virus replication centres, via an interaction with the regulatory subunits of PKA. Binding to PKA requires the N-terminus of E1A, which bears striking similarity to the amphipathic α-helical domain present in cellular AKAPs. E1A also targets the same docking-dimerization domain of PKA normally bound by cellular AKAPs. In addition, the AKAP like motif within E1A could restore PKA interaction to a cellular AKAP in which its normal interaction motif was deleted. During infection, E1A successfully competes with endogenous cellular AKAPs for PKA interaction. E1A’s role as a viral AKAP contributes to viral transcription, protein expression and progeny production. These data establish HAdV E1A as the first known viral AKAP. This represents a unique example of viral subversion of a crucial cellular regulatory pathway via structural mimicry of the PKA interaction domain of cellular AKAPs. PMID:27137912

  10. PSCDB: a database for protein structural change upon ligand binding.

    PubMed

    Amemiya, Takayuki; Koike, Ryotaro; Kidera, Akinori; Ota, Motonori

    2012-01-01

    Proteins are flexible molecules that undergo structural changes to function. The Protein Data Bank contains multiple entries for identical proteins determined under different conditions, e.g. with and without a ligand molecule, which provides important information for understanding the structural changes related to protein functions. We gathered 839 protein structural pairs of ligand-free and ligand-bound states from monomeric or homo-dimeric proteins, and constructed the Protein Structural Change DataBase (PSCDB). In the database, we focused on whether the motions were coupled with ligand binding. As a result, the protein structural changes were classified into seven classes, i.e. coupled domain motion (59 structural changes), independent domain motion (70), coupled local motion (125), independent local motion (135), burying ligand motion (104), no significant motion (311) and other type motion (35). PSCDB provides lists of each class. On each entry page, users can view detailed information about the motion, accompanied by a morphing animation of the structural changes. PSCDB is available at http://idp1.force.cs.is.nagoya-u.ac.jp/pscdb/. PMID:22080505

  11. Merging photolithography and robotic protein printing to create cellular microarrays.

    PubMed

    Lee, Ji Youn; Revzin, Alexander

    2011-01-01

    Photolithographic patterning of proteins on surfaces has been used extensively in the past to define cell adhesion domains with micrometer-scale resolution. However, photolithographic patterning is not amenable to depositing several different proteins on the same surface. We propose to merge photolithography with robotic printing of proteins in order to create arrays of protein spots (∼300-500 μm diameters) with encoded micrometer-scale cell adhesive domains. This method for biointerface design can employ standard positive tone resist lithography to create temporary stencils for printing of protein arrays. Alternatively, nonfouling poly(ethylene glycol) hydrogels can be micropatterned on top of protein spots. In both cases, cells become adherent on the underlying protein domains, but on-the-spot distribution of cells is defined by the photolithographic pattern. The ability to define multiple cell-substrate and cell-cell interaction scenarios on the same surface is applicable to high-throughput screening of the microenvironment components required for cellular differentiation, for example, for guiding stem cells toward the desired tissue type.

  12. Affinity labeling of GTP-binding proteins in cellular extracts.

    PubMed

    Löw, A; Faulhammer, H G; Sprinzl, M

    1992-05-25

    GTP-binding proteins in cellular extracts from Escherichia coli, Thermus thermophilus, yeast, wheat germ or calf thymus were identified using in situ periodate-oxidized [alpha-32P]GTP as affinity label. Site-specific reaction of individual GTP-binding proteins was achieved by cross-linking the protein-bound 2',3'-dialdehyde derivative of GTP with the single lysine residue of the conserved NKXD sequence through Schiff's base formation and subsequent cyanoborohydride reduction. Labeled GTP-binding proteins from prokaryotic or eukaryotic cell homogenates were separated by polyacrylamide gel electrophoresis and visualized by autoradiography. In addition cross-linking of [alpha-32P]GTP with GTP-binding proteins was demonstrated in model systems using different purified GTPases, human c-H-ras p21, transducin from bovine retina, polypeptide elongation factor Tu (EF-Tu) from T. thermophilus and initiation factor 2 (IF2) from T. thermophilus. The described affinity labeling technique can serve as an analytical method for the identification of GTPases belonging to the classes of ras-proteins, elongation and initiation factors, and heterotrimeric signal transducing G-proteins. PMID:1592117

  13. MALISAM: a database of structurally analogous motifs in proteins.

    PubMed

    Cheng, Hua; Kim, Bong-Hyun; Grishin, Nick V

    2008-01-01

    MALISAM (manual alignments for structurally analogous motifs) represents the first database containing pairs of structural analogs and their alignments. To find reliable analogs, we developed an approach based on three ideas. First, an insertion together with a part of the evolutionary core of one domain family (a hybrid motif) is analogous to a similar motif contained within the core of another domain family. Second, a motif at an interface, formed by secondary structural elements (SSEs) contributed by two or more domains or subunits contacting along that interface, is analogous to a similar motif present in the core of a single domain. Third, an artificial protein obtained through selection from random peptides or in sequence design experiments not biased by sequences of a particular homologous family, is analogous to a structurally similar natural protein. Each analogous pair is superimposed and aligned manually, as well as by several commonly used programs. Applications of this database may range from protein evolution studies, e.g. development of remote homology inference tools and discriminators between homologs and analogs, to protein-folding research, since in the absence of evolutionary reasons, similarity between proteins is caused by structural and folding constraints. The database is publicly available at http://prodata.swmed.edu/malisam. PMID:17855399

  14. Detection of protein similarities using nucleotide sequence databases.

    PubMed

    Henikoff, S; Wallace, J C

    1988-07-11

    A simple procedure is described for finding similarities between proteins using nucleotide sequence databases. The approach is illustrated by several examples of previously unknown correspondences with important biological implications: Drosophila elongation factor Tu is shown to be encoded by two genes that are differently expressed during development; a cluster of three Drosophila genes likely encode maltases; a flesh-fly fat body protein resembles the hypothesized Drosophila alcohol dehydrogenase ancestral protein; an unknown protein encoded at the multifunctional E. coli hisT locus resembles aspartate beta-semialdehyde dehydrogenase; and the E. coli tyrR protein is related to nitrogen regulatory proteins. These and other matches were discovered using a personal computer of the type available in most laboratories collecting DNA sequence data. As relatively few sequences were sampled to find these matches, it is likely that much of the existing data has not been adequately examined.

  15. PRINTS--a database of protein motif fingerprints.

    PubMed

    Attwood, T K; Beck, M E; Bleasby, A J; Parry-Smith, D J

    1994-09-01

    PRINTS is a compendium of protein motif 'fingerprints'. A fingerprint is defined as a group of motifs excised from conserved regions of a sequence alignment, whose diagnostic power or potency is refined by iterative databasescanning (in this case the OWL composite sequence database). Generally, the motifs do not overlap, but are separated along a sequence, though they may be contiguous in 3D-space. The use of groups of independent, linearly- or spatially-distinct motifs allows protein folds and functionalities to be characterised more flexibly and powerfully than conventional single-component patterns or regular expressions. The current version of the database contains 200 entries (encoding 950 motifs), covering a wide range of globular and membrane proteins, modular polypeptides, and so on. The growth of the databaseis influenced by a number of factors; e.g. the use of multiple motifs; the maximisation of sequence information through iterative database scanning; and the fact that the database searched is a large composite. The information contained within PRINTS is distinct from, but complementary to the consensus expressions stored in the widely-used PROSITE dictionary of patterns.

  16. CDD: a Conserved Domain Database for protein classification.

    PubMed

    Marchler-Bauer, Aron; Anderson, John B; Cherukuri, Praveen F; DeWeese-Scott, Carol; Geer, Lewis Y; Gwadz, Marc; He, Siqian; Hurwitz, David I; Jackson, John D; Ke, Zhaoxi; Lanczycki, Christopher J; Liebert, Cynthia A; Liu, Chunlei; Lu, Fu; Marchler, Gabriele H; Mullokandov, Mikhail; Shoemaker, Benjamin A; Simonyan, Vahan; Song, James S; Thiessen, Paul A; Yamashita, Roxanne A; Yin, Jodie J; Zhang, Dachuan; Bryant, Stephen H

    2005-01-01

    The Conserved Domain Database (CDD) is the protein classification component of NCBI's Entrez query and retrieval system. CDD is linked to other Entrez databases such as Proteins, Taxonomy and PubMed, and can be accessed at http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=cdd. CD-Search, which is available at http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi, is a fast, interactive tool to identify conserved domains in new protein sequences. CD-Search results for protein sequences in Entrez are pre-computed to provide links between proteins and domain models, and computational annotation visible upon request. Protein-protein queries submitted to NCBI's BLAST search service at http://www.ncbi.nlm.nih.gov/BLAST are scanned for the presence of conserved domains by default. While CDD started out as essentially a mirror of publicly available domain alignment collections, such as SMART, Pfam and COG, we have continued an effort to update, and in some cases replace these models with domain hierarchies curated at the NCBI. Here, we report on the progress of the curation effort and associated improvements in the functionality of the CDD information retrieval system. PMID:15608175

  17. PSORTdb: a protein subcellular localization database for bacteria

    PubMed Central

    Rey, Sébastien; Acab, Michael; Gardy, Jennifer L.; Laird, Matthew R.; deFays, Katalin; Lambert, Christophe; Brinkman, Fiona S. L.

    2005-01-01

    Information about bacterial subcellular localization (SCL) is important for protein function prediction and identification of suitable drug/vaccine/diagnostic targets. PSORTdb (http://db.psort.org/) is a web-accessible database of SCL for bacteria that contains both information determined through laboratory experimentation and computational predictions. The dataset of experimentally verified information (∼2000 proteins) was manually curated by us and represents the largest dataset of its kind. Earlier versions have been used for training SCL predictors, and its incorporation now into this new PSORTdb resource, with its associated additional annotation information and dataset version control, should aid researchers in future development of improved SCL predictors. The second component of this database contains computational analyses of proteins deduced from the most recent NCBI dataset of completely sequenced genomes. Analyses are currently calculated using PSORTb, the most precise automated SCL predictor for bacterial proteins. Both datasets can be accessed through the web using a very flexible text search engine, a data browser, or using BLAST, and the entire database or search results may be downloaded in various formats. Features such as GO ontologies and multiple accession numbers are incorporated to facilitate integration with other bioinformatics resources. PSORTdb is freely available under GNU General Public License. PMID:15608169

  18. Cellular factors modulating the mechanism of tau protein aggregation

    PubMed Central

    Fontaine, Sarah N.; Sabbagh, Jonathan J.; Baker, Jeremy; Martinez-Licha, Carlos R.; Darling, April

    2015-01-01

    Pathological accumulation of the microtubule-associated protein tau, in the form of neurofibrillary tangles, is a major hallmark of Alzheimer’s disease, the most prevalent neurodegenerative condition worldwide. In addition to Alzheimer’s disease, a number of neurodegenerative diseases, called tauopathies, are characterized by the accumulation of aggregated tau in a variety of brain regions. While tau normally plays an important role in stabilizing the microtubule network of the cytoskeleton, its dissociation from microtubules and eventual aggregation into pathological deposits is an area of intense focus for therapeutic development. Here we discuss the known cellular factors that affect tau aggregation, from post-translational modifications to molecular chaperones. PMID:25666877

  19. Cellular Localization of Engineered Human LINE-1 RNA and Proteins.

    PubMed

    Doucet, Aurélien J; Basyuk, Eugénia; Gilbert, Nicolas

    2016-01-01

    The human LINE-1 retrotransposon has the ability to mobilize into a new genomic location through an intracellular replication cycle. Immunofluorescence and in situ hybridization experiments have been developed to detect subcellular localization of retrotransposition intermediates (i.e., ORF1p, ORF2p, and L1 mRNA). Currently, these protocols are also used to validate the interaction between retrotransposition complex components and potential cellular partners involved in L1 replication. Here, we describe in details methods for the identification of LINE-1 proteins and/or RNA in cells transfected with vectors expressing engineered human LINE-1 elements. PMID:26895060

  20. P(3)DB: An Integrated Database for Plant Protein Phosphorylation.

    PubMed

    Yao, Qiuming; Bollinger, Curtis; Gao, Jianjiong; Xu, Dong; Thelen, Jay J

    2012-01-01

    Protein phosphorylation is widely recognized as the most widespread, enzyme-catalyzed post-translational modification in eukaryotes. In particular, plants have appropriated this signaling mechanism as evidenced by the twofold higher frequency of protein kinases within the genome compared to other eukaryotes. While all aspects of plant protein phosphorylation research have grown in the past 10 years; phosphorylation site mapping using high-resolution mass spectrometry has grown exponentially. In Arabidopsis alone there are thousands of experimentally determined phosphorylation sites. To archive these events in a user-intuitive format we have developed P(3)DB, the Plant Protein Phosphorylation Database (p3db.org). This database is a repository for plant protein phosphorylation site data, currently hosting information on 32,963 non-redundant sites collated from 23 experimental studies from six plant species. These data can be queried for a protein-of-interest using an integrated BLAST module to query similar sequences with known phosphorylation sites among the multiple plants currently investigated. The paper demonstrates how this resource can help identify functionally conserved phosphorylation sites in plants using a multi-system approach.

  1. Benchmarking NMR experiments: A relational database of protein pulse sequences

    NASA Astrophysics Data System (ADS)

    Senthamarai, Russell R. P.; Kuprov, Ilya; Pervushin, Konstantin

    2010-03-01

    Systematic benchmarking of multi-dimensional protein NMR experiments is a critical prerequisite for optimal allocation of NMR resources for structural analysis of challenging proteins, e.g. large proteins with limited solubility or proteins prone to aggregation. We propose a set of benchmarking parameters for essential protein NMR experiments organized into a lightweight (single XML file) relational database (RDB), which includes all the necessary auxiliaries (waveforms, decoupling sequences, calibration tables, setup algorithms and an RDB management system). The database is interfaced to the Spinach library ( http://spindynamics.org), which enables accurate simulation and benchmarking of NMR experiments on large spin systems. A key feature is the ability to use a single user-specified spin system to simulate the majority of deposited solution state NMR experiments, thus providing the (hitherto unavailable) unified framework for pulse sequence evaluation. This development enables predicting relative sensitivity of deposited implementations of NMR experiments, thus providing a basis for comparison, optimization and, eventually, automation of NMR analysis. The benchmarking is demonstrated with two proteins, of 170 amino acids I domain of αXβ2 Integrin and 440 amino acids NS3 helicase.

  2. [Identifying phosphopeptide by searching a site annotated protein database].

    PubMed

    Cheng, Kai; Wang, Fangjun; Bian, Yangyang; Ye, Mingling; Zou, Hanfa

    2015-01-01

    Phosphoproteome analysis is one of the important research fields in proteomics. In shotgun proteomics, phosphopeptides could be identified directly by setting phosphorylation as variable modifications in database search. However, search space increases significantly when variable modifications are set in post-translation modifications (PTMs) analysis, which will decrease the identification sensitivity. Because setting a variable modification on a specific type of amino acid residue means all of this amino acid residues in the database might be modified, which is not consistent with actual conditions. Phosphorylation and dephosphorylation are regulated by protein kinases and phosphatases, which can only occur on particular substrates. Therefore only residues within specific sequence are potential sites which may be modified. To address this issue, we extracted the characteristic sequence from the identified phosphorylation sites and created an annotated database containing phosphorylation site information, which allowed the searching engine to set variable modifications only on the serine, threonine and tyrosine residues that were identified to be phosphorylated previously. In this database only annotated serine, threonine and tyrosine can be modified. This strategy significantly reduced the search space. The performance of this new database searching strategy was evaluated by searching different types of data with Mascot, and higher sensitivity for phosphopeptide identification was achieved with high reliability.

  3. Derivation of rules for comparative protein modeling from a database of protein structure alignments.

    PubMed Central

    Sali, A.; Overington, J. P.

    1994-01-01

    We describe a database of protein structure alignments as well as methods and tools that use this database to improve comparative protein modeling. The current version of the database contains 105 alignments of similar proteins or protein segments. The database comprises 416 entries, 78,495 residues, 1,233 equivalent entry pairs, and 230,396 pairs of equivalent alignment positions. At present, the main application of the database is to improve comparative modeling by satisfaction of spatial restraints implemented in the program MODELLER (Sali A, Blundell TL, 1993, J Mol Biol 234:779-815). To illustrate the usefulness of the database, the restraints on the conformation of a disulfide bridge provided by an equivalent disulfide bridge in a related structure are derived from the alignments; the prediction success of the disulfide dihedral angle classes is increased to approximately 80%, compared to approximately 55% for modeling that relies on the stereochemistry of disulfide bridges alone. The second example of the use of the database is the derivation of the probability density function for comparative modeling of the cis/trans isomerism of the proline residues; the prediction success is increased from 0% to 82.9% for cis-proline and from 93.3% to 96.2% for trans-proline. The database is available via electronic mail. PMID:7833817

  4. SPIKE – a database, visualization and analysis tool of cellular signaling pathways

    PubMed Central

    Elkon, Ran; Vesterman, Rita; Amit, Nira; Ulitsky, Igor; Zohar, Idan; Weisz, Mali; Mass, Gilad; Orlev, Nir; Sternberg, Giora; Blekhman, Ran; Assa, Jackie; Shiloh, Yosef; Shamir, Ron

    2008-01-01

    Background Biological signaling pathways that govern cellular physiology form an intricate web of tightly regulated interlocking processes. Data on these regulatory networks are accumulating at an unprecedented pace. The assimilation, visualization and interpretation of these data have become a major challenge in biological research, and once met, will greatly boost our ability to understand cell functioning on a systems level. Results To cope with this challenge, we are developing the SPIKE knowledge-base of signaling pathways. SPIKE contains three main software components: 1) A database (DB) of biological signaling pathways. Carefully curated information from the literature and data from large public sources constitute distinct tiers of the DB. 2) A visualization package that allows interactive graphic representations of regulatory interactions stored in the DB and superposition of functional genomic and proteomic data on the maps. 3) An algorithmic inference engine that analyzes the networks for novel functional interplays between network components. SPIKE is designed and implemented as a community tool and therefore provides a user-friendly interface that allows registered users to upload data to SPIKE DB. Our vision is that the DB will be populated by a distributed and highly collaborative effort undertaken by multiple groups in the research community, where each group contributes data in its field of expertise. Conclusion The integrated capabilities of SPIKE make it a powerful platform for the analysis of signaling networks and the integration of knowledge on such networks with omics data. PMID:18289391

  5. Intrinsic Structural Disorder Confers Cellular Viability on Oncogenic Fusion Proteins

    PubMed Central

    Hegyi, Hedi; Buday, László; Tompa, Peter

    2009-01-01

    Chromosomal translocations, which often generate chimeric proteins by fusing segments of two distinct genes, represent the single major genetic aberration leading to cancer. We suggest that the unifying theme of these events is a high level of intrinsic structural disorder, enabling fusion proteins to evade cellular surveillance mechanisms that eliminate misfolded proteins. Predictions in 406 translocation-related human proteins show that they are significantly enriched in disorder (43.3% vs. 20.7% in all human proteins), they have fewer Pfam domains, and their translocation breakpoints tend to avoid domain splitting. The vicinity of the breakpoint is significantly more disordered than the rest of these already highly disordered fusion proteins. In the unlikely event of domain splitting in fusion it usually spares much of the domain or splits at locations where the newly exposed hydrophobic surface area approximates that of an intact domain. The mechanisms of action of fusion proteins suggest that in most cases their structural disorder is also essential to the acquired oncogenic function, enabling the long-range structural communication of remote binding and/or catalytic elements. In this respect, there are three major mechanisms that contribute to generating an oncogenic signal: (i) a phosphorylation site and a tyrosine-kinase domain are fused, and structural disorder of the intervening region enables intramolecular phosphorylation (e.g., BCR-ABL); (ii) a dimerisation domain fuses with a tyrosine kinase domain and disorder enables the two subunits within the homodimer to engage in permanent intermolecular phosphorylations (e.g., TFG-ALK); (iii) the fusion of a DNA-binding element to a transactivator domain results in an aberrant transcription factor that causes severe misregulation of transcription (e.g. EWS-ATF). Our findings also suggest novel strategies of intervention against the ensuing neoplastic transformations. PMID:19888473

  6. MannDB: A microbial annotation database for protein characterization

    SciTech Connect

    Zhou, C; Lam, M; Smith, J; Zemla, A; Dyer, M; Kuczmarski, T; Vitalis, E; Slezak, T

    2006-05-19

    MannDB was created to meet a need for rapid, comprehensive automated protein sequence analyses to support selection of proteins suitable as targets for driving the development of reagents for pathogen or protein toxin detection. Because a large number of open-source tools were needed, it was necessary to produce a software system to scale the computations for whole-proteome analysis. Thus, we built a fully automated system for executing software tools and for storage, integration, and display of automated protein sequence analysis and annotation data. MannDB is a relational database that organizes data resulting from fully automated, high-throughput protein-sequence analyses using open-source tools. Types of analyses provided include predictions of cleavage, chemical properties, classification, features, functional assignment, post-translational modifications, motifs, antigenicity, and secondary structure. Proteomes (lists of hypothetical and known proteins) are downloaded and parsed from Genbank and then inserted into MannDB, and annotations from SwissProt are downloaded when identifiers are found in the Genbank entry or when identical sequences are identified. Currently 36 open-source tools are run against MannDB protein sequences either on local systems or by means of batch submission to external servers. In addition, BLAST against protein entries in MvirDB, our database of microbial virulence factors, is performed. A web client browser enables viewing of computational results and downloaded annotations, and a query tool enables structured and free-text search capabilities. When available, links to external databases, including MvirDB, are provided. MannDB contains whole-proteome analyses for at least one representative organism from each category of biological threat organism listed by APHIS, CDC, HHS, NIAID, USDA, USFDA, and WHO. MannDB comprises a large number of genomes and comprehensive protein sequence analyses representing organisms listed as high

  7. PACRG, a protein linked to ciliary motility, mediates cellular signaling

    PubMed Central

    Loucks, Catrina M.; Bialas, Nathan J.; Dekkers, Martijn P. J.; Walker, Denise S.; Grundy, Laura J.; Li, Chunmei; Inglis, P. Nick; Kida, Katarzyna; Schafer, William R.; Blacque, Oliver E.; Jansen, Gert; Leroux, Michel R.

    2016-01-01

    Cilia are microtubule-based organelles that project from nearly all mammalian cell types. Motile cilia generate fluid flow, whereas nonmotile (primary) cilia are required for sensory physiology and modulate various signal transduction pathways. Here we investigate the nonmotile ciliary signaling roles of parkin coregulated gene (PACRG), a protein linked to ciliary motility. PACRG is associated with the protofilament ribbon, a structure believed to dictate the regular arrangement of motility-associated ciliary components. Roles for protofilament ribbon–associated proteins in nonmotile cilia and cellular signaling have not been investigated. We show that PACRG localizes to a small subset of nonmotile cilia in Caenorhabditis elegans, suggesting an evolutionary adaptation for mediating specific sensory/signaling functions. We find that it influences a learning behavior known as gustatory plasticity, in which it is functionally coupled to heterotrimeric G-protein signaling. We also demonstrate that PACRG promotes longevity in C. elegans by acting upstream of the lifespan-promoting FOXO transcription factor DAF-16 and likely upstream of insulin/IGF signaling. Our findings establish previously unrecognized sensory/signaling functions for PACRG and point to a role for this protein in promoting longevity. Furthermore, our work suggests additional ciliary motility-signaling connections, since EFHC1 (EF-hand containing 1), a potential PACRG interaction partner similarly associated with the protofilament ribbon and ciliary motility, also positively regulates lifespan. PMID:27193298

  8. PACRG, a protein linked to ciliary motility, mediates cellular signaling.

    PubMed

    Loucks, Catrina M; Bialas, Nathan J; Dekkers, Martijn P J; Walker, Denise S; Grundy, Laura J; Li, Chunmei; Inglis, P Nick; Kida, Katarzyna; Schafer, William R; Blacque, Oliver E; Jansen, Gert; Leroux, Michel R

    2016-07-01

    Cilia are microtubule-based organelles that project from nearly all mammalian cell types. Motile cilia generate fluid flow, whereas nonmotile (primary) cilia are required for sensory physiology and modulate various signal transduction pathways. Here we investigate the nonmotile ciliary signaling roles of parkin coregulated gene (PACRG), a protein linked to ciliary motility. PACRG is associated with the protofilament ribbon, a structure believed to dictate the regular arrangement of motility-associated ciliary components. Roles for protofilament ribbon-associated proteins in nonmotile cilia and cellular signaling have not been investigated. We show that PACRG localizes to a small subset of nonmotile cilia in Caenorhabditis elegans, suggesting an evolutionary adaptation for mediating specific sensory/signaling functions. We find that it influences a learning behavior known as gustatory plasticity, in which it is functionally coupled to heterotrimeric G-protein signaling. We also demonstrate that PACRG promotes longevity in C. elegans by acting upstream of the lifespan-promoting FOXO transcription factor DAF-16 and likely upstream of insulin/IGF signaling. Our findings establish previously unrecognized sensory/signaling functions for PACRG and point to a role for this protein in promoting longevity. Furthermore, our work suggests additional ciliary motility-signaling connections, since EFHC1 (EF-hand containing 1), a potential PACRG interaction partner similarly associated with the protofilament ribbon and ciliary motility, also positively regulates lifespan. PMID:27193298

  9. SoyProLow: A protein database enriched in low abundant soybean proteins

    PubMed Central

    Tavakolan, Mona; Alkharouf, Nadim W; Matthews, Benjamin F; Natarajan, Savithiry S

    2014-01-01

    Soybeans are an important legume crop that contain 2 major storage proteins, β-conglycinin and glycinin, which account about 70- 80% of total seed proteins. These abundant proteins hinder the isolation and characterization of several low abundant proteins in soybean seeds. Several protein extraction methodologies were developed in our laboratory to decrease these abundant storage proteins in seed extracts and to also decrease the amount of ribulose-1, 5-bisphosphate carboxylase/oxygenase (RuBisCO), which is normally very abundant in leaf extracts. One of the extraction methodologies used 40% isopropanol and was more effective in depleting soybean storage proteins and enhancing low abundant seed proteins than similar methods using 10-80% isopropanol. Extractions performed with 40% isopropanol decreased the amount of storage proteins and revealed 107 low abundant proteins when using the combined approaches of two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and Mass Spectrometry (MS). The separation of proteins was achieved by iso-electric focusing (IEF) and 2D-PAGE. The proteins were analyzed with MS techniques to provide amino acid sequence. The proteins were identified by comparing their amino acid sequences with those in different databases including NCBI-non redundant, UniprotKB and MSDB databases. In this investigation, previously published results on low abundant soybean seed proteins were used to create an online database (SoyProLow) to provide a data repository that can be used as a reference to identify and characterize low abundance proteins. This database is freely accessible to individuals using similar techniques and can be for the subsequent genetic manipulation to produce value added soybean traits. An intuitive user interface based on dynamic HTML enables users to browse the network and the profiles of the low abundant proteins. Availability http://bioinformatics.towson.edu/Soybean_low_abundance_proteins_2D_Gel_DB/Gel1.aspx PMID:25352730

  10. A protein relational database and protein family knowledge bases to facilitate structure-based design analyses.

    PubMed

    Mobilio, Dominick; Walker, Gary; Brooijmans, Natasja; Nilakantan, Ramaswamy; Denny, R Aldrin; Dejoannis, Jason; Feyfant, Eric; Kowticwar, Rupesh K; Mankala, Jyoti; Palli, Satish; Punyamantula, Sairam; Tatipally, Maneesh; John, Reji K; Humblet, Christine

    2010-08-01

    The Protein Data Bank is the most comprehensive source of experimental macromolecular structures. It can, however, be difficult at times to locate relevant structures with the Protein Data Bank search interface. This is particularly true when searching for complexes containing specific interactions between protein and ligand atoms. Moreover, searching within a family of proteins can be tedious. For example, one cannot search for some conserved residue as residue numbers vary across structures. We describe herein three databases, Protein Relational Database, Kinase Knowledge Base, and Matrix Metalloproteinase Knowledge Base, containing protein structures from the Protein Data Bank. In Protein Relational Database, atom-atom distances between protein and ligand have been precalculated allowing for millisecond retrieval based on atom identity and distance constraints. Ring centroids, centroid-centroid and centroid-atom distances and angles have also been included permitting queries for pi-stacking interactions and other structural motifs involving rings. Other geometric features can be searched through the inclusion of residue pair and triplet distances. In Kinase Knowledge Base and Matrix Metalloproteinase Knowledge Base, the catalytic domains have been aligned into common residue numbering schemes. Thus, by searching across Protein Relational Database and Kinase Knowledge Base, one can easily retrieve structures wherein, for example, a ligand of interest is making contact with the gatekeeper residue.

  11. Completion of autobuilt protein models using a database of protein fragments

    SciTech Connect

    Cowtan, Kevin

    2012-04-01

    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.

  12. NSort/DB: an intranuclear compartment protein database.

    PubMed

    Willadsen, Kai; Mohamad, Nurul; Bodén, Mikael

    2012-08-01

    Distinct substructures within the nucleus are associated with a wide variety of important nuclear processes. Structures such as chromatin and nuclear pores have specific roles, while others such as Cajal bodies are more functionally varied. Understanding the roles of these membraneless intra-nuclear compartments requires extensive data sets covering nuclear and compartment-associated proteins. NSort/DB is a database providing access to intra- or sub-nuclear compartment associations for the mouse nuclear proteome. Based on resources ranging from large-scale curated data sets to detailed experiments, this data set provides a high-quality set of annotations of non-exclusive association of nuclear proteins with structures such as promyelocytic leukaemia bodies and chromatin. The database is searchable by protein identifier or compartment, and has a documented web service API. The search interface, web service and data download are all freely available online at http://www.nsort.org/db/. Availability of this data set will enable systematic analyses of the protein complements of nuclear compartments, improving our understanding of the diverse functional repertoire of these structures.

  13. SynProt: A Database for Proteins of Detergent-Resistant Synaptic Protein Preparations

    PubMed Central

    Pielot, Rainer; Smalla, Karl-Heinz; Müller, Anke; Landgraf, Peter; Lehmann, Anne-Christin; Eisenschmidt, Elke; Haus, Utz-Uwe; Weismantel, Robert; Gundelfinger, Eckart D.; Dieterich, Daniela C.

    2012-01-01

    Chemical synapses are highly specialized cell–cell contacts for communication between neurons in the CNS characterized by complex and dynamic protein networks at both synaptic membranes. The cytomatrix at the active zone (CAZ) organizes the apparatus for the regulated release of transmitters from the presynapse. At the postsynaptic side, the postsynaptic density constitutes the machinery for detection, integration, and transduction of the transmitter signal. Both pre- and postsynaptic protein networks represent the molecular substrates for synaptic plasticity. Their function can be altered both by regulating their composition and by post-translational modification of their components. For a comprehensive understanding of synaptic networks the entire ensemble of synaptic proteins has to be considered. To support this, we established a comprehensive database for synaptic junction proteins (SynProt database) primarily based on proteomics data obtained from biochemical preparations of detergent-resistant synaptic junctions. The database currently contains 2,788 non-redundant entries of rat, mouse, and some human proteins, which mainly have been manually extracted from 12 proteomic studies and annotated for synaptic subcellular localization. Each dataset is completed with manually added information including protein classifiers as well as automatically retrieved and updated information from public databases (UniProt and PubMed). We intend that the database will be used to support modeling of synaptic protein networks and rational experimental design. PMID:22737123

  14. Cytoskeletal and cellular adhesion proteins in zebrafish (Danio rerio) myogenesis.

    PubMed

    Costa, M L; Escaleira, R; Manasfi, M; de Souza, L F; Mermelstein, C S

    2003-08-01

    The current myogenesis and myofibrillogenesis model has been based mostly on in vitro cell culture studies, and, to a lesser extent, on in situ studies in avian and mammalian embryos. While the more isolated artificial conditions of cells in culture permitted careful structural analysis, the actual in situ cellular structures have not been described in detail because the embryos are more difficult to section and manipulate. To overcome these difficulties, we used the optically clear and easy to handle embryos of the zebrafish Danio rerio. We monitored the expression of cytoskeletal and cell-adhesion proteins (actin, myosin, desmin, alpha-actinin, troponin, titin, vimentin and vinculin) using immunofluorescence microscopy and video-enhanced, background-subtracted, differential interference contrast of 24- to 48-h zebrafish embryos. In the mature myotome, the mononucleated myoblasts displayed periodic striations for all sarcomeric proteins tested. The changes in desmin distribution from aggregates to perinuclear and striated forms, although following the same sequence, occurred much faster than in other models. All desmin-positive cells were also positive for myofibrillar proteins and striated, in contrast to that which occurs in cell cultures. Vimentin appeared to be striated in mature cells, while it is developmentally down-regulated in vitro. The whole connective tissue septum between the somites was positive for adhesion proteins such as vinculin, instead of the isolated adhesion plaques observed in cell cultures. The differences in the myogenesis of zebrafish in situ and in cell culture in vitro suggest that some of the previously observed structures and protein distributions in cultures could be methodological artifacts.

  15. Separation and properties of cellular and scrapie prion proteins.

    PubMed Central

    Meyer, R K; McKinley, M P; Bowman, K A; Braunfeld, M B; Barry, R A; Prusiner, S B

    1986-01-01

    Purified preparations of scrapie prions contain a sialoglycoprotein of Mr 27,000-30,000, designated PrP 27-30, which is derived from the scrapie prion protein [Mr, 33,000-35,000 (PrP 33-35Sc)] by limited proteolysis. Under these same conditions of proteolysis, a cellular protein of the same size (PrP 33-35C) is completely degraded. Subcellular fractionation of hamster brain showed that both PrP 33-35Sc and PrP 33-35C were found only in membrane fractions. NaCl, EDTA, and osmotic shock failed to release the prion proteins from microsomal membranes. Electron microscopy of these microsomal fractions showed membrane vesicles but not prion amyloid rods. Detergent treatment of scrapie-infected membranes solubilized PrP 33-35C, while PrP 33-35Sc aggregated into amyloid rods; the concentration of PrP 33-35C was similar to that recovered from analogous fractions prepared from uninfected control brains. The apparent amphipathic character of the PrP 33-35Sc may explain the association of scrapie infectivity with both membranes and amyloid filaments. Images PMID:3085093

  16. Cellular FLICE-inhibitory protein: an attractive therapeutic target?

    PubMed Central

    Micheau, Olivier

    2003-01-01

    Cellular FLIP, also known as FLICE-inhibitory protein, has been identified as an inhibitor of apoptosis triggered by engagement of Death Receptor (DR) such as Fas or TRAIL. c-FLIP is recruited to DR signalling complexes, where it prevents caspase activation. Animal models indicated that c-FLIP plays an important role in T-cell proliferation and heart development. Abnormal c-FLIP expression has been identified in various diseases such as multiple sclerosis, Alzheimer’s disease, diabetes mellitus, rheumatoid arthritis and various cancers. The present review focuses on recent insights in c-FLIP dysregulation associated with human diseases, and addresses the possibilities of using c-FLIP as a therapeutic target. PMID:12885274

  17. The Cellular Prion Protein: A Player in Immunological Quiescence

    PubMed Central

    Bakkebø, Maren K.; Mouillet-Richard, Sophie; Espenes, Arild; Goldmann, Wilfred; Tatzelt, Jörg; Tranulis, Michael A.

    2015-01-01

    Despite intensive studies since the 1990s, the physiological role of the cellular prion protein (PrPC) remains elusive. Here, we present a novel concept suggesting that PrPC contributes to immunological quiescence in addition to cell protection. PrPC is highly expressed in diverse organs that by multiple means are particularly protected from inflammation, such as the brain, eye, placenta, pregnant uterus, and testes, while at the same time it is expressed in most cells of the lymphoreticular system. In this paradigm, PrPC serves two principal roles: to modulate the inflammatory potential of immune cells and to protect vulnerable parenchymal cells against noxious insults generated through inflammation. Here, we review studies of PrPC physiology in view of this concept. PMID:26388873

  18. Creation of a federated database of blood proteins: a powerful new tool for finding and characterizing biomarkers in serum.

    PubMed

    Marshall, John; Bowden, Peter; Schmit, Jean Claude; Betsou, Fay

    2014-01-29

    Protein biomarkers offer major benefits for diagnosis and monitoring of disease processes. Recent advances in protein mass spectrometry make it feasible to use this very sensitive technology to detect and quantify proteins in blood. To explore the potential of blood biomarkers, we conducted a thorough review to evaluate the reliability of data in the literature and to determine the spectrum of proteins reported to exist in blood with a goal of creating a Federated Database of Blood Proteins (FDBP). A unique feature of our approach is the use of a SQL database for all of the peptide data; the power of the SQL database combined with standard informatic algorithms such as BLAST and the statistical analysis system (SAS) allowed the rapid annotation and analysis of the database without the need to create special programs to manage the data. Our mathematical analysis and review shows that in addition to the usual secreted proteins found in blood, there are many reports of intracellular proteins and good agreement on transcription factors, DNA remodelling factors in addition to cellular receptors and their signal transduction enzymes. Overall, we have catalogued about 12,130 proteins identified by at least one unique peptide, and of these 3858 have 3 or more peptide correlations. The FDBP with annotations should facilitate testing blood for specific disease biomarkers.

  19. The National Center for Biotechnology Information's Protein Clusters Database.

    PubMed

    Klimke, William; Agarwala, Richa; Badretdin, Azat; Chetvernin, Slava; Ciufo, Stacy; Fedorov, Boris; Kiryutin, Boris; O'Neill, Kathleen; Resch, Wolfgang; Resenchuk, Sergei; Schafer, Susan; Tolstoy, Igor; Tatusova, Tatiana

    2009-01-01

    Rapid increases in DNA sequencing capabilities have led to a vast increase in the data generated from prokaryotic genomic studies, which has been a boon to scientists studying micro-organism evolution and to those who wish to understand the biological underpinnings of microbial systems. The NCBI Protein Clusters Database (ProtClustDB) has been created to efficiently maintain and keep the deluge of data up to date. ProtClustDB contains both curated and uncurated clusters of proteins grouped by sequence similarity. The May 2008 release contains a total of 285 386 clusters derived from over 1.7 million proteins encoded by 3806 nt sequences from the RefSeq collection of complete chromosomes and plasmids from four major groups: prokaryotes, bacteriophages and the mitochondrial and chloroplast organelles. There are 7180 clusters containing 376 513 proteins with curated gene and protein functional annotation. PubMed identifiers and external cross references are collected for all clusters and provide additional information resources. A suite of web tools is available to explore more detailed information, such as multiple alignments, phylogenetic trees and genomic neighborhoods. ProtClustDB provides an efficient method to aggregate gene and protein annotation for researchers and is available at http://www.ncbi.nlm.nih.gov/sites/entrez?db=proteinclusters.

  20. A pipeline for determining protein-protein interactions and proximities in the cellular milieu.

    PubMed

    Subbotin, Roman I; Chait, Brian T

    2014-11-01

    It remains extraordinarily challenging to elucidate endogenous protein-protein interactions and proximities within the cellular milieu. The dynamic nature and the large range of affinities of these interactions augment the difficulty of this undertaking. Among the most useful tools for extracting such information are those based on affinity capture of target bait proteins in combination with mass spectrometric readout of the co-isolated species. Although highly enabling, the utility of affinity-based methods is generally limited by difficulties in distinguishing specific from nonspecific interactors, preserving and isolating all unique interactions including those that are weak, transient, or rapidly exchanging, and differentiating proximal interactions from those that are more distal. Here, we have devised and optimized a set of methods to address these challenges. The resulting pipeline involves flash-freezing cells in liquid nitrogen to preserve the cellular environment at the moment of freezing; cryomilling to fracture the frozen cells into intact micron chunks to allow for rapid access of a chemical reagent and to stabilize the intact endogenous subcellular assemblies and interactors upon thawing; and utilizing the high reactivity of glutaraldehyde to achieve sufficiently rapid stabilization at low temperatures to preserve native cellular interactions. In the course of this work, we determined that relatively low molar ratios of glutaraldehyde to reactive amines within the cellular milieu were sufficient to preserve even labile and transient interactions. This mild treatment enables efficient and rapid affinity capture of the protein assemblies of interest under nondenaturing conditions, followed by bottom-up MS to identify and quantify the protein constituents. For convenience, we have termed this approach Stabilized Affinity Capture Mass Spectrometry. Here, we demonstrate that Stabilized Affinity Capture Mass Spectrometry allows us to stabilize and elucidate

  1. Biochemical properties and cellular localisation of STIM proteins.

    PubMed

    Dziadek, Marie A; Johnstone, Lorna S

    2007-08-01

    Human and murine STIM1 were originally discovered as candidate growth regulators in tumours and in the bone marrow stroma, and the structurally related vertebrate family members, STIM2 and the Drosophila homologue D-Stim, were subsequently identified. STIM proteins are ubiquitously expressed type I single-pass transmembrane proteins which have a unique combination of structural motifs within their polypeptide sequences. The extracellular regions contain an N-terminal unpaired EF-hand Ca(2+) binding motif adjacent to an unconventional glycosylated SAM domain, while the cytoplasmic regions contain alpha-helical coiled-coil domains within a region having homology to ERM domains adjacent to the transmembrane region, and phosphorylated proline-rich domains near the C-terminus. STIM1, STIM2 and D-Stim diverge significantly only in their structure C-terminal to the coiled-coil/ERM domains. The STIM structural domains were predicted to function in Ca(2+) binding as well as in mediating interactions between STIM proteins and other proteins, and homotypic STIM1-STIM1 and heterotypic STIM1-STIM2 interactions were demonstrated biochemically. However, the functional significance of the cellular localisation of STIM1 and its domain structure only became evident after recent breakthrough research identified STIM1 as a key regulator of store-operated calcium (SOC) entry into cells. It is now clear that STIM1 is both a sensor of Ca(2+) depletion in the endoplasmic reticulum (ER) lumen and an activator of Orai1-containing SOC channels in the plasma membrane. On the basis of recent functional studies a model can be proposed to explain how the biochemical properties of STIM1 contribute to its precise membrane localisation and its function in regulating SOC entry. PMID:17382385

  2. The cellular prion protein and its role in Alzheimer disease

    PubMed Central

    Irujo, A; Cuadrado-Tejedor, M; Paternain, B; Moleres, FJ; Ferrer, V

    2009-01-01

    The cellular prion protein (PrPC) is a membrane-bound glycoprotein especially abundant in the central nervous system (CNS). The scrapie prion protein (PrPSc, also termed prions) is responsible of transmissible spongiform encephalopathies (TSE), a group of neurodegenerative diseases which affect humans and other mammal species, although the presence of PrPC is needed for the establishment and further evolution of prions. The present work compares the expression and localization of PrPC between healthy human brains and those suffering from Alzheimer disease (AD). In both situations we have observed a rostrocaudal decrease in the amount of PrPC within the CNS, both by immunoblotting and immunohistochemistry techniques. PrPC is higher expressed in our control brains than in AD cases. There was a neuronal loss and astogliosis in our AD cases. There was a tendency of a lesser expression of PrPC in AD cases than in healthy ones. And in AD cases, the intensity of the expression of the unglycosylated band is higher than the di- and monoglycosylated bands. With regards to amyloid plaques, those present in AD cases were positively labeled for PrPC, a result which is further supported by the presence of PrPC in the amyloid plaques of a transgenic line of mice mimicking AD. The work was done according to Helsinki Declaration of 1975, and approved by the Ethics Committee of the Faculty of Medicine of the University of Navarre. PMID:19556894

  3. PTMcode: a database of known and predicted functional associations between post-translational modifications in proteins.

    PubMed

    Minguez, Pablo; Letunic, Ivica; Parca, Luca; Bork, Peer

    2013-01-01

    Post-translational modifications (PTMs) are involved in the regulation and structural stabilization of eukaryotic proteins. The combination of individual PTM states is a key to modulate cellular functions as became evident in a few well-studied proteins. This combinatorial setting, dubbed the PTM code, has been proposed to be extended to whole proteomes in eukaryotes. Although we are still far from deciphering such a complex language, thousands of protein PTM sites are being mapped by high-throughput technologies, thus providing sufficient data for comparative analysis. PTMcode (http://ptmcode.embl.de) aims to compile known and predicted PTM associations to provide a framework that would enable hypothesis-driven experimental or computational analysis of various scales. In its first release, PTMcode provides PTM functional associations of 13 different PTM types within proteins in 8 eukaryotes. They are based on five evidence channels: a literature survey, residue co-evolution, structural proximity, PTMs at the same residue and location within PTM highly enriched protein regions (hotspots). PTMcode is presented as a protein-based searchable database with an interactive web interface providing the context of the co-regulation of nearly 75 000 residues in >10 000 proteins.

  4. Protein structure determination using a database of interatomic distance probabilities.

    PubMed Central

    Wall, M. E.; Subramaniam, S.; Phillips, G. N.

    1999-01-01

    The accelerated pace of genomic sequencing has increased the demand for structural models of gene products. Improved quantitative methods are needed to study the many systems (e.g., macromolecular assemblies) for which data are scarce. Here, we describe a new molecular dynamics method for protein structure determination and molecular modeling. An energy function, or database potential, is derived from distributions of interatomic distances obtained from a database of known structures. X-ray crystal structures are refined by molecular dynamics with the new energy function replacing the Van der Waals potential. Compared to standard methods, this method improved the atomic positions, interatomic distances, and side-chain dihedral angles of structures randomized to mimic the early stages of refinement. The greatest enhancement in side-chain placement was observed for groups that are characteristically buried. More accurate calculated model phases will follow from improved interatomic distances. Details usually seen only in high-resolution refinements were improved, as is shown by an R-factor analysis. The improvements were greatest when refinements were carried out using X-ray data truncated at 3.5 A. The database potential should therefore be a valuable tool for determining X-ray structures, especially when only low-resolution data are available. PMID:10631988

  5. RepeatsDB: a database of tandem repeat protein structures

    PubMed Central

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

    2014-01-01

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

  6. PAT proteins, an ancient family of lipid droplet proteins that regulate cellular lipid stores

    PubMed Central

    Bickel, Perry E.; Tansey, John T.; Welte, Michael A.

    2009-01-01

    Summary The PAT family of lipid droplet proteins includes 5 members in mammals: perilipin, adipose differentiation-related protein (ADRP), tail-interacting protein of 47 kiloDaltons (TIP47), S3-12, and OXPAT. Members of this family are also present in evolutionarily distant organisms, including insects, slime molds and fungi. All PAT proteins share sequence similarity and the ability to bind intracellular lipid droplets, either constitutively or in response to metabolic stimuli, such as increased lipid flux into or out of lipid droplets. Positioned at the lipid droplet surface, PAT proteins manage access of other proteins (lipases) to the lipid esters within the lipid droplet core and can interact with cellular machinery important for lipid droplet biogenesis. Genetic variations in the gene for the best characterized of the mammalian PAT proteins, perilipin, have been associated with metabolic phenotypes, including type 2 diabetes mellitus and obesity. In this review, we discuss how the PAT proteins regulate cellular lipid metabolism both in mammals and in model organisms. PMID:19375517

  7. PAT proteins, an ancient family of lipid droplet proteins that regulate cellular lipid stores.

    PubMed

    Bickel, Perry E; Tansey, John T; Welte, Michael A

    2009-06-01

    The PAT family of lipid droplet proteins includes 5 members in mammals: perilipin, adipose differentiation-related protein (ADRP), tail-interacting protein of 47 kDa (TIP47), S3-12, and OXPAT. Members of this family are also present in evolutionarily distant organisms, including insects, slime molds and fungi. All PAT proteins share sequence similarity and the ability to bind intracellular lipid droplets, either constitutively or in response to metabolic stimuli, such as increased lipid flux into or out of lipid droplets. Positioned at the lipid droplet surface, PAT proteins manage access of other proteins (lipases) to the lipid esters within the lipid droplet core and can interact with cellular machinery important for lipid droplet biogenesis. Genetic variations in the gene for the best-characterized of the mammalian PAT proteins, perilipin, have been associated with metabolic phenotypes, including type 2 diabetes mellitus and obesity. In this review, we discuss how the PAT proteins regulate cellular lipid metabolism both in mammals and in model organisms. PMID:19375517

  8. Predict drug-protein interaction in cellular networking.

    PubMed

    Xiao, Xuan; Min, Jian-Liang; Wang, Pu; Chou, Kuo-Chen

    2013-01-01

    Involved with many diseases such as cancer, diabetes, neurodegenerative, inflammatory and respiratory disorders, GPCRs (G-protein-coupled receptors) are the most frequent targets for drug development: over 50% of all prescription drugs currently on the market are actually acting by targeting GPCRs directly or indirectly. Found in every living thing and nearly all cells, ion channels play crucial roles for many vital functions in life, such as heartbeat, sensory transduction, and central nervous system response. Their dysfunction may have significant impact to human health, and hence ion channels are deemed as "the next GPCRs". To develop GPCR-targeting or ion-channel-targeting drugs, the first important step is to identify the interactions between potential drug compounds with the two kinds of protein receptors in the cellular networking. In this minireview, we are to introduce two predictors. One is called iGPCR-Drug accessible at http://www.jci-bioinfo.cn/iGPCR-Drug/; the other called iCDI-PseFpt at http://www.jci-bioinfo.cn/iCDI-PseFpt. The former is for identifying the interactions of drug compounds with GPCRs; while the latter for that with ion channels. In both predictors, the drug compound was formulated by the two-dimensional molecular fingerprint, and the protein receptor by the pseudo amino acid composition generated with the grey model theory, while the operation engine was the fuzzy K-nearest neighbor algorithm. For the convenience of most experimental pharmaceutical and medical scientists, a step-bystep guide is provided on how to use each of the two web-servers to get the desired results without the need to follow the complicated mathematics involved originally for their establishment. PMID:23889048

  9. Predict drug-protein interaction in cellular networking.

    PubMed

    Xiao, Xuan; Min, Jian-Liang; Wang, Pu; Chou, Kuo-Chen

    2013-01-01

    Involved with many diseases such as cancer, diabetes, neurodegenerative, inflammatory and respiratory disorders, GPCRs (G-protein-coupled receptors) are the most frequent targets for drug development: over 50% of all prescription drugs currently on the market are actually acting by targeting GPCRs directly or indirectly. Found in every living thing and nearly all cells, ion channels play crucial roles for many vital functions in life, such as heartbeat, sensory transduction, and central nervous system response. Their dysfunction may have significant impact to human health, and hence ion channels are deemed as "the next GPCRs". To develop GPCR-targeting or ion-channel-targeting drugs, the first important step is to identify the interactions between potential drug compounds with the two kinds of protein receptors in the cellular networking. In this minireview, we are to introduce two predictors. One is called iGPCR-Drug accessible at http://www.jci-bioinfo.cn/iGPCR-Drug/; the other called iCDI-PseFpt at http://www.jci-bioinfo.cn/iCDI-PseFpt. The former is for identifying the interactions of drug compounds with GPCRs; while the latter for that with ion channels. In both predictors, the drug compound was formulated by the two-dimensional molecular fingerprint, and the protein receptor by the pseudo amino acid composition generated with the grey model theory, while the operation engine was the fuzzy K-nearest neighbor algorithm. For the convenience of most experimental pharmaceutical and medical scientists, a step-bystep guide is provided on how to use each of the two web-servers to get the desired results without the need to follow the complicated mathematics involved originally for their establishment.

  10. Cellular prion protein and NMDA receptor modulation: protecting against excitotoxicity

    PubMed Central

    Black, Stefanie A. G.; Stys, Peter K.; Zamponi, Gerald W.; Tsutsui, Shigeki

    2014-01-01

    Although it is well established that misfolding of the cellular prion protein (PrPC) into the β-sheet-rich, aggregated scrapie conformation (PrPSc) causes a variety of transmissible spongiform encephalopathies (TSEs), the physiological roles of PrPC are still incompletely understood. There is accumulating evidence describing the roles of PrPC in neurodegeneration and neuroinflammation. Recently, we identified a functional regulation of NMDA receptors by PrPC that involves formation of a physical protein complex between these proteins. Excessive NMDA receptor activity during conditions such as ischemia mediates enhanced Ca2+ entry into cells and contributes to excitotoxic neuronal death. In addition, NMDA receptors and/or PrPC play critical roles in neuroinflammation and glial cell toxicity. Inhibition of NMDA receptor activity protects against PrPSc-induced neuronal death. Moreover, in mice lacking PrPC, infarct size is increased after focal cerebral ischemia, and absence of PrPC increases susceptibility of neurons to NMDA receptor-dependent death. Recently, PrPC was found to be a receptor for oligomeric beta-amyloid (Aβ) peptides, suggesting a role for PrPC in Alzheimer's disease (AD). Our recent findings suggest that Aβ peptides enhance NMDA receptor current by perturbing the normal copper- and PrPC-dependent regulation of these receptors. Here, we review evidence highlighting a role for PrPC in preventing NMDA receptor-mediated excitotoxicity and inflammation. There is a need for more detailed molecular characterization of PrPC-mediated regulation of NMDA receptors, such as determining which NMDA receptor subunits mediate pathogenic effects upon loss of PrPC-mediated regulation and identifying PrPC binding site(s) on the receptor. This knowledge will allow development of novel therapeutic interventions for not only TSEs, but also for AD and other neurodegenerative disorders involving dysfunction of PrPC. PMID:25364752

  11. Dynamic circadian protein-protein interaction networks predict temporal organization of cellular functions.

    PubMed

    Wallach, Thomas; Schellenberg, Katja; Maier, Bert; Kalathur, Ravi Kiran Reddy; Porras, Pablo; Wanker, Erich E; Futschik, Matthias E; Kramer, Achim

    2013-03-01

    Essentially all biological processes depend on protein-protein interactions (PPIs). Timing of such interactions is crucial for regulatory function. Although circadian (~24-hour) clocks constitute fundamental cellular timing mechanisms regulating important physiological processes, PPI dynamics on this timescale are largely unknown. Here, we identified 109 novel PPIs among circadian clock proteins via a yeast-two-hybrid approach. Among them, the interaction of protein phosphatase 1 and CLOCK/BMAL1 was found to result in BMAL1 destabilization. We constructed a dynamic circadian PPI network predicting the PPI timing using circadian expression data. Systematic circadian phenotyping (RNAi and overexpression) suggests a crucial role for components involved in dynamic interactions. Systems analysis of a global dynamic network in liver revealed that interacting proteins are expressed at similar times likely to restrict regulatory interactions to specific phases. Moreover, we predict that circadian PPIs dynamically connect many important cellular processes (signal transduction, cell cycle, etc.) contributing to temporal organization of cellular physiology in an unprecedented manner. PMID:23555304

  12. Reduced native state stability in crowded cellular environment due to protein-protein interactions.

    PubMed

    Harada, Ryuhei; Tochio, Naoya; Kigawa, Takanori; Sugita, Yuji; Feig, Michael

    2013-03-01

    The effect of cellular crowding environments on protein structure and stability is a key issue in molecular and cellular biology. The classical view of crowding emphasizes the volume exclusion effect that generally favors compact, native states. Here, results from molecular dynamics simulations and NMR experiments show that protein crowders may destabilize native states via protein-protein interactions. In the model system considered here, mixtures of villin head piece and protein G at high concentrations, villin structures become increasingly destabilized upon increasing crowder concentrations. The denatured states observed in the simulation involve partial unfolding as well as more subtle conformational shifts. The unfolded states remain overall compact and only partially overlap with unfolded ensembles at high temperature and in the presence of urea. NMR measurements on the same systems confirm structural changes upon crowding based on changes of chemical shifts relative to dilute conditions. An analysis of protein-protein interactions and energetic aspects suggests the importance of enthalpic and solvation contributions to the crowding free energies that challenge an entropic-centered view of crowding effects.

  13. Network motifs in integrated cellular networks of transcription-regulation and protein-protein interaction

    NASA Astrophysics Data System (ADS)

    Yeger-Lotem, Esti; Sattath, Shmuel; Kashtan, Nadav; Itzkovitz, Shalev; Milo, Ron; Pinter, Ron Y.; Alon, Uri; Margalit, Hanah

    2004-04-01

    Genes and proteins generate molecular circuitry that enables the cell to process information and respond to stimuli. A major challenge is to identify characteristic patterns in this network of interactions that may shed light on basic cellular mechanisms. Previous studies have analyzed aspects of this network, concentrating on either transcription-regulation or protein-protein interactions. Here we search for composite network motifs: characteristic network patterns consisting of both transcription-regulation and protein-protein interactions that recur significantly more often than in random networks. To this end we developed algorithms for detecting motifs in networks with two or more types of interactions and applied them to an integrated data set of protein-protein interactions and transcription regulation in Saccharomyces cerevisiae. We found a two-protein mixed-feedback loop motif, five types of three-protein motifs exhibiting coregulation and complex formation, and many motifs involving four proteins. Virtually all four-protein motifs consisted of combinations of smaller motifs. This study presents a basic framework for detecting the building blocks of networks with multiple types of interactions.

  14. Prion search and cellular prion protein expression in stranded dolphins.

    PubMed

    Di Guardo, G; Cocumelli, C; Meoli, R; Barbaro, K; Terracciano, G; Di Francesco, C E; Mazzariol, S; Eleni, C

    2012-01-01

    The recent description of a prion disease (PD) case in a free-ranging bottlenose dolphin (Tursiops truncatus) prompted us to carry out an extensive search for the disease-associated isoform (PrPSc) of the cellular prion protein (PrPC) in the brain and in a range of lymphoid tissues from 23 striped dolphins (Stenella coeruleoalba), 5 bottlenose dolphins and 2 Risso s dolphins (Grampus griseus) found stranded between 2007 and 2012 along the Italian coastline. Three striped dolphins and one bottlenose dolphin showed microscopic lesions of encephalitis, with no evidence of spongiform brain lesions being detected in any of the 30 free-ranging cetaceans investigated herein. Nevertheless, we could still observe a prominent PrPC immunoreactivity in the brain as well as in lymphoid tissues from these dolphins. Although immunohistochemical and Western blot investigations yielded negative results for PrPSc deposition in all tissues from the dolphins under study, the reported occurrence of a spontaneous PD case in a wild dolphin is an intriguing issue and a matter of concern for both prion biology and intra/inter-species transmissibility, as well as for cetacean conservation medicine. PMID:23034277

  15. GPCRdb: the G protein-coupled receptor database - an introduction.

    PubMed

    Munk, C; Isberg, V; Mordalski, S; Harpsøe, K; Rataj, K; Hauser, A S; Kolb, P; Bojarski, A J; Vriend, G; Gloriam, D E

    2016-07-01

    GPCRs make up the largest family of human membrane proteins and of drug targets. Recent advances in GPCR pharmacology and crystallography have shed new light on signal transduction, allosteric modulation and biased signalling, translating into new mechanisms and principles for drug design. The GPCR database, GPCRdb, has served the community for over 20 years and has recently been extended to include a more multidisciplinary audience. This review is intended to introduce new users to the services in GPCRdb, which meets three overall purposes: firstly, to provide reference data in an integrated, annotated and structured fashion, with a focus on sequences, structures, single-point mutations and ligand interactions. Secondly, to equip the community with a suite of web tools for swift analysis of structures, sequence similarities, receptor relationships, and ligand target profiles. Thirdly, to facilitate dissemination through interactive diagrams of, for example, receptor residue topologies, phylogenetic relationships and crystal structure statistics. Herein, these services are described for the first time; visitors and guides are provided with good practices for their utilization. Finally, we describe complementary databases cross-referenced by GPCRdb and web servers with corresponding functionality. PMID:27155948

  16. GPCRdb: the G protein-coupled receptor database - an introduction.

    PubMed

    Munk, C; Isberg, V; Mordalski, S; Harpsøe, K; Rataj, K; Hauser, A S; Kolb, P; Bojarski, A J; Vriend, G; Gloriam, D E

    2016-07-01

    GPCRs make up the largest family of human membrane proteins and of drug targets. Recent advances in GPCR pharmacology and crystallography have shed new light on signal transduction, allosteric modulation and biased signalling, translating into new mechanisms and principles for drug design. The GPCR database, GPCRdb, has served the community for over 20 years and has recently been extended to include a more multidisciplinary audience. This review is intended to introduce new users to the services in GPCRdb, which meets three overall purposes: firstly, to provide reference data in an integrated, annotated and structured fashion, with a focus on sequences, structures, single-point mutations and ligand interactions. Secondly, to equip the community with a suite of web tools for swift analysis of structures, sequence similarities, receptor relationships, and ligand target profiles. Thirdly, to facilitate dissemination through interactive diagrams of, for example, receptor residue topologies, phylogenetic relationships and crystal structure statistics. Herein, these services are described for the first time; visitors and guides are provided with good practices for their utilization. Finally, we describe complementary databases cross-referenced by GPCRdb and web servers with corresponding functionality.

  17. Protein source and choice of anticoagulant decisively affect nanoparticle protein corona and cellular uptake

    NASA Astrophysics Data System (ADS)

    Schöttler, S.; Klein, Katja; Landfester, K.; Mailänder, V.

    2016-03-01

    Protein adsorption on nanoparticles has been a focus of the field of nanocarrier research in the past few years and more and more papers are dealing with increasingly detailed lists of proteins adsorbed to a plethora of nanocarriers. While there is an urgent need to understand the influence of this protein corona on nanocarriers' interactions with cells the strong impact of the protein source on corona formation and the consequence for interaction with different cell types are factors that are regularly neglected, but should be taken into account for a meaningful analysis. In this study, the importance of the choice of protein source used for in vitro protein corona analysis is concisely investigated. Major and decisive differences in cellular uptake of a polystyrene nanoparticle incubated in fetal bovine serum, human serum, human citrate and heparin plasma are reported. Furthermore, the protein compositions are determined for coronas formed in the respective incubation media. A strong influence of heparin, which is used as an anticoagulant for plasma generation, on cell interaction is demonstrated. While heparin enhances the uptake into macrophages, it prevents internalization into HeLa cells. Taken together we can give the recommendation that human plasma anticoagulated with citrate seems to give the most relevant results for in vitro studies of nanoparticle uptake.Protein adsorption on nanoparticles has been a focus of the field of nanocarrier research in the past few years and more and more papers are dealing with increasingly detailed lists of proteins adsorbed to a plethora of nanocarriers. While there is an urgent need to understand the influence of this protein corona on nanocarriers' interactions with cells the strong impact of the protein source on corona formation and the consequence for interaction with different cell types are factors that are regularly neglected, but should be taken into account for a meaningful analysis. In this study, the importance

  18. Ethanol Cellular Defense Induce Unfolded Protein Response in Yeast

    PubMed Central

    Pérez-Torrado, Roberto

    2016-01-01

    Ethanol is a valuable industrial product and a common metabolite used by many cell types. However, this molecule produces high levels of cytotoxicity affecting cellular performance at several levels. In the presence of ethanol, cells must adjust some of their components, such as the membrane lipids to maintain homeostasis. In the case of microorganism as Saccharomyces cerevisiae, ethanol is one of the principal products of their metabolism and is the main stress factor during fermentation. Although, many efforts have been made, mechanisms of ethanol tolerance are not fully understood and very little evidence is available to date for specific signaling by ethanol in the cell. This work studied two S. cerevisiae strains, CECT10094, and Temohaya-MI26, isolated from flor wine and agave fermentation (a traditional fermentation from Mexico) respectively, which differ in ethanol tolerance, in order to understand the molecular mechanisms underlying the ethanol stress response and the reasons for different ethanol tolerance. The transcriptome was analyzed after ethanol stress and, among others, an increased activation of genes related with the unfolded protein response (UPR) and its transcription factor, Hac1p, was observed in the tolerant strain CECT10094. We observed that this strain also resist more UPR agents than Temohaya-MI26 and the UPR-ethanol stress correlation was corroborated observing growth of 15 more strains and discarding UPR correlation with other stresses as thermal or oxidative stress. Furthermore, higher activation of UPR pathway in the tolerant strain CECT10094 was observed using a UPR mCherry reporter. Finally, we observed UPR activation in response to ethanol stress in other S. cerevisiae ethanol tolerant strains as the wine strains T73 and EC1118. This work demonstrates that the UPR pathway is activated under ethanol stress occurring in a standard fermentation and links this response to an enhanced ethanol tolerance. Thus, our data suggest that there

  19. Ethanol Cellular Defense Induce Unfolded Protein Response in Yeast.

    PubMed

    Navarro-Tapia, Elisabet; Nana, Rebeca K; Querol, Amparo; Pérez-Torrado, Roberto

    2016-01-01

    Ethanol is a valuable industrial product and a common metabolite used by many cell types. However, this molecule produces high levels of cytotoxicity affecting cellular performance at several levels. In the presence of ethanol, cells must adjust some of their components, such as the membrane lipids to maintain homeostasis. In the case of microorganism as Saccharomyces cerevisiae, ethanol is one of the principal products of their metabolism and is the main stress factor during fermentation. Although, many efforts have been made, mechanisms of ethanol tolerance are not fully understood and very little evidence is available to date for specific signaling by ethanol in the cell. This work studied two S. cerevisiae strains, CECT10094, and Temohaya-MI26, isolated from flor wine and agave fermentation (a traditional fermentation from Mexico) respectively, which differ in ethanol tolerance, in order to understand the molecular mechanisms underlying the ethanol stress response and the reasons for different ethanol tolerance. The transcriptome was analyzed after ethanol stress and, among others, an increased activation of genes related with the unfolded protein response (UPR) and its transcription factor, Hac1p, was observed in the tolerant strain CECT10094. We observed that this strain also resist more UPR agents than Temohaya-MI26 and the UPR-ethanol stress correlation was corroborated observing growth of 15 more strains and discarding UPR correlation with other stresses as thermal or oxidative stress. Furthermore, higher activation of UPR pathway in the tolerant strain CECT10094 was observed using a UPR mCherry reporter. Finally, we observed UPR activation in response to ethanol stress in other S. cerevisiae ethanol tolerant strains as the wine strains T73 and EC1118. This work demonstrates that the UPR pathway is activated under ethanol stress occurring in a standard fermentation and links this response to an enhanced ethanol tolerance. Thus, our data suggest that there

  20. Toxicological relationships between proteins obtained from protein target predictions of large toxicity databases

    SciTech Connect

    Nigsch, Florian; Mitchell, John B.O.

    2008-09-01

    The combination of models for protein target prediction with large databases containing toxicological information for individual molecules allows the derivation of 'toxiclogical' profiles, i.e., to what extent are molecules of known toxicity predicted to interact with a set of protein targets. To predict protein targets of drug-like and toxic molecules, we built a computational multiclass model using the Winnow algorithm based on a dataset of protein targets derived from the MDL Drug Data Report. A 15-fold Monte Carlo cross-validation using 50% of each class for training, and the remaining 50% for testing, provided an assessment of the accuracy of that model. We retained the 3 top-ranking predictions and found that in 82% of all cases the correct target was predicted within these three predictions. The first prediction was the correct one in almost 70% of cases. A model built on the whole protein target dataset was then used to predict the protein targets for 150 000 molecules from the MDL Toxicity Database. We analysed the frequency of the predictions across the panel of protein targets for experimentally determined toxicity classes of all molecules. This allowed us to identify clusters of proteins related by their toxicological profiles, as well as toxicities that are related. Literature-based evidence is provided for some specific clusters to show the relevance of the relationships identified.

  1. Cellular Binding of Anionic Nanoparticles is Inhibited by Serum Proteins Independent of Nanoparticle Composition.

    PubMed

    Fleischer, Candace C; Kumar, Umesh; Payne, Christine K

    2013-09-01

    Nanoparticles used in biological applications encounter a complex mixture of extracellular proteins. Adsorption of these proteins on the nanoparticle surface results in the formation of a "protein corona," which can dominate the interaction of the nanoparticle with the cellular environment. The goal of this research was to determine how nanoparticle composition and surface modification affect the cellular binding of protein-nanoparticle complexes. We examined the cellular binding of a collection of commonly used anionic nanoparticles: quantum dots, colloidal gold nanoparticles, and low-density lipoprotein particles, in the presence and absence of extracellular proteins. These experiments have the advantage of comparing different nanoparticles under identical conditions. Using a combination of fluorescence and dark field microscopy, flow cytometry, and spectroscopy, we find that cellular binding of these anionic nanoparticles is inhibited by serum proteins independent of nanoparticle composition or surface modification. We expect these results will aid in the design of nanoparticles for in vivo applications.

  2. iPPI-DB: an online database of modulators of protein-protein interactions.

    PubMed

    Labbé, Céline M; Kuenemann, Mélaine A; Zarzycka, Barbara; Vriend, Gert; Nicolaes, Gerry A F; Lagorce, David; Miteva, Maria A; Villoutreix, Bruno O; Sperandio, Olivier

    2016-01-01

    In order to boost the identification of low-molecular-weight drugs on protein-protein interactions (PPI), it is essential to properly collect and annotate experimental data about successful examples. This provides the scientific community with the necessary information to derive trends about privileged physicochemical properties and chemotypes that maximize the likelihood of promoting a given chemical probe to the most advanced stages of development. To this end we have developed iPPI-DB (freely accessible at http://www.ippidb.cdithem.fr), a database that contains the structure, some physicochemical characteristics, the pharmacological data and the profile of the PPI targets of several hundreds modulators of protein-protein interactions. iPPI-DB is accessible through a web application and can be queried according to two general approaches: using physicochemical/pharmacological criteria; or by chemical similarity to a user-defined structure input. In both cases the results are displayed as a sortable and exportable datasheet with links to external databases such as Uniprot, PubMed. Furthermore each compound in the table has a link to an individual ID card that contains its physicochemical and pharmacological profile derived from iPPI-DB data. This includes information about its binding data, ligand and lipophilic efficiencies, location in the PPI chemical space, and importantly similarity with known drugs, and links to external databases like PubChem, and ChEMBL.

  3. iPPI-DB: an online database of modulators of protein-protein interactions.

    PubMed

    Labbé, Céline M; Kuenemann, Mélaine A; Zarzycka, Barbara; Vriend, Gert; Nicolaes, Gerry A F; Lagorce, David; Miteva, Maria A; Villoutreix, Bruno O; Sperandio, Olivier

    2016-01-01

    In order to boost the identification of low-molecular-weight drugs on protein-protein interactions (PPI), it is essential to properly collect and annotate experimental data about successful examples. This provides the scientific community with the necessary information to derive trends about privileged physicochemical properties and chemotypes that maximize the likelihood of promoting a given chemical probe to the most advanced stages of development. To this end we have developed iPPI-DB (freely accessible at http://www.ippidb.cdithem.fr), a database that contains the structure, some physicochemical characteristics, the pharmacological data and the profile of the PPI targets of several hundreds modulators of protein-protein interactions. iPPI-DB is accessible through a web application and can be queried according to two general approaches: using physicochemical/pharmacological criteria; or by chemical similarity to a user-defined structure input. In both cases the results are displayed as a sortable and exportable datasheet with links to external databases such as Uniprot, PubMed. Furthermore each compound in the table has a link to an individual ID card that contains its physicochemical and pharmacological profile derived from iPPI-DB data. This includes information about its binding data, ligand and lipophilic efficiencies, location in the PPI chemical space, and importantly similarity with known drugs, and links to external databases like PubChem, and ChEMBL. PMID:26432833

  4. xComb: a cross-linked peptide database approach to protein-protein interaction analysis

    PubMed Central

    Panchaud, Alexandre; Singh, Pragya; Shaffer, Scott A.; Goodlett, David R.

    2010-01-01

    We developed an informatic method to identify tandem mass spectra composed of chemically cross-linked peptides from those of linear peptides and to assign sequence to each of the two unique peptide sequences. For a given set of proteins the key software tool, xComb, combs through all theoretically feasible cross-linked peptides to create a database consisting of a subset of all combinations represented as peptide FASTA files. The xComb library of select theoretical cross-linked peptides may then be used as a database that is examined by a standard proteomic search engine to match tandem mass spectral datasets to identify cross-linked peptides. The database search may be conducted against as many as 50 proteins with a number of common proteomic search engines, e.g. Phenyx, Sequest, OMSSA, Mascot and X!Tandem. By searching against a peptide library of linearized, cross-linked peptides, rather than a linearized protein library, search times are decreased and the process is decoupled from any specific search engine. A further benefit of decoupling from the search engine is that protein cross-linking studies may be conducted with readily available informatics tools for which scoring routines already exist within the proteomic community. PMID:20302351

  5. Studying cellular processes and detecting disease with protein microarrays

    SciTech Connect

    Zangar, Richard C.; Varnum, Susan M.; Bollinger, Nikki

    2005-10-31

    Protein microarrays are a rapidly developing analytic tool with diverse applications in biomedical research. These applications include profiling of disease markers or autoimmune responses, understanding molecular pathways, protein modifications and protein activities. One factor that is driving this expanding usage is the wide variety of experimental formats that protein microarrays can take. In this review, we provide a short, conceptual overview of the different approaches for protein microarray. We then examine some of the most significant applications of these microarrays to date, with an emphasis on how global protein analyses can be used to facilitate biomedical research.

  6. Protein Targeting and Transport as a Necessary Consequence of Increased Cellular Complexity

    PubMed Central

    Sommer, Maik S.; Schleiff, Enrico

    2014-01-01

    With increasing intracellular complexity, a new cell-biological problem that is the allocation of cytoplasmically synthesized proteins to their final destinations within the cell emerged. A special challenge is thereby the translocation of proteins into or across cellular membranes. The underlying mechanisms are only in parts well understood, but it can be assumed that the course of cellular evolution had a deep impact on the design of the required molecular machines. In this article, we aim to summarize the current knowledge and concepts of the evolutionary development of protein trafficking as a necessary premise and consequence of increased cellular complexity. PMID:25085907

  7. EKPD: a hierarchical database of eukaryotic protein kinases and protein phosphatases

    PubMed Central

    Wang, Yongbo; Liu, Zexian; Cheng, Han; Gao, Tianshun; Pan, Zhicheng; Yang, Qing; Guo, Anyuan; Xue, Yu

    2014-01-01

    We present here EKPD (http://ekpd.biocuckoo.org), a hierarchical database of eukaryotic protein kinases (PKs) and protein phosphatases (PPs), the key molecules responsible for the reversible phosphorylation of proteins that are involved in almost all aspects of biological processes. As extensive experimental and computational efforts have been carried out to identify PKs and PPs, an integrative resource with detailed classification and annotation information would be of great value for both experimentalists and computational biologists. In this work, we first collected 1855 PKs and 347 PPs from the scientific literature and various public databases. Based on previously established rationales, we classified all of the known PKs and PPs into a hierarchical structure with three levels, i.e. group, family and individual PK/PP. There are 10 groups with 149 families for the PKs and 10 groups with 33 families for the PPs. We constructed 139 and 27 Hidden Markov Model profiles for PK and PP families, respectively. Then we systematically characterized ∼50 000 PKs and >10 000 PPs in eukaryotes. In addition, >500 PKs and >400 PPs were computationally identified by ortholog search. Finally, the online service of the EKPD database was implemented in PHP + MySQL + JavaScript. PMID:24214991

  8. Protein fragment bimolecular fluorescence complementation analyses for the in vivo study of protein-protein interactions and cellular protein complex localizations

    PubMed Central

    Waadt, Rainer; Schlücking, Kathrin; Schroeder, Julian I.; Kudla, Jörg

    2014-01-01

    Summary The analyses of protein-protein interactions is crucial for understanding cellular processes including signal transduction, protein trafficking and movement. Protein fragment complementation assays are based on the reconstitution of protein function when non-active protein fragments are brought together by interacting proteins that were genetically fused to these protein fragments. Bimolecular fluorescence complementation (BiFC) relies on the reconstitution of fluorescent proteins and enables both the analysis of protein-protein interactions and the visualization of protein complex formations in vivo. Transient expression of proteins is a convenient approach to study protein functions in planta or in other organisms, and minimizes the need for time-consuming generation of stably expressing transgenic organisms. Here we describe protocols for BiFC analyses in Nicotiana benthamiana and Arabidopsis thaliana leaves transiently transformed by Agrobacterium infiltration. Further we discuss different BiFC applications and provide examples for proper BiFC analyses in planta. PMID:24057390

  9. Mitochondria tether protein trash to rejuvenate cellular environments.

    PubMed

    Mogk, Axel; Bukau, Bernd

    2014-10-23

    Protein damage segregates asymmetrically in dividing yeast cells, rejuvenating daughters at the expense of mother cells. Zhou et al. now show that newly synthesized proteins are particularly prone to aggregation and describe a mechanism that tethers aggregated proteins to mitochondria. This association constrains aggregate mobility, effectively retaining and sorting toxic aggregates away from younger cells. PMID:25417098

  10. Mitochondria tether protein trash to rejuvenate cellular environments.

    PubMed

    Mogk, Axel; Bukau, Bernd

    2014-10-23

    Protein damage segregates asymmetrically in dividing yeast cells, rejuvenating daughters at the expense of mother cells. Zhou et al. now show that newly synthesized proteins are particularly prone to aggregation and describe a mechanism that tethers aggregated proteins to mitochondria. This association constrains aggregate mobility, effectively retaining and sorting toxic aggregates away from younger cells.

  11. Protein import into complex plastids: Cellular organization of higher complexity.

    PubMed

    Maier, Uwe G; Zauner, Stefan; Hempel, Franziska

    2015-01-01

    Many protists with high ecological and medical relevance harbor plastids surrounded by four membranes. Thus, nucleus-encoded proteins of these complex plastids have to traverse these barriers. Here we report on the identification of the protein translocators located in two of the plastid surrounding membranes and present recent findings on the mechanisms of protein import into the plastids of diatoms.

  12. Cellular or viral protein binding to a cytomegalovirus promoter transcription initiation site: effects on transcription.

    PubMed Central

    Macias, M P; Huang, L; Lashmit, P E; Stinski, M F

    1996-01-01

    We have previously shown that the IE2 protein of human cytomegalovirus (CMV) represses its own synthesis by binding to the major immediate-early promoter (M. P. Macias and M. F. Stinski, Proc. Natl. Acad. Sci. USA 90:707-711, 1993). The binding of a viral protein (IE2) and a cellular protein in the region of the transcription start site was investigated by site-specific mutational analysis and electrophoretic mobility shift assay. The viral protein and the cellular protein require different but adjacent core DNA sequence elements for binding. In situ chemical footprinting analysis of DNA-protein interactions with purified CMV IE2 protein or HeLa cell nuclear extracts demonstrated binding sites that overlap the transcription start site. The IE2 protein footprint was between bp -15 and +2, relative to the transcription start site, and the cellular protein was between bp -16 and +7. The ability of the unknown human cellular protein of approximately 150 kDa to bind the CMV major immediate-early promoter correlates with an increase in the level of transcription efficiency. Mutations in the core DNA sequence element for cellular protein binding significantly reduced the level of in vitro transcription efficiency. Mutations upstream and downstream of the core sequence moderately reduced the transcription efficiency level. Negative autoregulation of the CMV promoter by the viral IE2 protein may involve both binding to the DNA template and interference with the function of a cellular protein that binds to the transcription start site and enhances transcription efficiency. PMID:8648697

  13. An antiviral disulfide compound blocks interaction between arenavirus Z protein and cellular promyelocytic leukemia protein

    SciTech Connect

    Garcia, C.C.; Topisirovic, I.; Djavani, M.; Borden, K.L.B.; Damonte, E.B.; Salvato, M.S.

    2010-03-19

    The promyelocytic leukemia protein (PML) forms nuclear bodies (NB) that can be redistributed by virus infection. In particular, lymphocytic choriomeningitis virus (LCMV) influences disruption of PML NB through the interaction of PML with the arenaviral Z protein. In a previous report, we have shown that the disulfide compound NSC20625 has antiviral and virucidal properties against arenaviruses, inducing unfolding and oligomerization of Z without affecting cellular RING-containing proteins such as the PML. Here, we further studied the effect of the zinc-finger-reactive disulfide NSC20625 on PML-Z interaction. In HepG2 cells infected with LCMV or transiently transfected with Z protein constructs, treatment with NSC20625 restored PML distribution from a diffuse-cytoplasmic pattern to punctate, discrete NB which appeared identical to NB found in control, uninfected cells. Similar results were obtained in cells transfected with a construct expressing a Z mutant in zinc-binding site 2 of the RING domain, confirming that this Z-PML interaction requires the integrity of only one zinc-binding site. Altogether, these results show that the compound NSC20625 suppressed Z-mediated PML NB disruption and may be used as a tool for designing novel antiviral strategies against arenavirus infection.

  14. Direct protein-protein interactions and substrate channeling between cellular retinoic acid binding proteins and CYP26B1.

    PubMed

    Nelson, Cara H; Peng, Chi-Chi; Lutz, Justin D; Yeung, Catherine K; Zelter, Alex; Isoherranen, Nina

    2016-08-01

    Cellular retinoic acid binding proteins (CRABPs) bind all-trans-retinoic acid (atRA) tightly. This study aimed to determine whether atRA is channeled directly to cytochrome P450 (CYP) CYP26B1 by CRABPs, and whether CRABPs interact directly with CYP26B1. atRA bound to CRABPs (holo-CRABP) was efficiently metabolized by CYP26B1. Isotope dilution experiments showed that delivery of atRA to CYP26B1 in solution was similar with or without CRABP. Holo-CRABPs had higher affinity for CYP26B1 than free atRA, but both apo-CRABPs inhibited the formation of 4-OH-RA by CYP26B1. Similar protein-protein interactions between soluble binding proteins and CYPs may be important for other lipophilic CYP substrates.

  15. Remote access to ACNUC nucleotide and protein sequence databases at PBIL.

    PubMed

    Gouy, Manolo; Delmotte, Stéphane

    2008-04-01

    The ACNUC biological sequence database system provides powerful and fast query and extraction capabilities to a variety of nucleotide and protein sequence databases. The collection of ACNUC databases served by the Pôle Bio-Informatique Lyonnais includes the EMBL, GenBank, RefSeq and UniProt nucleotide and protein sequence databases and a series of other sequence databases that support comparative genomics analyses: HOVERGEN and HOGENOM containing families of homologous protein-coding genes from vertebrate and prokaryotic genomes, respectively; Ensembl and Genome Reviews for analyses of prokaryotic and of selected eukaryotic genomes. This report describes the main features of the ACNUC system and the access to ACNUC databases from any internet-connected computer. Such access was made possible by the definition of a remote ACNUC access protocol and the implementation of Application Programming Interfaces between the C, Python and R languages and this communication protocol. Two retrieval programs for ACNUC databases, Query_win, with a graphical user interface and raa_query, with a command line interface, are also described. Altogether, these bioinformatics tools provide users with either ready-to-use means of querying remote sequence databases through a variety of selection criteria, or a simple way to endow application programs with an extensive access to these databases. Remote access to ACNUC databases is open to all and fully documented (http://pbil.univ-lyon1.fr/databases/acnuc/acnuc.html).

  16. 2P2Idb v2: update of a structural database dedicated to orthosteric modulation of protein-protein interactions.

    PubMed

    Basse, Marie-Jeanne; Betzi, Stéphane; Morelli, Xavier; Roche, Philippe

    2016-01-01

    2P2Idb is a hand-curated structural database dedicated to protein-protein interactions with known small molecule orthosteric modulators. It compiles the structural information related to orthosteric inhibitors and their target [i.e. related 3D structures available in the RCSB Protein Data Bank (PDB)] and provides links to other useful databases. 2P2Idb includes all interactions for which both the protein-protein and protein-inhibitor complexes have been structurally characterized. Since its first release in 2010, the database has grown constantly and the current version contains 27 protein-protein complexes and 274 protein-inhibitor complexes corresponding to 242 unique small molecule inhibitors which represent almost a 5-fold increase compared to the previous version. A number of new data have been added, including new protein-protein complexes, binding affinities, molecular descriptors, precalculated interface parameters and links to other webservers. A new query tool has been implemented to search for inhibitors within the database using standard molecular descriptors. A novel version of the 2P2I-inspector tool has been implemented to calculate a series of physical and chemical parameters of the protein interfaces. Several geometrical parameters including planarity, eccentricity and circularity have been added as well as customizable distance cutoffs. This tool has also been extended to protein-ligand interfaces. The 2P2I database thus represents a wealth of structural source of information for scientists interested in the properties of protein-protein interactions and the design of protein-protein interaction modulators. Database URL: http://2p2idb.cnrs-mrs.fr.

  17. Physiological enzymology: The next frontier in understanding protein structure and function at the cellular level.

    PubMed

    Lee, Irene; Berdis, Anthony J

    2016-01-01

    Historically, the study of proteins has relied heavily on characterizing the activity of a single purified protein isolated from other cellular components. This classic approach allowed scientists to unambiguously define the intrinsic kinetic and chemical properties of that protein. The ultimate hope was to extrapolate this information toward understanding how the enzyme or receptor behaves within its native cellular context. These types of detailed in vitro analyses were necessary to reduce the innate complexities of measuring the singular activity and biochemical properties of a specific enzyme without interference from other enzymes and potential competing substrates. However, recent developments in fields encompassing cell biology, molecular imaging, and chemical biology now provide the unique chemical tools and instrumentation to study protein structure, function, and regulation in their native cellular environment. These advancements provide the foundation for a new field, coined physiological enzymology, which quantifies the function and regulation of enzymes and proteins at the cellular level. In this Special Edition, we explore the area of Physiological Enzymology and Protein Function through a series of review articles that focus on the tools and techniques used to measure the cellular activity of proteins inside living cells. This article is part of a Special Issue entitled: Physiological Enzymology and Protein Functions.

  18. Cellular functions of gamma-secretase-related proteins.

    PubMed

    Haffner, Christof; Haass, Christian

    2006-01-01

    Amyloid-beta peptide (Abeta) is generated by gamma-secretase, a membrane protein complex with an unusual aspartyl protease activity consisting of the four components presenilin, nicastrin, APH-1 and PEN-2. Presenilin is considered the catalytic subunit of this complex since it represents the prototype of the new family of intramembrane-cleaving GxGD-type aspartyl proteases. Recently, five novel members of this family and a nicastrin-like protein were identified. Whereas one of the GxGD-type proteins was shown to be identical with signal peptide peptidase (SPP), the function of the others, now called SPP-like proteins (SPPLs), is not known. We therefore analyzed SPPL2b and SPPL3 and demonstrated that they localize to different subcellular compartments suggesting nonredundant functions. This was supported by different phenotypes obtained in knockdown studies in zebrafish embryos. In addition, these phenotypes could be phenocopied by ectopic expression of putative active site mutants, providing strong evidence for a proteolytic function of SPPL2b and SPPL3. We also identified and characterized the nicastrin-like protein nicalin which, together with the 130-kDa protein NOMO (Nodal modulator), forms a membrane protein complex different from gamma-secretase. We found that during zebrafish embryogenesis this complex is involved in the patterning of the axial mesendoderm, a process controlled by the Nodal signaling pathway. PMID:17047369

  19. CELLULAR MECHANISMS OF PROTEIN METABOLISM IN THE NEPHRON

    PubMed Central

    Oliver, Jean; MacDowell, Muriel

    1958-01-01

    1. The exaggeration of "hyaline droplet" formation observed in the renal lesion of epidemic hemorrhagic fever when treated with the infusion of large amounts of human serum albumin and the histochemical characteristics of the droplets so formed afford evidence towards their identification with the protein absorption droplets of experimental procedures and with those that occur in other renal diseases. 2. Protein absorption droplets (hyaline droplets) are the visible aspect of pathological modifications of a physiological process; i.e., the continuing reabsorption of plasma proteins by the proximal convolutions. 3. The mitochondria of the renal cells are directly involved in both the physiological and the abnormal reabsorption and disposal of the plasma proteins; the absorption droplets are a complex of reabsorbed proteins and mitochondrial substances and enzymes; they result whenever disposal is at a rate insufficient to prevent accumulation. 4. Failure of intracellular disposal of reabsorbed protein is determined by (a) the quantitative and qualitative characteristics of the protein and (b) by the functional state of renal cell. 5. Various factors in renal disease that result in disturbances of reabsorption and of intracellular disposal, both with and without droplet formation, are described. PMID:13525582

  20. Metabotropic glutamate receptor 5 couples cellular prion protein to intracellular signalling in Alzheimer's disease.

    PubMed

    Haas, Laura T; Salazar, Santiago V; Kostylev, Mikhail A; Um, Ji Won; Kaufman, Adam C; Strittmatter, Stephen M

    2016-02-01

    Alzheimer's disease-related phenotypes in mice can be rescued by blockade of either cellular prion protein or metabotropic glutamate receptor 5. We sought genetic and biochemical evidence that these proteins function cooperatively as an obligate complex in the brain. We show that cellular prion protein associates via transmembrane metabotropic glutamate receptor 5 with the intracellular protein mediators Homer1b/c, calcium/calmodulin-dependent protein kinase II, and the Alzheimer's disease risk gene product protein tyrosine kinase 2 beta. Coupling of cellular prion protein to these intracellular proteins is modified by soluble amyloid-β oligomers, by mouse brain Alzheimer's disease transgenes or by human Alzheimer's disease pathology. Amyloid-β oligomer-triggered phosphorylation of intracellular protein mediators and impairment of synaptic plasticity in vitro requires Prnp-Grm5 genetic interaction, being absent in transheterozygous loss-of-function, but present in either single heterozygote. Importantly, genetic coupling between Prnp and Grm5 is also responsible for signalling, for survival and for synapse loss in Alzheimer's disease transgenic model mice. Thus, the interaction between metabotropic glutamate receptor 5 and cellular prion protein has a central role in Alzheimer's disease pathogenesis, and the complex is a potential target for disease-modifying intervention.

  1. Binding affinity of amyloid oligomers to cellular membranes is a generic indicator of cellular dysfunction in protein misfolding diseases

    PubMed Central

    Evangelisti, Elisa; Cascella, Roberta; Becatti, Matteo; Marrazza, Giovanna; Dobson, Christopher M.; Chiti, Fabrizio; Stefani, Massimo; Cecchi, Cristina

    2016-01-01

    The conversion of peptides or proteins from their soluble native states into intractable amyloid deposits is associated with a wide range of human disorders. Misfolded protein oligomers formed during the process of aggregation have been identified as the primary pathogenic agents in many such conditions. Here, we show the existence of a quantitative relationship between the degree of binding to neuronal cells of different types of oligomers formed from a model protein, HypF-N, and the GM1 content of the plasma membranes. In addition, remarkably similar behavior is observed for oligomers of the Aβ42 peptide associated with Alzheimer’s disease. Further analysis has revealed the existence of a linear correlation between the level of the influx of Ca2+ across neuronal membranes that triggers cellular damage, and the fraction of oligomeric species bound to the membrane. Our findings indicate that the susceptibility of neuronal cells to different types of misfolded oligomeric assemblies is directly related to the extent of binding of such oligomers to the cellular membrane. PMID:27619987

  2. Binding affinity of amyloid oligomers to cellular membranes is a generic indicator of cellular dysfunction in protein misfolding diseases.

    PubMed

    Evangelisti, Elisa; Cascella, Roberta; Becatti, Matteo; Marrazza, Giovanna; Dobson, Christopher M; Chiti, Fabrizio; Stefani, Massimo; Cecchi, Cristina

    2016-01-01

    The conversion of peptides or proteins from their soluble native states into intractable amyloid deposits is associated with a wide range of human disorders. Misfolded protein oligomers formed during the process of aggregation have been identified as the primary pathogenic agents in many such conditions. Here, we show the existence of a quantitative relationship between the degree of binding to neuronal cells of different types of oligomers formed from a model protein, HypF-N, and the GM1 content of the plasma membranes. In addition, remarkably similar behavior is observed for oligomers of the Aβ42 peptide associated with Alzheimer's disease. Further analysis has revealed the existence of a linear correlation between the level of the influx of Ca(2+) across neuronal membranes that triggers cellular damage, and the fraction of oligomeric species bound to the membrane. Our findings indicate that the susceptibility of neuronal cells to different types of misfolded oligomeric assemblies is directly related to the extent of binding of such oligomers to the cellular membrane. PMID:27619987

  3. FERM family proteins and their importance in cellular movements and wound healing (review).

    PubMed

    Bosanquet, David C; Ye, Lin; Harding, Keith G; Jiang, Wen G

    2014-07-01

    Motility is a requirement for a number of biological processes, including embryonic development, neuronal development, immune responses, cancer progression and wound healing. Specific to wound healing is the migration of endothelial cells, fibroblasts and other key cellular players into the wound space. Aberrations in wound healing can result in either chronic wounds or abnormally healed wounds. The protein 4.1R, ezrin, radixin, moesin (FERM) superfamily consists of over 40 proteins all containing a three lobed N-terminal FERM domain which binds a variety of cell-membrane associated proteins and lipids. The C-terminal ends of these proteins typically contain an actin-binding domain (ABD). These proteins therefore mediate the linkage between the cell membrane and the actin cytoskeleton, and are involved in cellular movements and migration. Certain FERM proteins have been shown to promote cancer metastasis via this very mechanism. Herein we review the effects of a number of FERM proteins on wound healing and cancer. We show how these proteins typically aid wound healing through their effects on increasing cellular migration and movements, but also typically promote metastasis in cancer. We conclude that FERM proteins play important roles in cellular migration, with markedly different outcomes in the context of cancer and wound healing.

  4. TeloPIN: a database of telomeric proteins interaction network in mammalian cells

    PubMed Central

    Luo, Zhenhua; Dai, Zhiming; Xie, Xiaowei; Feng, Xuyang; Liu, Dan; Songyang, Zhou; Xiong, Yuanyan

    2015-01-01

    Interaction network surrounding telomeres has been intensively studied during the past two decades. However, no specific resource by integrating telomere interaction information data is currently available. To facilitate the understanding of the molecular interaction network by which telomeres are associated with biological process and diseases, we have developed TeloPIN (Telomeric Proteins Interaction Network) database (http://songyanglab.sysu.edu.cn/telopin/), a novel database that points to provide comprehensive information on protein–protein, protein–DNA and protein–RNA interaction of telomeres. TeloPIN database contains four types of interaction data, including (i) protein–protein interaction (PPI) data, (ii) telomeric proteins ChIP-seq data, (iii) telomere-associated proteins data and (iv) telomeric repeat-containing RNAs (TERRA)-interacting proteins data. By analyzing these four types of interaction data, we found that 358 and 199 proteins have more than one type of interaction information in human and mouse cells, respectively. We also developed table browser and TeloChIP genome browser to help researchers with better integrated visualization of interaction data from different studies. The current release of TeloPIN database includes 1111 PPI, eight telomeric protein ChIP-seq data sets, 1391 telomere-associated proteins and 183 TERRA-interacting proteins from 92 independent studies in mammalian cells. The interaction information provided by TeloPIN database will greatly expand our knowledge of telomeric proteins interaction network. Database URL: TeloPIN database address is http://songyanglab.sysu.edu.cn/telopin. TeloPIN database is freely available to non-commercial use. PMID:25792605

  5. Cellular antiviral responses against influenza A virus are countered at the posttranscriptional level by the viral NS1A protein via its binding to a cellular protein required for the 3' end processing of cellular pre-mRNAS.

    PubMed

    Noah, Diana L; Twu, Karen Y; Krug, Robert M

    2003-03-15

    The influenza A virus NS1 protein (NS1A protein) binds and inhibits the function of the 30-kDa subunit of CPSF, a cellular factor that is required for the 3'-end processing of cellular pre-mRNAs. Here we generate a recombinant influenza A/Udorn/72 virus that encodes an NS1A protein containing a mutated binding site for the 30-kDa subunit of CPSF. This mutant virus is substantially attenuated, indicating that this binding site in the NS1A protein is required for efficient virus replication. Using this mutant virus, we show that NS1A binding to CPSF mediates the viral posttranscriptional countermeasure against the initial cellular antiviral response--the interferon-alpha/beta (IFN-alpha/beta)-independent activation of the transcription of cellular antiviral genes, which requires the interferon regulatory factor-3 (IRF-3) transcription factor that is activated by virus infection. Whereas the posttranscriptional processing of these cellular antiviral pre-mRNAs is inhibited in cells infected by wild-type influenza A virus, functional antiviral mRNAs are produced in cells infected by the mutant virus. These results establish that the binding of 30-kDa CPSF to the NS1A protein is largely responsible for the posttranscriptional inhibition of the processing of these cellular antiviral pre-mRNAs. Mutation of this binding site in the NS1A protein also affects a second cellular antiviral response: in cells infected by the mutant virus, IFN-beta mRNA is produced earlier and in larger amounts.

  6. Imaging cellular dynamics in vivo with multicolor fluorescent proteins

    NASA Astrophysics Data System (ADS)

    Hoffman, Robert M.

    2005-04-01

    The new field of in vivo cell biology is being developed with multi-colored fluorescent proteins. With the use of fluorescent proteins, the behavior of individual cells can be visualized in the living animal. An example of the new cell biology is dual-color fluorescence imaging using red fluorescent protein (RFP)-expressing tumors transplanted in green fluorescent protein (GFP)-expressing transgenic mice. These models show with great clarity the details of the tumor-stroma cell-cell interaction especially tumor-induced angiogenesis, tumor-infiltrating lymphocytes, stromal fibroblasts and macrophages. Another example is the color-coding of cells with RFP or GFP such that both cell types and their interaction can be simultaneously visualized in vivo. Stem cells can also be visualized and tracked in vivo with fluorescent proteins. Mice, in which the regulatory elements of the stem-cell marker nestin drive GFP expression, can be used to visualize hair follicle stem cells including their ability to form hair follicles as well as blood vessels. Dual-color cells expressing GFP in the nucleus and RFP in the cytoplasm enable real-time visualization of nuclear-cytoplasm dynamics including cell cycle events and apoptosis. Dual-color cells also enable the in vivo imaging of cell and nuclear deformation as well as trafficking in capillaries in living animals. Multiple-color labeling of cells will enable multiple events to be simultaneously visualized in vivo including cell-cell interaction, gene expression, ion fluxes, protein and organelle trafficking, chromosome dynamics and numerous other processes currently still studied in vitro.

  7. Slight temperature changes affect protein affinity and cellular uptake/toxicity of nanoparticles.

    PubMed

    Mahmoudi, Morteza; Shokrgozar, Mohammad A; Behzadi, Shahed

    2013-04-21

    It is known that what the cell actually "sees" at the nanoscale is an outer shell formed of 'protein corona' on the surface of nanoparticles (NPs). The amount and composition of various proteins on the corona are strongly dependent on the biophysicochemical properties of NPs, which have been extensively studied. However, the effect of a small variation in temperature, due to the human circadian rhythm, on the composition of the protein corona and the affinity of various proteins to the surface of NPs, was ignored. Here, the effect of temperature on the composition of protein corona and the affinity of various proteins to the surface of NPs and, subsequently, cell responses to the protein coated NPs are probed. The results confirmed that cellular entrance, dispersion, and toxicity of NPs are strongly diverse with slight body temperature changes. This new finding can help scientists to maximise NP entrance to specific cells/organs with lower toxicity by adjusting the cellular/organ temperature.

  8. Protein-protein interaction networks identify targets which rescue the MPP+ cellular model of Parkinson’s disease

    NASA Astrophysics Data System (ADS)

    Keane, Harriet; Ryan, Brent J.; Jackson, Brendan; Whitmore, Alan; Wade-Martins, Richard

    2015-11-01

    Neurodegenerative diseases are complex multifactorial disorders characterised by the interplay of many dysregulated physiological processes. As an exemplar, Parkinson’s disease (PD) involves multiple perturbed cellular functions, including mitochondrial dysfunction and autophagic dysregulation in preferentially-sensitive dopamine neurons, a selective pathophysiology recapitulated in vitro using the neurotoxin MPP+. Here we explore a network science approach for the selection of therapeutic protein targets in the cellular MPP+ model. We hypothesised that analysis of protein-protein interaction networks modelling MPP+ toxicity could identify proteins critical for mediating MPP+ toxicity. Analysis of protein-protein interaction networks constructed to model the interplay of mitochondrial dysfunction and autophagic dysregulation (key aspects of MPP+ toxicity) enabled us to identify four proteins predicted to be key for MPP+ toxicity (P62, GABARAP, GBRL1 and GBRL2). Combined, but not individual, knockdown of these proteins increased cellular susceptibility to MPP+ toxicity. Conversely, combined, but not individual, over-expression of the network targets provided rescue of MPP+ toxicity associated with the formation of autophagosome-like structures. We also found that modulation of two distinct proteins in the protein-protein interaction network was necessary and sufficient to mitigate neurotoxicity. Together, these findings validate our network science approach to multi-target identification in complex neurological diseases.

  9. Protein-protein interaction networks identify targets which rescue the MPP+ cellular model of Parkinson’s disease

    PubMed Central

    Keane, Harriet; Ryan, Brent J.; Jackson, Brendan; Whitmore, Alan; Wade-Martins, Richard

    2015-01-01

    Neurodegenerative diseases are complex multifactorial disorders characterised by the interplay of many dysregulated physiological processes. As an exemplar, Parkinson’s disease (PD) involves multiple perturbed cellular functions, including mitochondrial dysfunction and autophagic dysregulation in preferentially-sensitive dopamine neurons, a selective pathophysiology recapitulated in vitro using the neurotoxin MPP+. Here we explore a network science approach for the selection of therapeutic protein targets in the cellular MPP+ model. We hypothesised that analysis of protein-protein interaction networks modelling MPP+ toxicity could identify proteins critical for mediating MPP+ toxicity. Analysis of protein-protein interaction networks constructed to model the interplay of mitochondrial dysfunction and autophagic dysregulation (key aspects of MPP+ toxicity) enabled us to identify four proteins predicted to be key for MPP+ toxicity (P62, GABARAP, GBRL1 and GBRL2). Combined, but not individual, knockdown of these proteins increased cellular susceptibility to MPP+ toxicity. Conversely, combined, but not individual, over-expression of the network targets provided rescue of MPP+ toxicity associated with the formation of autophagosome-like structures. We also found that modulation of two distinct proteins in the protein-protein interaction network was necessary and sufficient to mitigate neurotoxicity. Together, these findings validate our network science approach to multi-target identification in complex neurological diseases. PMID:26608097

  10. Protein structure determination by exhaustive search of Protein Data Bank derived databases.

    PubMed

    Stokes-Rees, Ian; Sliz, Piotr

    2010-12-14

    Parallel sequence and structure alignment tools have become ubiquitous and invaluable at all levels in the study of biological systems. We demonstrate the application and utility of this same parallel search paradigm to the process of protein structure determination, benefitting from the large and growing corpus of known structures. Such searches were previously computationally intractable. Through the method of Wide Search Molecular Replacement, developed here, they can be completed in a few hours with the aide of national-scale federated cyberinfrastructure. By dramatically expanding the range of models considered for structure determination, we show that small (less than 12% structural coverage) and low sequence identity (less than 20% identity) template structures can be identified through multidimensional template scoring metrics and used for structure determination. Many new macromolecular complexes can benefit significantly from such a technique due to the lack of known homologous protein folds or sequences. We demonstrate the effectiveness of the method by determining the structure of a full-length p97 homologue from Trichoplusia ni. Example cases with the MHC/T-cell receptor complex and the EmoB protein provide systematic estimates of minimum sequence identity, structure coverage, and structural similarity required for this method to succeed. We describe how this structure-search approach and other novel computationally intensive workflows are made tractable through integration with the US national computational cyberinfrastructure, allowing, for example, rapid processing of the entire Structural Classification of Proteins protein fragment database.

  11. Reprogramming cellular events by poly(ADP-ribose)-binding proteins

    PubMed Central

    Pic, Émilie; Ethier, Chantal; Dawson, Ted M.; Dawson, Valina L.; Masson, Jean-Yves; Poirier, Guy G.; Gagné, Jean-Philippe

    2013-01-01

    Poly(ADP-ribosyl)ation is a posttranslational modification catalyzed by the poly(ADP-ribose) polymerases (PARPs). These enzymes covalently modify glutamic, aspartic and lysine amino acid side chains of acceptor proteins by the sequential addition of ADP-ribose (ADPr) units. The poly(ADP-ribose) (pADPr) polymers formed alter the physico-chemical characteristics of the substrate with functional consequences on its biological activities. Recently, non-covalent binding to pADPr has emerged as a key mechanism to modulate and coordinate several intracellular pathways including the DNA damage response, protein stability and cell death. In this review, we describe the basis of non-covalent binding to pADPr that has led to the emerging concept of pADPr-responsive signaling pathways. This review emphasizes the structural elements and the modular strategies developed by pADPr-binding proteins to exert a fine-tuned control of a variety of pathways. Poly(ADP-ribosyl)ation reactions are highly regulated processes, both spatially and temporally, for which at least four specialized pADPr-binding modules accommodate different pADPr structures and reprogram protein functions. In this review, we highlight the role of well-characterized and newly discovered pADPr-binding modules in a diverse set of physiological functions. PMID:23268355

  12. The physiological role of the normal cellular prion protein (PrPC).

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The normal cellular prion protein (PrPC) is a highly conserved protein expressed by the prnp gene. It is an essential substrate for the propagation of transmissible spongiform encephalopathies (TSEs), but its natural function remains uncertain. Mice, genetically engineered to be devoid of the prnp...

  13. The human keratinocyte two-dimensional gel protein database (update 1992): towards an integrated approach to the study of cell proliferation, differentiation and skin diseases.

    PubMed

    Celis, J E; Rasmussen, H H; Madsen, P; Leffers, H; Honoré, B; Dejgaard, K; Gesser, B; Olsen, E; Gromov, P; Hoffmann, H J

    1992-12-01

    The master two-dimensional gel database of human keratinocytes currently lists 2980 cellular proteins (2098 isoelectric focusing, IEF; and 882 nonequilibrium pH gradient electrophoresis, NEPHGE) many of which correspond to posttranslational modifications. About 20% of all recorded proteins have been identified (protein name, organelle components, etc.) and they are listed in alphabetical order together with their M(r), pI, cellular localization and credit to the investigator(s) that aided in the identification. Also, we have listed 145 microsequenced proteins that are recorded in this database. As an aid in localizing the polypeptides we have included blow-ups of the master images (IEF, NEPHGE) displaying all the protein numbers. In the long run, the master keratinocyte database is expected to link protein and DNA sequencing and mapping information (Human Genome Program) and to provide an integrated picture of the expression levels and properties of the thousands of proteins that orchestrate various keratinocyte functions both in health and disease. PMID:1286666

  14. The fluorescent protein palette: tools for cellular imaging†

    PubMed Central

    Davidson, Michael W.

    2010-01-01

    This critical review provides an overview of the continually expanding family of fluorescent proteins (FPs) that have become essential tools for studies of cell biology and physiology. Here, we describe the characteristics of the genetically encoded fluorescent markers that now span the visible spectrum from deep blue to deep red. We identify some of the novel FPs that have unusual characteristics that make them useful reporters of the dynamic behaviors of proteins inside cells, and describe how many different optical methods can be combined with the FPs to provide quantitative measurements in living systems. “If wood is rubbed with the Pulmo marinus, it will have all the appearance of being on fire; so much so, indeed, that a walking-stick, thus treated, will light the way like a torch” (translation of Pliny the Elder from John Bostock, 1855). PMID:19771335

  15. A database of domain definitions for proteins with complex interdomain geometry.

    PubMed

    Majumdar, Indraneel; Kinch, Lisa N; Grishin, Nick V

    2009-01-01

    Protein structural domains are necessary for understanding evolution and protein folding, and may vary widely from functional and sequence based domains. Although, various structural domain databases exist, defining domains for some proteins is non-trivial, and definitions of their domain boundaries are not available. Here, we present a novel database of manually defined structural domains for a representative set of proteins from the SCOP "multi-domain proteins" class. (http://prodata.swmed.edu/multidom/). We consider our domains as mobile evolutionary units, which may rearrange during protein evolution. Additionally, they may be visualized as structurally compact and possibly independently folding units. We also found that representing domains as evolutionary and folding units do not always lead to a unique domain definition. However, unlike existing databases, we retain and refine these "alternate" domain definitions after careful inspection of structural similarity, functional sites and automated domain definition methods. We provide domain definitions, including actual residue boundaries, for proteins that well known databases like SCOP and CATH do not attempt to split. Our alternate domain definitions are suitable for sequence and structure searches by automated methods. Additionally, the database can be used for training and testing domain delineation algorithms. Since our domains represent structurally compact evolutionary units, the database may be useful for studying domain properties and evolution. PMID:19352501

  16. The Safety Dance: Biophysics of Membrane Protein Folding and Misfolding in a Cellular Context

    PubMed Central

    Schlebach, Jonathan P.; Sanders, Charles R.

    2015-01-01

    Most biological processes require the production and degradation of proteins, a task that weighs heavily on the cell. Mutations that compromise the conformational stability of proteins place both specific and general burdens on cellular protein homeostasis (proteostasis) in ways that contribute to numerous diseases. Efforts to elucidate the chain of molecular events responsible for diseases of protein folding address one of the foremost challenges in biomedical science. However, relatively little is known about the processes by which mutations prompt the misfolding of α-helical membrane proteins, which rely on an intricate network of cellular machinery to acquire and maintain their functional structures within cellular membranes. In this review, we summarize the current understanding of the physical principles that guide membrane protein biogenesis and folding in the context of mammalian cells. Additionally, we explore how pathogenic mutations that influence biogenesis may differ from those that disrupt folding and assembly, as well as how this may relate to disease mechanisms and therapeutic intervention. These perspectives indicate an imperative for the use of information from structural, cellular, and biochemical studies of membrane proteins in the design of novel therapeutics and in personalized medicine. PMID:25420508

  17. Multiple cellular proteins modulate the dynamics of K-ras association with the plasma membrane.

    PubMed

    Bhagatji, Pinkesh; Leventis, Rania; Rich, Rebecca; Lin, Chen-ju; Silvius, John R

    2010-11-17

    Although specific proteins have been identified that regulate the membrane association and facilitate intracellular transport of prenylated Rho- and Rab-family proteins, it is not known whether cellular proteins fulfill similar roles for other prenylated species, such as Ras-family proteins. We used a previously described method to evaluate how several cellular proteins, previously identified as potential binding partners (but not effectors) of K-ras4B, influence the dynamics of K-ras association with the plasma membrane. Overexpression of either PDEδ or PRA1 enhances, whereas knockdown of either protein reduces, the rate of dissociation of K-ras from the plasma membrane. Inhibition of calmodulin likewise reduces the rate of K-ras dissociation from the plasma membrane, in this case in a manner specific for the activated form of K-ras. By contrast, galectin-3 specifically reduces the rate of plasma membrane dissociation of activated K-ras, an effect that is blocked by the K-ras antagonist farnesylthiosalicylic acid (salirasib). Multiple cellular proteins thus control the dynamics of membrane association and intercompartmental movement of K-ras to an important degree even under basal cellular conditions.

  18. Domain fusion analysis by applying relational algebra to protein sequence and domain databases

    PubMed Central

    Truong, Kevin; Ikura, Mitsuhiko

    2003-01-01

    Background Domain fusion analysis is a useful method to predict functionally linked proteins that may be involved in direct protein-protein interactions or in the same metabolic or signaling pathway. As separate domain databases like BLOCKS, PROSITE, Pfam, SMART, PRINTS-S, ProDom, TIGRFAMs, and amalgamated domain databases like InterPro continue to grow in size and quality, a computational method to perform domain fusion analysis that leverages on these efforts will become increasingly powerful. Results This paper proposes a computational method employing relational algebra to find domain fusions in protein sequence databases. The feasibility of this method was illustrated on the SWISS-PROT+TrEMBL sequence database using domain predictions from the Pfam HMM (hidden Markov model) database. We identified 235 and 189 putative functionally linked protein partners in H. sapiens and S. cerevisiae, respectively. From scientific literature, we were able to confirm many of these functional linkages, while the remainder offer testable experimental hypothesis. Results can be viewed at . Conclusion As the analysis can be computed quickly on any relational database that supports standard SQL (structured query language), it can be dynamically updated along with the sequence and domain databases, thereby improving the quality of predictions over time. PMID:12734020

  19. Integrated Controlling System and Unified Database for High Throughput Protein Crystallography Experiments

    SciTech Connect

    Gaponov, Yu.A.; Igarashi, N.; Hiraki, M.; Sasajima, K.; Matsugaki, N.; Suzuki, M.; Kosuge, T.; Wakatsuki, S.

    2004-05-12

    An integrated controlling system and a unified database for high throughput protein crystallography experiments have been developed. Main features of protein crystallography experiments (purification, crystallization, crystal harvesting, data collection, data processing) were integrated into the software under development. All information necessary to perform protein crystallography experiments is stored (except raw X-ray data that are stored in a central data server) in a MySQL relational database. The database contains four mutually linked hierarchical trees describing protein crystals, data collection of protein crystal and experimental data processing. A database editor was designed and developed. The editor supports basic database functions to view, create, modify and delete user records in the database. Two search engines were realized: direct search of necessary information in the database and object oriented search. The system is based on TCP/IP secure UNIX sockets with four predefined sending and receiving behaviors, which support communications between all connected servers and clients with remote control functions (creating and modifying data for experimental conditions, data acquisition, viewing experimental data, and performing data processing). Two secure login schemes were designed and developed: a direct method (using the developed Linux clients with secure connection) and an indirect method (using the secure SSL connection using secure X11 support from any operating system with X-terminal and SSH support). A part of the system has been implemented on a new MAD beam line, NW12, at the Photon Factory Advanced Ring for general user experiments.

  20. Cellular oxido-reductive proteins of Chlamydomonas reinhardtii control the biosynthesis of silver nanoparticles

    PubMed Central

    2011-01-01

    Background Elucidation of molecular mechanism of silver nanoparticles (SNPs) biosynthesis is important to control its size, shape and monodispersity. The evaluation of molecular mechanism of biosynthesis of SNPs is of prime importance for the commercialization and methodology development for controlling the shape and size (uniform distribution) of SNPs. The unicellular algae Chlamydomonas reinhardtii was exploited as a model system to elucidate the role of cellular proteins in SNPs biosynthesis. Results The C. reinhardtii cell free extract (in vitro) and in vivo cells mediated synthesis of silver nanoparticles reveals SNPs of size range 5 ± 1 to 15 ± 2 nm and 5 ± 1 to 35 ± 5 nm respectively. In vivo biosynthesized SNPs were localized in the peripheral cytoplasm and at one side of flagella root, the site of pathway of ATP transport and its synthesis related enzymes. This provides an evidence for the involvement of oxidoreductive proteins in biosynthesis and stabilization of SNPs. Alteration in size distribution and decrease of synthesis rate of SNPs in protein-depleted fractions confirmed the involvement of cellular proteins in SNPs biosynthesis. Spectroscopic and SDS-PAGE analysis indicate the association of various proteins on C. reinhardtii mediated in vivo and in vitro biosynthesized SNPs. We have identified various cellular proteins associated with biosynthesized (in vivo and in vitro) SNPs by using MALDI-MS-MS, like ATP synthase, superoxide dismutase, carbonic anhydrase, ferredoxin-NADP+ reductase, histone etc. However, these proteins were not associated on the incubation of pre-synthesized silver nanoparticles in vitro. Conclusion Present study provides the indication of involvement of molecular machinery and various cellular proteins in the biosynthesis of silver nanoparticles. In this report, the study is mainly focused towards understanding the role of diverse cellular protein in the synthesis and capping of silver nanoparticles using C. reinhardtii as

  1. The Histone Database: a comprehensive resource for histones and histone fold-containing proteins.

    PubMed

    Mariño-Ramírez, Leonardo; Hsu, Benjamin; Baxevanis, Andreas D; Landsman, David

    2006-03-01

    The Histone Database is a curated and searchable collection of full-length sequences and structures of histones and nonhistone proteins containing histone-like folds, compiled from major public databases. Several new histone fold-containing proteins have been identified, including the huntingtin-interacting protein HYPM. Additionally, based on the recent crystal structure of the Son of Sevenless protein, an interpretation of the sequence analysis of the histone fold domain is presented. The database contains an updated collection of multiple sequence alignments for the four core histones (H2A, H2B, H3, and H4) and the linker histones (H1/H5) from a total of 975 organisms. The database also contains information on the human histone gene complement and provides links to three-dimensional structures of histone and histone fold-containing proteins. The Histone Database is a comprehensive bioinformatics resource for the study of structure and function of histones and histone fold-containing proteins. The database is available at http://research.nhgri.nih.gov/histones/.

  2. A Database of Domain Definitions for Proteins with Complex Interdomain Geometry

    PubMed Central

    Majumdar, Indraneel; Kinch, Lisa N.; Grishin, Nick V.

    2009-01-01

    Protein structural domains are necessary for understanding evolution and protein folding, and may vary widely from functional and sequence based domains. Although, various structural domain databases exist, defining domains for some proteins is non-trivial, and definitions of their domain boundaries are not available. Here, we present a novel database of manually defined structural domains for a representative set of proteins from the SCOP “multi-domain proteins” class. (http://prodata.swmed.edu/multidom/). We consider our domains as mobile evolutionary units, which may rearrange during protein evolution. Additionally, they may be visualized as structurally compact and possibly independently folding units. We also found that representing domains as evolutionary and folding units do not always lead to a unique domain definition. However, unlike existing databases, we retain and refine these “alternate” domain definitions after careful inspection of structural similarity, functional sites and automated domain definition methods. We provide domain definitions, including actual residue boundaries, for proteins that well known databases like SCOP and CATH do not attempt to split. Our alternate domain definitions are suitable for sequence and structure searches by automated methods. Additionally, the database can be used for training and testing domain delineation algorithms. Since our domains represent structurally compact evolutionary units, the database may be useful for studying domain properties and evolution. PMID:19352501

  3. PARPs database: A LIMS systems for protein-protein interaction data mining or laboratory information management system

    PubMed Central

    Droit, Arnaud; Hunter, Joanna M; Rouleau, Michèle; Ethier, Chantal; Picard-Cloutier, Aude; Bourgais, David; Poirier, Guy G

    2007-01-01

    Background In the "post-genome" era, mass spectrometry (MS) has become an important method for the analysis of proteins and the rapid advancement of this technique, in combination with other proteomics methods, results in an increasing amount of proteome data. This data must be archived and analysed using specialized bioinformatics tools. Description We herein describe "PARPs database," a data analysis and management pipeline for liquid chromatography tandem mass spectrometry (LC-MS/MS) proteomics. PARPs database is a web-based tool whose features include experiment annotation, protein database searching, protein sequence management, as well as data-mining of the peptides and proteins identified. Conclusion Using this pipeline, we have successfully identified several interactions of biological significance between PARP-1 and other proteins, namely RFC-1, 2, 3, 4 and 5. PMID:18093328

  4. Generic framework for mining cellular automata models on protein-folding simulations.

    PubMed

    Diaz, N; Tischer, I

    2016-01-01

    Cellular automata model identification is an important way of building simplified simulation models. In this study, we describe a generic architectural framework to ease the development process of new metaheuristic-based algorithms for cellular automata model identification in protein-folding trajectories. Our framework was developed by a methodology based on design patterns that allow an improved experience for new algorithms development. The usefulness of the proposed framework is demonstrated by the implementation of four algorithms, able to obtain extremely precise cellular automata models of the protein-folding process with a protein contact map representation. Dynamic rules obtained by the proposed approach are discussed, and future use for the new tool is outlined. PMID:27323045

  5. Interactions of cellular proteins involved in the transcriptional regulation of the human immunodeficiency virus.

    PubMed Central

    Garcia, J A; Wu, F K; Mitsuyasu, R; Gaynor, R B

    1987-01-01

    The human immunodeficiency virus (HIV) is a human retrovirus which is the etiologic agent of the acquired immunodeficiency syndrome. To study the cellular factors involved in the transcriptional regulation of this virus, we performed DNase I footprinting of the viral LTR using partially purified HeLa cell extracts. Five regions of the viral LTR appear critical for DNA binding of cellular proteins. These include the negative regulatory, enhancer, SP1, TATA and untranslated regions. Deletion mutagenesis of these binding domains has significant effects on the basal level of transcription and the ability to be induced by the viral tat protein. Mutations of either the negative regulatory or untranslated regions affect factor binding to the enhancer region. In addition, oligonucleotides complementary to several of the binding domains specifically compete for factor binding. These results suggest that interactions between several distinct cellular proteins are required for HIV transcriptional regulation. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 6. PMID:3428273

  6. Interactions of cellular proteins involved in the transcriptional regulation of the human immunodeficiency virus.

    PubMed

    Garcia, J A; Wu, F K; Mitsuyasu, R; Gaynor, R B

    1987-12-01

    The human immunodeficiency virus (HIV) is a human retrovirus which is the etiologic agent of the acquired immunodeficiency syndrome. To study the cellular factors involved in the transcriptional regulation of this virus, we performed DNase I footprinting of the viral LTR using partially purified HeLa cell extracts. Five regions of the viral LTR appear critical for DNA binding of cellular proteins. These include the negative regulatory, enhancer, SP1, TATA and untranslated regions. Deletion mutagenesis of these binding domains has significant effects on the basal level of transcription and the ability to be induced by the viral tat protein. Mutations of either the negative regulatory or untranslated regions affect factor binding to the enhancer region. In addition, oligonucleotides complementary to several of the binding domains specifically compete for factor binding. These results suggest that interactions between several distinct cellular proteins are required for HIV transcriptional regulation.

  7. SoyProDB: A database for the identification of soybean seed proteins

    PubMed Central

    Tavakolan, Mona; Alkharouf, Nadim W; Khan, Farooq H; Natarajan, Savithiry

    2013-01-01

    Soybean continues to serve as a rich and inexpensive source of protein for humans and animals. A substantial amount of information has been reported on the genotypic variation and beneficial genetic manipulation of soybeans. For better understanding of the consequences of genetic manipulation, elucidation of soybean protein composition is necessary, because of its direct relationship to phenotype. We have conducted studies to determine the composition of storage, allergen and anti-nutritional proteins in cultivated soybean using a combined proteomics approach. Two-dimensional polyacrylamide gel electrophoresis (2DPAGE) was implemented for the separation of proteins along with matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) and liquid chromatography mass spectrometry (LC-MS/MS) for the identification of proteins. Our analysis resulted in the identification of several proteins, and a web based database named soybean protein database (SoyProDB) was subsequently built to house and allow scientists to search the data. This database will be useful to scientists who wish to genetically alter soybean with higher quality storage proteins, and also helpful for consumers to get a greater understanding about proteins that compose soy products available in the market. The database is freely accessible. Availability http://bioinformatics.towson.edu/Soybean_Seed_Proteins_2D_Gel_DB/Home.aspx PMID:23423175

  8. OPM database and PPM web server: resources for positioning of proteins in membranes

    PubMed Central

    Lomize, Mikhail A.; Pogozheva, Irina D.; Joo, Hyeon; Mosberg, Henry I.; Lomize, Andrei L.

    2012-01-01

    The Orientations of Proteins in Membranes (OPM) database is a curated web resource that provides spatial positions of membrane-bound peptides and proteins of known three-dimensional structure in the lipid bilayer, together with their structural classification, topology and intracellular localization. OPM currently contains more than 1200 transmembrane and peripheral proteins and peptides from approximately 350 organisms that represent approximately 3800 Protein Data Bank entries. Proteins are classified into classes, superfamilies and families and assigned to 21 distinct membrane types. Spatial positions of proteins with respect to the lipid bilayer are optimized by the PPM 2.0 method that accounts for the hydrophobic, hydrogen bonding and electrostatic interactions of the proteins with the anisotropic water-lipid environment described by the dielectric constant and hydrogen-bonding profiles. The OPM database is freely accessible at http://opm.phar.umich.edu. Data can be sorted, searched or retrieved using the hierarchical classification, source organism, localization in different types of membranes. The database offers downloadable coordinates of proteins and peptides with membrane boundaries. A gallery of protein images and several visualization tools are provided. The database is supplemented by the PPM server (http://opm.phar.umich.edu/server.php) which can be used for calculating spatial positions in membranes of newly determined proteins structures or theoretical models. PMID:21890895

  9. iProClass: an integrated database of protein family, function and structure information.

    PubMed

    Huang, Hongzhan; Barker, Winona C; Chen, Yongxing; Wu, Cathy H

    2003-01-01

    The iProClass database provides comprehensive, value-added descriptions of proteins and serves as a framework for data integration in a distributed networking environment. The protein information in iProClass includes family relationships as well as structural and functional classifications and features. The current version consists of about 830 000 non-redundant PIR-PSD, SWISS-PROT, and TrEMBL proteins organized with more than 36 000 PIR superfamilies, 145 000 families, 4000 domains, 1300 motifs and 550 000 FASTA similarity clusters. It provides rich links to over 50 database of protein sequences, families, functions and pathways, protein-protein interactions, post-translational modifications, protein expressions, structures and structural classifications, genes and genomes, ontologies, literature and taxonomy. Protein and superfamily summary reports present extensive annotation information and include membership statistics and graphical display of domains and motifs. iProClass employs an open and modular architecture for interoperability and scalability. It is implemented in the Oracle object-relational database system and is updated biweekly. The database is freely accessible from the web site at http://pir.georgetown.edu/iproclass/ and searchable by sequence or text string. The data integration in iProClass supports exploration of protein relationships. Such knowledge is fundamental to the understanding of protein evolution, structure and function and crucial to functional genomic and proteomic research.

  10. Highly efficient extraction of cellular nucleic acid associated proteins in vitro with magnetic oxidized carbon nanotubes.

    PubMed

    Zhang, Yi; Hu, Zhengyan; Qin, Hongqiang; Wei, Xiaoluan; Cheng, Kai; Liu, Fangjie; Wu, Ren'an; Zou, Hanfa

    2012-12-01

    Nucleic acid associated proteins (NAaP) play the essential roles in gene regulation and protein expression. The global analysis of cellular NAaP would give a broad insight to understand the interaction between nucleic acids and the associated proteins, such as the important proteinous regulation factors on nucleic acids. Proteomic analysis presents a novel strategy to investigate a group of proteins. However, the large scale analysis of NAaP is yet impossible due to the lack of approaches to harvest target protein groups with a high efficiency. Herein, a simple and efficient method was developed to collect cellular NAaP using magnetic oxidized carbon nanotubes based on the strong interaction between carbon nanotubes and nucleic acids along with corresponding associated proteins. We found that the magnetic oxidized carbon nanotubes demonstrated a nearly 100% extraction efficiency for intracellular nucleic acids from cells in vitro. Importantly, the proteins associated on nucleic acids could be highly efficiently harvested using magnetic oxidized carbon nanotubes due to the binding of NAaP on nucleic acids. 1594 groups of nuclear NAaP and 2595 groups of cellular NAaP were extracted and identified from about 1,000,000 cells, and 803 groups of NAaP were analyzed with only about 10,000 cells, showing a promising performance for the proteomic analysis of NAaP from minute cellular samples. This highly efficient extraction strategy for NAaP is a simple approach to identify cellular nucleic acid associated proteome, and we believed this strategy could be further applied in systems biology to understand the gene expression and regulation.

  11. The Histone Database: an integrated resource for histones and histone fold-containing proteins.

    PubMed

    Mariño-Ramírez, Leonardo; Levine, Kevin M; Morales, Mario; Zhang, Suiyuan; Moreland, R Travis; Baxevanis, Andreas D; Landsman, David

    2011-01-01

    Eukaryotic chromatin is composed of DNA and protein components-core histones-that act to compactly pack the DNA into nucleosomes, the fundamental building blocks of chromatin. These nucleosomes are connected to adjacent nucleosomes by linker histones. Nucleosomes are highly dynamic and, through various core histone post-translational modifications and incorporation of diverse histone variants, can serve as epigenetic marks to control processes such as gene expression and recombination. The Histone Sequence Database is a curated collection of sequences and structures of histones and non-histone proteins containing histone folds, assembled from major public databases. Here, we report a substantial increase in the number of sequences and taxonomic coverage for histone and histone fold-containing proteins available in the database. Additionally, the database now contains an expanded dataset that includes archaeal histone sequences. The database also provides comprehensive multiple sequence alignments for each of the four core histones (H2A, H2B, H3 and H4), the linker histones (H1/H5) and the archaeal histones. The database also includes current information on solved histone fold-containing structures. The Histone Sequence Database is an inclusive resource for the analysis of chromatin structure and function focused on histones and histone fold-containing proteins.

  12. Quantitative analysis of regions of adenovirus E1A products involved in interactions with cellular proteins.

    PubMed

    Barbeau, D; Marcellus, R C; Bacchetti, S; Bayley, S T; Branton, P E

    1992-01-01

    Human adenovirus E1A proteins and oncogene products of several other DNA tumour viruses derive much of their oncogenic potential from interactions with cellular polypeptides. E1A proteins form complexes with p105Rb and a related p107 polypeptide, and with at least three other proteins (p60cycA, p130, and p300); all may be required for cell transformation. Using a series of E1A deletion mutants, we have carried out a quantitative analysis of the binding patterns of cellular proteins to E1A products. Binding of most of the proteins was affected at least partially by mutations within the amino terminal 25 residues, amino acids 36-69 within conserved region 1 (CR1), and residues 121-138 in conserved region 2 (CR2). However, the specific binding characteristics of each protein varied considerably. p300 was the only species for which binding was totally eliminated by deletions at the amino terminus. Removal of regions within CR1 eliminated binding of all species except p107 and p60cycA. Deletion of portions of CR2 reduced or eliminated binding of all proteins except p300. Thus, whereas cellular polypeptides generally were found to interact with the same three regions of E1A proteins, specific interactions varied considerably. PMID:1297336

  13. SurfaceomeDB: a cancer-orientated database for genes encoding cell surface proteins

    PubMed Central

    de Souza, Jorge Estefano Santana; Galante, Pedro Alexandre Favoretto; de Almeida, Renan Valieris Bueno; da Cunha, Julia Pinheiro Chagas; Ohara, Daniel Takatori; Ohno-Machado, Lucila; Old, Lloyd J.; de Souza, Sandro José

    2012-01-01

    Cell surface proteins (CSPs) are excellent targets for the development of diagnostic and therapeutic reagents, and it is estimated that 10–20% of all genes in the human genome encode CSPs. In an effort to integrate all data publicly available for genes encoding cell surface proteins, a database (SurfaceomeDB) was developed. SurfaceomeDB is a gene-centered portal containing different types of information, including annotation for gene expression, protein domains, somatic mutations in cancer, and protein-protein interactions for all human genes encoding CSPs. SurfaceomeDB was implemented as an integrative and relational database in a user-friendly web interface, where users can search for gene name, gene annotation, or keywords. There is also a streamlined graphical representation of all data provided and links to the most important data repositories and databases, such as NCBI, UCSC Genome Browser, and EBI. PMID:23390370

  14. MitoInteractome: Mitochondrial protein interactome database, and its application in 'aging network' analysis

    PubMed Central

    2009-01-01

    Background Mitochondria play a vital role in the energy production and apoptotic process of eukaryotic cells. Proteins in the mitochondria are encoded by nuclear and mitochondrial genes. Owing to a large increase in the number of identified mitochondrial protein sequences and completed mitochondrial genomes, it has become necessary to provide a web-based database of mitochondrial protein information. Results We present 'MitoInteractome', a consolidated web-based portal containing a wealth of information on predicted protein-protein interactions, physico-chemical properties, polymorphism, and diseases related to the mitochondrial proteome. MitoInteractome contains 6,549 protein sequences which were extracted from the following databases: SwissProt, MitoP, MitoProteome, HPRD and Gene Ontology database. The first general mitochondrial interactome has been constructed based on the concept of 'homologous interaction' using PSIMAP (Protein Structural Interactome MAP) and PEIMAP (Protein Experimental Interactome MAP). Using the above mentioned methods, protein-protein interactions were predicted for 74 species. The mitochondrial protein interaction data of humans was used to construct a network for the aging process. Analysis of the 'aging network' gave us vital insights into the interactions among proteins that influence the aging process. Conclusion MitoInteractome is a comprehensive database that would (1) aid in increasing our understanding of the molecular functions and interaction networks of mitochondrial proteins, (2) help in identifying new target proteins for experimental research using predicted protein-protein interaction information, and (3) help in identifying biomarkers for diagnosis and new molecular targets for drug development related to mitochondria. MitoInteractome is available at http://mitointeractome.kobic.kr/. PMID:19958484

  15. BioBuilder as a database development and functional annotation platform for proteins

    PubMed Central

    Navarro, J Daniel; Talreja, Naveen; Peri, Suraj; Vrushabendra, BM; Rashmi, BP; Padma, N; Surendranath, Vineeth; Jonnalagadda, Chandra Kiran; Kousthub, PS; Deshpande, Nandan; Shanker, K; Pandey, Akhilesh

    2004-01-01

    Background The explosion in biological information creates the need for databases that are easy to develop, easy to maintain and can be easily manipulated by annotators who are most likely to be biologists. However, deployment of scalable and extensible databases is not an easy task and generally requires substantial expertise in database development. Results BioBuilder is a Zope-based software tool that was developed to facilitate intuitive creation of protein databases. Protein data can be entered and annotated through web forms along with the flexibility to add customized annotation features to protein entries. A built-in review system permits a global team of scientists to coordinate their annotation efforts. We have already used BioBuilder to develop Human Protein Reference Database , a comprehensive annotated repository of the human proteome. The data can be exported in the extensible markup language (XML) format, which is rapidly becoming as the standard format for data exchange. Conclusions As the proteomic data for several organisms begins to accumulate, BioBuilder will prove to be an invaluable platform for functional annotation and development of customizable protein centric databases. BioBuilder is open source and is available under the terms of LGPL. PMID:15099404

  16. COMBREX-DB: an experiment centered database of protein function: knowledge, predictions and knowledge gaps.

    PubMed

    Chang, Yi-Chien; Hu, Zhenjun; Rachlin, John; Anton, Brian P; Kasif, Simon; Roberts, Richard J; Steffen, Martin

    2016-01-01

    The COMBREX database (COMBREX-DB; combrex.bu.edu) is an online repository of information related to (i) experimentally determined protein function, (ii) predicted protein function, (iii) relationships among proteins of unknown function and various types of experimental data, including molecular function, protein structure, and associated phenotypes. The database was created as part of the novel COMBREX (COMputational BRidges to EXperiments) effort aimed at accelerating the rate of gene function validation. It currently holds information on ∼ 3.3 million known and predicted proteins from over 1000 completely sequenced bacterial and archaeal genomes. The database also contains a prototype recommendation system for helping users identify those proteins whose experimental determination of function would be most informative for predicting function for other proteins within protein families. The emphasis on documenting experimental evidence for function predictions, and the prioritization of uncharacterized proteins for experimental testing distinguish COMBREX from other publicly available microbial genomics resources. This article describes updates to COMBREX-DB since an initial description in the 2011 NAR Database Issue.

  17. COMBREX-DB: an experiment centered database of protein function: knowledge, predictions and knowledge gaps.

    PubMed

    Chang, Yi-Chien; Hu, Zhenjun; Rachlin, John; Anton, Brian P; Kasif, Simon; Roberts, Richard J; Steffen, Martin

    2016-01-01

    The COMBREX database (COMBREX-DB; combrex.bu.edu) is an online repository of information related to (i) experimentally determined protein function, (ii) predicted protein function, (iii) relationships among proteins of unknown function and various types of experimental data, including molecular function, protein structure, and associated phenotypes. The database was created as part of the novel COMBREX (COMputational BRidges to EXperiments) effort aimed at accelerating the rate of gene function validation. It currently holds information on ∼ 3.3 million known and predicted proteins from over 1000 completely sequenced bacterial and archaeal genomes. The database also contains a prototype recommendation system for helping users identify those proteins whose experimental determination of function would be most informative for predicting function for other proteins within protein families. The emphasis on documenting experimental evidence for function predictions, and the prioritization of uncharacterized proteins for experimental testing distinguish COMBREX from other publicly available microbial genomics resources. This article describes updates to COMBREX-DB since an initial description in the 2011 NAR Database Issue. PMID:26635392

  18. COMBREX-DB: an experiment centered database of protein function: knowledge, predictions and knowledge gaps

    PubMed Central

    Chang, Yi-Chien; Hu, Zhenjun; Rachlin, John; Anton, Brian P.; Kasif, Simon; Roberts, Richard J.; Steffen, Martin

    2016-01-01

    The COMBREX database (COMBREX-DB; combrex.bu.edu) is an online repository of information related to (i) experimentally determined protein function, (ii) predicted protein function, (iii) relationships among proteins of unknown function and various types of experimental data, including molecular function, protein structure, and associated phenotypes. The database was created as part of the novel COMBREX (COMputational BRidges to EXperiments) effort aimed at accelerating the rate of gene function validation. It currently holds information on ∼3.3 million known and predicted proteins from over 1000 completely sequenced bacterial and archaeal genomes. The database also contains a prototype recommendation system for helping users identify those proteins whose experimental determination of function would be most informative for predicting function for other proteins within protein families. The emphasis on documenting experimental evidence for function predictions, and the prioritization of uncharacterized proteins for experimental testing distinguish COMBREX from other publicly available microbial genomics resources. This article describes updates to COMBREX-DB since an initial description in the 2011 NAR Database Issue. PMID:26635392

  19. AraPPISite: a database of fine-grained protein-protein interaction site annotations for Arabidopsis thaliana.

    PubMed

    Li, Hong; Yang, Shiping; Wang, Chuan; Zhou, Yuan; Zhang, Ziding

    2016-09-01

    Knowledge about protein interaction sites provides detailed information of protein-protein interactions (PPIs). To date, nearly 20,000 of PPIs from Arabidopsis thaliana have been identified. Nevertheless, the interaction site information has been largely missed by previously published PPI databases. Here, AraPPISite, a database that presents fine-grained interaction details for A. thaliana PPIs is established. First, the experimentally determined 3D structures of 27 A. thaliana PPIs are collected from the Protein Data Bank database and the predicted 3D structures of 3023 A. thaliana PPIs are modeled by using two well-established template-based docking methods. For each experimental/predicted complex structure, AraPPISite not only provides an interactive user interface for browsing interaction sites, but also lists detailed evolutionary and physicochemical properties of these sites. Second, AraPPISite assigns domain-domain interactions or domain-motif interactions to 4286 PPIs whose 3D structures cannot be modeled. In this case, users can easily query protein interaction regions at the sequence level. AraPPISite is a free and user-friendly database, which does not require user registration or any configuration on local machines. We anticipate AraPPISite can serve as a helpful database resource for the users with less experience in structural biology or protein bioinformatics to probe the details of PPIs, and thus accelerate the studies of plant genetics and functional genomics. AraPPISite is available at http://systbio.cau.edu.cn/arappisite/index.html .

  20. AraPPISite: a database of fine-grained protein-protein interaction site annotations for Arabidopsis thaliana.

    PubMed

    Li, Hong; Yang, Shiping; Wang, Chuan; Zhou, Yuan; Zhang, Ziding

    2016-09-01

    Knowledge about protein interaction sites provides detailed information of protein-protein interactions (PPIs). To date, nearly 20,000 of PPIs from Arabidopsis thaliana have been identified. Nevertheless, the interaction site information has been largely missed by previously published PPI databases. Here, AraPPISite, a database that presents fine-grained interaction details for A. thaliana PPIs is established. First, the experimentally determined 3D structures of 27 A. thaliana PPIs are collected from the Protein Data Bank database and the predicted 3D structures of 3023 A. thaliana PPIs are modeled by using two well-established template-based docking methods. For each experimental/predicted complex structure, AraPPISite not only provides an interactive user interface for browsing interaction sites, but also lists detailed evolutionary and physicochemical properties of these sites. Second, AraPPISite assigns domain-domain interactions or domain-motif interactions to 4286 PPIs whose 3D structures cannot be modeled. In this case, users can easily query protein interaction regions at the sequence level. AraPPISite is a free and user-friendly database, which does not require user registration or any configuration on local machines. We anticipate AraPPISite can serve as a helpful database resource for the users with less experience in structural biology or protein bioinformatics to probe the details of PPIs, and thus accelerate the studies of plant genetics and functional genomics. AraPPISite is available at http://systbio.cau.edu.cn/arappisite/index.html . PMID:27338257

  1. Identifying proteins from two-dimensional gels by molecular mass searching of peptide fragments in protein sequence databases.

    PubMed

    Henzel, W J; Billeci, T M; Stults, J T; Wong, S C; Grimley, C; Watanabe, C

    1993-06-01

    A rapid method for the identification of known proteins separated by two-dimensional gel electrophoresis is described in which molecular masses of peptide fragments are used to search a protein sequence database. The peptides are generated by in situ reduction, alkylation, and tryptic digestion of proteins electroblotted from two-dimensional gels. Masses are determined at the subpicomole level by matrix-assisted laser desorption/ionization mass spectrometry of the unfractionated digest. A computer program has been developed that searches the protein sequence database for multiple peptides of individual proteins that match the measured masses. To ensure that the most recent database updates are included, a theoretical digest of the entire database is generated each time the program is executed. This method facilitates simultaneous processing of a large number of two-dimensional gel spots. The method was applied to a two-dimensional gel of a crude Escherichia coli extract that was electroblotted onto poly(vinylidene difluoride) membrane. Ten randomly chosen spots were analyzed. With as few as three peptide masses, each protein was uniquely identified from over 91,000 protein sequences. All identifications were verified by concurrent N-terminal sequencing of identical spots from a second blot. One of the spots contained an N-terminally blocked protein that required enzymatic cleavage, peptide separation, and Edman degradation for confirmation of its identity.

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

    PubMed Central

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

    2014-01-01

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

  3. The Pfam protein families database: towards a more sustainable future.

    PubMed

    Finn, Robert D; Coggill, Penelope; Eberhardt, Ruth Y; Eddy, Sean R; Mistry, Jaina; Mitchell, Alex L; Potter, Simon C; Punta, Marco; Qureshi, Matloob; Sangrador-Vegas, Amaia; Salazar, Gustavo A; Tate, John; Bateman, Alex

    2016-01-01

    In the last two years the Pfam database (http://pfam.xfam.org) has undergone a substantial reorganisation to reduce the effort involved in making a release, thereby permitting more frequent releases. Arguably the most significant of these changes is that Pfam is now primarily based on the UniProtKB reference proteomes, with the counts of matched sequences and species reported on the website restricted to this smaller set. Building families on reference proteomes sequences brings greater stability, which decreases the amount of manual curation required to maintain them. It also reduces the number of sequences displayed on the website, whilst still providing access to many important model organisms. Matches to the full UniProtKB database are, however, still available and Pfam annotations for individual UniProtKB sequences can still be retrieved. Some Pfam entries (1.6%) which have no matches to reference proteomes remain; we are working with UniProt to see if sequences from them can be incorporated into reference proteomes. Pfam-B, the automatically-generated supplement to Pfam, has been removed. The current release (Pfam 29.0) includes 16 295 entries and 559 clans. The facility to view the relationship between families within a clan has been improved by the introduction of a new tool.

  4. Pyridalyl inhibits cellular protein synthesis in insect, but not mammalian, cell lines.

    PubMed

    Moriya, Koko; Hirakura, Setsuko; Kobayashi, Jun; Ozoe, Yoshihisa; Saito, Shigeru; Utsumi, Toshihiko

    2008-09-01

    To gain insight into the mechanism of action and selectivity of the insecticidal activity of pyridalyl, the cytotoxicity of pyridalyl against various insect and mammalian cell lines was characterized by measuring the inhibition of cellular protein synthesis. When the effect of pyridalyl on the cellular protein synthesis in Sf9 cells was evaluated by measuring the incorporation of [(3)H]leucine, rapid and significant inhibition of protein synthesis was observed. However, pyridalyl did not inhibit protein synthesis in a cell-free protein synthesis system, indicating that pyridalyl does not directly inhibit protein synthesis. No obvious cytotoxicity was observed against any of the mammalian cell lines tested. In the case of insect cell lines, remarkable differences in the cytotoxicity of pyridalyl were observed: the highest cytotoxicity (IC50 mM) was found against Sf9 cells derived from Spodoptera frugiperda, whereas no obvious cytotoxicity was observed against BmN4 cells derived from Bombyx mori. Measurements of the insecticidal activity of pyridalyl against Spodoptera litura and B. mori revealed a correlation between the cytotoxicity against cultured cell lines and the insecticidal activity. From these observations, it was concluded that the selective inhibition of cellular protein synthesis by pyridalyl might contribute significantly to the insecticidal activity and the selectivity of this compound. PMID:18454491

  5. Cellular Distribution of NDRG1 Protein in the Rat Kidney and Brain During Normal Postnatal Development

    PubMed Central

    Wakisaka, Yoshinobu; Furuta, Akiko; Masuda, Katsuaki; Morikawa, Wataru; Kuwano, Michihiko; Iwaki, Toru

    2003-01-01

    N-myc downregulated gene 1 (NDRG1) is a 43-kD protein whose mRNA is induced by DNA damage, hypoxia, or prolonged elevation of intracellular calcium. Although NDRG1 is also upregulated during cell differentiation, there are few studies on NDRG1 expression during postnatal development. Here we investigated the expression and cellular distribution of NDRG1 protein in rat kidney and brain during postnatal development. Immunohistochemical analysis revealed that the cellular localization of NDRG1 protein in the kidney changed from the proximal convoluted tubules to the collecting ducts between postnatal days 10 and 20. In the brain, a change in cellular expression was also found from the hippocampal pyramidal neurons to the astrocytes in the gray matter during the same postnatal period. These alterations in the cellular distribution of NDRG1 were associated with shifts in the molecular assembly on Western blots. Under non-reduced conditions, the main NDRG1 band was found only around 215 kD in both kidney and brain during the early postnatal stage. After postnatal day 10, the immunoreactive bands shifted to 43 kD in the kidney and 129 kD in the brain. These changes in the cellular distribution and state of assembly may correlate with the functional maturation of both organs. PMID:14566023

  6. MoonProt: a database for proteins that are known to moonlight

    PubMed Central

    Mani, Mathew; Chen, Chang; Amblee, Vaishak; Liu, Haipeng; Mathur, Tanu; Zwicke, Grant; Zabad, Shadi; Patel, Bansi; Thakkar, Jagravi; Jeffery, Constance J.

    2015-01-01

    Moonlighting proteins comprise a class of multifunctional proteins in which a single polypeptide chain performs multiple biochemical functions that are not due to gene fusions, multiple RNA splice variants or pleiotropic effects. The known moonlighting proteins perform a variety of diverse functions in many different cell types and species, and information about their structures and functions is scattered in many publications. We have constructed the manually curated, searchable, internet-based MoonProt Database (http://www.moonlightingproteins.org) with information about the over 200 proteins that have been experimentally verified to be moonlighting proteins. The availability of this organized information provides a more complete picture of what is currently known about moonlighting proteins. The database will also aid researchers in other fields, including determining the functions of genes identified in genome sequencing projects, interpreting data from proteomics projects and annotating protein sequence and structural databases. In addition, information about the structures and functions of moonlighting proteins can be helpful in understanding how novel protein functional sites evolved on an ancient protein scaffold, which can also help in the design of proteins with novel functions. PMID:25324305

  7. Extracting protein alignment models from the sequence database.

    PubMed Central

    Neuwald, A F; Liu, J S; Lipman, D J; Lawrence, C E

    1997-01-01

    Biologists often gain structural and functional insights into a protein sequence by constructing a multiple alignment model of the family. Here a program called Probe fully automates this process of model construction starting from a single sequence. Central to this program is a powerful new method to locate and align only those, often subtly, conserved patterns essential to the family as a whole. When applied to randomly chosen proteins, Probe found on average about four times as many relationships as a pairwise search and yielded many new discoveries. These include: an obscure subfamily of globins in the roundworm Caenorhabditis elegans ; two new superfamilies of metallohydrolases; a lipoyl/biotin swinging arm domain in bacterial membrane fusion proteins; and a DH domain in the yeast Bud3 and Fus2 proteins. By identifying distant relationships and merging families into superfamilies in this way, this analysis further confirms the notion that proteins evolved from relatively few ancient sequences. Moreover, this method automatically generates models of these ancient conserved regions for rapid and sensitive screening of sequences. PMID:9108146

  8. ANCHOR: a web server and database for analysis of protein-protein interaction binding pockets for drug discovery.

    PubMed

    Meireles, Lidio M C; Dömling, Alexander S; Camacho, Carlos J

    2010-07-01

    ANCHOR is a web-based tool whose aim is to facilitate the analysis of protein-protein interfaces with regard to its suitability for small molecule drug design. To this end, ANCHOR exploits the so-called anchor residues, i.e. amino acid side-chains deeply buried at protein-protein interfaces, to indicate possible druggable pockets to be targeted by small molecules. For a given protein-protein complex submitted by the user, ANCHOR calculates the change in solvent accessible surface area (DeltaSASA) upon binding for each side-chain, along with an estimate of its contribution to the binding free energy. A Jmol-based tool allows the user to interactively visualize selected anchor residues in their pockets as well as the stereochemical properties of the surrounding region such as hydrogen bonding. ANCHOR includes a Protein Data Bank (PDB) wide database of pre-computed anchor residues from more than 30,000 PDB entries with at least two protein chains. The user can query according to amino acids, buried area (SASA), energy or keywords related to indication areas, e.g. oncogene or diabetes. This database provides a resource to rapidly assess protein-protein interactions for the suitability of small molecules or fragments with bioisostere anchor analogues as possible compounds for pharmaceutical intervention. ANCHOR web server and database are freely available at http://structure.pitt.edu/anchor.

  9. Protein interaction patterns in different cellular environments are revealed by in-cell NMR

    PubMed Central

    Barbieri, Letizia; Luchinat, Enrico; Banci, Lucia

    2015-01-01

    In-cell NMR allows obtaining atomic-level information on biological macromolecules in their physiological environment. Soluble proteins may interact with the cellular environment in different ways: either specifically, with their functional partners, or non-specifically, with other cellular components. Such behaviour often causes the disappearance of the NMR signals. Here we show that by introducing mutations on the human protein profilin 1, used here as a test case, the in-cell NMR signals can be recovered. In human cells both specific and non-specific interactions are present, while in bacterial cells only the effect of non-specific interactions is observed. By comparing the NMR signal recovery pattern in human and bacterial cells, the relative contribution of each type of interaction can be assessed. This strategy allows detecting solution in-cell NMR spectra of soluble proteins without altering their fold, thus extending the applicability of in-cell NMR to a wider range of proteins. PMID:26399546

  10. Cellular transformation by the HTLV-I Tax protein, a jack-of-all-trades.

    PubMed

    Gatza, Michael L; Watt, Julie C; Marriott, Susan J

    2003-08-11

    The human T-cell leukemia virus type I (HTLV-I) is an oncogenic retrovirus that is responsible for adult T-cell leukemia and a neurological disease, HTLV-I-associated myelopathy/tropical spastic paraparesis. HTLV-I encodes an oncogenic protein, Tax, which affects a variety of cellular functions prompting it to be referred to as a jack-of-all trades. The ability of Tax to both transcriptionally regulate cellular gene expression and to functionally inactivate proteins involved in cell-cycle progression and DNA repair provide the basis for Tax-mediated transformation and leukemogenesis. This review will concentrate on the effects of Tax on the dysregulation of the G(1)/S and G(2)/M checkpoints as well as the suppression of DNA damage repair leading to cellular transformation. PMID:12910251

  11. Metabolic and cellular profile of wether goats: protein fractions and lactate dehydrogenase isoenzymes- reference values.

    PubMed

    Blackwell, J G; Libby, D W

    1982-06-01

    Blood serum concentrations of electrolytes; mineral, serum protein, and biochemical components; serum lactate dehydrogenase (LDH) activity; and blood cellular components were determined in 8 wether goats over a 59-day feeding period. The blood was collected on 6 sampling days, the blood cellular components were analyzed on collection days. The biochemical components were analyzed from frozen samples approximately 3 weeks after collection. The serum protein fractions and LDH isoenzymes were separated electrophoretically from frozen samples. The purpose was to determine the variability and changes in these values, using sample-day intervals over the feeding period. The blood serum concentrations of many of the biochemical and blood cellular components that were measured or calculated varied considerably, showing fluctuations over some portion of the period and ending with an erratic peak at period's end. The WBC count, neutrophil, and band neutrophil values were sharply decreased on sampling days 24 and 35, followed by fluctuations over the last half of the period.

  12. Hepatitis C virus NS2 protein activates cellular cyclic AMP-dependent pathways

    SciTech Connect

    Kim, Kyoung Mi; Kwon, Shi-Nae; Kang, Ju-Il; Lee, Song Hee; Jang, Sung Key; Ahn, Byung-Yoon; Kim, Yoon Ki . E-mail: yk-kim@korea.ac.kr

    2007-05-18

    Chronic infection of the hepatitis C virus (HCV) leads to liver cirrhosis and cancer. The mechanism leading to viral persistence and hepatocellular carcinoma, however, has not been fully understood. In this study, we show that the HCV infection activates cellular cAMP-dependent pathways. Expression of a luciferase reporter gene controlled by a basic promoter with the cAMP response element (CRE) was significantly elevated in human hepatoma Huh-7 cells infected with the HCV JFH1. Analysis with viral subgenomic replicons indicated that the HCV NS2 protein is responsible for the effect. Furthermore, the level of cellular transcripts whose stability is known to be regulated by cAMP was specifically reduced in cells harboring NS2-expressing replicons. These results allude to the HCV NS2 protein having a novel function of regulating cellular gene expression and proliferation through the cAMP-dependent pathway.

  13. The InterPro protein families database: the classification resource after 15 years.

    PubMed

    Mitchell, Alex; Chang, Hsin-Yu; Daugherty, Louise; Fraser, Matthew; Hunter, Sarah; Lopez, Rodrigo; McAnulla, Craig; McMenamin, Conor; Nuka, Gift; Pesseat, Sebastien; Sangrador-Vegas, Amaia; Scheremetjew, Maxim; Rato, Claudia; Yong, Siew-Yit; Bateman, Alex; Punta, Marco; Attwood, Teresa K; Sigrist, Christian J A; Redaschi, Nicole; Rivoire, Catherine; Xenarios, Ioannis; Kahn, Daniel; Guyot, Dominique; Bork, Peer; Letunic, Ivica; Gough, Julian; Oates, Matt; Haft, Daniel; Huang, Hongzhan; Natale, Darren A; Wu, Cathy H; Orengo, Christine; Sillitoe, Ian; Mi, Huaiyu; Thomas, Paul D; Finn, Robert D

    2015-01-01

    The InterPro database (http://www.ebi.ac.uk/interpro/) is a freely available resource that can be used to classify sequences into protein families and to predict the presence of important domains and sites. Central to the InterPro database are predictive models, known as signatures, from a range of different protein family databases that have different biological focuses and use different methodological approaches to classify protein families and domains. InterPro integrates these signatures, capitalizing on the respective strengths of the individual databases, to produce a powerful protein classification resource. Here, we report on the status of InterPro as it enters its 15th year of operation, and give an overview of new developments with the database and its associated Web interfaces and software. In particular, the new domain architecture search tool is described and the process of mapping of Gene Ontology terms to InterPro is outlined. We also discuss the challenges faced by the resource given the explosive growth in sequence data in recent years. InterPro (version 48.0) contains 36,766 member database signatures integrated into 26,238 InterPro entries, an increase of over 3993 entries (5081 signatures), since 2012.

  14. MSV3d: database of human MisSense Variants mapped to 3D protein structure.

    PubMed

    Luu, Tien-Dao; Rusu, Alin-Mihai; Walter, Vincent; Ripp, Raymond; Moulinier, Luc; Muller, Jean; Toursel, Thierry; Thompson, Julie D; Poch, Olivier; Nguyen, Hoan

    2012-01-01

    The elucidation of the complex relationships linking genotypic and phenotypic variations to protein structure is a major challenge in the post-genomic era. We present MSV3d (Database of human MisSense Variants mapped to 3D protein structure), a new database that contains detailed annotation of missense variants of all human proteins (20 199 proteins). The multi-level characterization includes details of the physico-chemical changes induced by amino acid modification, as well as information related to the conservation of the mutated residue and its position relative to functional features in the available or predicted 3D model. Major releases of the database are automatically generated and updated regularly in line with the dbSNP (database of Single Nucleotide Polymorphism) and SwissVar releases, by exploiting the extensive Décrypthon computational grid resources. The database (http://decrypthon.igbmc.fr/msv3d) is easily accessible through a simple web interface coupled to a powerful query engine and a standard web service. The content is completely or partially downloadable in XML or flat file formats. Database URL: http://decrypthon.igbmc.fr/msv3d.

  15. Myofibroblast Differentiation Modulates Keratocyte Crystallin Protein Expression, Concentration, and Cellular Light Scattering

    PubMed Central

    Brown, Donald; Pappa, Aglaia; Vasiliou, Vasilis

    2012-01-01

    Purpose. The purpose of this study was to determine whether myofibroblast differentiation altered keratocyte crystallin protein concentration and increased cellular light scattering. Methods. Serum-free cultured rabbit corneal keratocytes and TGFβ (5 ng/mL) induced myofibroblasts were harvested and counted and protein/RNA extracted. Expression of myofibroblast and keratocyte markers was determined by real-time PCR and Western blot analysis. The cell volume of calcein AM–loaded keratocytes and myofibroblasts was determined by using nonlinear optical microscopy. Cellular light scattering of transformed myofibroblasts expressing human keratocyte crystallins was measured by reflectance confocal microscopy. Results. Differentiated myofibroblasts showed a significant decrease in RNA levels for the keratocyte markers ALDH1A1, lumican, and keratocan and a significant increase in the myofibroblast marker α-smooth muscle actin. Volumetric and protein measurements showed that myofibroblast differentiation significantly increased cytoplasmic volume (293%; P < 0.001) and water-soluble and -insoluble protein content per cell (respectively, 442% and 431%; P < 0.002) compared to keratocytes. Western blot analysis showed that the level of ALDH1A1 protein per cell was similar between myofibroblasts and keratocytes, but was substantially reduced as a percentage of total water-soluble protein. Light scattering measurements showed that induced expression of corneal crystallins significantly decreased light scattering. Conclusions. These data suggest that myofibroblast differentiation leads to a marked increase in cell volume and dilution of corneal crystallins associated with an increase in cellular light scattering. PMID:22247459

  16. Slight temperature changes affect protein affinity and cellular uptake/toxicity of nanoparticles

    NASA Astrophysics Data System (ADS)

    Mahmoudi, Morteza; Shokrgozar, Mohammad A.; Behzadi, Shahed

    2013-03-01

    It is known that what the cell actually ``sees'' at the nanoscale is an outer shell formed of `protein corona' on the surface of nanoparticles (NPs). The amount and composition of various proteins on the corona are strongly dependent on the biophysicochemical properties of NPs, which have been extensively studied. However, the effect of a small variation in temperature, due to the human circadian rhythm, on the composition of the protein corona and the affinity of various proteins to the surface of NPs, was ignored. Here, the effect of temperature on the composition of protein corona and the affinity of various proteins to the surface of NPs and, subsequently, cell responses to the protein coated NPs are probed. The results confirmed that cellular entrance, dispersion, and toxicity of NPs are strongly diverse with slight body temperature changes. This new finding can help scientists to maximise NP entrance to specific cells/organs with lower toxicity by adjusting the cellular/organ temperature.It is known that what the cell actually ``sees'' at the nanoscale is an outer shell formed of `protein corona' on the surface of nanoparticles (NPs). The amount and composition of various proteins on the corona are strongly dependent on the biophysicochemical properties of NPs, which have been extensively studied. However, the effect of a small variation in temperature, due to the human circadian rhythm, on the composition of the protein corona and the affinity of various proteins to the surface of NPs, was ignored. Here, the effect of temperature on the composition of protein corona and the affinity of various proteins to the surface of NPs and, subsequently, cell responses to the protein coated NPs are probed. The results confirmed that cellular entrance, dispersion, and toxicity of NPs are strongly diverse with slight body temperature changes. This new finding can help scientists to maximise NP entrance to specific cells/organs with lower toxicity by adjusting the cellular

  17. Polyglutamine protein aggregation and toxicity are linked to the cellular stress response.

    PubMed

    Cowan, K J; Diamond, M I; Welch, W J

    2003-06-15

    Chronic exposure of cells to expanded polyglutamine proteins results in eventual cell demise. We constructed mouse cell lines expressing either the full-length androgen receptor (AR), or truncated forms of AR containing 25 or 65 glutamines to study the cellular consequences of chronic low-level exposure to these proteins. Expression of the polyglutamine-expanded truncated AR protein, but not the full-length expanded protein, resulted in the formation of cytoplasmic and nuclear aggregates and eventual cell death. Nuclear aggregates preferentially stained positive for heat shock protein (hsp)72, a sensitive indicator of a cellular stress response. Biochemical studies revealed that the presence of nuclear aggregates correlated with activation of the c-jun NH2-terminal kinase (JNK). Different metabolic insults, including heat shock treatment, and exposure to sodium arsenite or menadione, proved more toxic to those cells expressing the polyglutamine-expanded truncated protein than to cells expressing the non-expanded form. Cells containing cytoplasmic polyglutamine-protein aggregates exhibited a delayed expression of hsp72 after heat shock. Once expressed, hsp72 failed to localize normally and instead was sequestered within the protein aggregates. This was accompanied by an inability of the aggregate-containing cells to cease their stress response as evidenced by the continued presence of activated JNK. Finally, activation of the cellular stress response increased the overall extent of polyglutamine protein aggregation, especially within the nucleus. Inclusion of a JNK inhibitor reduced this stress-dependent increase in nuclear aggregates. Abnormal stress responses may contribute to enhanced cell vulnerability in cells expressing polyglutamine-expanded proteins and may increase the propensity of such cells to form cytoplasmic and nuclear inclusions. PMID:12783846

  18. Determination of de novo synthesized amino acids in cellular proteins revisited by 13C NMR spectroscopy.

    PubMed

    Flögel, U; Willker, W; Leibfritz, D

    1997-04-01

    13C nuclear magnetic resonance spectroscopy was used to determine the absolute amounts to de novo synthesized amino acids in both the perchloric acid extracts and the hydrolyzed protein fractions of F98 glioma cells incubated for 2 h with 5 mmol/l [U-13C]glucose. 13C NMR spectra of the hydrolyzed protein fraction revealed a marked incorporation of 13C-labelled alanine, aspartate and glutamate into the proteins of F98 cells within the incubation period. Additionally, small amounts of 13C-labelled glycine, proline and serine could unambiguously be identified in the protein fraction. Astonishingly, approximately equal amounts of 13C-labelled glutamate and aspartate were incorporated into the cellular proteins, although the cytosolic steady-state concentration of aspartate was below 13C NMR detectability. Hypertonic stress decreased the incorporation of 13C-labelled amino acids into the total protein, albeit their cytosolic concentrations were increased, which reflects an inhibition of protein synthesis under these conditions. On the other hand, hypotonic stress increased the amount of 13C-labelled proline incorporated into the cellular proteins even though the cytosolic concentration of 13C-labelled proline was largely decreased. Apparently, hypoosmotic conditions stimulate the synthesis of proteins or peptides with a high proline content. The results show that already after 2 h of incubation with [U-13C]glucose there is a pronounced flux of 13C label into the cellular proteins, which is usually disregarded if cytosolic fluids are examined only. This means that calculations of metabolic fluxes based on 13C NMR spectroscopic data obtained from perchloric acid extracts of cells or tissues and also from in vivo measurements consider only the labelled 'NMR visible' cytosolic metabolites, which may have to be corrected for fast label flowing off into other compartments.

  19. Using homology relations within a database markedly boosts protein sequence similarity search.

    PubMed

    Tong, Jing; Sadreyev, Ruslan I; Pei, Jimin; Kinch, Lisa N; Grishin, Nick V

    2015-06-01

    Inference of homology from protein sequences provides an essential tool for analyzing protein structure, function, and evolution. Current sequence-based homology search methods are still unable to detect many similarities evident from protein spatial structures. In computer science a search engine can be improved by considering networks of known relationships within the search database. Here, we apply this idea to protein-sequence-based homology search and show that it dramatically enhances the search accuracy. Our new method, COMPADRE (COmparison of Multiple Protein sequence Alignments using Database RElationships) assesses the relationship between the query sequence and a hit in the database by considering the similarity between the query and hit's known homologs. This approach increases detection quality, boosting the precision rate from 18% to 83% at half-coverage of all database homologs. The increased precision rate allows detection of a large fraction of protein structural relationships, thus providing structure and function predictions for previously uncharacterized proteins. Our results suggest that this general approach is applicable to a wide variety of methods for detection of biological similarities. The web server is available at prodata.swmed.edu/compadre.

  20. Using homology relations within a database markedly boosts protein sequence similarity search

    PubMed Central

    Tong, Jing; Sadreyev, Ruslan I.; Pei, Jimin; Kinch, Lisa N.; Grishin, Nick V.

    2015-01-01

    Inference of homology from protein sequences provides an essential tool for analyzing protein structure, function, and evolution. Current sequence-based homology search methods are still unable to detect many similarities evident from protein spatial structures. In computer science a search engine can be improved by considering networks of known relationships within the search database. Here, we apply this idea to protein-sequence–based homology search and show that it dramatically enhances the search accuracy. Our new method, COMPADRE (COmparison of Multiple Protein sequence Alignments using Database RElationships) assesses the relationship between the query sequence and a hit in the database by considering the similarity between the query and hit’s known homologs. This approach increases detection quality, boosting the precision rate from 18% to 83% at half-coverage of all database homologs. The increased precision rate allows detection of a large fraction of protein structural relationships, thus providing structure and function predictions for previously uncharacterized proteins. Our results suggest that this general approach is applicable to a wide variety of methods for detection of biological similarities. The web server is available at prodata.swmed.edu/compadre. PMID:26038555

  1. Using homology relations within a database markedly boosts protein sequence similarity search.

    PubMed

    Tong, Jing; Sadreyev, Ruslan I; Pei, Jimin; Kinch, Lisa N; Grishin, Nick V

    2015-06-01

    Inference of homology from protein sequences provides an essential tool for analyzing protein structure, function, and evolution. Current sequence-based homology search methods are still unable to detect many similarities evident from protein spatial structures. In computer science a search engine can be improved by considering networks of known relationships within the search database. Here, we apply this idea to protein-sequence-based homology search and show that it dramatically enhances the search accuracy. Our new method, COMPADRE (COmparison of Multiple Protein sequence Alignments using Database RElationships) assesses the relationship between the query sequence and a hit in the database by considering the similarity between the query and hit's known homologs. This approach increases detection quality, boosting the precision rate from 18% to 83% at half-coverage of all database homologs. The increased precision rate allows detection of a large fraction of protein structural relationships, thus providing structure and function predictions for previously uncharacterized proteins. Our results suggest that this general approach is applicable to a wide variety of methods for detection of biological similarities. The web server is available at prodata.swmed.edu/compadre. PMID:26038555

  2. MVsCarta: A protein database of matrix vesicles to aid understanding of biomineralization.

    PubMed

    Cui, Yazhou; Xu, Quan; Luan, Jing; Hu, Shichang; Pan, Jianbo; Han, Jinxiang; Ji, Zhiliang

    2015-06-01

    Matrix vesicles (MVs) are membranous nanovesicles released by chondrocytes, osteoblasts, and odontoblasts. They play a critical role in modulating mineralization. Here, we present a manually curated database of MV proteins, namely MVsCara to provide comprehensive information on MVs of protein components. In the current version, the database contains 2,713 proteins of six organisms identified in bone, cartilage, tooth tissues, and cells capable of producing a mineralized bone matrix. The MVsCarta database is now freely assessed at http://bioinf.xmu.edu.cn/MVsCarta. The search and browse methods were developed for better retrieval of data. In addition, bioinformatic tools like Gene Ontology (GO) analysis, network visualization and protein-protein interaction analysis were implemented for a functional understanding of MVs components. Similar database hasn't been reported yet. We believe that this free web-based database might serve as a useful repository to elucidate the novel function and regulation of MVs during mineralization, and to stimulate the advancement of MV studies. PMID:26166372

  3. The Biological Macromolecule Crystallization Database and NASA Protein Crystal Growth Archive.

    PubMed

    Gilliland, G L; Tung, M; Ladner, J

    1996-01-01

    The NIST/NASA/CARB Biological Macromolecule Crystallization Database (BMCD), NIST Standard Reference Database 21, contains crystal data and crystallization conditions for biological macromolecules. The database entries include data abstracted from published crystallographic reports. Each entry consists of information describing the biological macromolecule crystallized and crystal data and the crystallization conditions for each crystal form. The BMCD serves as the NASA Protein Crystal Growth Archive in that it contains protocols and results of crystallization experiments undertaken in microgravity (space). These database entries report the results, whether successful or not, from NASA-sponsored protein crystal growth experiments in microgravity and from microgravity crystallization studies sponsored by other international organizations. The BMCD was designed as a tool to assist x-ray crystallographers in the development of protocols to crystallize biological macromolecules, those that have previously been crystallized, and those that have not been crystallized.

  4. CancerResource: a comprehensive database of cancer-relevant proteins and compound interactions supported by experimental knowledge.

    PubMed

    Ahmed, Jessica; Meinel, Thomas; Dunkel, Mathias; Murgueitio, Manuela S; Adams, Robert; Blasse, Corinna; Eckert, Andreas; Preissner, Saskia; Preissner, Robert

    2011-01-01

    During the development of methods for cancer diagnosis and treatment, a vast amount of information is generated. Novel cancer target proteins have been identified and many compounds that activate or inhibit cancer-relevant target genes have been developed. This knowledge is based on an immense number of experimentally validated compound-target interactions in the literature, and excerpts from literature text mining are spread over numerous data sources. Our own analysis shows that the overlap between important existing repositories such as Comparative Toxicogenomics Database (CTD), Therapeutic Target Database (TTD), Pharmacogenomics Knowledge Base (PharmGKB) and DrugBank as well as between our own literature mining for cancer-annotated entries is surprisingly small. In order to provide an easy overview of interaction data, it is essential to integrate this information into a single, comprehensive data repository. Here, we present CancerResource, a database that integrates cancer-relevant relationships of compounds and targets from (i) our own literature mining and (ii) external resources complemented with (iii) essential experimental and supporting information on genes and cellular effects. In order to facilitate an overview of existing and supporting information, a series of novel information connections have been established. CancerResource addresses the spectrum of research on compound-target interactions in natural sciences as well as in individualized medicine; CancerResource is available at: http://bioinformatics.charite.de/cancerresource/.

  5. Protein turnover and cellular autophagy in growing and growth-inhibited 3T3 cells

    SciTech Connect

    Papadopoulos, T.; Pfeifer, U. )

    1987-07-01

    The relationship between growth, protein degradation, and cellular autophagy was tested in growing and in growth-inhibited 3T3 cell monolayers. For the biochemical evaluation of DNA and protein metabolism, growth-inhibited 3T3 cell monolayers with high cell density and growing 3T3 cell monolayers with low cell density were labeled simultaneously with ({sup 14}C)thymidine and ({sup 3}H)leucine. The evaluation of the DNA turnover and additional ({sup 3}H)thymidine autoradiography showed that 24 to 5% of 3T3 cells continue to replicate even in the growth-inhibited state, where no accumulation of protein and DNA can be observed. Cell loss, therefore, has to be assumed to compensate for the ongoing cell proliferation. When the data of protein turnover were corrected for cell loss, it was found that the rate constant of protein synthesis in nongrowing monolayers was reduced to half the value found in growing monolayers. Simultaneously, the rate constant of protein degradation in nongrowing monolayers was increased to about 1.5-fold the value of growing monolayers. These data are in agreement with the assumption that cellular autophagy represents a major pathway of regulating protein degradation in 3T3 cells and that the regulation of autophagic protein degradation is of relevance for the transition from a growing to a nongrowing state.

  6. Active Degradation Explains the Distribution of Nuclear Proteins during Cellular Senescence

    PubMed Central

    Giampieri, Enrico; De Cecco, Marco; Remondini, Daniel; Sedivy, John; Castellani, Gastone

    2015-01-01

    The amount of cellular proteins is a crucial parameter that is known to vary between cells as a function of the replicative passages, and can be important during physiological aging. The process of protein degradation is known to be performed by a series of enzymatic reactions, ranging from an initial step of protein ubiquitination to their final fragmentation by the proteasome. In this paper we propose a stochastic dynamical model of nuclear proteins concentration resulting from a balance between a constant production of proteins and their degradation by a cooperative enzymatic reaction. The predictions of this model are compared with experimental data obtained by fluorescence measurements of the amount of nuclear proteins in murine tail fibroblast (MTF) undergoing cellular senescence. Our model provides a three-parameter stationary distribution that is in good agreement with the experimental data even during the transition to the senescent state, where the nuclear protein concentration changes abruptly. The estimation of three parameters (cooperativity, saturation threshold, and maximal velocity of the reaction), and their evolution during replicative passages shows that only the maximal velocity varies significantly. Based on our modeling we speculate the reduction of functionality of the protein degradation mechanism as a possible competitive inhibition of the proteasome. PMID:26115222

  7. Cellular Noise Suppression by the Regulator of G Protein Signaling Sst2

    PubMed Central

    Dixit, Gauri; Kelley, Joshua B.; Houser, John R.; Elston, Timothy C.; Dohlman, Henrik G.

    2014-01-01

    Summary G proteins and their associated receptors process information from a variety of environmental stimuli to induce appropriate cellular responses. Generally speaking, each cell in a population responds within defined limits despite large variation in the expression of protein signaling components. Therefore we postulated that noise suppression is encoded within the signaling system. Using the yeast mating pathway as a model we evaluated the ability of a regulator of G protein signaling (RGS) protein to suppress noise. We found that the RGS protein Sst2 limits variability in transcription and morphogenesis in response to pheromone stimulation. While signal suppression is a result of both the GAP (GTPase accelerating) and receptor binding functions of Sst2, noise suppression requires only the GAP activity. Taken together our findings reveal a hitherto overlooked role of RGS proteins as noise suppressors, and demonstrate an ability to uncouple signal and noise in a prototypical stimulus-response pathway. PMID:24954905

  8. Cellular growth defects triggered by an overload of protein localization processes

    PubMed Central

    Kintaka, Reiko; Makanae, Koji; Moriya, Hisao

    2016-01-01

    High-level expression of a protein localized to an intracellular compartment is expected to cause cellular defects because it overloads localization processes. However, overloads of localization processes have never been studied systematically. Here, we show that the expression levels of green fluorescent proteins (GFPs) with localization signals were limited to the same degree as a toxic misfolded GFP in budding yeast cells, and that their high-level expression caused cellular defects associated with localization processes. We further show that limitation of the exportin Crm1 determined the expression limit of GFP with a nuclear export signal. Although misfolding of GFP with a vesicle-mediated transport signal triggered endoplasmic reticulum stress, it was not the primary determinant of its expression limit. The precursor of GFP with a mitochondrial targeting signal caused a cellular defect. Finally, we estimated the residual capacities of localization processes. High-level expression of a localized protein thus causes cellular defects by overloading the capacities of localization processes. PMID:27538565

  9. Cellular growth defects triggered by an overload of protein localization processes.

    PubMed

    Kintaka, Reiko; Makanae, Koji; Moriya, Hisao

    2016-01-01

    High-level expression of a protein localized to an intracellular compartment is expected to cause cellular defects because it overloads localization processes. However, overloads of localization processes have never been studied systematically. Here, we show that the expression levels of green fluorescent proteins (GFPs) with localization signals were limited to the same degree as a toxic misfolded GFP in budding yeast cells, and that their high-level expression caused cellular defects associated with localization processes. We further show that limitation of the exportin Crm1 determined the expression limit of GFP with a nuclear export signal. Although misfolding of GFP with a vesicle-mediated transport signal triggered endoplasmic reticulum stress, it was not the primary determinant of its expression limit. The precursor of GFP with a mitochondrial targeting signal caused a cellular defect. Finally, we estimated the residual capacities of localization processes. High-level expression of a localized protein thus causes cellular defects by overloading the capacities of localization processes. PMID:27538565

  10. Nucleotide binding database NBDB – a collection of sequence motifs with specific protein-ligand interactions

    PubMed Central

    Zheng, Zejun; Goncearenco, Alexander; Berezovsky, Igor N.

    2016-01-01

    NBDB database describes protein motifs, elementary functional loops (EFLs) that are involved in binding of nucleotide-containing ligands and other biologically relevant cofactors/coenzymes, including ATP, AMP, ATP, GMP, GDP, GTP, CTP, PAP, PPS, FMN, FAD(H), NAD(H), NADP, cAMP, cGMP, c-di-AMP and c-di-GMP, ThPP, THD, F-420, ACO, CoA, PLP and SAM. The database is freely available online at http://nbdb.bii.a-star.edu.sg. In total, NBDB contains data on 249 motifs that work in interactions with 24 ligands. Sequence profiles of EFL motifs were derived de novo from nonredundant Uniprot proteome sequences. Conserved amino acid residues in the profiles interact specifically with distinct chemical parts of nucleotide-containing ligands, such as nitrogenous bases, phosphate groups, ribose, nicotinamide, and flavin moieties. Each EFL profile in the database is characterized by a pattern of corresponding ligand–protein interactions found in crystallized ligand–protein complexes. NBDB database helps to explore the determinants of nucleotide and cofactor binding in different protein folds and families. NBDB can also detect fragments that match to profiles of particular EFLs in the protein sequence provided by user. Comprehensive information on sequence, structures, and interactions of EFLs with ligands provides a foundation for experimental and computational efforts on design of required protein functions. PMID:26507856

  11. Nucleotide binding database NBDB--a collection of sequence motifs with specific protein-ligand interactions.

    PubMed

    Zheng, Zejun; Goncearenco, Alexander; Berezovsky, Igor N

    2016-01-01

    NBDB database describes protein motifs, elementary functional loops (EFLs) that are involved in binding of nucleotide-containing ligands and other biologically relevant cofactors/coenzymes, including ATP, AMP, ATP, GMP, GDP, GTP, CTP, PAP, PPS, FMN, FAD(H), NAD(H), NADP, cAMP, cGMP, c-di-AMP and c-di-GMP, ThPP, THD, F-420, ACO, CoA, PLP and SAM. The database is freely available online at http://nbdb.bii.a-star.edu.sg. In total, NBDB contains data on 249 motifs that work in interactions with 24 ligands. Sequence profiles of EFL motifs were derived de novo from nonredundant Uniprot proteome sequences. Conserved amino acid residues in the profiles interact specifically with distinct chemical parts of nucleotide-containing ligands, such as nitrogenous bases, phosphate groups, ribose, nicotinamide, and flavin moieties. Each EFL profile in the database is characterized by a pattern of corresponding ligand-protein interactions found in crystallized ligand-protein complexes. NBDB database helps to explore the determinants of nucleotide and cofactor binding in different protein folds and families. NBDB can also detect fragments that match to profiles of particular EFLs in the protein sequence provided by user. Comprehensive information on sequence, structures, and interactions of EFLs with ligands provides a foundation for experimental and computational efforts on design of required protein functions.

  12. Web and database software for identification of intact proteins using "top down" mass spectrometry.

    PubMed

    Taylor, Gregory K; Kim, Yong-Bin; Forbes, Andrew J; Meng, Fanyu; McCarthy, Ryan; Kelleher, Neil L

    2003-08-15

    For the identification and characterization of proteins harboring posttranslational modifications (PTMs), a "top down" strategy using mass spectrometry has been forwarded recently but languishes without tailored software widely available. We describe a Web-based software and database suite called ProSight PTM constructed for large-scale proteome projects involving direct fragmentation of intact protein ions. Four main components of ProSight PTM are a database retrieval algorithm (Retriever), MySQL protein databases, a file/data manager, and a project tracker. Retriever performs probability-based identifications from absolute fragment ion masses, automatically compiled sequence tags, or a combination of the two, with graphical rendering and browsing of the results. The database structure allows known and putative protein forms to be searched, with prior or predicted PTM knowledge used during each search. Initial functionality is illustrated with a 36-kDa yeast protein identified from a processed cell extract after automated data acquisition using a quadrupole-FT hybrid mass spectrometer. A +142-Da delta(m) on glyceraldehyde-3-phosphate dehydrogenase was automatically localized between Asp90 and Asp192, consistent with its two cystine residues (149 and 153) alkylated by acrylamide (+71 Da each) during the gel-based sample preparation. ProSight PTM is the first search engine and Web environment for identification of intact proteins (https://prosightptm.scs.uiuc.edu/). PMID:14632120

  13. Nucleotide binding database NBDB--a collection of sequence motifs with specific protein-ligand interactions.

    PubMed

    Zheng, Zejun; Goncearenco, Alexander; Berezovsky, Igor N

    2016-01-01

    NBDB database describes protein motifs, elementary functional loops (EFLs) that are involved in binding of nucleotide-containing ligands and other biologically relevant cofactors/coenzymes, including ATP, AMP, ATP, GMP, GDP, GTP, CTP, PAP, PPS, FMN, FAD(H), NAD(H), NADP, cAMP, cGMP, c-di-AMP and c-di-GMP, ThPP, THD, F-420, ACO, CoA, PLP and SAM. The database is freely available online at http://nbdb.bii.a-star.edu.sg. In total, NBDB contains data on 249 motifs that work in interactions with 24 ligands. Sequence profiles of EFL motifs were derived de novo from nonredundant Uniprot proteome sequences. Conserved amino acid residues in the profiles interact specifically with distinct chemical parts of nucleotide-containing ligands, such as nitrogenous bases, phosphate groups, ribose, nicotinamide, and flavin moieties. Each EFL profile in the database is characterized by a pattern of corresponding ligand-protein interactions found in crystallized ligand-protein complexes. NBDB database helps to explore the determinants of nucleotide and cofactor binding in different protein folds and families. NBDB can also detect fragments that match to profiles of particular EFLs in the protein sequence provided by user. Comprehensive information on sequence, structures, and interactions of EFLs with ligands provides a foundation for experimental and computational efforts on design of required protein functions. PMID:26507856

  14. Web and database software for identification of intact proteins using "top down" mass spectrometry.

    PubMed

    Taylor, Gregory K; Kim, Yong-Bin; Forbes, Andrew J; Meng, Fanyu; McCarthy, Ryan; Kelleher, Neil L

    2003-08-15

    For the identification and characterization of proteins harboring posttranslational modifications (PTMs), a "top down" strategy using mass spectrometry has been forwarded recently but languishes without tailored software widely available. We describe a Web-based software and database suite called ProSight PTM constructed for large-scale proteome projects involving direct fragmentation of intact protein ions. Four main components of ProSight PTM are a database retrieval algorithm (Retriever), MySQL protein databases, a file/data manager, and a project tracker. Retriever performs probability-based identifications from absolute fragment ion masses, automatically compiled sequence tags, or a combination of the two, with graphical rendering and browsing of the results. The database structure allows known and putative protein forms to be searched, with prior or predicted PTM knowledge used during each search. Initial functionality is illustrated with a 36-kDa yeast protein identified from a processed cell extract after automated data acquisition using a quadrupole-FT hybrid mass spectrometer. A +142-Da delta(m) on glyceraldehyde-3-phosphate dehydrogenase was automatically localized between Asp90 and Asp192, consistent with its two cystine residues (149 and 153) alkylated by acrylamide (+71 Da each) during the gel-based sample preparation. ProSight PTM is the first search engine and Web environment for identification of intact proteins (https://prosightptm.scs.uiuc.edu/).

  15. Genomes to Life''Center for Molecular and Cellular Systems'': A research program for identification and characterization of protein complexes.

    SciTech Connect

    Buchanan, M V.; Larimer, Frank; Wiley, H S.; Kennel, S J.; Squier, Thomas C.; Ramsey, John M.; Rodland, Karin D.; Hurst, G B.; Smith, Richard D.; Xu, Ying; Dixon, David A.; Doktycz, M J.; Colson, Steve D.; Gesteland, R; Giometti, Carol S.; Young, Mark E.; Giddings, Ralph M.

    2002-02-01

    Goal 1 of Department of Energy's Genomes to Life (GTL) program seeks to identify and characterize the complete set of protein complexes within a cell. Goal 1 forms the foundation necessary to accomplish the other objectives of the GTL program, which focus on gene regulatory networks and molecular level characterization of interactions in microbial communities. Together this information would allow cells and their components to be understood in sufficient detail to predict, test, and understand the responses of a biological system to its environment. The Center for Molecular and Cellular Systems has been established to identify and characterize protein complexes using high through-put analytical technologies. A dynamic research program is being developed that supports the goals of the Center by focusing on the development of new capabilities for sample preparation and complex separations, molecular level identification of the protein complexes by mass spectrometry, characterization of the complexes in living cells by imaging techniques, and bioinformatics and computational tools for the collection and interpretation of data and formation of databases and tools to allow the data to be shared by the biological community.

  16. Searching for cellular partners of hantaviral nonstructural protein NSs: Y2H screening of mouse cDNA library and analysis of cellular interactome.

    PubMed

    Rönnberg, Tuomas; Jääskeläinen, Kirsi; Blot, Guillaume; Parviainen, Ville; Vaheri, Antti; Renkonen, Risto; Bouloy, Michele; Plyusnin, Alexander

    2012-01-01

    Hantaviruses (Bunyaviridae) are negative-strand RNA viruses with a tripartite genome. The small (S) segment encodes the nucleocapsid protein and, in some hantaviruses, also the nonstructural protein (NSs). The aim of this study was to find potential cellular partners for the hantaviral NSs protein. Toward this aim, yeast two-hybrid (Y2H) screening of mouse cDNA library was performed followed by a search for potential NSs protein counterparts via analyzing a cellular interactome. The resulting interaction network was shown to form logical, clustered structures. Furthermore, several potential binding partners for the NSs protein, for instance ACBD3, were identified and, to prove the principle, interaction between NSs and ACBD3 proteins was demonstrated biochemically.

  17. Searching for Cellular Partners of Hantaviral Nonstructural Protein NSs: Y2H Screening of Mouse cDNA Library and Analysis of Cellular Interactome

    PubMed Central

    Parviainen, Ville; Vaheri, Antti; Renkonen, Risto; Bouloy, Michele; Plyusnin, Alexander

    2012-01-01

    Hantaviruses (Bunyaviridae) are negative-strand RNA viruses with a tripartite genome. The small (S) segment encodes the nucleocapsid protein and, in some hantaviruses, also the nonstructural protein (NSs). The aim of this study was to find potential cellular partners for the hantaviral NSs protein. Toward this aim, yeast two-hybrid (Y2H) screening of mouse cDNA library was performed followed by a search for potential NSs protein counterparts via analyzing a cellular interactome. The resulting interaction network was shown to form logical, clustered structures. Furthermore, several potential binding partners for the NSs protein, for instance ACBD3, were identified and, to prove the principle, interaction between NSs and ACBD3 proteins was demonstrated biochemically. PMID:22506017

  18. Actin-binding proteins: the long road to understanding the dynamic landscape of cellular actin networks.

    PubMed

    Lappalainen, Pekka

    2016-08-15

    The actin cytoskeleton supports a vast number of cellular processes in nonmuscle cells. It is well established that the organization and dynamics of the actin cytoskeleton are controlled by a large array of actin-binding proteins. However, it was only 40 years ago that the first nonmuscle actin-binding protein, filamin, was identified and characterized. Filamin was shown to bind and cross-link actin filaments into higher-order structures and contribute to phagocytosis in macrophages. Subsequently many other nonmuscle actin-binding proteins were identified and characterized. These proteins regulate almost all steps of the actin filament assembly and disassembly cycles, as well as the arrangement of actin filaments into diverse three-dimensional structures. Although the individual biochemical activities of most actin-regulatory proteins are relatively well understood, knowledge of how these proteins function together in a common cytoplasm to control actin dynamics and architecture is only beginning to emerge. Furthermore, understanding how signaling pathways and mechanical cues control the activities of various actin-binding proteins in different cellular, developmental, and pathological processes will keep researchers busy for decades. PMID:27528696

  19. Contaminant loading in remote Arctic lakes affects cellular stress-related proteins expression in feral charr.

    USGS Publications Warehouse

    Wiseman, Steve; Jorgensen, Even H.; Maule, Alec G.; Vijayan, Mathilakath M.

    2011-01-01

    The remote Arctic lakes on Bjornoya Island, Norway, offer a unique opportunity to study possible affect of lifelong contaminant exposure in wild populations of landlocked Arctic charr (Salvelinus alpinus). This is because Lake Ellasjoen has persistent organic pollutant (POP) levels that are significantly greater than in the nearby Lake Oyangen. We examined whether this differential contaminant loading was reflected in the expression of protein markers of exposure and effect in the native fish. We assessed the expressions of cellular stress markers, including cytochrome P4501A (Cyp1A), heat shock protein 70 (hsp70), and glucocorticoid receptor (GR) in feral charr from the two lakes. The average polychlorinated biphenyl (PCB) load in the charr liver from Ellasjoen was approximately 25-fold higher than in individuals from Oyangen. Liver Cyp1A protein expression was significantly higher in individuals from Ellasjoen compared with Oyangen, confirming differential PCB exposure. There was no significant difference in hsp70 protein expression in charr liver between the two lakes. However, brain hsp70 protein expression was significantly elevated in charr from Ellasjoen compared with Oyangen. Also, liver GR protein expression was significantly higher in the Ellasjoen charr compared with Oyangen charr. Taken together, our results suggest changes to cellular stress-related protein expression as a possible adaptation to chronic-contaminant exposure in feral charr in the Norwegian high-Arctic.

  20. Molecular design and nanoparticle-mediated intracellular delivery of functional proteins to target cellular pathways

    NASA Astrophysics Data System (ADS)

    Shah, Dhiral Ashwin

    Intracellular delivery of specific proteins and peptides represents a novel method to influence stem cells for gain-of-function and loss-of-function. Signaling control is vital in stem cells, wherein intricate control of and interplay among critical pathways directs the fate of these cells into either self-renewal or differentiation. The most common route to manipulate cellular function involves the introduction of genetic material such as full-length genes and shRNA into the cell to generate (or prevent formation of) the target protein, and thereby ultimately alter cell function. However, viral-mediated gene delivery may result in relatively slow expression of proteins and prevalence of oncogene insertion into the cell, which can alter cell function in an unpredictable fashion, and non-viral delivery may lead to low efficiency of genetic delivery. For example, the latter case plagues the generation of induced pluripotent stem cells (iPSCs) and hinders their use for in vivo applications. Alternatively, introducing proteins into cells that specifically recognize and influence target proteins, can result in immediate deactivation or activation of key signaling pathways within the cell. In this work, we demonstrate the cellular delivery of functional proteins attached to hydrophobically modified silica (SiNP) nanoparticles to manipulate specifically targeted cell signaling proteins. In the Wnt signaling pathway, we have targeted the phosphorylation activity of glycogen synthase kinase-3beta (GSK-3beta) by designing a chimeric protein and delivering it in neural stem cells. Confocal imaging indicates that the SiNP-chimeric protein conjugates were efficiently delivered to the cytosol of human embryonic kidney cells and rat neural stem cells, presumably via endocytosis. This uptake impacted the Wnt signaling cascade, indicated by the elevation of beta-catenin levels, and increased transcription of Wnt target genes, such as c-MYC. The results presented here suggest that

  1. Retinoblastoma-binding Protein 4-regulated Classical Nuclear Transport Is Involved in Cellular Senescence.

    PubMed

    Tsujii, Akira; Miyamoto, Yoichi; Moriyama, Tetsuji; Tsuchiya, Yuko; Obuse, Chikashi; Mizuguchi, Kenji; Oka, Masahiro; Yoneda, Yoshihiro

    2015-12-01

    Nucleocytoplasmic trafficking is a fundamental cellular process in eukaryotic cells. Here, we demonstrated that retinoblastoma-binding protein 4 (RBBP4) functions as a novel regulatory factor to increase the efficiency of importin α/β-mediated nuclear import. RBBP4 accelerates the release of importin β1 from importin α via competitive binding to the importin β-binding domain of importin α in the presence of RanGTP. Therefore, it facilitates importin α/β-mediated nuclear import. We showed that the importin α/β pathway is down-regulated in replicative senescent cells, concomitant with a decrease in RBBP4 level. Knockdown of RBBP4 caused both suppression of nuclear transport and induction of cellular senescence. This is the first report to identify a factor that competes with importin β1 to bind to importin α, and it demonstrates that the loss of this factor can trigger cellular senescence.

  2. An Arabidopsis WDR protein coordinates cellular networks involved in light, stress response and hormone signals.

    PubMed

    Chuang, Huey-Wen; Feng, Ji-Huan; Feng, Yung-Lin; Wei, Miam-Ju

    2015-12-01

    The WD-40 repeat (WDR) protein acts as a scaffold for protein interactions in various cellular events. An Arabidopsis WDR protein exhibited sequence similarity with human WDR26, a scaffolding protein implicated in H2O2-induced cell death in neural cells. The AtWDR26 transcript was induced by auxin, abscisic acid (ABA), ethylene (ET), osmostic stress and salinity. The expression of AtWDR26 was regulated by light, and seed germination of the AtWDR26 overexpression (OE) and seedling growth of the T-DNA knock-out (KO) exhibited altered sensitivity to light. Root growth of the OE seedlings increased tolerance to ZnSO4 and NaCl stresses and were hypersensitive to inhibition of osmotic stress. Seedlings of OE and KO altered sensitivities to multiple hormones. Transcriptome analysis of the transgenic plants overexpressing AtWDR26 showed that genes involved in the chloroplast-related metabolism constituted the largest group of the up-regulated genes. AtWDR26 overexpression up-regulated a large number of genes related to defense cellular events including biotic and abiotic stress response. Furthermore, several members of genes functioning in the regulation of Zn homeostasis, and hormone synthesis and perception of auxin and JA were strongly up-regulated in the transgenic plants. Our data provide physiological and transcriptional evidence for AtWDR26 role in hormone, light and abiotic stress cellular events.

  3. MobiDB 2.0: an improved database of intrinsically disordered and mobile proteins

    PubMed Central

    Potenza, Emilio; Domenico, Tomás Di; Walsh, Ian; Tosatto, Silvio C.E.

    2015-01-01

    MobiDB (http://mobidb.bio.unipd.it/) is a database of intrinsically disordered and mobile proteins. Intrinsically disordered regions are key for the function of numerous proteins. Here we provide a new version of MobiDB, a centralized source aimed at providing the most complete picture on different flavors of disorder in protein structures covering all UniProt sequences (currently over 80 million). The database features three levels of annotation: manually curated, indirect and predicted. Manually curated data is extracted from the DisProt database. Indirect data is inferred from PDB structures that are considered an indication of intrinsic disorder. The 10 predictors currently included (three ESpritz flavors, two IUPred flavors, two DisEMBL flavors, GlobPlot, VSL2b and JRONN) enable MobiDB to provide disorder annotations for every protein in absence of more reliable data. The new version also features a consensus annotation and classification for long disordered regions. In order to complement the disorder annotations, MobiDB features additional annotations from external sources. Annotations from the UniProt database include post-translational modifications and linear motifs. Pfam annotations are displayed in graphical form and are link-enabled, allowing the user to visit the corresponding Pfam page for further information. Experimental protein–protein interactions from STRING are also classified for disorder content. PMID:25361972

  4. Chapter Three - Ubiquitination and Protein Turnover of G-Protein-Coupled Receptor Kinases in GPCR Signaling and Cellular Regulation.

    PubMed

    Penela, P

    2016-01-01

    G-protein-coupled receptors (GPCRs) are responsible for regulating a wide variety of physiological processes, and distinct mechanisms for GPCR inactivation exist to guarantee correct receptor functionality. One of the widely used mechanisms is receptor phosphorylation by specific G-protein-coupled receptor kinases (GRKs), leading to uncoupling from G proteins (desensitization) and receptor internalization. GRKs and β-arrestins also participate in the assembly of receptor-associated multimolecular complexes, thus initiating alternative G-protein-independent signaling events. In addition, the abundant GRK2 kinase has diverse "effector" functions in cellular migration, proliferation, and metabolism homeostasis by means of the phosphorylation or interaction with non-GPCR partners. Altered expression of GRKs (particularly of GRK2 and GRK5) occurs during pathological conditions characterized by impaired GPCR signaling including inflammatory syndromes, cardiovascular disease, and tumor contexts. It is increasingly appreciated that different pathways governing GRK protein stability play a role in the modulation of kinase levels in normal and pathological conditions. Thus, enhanced GRK2 degradation by the proteasome pathway occurs upon GPCR stimulation, what allows cellular adaptation to chronic stimulation in a physiological setting. β-arrestins participate in this process by facilitating GRK2 phosphorylation by different kinases and by recruiting diverse E3 ubiquitin ligase to the receptor complex. Different proteolytic systems (ubiquitin-proteasome, calpains), chaperone activities and signaling pathways influence the stability of GRKs in different ways, thus endowing specificity to GPCR regulation as protein turnover of GRKs can be differentially affected. Therefore, modulation of protein stability of GRKs emerges as a versatile mechanism for feedback regulation of GPCR signaling and basic cellular processes. PMID:27378756

  5. Chapter Three - Ubiquitination and Protein Turnover of G-Protein-Coupled Receptor Kinases in GPCR Signaling and Cellular Regulation.

    PubMed

    Penela, P

    2016-01-01

    G-protein-coupled receptors (GPCRs) are responsible for regulating a wide variety of physiological processes, and distinct mechanisms for GPCR inactivation exist to guarantee correct receptor functionality. One of the widely used mechanisms is receptor phosphorylation by specific G-protein-coupled receptor kinases (GRKs), leading to uncoupling from G proteins (desensitization) and receptor internalization. GRKs and β-arrestins also participate in the assembly of receptor-associated multimolecular complexes, thus initiating alternative G-protein-independent signaling events. In addition, the abundant GRK2 kinase has diverse "effector" functions in cellular migration, proliferation, and metabolism homeostasis by means of the phosphorylation or interaction with non-GPCR partners. Altered expression of GRKs (particularly of GRK2 and GRK5) occurs during pathological conditions characterized by impaired GPCR signaling including inflammatory syndromes, cardiovascular disease, and tumor contexts. It is increasingly appreciated that different pathways governing GRK protein stability play a role in the modulation of kinase levels in normal and pathological conditions. Thus, enhanced GRK2 degradation by the proteasome pathway occurs upon GPCR stimulation, what allows cellular adaptation to chronic stimulation in a physiological setting. β-arrestins participate in this process by facilitating GRK2 phosphorylation by different kinases and by recruiting diverse E3 ubiquitin ligase to the receptor complex. Different proteolytic systems (ubiquitin-proteasome, calpains), chaperone activities and signaling pathways influence the stability of GRKs in different ways, thus endowing specificity to GPCR regulation as protein turnover of GRKs can be differentially affected. Therefore, modulation of protein stability of GRKs emerges as a versatile mechanism for feedback regulation of GPCR signaling and basic cellular processes.

  6. Efficient isolation and elution of cellular proteins using aptamer-mediated protein precipitation assay.

    PubMed

    Kim, Kiseok; Lee, SeungJin; Ryu, Sungho; Han, Dongil

    2014-05-23

    Protein precipitation is one of the most widely used methods for antigen detection and purification in biological research. We developed a reproducible aptamer-mediated magnetic protein precipitation method that is able to efficiently capture, purify and isolate the target proteins. We discovered DNA aptamers having individually high affinity and specificity against human epidermal growth factor receptor (EGFR) and human insulin receptor (INSR). Using aptamers and magnetic beads, we showed it is highly efficient technique to enrich endogenous proteins complex and is applicable to identify physiologically relevant protein-protein interactions with minimized nonspecific binding of proteins. The results presented here indicate that aptamers would be applicable as a useful and cost-effective tool to identify the presence of the particular target protein with their specific protein partners.

  7. Proteomic analysis of cellular protein expression profiles in response to grass carp reovirus infection.

    PubMed

    Xu, Dan; Song, Lang; Wang, Hao; Xu, Xiaoyan; Wang, Tu; Lu, Liqun

    2015-06-01

    Grass carp (Ctenopharyngodon idella) hemorrhagic disease, caused by grass carp reovirus (GCRV), is emerging as a serious problem in grass carp aquaculture. To better understand the molecular responses to GCRV infection, two-dimensional electrophoresis (2-DE) and matrix-assisted laser desorption/ionization tandem mass spectroscopy were performed to investigate altered proteins in C. idella kidney (CIK) cells. Differentially expressed proteins in mock infected CIK cells and GCRV-infected CIK cells were compared. Twenty-three differentially expressed spots were identified (22 upregulated spots and 1 downregulated spot), which included cytoskeleton proteins, macromolecular biosynthesis-associated proteins, stress response proteins, signal transduction proteins, energy metabolism-associated proteins and ubiquitin proteasome pathway-associated proteins. Moreover, 10 of the corresponding genes of the differentially expressed proteins were quantified by real-time reverse transcription polymerase chain reaction to examine their transcriptional profiles. The T cell internal antigen 1 (TIA1) and Ras-GTPase-activating SH3-domain-binding protein1 (G3BP1) of the cellular stress granule pathway from grass carp C. idella (designated as CiTIA1 and CiG3BP1) were upregulated and downregulated during GCRV infection, respectively. The full-length cDNA of CiTIA1 was 2753 bp, with an open reading frame (ORF) of 1155bp, which encodes a putative 385-amino acid protein. The 2271 bp full-length cDNA of CiG3BP1 comprised an ORF of 1455 bp that encodes a putative 485-amino acid protein. Phylogenetic analysis revealed that the complete ORFs of CiTIA1 and CiG3BP1 were very similar to zebrafish and well-characterized mammalian homologs. The expressions of the cellular proteins CiTIA1 and CiG3BP1 in response to GCRV were validated by western blotting, which indicated that the GCRV should unlink TIA1 aggregation and stress granule formation. This study provides useful information on the proteomic

  8. Papillomavirus E2 Proteins and the Host Brd4 Protein Associate with Transcriptionally Active Cellular Chromatin▿ †

    PubMed Central

    Jang, Moon Kyoo; Kwon, Deukwoo; McBride, Alison A.

    2009-01-01

    The interaction of papillomavirus E2 proteins with cellular Brd4 protein is important for transcriptional regulation of viral genes and partitioning of viral genomes. Bovine papillomavirus type 1 (BPV-1) E2 binds cellular chromatin in complex with Brd4 in both mitotic and interphase cells. To identify specific sites of E2 interaction on cellular chromatin, a genome-wide chromatin immunoprecipitation-on-chip analysis was carried out using human promoter sequences. Both E2 and Brd4 were found bound to most transcriptionally active promoters in C33A cells. These promoters were also bound by RNA polymerase II and were modified by histone H3 acetylation and K4 trimethylation, all indicators of active transcription. E2 binding strongly correlated with Brd4 and RNA polymerase II occupancy and H3K4me3 modification at all human promoters, indicating that E2 bound to active promoters. E2 binding did not correlate with the presence of consensus E2 binding sites in the promoters. Furthermore, the mRNA levels of E2-bound cellular genes were not significantly changed by E2 expression. Thus, the papillomavirus E2 proteins bind to transcriptionally active cellular genes but do not change their activity. We propose that this may be a way for the virus to ensure that the viral genome is retained in transcriptionally active regions of the nucleus to escape silencing. Therefore, E2-mediated tethering of viral genomes to host chromatin has multiple roles: to partition the viral genome to daughter cells, to ensure that the genomes are retained in the nucleus, and to make certain that the genomes are retained in functionally active nuclear domains. PMID:19129460

  9. Cellular Solid-State NMR Investigation of a Membrane Protein Using Dynamic Nuclear Polarization

    PubMed Central

    Yamamoto, Kazutoshi; Caporini, Marc A.; Im, Sang-Choul; Waskell, Lucy; Ramamoorthy, Ayyalusamy

    2014-01-01

    While an increasing number of structural biology studies successfully demonstrate the power of high-resolution structures and dynamics of membrane proteins in fully understanding their function, there is considerable interest in developing NMR approaches to obtain such information in a cellular setting. As long as the proteins inside the living cell tumble rapidly in the NMR timescale, recently developed in-cell solution NMR approaches can be applied towards the determination of 3D structural information. However, there are numerous challenges that need to be overcome to study membrane proteins inside a cell. Research in our laboratory is focused on developing a combination of solid-state NMR and biological approaches to overcome these challenges with a specific emphasis on obtaining high-resolution structural insights into electron transfer biological processes mediated by membrane-bound proteins like mammalian cytochrome b5, cytochrome P450 and cytochrome P450 reductase. In this study, we demonstrate the feasibility of using the signal-enhancement rendered by dynamic nuclear polarization (DNP) magic angle spinning (MAS) NMR spectroscopy for in-cell studies on a membrane-anchored protein. Our experimental results obtained from 13C-labeled membrane-anchored cytochrome b5 in native Escherichia coli cells show a ~16-fold DNP signal enhancement (ε). Further, results obtained from a 2D 13C/13C chemical shift correlation MAS experiment demonstrates that it is highly possible to suppress the background signals from other cellular contents for high-resolution structural studies on membrane proteins. We believe that this study would pave new avenues for high-resolution 3D structural studies on a variety of membrane-associated proteins and their complexes in the cellular context to fully understand their functional roles in physiological processes. PMID:25017802

  10. SCNProDB: A database for the identification of soybean cyst nematode proteins

    PubMed Central

    Natarajan, Savithiry; Tavakolan, Mona; Alkharouf, Nadim W; Matthews, Benjamin F

    2014-01-01

    Soybean cyst nematode (Heterodera glycines, SCN) is the most destructive pathogen of soybean around the world. Crop rotation and resistant cultivars are used to mitigate the damage of SCN, but these approaches are not completely successful because of the varied SCN populations. Thus, the limitations of these practices with soybean dictate investigation of other avenues of protection of soybean against SCN, perhaps through genetically engineering of broad resistance to SCN. For better understanding of the consequences of genetic manipulation, elucidation of SCN protein composition at the subunit level is necessary. We have conducted studies to determine the composition of SCN proteins using a proteomics approach in our laboratory using twodimensional polyacrylamide gel electrophoresis (2D-PAGE) to separate SCN proteins and to characterize the proteins further using mass spectrometry. Our analysis resulted in the identification of several hundred proteins. In this investigation, we developed a web based database (SCNProDB) containing protein information obtained from our previous published studies. This database will be useful to scientists who wish to develop SCN resistant soybean varieties through genetic manipulation and breeding efforts. The database is freely accessible from: http://bioinformatics.towson.edu/Soybean_SCN_proteins_2D_Gel_DB/Gel1.aspx PMID:25097385

  11. KnotProt: a database of proteins with knots and slipknots

    PubMed Central

    Jamroz, Michal; Niemyska, Wanda; Rawdon, Eric J.; Stasiak, Andrzej; Millett, Kenneth C.; Sułkowski, Piotr; Sulkowska, Joanna I.

    2015-01-01

    The protein topology database KnotProt, http://knotprot.cent.uw.edu.pl/, collects information about protein structures with open polypeptide chains forming knots or slipknots. The knotting complexity of the cataloged proteins is presented in the form of a matrix diagram that shows users the knot type of the entire polypeptide chain and of each of its subchains. The pattern visible in the matrix gives the knotting fingerprint of a given protein and permits users to determine, for example, the minimal length of the knotted regions (knot's core size) or the depth of a knot, i.e. how many amino acids can be removed from either end of the cataloged protein structure before converting it from a knot to a different type of knot. In addition, the database presents extensive information about the biological functions, families and fold types of proteins with non-trivial knotting. As an additional feature, the KnotProt database enables users to submit protein or polymer chains and generate their knotting fingerprints. PMID:25361973

  12. A METHOD FOR DETERMINING TOTAL PROTEIN OF ISOLATED CELLULAR ELEMENTS AND CORRESPONDING TRITIUM RADIOACTIVITY

    PubMed Central

    Koenig, Edward

    1968-01-01

    A method is described for the microanalysis of protein, obtained from isolated tissue elements, in the range of 500 µµg-500 mµg. The method entails solubilization of cellular protein with phosphoric acid and heat after extraction of acid-soluble compounds, lipids, and RNA. A procedure for the extraction and recovery of cellular RNA by the use of 40% trichloroacetic acid is presented. The solubilized protein, in the form of a microdroplet, is photomicrographed with monochromatic light at 230 mµ. Total density in the microdroplet is determined from calibrated photographic plates by microdensitometry, and is converted to protein mass by using an experimentally determined average specific absorbance value. A solubilized protein labeled with tritium can be recovered after photomicrography, combusted, and reduced to generate tritiated gas for high-efficiency tritium radiometry. Total protein was analyzed in (a) nerve cells of three different sizes from Deiters' nucleus of the rabbit; and the whole rod cell and rod cell nucleus of the rabbit retina. PMID:5664225

  13. A High-Content Imaging Screen for Cellular Regulators of β-Catenin Protein Abundance.

    PubMed

    Zeng, Xin; Montoute, Monica; Bee, Tiger W; Lin, Hong; Kallal, Lorena A; Liu, Yan; Agarwal, Pankaj; Wang, Dayuan; Lu, Quinn; Morrow, Dwight; Pope, Andrew J; Wu, Zining

    2016-03-01

    Abnormal accumulation of β-catenin protein, a key transcriptional activator required for Wnt signaling, is the hallmark of many tumor types, including colon cancer. In normal cells, β-catenin protein level is tightly controlled by a multiprotein complex through the proteosome pathway. Mutations in the components of the β-catenin degradation complex, such as adenomatous polyposis coli (APC) and Axin, lead to β-catenin stabilization and the constitutive activation of target genes. Since the signal transduction of Wnt/β-catenin is mainly mediated by protein-protein interactions, this pathway has been particularly refractory to conventional target-based small-molecule screening. Here we designed a cellular high-content imaging assay to detect β-catenin protein through immunofluorescent staining in the SW480 colon cancer cell line, which has elevated β-catenin endogenously. We demonstrate that the assay is robust and specific to screen a focused biologically diverse chemical library set against known targets that play diverse cellular functions. We identified a number of hits that reduce β-catenin levels without causing cell death. These hits may serve as tools to understand the dynamics of β-catenin degradation. This study demonstrates that detecting cell-based β-catenin protein stability is a viable approach to identifying novel mechanisms of β-catenin regulation as well as small molecules of therapeutic potential. PMID:26656867

  14. A Method of Extracting Sentences Related to Protein Interaction from Literature using a Structure Database

    NASA Astrophysics Data System (ADS)

    Kaneta, Yoshikazu; Munna, Md. Ahaduzzaman; Ohkawa, Takenao

    Because a protein expresses its function through interaction with other substrates, it is vital to create a database of protein interaction. Since the total volume of information on protein interaction is described in terms of thousands of literatures, it is nearly impossible to extract all this information manually. Although extraction systems for interaction information based on the template matching method have already been developed, it is not possible to match all the sentences with interaction information due to the extent of sentence complexity. We propose a method of extracting sentences with interaction information independent of sentence structure. In a protein-compound complex structure, the interacting residue is near to its partner. The distance between them can be calculated by using the structure data in the PDB database, with a short distance indicating that the sentences associated with them might describe the interaction information. In a free-protein structure, the distance cannot be calculated because the coordinates of the protein's partner are not registered in the structure data. Hence, we use the homology protein structure data, which is complexed with the protein's parter. The proposed method was applied to seven literatures written about protein-compound complexes and four literatures written about free proteins, obtaining F-measures of 71% and 72%, respectively.

  15. Modulation of cellular signaling by herpesvirus-encoded G protein-coupled receptors

    PubMed Central

    de Munnik, Sabrina M.; Smit, Martine J.; Leurs, Rob; Vischer, Henry F.

    2015-01-01

    Human herpesviruses (HHVs) are widespread infectious pathogens that have been associated with proliferative and inflammatory diseases. During viral evolution, HHVs have pirated genes encoding viral G protein-coupled receptors (vGPCRs), which are expressed on infected host cells. These vGPCRs show highest homology to human chemokine receptors, which play a key role in the immune system. Importantly, vGPCRs have acquired unique properties such as constitutive activity and the ability to bind a broad range of human chemokines. This allows vGPCRs to hijack human proteins and modulate cellular signaling for the benefit of the virus, ultimately resulting in immune evasion and viral dissemination to establish a widespread and lifelong infection. Knowledge on the mechanisms by which herpesviruses reprogram cellular signaling might provide insight in the contribution of vGPCRs to viral survival and herpesvirus-associated pathologies. PMID:25805993

  16. The Rift Valley Fever virus protein NSm and putative cellular protein interactions

    PubMed Central

    2012-01-01

    Rift Valley Fever is an infectious viral disease and an emerging problem in many countries of Africa and on the Arabian Peninsula. The causative virus is predominantly transmitted by mosquitoes and high mortality and abortion rates characterize outbreaks in animals while symptoms ranging from mild to life-threatening encephalitis and hemorrhagic fever are noticed among infected humans. For a better prevention and treatment of the infection, an increased knowledge of the infectious process of the virus is required. The focus of this work was to identify protein-protein interactions between the non-structural protein (NSm), encoded by the M-segment of the virus, and host cell proteins. This study was initiated by screening approximately 26 million cDNA clones of a mouse embryonic cDNA library for interactions with the NSm protein using a yeast two-hybrid system. We have identified nine murine proteins that interact with NSm protein of Rift Valley Fever virus, and the putative protein-protein interactions were confirmed by growth selection procedures and β-gal activity measurements. Our results suggest that the cleavage and polyadenylation specificity factor subunit 2 (Cpsf2), the peptidyl-prolyl cis-trans isomerase (cyclophilin)-like 2 protein (Ppil2), and the synaptosome-associated protein of 25 kDa (SNAP-25) are the most promising targets for the NSm protein of the virus during an infection. PMID:22838834

  17. Encapsulated cellular implants for recombinant protein delivery and therapeutic modulation of the immune system.

    PubMed

    Lathuilière, Aurélien; Mach, Nicolas; Schneider, Bernard L

    2015-05-08

    Ex vivo gene therapy using retrievable encapsulated cellular implants is an effective strategy for the local and/or chronic delivery of therapeutic proteins. In particular, it is considered an innovative approach to modulate the activity of the immune system. Two recently proposed therapeutic schemes using genetically engineered encapsulated cells are discussed here: the chronic administration of monoclonal antibodies for passive immunization against neurodegenerative diseases and the local delivery of a cytokine as an adjuvant for anti-cancer vaccines.

  18. Encapsulated Cellular Implants for Recombinant Protein Delivery and Therapeutic Modulation of the Immune System

    PubMed Central

    Lathuilière, Aurélien; Mach, Nicolas; Schneider, Bernard L.

    2015-01-01

    Ex vivo gene therapy using retrievable encapsulated cellular implants is an effective strategy for the local and/or chronic delivery of therapeutic proteins. In particular, it is considered an innovative approach to modulate the activity of the immune system. Two recently proposed therapeutic schemes using genetically engineered encapsulated cells are discussed here: the chronic administration of monoclonal antibodies for passive immunization against neurodegenerative diseases and the local delivery of a cytokine as an adjuvant for anti-cancer vaccines. PMID:26006227

  19. Structural basis of lentiviral subversion of a cellular protein degradation pathway

    NASA Astrophysics Data System (ADS)

    Schwefel, David; Groom, Harriet C. T.; Boucherit, Virginie C.; Christodoulou, Evangelos; Walker, Philip A.; Stoye, Jonathan P.; Bishop, Kate N.; Taylor, Ian A.

    2014-01-01

    Lentiviruses contain accessory genes that have evolved to counteract the effects of host cellular defence proteins that inhibit productive infection. One such restriction factor, SAMHD1, inhibits human immunodeficiency virus (HIV)-1 infection of myeloid-lineage cells as well as resting CD4+ T cells by reducing the cellular deoxynucleoside 5'-triphosphate (dNTP) concentration to a level at which the viral reverse transcriptase cannot function. In other lentiviruses, including HIV-2 and related simian immunodeficiency viruses (SIVs), SAMHD1 restriction is overcome by the action of viral accessory protein x (Vpx) or the related viral protein r (Vpr) that target and recruit SAMHD1 for proteasomal degradation. The molecular mechanism by which these viral proteins are able to usurp the host cell's ubiquitination machinery to destroy the cell's protection against these viruses has not been defined. Here we present the crystal structure of a ternary complex of Vpx with the human E3 ligase substrate adaptor DCAF1 and the carboxy-terminal region of human SAMHD1. Vpx is made up of a three-helical bundle stabilized by a zinc finger motif, and wraps tightly around the disc-shaped DCAF1 molecule to present a new molecular surface. This adapted surface is then able to recruit SAMHD1 via its C terminus, making it a competent substrate for the E3 ligase to mark for proteasomal degradation. The structure reported here provides a molecular description of how a lentiviral accessory protein is able to subvert the cell's normal protein degradation pathway to inactivate the cellular viral defence system.

  20. The effect of blood protein adsorption on cellular uptake of anatase TiO2 nanoparticles

    PubMed Central

    Allouni, Zouhir E; Gjerdet, Nils R; Cimpan, Mihaela R; Høl, Paul J

    2015-01-01

    Protein adsorption onto nanoparticles (NPs) in biological fluids has emerged as an important factor when testing biological responses to NPs, as this may influence both uptake and subsequent toxicity. The aim of the present study was to quantify the adsorption of proteins onto TiO2 NPs and to test the influence on cellular uptake. The surface composition of the particles was characterized by thermal analysis and by X-ray photoelectron spectroscopy. The adsorption of three blood proteins, ie, human serum albumin (HSA), γ-globulins (Glbs), and fibrinogen (Fib), onto three types of anatase NPs of different sizes was quantified for each protein. The concentration of the adsorbed protein was measured by ultraviolet-visible spectrophotometry using the Bradford method. The degree of cellular uptake was quantified by inductivity coupled plasma mass spectroscopy, and visualized by an ultra-high resolution imaging system. The proteins were adsorbed onto all of the anatase NPs. The quantity adsorbed increased with time and was higher for the smaller particles. Fib and Glbs showed the highest affinity to TiO2 NPs, while the lowest was seen for HSA. The adsorption of proteins affected the surface charge and the hydrodynamic diameter of the NPs in cell culture medium. The degree of particle uptake was highest in protein-free medium and in the presence HSA, followed by culture medium supplemented with Glbs, and lowest in the presence of Fib. The results indicate that the uptake of anatase NPs by fibroblasts is influenced by the identity of the adsorbed protein. PMID:25632230

  1. The effect of blood protein adsorption on cellular uptake of anatase TiO2 nanoparticles.

    PubMed

    Allouni, Zouhir E; Gjerdet, Nils R; Cimpan, Mihaela R; Høl, Paul J

    2015-01-01

    Protein adsorption onto nanoparticles (NPs) in biological fluids has emerged as an important factor when testing biological responses to NPs, as this may influence both uptake and subsequent toxicity. The aim of the present study was to quantify the adsorption of proteins onto TiO2 NPs and to test the influence on cellular uptake. The surface composition of the particles was characterized by thermal analysis and by X-ray photoelectron spectroscopy. The adsorption of three blood proteins, ie, human serum albumin (HSA), γ-globulins (Glbs), and fibrinogen (Fib), onto three types of anatase NPs of different sizes was quantified for each protein. The concentration of the adsorbed protein was measured by ultraviolet-visible spectrophotometry using the Bradford method. The degree of cellular uptake was quantified by inductivity coupled plasma mass spectroscopy, and visualized by an ultra-high resolution imaging system. The proteins were adsorbed onto all of the anatase NPs. The quantity adsorbed increased with time and was higher for the smaller particles. Fib and Glbs showed the highest affinity to TiO2 NPs, while the lowest was seen for HSA. The adsorption of proteins affected the surface charge and the hydrodynamic diameter of the NPs in cell culture medium. The degree of particle uptake was highest in protein-free medium and in the presence HSA, followed by culture medium supplemented with Glbs, and lowest in the presence of Fib. The results indicate that the uptake of anatase NPs by fibroblasts is influenced by the identity of the adsorbed protein. PMID:25632230

  2. Stem-loop binding protein is a multifaceted cellular regulator of HIV-1 replication

    PubMed Central

    Tucker, Lynne D.; Asara, John M.; Cheruiyot, Collins K.; Lu, Huafei; Wu, Zhijin J.; Newstein, Michael C.; Dooner, Mark S.; Friedman, Jennifer; Lally, Michelle A.; Ramratnam, Bharat

    2016-01-01

    A rare subset of HIV-1–infected individuals is able to maintain plasma viral load (VL) at low levels without antiretroviral treatment. Identifying the mechanisms underlying this atypical response to infection may lead to therapeutic advances for treating HIV-1. Here, we developed a proteomic analysis to compare peripheral blood cell proteomes in 20 HIV-1–infected individuals who maintained either high or low VL with the aim of identifying host factors that impact HIV-1 replication. We determined that the levels of multiple histone proteins were markedly decreased in cohorts of individuals with high VL. This reduction was correlated with lower levels of stem-loop binding protein (SLBP), which is known to control histone metabolism. Depletion of cellular SLBP increased promoter engagement with the chromatin structures of the host gene high mobility group protein A1 (HMGA1) and viral long terminal repeat (LTR), which led to higher levels of HIV-1 genomic integration and proviral transcription. Further, we determined that TNF-α regulates expression of SLBP and observed that plasma TNF-α levels in HIV-1–infected individuals correlated directly with VL levels and inversely with cellular SLBP levels. Our findings identify SLBP as a potentially important cellular regulator of HIV-1, thereby establishing a link between histone metabolism, inflammation, and HIV-1 infection. PMID:27454292

  3. Sentra : a database of signal transduction proteins for comparative genome analysis.

    SciTech Connect

    D'Souza, M.; Glass, E. M.; Syed, M. H.; Zhang, Y.; Rodriguez, A.; Maltsev, N.; Galerpin, M. Y.; Mathematics and Computer Science; Univ. of Chicago; NIH

    2007-01-01

    Sentra (http://compbio.mcs.anl.gov/sentra), a database of signal transduction proteins encoded in completely sequenced prokaryotic genomes, has been updated to reflect recent advances in understanding signal transduction events on a whole-genome scale. Sentra consists of two principal components, a manually curated list of signal transduction proteins in 202 completely sequenced prokaryotic genomes and an automatically generated listing of predicted signaling proteins in 235 sequenced genomes that are awaiting manual curation. In addition to two-component histidine kinases and response regulators, the database now lists manually curated Ser/Thr/Tyr protein kinases and protein phosphatases, as well as adenylate and diguanylate cyclases and c-di-GMP phosphodiesterases, as defined in several recent reviews. All entries in Sentra are extensively annotated with relevant information from public databases (e.g. UniProt, KEGG, PDB and NCBI). Sentra's infrastructure was redesigned to support interactive cross-genome comparisons of signal transduction capabilities of prokaryotic organisms from a taxonomic and phenotypic perspective and in the framework of signal transduction pathways from KEGG. Sentra leverages the PUMA2 system to support interactive analysis and annotation of signal transduction proteins by the users.

  4. Super-resolution imaging and tracking of protein-protein interactions in sub-diffraction cellular space.

    PubMed

    Liu, Zhen; Xing, Dong; Su, Qian Peter; Zhu, Yun; Zhang, Jiamei; Kong, Xinyu; Xue, Boxin; Wang, Sheng; Sun, Hao; Tao, Yile; Sun, Yujie

    2014-07-17

    Imaging the location and dynamics of individual interacting protein pairs is essential but often difficult because of the fluorescent background from other paired and non-paired molecules, particularly in the sub-diffraction cellular space. Here we develop a new method combining bimolecular fluorescence complementation and photoactivated localization microscopy for super-resolution imaging and single-molecule tracking of specific protein-protein interactions. The method is used to study the interaction of two abundant proteins, MreB and EF-Tu, in Escherichia coli cells. The super-resolution imaging shows interesting distribution and domain sizes of interacting MreB-EF-Tu pairs as a subpopulation of total EF-Tu. The single-molecule tracking of MreB, EF-Tu and MreB-EF-Tu pairs reveals intriguing localization-dependent heterogonous dynamics and provides valuable insights to understanding the roles of MreB-EF-Tu interactions.

  5. Super-resolution imaging and tracking of protein-protein interactions in sub-diffraction cellular space

    NASA Astrophysics Data System (ADS)

    Liu, Zhen; Xing, Dong; Su, Qian Peter; Zhu, Yun; Zhang, Jiamei; Kong, Xinyu; Xue, Boxin; Wang, Sheng; Sun, Hao; Tao, Yile; Sun, Yujie

    2014-07-01

    Imaging the location and dynamics of individual interacting protein pairs is essential but often difficult because of the fluorescent background from other paired and non-paired molecules, particularly in the sub-diffraction cellular space. Here we develop a new method combining bimolecular fluorescence complementation and photoactivated localization microscopy for super-resolution imaging and single-molecule tracking of specific protein-protein interactions. The method is used to study the interaction of two abundant proteins, MreB and EF-Tu, in Escherichia coli cells. The super-resolution imaging shows interesting distribution and domain sizes of interacting MreB-EF-Tu pairs as a subpopulation of total EF-Tu. The single-molecule tracking of MreB, EF-Tu and MreB-EF-Tu pairs reveals intriguing localization-dependent heterogonous dynamics and provides valuable insights to understanding the roles of MreB-EF-Tu interactions.

  6. Interacting factors and cellular localization of SR protein-specific kinase Dsk1

    SciTech Connect

    Tang, Zhaohua; Luca, Maria; Taggart-Murphy, Laura; Portillio, Jessica; Chang, Cathey; Guven, Ayse; Lin, Ren-Jang; Murray, Johanne; Carr, Antony

    2012-10-01

    Schizosaccharomyces pombe Dsk1 is an SR protein-specific kinase (SRPK), whose homologs have been identified in every eukaryotic organism examined. Although discovered as a mitotic regulator with protein kinase activity toward SR splicing factors, it remains largely unknown about what and how Dsk1 contributes to cell cycle and pre-mRNA splicing. In this study, we investigated the Dsk1 function by determining interacting factors and cellular localization of the kinase. Consistent with its reported functions, we found that pre-mRNA processing and cell cycle factors are prominent among the proteins co-purified with Dsk1. The identification of these factors led us to find Rsd1 as a novel Dsk1 substrate, as well as the involvement of Dsk1 in cellular distribution of poly(A){sup +} RNA. In agreement with its role in nuclear events, we also found that Dsk1 is mainly localized in the nucleus during G{sub 2} phase and at mitosis. Furthermore, we revealed the oscillation of Dsk1 protein in a cell cycle-dependent manner. This paper marks the first comprehensive analysis of in vivo Dsk1-associated proteins in fission yeast. Our results reflect the conserved role of SRPK family in eukaryotic organisms, and provide information about how Dsk1 functions in pre-mRNA processing and cell-division cycle.

  7. Nitric oxide blocks cellular heme insertion into a broad range of heme proteins

    PubMed Central

    Waheed, Syed Mohsin; Ghosh, Arnab; Chakravarti, Ritu; Biswas, Ashis; Haque, Mohammad Mahfuzul; Panda, Koustubh; Stuehr, Dennis J.

    2010-01-01

    Although heme insertion into proteins enables their function in bioenergetics, metabolism, and signaling, the mechanisms and regulation of this process is not fully understood. We developed a means to study cellular heme insertion into apo-protein targets over a 3 h time period, and then investigated how nitric oxide (NO) released from a chemical donor (NOC-18) might influence heme (protoporphyrin IX) insertion into seven targets that present a range of protein structure, heme ligation, and function (three NO synthases, two cytochrome P450’s, catalase, and hemoglobin). NO blocked cellular heme insertion into all seven apo-protein targets. The inhibition occurred at relatively low (nM/min) fluxes of NO, was reversible, and did not involve changes in intracellular heme level, activation of guanylate cyclase, or inhibition of mitochondrial ATP production. These aspects and the range of protein targets suggest that NO can act as a global inhibitor of heme insertion, possibly by inhibiting a common step in the process. PMID:20211245

  8. Identification of SNPs in Cellular Retinol Binding Protein 1 and Cellular Retinol Binding Protein 3 Genes and Their Associations with Laying Performance Traits in Erlang Mountainous Chicken

    PubMed Central

    Wang, Yan; Xiao, Li-Hua; Zhao, Xiao-Ling; Liu, Yi-Ping; Zhu, Qing

    2014-01-01

    CRBP1 (cellular retinol binding protein 1) and CRBP3 (cellular retinol binding protein 3), are important components of the retinoid signaling pathway and take part in vitamin A absorption, transport and metabolism. Based on the role of vitamin A in chicken laying performance, we investigated the polymorphism of CRBP1 and CRBP3 genes in 349 chickens using single strand conformation polymorphism and DNA sequencing methods. Only one polymorphism was identified in the third intron of CRBP1, two polymorphisms were detected in CRBP3; they were located in the second intron and the third intron respectively. The association studies between these three SNPs and laying performance traits were performed in Erlang mountainous chicken. Notably, the SNP g.14604G>T of CRBP1 was shown to be significantly associated with body weight at first egg (BWFE), age at first egg (AFE), weight at first egg (WFE) and total number of eggs with 300 age (EN). The CRBP3 polymorphism g.934C>G was associated with AFE, and the g.1324A>G was associated with AFE and BWFE, but none of these polymorphisms were associated with egg quality traits. Haplotype combinations constructed on these two SNPs of CRBP3 gene were associated with BWFE and AFE. In particular, diplotype H2H2 had positive effect on AFE, BWFE, EN, and average egg-laying interval. We herein describe for the first time basic research on the polymorphism of chicken CRBP1 and CRBP3 genes that is predictive of genetic potential for laying performance in chicken. PMID:25083100

  9. Cellular fatty acid and soluble protein composition of Actinobacillus actinomycetemcomitans and related organisms.

    PubMed Central

    Calhoon, D A; Mayberry, W R; Slots, J

    1981-01-01

    The cellular fatty acid and protein content of twenty-five representative strains of Actinobacillus actinomycetecomitans isolated from juvenile and adult periodontitis patients was compared to that of 15 reference strains of oral and nonoral Actinobacillus species and Haemophilus aphrophilus. Trimethylsilyl derivatives of the fatty acid methyl esters were analyzed by gas-liquid chromatography. The predominant fatty acids of all 40 strains examined were 14:0, 3-OH 14:0, 16 delta, and 16:0. Actinobacillus seminis (ATCC 15768) was unlike the other strains examined because of a greater amount of 14:0 detected. The soluble protein analysis using polyacrylamide gel electrophoresis revealed that A. actinomycetemcomitans, H. aphrophilus, and nonoral Actinobacillus species possessed distinct protein profiles attesting to the validity of separating these organisms into different species. Established biotypes of A. actinomycetemcomitans could not be differentiated on the basis of fatty acid or protein profiles. PMID:7287893

  10. Molecular modeling of the conformational dynamics of the cellular prion protein

    NASA Astrophysics Data System (ADS)

    Nguyen, Charles; Colling, Ian; Bartz, Jason; Soto, Patricia

    2014-03-01

    Prions are infectious agents responsible for transmissible spongiform encephalopathies (TSEs), a type of fatal neurodegenerative disease in mammals. Prions propagate biological information by conversion of the non-pathological version of the prion protein to the infectious conformation, PrPSc. A wealth of knowledge has shed light on the nature and mechanism of prion protein conversion. In spite of the significance of this problem, we are far from fully understanding the conformational dynamics of the cellular isoform. To remedy this situation we employ multiple biomolecular modeling techniques such as docking and molecular dynamics simulations to map the free energy landscape and determine what specific regions of the prion protein are most conductive to binding. The overall goal is to characterize the conformational dynamics of the cell form of the prion protein, PrPc, to gain insight into inhibition pathways against misfolding. NE EPSCoR FIRST Award to Patricia Soto.

  11. Protein kinase–inhibitor database: Structural variability of and inhibitor interactions with the protein kinase P-loop

    PubMed Central

    Patel, Ronak Y.; Doerksen, Robert J.

    2011-01-01

    Structure based drug design of protein-kinase inhibitors has been facilitated by availability of an enormous number of structures in the Protein Databank (PDB), systematic analyses of which can provide insight into the factors which govern ligand–protein kinase interactions and into the conformational variability of the protein kinases. In this study, a non-redundant database containing 755 unique, curated and annotated PDB protein kinase–inhibitor complexes (each consisting of a single protein kinase chain, a ligand and water molecules around the ligand) was created. With this dataset, analyses were performed of protein conformational variability and interactions of ligands with 11 P-loop residues. Analysis of ligand–protein interactions included ligand atom preference, ligand–protein hydrogen bonds and the number and position of crystallographic water molecules around important P-loop residues. Analysis of variability in the conformation of the P-loop considered backbone and side-chain dihedral angles, and solvent accessible surface area (SASA). A distorted conformation of the P-loop was observed for some of the protein kinase structures. Lower SASA was observed for the hydrophobic residue in β1 of several members of the AGC family of protein kinases. Our systematic studies were performed amino-acid by amino-acid, which is unusual for analyses of protein kinase–inhibitor complexes. PMID:20681595

  12. Computer-Aided Protein Directed Evolution: a Review of Web Servers, Databases and other Computational Tools for Protein Engineering.

    PubMed

    Verma, Rajni; Schwaneberg, Ulrich; Roccatano, Danilo

    2012-01-01

    The combination of computational and directed evolution methods has proven a winning strategy for protein engineering. We refer to this approach as computer-aided protein directed evolution (CAPDE) and the review summarizes the recent developments in this rapidly growing field. We will restrict ourselves to overview the availability, usability and limitations of web servers, databases and other computational tools proposed in the last five years. The goal of this review is to provide concise information about currently available computational resources to assist the design of directed evolution based protein engineering experiment.

  13. SIRT3 regulates cellular iron metabolism and cancer growth by repressing iron regulatory protein 1.

    PubMed

    Jeong, S M; Lee, J; Finley, L W S; Schmidt, P J; Fleming, M D; Haigis, M C

    2015-04-16

    Iron metabolism is essential for many cellular processes, including oxygen transport, respiration and DNA synthesis, and many cancer cells exhibit dysregulation in iron metabolism. Maintenance of cellular iron homeostasis is regulated by iron regulatory proteins (IRPs), which control the expression of iron-related genes by binding iron-responsive elements (IREs) of target mRNAs. Here, we report that mitochondrial SIRT3 regulates cellular iron metabolism by modulating IRP1 activity. SIRT3 loss increases reactive oxygen species production, leading to elevated IRP1 binding to IREs. As a consequence, IRP1 target genes, such as the transferrin receptor (TfR1), a membrane-associated glycoprotein critical for iron uptake and cell proliferation, are controlled by SIRT3. Importantly, SIRT3 deficiency results in a defect in cellular iron homeostasis. SIRT3 null cells contain high levels of iron and lose iron-dependent TfR1 regulation. Moreover, SIRT3 null mice exhibit higher levels of iron and TfR1 expression in the pancreas. We found that the regulation of iron uptake and TfR1 expression contribute to the tumor-suppressive activity of SIRT3. Indeed, SIRT3 expression is negatively correlated with TfR1 expression in human pancreatic cancers. SIRT3 overexpression decreases TfR1 expression by inhibiting IRP1 and represses proliferation in pancreatic cancer cells. Our data uncover a novel role of SIRT3 in cellular iron metabolism through IRP1 regulation and suggest that SIRT3 functions as a tumor suppressor, in part, by modulating cellular iron metabolism. PMID:24909164

  14. Identification of shed proteins from Chinese hamster ovary cells: Application of statistical confidence using human and mouse protein databases

    SciTech Connect

    Ahram, Mamoun; Strittmatter, Eric F.; Monroe, Matthew E.; Adkins, Joshua N.; Hunter, Joel C.; Miller, John H.; Springer, David L.

    2005-05-01

    The shedding process releases ligands, receptors, and other proteins from the surface of the cell and is a mechanism whereby cells communicate. Even though altered regulation of this process has been implicated in several diseases, global approaches to evaluate shed proteins have not been developed. A goal of this study was to identify global changes in shed proteins in media taken from cells exposed to low-doses of radiation in an effort to develop a fundamental understanding of the bystander response. CHO cells were chosen for this study because they have been widely used for radiation studies and since they have been reported to respond to radiation by releasing factors into the media that cause genomic instability and cytotoxicity in unexposed cells, i.e., a bystander effect. Media samples taken for irradiated cells were evaluated using a combination of tandem- and FTICR-mass spectrometry analysis. Since the hamster genome has not been sequenced, mass spectrometry data was searched against the mouse and human proteins databases. Nearly 150 proteins that were identified by tandem mass spectrometry were confirmed by FTICR. When both types of mass spectrometry data were evaluated with a new confidence scoring tool, which is based on discriminant analyses, about 500 protein were identified. Approximately 20% of these identifications were either integral membrane proteins or membrane associated proteins, suggesting that they were derived from the cell surface, hence were likely shed. However, estimates of quantitative changes, based on two independent mass spectrometry approaches, did not identify any protein abundance changes attributable to the bystander effect. Results from this study demonstrate the feasibility of global evaluation of shed proteins using mass spectrometry in conjunction with cross-species protein databases and that significant improvement in peptide/protein identifications is provided by the confidence scoring tool.

  15. CPAD, Curated Protein Aggregation Database: A Repository of Manually Curated Experimental Data on Protein and Peptide Aggregation.

    PubMed

    Thangakani, A Mary; Nagarajan, R; Kumar, Sandeep; Sakthivel, R; Velmurugan, D; Gromiha, M Michael

    2016-01-01

    Accurate distinction between peptide sequences that can form amyloid-fibrils or amorphous β-aggregates, identification of potential aggregation prone regions in proteins, and prediction of change in aggregation rate of a protein upon mutation(s) are critical to research on protein misfolding diseases, such as Alzheimer's and Parkinson's, as well as biotechnological production of protein based therapeutics. We have developed a Curated Protein Aggregation Database (CPAD), which has collected results from experimental studies performed by scientific community aimed at understanding protein/peptide aggregation. CPAD contains more than 2300 experimentally observed aggregation rates upon mutations in known amyloidogenic proteins. Each entry includes numerical values for the following parameters: change in rate of aggregation as measured by fluorescence intensity or turbidity, name and source of the protein, Uniprot and Protein Data Bank codes, single point as well as multiple mutations, and literature citation. The data in CPAD has been supplemented with five different types of additional information: (i) Amyloid fibril forming hexa-peptides, (ii) Amorphous β-aggregating hexa-peptides, (iii) Amyloid fibril forming peptides of different lengths, (iv) Amyloid fibril forming hexa-peptides whose crystal structures are available in the Protein Data Bank (PDB) and (v) Experimentally validated aggregation prone regions found in amyloidogenic proteins. Furthermore, CPAD is linked to other related databases and resources, such as Uniprot, Protein Data Bank, PUBMED, GAP, TANGO, WALTZ etc. We have set up a web interface with different search and display options so that users have the ability to get the data in multiple ways. CPAD is freely available at http://www.iitm.ac.in/bioinfo/CPAD/. The potential applications of CPAD have also been discussed. PMID:27043825

  16. circRNADb: A comprehensive database for human circular RNAs with protein-coding annotations

    PubMed Central

    Chen, Xiaoping; Han, Ping; Zhou, Tao; Guo, Xuejiang; Song, Xiaofeng; Li, Yan

    2016-01-01

    It has been known that circular RNAs are widely expressed in human tissues and cells, and play important regulatory roles in physiological or pathological processes. However, there is lack of comprehensively annotated human circular RNAs database. In this study we established a circRNA database, named as circRNADb, containing 32,914 human exonic circRNAs carefully selected from diversified sources. The detailed information of the circRNA, including genomic information, exon splicing, genome sequence, internal ribosome entry site (IRES), open reading frame (ORF) and references were provided in circRNADb. In addition, circRNAs were found to be able to encode proteins, which have not been reported in any species. 16328 circRNAs were annotated to have ORF longer than 100 amino acids, of which 7170 have IRES elements. 46 circRNAs from 37 genes were found to have their corresponding proteins expressed according mass spectrometry. The database provides the function of data search, browse, download, submit and feedback for the user to study particular circular RNA of interest and update the database continually. circRNADb will be built to be a biological information platform for circRNA molecules and related biological functions in the future. The database can be freely available through the web server at http://reprod.njmu.edu.cn/circrnadb. PMID:27725737

  17. Functional domains and sub-cellular distribution of the Hedgehog transducing protein Smoothened in Drosophila.

    PubMed

    Nakano, Y; Nystedt, S; Shivdasani, A A; Strutt, H; Thomas, C; Ingham, P W

    2004-06-01

    The Hedgehog signalling pathway is deployed repeatedly during normal animal development and its inappropriate activity is associated with various tumours in human. The serpentine protein Smoothened (Smo) is essential for cells to respond to the Hedeghog (Hh) signal; oncogenic forms of Smo have been isolated from human basal cell carcinomas. Despite similarities with ligand binding G-protein coupled receptors, the molecular basis of Smo activity and its regulation remains unclear. In non-responding cells, Smo is suppressed by the activity of another multipass membrane spanning protein Ptc, which acts as the Hh receptor. In Drosophila, binding of Hh to Ptc has been shown to cause an accumulation of phosphorylated Smo protein and a concomitant stabilisation of the activated form of the Ci transcription factor. Here, we identify domains essential for Smo activity and investigate the sub-cellular distribution of the wild type protein in vivo. We find that deletion of the amino terminus and the juxtamembrane region of the carboxy terminus of the protein result in the loss of normal Smo activity. Using Green Fluorescent Protein (GFP) and horseradish peroxidase fusion proteins we show that Smo accumulates in the plasma membrane of cells in which Ptc activity is abrogated by Hh but is targeted to the degradative pathway in cells where Ptc is active. We further demonstrate that Smo accumulation is likely to be a cause, rather than a consequence, of Hh signal transduction.

  18. Topology of Endoplasmic Reticulum-Associated Cellular and Viral Proteins Determined with Split-GFP.

    PubMed

    Hyun, Seong-In; Maruri-Avidal, Liliana; Moss, Bernard

    2015-07-01

    The split green fluorescent protein (GFP) system was adapted for investigation of the topology of ER-associated proteins. A 215-amino acid fragment of GFP (S1-10) was expressed in the cytoplasm as a free protein or fused to the N-terminus of calnexin and in the ER as an intraluminal protein or fused to the C-terminus of calnexin. A 16-amino acid fragment of GFP (S11) was fused to the N- or C-terminus of the target protein. Fluorescence occurred when both GFP fragments were in the same intracellular compartment. After validation with the cellular proteins PDI and tapasin, we investigated two vaccinia virus proteins (L2 and A30.5) of unknown topology that localize to the ER and are required for assembly of the viral membrane. Our results indicated that the N- and C-termini of L2 faced the cytoplasmic and luminal sides of the ER, respectively. In contrast both the N- and C-termini of A30.5 faced the cytoplasm. The system offers advantages for quickly determining the topology of intracellular proteins: the S11 tag is similar in length to commonly used epitope tags; multiple options are available for detecting fluorescence in live or fixed cells; transfection protocols are adaptable to numerous expression systems and can enable high throughput applications.

  19. Cellular uptake and intracellular fate of protein releasing bacterial amyloids in mammalian cells.

    PubMed

    Seras-Franzoso, Joaquin; Sánchez-Chardi, Alejandro; Garcia-Fruitós, Elena; Vázquez, Esther; Villaverde, Antonio

    2016-04-14

    Bacterial Inclusion Bodies (IBs) are amyloidal protein deposits that functionally mimic secretory granules from the endocrine system. When formed by therapeutically relevant proteins, they complement missing intracellular activities in jeopardized cell cultures, offering an intriguing platform for protein drug delivery in substitutive therapies. Despite the therapeutic potential of IBs, their capability to interact with eukaryotic cells, cross the cell membrane and release their functional building blocks into the cytosolic space remains essentially unexplored. We have systematically dissected the process by which bacterial amyloids interact with mammalian cells. An early and tight cell membrane anchorage of IBs is followed by cellular uptake of single or grouped IBs of variable sizes by macropinocytosis. Although an important fraction of the penetrating particles is led to lysosomal degradation, biologically significant amounts of protein are released into the cytosol. In addition, our data suggest the involvement of the bacterial cell folding modulator DnaK in the release of functional proteins from these amyloidal reservoirs. The mechanisms supporting the internalization of disintegrable protein nanoparticles revealed here offer clues to implement novel approaches for protein drug delivery based on controlled protein packaging as bacterial IBs.

  20. Protein backbone angle restraints from searching a database for chemical shift and sequence homology.

    PubMed

    Cornilescu, G; Delaglio, F; Bax, A

    1999-03-01

    Chemical shifts of backbone atoms in proteins are exquisitely sensitive to local conformation, and homologous proteins show quite similar patterns of secondary chemical shifts. The inverse of this relation is used to search a database for triplets of adjacent residues with secondary chemical shifts and sequence similarity which provide the best match to the query triplet of interest. The database contains 13C alpha, 13C beta, 13C', 1H alpha and 15N chemical shifts for 20 proteins for which a high resolution X-ray structure is available. The computer program TALOS was developed to search this database for strings of residues with chemical shift and residue type homology. The relative importance of the weighting factors attached to the secondary chemical shifts of the five types of resonances relative to that of sequence similarity was optimized empirically. TALOS yields the 10 triplets which have the closest similarity in secondary chemical shift and amino acid sequence to those of the query sequence. If the central residues in these 10 triplets exhibit similar phi and psi backbone angles, their averages can reliably be used as angular restraints for the protein whose structure is being studied. Tests carried out for proteins of known structure indicate that the root-mean-square difference (rmsd) between the output of TALOS and the X-ray derived backbone angles is about 15 degrees. Approximately 3% of the predictions made by TALOS are found to be in error.

  1. Protein backbone torsion angle-based structure comparison and secondary structure database web server.

    PubMed

    Jung, Sunghoon; Bae, Se-Eun; Ahn, Insung; Son, Hyeon S

    2013-09-01

    Structural information has been a major concern for biological and pharmaceutical studies for its intimate relationship to the function of a protein. Three-dimensional representation of the positions of protein atoms is utilized among many structural information repositories that have been published. The reliability of the torsional system, which represents the native processes of structural change in the structural analysis, was partially proven with previous structural alignment studies. Here, a web server providing structural information and analysis based on the backbone torsional representation of a protein structure is newly introduced. The web server offers functions of secondary structure database search, secondary structure calculation, and pair-wise protein structure comparison, based on a backbone torsion angle representation system. Application of the implementation in pair-wise structural alignment showed highly accurate results. The information derived from this web server might be further utilized in the field of ab initio protein structure modeling or protein homology-related analyses.

  2. Ancient conserved regions in new gene sequences and the protein databases

    SciTech Connect

    Green, P.; Hillier, L.; Waterston, R. ); Lipman, D.; States, D.; Claverie, J.M. )

    1993-03-19

    Sets of new gene sequences from human, nematode, and yeast were compared with each other and with a set of Escherichia coli genes in order to detect ancient evolutionarily conserved regions (ACRs) in the encoded proteins. Nearly all of the ACRs so identified were found to be homologous to sequences in the protein databases. This suggests that currently known proteins may already include representatives of most ACRs and that new sequences not similar to any database sequence are unlikely to contain ACRs. Preliminary analyses indicate that moderately expressed genes may be more likely to contain ACRs than rarely expressed genes. It is estimated that there are fewer than 900 ACRs in all. 20 refs., 2 figs., 4 tabs.

  3. Recent improvements of the ProDom database of protein domain families.

    PubMed

    Corpet, F; Gouzy, J; Kahn, D

    1999-01-01

    The ProDom database contains protein domain families generated from the SWISS-PROT database by automated sequence comparisons. The current version was built with a new improved procedure based on recursive PSI-BLAST homology searches. ProDom can be searched on the World Wide Web to study domain arrangements within either known families or new proteins, with the help of a user-friendly graphical interface (http://www.toulouse.inra.fr/prodom.html). Recent improvements to the ProDom server include: ProDom queries under the SRS Sequence Retrieval System; links to the PredictProtein server; phylogenetic trees and condensed multiple alignments for a better representation of large domain families, with zooming in and out capabilities. In addition, a similar server was set up to display the outcome of whole genome domain analysis as applied to 17 completed microbial genomes (http://www.toulouse.inra.fr/prodomCG.html ).

  4. Role for zinc in a cellular response mediated by protein kinase C in human B lymphocytes

    SciTech Connect

    Forbes, I.J.; Zalewski, P.D.; Giannakis, C. )

    1991-07-01

    Recent studies have suggested a role for Zn{sup 2+}, distinct from that of CA{sup 2+}, in the subcellular distribution and activation of protein kinase C (PKC). Here the author show that Zn{sup 2+} is required for a cellular response mediated by PKC, the rapid loss of expression of a human B cell receptor MER, detected by resetting with mouse erythrocytes. Zn{sup 2+}, in the presence of the Zn{sup 2+} ionophore pyrithione, caused rapid inhibition of MER rosetting at concentrations which induce the translocation and activation of PKC. This required cellular uptake of Zn{sup 2+} and was blocked by 1,10-phenanthroline and TPEN which chelate Zn{sup 2+} but not Ca{sup 2+}. Gold, a metal with similar properties, also induced translocation of PKC and inhibition of MER. Phenanthroline and TPEN also blocked the inhibition of MER induced by the PKC activators phorbol ester and sodium fluoride, suggesting that endogenous cellular Zn{sup 2+} is required. They propose that some cellular actions of PKC require a Zn{sup 2+}-dependent event and that these may be a target for gold during chrysotherapy in rheumatoid arthritis.

  5. Viral and Cellular Proteins Containing FGDF Motifs Bind G3BP to Block Stress Granule Formation

    PubMed Central

    Panas, Marc D.; Schulte, Tim; Thaa, Bastian; Sandalova, Tatiana; Kedersha, Nancy; Achour, Adnane; McInerney, Gerald M.

    2015-01-01

    The Ras-GAP SH3 domain–binding proteins (G3BP) are essential regulators of the formation of stress granules (SG), cytosolic aggregates of proteins and RNA that are induced upon cellular stress, such as virus infection. Many viruses, including Semliki Forest virus (SFV), block SG induction by targeting G3BP. In this work, we demonstrate that the G3BP-binding motif of SFV nsP3 consists of two FGDF motifs, in which both phenylalanine and the glycine residue are essential for binding. In addition, we show that binding of the cellular G3BP-binding partner USP10 is also mediated by an FGDF motif. Overexpression of wt USP10, but not a mutant lacking the FGDF-motif, blocks SG assembly. Further, we identified FGDF-mediated G3BP binding site in herpes simplex virus (HSV) protein ICP8, and show that ICP8 binding to G3BP also inhibits SG formation, which is a novel function of HSV ICP8. We present a model of the three-dimensional structure of G3BP bound to an FGDF-containing peptide, likely representing a binding mode shared by many proteins to target G3BP. PMID:25658430

  6. Viral and cellular proteins containing FGDF motifs bind G3BP to block stress granule formation.

    PubMed

    Panas, Marc D; Schulte, Tim; Thaa, Bastian; Sandalova, Tatiana; Kedersha, Nancy; Achour, Adnane; McInerney, Gerald M

    2015-02-01

    The Ras-GAP SH3 domain-binding proteins (G3BP) are essential regulators of the formation of stress granules (SG), cytosolic aggregates of proteins and RNA that are induced upon cellular stress, such as virus infection. Many viruses, including Semliki Forest virus (SFV), block SG induction by targeting G3BP. In this work, we demonstrate that the G3BP-binding motif of SFV nsP3 consists of two FGDF motifs, in which both phenylalanine and the glycine residue are essential for binding. In addition, we show that binding of the cellular G3BP-binding partner USP10 is also mediated by an FGDF motif. Overexpression of wt USP10, but not a mutant lacking the FGDF-motif, blocks SG assembly. Further, we identified FGDF-mediated G3BP binding site in herpes simplex virus (HSV) protein ICP8, and show that ICP8 binding to G3BP also inhibits SG formation, which is a novel function of HSV ICP8. We present a model of the three-dimensional structure of G3BP bound to an FGDF-containing peptide, likely representing a binding mode shared by many proteins to target G3BP.

  7. Interactions of the p53 protein family in cellular stress response in gastrointestinal tumors

    PubMed Central

    Vilgelm, Anna E.; Washington, Mary K.; Wei, Jinxiong; Chen, Heidi; Prassolov, Vladimir S.; Zaika, Alexander I.

    2010-01-01

    p53, p63 and p73 are members of the p53 protein family involved in regulation of cell cycle, apoptosis, differentiation and other critical cellular processes. Here we investigated the contribution of the entire p53 family in chemotherapeutic drug response in gastrointestinal tumors. Real-time PCR and immunohistochemistry revealed complexity and variability of expression profiles of the p53 protein family. Using colon and esophageal cancer cells, we found that the integral transcription activity of the entire p53 family, as measured by the reporter analysis, associated with response to drug treatment in studied cells. We also found that p53 and p73, as well as p63 and p73, bind simultaneously to the promoters of p53 target genes. Taken together, our results support the view that the p53 protein family functions as an interacting network of proteins and show that cellular responses to chemotherapeutic drug treatment are determined by the total activity of the entire p53 family, rather than p53 alone. PMID:20197393

  8. Interactions of the p53 protein family in cellular stress response in gastrointestinal tumors.

    PubMed

    Vilgelm, Anna E; Washington, Mary K; Wei, Jinxiong; Chen, Heidi; Prassolov, Vladimir S; Zaika, Alexander I

    2010-03-01

    p53, p63, and p73 are members of the p53 protein family involved in regulation of cell cycle, apoptosis, differentiation, and other critical cellular processes. Here, we investigated the contribution of the entire p53 family in chemotherapeutic drug response in gastrointestinal tumors. Real-time PCR and immunohistochemistry revealed complexity and variability of expression profiles of the p53 protein family. Using colon and esophageal cancer cells, we found that the integral transcription activity of the entire p53 family, as measured by the reporter analysis, associated with response to drug treatment in studied cells. We also found that p53 and p73, as well as p63 and p73, bind simultaneously to the promoters of p53 target genes. Taken together, our results support the view that the p53 protein family functions as an interacting network of proteins and show that cellular responses to chemotherapeutic drug treatment are determined by the total activity of the entire p53 family rather than p53 alone.

  9. Amyloid beta-protein induces the cerebrovascular cellular pathology of Alzheimer's disease and related disorders.

    PubMed

    Van Nostrand, W E; Davis-Salinas, J; Saporito-Irwin, S M

    1996-01-17

    One of the hallmark pathologic characteristics of Alzheimer's disease (AD) and related disorders is deposition of the 39-42 amino acid amyloid beta-protein (A beta) in the walls of cerebral blood vessels. The cerebrovascular A beta deposits in these disorders are associated with degenerating smooth muscle cells in the vessel wall which have been implicated in the expression of the amyloid beta-protein precursor (A beta PP) and formation of A beta. We have established primary cultures of human cerebrovascular smooth muscle cells as a model for investigating the cellular pathologic processes involved in the cerebral amyloid angiopathy of AD and related disorders. Recently, we have shown that A beta 1-42, the predominant pathologic cerebrovascular form of A beta, causes extensive cellular degeneration that is accompanied by a striking increase in the levels of cellular A beta PP, potentially amyloidogenic carboxyl terminal A beta PP fragments, and soluble A beta peptide in the cultured human cerebrovascular smooth muscle cells. Together, these studies provide evidence that A beta contributes to the onset and progression of the cerebrovascular pathology associated with AD and related disorders and suggests the mechanism involves a molecular cascade with a novel product-precursor relationship that results in the adverse production and accumulation of A beta.

  10. Differential activities of cellular and viral macro domain proteins in binding of ADP-ribose metabolites.

    PubMed

    Neuvonen, Maarit; Ahola, Tero

    2009-01-01

    Macro domain is a highly conserved protein domain found in both eukaryotes and prokaryotes. Macro domains are also encoded by a set of positive-strand RNA viruses that replicate in the cytoplasm of animal cells, including coronaviruses and alphaviruses. The functions of the macro domain are poorly understood, but it has been suggested to be an ADP-ribose-binding module. We have here characterized three novel human macro domain proteins that were found to reside either in the cytoplasm and nucleus [macro domain protein 2 (MDO2) and ganglioside-induced differentiation-associated protein 2] or in mitochondria [macro domain protein 1 (MDO1)], and compared them with viral macro domains from Semliki Forest virus, hepatitis E virus, and severe acute respiratory syndrome coronavirus, and with a yeast macro protein, Poa1p. MDO2 specifically bound monomeric ADP-ribose with a high affinity (K(d)=0.15 microM), but did not bind poly(ADP-ribose) efficiently. MDO2 also hydrolyzed ADP-ribose-1'' phosphate, resembling Poa1p in all these properties. Ganglioside-induced differentiation-associated protein 2 did not show affinity for ADP-ribose or its derivatives, but instead bound poly(A). MDO1 was generally active in these reactions, including poly(A) binding. Individual point mutations in MDO1 abolished monomeric ADP-ribose binding, but not poly(ADP-ribose) binding; in poly(ADP-ribose) binding assays, the monomer did not compete against polymer binding. The viral macro proteins bound poly(ADP-ribose) and poly(A), but had a low affinity for monomeric ADP-ribose. Thus, the viral proteins do not closely resemble any of the human proteins in their biochemical functions. The differential activity profiles of the human proteins implicate them in different cellular pathways, some of which may involve RNA rather than ADP-ribose derivatives.

  11. A fluorescent reporter for mapping cellular protein-protein interactions in time and space.

    PubMed

    Moreno, Daniel; Neller, Joachim; Kestler, Hans A; Kraus, Johann; Dünkler, Alexander; Johnsson, Nils

    2013-01-01

    We introduce a fluorescent reporter for monitoring protein-protein interactions in living cells. The method is based on the Split-Ubiquitin method and uses the ratio of two auto-fluorescent reporter proteins as signal for interaction (SPLIFF). The mating of two haploid yeast cells initiates the analysis and the interactions are followed online by two-channel time-lapse microscopy of the diploid cells during their first cell cycle. Using this approach we could with high spatio-temporal resolution visualize the differences between the interactions of the microtubule binding protein Stu2p with two of its binding partners, monitor the transient association of a Ran-GTPase with its receptors at the nuclear pore, and distinguish between protein interactions at the polar cortical domain at different phases of polar growth. These examples further demonstrate that protein-protein interactions identified from large-scale screens can be effectively followed up by high-resolution single-cell analysis. PMID:23511205

  12. Impact of protein pre-coating on the protein corona composition and nanoparticle cellular uptake.

    PubMed

    Mirshafiee, Vahid; Kim, Raehyun; Park, Soyun; Mahmoudi, Morteza; Kraft, Mary L

    2016-01-01

    Nanoparticles (NPs) are functionalized with targeting ligands to enable selectively delivering drugs to desired locations in the body. When these functionalized NPs enter the blood stream, plasma proteins bind to their surfaces, forming a protein corona that affects NP uptake and targeting efficiency. To address this problem, new strategies for directing the formation of a protein corona that has targeting capabilities are emerging. Here, we have investigated the feasibility of directing corona composition to promote targeted NP uptake by specific types of cells. We used the well-characterized process of opsonin-induced phagocytosis by macrophages as a simplified model of corona-mediated NP uptake by a desired cell type. We demonstrate that pre-coating silica NPs with gamma-globulins (γ-globulins) produced a protein corona that was enriched with opsonins, such as immunoglobulins. Although immunoglobulins are ligands that bind to receptors on macrophages and elicit phagocytois, the opsonin-rich protein corona did not increase NP uptake by macrophage RAW 264.7 cells. Immunolabeling experiments indicated that the binding of opsonins to their target cell surface receptors was impeded by other proteins in the corona. Thus, corona-mediated NP targeting strategies must optimize both the recruitment of the desired plasma proteins as well as their accessibility and orientation in the corona layer.

  13. Start2Fold: a database of hydrogen/deuterium exchange data on protein folding and stability

    PubMed Central

    Pancsa, Rita; Varadi, Mihaly; Tompa, Peter; Vranken, Wim F.

    2016-01-01

    Proteins fulfil a wide range of tasks in cells; understanding how they fold into complex three-dimensional (3D) structures and how these structures remain stable while retaining sufficient dynamics for functionality is essential for the interpretation of overall protein behaviour. Since the 1950's, solvent exchange-based methods have been the most powerful experimental means to obtain information on the folding and stability of proteins. Considerable expertise and care were required to obtain the resulting datasets, which, despite their importance and intrinsic value, have never been collected, curated and classified. Start2Fold is an openly accessible database (http://start2fold.eu) of carefully curated hydrogen/deuterium exchange (HDX) data extracted from the literature that is open for new submissions from the community. The database entries contain (i) information on the proteins investigated and the underlying experimental procedures and (ii) the classification of the residues based on their exchange protection levels, also allowing for the instant visualization of the relevant residue groups on the 3D structures of the corresponding proteins. By providing a clear hierarchical framework for the easy sharing, comparison and (re-)interpretation of HDX data, Start2Fold intends to promote a better understanding of how the protein sequence encodes folding and structure as well as the development of new computational methods predicting protein folding and stability. PMID:26582925

  14. Ultrasensitive proteomic quantitation of cellular signaling by digitized nanoparticle-protein counting

    PubMed Central

    Jacob, Thomas; Agarwal, Anupriya; Ramunno-Johnson, Damien; O’Hare, Thomas; Gönen, Mehmet; Tyner, Jeffrey W.; Druker, Brian J.; Vu, Tania Q.

    2016-01-01

    Many important signaling and regulatory proteins are expressed at low abundance and are difficult to measure in single cells. We report a molecular imaging approach to quantitate protein levels by digitized, discrete counting of nanoparticle-tagged proteins. Digitized protein counting provides ultrasensitive molecular detection of proteins in single cells that surpasses conventional methods of quantitating total diffuse fluorescence, and offers a substantial improvement in protein quantitation. We implement this digitized proteomic approach in an integrated imaging platform, the single cell-quantum dot platform (SC-QDP), to execute sensitive single cell phosphoquantitation in response to multiple drug treatment conditions and using limited primary patient material. The SC-QDP: 1) identified pAKT and pERK phospho-heterogeneity and insensitivity in individual leukemia cells treated with a multi-drug panel of FDA-approved kinase inhibitors, and 2) revealed subpopulations of drug-insensitive CD34+ stem cells with high pCRKL and pSTAT5 signaling in chronic myeloid leukemia patient blood samples. This ultrasensitive digitized protein detection approach is valuable for uncovering subtle but important differences in signaling, drug insensitivity, and other key cellular processes amongst single cells. PMID:27320899

  15. Cellular and functional specificity among ferritin-like proteins in the multicellular cyanobacterium Nostoc punctiforme.

    PubMed

    Ekman, Martin; Sandh, Gustaf; Nenninger, Anja; Oliveira, Paulo; Stensjö, Karin

    2014-03-01

    Ferritin-like proteins constitute a remarkably heterogeneous protein family, including ferritins, bacterioferritins and Dps proteins. The genome of the filamentous heterocyst-forming cyanobacterium Nostoc punctiforme encodes five ferritin-like proteins. In the present paper, we report a multidimensional characterization of these proteins. Our phylogenetic and bioinformatics analyses suggest both structural and physiological differences among the ferritin-like proteins. The expression of these five genes responded differently to hydrogen peroxide treatment, with a significantly higher rise in transcript level for Npun_F3730 as compared with the other four genes. A specific role for Npun_F3730 in the cells tolerance against hydrogen peroxide was also supported by the inactivation of Npun_F3730, Npun_R5701 and Npun_R6212; among these, only the ΔNpun_F3730 strain showed an increased sensitivity to hydrogen peroxide compared with wild type. Analysis of promoter-GFP reporter fusions of the ferritin-like genes indicated that Npun_F3730 and Npun_R5701 were expressed in all cell types of a diazotrophic culture, while Npun_F6212 was expressed specifically in heterocysts. Our study provides the first comprehensive analysis combining functional differentiation and cellular specificity within this important group of proteins in a multicellular cyanobacterium. PMID:23992552

  16. ERp57 as a novel cellular factor controlling prion protein biosynthesis: Therapeutic potential of protein disulfide isomerases.

    PubMed

    Sepulveda, Martin; Rozas, Pablo; Hetz, Claudio; Medinas, Danilo B

    2016-01-01

    Disturbance of endoplasmic reticulum (ER) proteostasis is observed in Prion-related disorders (PrDs). The protein disulfide isomerase ERp57 is a stress-responsive ER chaperone up-regulated in the brain of Creutzfeldt-Jakob disease patients. However, the actual role of ERp57 in prion protein (PrP) biogenesis and the ER stress response remained poorly defined. We have recently addressed this question using gain- and loss-of-function approaches in vitro and animal models, observing that ERp57 regulates steady-state levels of PrP. Our results revealed that ERp57 modulates the biosynthesis and maturation of PrP but, surprisingly, does not contribute to the global cellular reaction against ER stress in neurons. Here we discuss the relevance of ERp57 as a possible therapeutic target in PrDs and other protein misfolding disorders. PMID:26864548

  17. SELDI-TOF analysis of glioblastoma cyst fluid is an approach for assessing cellular protein expression

    PubMed Central

    Hoelscher, Martin; Richter, Nina; Melle, Christian; von Eggeling, Ferdinand; Schaenzer, Anne; Nestler, Ulf

    2013-01-01

    Objectives: In about 10% of glioblastoma patients, preoperative MRI discloses the presence of tumor cysts. Whereas the impact of cystic appearance on prognosis has been discussed extensively, only little is known about the tumor cyst fluid. In this study, we tested the feasibility of the surface enhanced laser desorption ionization time of flight (SELDI-TOF) technique to detect cyst fluid proteins. Methods: Cyst fluid was collected from 21 glioblastoma patients for SELDI-TOF analysis and compared to control cerebrospinal fluids from 15 patients with spinal stenosis. Resulting protein peaks with significant differences between groups were further described, using the molecular weight in an internet search of protein databases and publications. Two potential cyst fluid proteins, basigin and ferritin light chain, were selected for immunohistological detection in the histologic slides of the patients, metallothionein (MT) served as negative control. Results: As supposed from the results of the SELDI-TOF analysis, basigin and ferritin were detected immunohistochemically in the cyst wall, whereas MT was more equally distributed between the cyst wall and the surrounding tumor tissue. Median survival time of the patients was 20 months (range 2 to 102 months) and correlated with age, but not with expression of the three proteins. Discussion: The SELDI-TOF approach reveals a number of proteins, potentially present in glioblastoma cyst fluid. Identification of these proteins in tumor cells may help understand the pathogenetic pathways and the prognostic value of cystic changes. PMID:24225180

  18. Mps1 (Monopolar Spindle 1) Protein Inhibition Affects Cellular Growth and Pro-Embryogenic Masses Morphology in Embryogenic Cultures of Araucaria angustifolia (Araucariaceae).

    PubMed

    Douétts-Peres, Jackellinne C; Cruz, Marco Antônio L; Reis, Ricardo S; Heringer, Angelo S; de Oliveira, Eduardo A G; Elbl, Paula M; Floh, Eny I S; Silveira, Vanildo; Santa-Catarina, Claudete

    2016-01-01

    Somatic embryogenesis has been shown to be an efficient tool for studying processes based on cell growth and development. The fine regulation of the cell cycle is essential for proper embryo formation during the process of somatic embryogenesis. The aims of the present work were to identify and perform a structural and functional characterization of Mps1 and to analyze the effects of the inhibition of this protein on cellular growth and pro-embryogenic mass (PEM) morphology in embryogenic cultures of A. angustifolia. A single-copy Mps1 gene named AaMps1 was retrieved from the A. angustifolia transcriptome database, and through a mass spectrometry approach, AaMps1 was identified and quantified in embryogenic cultures. The Mps1 inhibitor SP600125 (10 μM) inhibited cellular growth and changed PEMs, and these effects were accompanied by a reduction in AaMps1 protein levels in embryogenic cultures. Our work has identified the Mps1 protein in a gymnosperm species for the first time, and we have shown that inhibiting Mps1 affects cellular growth and PEM differentiation during A. angustifolia somatic embryogenesis. These data will be useful for better understanding cell cycle control during somatic embryogenesis in plants. PMID:27064899

  19. Mps1 (Monopolar Spindle 1) Protein Inhibition Affects Cellular Growth and Pro-Embryogenic Masses Morphology in Embryogenic Cultures of Araucaria angustifolia (Araucariaceae)

    PubMed Central

    Douétts-Peres, Jackellinne C.; Cruz, Marco Antônio L.; Reis, Ricardo S.; Heringer, Angelo S.; de Oliveira, Eduardo A. G.; Elbl, Paula M.; Floh, Eny I. S.; Silveira, Vanildo

    2016-01-01

    Somatic embryogenesis has been shown to be an efficient tool for studying processes based on cell growth and development. The fine regulation of the cell cycle is essential for proper embryo formation during the process of somatic embryogenesis. The aims of the present work were to identify and perform a structural and functional characterization of Mps1 and to analyze the effects of the inhibition of this protein on cellular growth and pro-embryogenic mass (PEM) morphology in embryogenic cultures of A. angustifolia. A single-copy Mps1 gene named AaMps1 was retrieved from the A. angustifolia transcriptome database, and through a mass spectrometry approach, AaMps1 was identified and quantified in embryogenic cultures. The Mps1 inhibitor SP600125 (10 μM) inhibited cellular growth and changed PEMs, and these effects were accompanied by a reduction in AaMps1 protein levels in embryogenic cultures. Our work has identified the Mps1 protein in a gymnosperm species for the first time, and we have shown that inhibiting Mps1 affects cellular growth and PEM differentiation during A. angustifolia somatic embryogenesis. These data will be useful for better understanding cell cycle control during somatic embryogenesis in plants. PMID:27064899

  20. Mps1 (Monopolar Spindle 1) Protein Inhibition Affects Cellular Growth and Pro-Embryogenic Masses Morphology in Embryogenic Cultures of Araucaria angustifolia (Araucariaceae).

    PubMed

    Douétts-Peres, Jackellinne C; Cruz, Marco Antônio L; Reis, Ricardo S; Heringer, Angelo S; de Oliveira, Eduardo A G; Elbl, Paula M; Floh, Eny I S; Silveira, Vanildo; Santa-Catarina, Claudete

    2016-01-01

    Somatic embryogenesis has been shown to be an efficient tool for studying processes based on cell growth and development. The fine regulation of the cell cycle is essential for proper embryo formation during the process of somatic embryogenesis. The aims of the present work were to identify and perform a structural and functional characterization of Mps1 and to analyze the effects of the inhibition of this protein on cellular growth and pro-embryogenic mass (PEM) morphology in embryogenic cultures of A. angustifolia. A single-copy Mps1 gene named AaMps1 was retrieved from the A. angustifolia transcriptome database, and through a mass spectrometry approach, AaMps1 was identified and quantified in embryogenic cultures. The Mps1 inhibitor SP600125 (10 μM) inhibited cellular growth and changed PEMs, and these effects were accompanied by a reduction in AaMps1 protein levels in embryogenic cultures. Our work has identified the Mps1 protein in a gymnosperm species for the first time, and we have shown that inhibiting Mps1 affects cellular growth and PEM differentiation during A. angustifolia somatic embryogenesis. These data will be useful for better understanding cell cycle control during somatic embryogenesis in plants.

  1. Three-dimensional super-resolution protein localization correlated with vitrified cellular context.

    PubMed

    Liu, Bei; Xue, Yanhong; Zhao, Wei; Chen, Yan; Fan, Chunyan; Gu, Lusheng; Zhang, Yongdeng; Zhang, Xiang; Sun, Lei; Huang, Xiaojun; Ding, Wei; Sun, Fei; Ji, Wei; Xu, Tao

    2015-10-14

    We demonstrate the use of cryogenic super-resolution correlative light and electron microscopy (csCLEM) to precisely determine the spatial relationship between proteins and their native cellular structures. Several fluorescent proteins (FPs) were found to be photoswitchable and emitted far more photons under our cryogenic imaging condition, resulting in higher localization precision which is comparable to ambient super-resolution imaging. Vitrified specimens were prepared by high pressure freezing and cryo-sectioning to maintain a near-native state with better fluorescence preservation. A 2-3-fold improvement of resolution over the recent reports was achieved due to the photon budget performance of screening out Dronpa and optimized imaging conditions, even with thin sections which is at a disadvantage when calculate the structure resolution from label density. We extended csCLEM to mammalian cells by introducing cryo-sectioning and observed good correlation of a mitochondrial protein with the mitochondrial outer membrane at nanometer resolution in three dimensions.

  2. Kirkwood–Buff theory of molecular and protein association, aggregation, and cellular crowding

    PubMed Central

    Gee, Moon Bae; Smith, Paul E.

    2009-01-01

    An analysis of the effect of a cosolvent on the association of a solute in solution using the Kirkwood–Buff theory of solutions is presented. The approach builds on the previous results of Ben-Naim by extending the range of applicability to include any number of components at finite concentrations in both closed and semiopen systems. The derived expressions, which are exact, provide a foundation for the analysis and rationalization of cosolvent effects on molecular and biomolecular equilibria including protein association, aggregation, and cellular crowding. A slightly different view of cellular crowding is subsequently obtained. In particular, it is observed that the addition of large cosolvents still favors the associated form even when traditional excluded volume effects are absent. PMID:19894976

  3. Cellular inhibitor of apoptosis proteins prevent clearance of hepatitis B virus

    PubMed Central

    Ebert, Gregor; Preston, Simon; Allison, Cody; Cooney, James; Toe, Jesse G.; Stutz, Michael D.; Ojaimi, Samar; Scott, Hamish W.; Baschuk, Nikola; Nachbur, Ueli; Torresi, Joseph; Chin, Ruth; Colledge, Danielle; Li, Xin; Warner, Nadia; Revill, Peter; Bowden, Scott; Silke, John; Begley, C. Glenn; Pellegrini, Marc

    2015-01-01

    Hepatitis B virus (HBV) infection can result in a spectrum of outcomes from immune-mediated control to disease progression, cirrhosis, and liver cancer. The host molecular pathways that influence and contribute to these outcomes need to be defined. Using an immunocompetent mouse model of chronic HBV infection, we identified some of the host cellular and molecular factors that impact on infection outcomes. Here, we show that cellular inhibitor of apoptosis proteins (cIAPs) attenuate TNF signaling during hepatitis B infection, and they restrict the death of infected hepatocytes, thus allowing viral persistence. Animals with a liver-specific cIAP1 and total cIAP2 deficiency efficiently control HBV infection compared with WT mice. This phenotype was partly recapitulated in mice that were deficient in cIAP2 alone. These results indicate that antagonizing the function of cIAPs may promote the clearance of HBV infection. PMID:25902529

  4. UET: a database of evolutionarily-predicted functional determinants of protein sequences that cluster as functional sites in protein structures.

    PubMed

    Lua, Rhonald C; Wilson, Stephen J; Konecki, Daniel M; Wilkins, Angela D; Venner, Eric; Morgan, Daniel H; Lichtarge, Olivier

    2016-01-01

    The structure and function of proteins underlie most aspects of biology and their mutational perturbations often cause disease. To identify the molecular determinants of function as well as targets for drugs, it is central to characterize the important residues and how they cluster to form functional sites. The Evolutionary Trace (ET) achieves this by ranking the functional and structural importance of the protein sequence positions. ET uses evolutionary distances to estimate functional distances and correlates genotype variations with those in the fitness phenotype. Thus, ET ranks are worse for sequence positions that vary among evolutionarily closer homologs but better for positions that vary mostly among distant homologs. This approach identifies functional determinants, predicts function, guides the mutational redesign of functional and allosteric specificity, and interprets the action of coding sequence variations in proteins, people and populations. Now, the UET database offers pre-computed ET analyses for the protein structure databank, and on-the-fly analysis of any protein sequence. A web interface retrieves ET rankings of sequence positions and maps results to a structure to identify functionally important regions. This UET database integrates several ways of viewing the results on the protein sequence or structure and can be found at http://mammoth.bcm.tmc.edu/uet/.

  5. Identification of cyclophilin-40 interacting proteins reveals potential cellular function of cyclophilin-40

    PubMed Central

    Park, Miki Susanto; Chu, Feixia; Xie, Jinghang; Wang, Yu; Bhattacharya, Pompeya; Chan, William K.

    2010-01-01

    Cyclophilin-40 (CyP40) is part of the immunophilin family and is found in Hsp90-containing protein complexes. We are interested in identifying proteins that interact with CyP40. CyP40 interacting proteins in HeLa cells were identified using the tandem affinity purification approach. Adenovirus (AdCyP40) expressing human CyP40 protein, fused with a streptavidin and a calmodulin binding peptides at the N-terminus, was generated. Proteins were separated on a SDS-PAGE gel after tandem affinity purification. Ten silver-stained protein bands that were enriched in the AdCyP40-infected lysate and the corresponding regions in the control lysate were excised, digested by trypsin and identified by tandem mass spectrometric analysis. Eleven interacting proteins were identified and four of which (RACK1, Ku70, RPS3, and NF45) were expressed in rabbit reticulocyte lysates, bacteria, and MCF-7 cells. We confirmed that these proteins interact with CyP40. We observed that RACK1 suppressed the cobalt chloride-induced, HRE-dependent luciferase activity in MCF-7 cells but not in MCF-7 stable cells expressing about 5% of the cellular CyP40 content. In addition, RACK1 reduced the HIF-1α protein accumulation after cobalt chloride treatment which was not observed when the CyP40 content was down-regulated. Collectively, we conclude that reduction of the HIF-1α protein by RACK1 is CyP40-mediated. PMID:21146485

  6. [Neurofibromin - protein structure and cellular functions in the context of neurofibromatosis type I pathogenesis].

    PubMed

    Abramowicz, Anna; Gos, Monika

    2015-12-09

    Neurofibromatosis type I (NF1) is multisystemic disease characterized by pigmentary skin changes, increased susceptibility to tumor formation, neurological deficits and skeletal defects. The disease is a monogenic, autosomal dominant disorder, caused by the presence of mutations in the NF1 gene encoding neurofibromin - a multifunctional regulatory protein. The basic function of neurofibromin protein is modulation of the RAS protein activity necessary for regulation of cell proliferation and differentiation by the RAS/MAPK and RAS/PI3K/AKT signal transduction pathways. In addition, neurofibromin is a regulator of adenylate cyclase activity and therefore may interfere with signaling by the cAMP/protein kinase A pathway that regulates cell cycle progression or learning and memory formation processes. Neurofibromin also interacts with many other proteins that are engaged in intracellular transport (tubulin, kinesin), actin cytoskeleton rearrangements (LIMK2, Rho and Rac) or morphogenesis of neural cells (syndecans, CRMP proteins). The activity of neurofibromin is strictly regulated by the expression of different NF1 mRNA isoforms depending on tissue type or period in organism development, the protein localization, posttranslational modifications (phosphorylation, ubiquitination) or interactions with other proteins (e.g. 14-3-3). The fact that neurofibromin is engaged in many cellular processes has significant consequences when the proper protein functioning is impaired due to decreased protein level or activity. It affects the normal cell function and results in disturbances of organism development that lead to the occurrence of clinical signs specific for NF1. In the article, the basic neurofibromin functions are presented in the context of the molecular pathogenesis of NF1.

  7. Modification of an acetone-sodium dodecyl sulfate disruption method for cellular protein extraction from neuropathogenic Clostridium botulinum

    Technology Transfer Automated Retrieval System (TEKTRAN)

    An acetone-sodium dodecyl sulfate (SDS) disruption method was used for the extraction of cellular proteins from neurotoxigenic Clostridium botulinum. The amount of protein extracted per gram of dry weight and the protein profile as revealed by polyacrylamide gel electrophoresis (PAGE) was comparabl...

  8. PATtyFams: Protein Families for the Microbial Genomes in the PATRIC Database

    PubMed Central

    Davis, James J.; Gerdes, Svetlana; Olsen, Gary J.; Olson, Robert; Pusch, Gordon D.; Shukla, Maulik; Vonstein, Veronika; Wattam, Alice R.; Yoo, Hyunseung

    2016-01-01

    The ability to build accurate protein families is a fundamental operation in bioinformatics that influences comparative analyses, genome annotation, and metabolic modeling. For several years we have been maintaining protein families for all microbial genomes in the PATRIC database (Pathosystems Resource Integration Center, patricbrc.org) in order to drive many of the comparative analysis tools that are available through the PATRIC website. However, due to the burgeoning number of genomes, traditional approaches for generating protein families are becoming prohibitive. In this report, we describe a new approach for generating protein families, which we call PATtyFams. This method uses the k-mer-based function assignments available through RAST (Rapid Annotation using Subsystem Technology) to rapidly guide family formation, and then differentiates the function-based groups into families using a Markov Cluster algorithm (MCL). This new approach for generating protein families is rapid, scalable and has properties that are consistent with alignment-based methods. PMID:26903996

  9. Divalent metals stabilize cellular prion proteins and alter the rate of proteinase-K dependent limited proteolysis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background: The key biochemical event in the pathogenesis of prion diseases is the conversion of normal cellular prion proteins (PrP**c) to the proteinase K (PK) resistant, abnormal form (PrP**sc); however, the cellular mechanisms underlying the conversion remain enigmatic. Binding of divalent ca...

  10. Fast kinase domain-containing protein 3 is a mitochondrial protein essential for cellular respiration

    SciTech Connect

    Simarro, Maria; Gimenez-Cassina, Alfredo; Kedersha, Nancy; Lazaro, Jean-Bernard; Adelmant, Guillaume O.; Marto, Jarrod A.; Rhee, Kirsten; Tisdale, Sarah; Danial, Nika; Benarafa, Charaf; Orduna, Anonio; Anderson, Paul

    2010-10-22

    Research highlights: {yields} Five members of the FAST kinase domain-containing proteins are localized to mitochondria in mammalian cells. {yields} The FASTKD3 interactome includes proteins involved in various aspects of mitochondrial metabolism. {yields} Targeted knockdown of FASTKD3 significantly reduces basal and maximal mitochondrial oxygen consumption. -- Abstract: Fas-activated serine/threonine phosphoprotein (FAST) is the founding member of the FAST kinase domain-containing protein (FASTKD) family that includes FASTKD1-5. FAST is a sensor of mitochondrial stress that modulates protein translation to promote the survival of cells exposed to adverse conditions. Mutations in FASTKD2 have been linked to a mitochondrial encephalomyopathy that is associated with reduced cytochrome c oxidase activity, an essential component of the mitochondrial electron transport chain. We have confirmed the mitochondrial localization of FASTKD2 and shown that all FASTKD family members are found in mitochondria. Although human and mouse FASTKD1-5 genes are expressed ubiquitously, some of them are most abundantly expressed in mitochondria-enriched tissues. We have found that RNA interference-mediated knockdown of FASTKD3 severely blunts basal and stress-induced mitochondrial oxygen consumption without disrupting the assembly of respiratory chain complexes. Tandem affinity purification reveals that FASTKD3 interacts with components of mitochondrial respiratory and translation machineries. Our results introduce FASTKD3 as an essential component of mitochondrial respiration that may modulate energy balance in cells exposed to adverse conditions by functionally coupling mitochondrial protein synthesis to respiration.

  11. Functions of the cellular prion protein, the end of Moore's law, and Ockham's razor theory

    PubMed Central

    del Río, José A.; Gavín, Rosalina

    2016-01-01

    ABSTRACT Since its discovery the cellular prion protein (encoded by the Prnp gene) has been associated with a large number of functions. The proposed functions rank from basic cellular processes such as cell cycle and survival to neural functions such as behavior and neuroprotection, following a pattern similar to that of Moore's law for electronics. In addition, particular interest is increasing in the participation of Prnp in neurodegeneration. However, in recent years a redefinition of these functions has begun, since examples of previously attributed functions were increasingly re-associated with other proteins. Most of these functions are linked to so-called “Prnp-flanking genes” that are close to the genomic locus of Prnp and which are present in the genome of some Prnp mouse models. In addition, their role in neuroprotection against convulsive insults has been confirmed in recent studies. Lastly, in recent years a large number of models indicating the participation of different domains of the protein in apoptosis have been uncovered. However, after more than 10 years of molecular dissection our view is that the simplest mechanistic model in PrPC-mediated cell death should be considered, as Ockham's razor theory suggested. PMID:26890218

  12. Contrasting roles for Myc and Mad proteins in cellular growth and differentiation.

    PubMed Central

    Chin, L; Schreiber-Agus, N; Pellicer, I; Chen, K; Lee, H W; Dudast, M; Cordon-Cardo, C; DePinho, R A

    1995-01-01

    The positive effects of Myc on cellular growth and gene expression are antagonized by activities of another member of the Myc superfamily, Mad. Characterization of the mouse homolog of human mad on the structural level revealed that domains shown previously to be required in the human protein for anti-Myc repression, sequence-specific DNA-binding activity, and dimerization with its partner Max are highly conserved. Conservation is also evident on the biological level in that both human and mouse mad can antagonize the ability of c-myc to cooperate with ras in the malignant transformation of cultured cells. An analysis of c-myc and mad gene expression in the developing mouse showed contrasting patterns with respect to tissue distribution and developmental stage. Regional differences in expression were more striking on the cellular level, particularly in the mouse and human gastrointestinal system, wherein c-Myc protein was readily detected in immature proliferating cells at the base of the colonic crypts, while Mad protein distribution was restricted to the postmitotic differentiated cells in the apex of the crypts. An increasing gradient of Mad was also evident in the more differentiated subcorneal layers of the stratified squamous epithelium of the skin. Together, these observations support the view that both downregulation of Myc and accumulation of Mad may be necessary for progression of precursor cells to a growth-arrested, terminally differentiated state. Images Fig. 1 Fig. 2 Fig. 3 PMID:7667316

  13. Functions of the cellular prion protein, the end of Moore's law, and Ockham's razor theory.

    PubMed

    del Río, José A; Gavín, Rosalina

    2016-01-01

    Since its discovery the cellular prion protein (encoded by the Prnp gene) has been associated with a large number of functions. The proposed functions rank from basic cellular processes such as cell cycle and survival to neural functions such as behavior and neuroprotection, following a pattern similar to that of Moore's law for electronics. In addition, particular interest is increasing in the participation of Prnp in neurodegeneration. However, in recent years a redefinition of these functions has begun, since examples of previously attributed functions were increasingly re-associated with other proteins. Most of these functions are linked to so-called "Prnp-flanking genes" that are close to the genomic locus of Prnp and which are present in the genome of some Prnp mouse models. In addition, their role in neuroprotection against convulsive insults has been confirmed in recent studies. Lastly, in recent years a large number of models indicating the participation of different domains of the protein in apoptosis have been uncovered. However, after more than 10 years of molecular dissection our view is that the simplest mechanistic model in PrP(C)-mediated cell death should be considered, as Ockham's razor theory suggested.

  14. Turning gold into 'junk': transposable elements utilize central proteins of cellular networks.

    PubMed

    Abrusán, György; Szilágyi, András; Zhang, Yang; Papp, Balázs

    2013-03-01

    The numerous discovered cases of domesticated transposable element (TE) proteins led to the recognition that TEs are a significant source of evolutionary innovation. However, much less is known about the reverse process, whether and to what degree the evolution of TEs is influenced by the genome of their hosts. We addressed this issue by searching for cases of incorporation of host genes into the sequence of TEs and examined the systems-level properties of these genes using the Saccharomyces cerevisiae and Drosophila melanogaster genomes. We identified 51 cases where the evolutionary scenario was the incorporation of a host gene fragment into a TE consensus sequence, and we show that both the yeast and fly homologues of the incorporated protein sequences have central positions in the cellular networks. An analysis of selective pressure (Ka/Ks ratio) detected significant selection in 37% of the cases. Recent research on retrovirus-host interactions shows that virus proteins preferentially target hubs of the host interaction networks enabling them to take over the host cell using only a few proteins. We propose that TEs face a similar evolutionary pressure to evolve proteins with high interacting capacities and take some of the necessary protein domains directly from their hosts.

  15. The bacterial virulence factor NleA inhibits cellular protein secretion by disrupting mammalian COPII function.

    PubMed

    Kim, Jinoh; Thanabalasuriar, Ajitha; Chaworth-Musters, Tessa; Fromme, J Chris; Frey, Elizabeth A; Lario, Paula I; Metalnikov, Pavel; Rizg, Keyrillos; Thomas, Nikhil A; Lee, Sau Fung; Hartland, Elizabeth L; Hardwidge, Philip R; Pawson, Tony; Strynadka, Natalie C; Finlay, B Brett; Schekman, Randy; Gruenheid, Samantha

    2007-09-13

    Enterohemorrhagic and enteropathogenic Escherichia coli (EHEC and EPEC) maintain an extracellular lifestyle and use a type III secretion system to translocate effector proteins into the host cytosol. These effectors manipulate host pathways to favor bacterial replication and survival. NleA is an EHEC/EPEC- and related species-specific translocated effector protein that is essential for bacterial virulence. However, the mechanism by which NleA impacts virulence remains undetermined. Here we demonstrate that NleA compromises the Sec23/24 complex, a component of the mammalian COPII protein coat that shapes intracellular protein transport vesicles, by directly binding Sec24. Expression of an NleA-GFP fusion protein reduces the efficiency of cellular secretion by 50%, and secretion is inhibited in EPEC-infected cells. Direct biochemical experiments show that NleA inhibits COPII-dependent protein export from the endoplasmic reticulum. Collectively, these findings indicate that disruption of COPII function in host cells contributes to the virulence of EPEC and EHEC.

  16. Suppression of cellular proliferation and invasion by the concerted lipid and protein phosphatase activities of PTEN

    PubMed Central

    Davidson, Lindsay; Maccario, Helene; Perera, Nevin M.; Yang, Xuesong; Spinelli, Laura; Tibarewal, Priyanka; Glancy, Ben; Gray, Alex; Weijer, Cornelis J.; Downes, C. Peter; Leslie, Nick R.

    2009-01-01

    PTEN is a tumour suppressor with phosphatase activity in vitro against both lipids and proteins and other potential non-enzymatic mechanisms of action. Although the importance of PTEN’s lipid phosphatase activity in regulating the PI3K signalling pathway is recognised, the significance of PTEN’s other mechanisms of action is currently unclear. Here, we describe the systematic identification of a PTEN mutant, PTEN Y138L, with activity against lipid, but not soluble substrates. Using this mutant we provide evidence for the interfacial activation of PTEN against lipid substrates. We also show that when re-expressed at physiological levels in PTEN null U87MG glioblastoma cells the protein phosphatase activity of PTEN is not required to regulate cellular PtdInsP3 levels or the downstream protein kinase Akt/PKB. Finally, in 3D Matrigel cultures of U87MG cells similarly re-expressing PTEN mutants, both the protein and lipid phosphatase activities were required to inhibit invasion, but either activity alone significantly inhibited proliferation, albeit only weakly for the protein phosphatase activity. Our data provides a novel tool to address the significance of PTEN’s separable lipid and protein phosphatase activities and suggest that both activities act to suppress proliferation and act together to suppress invasion. PMID:19915616

  17. VHLdb: A database of von Hippel-Lindau protein interactors and mutations.

    PubMed

    Tabaro, Francesco; Minervini, Giovanni; Sundus, Faiza; Quaglia, Federica; Leonardi, Emanuela; Piovesan, Damiano; Tosatto, Silvio C E

    2016-01-01

    Mutations in von Hippel-Lindau tumor suppressor protein (pVHL) predispose to develop tumors affecting specific target organs, such as the retina, epididymis, adrenal glands, pancreas and kidneys. Currently, more than 400 pVHL interacting proteins are either described in the literature or predicted in public databases. This data is scattered among several different sources, slowing down the comprehension of pVHL's biological role. Here we present VHLdb, a novel database collecting available interaction and mutation data on pVHL to provide novel integrated annotations. In VHLdb, pVHL interactors are organized according to two annotation levels, manual and automatic. Mutation data are easily accessible and a novel visualization tool has been implemented. A user-friendly feedback function to improve database content through community-driven curation is also provided. VHLdb presently contains 478 interactors, of which 117 have been manually curated, and 1,074 mutations. This makes it the largest available database for pVHL-related information. VHLdb is available from URL: http://vhldb.bio.unipd.it/. PMID:27511743

  18. VHLdb: A database of von Hippel-Lindau protein interactors and mutations

    PubMed Central

    Tabaro, Francesco; Minervini, Giovanni; Sundus, Faiza; Quaglia, Federica; Leonardi, Emanuela; Piovesan, Damiano; Tosatto, Silvio C. E.

    2016-01-01

    Mutations in von Hippel-Lindau tumor suppressor protein (pVHL) predispose to develop tumors affecting specific target organs, such as the retina, epididymis, adrenal glands, pancreas and kidneys. Currently, more than 400 pVHL interacting proteins are either described in the literature or predicted in public databases. This data is scattered among several different sources, slowing down the comprehension of pVHL’s biological role. Here we present VHLdb, a novel database collecting available interaction and mutation data on pVHL to provide novel integrated annotations. In VHLdb, pVHL interactors are organized according to two annotation levels, manual and automatic. Mutation data are easily accessible and a novel visualization tool has been implemented. A user-friendly feedback function to improve database content through community-driven curation is also provided. VHLdb presently contains 478 interactors, of which 117 have been manually curated, and 1,074 mutations. This makes it the largest available database for pVHL-related information. VHLdb is available from URL: http://vhldb.bio.unipd.it/. PMID:27511743

  19. InterEvol database: exploring the structure and evolution of protein complex interfaces.

    PubMed

    Faure, Guilhem; Andreani, Jessica; Guerois, Raphaël

    2012-01-01

    Capturing how the structures of interacting partners evolved at their binding interfaces is a fundamental issue for understanding interactomes evolution. In that scope, the InterEvol database was designed for exploring 3D structures of homologous interfaces of protein complexes. For every chain forming a complex in the protein data bank (PDB), close and remote structural interologs were identified providing essential snapshots for studying interfaces evolution. The database provides tools to retrieve and visualize these structures. In addition, pre-computed multiple sequence alignments of most likely interologs retrieved from a wide range of species can be downloaded to enrich the analysis. The database can be queried either directly by pdb code or keyword but also from the sequence of one or two partners. Interologs multiple sequence alignments can also be recomputed online with tailored parameters using the InterEvolAlign facility. Last, an InterEvol PyMol plugin was developed to improve interactive exploration of structures versus sequence alignments at the interfaces of complexes. Based on a series of automatic methods to extract structural and sequence data, the database will be monthly updated. Structures coordinates and sequence alignments can be queried and downloaded from the InterEvol web interface at http://biodev.cea.fr/interevol/.

  20. Investigating the Cellular Distribution and Interactions of HIV-1 Nucleocapsid Protein by Quantitative Fluorescence Microscopy

    PubMed Central

    Anton, Halina; Taha, Nedal; Boutant, Emmanuel; Richert, Ludovic; Khatter, Heena; Klaholz, Bruno; Rondé, Philippe; Réal, Eléonore; de Rocquigny, Hugues; Mély, Yves

    2015-01-01

    The nucleocapsid protein (NCp7) of the Human immunodeficiency virus type 1 (HIV-1) is a small basic protein containing two zinc fingers. About 2000 NCp7 molecules coat the genomic RNA in the HIV-1 virion. After infection of a target cell, the viral core enters into the cytoplasm, where NCp7 chaperones the reverse transcription of the genomic RNA into the proviral DNA. As a consequence of their much lower affinity for double-stranded DNA as compared to single-stranded RNAs, NCp7 molecules are thought to be released in the cytoplasm and the nucleus of infected cells in the late steps of reverse transcription. Yet, little is known on the cellular distribution of the released NCp7 molecules and on their possible interactions with cell components. Hence, the aim of this study was to identify potential cellular partners of NCp7 and to monitor its intracellular distribution and dynamics by means of confocal fluorescence microscopy, fluorescence lifetime imaging microscopy, fluorescence recovery after photobleaching, fluorescence correlation and cross-correlation spectroscopy, and raster imaging correlation spectroscopy. HeLa cells transfected with eGFP-labeled NCp7 were used as a model system. We found that NCp7-eGFP localizes mainly in the cytoplasm and the nucleoli, where it binds to cellular RNAs, and notably to ribosomal RNAs which are the most abundant. The binding of NCp7 to ribosomes was further substantiated by the intracellular co-diffusion of NCp7 with the ribosomal protein 26, a component of the large ribosomal subunit. Finally, gradient centrifugation experiments demonstrate a direct association of NCp7 with purified 80S ribosomes. Thus, our data suggest that NCp7 molecules released in newly infected cells may primarily bind to ribosomes, where they may exert a new potential role in HIV-1 infection. PMID:25723396

  1. Humoral and cellular immune responses to matrix protein of measles virus in subacute sclerosing panencephalitis.

    PubMed Central

    Dhib-Jalbut, S; McFarland, H F; Mingioli, E S; Sever, J L; McFarlin, D E

    1988-01-01

    The immune response to matrix (M) protein of measles virus was examined in patients with subacute sclerosing panencephalitis (SSPE) and controls. Antibodies specific for M and nucleocapsid (NC) proteins in 11 serum and 8 cerebrospinal fluid (CSF) samples from patients with SSPE were quantitated by enzyme-linked immunosorbent assay by using affinity-purified measles virus proteins. Geometric mean anti-NC antibody titers were higher in the serum (6.58 +/- 0.98 [mean +/- standard deviation]) and CSF (4.38 +/- 0.74) of SSPE patients compared with controls. Anti-M antibodies were present in the serum and CSF of all SSPE samples tested but in titers lower than those of anti-NC antibodies. Geometric mean anti-M antibody titer was 3.35 +/- 0.53 in sera from patients with SSPE compared with 3.05 +/- 0.66 in sera from patients with other neurological diseases and 3.12 +/- 0.74 in sera from healthy individuals. Geometric mean anti-M antibody titer was 2.59 +/- 0.86 in the CSF of eight patients with SSPE compared with a mean less than 1.00 for patients with other neurological disease (controls). Intrathecal synthesis of anti-M or anti-NC antibodies was established in four patients with SSPE. The cellular immune responses to M, F, HA, and NC proteins were examined in four of the patients with SSPE by lymphoproliferation and were not significantly different from those in five healthy controls. The results demonstrate humoral and cellular immune responses to M protein in patients with SSPE and indicate that it is unlikely that a defect in the immune response to this virus component accounts for the disease process in the patients studied. Images PMID:3373575

  2. Negative Example Selection for Protein Function Prediction: The NoGO Database

    PubMed Central

    Youngs, Noah; Penfold-Brown, Duncan; Bonneau, Richard; Shasha, Dennis

    2014-01-01

    Negative examples – genes that are known not to carry out a given protein function – are rarely recorded in genome and proteome annotation databases, such as the Gene Ontology database. Negative examples are required, however, for several of the most powerful machine learning methods for integrative protein function prediction. Most protein function prediction efforts have relied on a variety of heuristics for the choice of negative examples. Determining the accuracy of methods for negative example prediction is itself a non-trivial task, given that the Open World Assumption as applied to gene annotations rules out many traditional validation metrics. We present a rigorous comparison of these heuristics, utilizing a temporal holdout, and a novel evaluation strategy for negative examples. We add to this comparison several algorithms adapted from Positive-Unlabeled learning scenarios in text-classification, which are the current state of the art methods for generating negative examples in low-density annotation contexts. Lastly, we present two novel algorithms of our own construction, one based on empirical conditional probability, and the other using topic modeling applied to genes and annotations. We demonstrate that our algorithms achieve significantly fewer incorrect negative example predictions than the current state of the art, using multiple benchmarks covering multiple organisms. Our methods may be applied to generate negative examples for any type of method that deals with protein function, and to this end we provide a database of negative examples in several well-studied organisms, for general use (The NoGO database, available at: bonneaulab.bio.nyu.edu/nogo.html). PMID:24922051

  3. Negative example selection for protein function prediction: the NoGO database.

    PubMed

    Youngs, Noah; Penfold-Brown, Duncan; Bonneau, Richard; Shasha, Dennis

    2014-06-01

    Negative examples - genes that are known not to carry out a given protein function - are rarely recorded in genome and proteome annotation databases, such as the Gene Ontology database. Negative examples are required, however, for several of the most powerful machine learning methods for integrative protein function prediction. Most protein function prediction efforts have relied on a variety of heuristics for the choice of negative examples. Determining the accuracy of methods for negative example prediction is itself a non-trivial task, given that the Open World Assumption as applied to gene annotations rules out many traditional validation metrics. We present a rigorous comparison of these heuristics, utilizing a temporal holdout, and a novel evaluation strategy for negative examples. We add to this comparison several algorithms adapted from Positive-Unlabeled learning scenarios in text-classification, which are the current state of the art methods for generating negative examples in low-density annotation contexts. Lastly, we present two novel algorithms of our own construction, one based on empirical conditional probability, and the other using topic modeling applied to genes and annotations. We demonstrate that our algorithms achieve significantly fewer incorrect negative example predictions than the current state of the art, using multiple benchmarks covering multiple organisms. Our methods may be applied to generate negative examples for any type of method that deals with protein function, and to this end we provide a database of negative examples in several well-studied organisms, for general use (The NoGO database, available at: bonneaulab.bio.nyu.edu/nogo.html).

  4. pE-DB: a database of structural ensembles of intrinsically disordered and of unfolded proteins.

    PubMed

    Varadi, Mihaly; Kosol, Simone; Lebrun, Pierre; Valentini, Erica; Blackledge, Martin; Dunker, A Keith; Felli, Isabella C; Forman-Kay, Julie D; Kriwacki, Richard W; Pierattelli, Roberta; Sussman, Joel; Svergun, Dmitri I; Uversky, Vladimir N; Vendruscolo, Michele; Wishart, David; Wright, Peter E; Tompa, Peter

    2014-01-01

    The goal of pE-DB (http://pedb.vib.be) is to serve as an openly accessible database for the deposition of structural ensembles of intrinsically disordered proteins (IDPs) and of denatured proteins based on nuclear magnetic resonance spectroscopy, small-angle X-ray scattering and other data measured in solution. Owing to the inherent flexibility of IDPs, solution techniques are particularly appropriate for characterizing their biophysical properties, and structural ensembles in agreement with these data provide a convenient tool for describing the underlying conformational sampling. Database entries consist of (i) primary experimental data with descriptions of the acquisition methods and algorithms used for the ensemble calculations, and (ii) the structural ensembles consistent with these data, provided as a set of models in a Protein Data Bank format. PE-DB is open for submissions from the community, and is intended as a forum for disseminating the structural ensembles and the methodologies used to generate them. While the need to represent the IDP structures is clear, methods for determining and evaluating the structural ensembles are still evolving. The availability of the pE-DB database is expected to promote the development of new modeling methods and leads to a better understanding of how function arises from disordered states.

  5. pE-DB: a database of structural ensembles of intrinsically disordered and of unfolded proteins

    PubMed Central

    Varadi, Mihaly; Kosol, Simone; Lebrun, Pierre; Valentini, Erica; Blackledge, Martin; Dunker, A. Keith; Felli, Isabella C.; Forman-Kay, Julie D.; Kriwacki, Richard W.; Pierattelli, Roberta; Sussman, Joel; Svergun, Dmitri I.; Uversky, Vladimir N.; Vendruscolo, Michele; Wishart, David; Wright, Peter E.; Tompa, Peter

    2014-01-01

    The goal of pE-DB (http://pedb.vib.be) is to serve as an openly accessible database for the deposition of structural ensembles of intrinsically disordered proteins (IDPs) and of denatured proteins based on nuclear magnetic resonance spectroscopy, small-angle X-ray scattering and other data measured in solution. Owing to the inherent flexibility of IDPs, solution techniques are particularly appropriate for characterizing their biophysical properties, and structural ensembles in agreement with these data provide a convenient tool for describing the underlying conformational sampling. Database entries consist of (i) primary experimental data with descriptions of the acquisition methods and algorithms used for the ensemble calculations, and (ii) the structural ensembles consistent with these data, provided as a set of models in a Protein Data Bank format. PE-DB is open for submissions from the community, and is intended as a forum for disseminating the structural ensembles and the methodologies used to generate them. While the need to represent the IDP structures is clear, methods for determining and evaluating the structural ensembles are still evolving. The availability of the pE-DB database is expected to promote the development of new modeling methods and leads to a better understanding of how function arises from disordered states. PMID:24174539

  6. Use of the BioGRID Database for Analysis of Yeast Protein and Genetic Interactions.

    PubMed

    Oughtred, Rose; Chatr-aryamontri, Andrew; Breitkreutz, Bobby-Joe; Chang, Christie S; Rust, Jennifer M; Theesfeld, Chandra L; Heinicke, Sven; Breitkreutz, Ashton; Chen, Daici; Hirschman, Jodi; Kolas, Nadine; Livstone, Michael S; Nixon, Julie; O'Donnell, Lara; Ramage, Lindsay; Winter, Andrew; Reguly, Teresa; Sellam, Adnane; Stark, Chris; Boucher, Lorrie; Dolinski, Kara; Tyers, Mike

    2016-01-01

    The BioGRID database is an extensive repository of curated genetic and protein interactions for the budding yeast Saccharomyces cerevisiae, the fission yeast Schizosaccharomyces pombe, and the yeast Candida albicans SC5314, as well as for several other model organisms and humans. This protocol describes how to use the BioGRID website to query genetic or protein interactions for any gene of interest, how to visualize the associated interactions using an embedded interactive network viewer, and how to download data files for either selected interactions or the entire BioGRID interaction data set. PMID:26729909

  7. Use of the BioGRID Database for Analysis of Yeast Protein and Genetic Interactions.

    PubMed

    Oughtred, Rose; Chatr-aryamontri, Andrew; Breitkreutz, Bobby-Joe; Chang, Christie S; Rust, Jennifer M; Theesfeld, Chandra L; Heinicke, Sven; Breitkreutz, Ashton; Chen, Daici; Hirschman, Jodi; Kolas, Nadine; Livstone, Michael S; Nixon, Julie; O'Donnell, Lara; Ramage, Lindsay; Winter, Andrew; Reguly, Teresa; Sellam, Adnane; Stark, Chris; Boucher, Lorrie; Dolinski, Kara; Tyers, Mike

    2016-01-04

    The BioGRID database is an extensive repository of curated genetic and protein interactions for the budding yeast Saccharomyces cerevisiae, the fission yeast Schizosaccharomyces pombe, and the yeast Candida albicans SC5314, as well as for several other model organisms and humans. This protocol describes how to use the BioGRID website to query genetic or protein interactions for any gene of interest, how to visualize the associated interactions using an embedded interactive network viewer, and how to download data files for either selected interactions or the entire BioGRID interaction data set.

  8. PANDIT: an evolution-centric database of protein and associated nucleotide domains with inferred trees.

    PubMed

    Whelan, Simon; de Bakker, Paul I W; Quevillon, Emmanuel; Rodriguez, Nicolas; Goldman, Nick

    2006-01-01

    PANDIT is a database of homologous sequence alignments accompanied by estimates of their corresponding phylogenetic trees. It provides a valuable resource to those studying phylogenetic methodology and the evolution of coding-DNA and protein sequences. Currently in version 17.0, PANDIT comprises 7738 families of homologous protein domains; for each family, DNA and corresponding amino acid sequence multiple alignments are available together with high quality phylogenetic tree estimates. Recent improvements include expanded methods for phylogenetic tree inference, assessment of alignment quality and a redesigned web interface, available at the URL http://www.ebi.ac.uk/goldman-srv/pandit.

  9. Rapamycin reverses cellular phenotypes and enhances mutant protein clearance in Hutchinson-Gilford progeria syndrome cells.

    PubMed

    Cao, Kan; Graziotto, John J; Blair, Cecilia D; Mazzulli, Joseph R; Erdos, Michael R; Krainc, Dimitri; Collins, Francis S

    2011-06-29

    Hutchinson-Gilford progeria syndrome (HGPS) is a lethal genetic disorder characterized by premature aging. HGPS is most commonly caused by a de novo single-nucleotide substitution in the lamin A/C gene (LMNA) that partially activates a cryptic splice donor site in exon 11, producing an abnormal lamin A protein termed progerin. Accumulation of progerin in dividing cells adversely affects the integrity of the nuclear scaffold and leads to nuclear blebbing in cultured cells. Progerin is also produced in normal cells, increasing in abundance as senescence approaches. Here, we report the effect of rapamycin, a macrolide antibiotic that has been implicated in slowing cellular and organismal aging, on the cellular phenotypes of HGPS fibroblasts. Treatment with rapamycin abolished nuclear blebbing, delayed the onset of cellular senescence, and enhanced the degradation of progerin in HGPS cells. Rapamycin also decreased the formation of insoluble progerin aggregates and induced clearance through autophagic mechanisms in normal fibroblasts. Our findings suggest an additional mechanism for the beneficial effects of rapamycin on longevity and encourage the hypothesis that rapamycin treatment could provide clinical benefit for children with HGPS.

  10. Anterior Gradient Protein-2 Is a Regulator of Cellular Adhesion in Prostate Cancer

    PubMed Central

    Chanda, Diptiman; Lee, Joo Hyoung; Sawant, Anandi; Hensel, Jonathan A.; Isayeva, Tatyana; Reilly, Stephanie D.; Siegal, Gene P.; Smith, Claire; Grizzle, William; Singh, Raj; Ponnazhagan, Selvarangan

    2014-01-01

    Anterior Gradient Protein (AGR-2) is reported to be over-expressed in many epithelial cancers and promotes metastasis. A clear-cut mechanism for its observed function(s) has not been previously identified. We found significant upregulation of AGR-2 expression in a bone metastatic prostate cancer cell line, PC3, following culturing in bone marrow-conditioned medium. Substantial AGR-2 expression was also confirmed in prostate cancer tissue specimens in patients with bone lesions. By developing stable clones of PC3 cells with varying levels of AGR-2 expression, we identified that abrogation of AGR-2 significantly reduced cellular attachment to fibronectin, collagen I, collagen IV, laminin I and fibrinogen. Loss of cellular adhesion was associated with sharp decrease in the expression of α4, α5, αV, β3 and β4 integrins. Failure to undergo apoptosis following detachment is a hallmark of epithelial cancer metastasis. The AGR-2-silenced PC3 cells showed higher resistance to Tumor necrosis factor-related apoptosis- inducing ligand (TRAIL) induced apoptosis in vitro. This observation was also supported by significantly reduced Caspase-3 expression in AGR-2-silenced PC3 cells, which is a key effector of both extrinsic and intrinsic death signaling pathways. These data suggest that AGR-2 influence prostate cancer metastasis by regulation of cellular adhesion and apoptosis. PMID:24587138

  11. Transient expression of protein tyrosine phosphatases encoded in Cotesia plutellae bracovirus inhibits insect cellular immune responses

    NASA Astrophysics Data System (ADS)

    Ibrahim, Ahmed M. A.; Kim, Yonggyun

    2008-01-01

    Several immunosuppressive factors are associated with parasitism of an endoparasitoid wasp, Cotesia plutellae, on the diamondback moth, Plutella xylostella. C. plutellae bracovirus (CpBV) encodes a large number of putative protein tyrosine phosphatases (PTPs), which may play a role in inhibiting host cellular immunity. To address this inhibitory hypothesis of CpBV-PTPs, we performed transient expression of individual CpBV-PTPs in hemocytes of the beet armyworm, Spodoptera exigua, and analyzed their cellular immune responses. Two different forms of CpBV-PTPs were chosen and cloned into a eukaryotic expression vector under the control of the p10 promoter of baculovirus: one with the normal cysteine active site (CpBV-PTP1) and the other with a mutated active site (CpBV-PTP5). The hemocytes transfected with CpBV-PTP1 significantly increased in PTP activity compared to control hemocytes, but those with CpBV-PTP5 exhibited a significant decrease in the PTP activity. All transfected hemocytes exhibited a significant reduction in both cell spreading and encapsulation activities compared to control hemocytes. Co-transfection of CpBV-PTP1 together with its double-stranded RNA reduced the messenger RNA (mRNA) level of CpBV-PTP1 and resulted in recovery of both hemocyte behaviors. This is the first report demonstrating that the polydnaviral PTPs can manipulate PTP activity of the hemocytes to interrupt cellular immune responses.

  12. Multiple cellular proteins interact with LEDGF/p75 through a conserved unstructured consensus motif.

    PubMed

    Tesina, Petr; Čermáková, Kateřina; Hořejší, Magdalena; Procházková, Kateřina; Fábry, Milan; Sharma, Subhalakshmi; Christ, Frauke; Demeulemeester, Jonas; Debyser, Zeger; De Rijck, Jan; Veverka, Václav; Řezáčová, Pavlína

    2015-01-01

    Lens epithelium-derived growth factor (LEDGF/p75) is an epigenetic reader and attractive therapeutic target involved in HIV integration and the development of mixed lineage leukaemia (MLL1) fusion-driven leukaemia. Besides HIV integrase and the MLL1-menin complex, LEDGF/p75 interacts with various cellular proteins via its integrase binding domain (IBD). Here we present structural characterization of IBD interactions with transcriptional repressor JPO2 and domesticated transposase PogZ, and show that the PogZ interaction is nearly identical to the interaction of LEDGF/p75 with MLL1. The interaction with the IBD is maintained by an intrinsically disordered IBD-binding motif (IBM) common to all known cellular partners of LEDGF/p75. In addition, based on IBM conservation, we identify and validate IWS1 as a novel LEDGF/p75 interaction partner. Our results also reveal how HIV integrase efficiently displaces cellular binding partners from LEDGF/p75. Finally, the similar binding modes of LEDGF/p75 interaction partners represent a new challenge for the development of selective interaction inhibitors.

  13. CentrosomeDB: a new generation of the centrosomal proteins database for Human and Drosophila melanogaster.

    PubMed

    Alves-Cruzeiro, Joao Miguel da Conceiçao; Nogales-Cadenas, Rubén; Pascual-Montano, Alberto Domingo

    2014-01-01

    We present the second generation of centrosomeDB, available online at http://centrosome.cnb.csic.es, with a significant expansion of 1357 human and drosophila centrosomal genes and their corresponding information. The centrosome of animal cells takes part in important biological processes such as the organization of the interphase microtubule cytoskeleton and the assembly of the mitotic spindle. The active research done during the past decades has produced lots of data related to centrosomal proteins. Unfortunately, the accumulated data are dispersed among diverse and heterogeneous sources of information. We believe that the availability of a repository collecting curated evidences of centrosomal proteins would constitute a key resource for the scientific community. This was our first motivation to introduce CentrosomeDB in NAR database issue in 2009, collecting a set of human centrosomal proteins that were reported in the literature and other sources. The intensive use of this resource during these years has encouraged us to present this new expanded version. Using our database, the researcher is offered the possibility to study the evolution, function and structure of the centrosome. We have compiled information from many sources, including Gene Ontology, disease-association, single nucleotide polymorphisms and associated gene expression experiments. Special interest has been paid to protein-protein interaction.

  14. Depletion of cellular poly (A) binding protein prevents protein synthesis and leads to apoptosis in HeLa cells

    SciTech Connect

    Thangima Zannat, Mst.; Bhattacharjee, Rumpa B.; Bag, Jnanankur

    2011-05-13

    Highlights: {yields} Depletion of cellular PABP level arrests mRNA translation in HeLa cells. {yields} PABP knock down leads to apoptotic cell death. {yields} PABP depletion does not affect transcription. {yields} PABP depletion does not lead to nuclear accumulation of mRNA. -- Abstract: The cytoplasmic poly (A) binding protein (PABP) is important in mRNA translation and stability. In yeast, depletion of PABP leads to translation arrest. Similarly, the PABP gene in Drosophila is important for proper development. It is however uncertain, whether mammalian PABP is essential for mRNA translation. Here we showed the effect of PABP depletion on mRNA metabolism in HeLa cells by using a small interfering RNA. Our results suggest that depletion of PABP prevents protein synthesis and consequently leads to cell death through apoptosis. Interestingly, no detectable effect of PABP depletion on transcription, transport and stability of mRNA was observed.

  15. Microbial Protein-Protein Interactions (MiPPI) Data from the Genomics: GTL Center for Molecular and Cellular Systems (CMCS)

    DOE Data Explorer

    The Genomic Science Center for Molecular and Cellular Systems (CMCS), established in 2002, seeks to identify and characterize the complete set of protein complexes within a cell to provide a mechanistic basis for the understanding of biochemical functions. The CMCS is anchored at ORNL and PNNL. CMCS initially focused on the identification and characterization of protein complexes in two microbial systems,Rhodopseudomonas palustris (R. palustris) and Shewanella oneidensis (S. oneidensis). These two organisms have also been the focus of major DOE Genomic Science/Microbial Cell Program (MCP) projects. To develop an approach for identifying the diverse types of complexes present in microbial organisms, CMCS incorporates a number of molecular biology, microbiology, analytical and computational tools in an integrated pipeline.

  16. N-terminal modifications of cellular proteins: The enzymes involved, their substrate specificities and biological effects

    PubMed Central

    Varland, Sylvia; Osberg, Camilla; Arnesen, Thomas

    2015-01-01

    The vast majority of eukaryotic proteins are N-terminally modified by one or more processing enzymes. Enzymes acting on the very first amino acid of a polypeptide include different peptidases, transferases, and ligases. Methionine aminopeptidases excise the initiator methionine leaving the nascent polypeptide with a newly exposed amino acid that may be further modified. N-terminal acetyl-, methyl-, myristoyl-, and palmitoyltransferases may attach an acetyl, methyl, myristoyl, or palmitoyl group, respectively, to the α-amino group of the target protein N-terminus. With the action of ubiquitin ligases, one or several ubiquitin molecules are transferred, and hence, constitute the N-terminal modification. Modifications at protein N-termini represent an important contribution to proteomic diversity and complexity, and are essential for protein regulation and cellular signaling. Consequently, dysregulation of the N-terminal modifying enzymes is implicated in human diseases. We here review the different protein N-terminal modifications occurring co- or post-translationally with emphasis on the responsible enzymes and their substrate specificities. PMID:25914051

  17. Cellular and subcellular localization of Ras guanyl nucleotide-releasing protein in the rat hippocampus.

    PubMed

    Pierret, P; Vallée, A; Mechawar, N; Dower, N A; Stone, J C; Richardson, P M; Dunn, R J

    2001-01-01

    Ras guanyl nucleotide-releasing protein (RasGRP) is a recently discovered Ras guanyl nucleotide exchange factor that is expressed in selected regions of the rodent CNS, with high levels of expression in the hippocampus. Biochemical studies suggest that RasGRP can activate the Ras signal pathway in response to changes in diacylglycerol and possibly calcium. To investigate potential sites for RasGRP signaling, we have determined the cellular and subcellular localization of RasGRP protein in adult rat hippocampus, and have also examined the appearance of RasGRP mRNA and protein during hippocampal development. RasGRP immunoreactivity is predominately localized to those neurons participating in the direct cortico-hippocampo-cortical loop. In both hippocampal and entorhinal neurons, RasGRP protein appeared to be localized to both dendrites and somata, but not to axons. Electron microscopy of hippocampal pyramidal cells confirmed RasGRP immunoreactivity in neuronal cell bodies and dendrites, where it appeared to be associated with microtubules. The localization of RasGRP to dendrites suggests a role for this pathway in the regulation of dendritic function. Examination of developing hippocampal structures indicated that RasGRP mRNA and protein appear synchronously during the first 2 weeks of postnatal development as these neurons become fully mature. This result indicates that the RasGRP signal transduction pathway is not required during early hippocampal development, but is a feature of mature neurons during the later stages of development.

  18. Identification and characterization of thymus LIM protein: targeted disruption reduces thymus cellularity.

    PubMed

    Kirchner, J; Forbush, K A; Bevan, M J

    2001-12-01

    We have identified a novel LIM gene encoding the thymus LIM protein (TLP), expressed specifically in the thymus in a subset of cortical epithelial cells. TLP was identified as a gene product which is upregulated in a thymus in which selection of T cells is occurring (Rag(-/-) OT-1) compared to its expression in a thymus in which selection is blocked at the CD4+ CD8+ stage of T-cell development (Rag(-/-) Tap(-/-) OT-1). TLP has an apparent molecular mass of 23 kDa and exists as two isomers (TLP-A and TLP-B), which are generated by alternative splicing of the message. The sequences of TLP-A and TLP-B are identical except for the C-terminal 19 or 20 amino acids. Based on protein sequence alignment, TLP is most closely related to the cysteine-rich proteins, a subclass of the family of LIM-only proteins. In both medullary and cortical thymic epithelial cell lines transduced with TLP, the protein localizes to the cytoplasm but does not appear to be strongly associated with actin. In immunohistochemical studies, TLP seems to be localized in a subset of epithelial cells in the cortex and is most abundant near the corticomedullary junction. We generated mice with a targeted disruption of the Tlp locus. In the absence of TLP, thymocyte development and thymus architecture appear to be normal but thymocyte cellularity is reduced by approximately 30%, with a proportional reduction in each subpopulation.

  19. A quantitative chaperone interaction network reveals the architecture of cellular protein homeostasis pathways

    PubMed Central

    Taipale, Mikko; Tucker, George; Peng, Jian; Krykbaeva, Irina; Lin, Zhen-Yuan; Larsen, Brett; Choi, Hyungwon; Berger, Bonnie; Gingras, Anne-Claude; Lindquist, Susan

    2014-01-01

    Chaperones are abundant cellular proteins that promote the folding and function of their substrate proteins (clients). In vivo, chaperones also associate with a large and diverse set of co-factors (co-chaperones) that regulate their specificity and function. However, how these co-chaperones regulate protein folding and whether they have chaperone-independent biological functions is largely unknown. We have combined mass spectrometry and quantitative high-throughput LUMIER assays to systematically characterize the chaperone/co-chaperone/client interaction network in human cells. We uncover hundreds of novel chaperone clients, delineate their participation in specific co-chaperone complexes, and establish a surprisingly distinct network of protein/protein interactions for co-chaperones. As a salient example of the power of such analysis, we establish that NUDC family co-chaperones specifically associate with structurally related but evolutionarily distinct β-propeller folds. We provide a framework for deciphering the proteostasis network, its regulation in development and disease, and expand the use of chaperones as sensors for drug/target engagement. PMID:25036637

  20. FAST KINASE DOMAIN-CONTAINING PROTEIN 3 IS A MITOCHONDRIAL PROTEIN ESSENTIAL FOR CELLULAR RESPIRATION

    PubMed Central

    Simarro, Maria; Gimenez-Cassina, Alfredo; Kedersha, Nancy; Lazaro, Jean-Bernard; Adelmant, Guillaume O; Marto, Jarrod A; Rhee, Kirsten; Tisdale, Sarah; Danial, Nika; Benarafa, Charaf; Orduña, Anonio; Anderson, Paul

    2010-01-01

    Fas-activated serine/threonine phosphoprotein (FAST) is the founding member of the FAST kinase domain-containing protein (FASTKD) family that includes FASTKD1-5. FAST is a sensor of mitochondrial stress that modulates protein translation to promote the survival of cells exposed to adverse conditions. Mutations in FASTKD2 have been linked to a mitochondrial encephalomyopathy that is associated with reduced cytochrome c oxidase activity, an essential component of the mitochondrial electron transport chain. We have confirmed the mitochondrial localization of FASTKD2 and shown that all FASTKD family members are found in mitochondria. Although human and mouse FASTKD1-5 genes are expressed ubiquitously, some of them are most abundantly expressed in mitochondria-enriched tissues. We have found that RNA interference-mediated knockdown of FASTKD3 severely blunts basal and stress-induced mitochondrial oxygen consumption without disrupting the assembly of respiratory chain complexes. Tandem affinity purification reveals that FASTKD3 interacts with components of mitochondrial respiratory and translation machineries. Our results introduce FASTKD3 as an essential component of mitochondrial respiration that may modulate energy balance in cells exposed to adverse conditions by functionally coupling mitochondrial protein synthesis to respiration. PMID:20869947

  1. Molecular basis of cellular localization of poly C binding protein 1 in neuronal cells

    SciTech Connect

    Berry, Andrea M.; Flock, Kelly E.; Loh, Horace H.; Ko, Jane L. . E-mail: kojane@shu.edu

    2006-11-03

    Poly C binding protein 1 (PCBP) is involved in the transcriptional regulation of neuronal mu-opioid receptor gene. In this study, we examined the molecular basis of PCBP cellular/nuclear localization in neuronal cells using EGFP fusion protein. PCBP, containing three KH domains and a variable domain, distributed in cytoplasm and nucleus with a preferential nuclear expression. Domain-deletional analyses suggested the requirement of variable and KH3 domains for strong PCBP nuclear expression. Within the nucleus, a low nucleolar PCBP expression was observed, and PCBP variable domain contributed to this restricted nucleolar expression. Furthermore, the punctate nuclear pattern of PCBP was correlated to its single-stranded (ss) DNA binding ability, with both requiring cooperativity of at least three sequential domains. Collectively, certain PCBP domains thus govern its nuclear distribution and transcriptional regulatory activity in the nucleus of neurons, whereas the low nucleolar expression implicates the disengagement of PCBP in the ribosomal RNA synthesis.

  2. Detection of cellular prion protein in exosomes derived from ovine plasma.

    PubMed

    Berrone, Elena; Corona, Cristiano; Mazza, Maria; Vallino Costassa, Elena; Faro, Monica Lo; Properzi, Francesca; Guglielmetti, Chiara; Maurella, Cristiana; Caramelli, Maria; Deregibus, Maria Chiara; Camussi, Giovanni; Casalone, Cristina

    2015-12-01

    Prion protein (PrP) is present at extremely low levels in the blood of animals and its detection is complicated by the poor sensitivity of current standard methodologies. Interesting results have been obtained with recent advanced technologies that are able to detect minute amounts of the pathological PrP (PrPSc), but their efficiency is reduced by various factors present in blood. In this study, we were able to extract cellular PrP (PrPC) from plasma-derived exosomes by a simple, fast method without the use of differential ultracentrifugation and to visualize it by Western blotting, reducing the presence of most plasma proteins. This result confirms that blood is capable of releasing PrP in association with exosomes and could be useful to better study its role in the pathogenesis of transmissible spongiform encephalopathies. PMID:26399471

  3. Cellular prion protein ablation impairs behavior as a function of age.

    PubMed

    Coitinho, Adriana S; Roesler, Rafael; Martins, Vilma R; Brentani, Ricardo R; Izquierdo, Ivan

    2003-07-18

    Cellular prion protein (PrPc) has been associated with some physiological functions in recent reports. Here we investigate behavioral parameters in 3- and 9-month-old mice lacking PrPc protein (Prnp0/0) and in rats after intrahippocampal administration of affinity purified anti-PrPc IgG (0.09 microg/side). No differences were observed between 3-month-old animals. However, 9-month-old Prnp0/0 mice and rats infused with anti-PrPc antibody showed a clear impairment of short- and long-term memory retention of a step-down inhibitory avoidance task. A decreased locomotor activity during exploration of an open field was also observed. These results suggest that systems involved in memory formation become more susceptible to mechanisms that require PrPc between the ages of 3 and 9 months in both mice and rats.

  4. Addressing Statistical Biases in Nucleotide-Derived Protein Databases for Proteogenomic Search Strategies

    PubMed Central

    2012-01-01

    Proteogenomics has the potential to advance genome annotation through high quality peptide identifications derived from mass spectrometry experiments, which demonstrate a given gene or isoform is expressed and translated at the protein level. This can advance our understanding of genome function, discovering novel genes and gene structure that have not yet been identified or validated. Because of the high-throughput shotgun nature of most proteomics experiments, it is essential to carefully control for false positives and prevent any potential misannotation. A number of statistical procedures to deal with this are in wide use in proteomics, calculating false discovery rate (FDR) and posterior error probability (PEP) values for groups and individual peptide spectrum matches (PSMs). These methods control for multiple testing and exploit decoy databases to estimate statistical significance. Here, we show that database choice has a major effect on these confidence estimates leading to significant differences in the number of PSMs reported. We note that standard target:decoy approaches using six-frame translations of nucleotide sequences, such as assembled transcriptome data, apparently underestimate the confidence assigned to the PSMs. The source of this error stems from the inflated and unusual nature of the six-frame database, where for every target sequence there exists five “incorrect” targets that are unlikely to code for protein. The attendant FDR and PEP estimates lead to fewer accepted PSMs at fixed thresholds, and we show that this effect is a product of the database and statistical modeling and not the search engine. A variety of approaches to limit database size and remove noncoding target sequences are examined and discussed in terms of the altered statistical estimates generated and PSMs reported. These results are of importance to groups carrying out proteogenomics, aiming to maximize the validation and discovery of gene structure in sequenced genomes

  5. Cellular COPII Proteins Are Involved in Production of the Vesicles That Form the Poliovirus Replication Complex

    PubMed Central

    Rust, René C.; Landmann, Lukas; Gosert, Rainer; Tang, Bor Luen; Hong, Wanjin; Hauri, Hans-Peter; Egger, Denise; Bienz, Kurt

    2001-01-01

    Poliovirus (PV) replicates its genome in association with membranous vesicles in the cytoplasm of infected cells. To elucidate the origin and mode of formation of PV vesicles, immunofluorescence labeling with antibodies against the viral vesicle marker proteins 2B and 2BC, as well as cellular markers of the endoplasmic reticulum (ER), anterograde transport vesicles, and the Golgi complex, was performed in BT7-H cells. Optical sections obtained by confocal laser scanning microscopy were subjected to a deconvolution process to enhance resolution and signal-to-noise ratio and to allow for a three-dimensional representation of labeled membrane structures. The mode of formation of the PV vesicles was, on morphological grounds, similar to the formation of anterograde membrane traffic vesicles in uninfected cells. ER-resident membrane markers were excluded from both types of vesicles, and the COPII components Sec13 and Sec31 were both found to be colocalized on the vesicular surface, indicating the presence of a functional COPII coat. PV vesicle formation during early time points of infection did not involve the Golgi complex. The expression of PV protein 2BC or the entire P2 and P3 genomic region led to the production of vesicles carrying a COPII coat and showing the same mode of formation as vesicles produced after PV infection. These results indicate that PV vesicles are formed at the ER by the cellular COPII budding mechanism and thus are homologous to the vesicles of the anterograde membrane transport pathway. PMID:11559814

  6. Functional Modification of Fibrous PCL Scaffolds with Fusion Protein VEGF-HGFI Enhanced Cellularization and Vascularization.

    PubMed

    Zhao, Liqiang; Ma, Shaoyang; Pan, Yiwa; Zhang, Qiuying; Wang, Kai; Song, Dongmin; Wang, Xiangxiang; Feng, Guowei; Liu, Ruming; Xu, Haijin; Zhang, Jun; Qiao, Mingqiang; Kong, Deling

    2016-09-01

    The lack of efficient vascularization within frequently used poly(ε-caprolactone) (PCL) scaffolds has hindered their application in tissue engineering. Hydrophobin HGFI, an amphiphilic protein, can form a self-assembly layer on the surface of PCL scaffolds and convert their wettability. In this study, a fusion protein consisting of HGFI and vascular endothelial growth factor (VEGF) is prepared by Pichia pastoris expression system. Sodium dodecyl sulface-polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting confirm that the VEGF-HGFI is successfully isolated and purified. Transmission electron microscope and water contact angle measurement demonstrate that VEGF-HGFI can form a self-assembly layer with about 25 nm in thickness on electrospun PCL fibers and increase their hydrophilicity. VEGF-HGFI modification can effectively enhance the adhesion, migration, and proliferation of human umbilical vein endothelial cells. Near-infrared fluorescence imaging shows that the VEGF-HGFI modification on PCL scaffolds can exist at least 21 d in vitro and at least 14 d in vivo. Bioluminescence imaging shows that VEGF-HGFI can effectively activate vascular endothelial growth factor receptor 2 receptors. Subcutaneous implantation in mice and rats reveal that cellularization and vascularization are significantly improved in VEGF-HGFI modified PCL scaffolds. These results suggest that VEGF-HGFI is a useful molecule for functional modification of scaffolds to enhance cellularization and vascularization in tissue engineering. PMID:27391702

  7. The cellular prion protein traps Alzheimer's Aβ in an oligomeric form and disassembles amyloid fibers

    PubMed Central

    Younan, Nadine D.; Sarell, Claire J.; Davies, Paul; Brown, David R.; Viles, John H.

    2013-01-01

    There is now strong evidence to show that the presence of the cellular prion protein (PrPC) mediates amyloid-β (Aβ) neurotoxicity in Alzheimer's disease (AD). Here, we probe the molecular details of the interaction between PrPC and Aβ and discover that substoichiometric amounts of PrPC, as little as 1/20, relative to Aβ will strongly inhibit amyloid fibril formation. This effect is specific to the unstructured N-terminal domain of PrPC. Electron microscopy indicates PrPC is able to trap Aβ in an oligomeric form. Unlike fibers, this oligomeric Aβ contains antiparallel β sheet and binds to a oligomer specific conformational antibody. Our NMR studies show that a specific region of PrPC, notably residues 95–113, binds to Aβ oligomers, but only once Aβ misfolds. The ability of PrPC to trap and concentrate Aβ in an oligomeric form and disassemble mature fibers suggests a mechanism by which PrPC might confer Aβ toxicity in AD, as oligomers are thought to be the toxic form of Aβ. Identification of a specific recognition site on PrPC that traps Aβ in an oligomeric form is potentially a therapeutic target for the treatment of Alzheimer's disease.—Younan, N. D., Sarell, C. J., Davies, P., Brown, D. R., Viles, J. H. The cellular prion protein traps Alzheimer's Aβ in an oligomeric form and disassembles amyloid fibers. PMID:23335053

  8. PepX: a structural database of non-redundant protein-peptide complexes.

    PubMed

    Vanhee, Peter; Reumers, Joke; Stricher, Francois; Baeten, Lies; Serrano, Luis; Schymkowitz, Joost; Rousseau, Frederic

    2010-01-01

    Although protein-peptide interactions are estimated to constitute up to 40% of all protein interactions, relatively little information is available for the structural details of these interactions. Peptide-mediated interactions are a prime target for drug design because they are predominantly present in signaling and regulatory networks. A reliable data set of nonredundant protein-peptide complexes is indispensable as a basis for modeling and design, but current data sets for protein-peptide interactions are often biased towards specific types of interactions or are limited to interactions with small ligands. In PepX (http://pepx.switchlab.org), we have designed an unbiased and exhaustive data set of all protein-peptide complexes available in the Protein Data Bank with peptide lengths up to 35 residues. In addition, these complexes have been clustered based on their binding interfaces rather than sequence homology, providing a set of structurally diverse protein-peptide interactions. The final data set contains 505 unique protein-peptide interface clusters from 1431 complexes. Thorough annotation of each complex with both biological and structural information facilitates searching for and browsing through individual complexes and clusters. Moreover, we provide an additional source of data for peptide design by annotating peptides with naturally occurring backbone variations using fragment clusters from the BriX database.

  9. AraPerox. A Database of Putative Arabidopsis Proteins from Plant Peroxisomes1[w

    PubMed Central

    Reumann, Sigrun; Ma, Changle; Lemke, Steffen; Babujee, Lavanya

    2004-01-01

    To identify unknown proteins from plant peroxisomes, the Arabidopsis genome was screened for proteins with putative major or minor peroxisome targeting signals type 1 or 2 (PTS1 or PTS2), as defined previously (Reumann S [2004] Plant Physiol 135: 783–800). About 220 and 60 proteins were identified that carry a putative PTS1 or PTS2, respectively. To further support postulated targeting to peroxisomes, several prediction programs were applied and the putative targeting domains analyzed for properties conserved in peroxisomal proteins and for PTS conservation in homologous plant expressed sequence tags. The majority of proteins with a major PTS and medium to high overall probability of peroxisomal targeting represent novel nonhypothetical proteins and include several enzymes involved in β-oxidation of unsaturated fatty acids and branched amino acids, and 2-hydroxy acid oxidases with a predicted function in fatty acid α-oxidation, as well as NADP-dependent dehydrogenases and reductases. In addition, large protein families with many putative peroxisomal isoforms were recognized, including acyl-activating enzymes, GDSL lipases, and small thioesterases. Several proteins are homologous to prokaryotic enzymes of a novel aerobic hybrid degradation pathway for aromatic compounds and proposed to be involved in peroxisomal biosynthesis of plant hormones like jasmonic acid, auxin, and salicylic acid. Putative regulatory proteins of plant peroxisomes include protein kinases, small heat shock proteins, and proteases. The information on subcellular targeting prediction, homology, and in silico expression analysis for these Arabidopsis proteins has been compiled in the public database AraPerox to accelerate discovery and experimental investigation of novel metabolic and regulatory pathways of plant peroxisomes. PMID:15333753

  10. Activation of human natural killer cells by the soluble form of cellular prion protein

    SciTech Connect

    Seong, Yeon-Jae; Sung, Pil Soo; Jang, Young-Soon; Choi, Young Joon; Park, Bum-Chan; Park, Su-Hyung; Park, Young Woo; Shin, Eui-Cheol

    2015-08-21

    Cellular prion protein (PrP{sup C}) is widely expressed in various cell types, including cells of the immune system. However, the specific roles of PrP{sup C} in the immune system have not been clearly elucidated. In the present study, we investigated the effects of a soluble form of recombinant PrP{sup C} protein on human natural killer (NK) cells. Recombinant soluble PrP{sup C} protein was generated by fusion of human PrP{sup C} with the Fc portion of human IgG{sub 1} (PrP{sup C}-Fc). PrP{sup C}-Fc binds to the surface of human NK cells, particularly to CD56{sup dim} NK cells. PrP{sup C}-Fc induced the production of cytokines and chemokines and the degranulation of granzyme B from NK cells. In addition, PrP{sup C}-Fc facilitated the IL-15-induced proliferation of NK cells. PrP{sup C}-Fc induced phosphorylation of ERK-1/2 and JNK in NK cells, and inhibitors of the ERK or the JNK pathways abrogated PrP{sup C}-Fc-induced cytokine production in NK cells. In conclusion, the soluble form of recombinant PrP{sup C}-Fc protein activates human NK cells via the ERK and JNK signaling pathways. - Highlights: • Recombinant soluble PrP{sup C} (PrP{sup C}-Fc) was generated by fusion of human PrP{sup C} with IgG1 Fc portion. • PrP{sup C}-Fc protein induces the production of cytokines and degranulation from human NK cells. • PrP{sup C}-Fc protein enhances the IL-15-induced proliferation of human NK cells. • PrP{sup C}-Fc protein activates human NK cells via the ERK and JNK signaling pathways.

  11. UniPROBE, update 2011: expanded content and search tools in the online database of protein-binding microarray data on protein-DNA interactions.

    PubMed

    Robasky, Kimberly; Bulyk, Martha L

    2011-01-01

    The Universal PBM Resource for Oligonucleotide-Binding Evaluation (UniPROBE) database is a centralized repository of information on the DNA-binding preferences of proteins as determined by universal protein-binding microarray (PBM) technology. Each entry for a protein (or protein complex) in UniPROBE provides the quantitative preferences for all possible nucleotide sequence variants ('words') of length k ('k-mers'), as well as position weight matrix (PWM) and graphical sequence logo representations of the k-mer data. In this update, we describe >130% expansion of the database content, incorporation of a protein BLAST (blastp) tool for finding protein sequence matches in UniPROBE, the introduction of UniPROBE accession numbers and additional database enhancements. The UniPROBE database is available at http://uniprobe.org.

  12. Protein Secondary Structures (alpha-helix and beta-sheet) at a Cellular Levle and Protein Fractions in Relation to Rumen Degradation Behaviours of Protein: A New Approach

    SciTech Connect

    Yu,P.

    2007-01-01

    Studying the secondary structure of proteins leads to an understanding of the components that make up a whole protein, and such an understanding of the structure of the whole protein is often vital to understanding its digestive behaviour and nutritive value in animals. The main protein secondary structures are the {alpha}-helix and {beta}-sheet. The percentage of these two structures in protein secondary structures influences protein nutritive value, quality and digestive behaviour. A high percentage of {beta}-sheet structure may partly cause a low access to gastrointestinal digestive enzymes, which results in a low protein value. The objectives of the present study were to use advanced synchrotron-based Fourier transform IR (S-FTIR) microspectroscopy as a new approach to reveal the molecular chemistry of the protein secondary structures of feed tissues affected by heat-processing within intact tissue at a cellular level, and to quantify protein secondary structures using multicomponent peak modelling Gaussian and Lorentzian methods, in relation to protein digestive behaviours and nutritive value in the rumen, which was determined using the Cornell Net Carbohydrate Protein System. The synchrotron-based molecular chemistry research experiment was performed at the National Synchrotron Light Source at Brookhaven National Laboratory, US Department of Energy. The results showed that, with S-FTIR microspectroscopy, the molecular chemistry, ultrastructural chemical make-up and nutritive characteristics could be revealed at a high ultraspatial resolution ({approx}10 {mu}m). S-FTIR microspectroscopy revealed that the secondary structure of protein differed between raw and roasted golden flaxseeds in terms of the percentages and ratio of {alpha}-helixes and {beta}-sheets in the mid-IR range at the cellular level. By using multicomponent peak modelling, the results show that the roasting reduced (P <0.05) the percentage of {alpha}-helixes (from 47.1% to 36.1%: S

  13. A Database of Plastid Protein Families from Red Algae and Apicomplexa and Expression Regulation of the moeB Gene

    PubMed Central

    2015-01-01

    We report the database of plastid protein families from red algae, secondary and tertiary rhodophyte-derived plastids, and Apicomplexa constructed with the novel method to infer orthology. The families contain proteins with maximal sequence similarity and minimal paralogous content. The database contains 6509 protein entries, 513 families and 278 nonsingletons (from which 230 are paralog-free, and among the remaining 48, 46 contain at maximum two proteins per species, and 2 contain at maximum three proteins per species). The method is compared with other approaches. Expression regulation of the moeB gene is studied using this database and the model of RNA polymerase competition. An analogous database obtained for green algae and their symbiotic descendants, and applications based on it are published earlier. PMID:26114108

  14. A Database of Plastid Protein Families from Red Algae and Apicomplexa and Expression Regulation of the moeB Gene.

    PubMed

    Zverkov, Oleg A; Seliverstov, Alexandr V; Lyubetsky, Vassily A

    2015-01-01

    We report the database of plastid protein families from red algae, secondary and tertiary rhodophyte-derived plastids, and Apicomplexa constructed with the novel method to infer orthology. The families contain proteins with maximal sequence similarity and minimal paralogous content. The database contains 6509 protein entries, 513 families and 278 nonsingletons (from which 230 are paralog-free, and among the remaining 48, 46 contain at maximum two proteins per species, and 2 contain at maximum three proteins per species). The method is compared with other approaches. Expression regulation of the moeB gene is studied using this database and the model of RNA polymerase competition. An analogous database obtained for green algae and their symbiotic descendants, and applications based on it are published earlier.

  15. Methylated arsenic metabolites bind to PML protein but do not induce cellular differentiation and PML-RARα protein degradation.

    PubMed

    Wang, Qian Qian; Zhou, Xin Yi; Zhang, Yan Fang; Bu, Na; Zhou, Jin; Cao, Feng Lin; Naranmandura, Hua

    2015-09-22

    Arsenic trioxide (As2O3) is one of the most effective therapeutic agents used for patients with acute promyelocytic leukemia (APL). The probable explanation for As2O3-induced cell differentiation is the direct targeting of PML-RARα oncoprotein by As2O3, which results in initiation of PML-RARα degradation. However, after injection, As2O3 is rapidly methylated in body to different intermediate metabolites such as trivalent monomethylarsonous acid (MMA(III)) and dimethylarsinous acid (DMA(III)), therefore, it remains unknown that which arsenic specie is actually responsible for the therapeutic effects against APL. Here we have shown the role of As2O3 (as iAs(III)) and its intermediate metabolites (i.e., MMA(III)/DMA(III)) in NB4 cells. Inorganic iAs(III) predominantly showed induction of cell differentiation, while MMA(III) and DMA(III) specifically showed to induce mitochondria and endoplasmic reticulum-mediated apoptosis, respectively. On the other hand, in contrast to iAs(III), MMA(III) showed stronger binding affinity for ring domain of PML recombinant protein, however, could not induce PML protein SUMOylation and ubiquitin/proteasome degradation. In summary, our results suggest that the binding of arsenicals to the ring domain of PML proteins is not associated with the degradation of PML-RARα fusion protein. Moreover, methylated arsenicals can efficiently lead to cellular apoptosis, however, they are incapable of inducing NB4 cell differentiation. PMID:26213848

  16. PDBj Mine: design and implementation of relational database interface for Protein Data Bank Japan.

    PubMed

    Kinjo, Akira R; Yamashita, Reiko; Nakamura, Haruki

    2010-01-01

    This article is a tutorial for PDBj Mine, a new database and its interface for Protein Data Bank Japan (PDBj). In PDBj Mine, data are loaded from files in the PDBMLplus format (an extension of PDBML, PDB's canonical XML format, enriched with annotations), which are then served for the user of PDBj via the worldwide web (WWW). We describe the basic design of the relational database (RDB) and web interfaces of PDBj Mine. The contents of PDBMLplus files are first broken into XPath entities, and these paths and data are indexed in the way that reflects the hierarchical structure of the XML files. The data for each XPath type are saved into the corresponding relational table that is named as the XPath itself. The generation of table definitions from the PDBMLplus XML schema is fully automated. For efficient search, frequently queried terms are compiled into a brief summary table. Casual users can perform simple keyword search, and 'Advanced Search' which can specify various conditions on the entries. More experienced users can query the database using SQL statements which can be constructed in a uniform manner. Thus, PDBj Mine achieves a combination of the flexibility of XML documents and the robustness of the RDB. Database URL: http://www.pdbj.org/ PMID:20798081

  17. Loops In Proteins (LIP)--a comprehensive loop database for homology modelling.

    PubMed

    Michalsky, E; Goede, A; Preissner, R

    2003-12-01

    One of the most important and challenging tasks in protein modelling is the prediction of loops, as can be seen in the large variety of existing approaches. Loops In Proteins (LIP) is a database that includes all protein segments of a length up to 15 residues contained in the Protein Data Bank (PDB). In this study, the applicability of LIP to loop prediction in the framework of homology modelling is investigated. Searching the database for loop candidates takes less than 1 s on a desktop PC, and ranking them takes a few minutes. This is an order of magnitude faster than most existing procedures. The measure of accuracy is the root mean square deviation (RMSD) with respect to the main-chain atoms after local superposition of target loop and predicted loop. Loops of up to nine residues length were modelled with a local RMSD <1 A and those of length up to 14 residues with an accuracy better than 2 A. The results were compared in detail with a thoroughly evaluated and tested ab initio method published recently and additionally with two further methods for a small loop test set. The LIP method produced very good predictions. In particular for longer loops it outperformed other methods.

  18. ANGIOGENES: knowledge database for protein-coding and noncoding RNA genes in endothelial cells.

    PubMed

    Müller, Raphael; Weirick, Tyler; John, David; Militello, Giuseppe; Chen, Wei; Dimmeler, Stefanie; Uchida, Shizuka

    2016-09-01

    Increasing evidence indicates the presence of long noncoding RNAs (lncRNAs) is specific to various cell types. Although lncRNAs are speculated to be more numerous than protein-coding genes, the annotations of lncRNAs remain primitive due to the lack of well-structured schemes for their identification and description. Here, we introduce a new knowledge database "ANGIOGENES" (http://angiogenes.uni-frankfurt.de) to allow for in silico screening of protein-coding genes and lncRNAs expressed in various types of endothelial cells, which are present in all tissues. Using the latest annotations of protein-coding genes and lncRNAs, publicly-available RNA-seq data was analyzed to identify transcripts that are expressed in endothelial cells of human, mouse and zebrafish. The analyzed data were incorporated into ANGIOGENES to provide a one-stop-shop for transcriptomics data to facilitate further biological validation. ANGIOGENES is an intuitive and easy-to-use database to allow in silico screening of expressed, enriched and/or specific endothelial transcripts under various conditions. We anticipate that ANGIOGENES serves as a starting point for functional studies to elucidate the roles of protein-coding genes and lncRNAs in angiogenesis.

  19. ANGIOGENES: knowledge database for protein-coding and noncoding RNA genes in endothelial cells

    PubMed Central

    Müller, Raphael; Weirick, Tyler; John, David; Militello, Giuseppe; Chen, Wei; Dimmeler, Stefanie; Uchida, Shizuka

    2016-01-01

    Increasing evidence indicates the presence of long noncoding RNAs (lncRNAs) is specific to various cell types. Although lncRNAs are speculated to be more numerous than protein-coding genes, the annotations of lncRNAs remain primitive due to the lack of well-structured schemes for their identification and description. Here, we introduce a new knowledge database “ANGIOGENES” (http://angiogenes.uni-frankfurt.de) to allow for in silico screening of protein-coding genes and lncRNAs expressed in various types of endothelial cells, which are present in all tissues. Using the latest annotations of protein-coding genes and lncRNAs, publicly-available RNA-seq data was analyzed to identify transcripts that are expressed in endothelial cells of human, mouse and zebrafish. The analyzed data were incorporated into ANGIOGENES to provide a one-stop-shop for transcriptomics data to facilitate further biological validation. ANGIOGENES is an intuitive and easy-to-use database to allow in silico screening of expressed, enriched and/or specific endothelial transcripts under various conditions. We anticipate that ANGIOGENES serves as a starting point for functional studies to elucidate the roles of protein-coding genes and lncRNAs in angiogenesis. PMID:27582018

  20. ANGIOGENES: knowledge database for protein-coding and noncoding RNA genes in endothelial cells.

    PubMed

    Müller, Raphael; Weirick, Tyler; John, David; Militello, Giuseppe; Chen, Wei; Dimmeler, Stefanie; Uchida, Shizuka

    2016-01-01

    Increasing evidence indicates the presence of long noncoding RNAs (lncRNAs) is specific to various cell types. Although lncRNAs are speculated to be more numerous than protein-coding genes, the annotations of lncRNAs remain primitive due to the lack of well-structured schemes for their identification and description. Here, we introduce a new knowledge database "ANGIOGENES" (http://angiogenes.uni-frankfurt.de) to allow for in silico screening of protein-coding genes and lncRNAs expressed in various types of endothelial cells, which are present in all tissues. Using the latest annotations of protein-coding genes and lncRNAs, publicly-available RNA-seq data was analyzed to identify transcripts that are expressed in endothelial cells of human, mouse and zebrafish. The analyzed data were incorporated into ANGIOGENES to provide a one-stop-shop for transcriptomics data to facilitate further biological validation. ANGIOGENES is an intuitive and easy-to-use database to allow in silico screening of expressed, enriched and/or specific endothelial transcripts under various conditions. We anticipate that ANGIOGENES serves as a starting point for functional studies to elucidate the roles of protein-coding genes and lncRNAs in angiogenesis. PMID:27582018

  1. CentrosomeDB: a new generation of the centrosomal proteins database for Human and Drosophila melanogaster

    PubMed Central

    Alves-Cruzeiro, Joao Miguel da Conceiçao; Nogales-Cadenas, Rubén; Pascual-Montano, Alberto Domingo

    2014-01-01

    We present the second generation of centrosomeDB, available online at http://centrosome.cnb.csic.es, with a significant expansion of 1357 human and drosophila centrosomal genes and their corresponding information. The centrosome of animal cells takes part in important biological processes such as the organization of the interphase microtubule cytoskeleton and the assembly of the mitotic spindle. The active research done during the past decades has produced lots of data related to centrosomal proteins. Unfortunately, the accumulated data are dispersed among diverse and heterogeneous sources of information. We believe that the availability of a repository collecting curated evidences of centrosomal proteins would constitute a key resource for the scientific community. This was our first motivation to introduce CentrosomeDB in NAR database issue in 2009, collecting a set of human centrosomal proteins that were reported in the literature and other sources. The intensive use of this resource during these years has encouraged us to present this new expanded version. Using our database, the researcher is offered the possibility to study the evolution, function and structure of the centrosome. We have compiled information from many sources, including Gene Ontology, disease-association, single nucleotide polymorphisms and associated gene expression experiments. Special interest has been paid to protein–protein interaction. PMID:24270791

  2. Zebrafish cellular nucleic acid-binding protein: gene structure and developmental behaviour.

    PubMed

    Armas, Pablo; Cachero, Sebastián; Lombardo, Verónica A; Weiner, Andrea; Allende, Miguel L; Calcaterra, Nora B

    2004-08-01

    Here we analyse the structural organisation and expression of the zebrafish cellular nucleic acid-binding protein (zCNBP) gene and protein. The gene is organised in five exons and four introns. A noteworthy feature of the gene is the absence of a predicted promoter region. The coding region encodes a 163-amino acid polypeptide with the highly conserved general structural organisation of seven CCHC Zn knuckle domains and an RGG box between the first and the second Zn knuckles. Although theoretical alternative splicing is possible, only one form of zCNBP is actually detected. This form is able to bind to single-stranded DNA and RNA probes in vitro. The analysis of zCNBP developmental expression shows a high amount of CNBP-mRNA in ovary and during the first developmental stages. CNBP-mRNA levels decrease while early development progresses until the midblastula transition (MBT) stage and increases again thereafter. The protein is localised in the cytoplasm of blastomeres whereas it is mainly nuclear in developmental stages after the MBT. These findings suggest that CNBP is a strikingly conserved single-stranded nucleic acid-binding protein which might interact with maternal mRNA during its storage in the embryo cell cytoplasm. It becomes nuclear once MBT takes place possibly in order to modulate zygotic transcription and/or to associate with newly synthesised transcripts.

  3. Conserved cellular function and stress-mediated regulation among members of the proteolipid protein family.

    PubMed

    Fernández, María E; Alfonso, Julieta; Brocco, Marcela A; Frasch, Alberto C

    2010-05-01

    Chronic stress causes morphological alterations in the hippocampus of rodents and tree shrews, including atrophy of CA3 dendrites and loss of synapses. The molecular mechanisms underlying these structural changes remain largely unknown. We have previously identified M6a as a stress responsive gene and shown that M6a is involved in filopodium/spine outgrowth and, likely, synapse formation. M6a belongs to the proteolipid protein (PLP) family, all of their members having four transmembrane domains that allow their localization at the plasma membrane. In the present work, we analyzed other members of this family, the closely related M6b as well as PLP and its splice variant DM20. We found that chronic restraint stress in mice reduces M6b and DM20, but not PLP, mRNA levels in the hippocampus. In addition, M6b and DM20, but again not PLP, induce filopodium formation in primary cultures of hippocampal neurons. Several M6b protein isoforms were studied, all of them having similar effects except for the one lacking the transmembrane domains. Our results reveal a conserved cellular function and a stress-mediated regulation among members of the proteolipid protein family, suggesting an involvement of proteolipid proteins in the stress response. PMID:19937804

  4. Protein arginylation regulates cellular stress response by stabilizing HSP70 and HSP40 transcripts

    PubMed Central

    Deka, Kamalakshi; Singh, Archana; Chakraborty, Surajit; Mukhopadhyay, Rupak; Saha, Sougata

    2016-01-01

    ATE1-mediated post-translational addition of arginine to a protein has been shown to regulate activity, interaction, and stability of the protein substrates. Arginylation has been linked to many different stress conditions, namely ER stress, cytosolic misfolded protein stress, and nitrosative stress. However, clear understanding about the effect of arginylation in cellular stress responses is yet to emerge. In this study, we investigated the role of arginylation in heat-stress response. Our findings suggest that Ate1 knock out (KO) cells are more susceptible to heat stress compared with its wild-type counterparts due to the induction of apoptosis in KO cells. Gene expression analysis of inducible heat-shock proteins (HSP70.1, HSP70.3, and HSP40) showed induction of these genes in KO cells early in the heat shock, but were drastically diminished at the later period of heat shock. Further analysis revealed that loss of ATE1 drastically reduced the stability of all three HSP mRNAs. These phenotypes were greatly restored by overexpression of Ate1 in KO cells. Our findings show that arginylation plays a protective role during heat stress by regulating HSP gene expression and mRNA stability. PMID:27752365

  5. Diurnal Rhythms Result in Significant Changes in the Cellular Protein Complement in the Cyanobacterium Cyanothece 51142

    SciTech Connect

    Stockel, Jana; Jacobs, Jon M.; Elvitigala, Thanura R.; Liberton, Michelle L.; Welsh, Eric A.; Polpitiya, Ashoka D.; Gritsenko, Marina A.; Nicora, Carrie D.; Koppenaal, David W.; Smith, Richard D.; Pakrasi, Himadri B.

    2011-02-22

    Cyanothece sp. ATCC 51142 is a diazotrophic cyanobacterium notable for its ability to perform oxygenic photosynthesis and dinitrogen fixation in the same single cell. Previous transcriptional analysis revealed that the existence of these incompatible cellular processes largely depends on tightly synchronized expression programs involving ,30% of genes in the genome. To expand upon current knowledge, we have utilized sensitive proteomic approaches to examine the impact of diurnal rhythms on the protein complement in Cyanothece 51142. We found that 250 proteins accounting for,5% of the predicted ORFs from the Cyanothece 51142 genome and 20% of proteins detected under alternating light/dark conditions exhibited periodic oscillations in their abundances. Our results suggest that altered enzyme activities at different phases during the diurnal cycle can be attributed to changes in the abundance of related proteins and key compounds. The integration of global proteomics and transcriptomic data further revealed that post-transcriptional events are important for temporal regulation of processes such as photosynthesis in Cyanothece 51142. This analysis is the first comprehensive report on global quantitative proteomics in a unicellular diazotrophic cyanobacterium and uncovers novel findings about diurnal rhythms.

  6. VISUALIZIATION OF CELLULAR PHOSPHOINOSITIDE POOLS WITH GFP-FUSED PROTEIN-DOMAINS

    PubMed Central

    Balla, Tamas; Várnai, Péter

    2011-01-01

    This unit describes the method of following phosphoinositide dynamics in live cells. Inositol phospholipids have emerged as universal signaling molecules present in virtually every membrane of eukaryotic cells. Phosphoinositides are present only in tiny amounts compared to structural lipids but are metabolically very active as they are produced and degraded by the numerous inositide kinase and phosphatase enzymes. Phosphoinositides control the membrane-recruitment and activity of many protein signaling-complexes in specific membrane compartments and have been implicated in the regulation of a variety of signaling and trafficking pathways. It has been a challenge to develop methods that allow detection of phosphoinositides at the single cell level. The only available technique in live cell application is based on the use of the same protein domains selected by evolution to recognize cellular phosphoinositides. Some of these isolated protein modules when fused to fluorescent proteins can follow dynamic changes in phosphoinositides. While this technique can provide information on phosphoinositide dynamics in live cells with subcellular resolution and rapidly gained popularity, it also has several limitations that must be taken into account when interpreting the data. Here, we summarize the design and practical use of these constructs and also review important considerations for the interpretation of the data obtained by this technique. PMID:19283730

  7. iLIR database: A web resource for LIR motif-containing proteins in eukaryotes

    PubMed Central

    Jacomin, Anne-Claire; Samavedam, Siva; Promponas, Vasilis; Nezis, Ioannis P.

    2016-01-01

    ABSTRACT Atg8-family proteins are the best-studied proteins of the core autophagic machinery. They are essential for the elongation and closure of the phagophore into a proper autophagosome. Moreover, Atg8-family proteins are associated with the phagophore from the initiation of the autophagic process to, or just prior to, the fusion between autophagosomes with lysosomes. In addition to their implication in autophagosome biogenesis, they are crucial for selective autophagy through their ability to interact with selective autophagy receptor proteins necessary for the specific targeting of substrates for autophagic degradation. In the past few years it has been revealed that Atg8-interacting proteins include not only receptors but also components of the core autophagic machinery, proteins associated with vesicles and their transport, and specific proteins that are selectively degraded by autophagy. Atg8-interacting proteins contain a short linear LC3-interacting region/LC3 recognition sequence/Atg8-interacting motif (LIR/LRS/AIM) motif which is responsible for their interaction with Atg8-family proteins. These proteins are referred to as LIR-containing proteins (LIRCPs). So far, many experimental efforts have been carried out to identify new LIRCPs, leading to the characterization of some of them in the past 10 years. Given the need for the identification of LIRCPs in various organisms, we developed the iLIR database (https://ilir.warwick.ac.uk) as a freely available web resource, listing all the putative canonical LIRCPs identified in silico in the proteomes of 8 model organisms using the iLIR server, combined with a Gene Ontology (GO) term analysis. Additionally, a curated text-mining analysis of the literature permitted us to identify novel putative LICRPs in mammals that have not previously been associated with autophagy. PMID:27484196

  8. The CATH extended protein-family database: providing structural annotations for genome sequences.

    PubMed

    Pearl, Frances M G; Lee, David; Bray, James E; Buchan, Daniel W A; Shepherd, Adrian J; Orengo, Christine A

    2002-02-01

    An automatic sequence search and analysis protocol (DomainFinder) based on PSI-BLAST and IMPALA, and using conservative thresholds, has been developed for reliably integrating gene sequences from GenBank into their respective structural families within the CATH domain database (http://www.biochem.ucl.ac.uk/bsm/cath_new). DomainFinder assigns a new gene sequence to a CATH homologous superfamily provided that PSI-BLAST identifies a clear relationship to at least one other Protein Data Bank sequence within that superfamily. This has resulted in an expansion of the CATH protein family database (CATH-PFDB v1.6) from 19,563 domain structures to 176,597 domain sequences. A further 50,000 putative homologous relationships can be identified using less stringent cut-offs and these relationships are maintained within neighbour tables in the CATH Oracle database, pending further evidence of their suggested evolutionary relationship. Analysis of the CATH-PFDB has shown that only 15% of the sequence families are close enough to a known structure for reliable homology modeling. IMPALA/PSI-BLAST profiles have been generated for each of the sequence families in the expanded CATH-PFDB and a web server has been provided so that new sequences may be scanned against the profile library and be assigned to a structure and homologous superfamily.

  9. Quantitative MS-based enzymology of caspases reveals distinct protein substrate specificities, hierarchies, and cellular roles

    PubMed Central

    Zhuang, Min; Wiita, Arun P.; O’Donoghue, Anthony J.; Knudsen, Giselle M.; Craik, Charles S.; Wells, James A.

    2016-01-01

    Proteases constitute the largest enzyme family, yet their biological roles are obscured by our rudimentary understanding of their cellular substrates. There are 12 human caspases that play crucial roles in inflammation and cell differentiation and drive the terminal stages of cell death. Recent N-terminomics technologies have begun to enumerate the diverse substrates individual caspases can cleave in complex cell lysates. It is clear that many caspases have shared substrates; however, few data exist about the catalytic efficiencies (kcat/KM) of these substrates, which is critical to understanding their true substrate preferences. In this study, we use quantitative MS to determine the catalytic efficiencies for hundreds of natural protease substrates in cellular lysate for two understudied members: caspase-2 and caspase-6. Most substrates are new, and the cleavage rates vary up to 500-fold. We compare the cleavage rates for common substrates with those found for caspase-3, caspase-7, and caspase-8, involved in apoptosis. There is little correlation in catalytic efficiencies among the five caspases, suggesting each has a unique set of preferred substrates, and thus more specialized roles than previously understood. We synthesized peptide substrates on the basis of protein cleavage sites and found similar catalytic efficiencies between the protein and peptide substrates. These data suggest the rates of proteolysis are dominated more by local primary sequence, and less by the tertiary protein fold. Our studies highlight that global quantitative rate analysis for posttranslational modification enzymes in complex milieus for native substrates is critical to better define their functions and relative sequence of events. PMID:27006500

  10. Cellular IAP proteins and LUBAC differentially regulate necrosome-associated RIP1 ubiquitination

    PubMed Central

    de Almagro, M C; Goncharov, T; Newton, K; Vucic, D

    2015-01-01

    Necroptosis is a caspase-independent regulated type of cell death that relies on receptor-interacting protein kinases RIP1 (receptor-interacting protein kinases 1) and RIP3. Tumor necrosis factor-α (TNFα)-stimulated assembly of the TNFR1 (TNF receptor 1)-associated signaling complex leads to the recruitment of RIP1, whose ubiquitination is mediated by the cellular inhibitors of apoptosis (c-IAPs). Translocation of RIP1 to the cytoplasm and association of RIP1 with the necrosome is believed to correlate with deubiquitination of RIP1. However, we found that RIP1 is ubiquitinated with K63 and linear polyubiquitin chains during TNFα, IAP antagonist BV6 and caspase inhibitor zVAD-fmk-induced necroptotic signaling. Furthermore, ubiquitinated RIP1 is associated with the necrosome, and RIP1 ubiquitination in the necrosome coincides with RIP3 phosphorylation. Both cellular IAPs and LUBAC (linear ubiquitin chain assembly complex) modulate RIP1 ubiquitination in IAP antagonist-treated necrotic cells, but they use different mechanisms. c-IAP1 regulates RIP1 recruitment to the necrosome without directly affecting RIP1 ubiquitination, whereas HOIP and HOIL1 mediate linear ubiquitination of RIP1 in the necrosome, but are not essential for necrosome formation. Knockdown of the E3 ligase c-IAP1 decreased RIP1 ubiquitination, necrosome assembly and necroptosis induced by TNFα, BV6 and zVAD-fmk. c-IAP1 deficiency likely decreases necroptotic cell death through the activation of the noncanonical NF-κB pathway and consequent c-IAP2 upregulation. The ability to upregulate c-IAP2 could determine whether c-IAP1 absence will have a positive or negative impact on TNFα-induced necroptotic cell death and necrosome formation. Collectively, these results reveal unexpected complexity of the roles of IAP proteins, IAP antagonists and LUBAC in the regulation of necrosome assembly. PMID:26111062

  11. PALI-a database of Phylogeny and ALIgnment of homologous protein structures.

    PubMed

    Balaji, S; Sujatha, S; Kumar, S S; Srinivasan, N

    2001-01-01

    PALI (release 1.2) contains three-dimensional (3-D) structure-dependent sequence alignments as well as structure-based phylogenetic trees of homologous protein domains in various families. The data set of homologous protein structures has been derived by consulting the SCOP database (release 1.50) and the data set comprises 604 families of homologous proteins involving 2739 protein domain structures with each family made up of at least two members. Each member in a family has been structurally aligned with every other member in the same family (pairwise alignment) and all the members in the family are also aligned using simultaneous super-position (multiple alignment). The structural alignments are performed largely automatically, with manual interventions especially in the cases of distantly related proteins, using the program STAMP (version 4.2). Every family is also associated with two dendrograms, calculated using PHYLIP (version 3.5), one based on a structural dissimilarity metric defined for every pairwise alignment and the other based on similarity of topologically equivalent residues. These dendrograms enable easy comparison of sequence and structure-based relationships among the members in a family. Structure-based alignments with the details of structural and sequence similarities, superposed coordinate sets and dendrograms can be accessed conveniently using a web interface. The database can be queried for protein pairs with sequence or structural similarities falling within a specified range. Thus PALI forms a useful resource to help in analysing the relationship between sequence and structure variation at a given level of sequence similarity. PALI also contains over 653 'orphans' (single member families). Using the web interface involving PSI_BLAST and PHYLIP it is possible to associate the sequence of a new protein with one of the families in PALI and generate a phylogenetic tree combining the query sequence and proteins of known 3-D structure. The

  12. Interaction between core protein of classical swine fever virus with cellular IQGAP1 proetin appears essential for virulence in swine

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Here we show that IQGAP1, a cellular protein that plays a pivotal role as a regulator of the cytoskeleton affecting cell adhesion, polarization and migration, interacts with Classical Swine Fever Virus (CSFV) Core protein. Sequence analyses identified a defined set of residues within CSFV Core prote...

  13. Effect of cleavage enzyme, search algorithm and decoy database on mass spectrometric identification of wheat gluten proteins

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Tandem mass spectrometry (MS/MS) is routinely used to identify proteins by comparing peptide spectra to those generated in silico from protein sequence databases. Wheat storage proteins (gliadins and glutenins) are difficult to distinguish by MS/MS as they have few cleavable tryptic sites, often res...

  14. The Perilipins: Major Cytosolic Lipid Droplet-Associated Proteins and Their Roles in Cellular Lipid Storage, Mobilization, and Systemic Homeostasis.

    PubMed

    Kimmel, Alan R; Sztalryd, Carole

    2016-07-17

    The discovery by Dr. Constantine Londos of perilipin 1, the major scaffold protein at the surface of cytosolic lipid droplets in adipocytes, marked a fundamental conceptual change in the understanding of lipolytic regulation. Focus then shifted from the enzymatic activation of lipases to substrate accessibility, mediated by perilipin-dependent protein sequestration and recruitment. Consequently, the lipid droplet became recognized as a unique, metabolically active cellular organelle and its surface as the active site for novel protein-protein interactions. A new area of investigation emerged, centered on lipid droplets' biology and their role in energy homeostasis. The perilipin family is of ancient origin and has expanded to include five mammalian genes and a growing list of evolutionarily conserved members. Universally, the perilipins modulate cellular lipid storage. This review provides a summary that connects the perilipins to both cellular and whole-body homeostasis. PMID:27431369

  15. Localization of Cellular Retinol-Binding Protein and Retinol-Binding Protein in Cells Comprising the Blood-Brain Barrier of Rat and Human

    NASA Astrophysics Data System (ADS)

    MacDonald, Paul N.; Bok, Dean; Ong, David E.

    1990-06-01

    Brain is not generally recognized as an organ that requiries vitamin A, perhaps because no obvious histologic lesions have been observed in severely vitamin A-deficient animals. However, brain tissue does contain cellular vitamin A-binding proteins and a nuclear receptor protein for retinoic acid. In the present study, immunohistochemical techniques were used to determine the cell-specific location of cellular retinol-binding protein in human and rat brain tissue. Cellular retinol-binding protein was localized specifically within the endothelial cells of the brain microvasculature and within the cuboidal epithelial cells of the choroid plexus, two primary sites of the mammalian blood-brain barrier. In addition, autoradiographic procedures demonstrated binding sites for serum retinol-binding protein in the choroidal epithelium. These observations suggest that a significant movement of retinol across the blood-brain barrier may occur.

  16. Localization of cellular retinol-binding protein and retinol-binding protein in cells comprising the blood-brain barrier of rat and human

    SciTech Connect

    MacDonald, P.N.; Ong, D.E. ); Bok, D. )

    1990-06-01

    Brain is not generally recognized as an organ that requires vitamin A, perhaps because no obvious histologic lesions have been observed in severely vitamin A-deficient animals. However, brain tissue does contain cellular vitamin A-binding proteins and a nuclear receptor protein for retinoic acid. In the present study, immunohistochemical techniques were used to determine the cell-specific location of cellular retinol-binding protein in human and rat brain tissue. Cellular retinol-binding protein was localized specifically within the cuboidal epithelial cells of the choroid plexus, two primary sites of the mammalian blood-brain barrier. In addition, autoradiographic procedures demonstrated binding sites for serum retinol-binding protein in the choroidal epithelium. These observations suggest that a significant movement of retinol across the blood-brain barrier may occur.

  17. Identification of novel putative-binding proteins for cellular prion protein and a specific interaction with the STIP1 homology and U-Box-containing protein 1.

    PubMed

    Gimenez, Ana Paula Lappas; Richter, Larissa Morato Luciani; Atherino, Mariana Campos; Beirão, Breno Castello Branco; Fávaro, Celso; Costa, Michele Dietrich Moura; Zanata, Silvio Marques; Malnic, Bettina; Mercadante, Adriana Frohlich

    2015-01-01

    Prion diseases involve the conversion of the endogenous cellular prion protein, PrP(C), into a misfolded infectious isoform, PrP(Sc). Several functions have been attributed to PrP(C), and its role has also been investigated in the olfactory system. PrP(C) is expressed in both the olfactory bulb (OB) and olfactory epithelium (OE) and the nasal cavity is an important route of transmission of diseases caused by prions. Moreover, Prnp(-/-) mice showed impaired behavior in olfactory tests. Given the high PrP(C) expression in OE and its putative role in olfaction, we screened a mouse OE cDNA library to identify novel PrP(C)-binding partners. Ten different putative PrP(C) ligands were identified, which were involved in functions such as cellular proliferation and apoptosis, cytoskeleton and vesicle transport, ubiquitination of proteins, stress response, and other physiological processes. In vitro binding assays confirmed the interaction of PrP(C) with STIP1 homology and U-Box containing protein 1 (Stub1) and are reported here for the first time. Stub1 is a co-chaperone with ubiquitin E3-ligase activity, which is associated with neurodegenerative diseases characterized by protein misfolding and aggregation. Physiological and pathological implications of PrP(C)-Stub1 interaction are under investigation. The PrP(C)-binding proteins identified here are not exclusive to the OE, suggesting that these interactions may occur in other tissues and play general biological roles. These data corroborate the proposal that PrP(C) is part of a multiprotein complex that modulates several cellular functions and provide a platform for further studies on the physiological and pathological roles of prion protein.

  18. Cellular stress conditions are reflected in the protein and RNA content of endothelial cell-derived exosomes

    PubMed Central

    de Jong, Olivier G.; Verhaar, Marianne C.; Chen, Yong; Vader, Pieter; Gremmels, Hendrik; Posthuma, George; Schiffelers, Raymond M.; Gucek, Marjan; van Balkom, Bas W.M.

    2012-01-01

    Background The healthy vascular endothelium, which forms the barrier between blood and the surrounding tissues, is known to efficiently respond to stress signals like hypoxia and inflammation by adaptation of cellular physiology and the secretion of (soluble) growth factors and cytokines. Exosomes are potent mediators of intercellular communication. Their content consists of RNA and proteins from the cell of origin, and thus depends on the condition of these cells at the time of exosome biogenesis. It has been suggested that exosomes protect their target cells from cellular stress through the transfer of RNA and proteins. We hypothesized that endothelium-derived exosomes are involved in the endothelial response to cellular stress, and that exosome RNA and protein content reflect the effects of cellular stress induced by hypoxia, inflammation or hyperglycemia. Methods We exposed cultured endothelial cells to different types of cellular stress (hypoxia, TNF-α-induced activation, high glucose and mannose concentrations) and compared mRNA and protein content of exosomes produced by these cells by microarray analysis and a quantitative proteomics approach. Results We identified 1,354 proteins and 1,992 mRNAs in endothelial cell-derived exosomes. Several proteins and mRNAs showed altered abundances after exposure of their producing cells to cellular stress, which were confirmed by immunoblot or qPCR analysis. Conclusion Our data show that hypoxia and endothelial activation are reflected in RNA and protein exosome composition, and that exposure to high sugar concentrations alters exosome protein composition only to a minor extend, and does not affect exosome RNA composition. PMID:24009886

  19. Binding of cellular repressor protein or the IE2 protein to a cis-acting negative regulatory element upstream of a human cytomegalovirus early promoter.

    PubMed Central

    Huang, L; Stinski, M F

    1995-01-01

    We have previously shown that the human cytomegalovirus early UL4 promoter has upstream negative and positive cis-acting regulatory elements. In the absence of the upstream negative regulatory region, the positive element confers strong transcriptional activity. The positive element contains a CCAAT box dyad symmetry and binds the cellular transcription factor NF-Y. The effect of the negative regulatory element is negated by the viral IE2 protein (L. Huang, C.L. Malone, and M.F. Stinski, J. Virol. 68:2108, 1994). We investigated the binding of cellular or viral IE2 protein to the negative regulatory region. The major cis-acting negative regulatory element was located between -168 and -134 bp relative to the transcription start site. This element could be transferred to a heterologous promoter, and it functioned in either orientation. Mutational analysis demonstrated that a core DNA sequence in the cis-acting negative regulatory element, 5'-GTTTGGAATCGTT-3', was required for the binding of either a cellular repressor protein(s) or the viral IE2 protein. The cellular DNA binding activity was present in both nonpermissive HeLa and permissive human fibroblast cells but more abundant in HeLa cells. Binding of the cellular repressor protein to the upstream cis-acting negative regulatory element correlates with repression of transcription from the early UL4 promoter. Binding of the viral IE2 protein correlates with negation of the repressive effect. PMID:7494269

  20. Standard trivalent influenza virus protein vaccination does not prime antibody-dependent cellular cytotoxicity in macaques.

    PubMed

    Jegaskanda, Sinthujan; Amarasena, Thakshila H; Laurie, Karen L; Tan, Hyon-Xhi; Butler, Jeff; Parsons, Matthew S; Alcantara, Sheilajen; Petravic, Janka; Davenport, Miles P; Hurt, Aeron C; Reading, Patrick C; Kent, Stephen J

    2013-12-01

    Yearly vaccination with the trivalent inactivated influenza vaccine (TIV) is recommended, since current vaccines induce little cross neutralization to divergent influenza strains. Whether the TIV can induce antibody-dependent cellular cytotoxicity (ADCC) responses that can cross-recognize divergent influenza virus strains is unknown. We immunized 6 influenza-naive pigtail macaques twice with the 2011-2012 season TIV and then challenged the macaques, along with 12 control macaques, serially with H1N1 and H3N2 viruses. We measured ADCC responses in plasma to a panel of H1 and H3 hemagglutinin (HA) proteins and influenza virus-specific CD8 T cell (CTL) responses using a sensitive major histocompatibility complex (MHC) tetramer reagent. The TIV was weakly immunogenic and, although binding antibodies were detected by enzyme-linked immunosorbent assay (ELISA), did not induce detectable influenza virus-specific ADCC or CTL responses. The H1N1 challenge elicited robust ADCC to both homologous and heterologous H1 HA proteins, but not influenza virus HA proteins from different subtypes (H2 to H7). There was no anamnestic influenza virus-specific ADCC or CTL response in vaccinated animals. The subsequent H3N2 challenge did not induce or boost ADCC either to H1 HA proteins or to divergent H3 proteins but did boost CTL responses. ADCC or CTL responses were not induced by TIV vaccination in influenza-naive macaques. There was a marked difference in the ability of infection compared to that of vaccination to induce cross-reactive ADCC and CTL responses. Improved vaccination strategies are needed to induce broad-based ADCC immunity to influenza.

  1. The PhosphoGRID Saccharomyces cerevisiae protein phosphorylation site database: version 2.0 update

    PubMed Central

    Sadowski, Ivan; Breitkreutz, Bobby-Joe; Stark, Chris; Su, Ting-Cheng; Dahabieh, Matthew; Raithatha, Sheetal; Bernhard, Wendy; Oughtred, Rose; Dolinski, Kara; Barreto, Kris; Tyers, Mike

    2013-01-01

    PhosphoGRID is an online database that curates and houses experimentally verified in vivo phosphorylation sites in the Saccharomyces cerevisiae proteome (www.phosphogrid.org). Phosphosites are annotated with specific protein kinases and/or phosphatases, along with the condition(s) under which the phosphorylation occurs and/or the effects on protein function. We report here an updated data set, including nine additional high-throughput (HTP) mass spectrometry studies. The version 2.0 data set contains information on 20 177 unique phosphorylated residues, representing a 4-fold increase from version 1.0, and includes 1614 unique phosphosites derived from focused low-throughput (LTP) studies. The overlap between HTP and LTP studies represents only ∼3% of the total unique sites, but importantly 45% of sites from LTP studies with defined function were discovered in at least two independent HTP studies. The majority of new phosphosites in this update occur on previously documented proteins, suggesting that coverage of phosphoproteins in the yeast proteome is approaching saturation. We will continue to update the PhosphoGRID data set, with the expectation that the integration of information from LTP and HTP studies will enable the development of predictive models of phosphorylation-based signaling networks. Database URL: http://www.phosphogrid.org/ PMID:23674503

  2. Three-dimensional super-resolution protein localization correlated with vitrified cellular context

    PubMed Central

    Liu, Bei; Xue, Yanhong; Zhao, Wei; Chen, Yan; Fan, Chunyan; Gu, Lusheng; Zhang, Yongdeng; Zhang, Xiang; Sun, Lei; Huang, Xiaojun; Ding, Wei; Sun, Fei; Ji, Wei; Xu, Tao

    2015-01-01

    We demonstrate the use of cryogenic super-resolution correlative light and electron microscopy (csCLEM) to precisely determine the spatial relationship between proteins and their native cellular structures. Several fluorescent proteins (FPs) were found to be photoswitchable and emitted far more photons under our cryogenic imaging condition, resulting in higher localization precision which is comparable to ambient super-resolution imaging. Vitrified specimens were prepared by high pressure freezing and cryo-sectioning to maintain a near-native state with better fluorescence preservation. A 2-3-fold improvement of resolution over the recent reports was achieved due to the photon budget performance of screening out Dronpa and optimized imaging conditions, even with thin sections which is at a disadvantage when calculate the structure resolution from label density. We extended csCLEM to mammalian cells by introducing cryo-sectioning and observed good correlation of a mitochondrial protein with the mitochondrial outer membrane at nanometer resolution in three dimensions. PMID:26462878

  3. EBNA-LP Associates with Cellular Proteins Including DNA-PK and HA95

    PubMed Central

    Han, Innoc; Harada, Shizuko; Weaver, David; Xue, Yong; Lane, William; Orstavik, Sigurd; Skalhegg, Bjorn; Kieff, Elliott

    2001-01-01

    EBNA-LP-associated proteins were identified by sequencing proteins that immunoprecipitated with Flag epitope-tagged EBNA-LP (FLP) from lymphoblasts in which FLP was stably expressed. The association of EBNA-LP with Hsp70 (72/73) was confirmed, and sequences of DNA-PK catalytic subunit (DNA-PKcs), HA95, Hsp27, prolyl 4-hydroxylase α-1 subunit, α-tubulin, and β-tubulin were identified. The fraction of total cellular HA95 that associated with FLP was very high, while progressively lower fractions of the total DNA-PKcs, Hsp70, Hsp 27, α-tubulin, and β-tubulin specifically associated with EBNA-LP as determined by immunoblotting with antibodies to these proteins. EBNA-LP bound to two domains in the DNA-PKcs C terminus and DNA-PKcs associated with the EBNA-LP repeat domain. DNA-PKcs that was bound to EBNA-LP phosphorylated p53 or EBNA-LP in vitro, and the phosphorylation of EBNA-LP was inhibited by Wortmannin, a specific in vitro inhibitor of DNA-PKcs. PMID:11160753

  4. Simian virus 40 late proteins possess lytic properties that render them capable of permeabilizing cellular membranes.

    PubMed

    Daniels, Robert; Rusan, Nasser M; Wilbuer, Anne-Kathrin; Norkin, Leonard C; Wadsworth, Patricia; Hebert, Daniel N

    2006-07-01

    Many nonenveloped viruses have evolved an infectious cycle that culminates in the lysis or permeabilization of the host to enable viral release. How these viruses initiate the lytic event is largely unknown. Here, we demonstrated that the simian virus 40 progeny accumulated at the nuclear envelope prior to the permeabilization of the nuclear, endoplasmic reticulum, and plasma membranes at a time which corresponded with the release of the progeny. The permeabilization of these cellular membranes temporally correlated with late protein expression and was not observed upon the inhibition of their synthesis. To address whether one or more of the late proteins possessed an inherent capacity to induce membrane permeabilization, we examined the permeability of Escherichia coli that separately expressed the late proteins. VP2 and VP3, but not VP1, caused the permeabilization of bacterial membranes. Additionally, VP3 expression resulted in bacterial cell lysis. These findings demonstrate that VP3 possesses an inherent lytic property that is independent of eukaryotic signaling or cell death pathways.

  5. Quantitation of the Noncovalent Cellular Retinol-Binding Protein, Type 1 Complex Through Native Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Li, Wenjing; Yu, Jianshi; Kane, Maureen A.

    2016-10-01

    Native mass spectrometry (MS) has become a valuable tool in probing noncovalent protein-ligand interactions in a sample-efficient way, yet the quantitative application potential of native MS has not been fully explored. Cellular retinol binding protein, type I (CrbpI) chaperones retinol and retinal in the cell, protecting them from nonspecific oxidation and delivering them to biosynthesis enzymes where the bound (holo-) and unbound (apo-) forms of CrbpI exert distinct biological functions. Using nanoelectrospray, we developed a native MS assay for probing apo- and holo-CrbpI abundance to facilitate exploring their biological functions in retinoid metabolism and signaling. The methods were developed on two platforms, an Orbitrap-based Thermo Exactive and a Q-IMS-TOF-based Waters Synapt G2S, where similar ion behaviors under optimized conditions were observed. Overall, our results suggested that within the working range (~1-10 μM), gas-phase ions in the native state linearly correspond to solution concentration and relative ion intensities of the apo- and holo-protein ions can linearly respond to the solution ratios, suggesting native MS is a viable tool for relative quantitation in this system.

  6. The cellular membrane as a mediator for small molecule interaction with membrane proteins.

    PubMed

    Mayne, Christopher G; Arcario, Mark J; Mahinthichaichan, Paween; Baylon, Javier L; Vermaas, Josh V; Navidpour, Latifeh; Wen, Po-Chao; Thangapandian, Sundarapandian; Tajkhorshid, Emad

    2016-10-01

    The cellular membrane constitutes the first element that encounters a wide variety of molecular species to which a cell might be exposed. Hosting a large number of structurally and functionally diverse proteins associated with this key metabolic compartment, the membrane not only directly controls the traffic of various molecules in and out of the cell, it also participates in such diverse and important processes as signal transduction and chemical processing of incoming molecular species. In this article, we present a number of cases where details of interaction of small molecular species such as drugs with the membrane, which are often experimentally inaccessible, have been studied using advanced molecular simulation techniques. We have selected systems in which partitioning of the small molecule with the membrane constitutes a key step for its final biological function, often binding to and interacting with a protein associated with the membrane. These examples demonstrate that membrane partitioning is not only important for the overall distribution of drugs and other small molecules into different compartments of the body, it may also play a key role in determining the efficiency and the mode of interaction of the drug with its target protein. This article is part of a Special Issue entitled: Biosimulations edited by Ilpo Vattulainen and Tomasz Róg. PMID:27163493

  7. Highly efficient enzyme encapsulation in a protein nanocage: towards enzyme catalysis in a cellular nanocompartment mimic

    NASA Astrophysics Data System (ADS)

    Schoonen, Lise; Nolte, Roeland J. M.; van Hest, Jan C. M.

    2016-07-01

    The study of enzyme behavior in small nanocompartments is crucial for the understanding of biocatalytic processes in the cellular environment. We have developed an enzymatic conjugation strategy to attach a model enzyme to the interior of a cowpea chlorotic mottle virus capsid. It is shown that with this methodology high encapsulation efficiencies can be achieved. Additionally, we demonstrate that the encapsulation does not affect the enzyme performance in terms of a decreased activity or a hampered substrate diffusion. Finally, it is shown that the encapsulated enzymes are protected against proteases. We believe that our strategy can be used to study enzyme kinetics in an environment that approaches physiological conditions.The study of enzyme behavior in small nanocompartments is crucial for the understanding of biocatalytic processes in the cellular environment. We have developed an enzymatic conjugation strategy to attach a model enzyme to the interior of a cowpea chlorotic mottle virus capsid. It is shown that with this methodology high encapsulation efficiencies can be achieved. Additionally, we demonstrate that the encapsulation does not affect the enzyme performance in terms of a decreased activity or a hampered substrate diffusion. Finally, it is shown that the encapsulated enzymes are protected against proteases. We believe that our strategy can be used to study enzyme kinetics in an environment that approaches physiological conditions. Electronic supplementary information (ESI) available: Experimental procedures for the cloning, expression, and purification of all proteins, as well as supplementary figures and calculations. See DOI: 10.1039/c6nr04181g

  8. Small heat shock proteins in cellular adhesion and migration: evidence from Plasmodium genetics.

    PubMed

    Montagna, Georgina N; Matuschewski, Kai; Buscaglia, Carlos A

    2012-01-01

    Cellular locomotion and adhesion critically depend on regulated turnover of filamentous actin. Biochemical data from diverse model systems support a role for the family of small heat shock proteins (HSPBs) in microfilament regulation. The small chaperones could either act directly, through competition with the motor myosin, or indirectly, through modulation of actin depolymerizing factor/cofilin activity. However, a direct link between HSPBs and actin-based cellular motility remained to be established. In a recent experimental genetics study, we provided evidence for regulation of Plasmodium motility by HSPB6/Hsp20. The infectious forms of malaria parasites, termed sporozoites, display fast and continuous substrate-dependent motility, which is largely driven by turnover of actin microfilaments. Sporozoite gliding locomotion is essential to avoid destruction by host defense mechanisms and to ultimately reach a hepatocyte, the target cell, where to transform and replicate. Genetic ablation of Plasmodium HSP20 dramatically changed sporozoite speed and substrate adhesion, resulting in impaired natural malaria transmission. In this article, we discuss the function of Hsp20 in this fast-moving unicellular protozoan and implications for the roles of HSPBs in adhesion and migration of eukaryotic cells.

  9. Amyloid Oligomers and Mature Fibrils Prepared from an Innocuous Protein Cause Diverging Cellular Death Mechanisms.

    PubMed

    Harte, Níal P; Klyubin, Igor; McCarthy, Eoin K; Min, Soyoung; Garrahy, Sarah Ann; Xie, Yongjing; Davey, Gavin P; Boland, John J; Rowan, Michael J; Mok, K Hun

    2015-11-20

    Despite significant advances, the molecular identity of the cytotoxic species populated during in vivo amyloid formation crucial for the understanding of neurodegenerative disorders is yet to be revealed. In this study lysozyme prefibrillar oligomers and fibrils in both mature and sonicated states have been isolated through an optimized ultrafiltration/ultracentrifugation method and characterized with various optical spectroscopic techniques, atomic force microscopy, and transmission electron microscopy. We examined their level and mode of toxicity on rat pheochromocytoma (PC12) cells in both differentiated and undifferentiated states. We find that oligomers and fibrils display cytotoxic capabilities toward cultured cells in vitro, with oligomers producing elevated levels of cellular injury toward undifferentiated PC12 cells (PC12(undiff)). Furthermore, dual flow cytometry staining experiments demonstrate that the oligomers and mature fibrils induce divergent cellular death pathways (apoptosis and secondary necrosis, respectively) in these PC12 cells. We have also shown that oligomers but not sonicated mature fibrils inhibit hippocampal long term potentiation, a form of synaptic plasticity implicated in learning and memory, in vivo. We conclude that our in vitro and in vivo findings confer a level of resistance toward amyloid fibrils, and that the PC 12-based comparative cytotoxicity assay can provide insights into toxicity differences between differently aggregated protein species.

  10. Protein coronas on gold nanorods passivated with amphiphilic ligands affect cytotoxicity and cellular response to penicillin/streptomycin.

    PubMed

    Kah, James Chen Yong; Grabinski, Christin; Untener, Emily; Garrett, Carol; Chen, John; Zhu, David; Hussain, Saber M; Hamad-Schifferli, Kimberly

    2014-05-27

    We probe how amphiphilic ligands (ALs) of four different types affect the formation of protein coronas on gold nanorods (NRs) and their impact on cellular response. NRs coated with cetyltrimethylammonium bromide were ligand exchanged with polyoxyethylene[10]cetyl ether, oligofectamine, and phosphatidylserine (PS). Protein coronas from equine serum (ES) were formed on these NR-ALs, and their colloidal stability, as well as cell uptake, proliferation, oxidative stress, and gene expression, were examined. We find that the protein corona that forms and its colloidal stability are affected by AL type and that the cellular response to these NR-AL-coronas (NR-AL-ES) is both ligand and corona dependent. We also find that the presence of common cell culture supplement penicillin/streptomycin can impact the colloidal stability and cellular response of NR-AL and NR-AL-ES, showing that the cell response is not necessarily inert to pen/strep when in the presence of nanoparticles. Although the protein corona is what the cells see, the underlying surface ligands evidently play an important role in shaping and defining the physical characteristics of the corona, which ultimately impacts the cellular response. Further, the results of this study suggest that the cellular behavior toward NR-AL is mediated by not only the type of AL and the protein corona it forms but also its resulting colloidal stability and interaction with cell culture supplements.

  11. PolyQ 2.0: an improved version of PolyQ, a database of human polyglutamine proteins.

    PubMed

    Li, Chen; Nagel, Jeremy; Androulakis, Steve; Song, Jiangning; Buckle, Ashley M

    2016-01-01

    Proteins with expanded polyglutamine (polyQ) repeats are involved in human neurodegenerative diseases, via a gain-of-function mechanism of neuronal toxicity involving protein conformational changes that result in the formation and deposition of β-sheet-rich aggregates. Aggregation is dependent on the context and properties of the host protein, such as domain context and location of the repeat tract. In order to explore this relationship in greater detail, here we describe PolyQ 2.0, an updated database that provides a comprehensive knowledgebase for human polyQ proteins. Compared with the previous PolyQ database, our new database provides a variety of substantial updates including detailed biological annotations and search options. Biological annotations in terms of domain context information, protein structural and functional annotation, single point mutations, predicted disordered regions, protein-protein interaction partners, metabolic/signaling pathways, post-translational modification sites and evolutionary information are made available. Several new database functionalities have also been provided, including search using multiple/combinatory keywords, and submission of new data entries. Also, several third-party plug-ins are employed to enhance data visualization in PolyQ 2.0. In PolyQ 2.0 the proteins are reclassified into 3 new categories and contain 9 reviewed disease-associated polyQ proteins, 105 reviewed non-disease polyQ proteins and 146 un-reviewed polyQ proteins (reviewed by UniProt curators). We envisage that this updated database will be a useful resource for functional and structural investigation of human polyQ proteins. Database URL: http://lightning.med.monash.edu/polyq2/.

  12. PolyQ 2.0: an improved version of PolyQ, a database of human polyglutamine proteins.

    PubMed

    Li, Chen; Nagel, Jeremy; Androulakis, Steve; Song, Jiangning; Buckle, Ashley M

    2016-01-01

    Proteins with expanded polyglutamine (polyQ) repeats are involved in human neurodegenerative diseases, via a gain-of-function mechanism of neuronal toxicity involving protein conformational changes that result in the formation and deposition of β-sheet-rich aggregates. Aggregation is dependent on the context and properties of the host protein, such as domain context and location of the repeat tract. In order to explore this relationship in greater detail, here we describe PolyQ 2.0, an updated database that provides a comprehensive knowledgebase for human polyQ proteins. Compared with the previous PolyQ database, our new database provides a variety of substantial updates including detailed biological annotations and search options. Biological annotations in terms of domain context information, protein structural and functional annotation, single point mutations, predicted disordered regions, protein-protein interaction partners, metabolic/signaling pathways, post-translational modification sites and evolutionary information are made available. Several new database functionalities have also been provided, including search using multiple/combinatory keywords, and submission of new data entries. Also, several third-party plug-ins are employed to enhance data visualization in PolyQ 2.0. In PolyQ 2.0 the proteins are reclassified into 3 new categories and contain 9 reviewed disease-associated polyQ proteins, 105 reviewed non-disease polyQ proteins and 146 un-reviewed polyQ proteins (reviewed by UniProt curators). We envisage that this updated database will be a useful resource for functional and structural investigation of human polyQ proteins. Database URL: http://lightning.med.monash.edu/polyq2/. PMID:26980520

  13. The Ubiquitin-associated Domain of Cellular Inhibitor of Apoptosis Proteins Facilitates Ubiquitylation*

    PubMed Central

    Budhidarmo, Rhesa; Day, Catherine L.

    2014-01-01

    The cellular inhibitor of apoptosis (cIAP) proteins are essential RING E3 ubiquitin ligases that regulate apoptosis and inflammatory responses. cIAPs contain a ubiquitin-associated (UBA) domain that binds ubiquitin and is implicated in the regulation of cell survival and proteasomal degradation. Here we show that mutation of the MGF and LL motifs in the UBA domain of cIAP1 caused unfolding and increased cIAP1 multimonoubiquitylation. By developing a UBA mutant that disrupted ubiquitin binding but not the structure of the UBA domain, we found that the UBA domain enhances cIAP1 and cIAP2 ubiquitylation. We demonstrate that the UBA domain binds to the UbcH5b∼Ub conjugate, and this promotes RING domain-dependent monoubiquitylation. This study establishes ubiquitin-binding modules, such as the UBA domain, as important regulatory modules that can fine tune the activity of E3 ligases. PMID:25065467

  14. Protein-based virtual screening of chemical databases. II. Are homology models of G-Protein Coupled Receptors suitable targets?

    PubMed

    Bissantz, Caterina; Bernard, Philippe; Hibert, Marcel; Rognan, Didier

    2003-01-01

    The aim of the current study is to investigate whether homology models of G-Protein-Coupled Receptors (GPCRs) that are based on bovine rhodopsin are reliable enough to be used for virtual screening of chemical databases. Starting from the recently described 2.8 A-resolution X-ray structure of bovine rhodopsin, homology models of an "antagonist-bound" form of three human GPCRs (dopamine D3 receptor, muscarinic M1 receptor, vasopressin V1a receptor) were constructed. The homology models were used to screen three-dimensional databases using three different docking programs (Dock, FlexX, Gold) in combination with seven scoring functions (ChemScore, Dock, FlexX, Fresno, Gold, Pmf, Score). Rhodopsin-based homology models turned out to be suitable, indeed, for virtual screening since known antagonists seeded in the test databases could be distinguished from randomly chosen molecules. However, such models are not accurate enough for retrieving known agonists. To generate receptor models better suited for agonist screening, we developed a new knowledge- and pharmacophore-based modeling procedure that might partly simulate the conformational changes occurring in the active site during receptor activation. Receptor coordinates generated by this new procedure are now suitable for agonist screening. We thus propose two alternative strategies for the virtual screening of GPCR ligands, relying on a different set of receptor coordinates (antagonist-bound and agonist-bound states).

  15. Inhibition of host protein synthesis and degradation of cellular mRNAs during infection by influenza and herpes simplex virus

    SciTech Connect

    Inglis, S.C.

    1982-12-01

    Cloned DNA copies of two cellular genes were used to monitor, by blot hybridization, the stability of particular cell mRNAs after infection by influenza virus and herpes virus. The results indicated that the inhibition of host cell protein synthesis that accompanied infection by each virus could be explained by a reduction in the amounts of cellular mRN As in the cytoplasm, and they suggested that this decrease was due to virus-mediated mRNA degradation.

  16. Viral Replication Protein Inhibits Cellular Cofilin Actin Depolymerization Factor to Regulate the Actin Network and Promote Viral Replicase Assembly

    PubMed Central

    Kovalev, Nikolay; de Castro Martín, Isabel Fernández; Barajas, Daniel; Risco, Cristina; Nagy, Peter D.

    2016-01-01

    RNA viruses exploit host cells by co-opting host factors and lipids and escaping host antiviral responses. Previous genome-wide screens with Tomato bushy stunt virus (TBSV) in the model host yeast have identified 18 cellular genes that are part of the actin network. In this paper, we show that the p33 viral replication factor interacts with the cellular cofilin (Cof1p), which is an actin depolymerization factor. Using temperature-sensitive (ts) Cof1p or actin (Act1p) mutants at a semi-permissive temperature, we find an increased level of TBSV RNA accumulation in yeast cells and elevated in vitro activity of the tombusvirus replicase. We show that the large p33 containing replication organelle-like structures are located in the close vicinity of actin patches in yeast cells or around actin cable hubs in infected plant cells. Therefore, the actin filaments could be involved in VRC assembly and the formation of large viral replication compartments containing many individual VRCs. Moreover, we show that the actin network affects the recruitment of viral and cellular components, including oxysterol binding proteins and VAP proteins to form membrane contact sites for efficient transfer of sterols to the sites of replication. Altogether, the emerging picture is that TBSV, via direct interaction between the p33 replication protein and Cof1p, controls cofilin activities to obstruct the dynamic actin network that leads to efficient subversion of cellular factors for pro-viral functions. In summary, the discovery that TBSV interacts with cellular cofilin and blocks the severing of existing filaments and the formation of new actin filaments in infected cells opens a new window to unravel the way by which viruses could subvert/co-opt cellular proteins and lipids. By regulating the functions of cofilin and the actin network, which are central nodes in cellular pathways, viruses could gain supremacy in subversion of cellular factors for pro-viral functions. PMID:26863541

  17. The drug-minded protein interaction database (DrumPID) for efficient target analysis and drug development

    PubMed Central

    Kunz, Meik; Liang, Chunguang; Nilla, Santosh; Cecil, Alexander; Dandekar, Thomas

    2016-01-01

    The drug-minded protein interaction database (DrumPID) has been designed to provide fast, tailored information on drugs and their protein networks including indications, protein targets and side-targets. Starting queries include compound, target and protein interactions and organism-specific protein families. Furthermore, drug name, chemical structures and their SMILES notation, affected proteins (potential drug targets), organisms as well as diseases can be queried including various combinations and refinement of searches. Drugs and protein interactions are analyzed in detail with reference to protein structures and catalytic domains, related compound structures as well as potential targets in other organisms. DrumPID considers drug functionality, compound similarity, target structure, interactome analysis and organismic range for a compound, useful for drug development, predicting drug side-effects and structure–activity relationships. Database URL: http://drumpid.bioapps.biozentrum.uni-wuerzburg.de PMID:27055828

  18. Solvent accessible surface representation in a database system for protein docking

    SciTech Connect

    Seidl, T.; Kriegel, H.P.

    1995-12-31

    Protein docking is a new and challenging application for query processing in database systems. Our architecture for an efficient support of docking queries is based on the multistep query processing paradigm, a technique well-known from spatial database systems. Along with physicochemical parameters, the geometry of the molecules plays a fundamental role for docking retrieval. Thus, 3D structures and 3D surfaces of molecules are basic objects in molecular databases. We specify a molecular surface representation based on topology, define a class of neighborhood queries, and sketch some applications with respect to the docking problem. We suggest a patch-based data structure called the TriEdge structure, first, to efficiently support topological query processing, and second, to save space in comparison to common planar graph representations such as the quad-edge structure. In analogy to the quad-edge structure, the TriEdge structure has an algebraic interface and is implemented via complex pointers. However, we achieve a reduction of the space requirement by a factor of four. Finally, we investigate the time performance of our prototype.

  19. Cucumber Necrosis Virus Recruits Cellular Heat Shock Protein 70 Homologs at Several Stages of Infection

    PubMed Central

    Alam, Syed Benazir

    2015-01-01

    ABSTRACT RNA viruses often depend on host factors for multiplication inside cells due to the constraints of their small genome size and limited coding capacity. One such factor that has been exploited by several plant and animal viruses is heat shock protein 70 (HSP70) family homologs which have been shown to play roles for different viruses in viral RNA replication, viral assembly, disassembly, and cell-to-cell movement. Using next generation sequence analysis, we reveal that several isoforms of Hsp70 and Hsc70 transcripts are induced to very high levels during cucumber necrosis virus (CNV) infection of Nicotiana benthamiana and that HSP70 proteins are also induced by at least 10-fold. We show that HSP70 family protein homologs are co-opted by CNV at several stages of infection. We have found that overexpression of Hsp70 or Hsc70 leads to enhanced CNV genomic RNA, coat protein (CP), and virion accumulation, whereas downregulation leads to a corresponding decrease. Hsc70-2 was found to increase solubility of CNV CP in vitro and to increase accumulation of CNV CP independently of viral RNA replication during coagroinfiltration in N. benthamiana. In addition, virus particle assembly into virus-like particles in CP agroinfiltrated plants was increased in the presence of Hsc70-2. HSP70 was found to increase the targeting of CNV CP to chloroplasts during infection, reinforcing the role of HSP70 in chloroplast targeting of host proteins. Hence, our findings have led to the discovery of a highly induced host factor that has been co-opted to play multiple roles during several stages of the CNV infection cycle. IMPORTANCE Because of the small size of its RNA genome, CNV is dependent on interaction with host cellular components to successfully complete its multiplication cycle. We have found that CNV induces HSP70 family homologs to a high level during infection, possibly as a result of the host response to the high levels of CNV proteins that accumulate during infection

  20. Vaccines displaying mycobacterial proteins on biopolyester beads stimulate cellular immunity and induce protection against tuberculosis.

    PubMed

    Parlane, Natalie A; Grage, Katrin; Mifune, Jun; Basaraba, Randall J; Wedlock, D Neil; Rehm, Bernd H A; Buddle, Bryce M

    2012-01-01

    New improved vaccines are needed for control of both bovine and human tuberculosis. Tuberculosis protein vaccines have advantages with regard to safety and ease of manufacture, but efficacy against tuberculosis has been difficult to achieve. Protective cellular immune responses can be preferentially induced when antigens are displayed on small particles. In this study, Escherichia coli and Lactococcus lactis were engineered to produce spherical polyhydroxybutyrate (PHB) inclusions which displayed a fusion protein of Mycobacterium tuberculosis, antigen 85A (Ag85A)-early secreted antigenic target 6-kDa protein (ESAT-6). L. lactis was chosen as a possible production host due its extensive use in the food industry and reduced risk of lipopolysaccharide contamination. Mice were vaccinated with PHB bead vaccines with or without displaying Ag85A-ESAT-6, recombinant Ag85A-ESAT-6, or M. bovis BCG. Separate groups of mice were used to measure immune responses and assess protection against an aerosol M. bovis challenge. Increased amounts of antigen-specific gamma interferon, interleukin-17A (IL-17A), IL-6, and tumor necrosis factor alpha were produced from splenocytes postvaccination, but no or minimal IL-4, IL-5, or IL-10 was produced, indicating Th1- and Th17-biased T cell responses. Decreased lung bacterial counts and less extensive foci of inflammation were observed in lungs of mice receiving BCG or PHB bead vaccines displaying Ag85A-ESAT-6 produced in either E. coli or L. lactis compared to those observed in the lungs of phosphate-buffered saline-treated control mice. No differences between those receiving wild-type PHB beads and those receiving recombinant Ag85A-ESAT-6 were observed. This versatile particulate vaccine delivery system incorporates a relatively simple production process using safe bacteria, and the results show that it is an effective delivery system for a tuberculosis protein vaccine. PMID:22072720

  1. PATtyFams: Protein families for the microbial genomes in the PATRIC database

    DOE PAGESBeta

    Davis, James J.; Gerdes, Svetlana; Olsen, Gary J.; Olson, Robert; Pusch, Gordon D.; Shukla, Maulik; Vonstein, Veronika; Wattam, Alice R.; Yoo, Hyunseung

    2016-02-08

    The ability to build accurate protein families is a fundamental operation in bioinformatics that influences comparative analyses, genome annotation, and metabolic modeling. For several years we have been maintaining protein families for all microbial genomes in the PATRIC database (Pathosystems Resource Integration Center, patricbrc.org) in order to drive many of the comparative analysis tools that are available through the PATRIC website. However, due to the burgeoning number of genomes, traditional approaches for generating protein families are becoming prohibitive. In this report, we describe a new approach for generating protein families, which we call PATtyFams. This method uses the k-mer-based functionmore » assignments available through RAST (Rapid Annotation using Subsystem Technology) to rapidly guide family formation, and then differentiates the function-based groups into families using a Markov Cluster algorithm (MCL). In conclusion, this new approach for generating protein families is rapid, scalable and has properties that are consistent with alignment-based methods.« less

  2. ModBase, a database of annotated comparative protein structure models, and associated resources.

    PubMed

    Pieper, Ursula; Webb, Benjamin M; Barkan, David T; Schneidman-Duhovny, Dina; Schlessinger, Avner; Braberg, Hannes; Yang, Zheng; Meng, Elaine C; Pettersen, Eric F; Huang, Conrad C; Datta, Ruchira S; Sampathkumar, Parthasarathy; Madhusudhan, Mallur S; Sjölander, Kimmen; Ferrin, Thomas E; Burley, Stephen K; Sali, Andrej

    2011-01-01

    ModBase (http://salilab.org/modbase) is a database of annotated comparative protein structure models. The models are calculated by ModPipe, an automated modeling pipeline that relies primarily on Modeller for fold assignment, sequence-structure alignment, model building and model assessment (http://salilab.org/modeller/). ModBase currently contains 10,355,444 reliable models for domains in 2,421,920 unique protein sequences. ModBase allows users to update comparative models on demand, and request modeling of additional sequences through an interface to the ModWeb modeling server (http://salilab.org/modweb). ModBase models are available through the ModBase interface as well as the Protein Model Portal (http://www.proteinmodelportal.org/). Recently developed associated resources include the SALIGN server for multiple sequence and structure alignment (http://salilab.org/salign), the ModEval server for predicting the accuracy of protein structure models (http://salilab.org/modeval), the PCSS server for predicting which peptides bind to a given protein (http://salilab.org/pcss) and the FoXS server for calculating and fitting Small Angle X-ray Scattering profiles (http://salilab.org/foxs). PMID:21097780

  3. Computational methodologies for compound database searching that utilize experimental protein-ligand interaction information.

    PubMed

    Tan, Lu; Batista, Jose; Bajorath, Jürgen

    2010-09-01

    Ligand- and target structure-based methods are widely used in virtual screening, but there is currently no methodology available that fully integrates these different approaches. Herein, we provide an overview of various attempts that have been made to combine ligand- and structure-based computational screening methods. We then review different types of approaches that utilize protein-ligand interaction information for database screening and filtering. Interaction-based approaches make use of a variety of methodological concepts including pharmacophore modeling and direct or indirect encoding of protein-ligand interactions in fingerprint formats. These interaction-based methods have been successfully applied to tackle different tasks related to virtual screening including postprocessing of docking poses, prioritization of binding modes, selectivity analysis, or similarity searching. Furthermore, we discuss the recently developed interacting fragment approach that indirectly incorporates 3D interaction information into 2D similarity searching and bridges between ligand- and structure-based methods.

  4. FastaHerder2: Four Ways to Research Protein Function and Evolution with Clustering and Clustered Databases.

    PubMed

    Mier, Pablo; Andrade-Navarro, Miguel A

    2016-04-01

    The accelerated growth of protein databases offers great possibilities for the study of protein function using sequence similarity and conservation. However, the huge number of sequences deposited in these databases requires new ways of analyzing and organizing the data. It is necessary to group the many very similar sequences, creating clusters with automated derived annotations useful to understand their function, evolution, and level of experimental evidence. We developed an algorithm called FastaHerder2, which can cluster any protein database, putting together very similar protein sequences based on near-full-length similarity and/or high threshold of sequence identity. We compressed 50 reference proteomes, along with the SwissProt database, which we could compress by 74.7%. The clustering algorithm was benchmarked using OrthoBench and compared with FASTA HERDER, a previous version of the algorithm, showing that FastaHerder2 can cluster a set of proteins yielding a high compression, with a lower error rate than its predecessor. We illustrate the use of FastaHerder2 to detect biologically relevant functional features in protein families. With our approach we seek to promote a modern view and usage of the protein sequence databases more appropriate to the postgenomic era. PMID:26828375

  5. The role of mitochondria in cytosolic-nuclear iron–sulfur protein biogenesis and in cellular iron regulation.

    PubMed

    Lill, Roland; Srinivasan, Vasundara; Mühlenhoff, Ulrich

    2014-12-01

    Mitochondria are indispensable in eukaryotes because of their function in the maturation of cytosolic and nuclear iron–sulfur proteins that are essential for DNA synthesis and repair, tRNA modification, and protein translation. The mitochondrial Fe/S cluster assembly machinery not only generates the organelle's iron–sulfur proteins, but also extra-mitochondrial ones. Biogenesis of the latter proteins requires the mitochondrial ABC transporter Atm1 that exports a sulfur-containing compound in a glutathione-dependent fashion. The process is further assisted by the cytosolic iron–sulfur protein assembly machinery. Here, we discuss the knowns and unknowns of the mitochondrial export process that is also crucial for signaling the cellular iron status to the regulatory systems involved in the maintenance of cellular iron homeostasis.

  6. Investigation of protein functions through data-mining on integrated human transcriptome database, H-Invitational database (H-InvDB).

    PubMed

    Yamasaki, Chisato; Koyanagi, Kanako O; Fujii, Yasuyuki; Itoh, Takeshi; Barrero, Roberto; Tamura, Takuro; Yamaguchi-Kabata, Yumi; Tanino, Motohiko; Takeda, Jun-Ichi; Fukuchi, Satoshi; Miyazaki, Satoru; Nomura, Nobuo; Sugano, Sumio; Imanishi, Tadashi; Gojobori, Takashi

    2005-12-30

    H-Invitational Database (H-InvDB; ) is a human transcriptome database, containing integrative annotation of 41,118 full-length cDNA clones originated from 21,037 loci. H-InvDB is a product of the H-Invitational project, an international collaboration to systematically and functionally validate human genes by analysis of a unique set of high quality full-length cDNA clones using automatic annotation and human curation under unified criteria. Here, 19,574 proteins encoded by these cDNAs were classified into 11,709 function-known and 7865 function-unknown hypothetical proteins by similarity with protein databases and motif prediction (InterProScan). The proportion of "hypothetical proteins" in H-InvDB was as high as 40.4%. In this study, we thus conducted data-mining in H-InvDB with the aim of assigning advanced functional annotations to those hypothetical proteins. First, by data-mining in the H-InvDB version of GTOP, we identified 337 SCOP domains within 7865 H-Inv hypothetical proteins. Second, by data-mining of predicted subcellular localization by SOSUI and TMHMM in H-InvDB, we found 1032 transmembrane proteins within H-Inv hypothetical proteins. These results clearly demonstrate that structural prediction is effective for functional annotation of proteins with unknown functions. All the data in H-InvDB are shown in two main views, the cDNA view and the Locus view, and five auxiliary databases with web-based viewers; DiseaseInfo Viewer, H-ANGEL, Clustering Viewer, G-integra and TOPO Viewer; the data also are provided as flat files and XML files. The data consists of descriptions of their gene structures, novel alternative splicing isoforms, functional RNAs, functional domains, subcellular localizations, metabolic pathways, predictions of protein 3D structure, mapping of SNPs and microsatellite repeat motifs in relation with orphan diseases, gene expression profiling, and comparisons with mouse full-length cDNAs in the context of molecular evolution. This unique

  7. Adolescent Risperidone treatment alters protein expression associated with protein trafficking and cellular metabolism in the adult rat prefrontal cortex.

    PubMed

    Farrelly, Lorna A; Dicker, Patrick; Wynne, Kieran; English, Jane; Cagney, Gerard; Föcking, Melanie; Cotter, David R

    2014-06-01

    The prefrontal cortex (PFC) is associated with mental health illnesses including schizophrenia, depression, bipolar disorder, and autism spectrum disorders. It richly expresses neuroreceptors which are the target for antipsychotics. However, as the precise mechanism of action of antipsychotic medications is not known, proteomic studies of the effects of antipsychotic drugs on the brain are warranted. In the current study, we aimed to characterize protein expression in the adult rodent PFC (n = 5 per group) following low-dose treatment with Risperidone or saline in adolescence (postnatal days 34-47). The PFC was examined by triplicate 1 h runs of label-free LC-MS/MS. The raw mass spectral data were analyzed with the MaxQuant(TM) software. Statistical analysis was carried out using SAS® Version 9.1. Pathway and functional analysis was performed with IngenuityPathway Analysis and in the Database for Annotation, Visualization and Integrated Discovery (DAVID), respectively, the most implicated pathways were found to be related to mitochondrial function, protein trafficking, and the cytoskeleton. This report adds to the current repertoire of data available concerning the effects of antipsychotic drugs on the brain and sheds light on their biological mechanisms. The MS data have been deposited with the ProteomeXchange Consortium with dataset identifier PXD000480. PMID:24733778

  8. CoDNaS 2.0: a comprehensive database of protein conformational diversity in the native state

    PubMed Central

    Monzon, Alexander Miguel; Rohr, Cristian Oscar; Fornasari, María Silvina; Parisi, Gustavo

    2016-01-01

    CoDNaS (conformational diversity of the native state) is a protein conformational diversity database. Conformational diversity describes structural differences between conformers that define the native state of proteins. It is a key concept to understand protein function and biological processes related to protein functions. CoDNaS offers a well curated database that is experimentally driven, thoroughly linked, and annotated. CoDNaS facilitates the extraction of key information on small structural differences based on protein movements. CoDNaS enables users to easily relate the degree of conformational diversity with physical, chemical and biological properties derived from experiments on protein structure and biological characteristics. The new version of CoDNaS includes ∼70% of all available protein structures, and new tools have been added that run sequence searches, display structural flexibility profiles and allow users to browse the database for different structural classes. These tools facilitate the exploration of protein conformational diversity and its role in protein function. Database URL: http://ufq.unq.edu.ar/codnas PMID:27022160

  9. CoDNaS 2.0: a comprehensive database of protein conformational diversity in the native state.

    PubMed

    Monzon, Alexander Miguel; Rohr, Cristian Oscar; Fornasari, María Silvina; Parisi, Gustavo

    2016-01-01

    CoDNaS (conformational diversity of the native state) is a protein conformational diversity database. Conformational diversity describes structural differences between conformers that define the native state of proteins. It is a key concept to understand protein function and biological processes related to protein functions. CoDNaS offers a well curated database that is experimentally driven, thoroughly linked, and annotated. CoDNaS facilitates the extraction of key information on small structural differences based on protein movements. CoDNaS enables users to easily relate the degree of conformational diversity with physical, chemical and biological properties derived from experiments on protein structure and biological characteristics. The new version of CoDNaS includes ∼70% of all available protein structures, and new tools have been added that run sequence searches, display structural flexibility profiles and allow users to browse the database for different structural classes. These tools facilitate the exploration of protein conformational diversity and its role in protein function. Database URL:http://ufq.unq.edu.ar/codnas. PMID:27022160

  10. SuperLigands – a database of ligand structures derived from the Protein Data Bank

    PubMed Central

    Michalsky, Elke; Dunkel, Mathias; Goede, Andrean; Preissner, Robert

    2005-01-01

    Background Currently, the PDB contains approximately 29,000 protein structures comprising over 70,000 experimentally determined three-dimensional structures of over 5,000 different low molecular weight compounds. Information about these PDB ligands can be very helpful in the field of molecular modelling and prediction, particularly for the prediction of protein binding sites and function. Description Here we present an Internet accessible database delivering PDB ligands in the MDL Mol file format which, in contrast to the PDB format, includes information about bond types. Structural similarity of the compounds can be detected by calculation of Tanimoto coefficients and by three-dimensional superposition. Topological similarity of PDB ligands to known drugs can be assessed via Tanimoto coefficients. Conclusion SuperLigands supplements the set of existing resources of information about small molecules bound to PDB structures. Allowing for three-dimensional comparison of the compounds as a novel feature, this database represents a valuable means of analysis and prediction in the field of biological and medical research. PMID:15943884

  11. The Candida Genome Database: the new homology information page highlights protein similarity and phylogeny.

    PubMed

    Binkley, Jonathan; Arnaud, Martha B; Inglis, Diane O; Skrzypek, Marek S; Shah, Prachi; Wymore, Farrell; Binkley, Gail; Miyasato, Stuart R; Simison, Matt; Sherlock, Gavin

    2014-01-01

    The Candida Genome Database (CGD, http://www.candidagenome.org/) is a freely available online resource that provides gene, protein and sequence information for multiple Candida species, along with web-based tools for accessing, analyzing and exploring these data. The goal of CGD is to facilitate and accelerate research into Candida pathogenesis and biology. The CGD Web site is organized around Locus pages, which display information collected about individual genes. Locus pages have multiple tabs for accessing different types of information; the default Summary tab provides an overview of the gene name, aliases, phenotype and Gene Ontology curation, whereas other tabs display more in-depth information, including protein product details for coding genes, notes on changes to the sequence or structure of the gene and a comprehensive reference list. Here, in this update to previous NAR Database articles featuring CGD, we describe a new tab that we have added to the Locus page, entitled the Homology Information tab, which displays phylogeny and gene similarity information for each locus.

  12. Electrochemical aptasensor of cellular prion protein based on modified polypyrrole with redox dendrimers.

    PubMed

    Miodek, A; Castillo, G; Hianik, T; Korri-Youssoufi, H

    2014-06-15

    This work consists of the development of an electrochemical aptasensor based on polyprrole modified with redox dendrimers, able to detect human cellular prions PrP(C) with high sensitivity. The gold surface was modified by conductive polypyrrole film coupled to polyamidoamine dendrimers of fourth generation (PAMAM G4) and ferrocenyl group as redox marker. The aptamers were immobilized on the surface via biotin/streptavidin chemistry. Electrochemical signal was detected by ferrocenyl group incorporated between dendrimers and aptamers layers. We demonstrated that the interaction between aptamer and prion protein led to variation in electrochemical signal of the ferrocenyl group. The kinetics parameters (diffusion coefficient D and heterogeneous constant transfer ket) calculated from electrochemical signals demonstrate that the variation in redox signal results from the lower diffusion process of ions during redox reaction after prion interaction due to bulk effect of larger protein. The association of redox dendrimers with conducting polypyrrole leads to high sensitivity of PrP(C) determination with detection limit of 0.8 pM, which is three orders of magnitude lower, compared to flat ferrocene-functionalized polypyrrole. Detection of PrP(C) in spiked blood plasma has been achieved and demonstrated a recovery up to 90%. PMID:24480126

  13. Distribution of the cellular prion protein in the central nervous system of the chicken.

    PubMed

    Atoji, Yasuro; Ishiguro, Naotaka

    2009-12-01

    The cellular prion protein (PrP), a cell membrane-bound glycoprotein mainly located in the dendrites and axons of the central nervous system (CNS), is responsible for transmissible spongiform encephalopathies in mammals. PrP genes are widely conserved in vertebrates. In birds, the presence of PrP mRNA has been confirmed in neurons of the chicken brain, but localization of the protein remains to be determined. In the present study, we demonstrated the regional distribution of PrP in the CNS of adult chickens by immunohistochemical staining with a monoclonal antibody that recognizes chicken PrP 161-164. Immunoreactivity was observed in the neuropil, but not in neuronal somata or glial cells. It was preferentially intense in the olfactory bulb, the dorsal thalamus, the hypothalamus, and most regions of the telencephalon. Immunostaining became less intense toward the brainstem, but many nuclei were immunoreactive. Among brainstem nuclei, moderate immunostaining was observed in the nucleus of the solitary tract, dorsal motor nucleus of the vagus nerve, and substantia gelatinosa Rolandi. The cerebellar cortex was devoid of PrP immunoreactivity. The dorsal horn in the spinal cord was strongly immunoreactive. In situ hybridization with two probes of the C-terminal portion demonstrated localization of PrP mRNA in neurons of the brain and spinal cord. These findings suggest that PrP in the chicken CNS is localized in the dendrites and axons of neurons and that it is associated with certain sensory systems.

  14. EDTA treatment alters protein glycosylation in the cellular slime mold Dictyostelium discoideum

    SciTech Connect

    West, C.M.; Brownstein, S.A. )

    1988-03-01

    The authors have found that treatment of cells with EDTA resulted in the accumulation of lower molecular weight forms of two cell-type-specific glycoproteins. These new glycoproteins lacked a developmentally regulated glycoantigen defined by monoclonal antibody 54.2. Since EDTA dissociated the cells, the possible involvement of cell separation was tested by immobilizing cells in soft agarose. Glycoantigen expression on these proteins was found to be dependent on cAMP and high oxygen tension but not on cell contact, and was reversibly sensitive to EDTA regardless of the state of cell association. The EDTA effect was mimicked by other soluble, but not particulate, membrane impermeable chelators, could be completed by Zn{sup 2+} better than Mg{sup 2+}, and appeared to involve an intracellular mechanism. Studies with ({sup 14}C)EDTA showed that EDTA equilibrated with a cellular compartment in a temperature-dependent, Zn{sup 2+}-insensitive fashion with half-time kinetics of loading and unloading of 30-40 min. The data suggest that this step in glycosylation, which was found to be delayed 1 or more hours subsequent to protein synthesis, involves an intracellular, transition metal-ion-dependent process which can be modulated by chelators entering the cell through the endocytic pathway.

  15. The Dengue Virus NS5 Protein Intrudes in the Cellular Spliceosome and Modulates Splicing.

    PubMed

    De Maio, Federico A; Risso, Guillermo; Iglesias, Nestor G; Shah, Priya; Pozzi, Berta; Gebhard, Leopoldo G; Mammi, Pablo; Mancini, Estefania; Yanovsky, Marcelo J; Andino, Raul; Krogan, Nevan; Srebrow, Anabella; Gamarnik, Andrea V

    2016-08-01

    Dengue virus NS5 protein plays multiple functions in the cytoplasm of infected cells, enabling viral RNA replication and counteracting host antiviral responses. Here, we demonstrate a novel function of NS5 in the nucleus where it interferes with cellular splicing. Using global proteomic analysis of infected cells together with functional studies, we found that NS5 binds spliceosome complexes and modulates endogenous splicing as well as minigene-derived alternative splicing patterns. In particular, we show that NS5 alone, or in the context of viral infection, interacts with core components of the U5 snRNP particle, CD2BP2 and DDX23, alters the inclusion/exclusion ratio of alternative splicing events, and changes mRNA isoform abundance of known antiviral factors. Interestingly, a genome wide transcriptome analysis, using recently developed bioinformatics tools, revealed an increase of intron retention upon dengue virus infection, and viral replication was improved by silencing specific U5 components. Different mechanistic studies indicate that binding of NS5 to the spliceosome reduces the efficiency of pre-mRNA processing, independently of NS5 enzymatic activities. We propose that NS5 binding to U5 snRNP proteins hijacks the splicing machinery resulting in a less restrictive environment for viral replication. PMID:27575636

  16. The Dengue Virus NS5 Protein Intrudes in the Cellular Spliceosome and Modulates Splicing.

    PubMed

    De Maio, Federico A; Risso, Guillermo; Iglesias, Nestor G; Shah, Priya; Pozzi, Berta; Gebhard, Leopoldo G; Mammi, Pablo; Mancini, Estefania; Yanovsky, Marcelo J; Andino, Raul; Krogan, Nevan; Srebrow, Anabella; Gamarnik, Andrea V

    2016-08-01

    Dengue virus NS5 protein plays multiple functions in the cytoplasm of infected cells, enabling viral RNA replication and counteracting host antiviral responses. Here, we demonstrate a novel function of NS5 in the nucleus where it interferes with cellular splicing. Using global proteomic analysis of infected cells together with functional studies, we found that NS5 binds spliceosome complexes and modulates endogenous splicing as well as minigene-derived alternative splicing patterns. In particular, we show that NS5 alone, or in the context of viral infection, interacts with core components of the U5 snRNP particle, CD2BP2 and DDX23, alters the inclusion/exclusion ratio of alternative splicing events, and changes mRNA isoform abundance of known antiviral factors. Interestingly, a genome wide transcriptome analysis, using recently developed bioinformatics tools, revealed an increase of intron retention upon dengue virus infection, and viral replication was improved by silencing specific U5 components. Different mechanistic studies indicate that binding of NS5 to the spliceosome reduces the efficiency of pre-mRNA processing, independently of NS5 enzymatic activities. We propose that NS5 binding to U5 snRNP proteins hijacks the splicing machinery resulting in a less restrictive environment for viral replication.

  17. The Dengue Virus NS5 Protein Intrudes in the Cellular Spliceosome and Modulates Splicing

    PubMed Central

    Shah, Priya; Pozzi, Berta; Gebhard, Leopoldo G.; Mammi, Pablo; Yanovsky, Marcelo J.; Andino, Raul; Krogan, Nevan; Srebrow, Anabella; Gamarnik, Andrea V.

    2016-01-01

    Dengue virus NS5 protein plays multiple functions in the cytoplasm of infected cells, enabling viral RNA replication and counteracting host antiviral responses. Here, we demonstrate a novel function of NS5 in the nucleus where it interferes with cellular splicing. Using global proteomic analysis of infected cells together with functional studies, we found that NS5 binds spliceosome complexes and modulates endogenous splicing as well as minigene-derived alternative splicing patterns. In particular, we show that NS5 alone, or in the context of viral infection, interacts with core components of the U5 snRNP particle, CD2BP2 and DDX23, alters the inclusion/exclusion ratio of alternative splicing events, and changes mRNA isoform abundance of known antiviral factors. Interestingly, a genome wide transcriptome analysis, using recently developed bioinformatics tools, revealed an increase of intron retention upon dengue virus infection, and viral replication was improved by silencing specific U5 components. Different mechanistic studies indicate that binding of NS5 to the spliceosome reduces the efficiency of pre-mRNA processing, independently of NS5 enzymatic activities. We propose that NS5 binding to U5 snRNP proteins hijacks the splicing machinery resulting in a less restrictive environment for viral replication. PMID:27575636

  18. SED1/MFG-E8: a bi-motif protein that orchestrates diverse cellular interactions.

    PubMed

    Raymond, Adam; Ensslin, Michael A; Shur, Barry D

    2009-04-15

    MFG-E8 was initially identified as a principle component of the Milk Fat Globule, a membrane-encased collection of proteins and triglycerides that bud from the apical surface of mammary epithelia during lactation. It has since been independently identified in many species and by many investigators and given a variety of names, including p47, lactadherin, rAGS, PAS6/7, and BA-46. The acronym SED1 was proposed to bring cohesion to this nomenclature based upon it being a Secreted protein that contains two distinct functional domains: an N-terminal domain with two EGF-repeats, the second of which has an integrin-binding RGD motif, and a C-terminal domain with two Discoidin/F5/8C domains that bind to anionic phospholipids and/or extracellular matrices. SED1/MFG-E8 is now known to participate in a wide variety of cellular interactions, including phagocytosis of apoptotic lymphocytes and other apoptotic cells, adhesion between sperm and the egg coat, repair of intestinal mucosa, mammary gland branching morphogenesis, angiogenesis, among others. This article will explore the various roles proposed for SED1/MFG-E8, as well as its provocative therapeutic potential. PMID:19204935

  19. Electrochemical aptasensor of cellular prion protein based on modified polypyrrole with redox dendrimers.

    PubMed

    Miodek, A; Castillo, G; Hianik, T; Korri-Youssoufi, H

    2014-06-15

    This work consists of the development of an electrochemical aptasensor based on polyprrole modified with redox dendrimers, able to detect human cellular prions PrP(C) with high sensitivity. The gold surface was modified by conductive polypyrrole film coupled to polyamidoamine dendrimers of fourth generation (PAMAM G4) and ferrocenyl group as redox marker. The aptamers were immobilized on the surface via biotin/streptavidin chemistry. Electrochemical signal was detected by ferrocenyl group incorporated between dendrimers and aptamers layers. We demonstrated that the interaction between aptamer and prion protein led to variation in electrochemical signal of the ferrocenyl group. The kinetics parameters (diffusion coefficient D and heterogeneous constant transfer ket) calculated from electrochemical signals demonstrate that the variation in redox signal results from the lower diffusion process of ions during redox reaction after prion interaction due to bulk effect of larger protein. The association of redox dendrimers with conducting polypyrrole leads to high sensitivity of PrP(C) determination with detection limit of 0.8 pM, which is three orders of magnitude lower, compared to flat ferrocene-functionalized polypyrrole. Detection of PrP(C) in spiked blood plasma has been achieved and demonstrated a recovery up to 90%.

  20. The Molecular Chaperone Binding Protein BiP Prevents Leaf Dehydration-Induced Cellular Homeostasis Disruption

    PubMed Central

    Carvalho, Humberto H.; Brustolini, Otávio J. B.; Pimenta, Maiana R.; Mendes, Giselle C.; Gouveia, Bianca C.; Silva, Priscila A.; Silva, José Cleydson F.; Mota, Clenilso S.; Soares-Ramos, Juliana R. L.; Fontes, Elizabeth P. B.

    2014-01-01

    BiP overexpression improves leaf water relations during droughts and delays drought-induced leaf senescence. However, whether BiP controls cellular homeostasis under drought conditions or simply delays dehydration-induced leaf senescence as the primary cause for water stress tolerance remains to be determined. To address this issue, we examined the drought-induced transcriptomes of BiP-overexpressing lines and wild-type (WT) lines under similar leaf water potential (ψw) values. In the WT leaves, a ψw reduction of −1.0 resulted in 1339 up-regulated and 2710 down-regulated genes; in the BiP-overexpressing line 35S::BiP-4, only 334 and 420 genes were induced and repressed, respectively, at a similar leaf ψw = −1.0 MPa. This level of leaf dehydration was low enough to induce a repertory of typical drought-responsive genes in WT leaves but not in 35S::BiP-4 dehydrated leaves. The responders included hormone-related genes, functional and regulatory genes involved in drought protection and senescence-associated genes. The number of differentially expressed genes in the 35S::BiP-4 line approached the wild type number at a leaf ψw = −1.6 MPa. However, N-rich protein (NRP)- mediated cell death signaling genes and unfolded protein response (UPR) genes were induced to a much lower extent in the 35S::BiP-4 line than in the WT even at ψw = −1.6 MPa. The heatmaps for UPR, ERAD (ER-associated degradation protein system), drought-responsive and cell death-associated genes revealed that the leaf transcriptome of 35S::BiP-4 at ψw = −1.0 MPa clustered together with the transcriptome of well-watered leaves and they diverged considerably from the drought-induced transcriptome of the WT (ψw = −1.0, −1.7 and −2.0 MPa) and 35S::BiP-4 leaves at ψw = −1.6 MPa. Taken together, our data revealed that BiP-overexpressing lines requires a much higher level of stress (ψw = −1.6 MPa) to respond to drought than that of WT (ψw = −1

  1. The molecular chaperone binding protein BiP prevents leaf dehydration-induced cellular homeostasis disruption.

    PubMed

    Carvalho, Humberto H; Brustolini, Otávio J B; Pimenta, Maiana R; Mendes, Giselle C; Gouveia, Bianca C; Silva, Priscila A; Silva, José Cleydson F; Mota, Clenilso S; Soares-Ramos, Juliana R L; Fontes, Elizabeth P B

    2014-01-01

    BiP overexpression improves leaf water relations during droughts and delays drought-induced leaf senescence. However, whether BiP controls cellular homeostasis under drought conditions or simply delays dehydration-induced leaf senescence as the primary cause for water stress tolerance remains to be determined. To address this issue, we examined the drought-induced transcriptomes of BiP-overexpressing lines and wild-type (WT) lines under similar leaf water potential (ψw) values. In the WT leaves, a ψw reduction of -1.0 resulted in 1339 up-regulated and 2710 down-regulated genes; in the BiP-overexpressing line 35S::BiP-4, only 334 and 420 genes were induced and repressed, respectively, at a similar leaf ψw = -1.0 MPa. This level of leaf dehydration was low enough to induce a repertory of typical drought-responsive genes in WT leaves but not in 35S::BiP-4 dehydrated leaves. The responders included hormone-related genes, functional and regulatory genes involved in drought protection and senescence-associated genes. The number of differentially expressed genes in the 35S::BiP-4 line approached the wild type number at a leaf ψw = -1.6 MPa. However, N-rich protein (NRP)- mediated cell death signaling genes and unfolded protein response (UPR) genes were induced to a much lower extent in the 35S::BiP-4 line than in the WT even at ψw = -1.6 MPa. The heatmaps for UPR, ERAD (ER-associated degradation protein system), drought-responsive and cell death-associated genes revealed that the leaf transcriptome of 35S::BiP-4 at ψw = -1.0 MPa clustered together with the transcriptome of well-watered leaves and they diverged considerably from the drought-induced transcriptome of the WT (ψw = -1.0, -1.7 and -2.0 MPa) and 35S::BiP-4 leaves at ψw = -1.6 MPa. Taken together, our data revealed that BiP-overexpressing lines requires a much higher level of stress (ψw = -1.6 MPa) to respond to drought than that of WT (ψw = -1.0). Therefore, Bi

  2. Dengue Virus NS1 Protein Modulates Cellular Energy Metabolism by Increasing Glyceraldehyde-3-Phosphate Dehydrogenase Activity

    PubMed Central

    Allonso, Diego; Andrade, Iamara S.; Conde, Jonas N.; Coelho, Diego R.; Rocha, Daniele C. P.; da Silva, Manuela L.; Ventura, Gustavo T.

    2015-01-01

    ABSTRACT Dengue is one of the main public health concerns worldwide. Recent estimates indicate that over 390 million people are infected annually with the dengue virus (DENV), resulting in thousands of deaths. Among the DENV nonstructural proteins, the NS1 protein is the only one whose function during replication is still unknown. NS1 is a 46- to 55-kDa glycoprotein commonly found as both a membrane-associated homodimer and a soluble hexameric barrel-shaped lipoprotein. Despite its role in the pathogenic process, NS1 is essential for proper RNA accumulation and virus production. In the present study, we identified that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) interacts with intracellular NS1. Molecular docking revealed that this interaction occurs through the hydrophobic protrusion of NS1 and the hydrophobic residues located at the opposite side of the catalytic site. Moreover, addition of purified recombinant NS1 enhanced the glycolytic activity of GAPDH in vitro. Interestingly, we observed that DENV infection promoted the relocalization of GAPDH to the perinuclear region, where NS1 is commonly found. Both DENV infection and expression of NS1 itself resulted in increased GAPDH activity. Our findings indicate that the NS1 protein acts to increase glycolytic flux and, consequently, energy production, which is consistent with the recent finding that DENV induces and requires glycolysis for proper replication. This is the first report to propose that NS1 is an important modulator of cellular energy metabolism. The data presented here provide new insights that may be useful for further drug design and the development of alternative antiviral therapies against DENV. IMPORTANCE Dengue represents a serious public health problem worldwide and is caused by infection with dengue virus (DENV). Estimates indicate that half of the global population is at risk of infection, with almost 400 million cases occurring per year. The NS1 glycoprotein is found in both the

  3. Direct identification of human cellular microRNAs by nanoflow liquid chromatography-high-resolution tandem mass spectrometry and database searching.

    PubMed

    Nakayama, Hiroshi; Yamauchi, Yoshio; Taoka, Masato; Isobe, Toshiaki

    2015-03-01

    MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene networks and participate in many physiological and pathological pathways. To date, miRNAs have been characterized mostly by genetic technologies, which have the advantages of being very sensitive and using high-throughput instrumentation; however, these techniques cannot identify most post-transcriptional modifications of miRNAs that would affect their functions. Herein, we report an analytical system for the direct identification of miRNAs that incorporates nanoflow liquid chromatography-high-resolution tandem mass spectrometry and RNA-sequence database searching. By introducing a spray-assisting device that stabilizes negative nanoelectrospray ionization of RNAs and by searching an miRNA sequence database using the obtained tandem mass spectrometry data for the RNA mixture, we successfully identified femtomole quantities of human cellular miRNAs and their 3'-terminal variants. This is the first report of a fully automated, and thus objective, tandem mass spectrometry-based analytical system that can be used to identify miRNAs.

  4. When a domain isn’t a domain, and why it’s important to properly filter proteins in databases

    PubMed Central

    Towse, Clare-Louise; Daggett, Valerie

    2013-01-01

    Summary Membership in a protein domain database does not a domain make; a feature we realized when generating a consensus view of protein fold space with our Consensus Domain Dictionary (CDD). This dictionary was used to select representative structures for characterization of the protein dynameome: the Dynameomics initiative. Through this endeavor we rejected a surprising 40% of the 1695 folds in the CDD as being non-autonomous folding units. Although some of this was due to the challenges of grouping similar fold topologies, the dissonance between the cataloguing and structural qualification of protein domains remains surprising. Another potential factor is previously overlooked intrinsic disorder; predicted estimates suggest 40% of proteins to have either local or global disorder. One thing is clear, filtering a structural database and ensuring a consistent definition for protein domains is crucial, and caution is prescribed when generalizations of globular domains are drawn from unfiltered protein domain datasets. PMID:23108912

  5. Nitric oxide-mediated modulation of iron regulatory proteins: implication for cellular iron homeostasis.

    PubMed

    Kim, Sangwon; Ponka, Prem

    2002-01-01

    Iron regulatory proteins (IRP1 and IRP2) control the synthesis of transferrin receptors (TfR) and ferritin by binding to iron-responsive elements (IREs) that are located in the 3' untranslated region (UTR) and the 5' UTR of their respective mRNAs. Cellular iron levels affect binding of IRPs to IREs and consequently expression of TfR and ferritin. Moreover, NO(.), a redox species of nitric oxide that interacts primarily with iron, can activate IRP1 RNA-binding activity resulting in an increase in TfR mRNA levels and a decrease in ferritin synthesis. We have shown that treatment of RAW 264.7 cells (a murine macrophage cell line) with NO(+) (nitrosonium ion, which causes S-nitrosylation of thiol groups) resulted in a rapid decrease in RNA-binding of IRP2, followed by IRP2 degradation, and these changes were associated with a decrease in TfR mRNA levels and a dramatic increase in ferritin synthesis. Moreover, we demonstrated that stimulation of RAW 264.7 cells with lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma) increased IRP1 binding activity, whereas RNA-binding of IRP2 decreased and was followed by a degradation of this protein. Furthermore, the decrease of IRP2 binding/protein levels was associated with a decrease in TfR mRNA levels and an increase in ferritin synthesis in LPS/IFN-gamma-treated cells, and these changes were prevented by inhibitors of inducible nitric oxide synthase. These results suggest that NO(+)-mediated degradation of IRP2 plays a major role in iron metabolism during inflammation.

  6. The comparison of protein-entrapped liposomes and lipoparticles: preparation, characterization, and efficacy of cellular uptake

    PubMed Central

    Chang, Wei-Kuo; Tai, Yu-Ju; Chiang, Chiao-Hsi; Hu, Chieh-Shen; Hong, Po-Da; Yeh, Ming-Kung

    2011-01-01

    Fluorescein isothiocyanate-conjugated bovine serum albumin (FITC-BSA)-loaded polyethylene glycol (PEG)-modified liposomes and lipoparticles with high protein entrapment were developed. The lipid formula of the liposomes contained PEGylated lipids and unsaturated fatty acids for enhancing membrane fluidity and effective delivery into cells. The preparation techniques, lipid content, and PEG-modified lipoparticle ratios were evaluated. The PEG-modified lipoparticles prepared by ethanol injection extrusion (100 nm pore size) achieve a population of blank liposomes with a mean size of 125 ± 2.3 nm and a zeta potential of −12.4 ± 1.5 mV. The average particle size of the PEG-modified lipoparticles was 133.7 ± 8.6 nm with a zeta potential of +13.3 mV. Lipoparticle conformation was determined using transmission electron microscopy and field-emission scanning electron microscopy. The FITC-BSA encapsulation efficiency was dramatically increased from 19.0% for liposomes to 59.7% for lipoparticles. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) results confirmed the preparation process, and an 8-hour leaching test did not harm the protein structure. Once prepared, the physical and chemical stability of the PEG-modified lipoparticle formulations was satisfactory over 90 days. In vitro retention tests indicated that the 50% retention time for the protein-containing lipoparticles was 7.9 hours, substantially longer than the liposomes at 3.3 hours. A Caco-2 cell model was used for evaluating the cytotoxicity and cell uptake efficiency of the PEG-modified lipoparticles. At a lipid content below 0.25 mM, neither the liposomes nor the lipoparticles caused significant cellular cytotoxicity (P < 0.01) and FITC-BSA was significantly taken up into cells within 60 minutes (P < 0.01). PMID:22072876

  7. CLIPZ: a database and analysis environment for experimentally determined binding sites of RNA-binding proteins.

    PubMed

    Khorshid, Mohsen; Rodak, Christoph; Zavolan, Mihaela

    2011-01-01

    The stability, localization and translation rate of mRNAs are regulated by a multitude of RNA-binding proteins (RBPs) that find their targets directly or with the help of guide RNAs. Among the experimental methods for mapping RBP binding sites, cross-linking and immunoprecipitation (CLIP) coupled with deep sequencing provides transcriptome-wide coverage as well as high resolution. However, partly due to their vast volume, the data that were so far generated in CLIP experiments have not been put in a form that enables fast and interactive exploration of binding sites. To address this need, we have developed the CLIPZ database and analysis environment. Binding site data for RBPs such as Argonaute 1-4, Insulin-like growth factor II mRNA-binding protein 1-3, TNRC6 proteins A-C, Pumilio 2, Quaking and Polypyrimidine tract binding protein can be visualized at the level of the genome and of individual transcripts. Individual users can upload their own sequence data sets while being able to limit the access to these data to specific users, and analyses of the public and private data sets can be performed interactively. CLIPZ, available at http://www.clipz.unibas.ch, aims to provide an open access repository of information for post-transcriptional regulatory elements.

  8. CLIPZ: a database and analysis environment for experimentally determined binding sites of RNA-binding proteins

    PubMed Central

    Khorshid, Mohsen; Rodak, Christoph; Zavolan, Mihaela

    2011-01-01

    The stability, localization and translation rate of mRNAs are regulated by a multitude of RNA-binding proteins (RBPs) that find their targets directly or with the help of guide RNAs. Among the experimental methods for mapping RBP binding sites, cross-linking and immunoprecipitation (CLIP) coupled with deep sequencing provides transcriptome-wide coverage as well as high resolution. However, partly due to their vast volume, the data that were so far generated in CLIP experiments have not been put in a form that enables fast and interactive exploration of binding sites. To address this need, we have developed the CLIPZ database and analysis environment. Binding site data for RBPs such as Argonaute 1-4, Insulin-like growth factor II mRNA-binding protein 1-3, TNRC6 proteins A-C, Pumilio 2, Quaking and Polypyrimidine tract binding protein can be visualized at the level of the genome and of individual transcripts. Individual users can upload their own sequence data sets while being able to limit the access to these data to specific users, and analyses of the public and private data sets can be performed interactively. CLIPZ, available at http://www.clipz.unibas.ch, aims to provide an open access repository of information for post-transcriptional regulatory elements. PMID:21087992

  9. Sulfolobus Spindle-Shaped Virus 1 Contains Glycosylated Capsid Proteins, a Cellular Chromatin Protein, and Host-Derived Lipids

    PubMed Central

    Quemin, Emmanuelle R. J.; Pietilä, Maija K.; Oksanen, Hanna M.; Forterre, Patrick; Rijpstra, W. Irene C.; Schouten, Stefan; Bamford, Dennis H.; Prangishvili, David

    2015-01-01

    ABSTRACT Geothermal and hypersaline environments are rich in virus-like particles, among which spindle-shaped morphotypes dominate. Currently, viruses with spindle- or lemon-shaped virions are exclusive to Archaea and belong to two distinct viral families. The larger of the two families, the Fuselloviridae, comprises tail-less, spindle-shaped viruses, which infect hosts from phylogenetically distant archaeal lineages. Sulfolobus spindle-shaped virus 1 (SSV1) is the best known member of the family and was one of the first hyperthermophilic archaeal viruses to be isolated. SSV1 is an attractive model for understanding virus-host interactions in Archaea; however, the constituents and architecture of SSV1 particles remain only partially characterized. Here, we have conducted an extensive biochemical characterization of highly purified SSV1 virions and identified four virus-encoded structural proteins, VP1 to VP4, as well as one DNA-binding protein of cellular origin. The virion proteins VP1, VP3, and VP4 undergo posttranslational modification by glycosylation, seemingly at multiple sites. VP1 is also proteolytically processed. In addition to the viral DNA-binding protein VP2, we show that viral particles contain the Sulfolobus solfataricus chromatin protein Sso7d. Finally, we provide evidence indicating that SSV1 virions contain glycerol dibiphytanyl glycerol tetraether (GDGT) lipids, resolving a long-standing debate on the presence of lipids within SSV1 virions. A comparison of the contents of lipids isolated from the virus and its host cell suggests that GDGTs are acquired by the virus in a selective manner from the host cytoplasmic membrane, likely during progeny egress. IMPORTANCE Although spindle-shaped viruses represent one of the most prominent viral groups in Archaea, structural data on their virion constituents and architecture still are scarce. The comprehensive biochemical characterization of the hyperthermophilic virus SSV1 presented here brings novel and

  10. Signal transduction across cellular membranes can be mediated by coupling of the clustering of anchored proteins in both leaflets

    NASA Astrophysics Data System (ADS)

    Yue, Tongtao; Zhang, Xianren

    2012-01-01

    One key question in signal transduction is how the signal is relayed from the outer leaflet of a cellular membrane to the inner leaflet. Using a simulation model, a mechanism for the mediation of signal transduction is proposed here in which the coupling between membrane proteins in different leaflets can be achieved by the clustering of anchored proteins, without recruiting transmembrane proteins. Depending on the hydrophobic length of the anchored proteins, three coupling patterns, including face-to-face clustering, interdigitated clustering, and weak-coupled clustering, are observed in this work. This observation provides a possible explanation of how a particular downstream signaling pathway is selected.

  11. An improved toolbox to unravel the plant cellular machinery by tandem affinity purification of Arabidopsis protein complexes.

    PubMed

    Van Leene, Jelle; Eeckhout, Dominique; Cannoot, Bernard; De Winne, Nancy; Persiau, Geert; Van De Slijke, Eveline; Vercruysse, Leen; Dedecker, Maarten; Verkest, Aurine; Vandepoele, Klaas; Martens, Lennart; Witters, Erwin; Gevaert, Kris; De Jaeger, Geert

    2015-01-01

    Tandem affinity purification coupled to mass spectrometry (TAP-MS) is one of the most advanced methods to characterize protein complexes in plants, giving a comprehensive view on the protein-protein interactions (PPIs) of a certain protein of interest (bait). The bait protein is fused to a double affinity tag, which consists of a protein G tag and a streptavidin-binding peptide separated by a very specific protease cleavage site, allowing highly specific protein complex isolation under near-physiological conditions. Implementation of this optimized TAP tag, combined with ultrasensitive MS, means that these experiments can be performed on small amounts (25 mg of total protein) of protein extracts from Arabidopsis cell suspension cultures. It is also possible to use this approach to isolate low abundant protein complexes from Arabidopsis seedlings, thus opening perspectives for the exploration of protein complexes in a plant developmental context. Next to protocols for efficient biomass generation of seedlings (∼7.5 months), we provide detailed protocols for TAP (1 d), and for sample preparation and liquid chromatography-tandem MS (LC-MS/MS; ∼5 d), either from Arabidopsis seedlings or from cell cultures. For the identification of specific co-purifying proteins, we use an extended protein database and filter against a list of nonspecific proteins on the basis of the occurrence of a co-purified protein among 543 TAP experiments. The value of the provided protocols is illustrated through numerous applications described in recent literature.

  12. Successful Interference with Cellular Immune Responses to Immunogenic Proteins Encoded by Recombinant Viral Vectors

    PubMed Central

    Sarukhan, Adelaida; Camugli, Sabine; Gjata, Bernard; von Boehmer, Harald; Danos, Olivier; Jooss, Karin

    2001-01-01

    Vectors derived from the adeno-associated virus (AAV) have been successfully used for the long-term expression of therapeutic genes in animal models and patients. One of the major advantages of these vectors is the absence of deleterious immune responses following gene transfer. However, AAV vectors, when used in vaccination studies, can result in efficient humoral and cellular responses against the transgene product. It is therefore important to understand the factors which influence the establishment of these immune responses in order to design safe and efficient procedures for AAV-based gene therapies. We have compared T-cell activation against a strongly immunogenic protein, the influenza virus hemagglutinin (HA), which is synthesized in skeletal muscle following gene transfer with an adenovirus (Ad) or an AAV vector. In both cases, cellular immune responses resulted in the elimination of transduced muscle fibers within 4 weeks. However, the kinetics of CD4+ T-cell activation were markedly delayed when AAV vectors were used. Upon recombinant Ad (rAd) gene transfer, T cells were activated both by direct transduction of dendritic cells and by cross-presentation of the transgene product, while upon rAAV gene transfer T cells were only activated by the latter mechanism. These results suggested that activation of the immune system by the transgene product following rAAV-mediated gene transfer might be easier to control than that following rAd-mediated gene transfer. Therefore, we tested protocols aimed at interfering with either antigen presentation by blocking the CD40/CD40L pathway or with the T-cell response by inducing transgene-specific tolerance. Long-term expression of the AAV-HA was achieved in both cases, whereas immune responses against Ad-HA could not be prevented. These data clearly underline the importance of understanding the mechanisms by which vector-encoded proteins are recognized by the immune system in order to specifically interfere with them and

  13. 2P2Idb v2: update of a structural database dedicated to orthosteric modulation of protein–protein interactions

    PubMed Central

    Basse, Marie-Jeanne; Betzi, Stéphane; Morelli, Xavier; Roche, Philippe

    2016-01-01

    2P2Idb is a hand-curated structural database dedicated to protein–protein interactions with known small molecule orthosteric modulators. It compiles the structural information related to orthosteric inhibitors and their target [i.e. related 3D structures available in the RCSB Protein Data Bank (PDB)] and provides links to other useful databases. 2P2Idb includes all interactions for which both the protein–protein and protein–inhibitor complexes have been structurally characterized. Since its first release in 2010, the database has grown constantly and the current version contains 27 protein–protein complexes and 274 protein–inhibitor complexes corresponding to 242 unique small molecule inhibitors which represent almost a 5-fold increase compared to the previous version. A number of new data have been added, including new protein–protein complexes, binding affinities, molecular descriptors, precalculated interface parameters and links to other webservers. A new query tool has been implemented to search for inhibitors within the database using standard molecular descriptors. A novel version of the 2P2I-inspector tool has been implemented to calculate a series of physical and chemical parameters of the protein interfaces. Several geometrical parameters including planarity, eccentricity and circularity have been added as well as customizable distance cutoffs. This tool has also been extended to protein–ligand interfaces. The 2P2I database thus represents a wealth of structural source of information for scientists interested in the properties of protein–protein interactions and the design of protein–protein interaction modulators. Database URL: http://2p2idb.cnrs-mrs.fr PMID:26980515

  14. The relationship between redox enzyme activity and electrochemical potential-cellular and mechanistic implications from protein film electrochemistry.

    PubMed

    Gates, Andrew J; Kemp, Gemma L; To, Chun Yip; Mann, James; Marritt, Sophie J; Mayes, Andrew G; Richardson, David J; Butt, Julea N

    2011-05-01

    In protein film electrochemistry a redox protein of interest is studied as an electroactive film adsorbed on an electrode surface. For redox enzymes this configuration allows quantification of the relationship between catalytic activity and electrochemical potential. Considered as a function of enzyme environment, i.e., pH, substrate concentration etc., the activity-potential relationship provides a fingerprint of activity unique to a given enzyme. Here we consider the nature of the activity-potential relationship in terms of both its cellular impact and its origin in the structure and catalytic mechanism of the enzyme. We propose that the activity-potential relationship of a redox enzyme is tuned to facilitate cellular function and highlight opportunities to test this hypothesis through computational, structural, biochemical and cellular studies.

  15. The relationship between redox enzyme activity and electrochemical potential-cellular and mechanistic implications from protein film electrochemistry.

    PubMed

    Gates, Andrew J; Kemp, Gemma L; To, Chun Yip; Mann, James; Marritt, Sophie J; Mayes, Andrew G; Richardson, David J; Butt, Julea N

    2011-05-01

    In protein film electrochemistry a redox protein of interest is studied as an electroactive film adsorbed on an electrode surface. For redox enzymes this configuration allows quantification of the relationship between catalytic activity and electrochemical potential. Considered as a function of enzyme environment, i.e., pH, substrate concentration etc., the activity-potential relationship provides a fingerprint of activity unique to a given enzyme. Here we consider the nature of the activity-potential relationship in terms of both its cellular impact and its origin in the structure and catalytic mechanism of the enzyme. We propose that the activity-potential relationship of a redox enzyme is tuned to facilitate cellular function and highlight opportunities to test this hypothesis through computational, structural, biochemical and cellular studies. PMID:21423952

  16. Resolution of the cellular proteome of the nucleocapsid protein from a highly pathogenic isolate of porcine reproductive and respiratory syndrome virus identifies PARP-1 as a cellular target whose interaction is critical for virus biology.

    PubMed

    Liu, Long; Lear, Zoe; Hughes, David J; Wu, Weining; Zhou, En-min; Whitehouse, Adrian; Chen, Hongying; Hiscox, Julian A

    2015-03-23

    Porcine reproductive and respiratory syndrome virus (PRRSV) is a major threat to the swine industry and food security worldwide. The nucleocapsid (N) protein is a major structural protein of PRRSV. The primary function of this protein is to encapsidate the viral RNA genome, and it is also thought to participate in the modulation of host cell biology and recruitment of cellular factors to facilitate virus infection. In order to the better understand these latter roles the cellular interactome of PRRSV N protein was defined using label free quantitative proteomics. This identified several cellular factors that could interact with the N protein including poly [ADP-ribose] polymerase 1 (PARP-1), a cellular protein, which can add adenosine diphosphate ribose to a protein. Use of the PARP-1 small molecule inhibitor, 3-AB, in PRRSV infected cells demonstrated that PARP-1 was required and acted as an enhancer factor for virus biology. Serial growth of PRRSV in different concentrations of 3-AB did not yield viruses that were able to grow with wild type kinetics, suggesting that by targeting a cellular protein crucial for virus biology, resistant phenotypes did not emerge. This study provides further evidence that cellular proteins, which are critical for virus biology, can also be targeted to ablate virus growth and provide a high barrier for the emergence of drug resistance.

  17. PPD v1.0--an integrated, web-accessible database of experimentally determined protein pKa values.

    PubMed

    Toseland, Christopher P; McSparron, Helen; Davies, Matthew N; Flower, Darren R

    2006-01-01

    The Protein pK(a) Database (PPD) v1.0 provides a compendium of protein residue-specific ionization equilibria (pK(a) values), as collated from the primary literature, in the form of a web-accessible postgreSQL relational database. Ionizable residues play key roles in the molecular mechanisms that underlie many biological phenomena, including protein folding and enzyme catalysis. The PPD serves as a general protein pK(a) archive and as a source of data that allows for the development and improvement of pK(a) prediction systems. The database is accessed through an HTML interface, which offers two fast, efficient search methods: an amino acid-based query and a Basic Local Alignment Search Tool search. Entries also give details of experimental techniques and links to other key databases, such as National Center for Biotechnology Information and the Protein Data Bank, providing the user with considerable background information. The database can be found at the following URL: http://www.jenner.ac.uk/PPD. PMID:16381845

  18. A general path for large-scale solubilization of cellular proteins: From membrane receptors to multiprotein complexes

    PubMed Central

    Pullara, Filippo; Guerrero-Santoro, Jennifer; Calero, Monica; Zhang, Qiangmin; Peng, Ye; Spåhr, Henrik; Kornberg, Guy L.; Cusimano, Antonella; Stevenson, Hilary P.; Santamaria-Suarez, Hugo; Reynolds, Shelley L.; Brown, Ian S.; Monga, Satdarshan P.S.; Van Houten, Bennett; Rapić-Otrin, Vesna; Calero, Guillermo; Levine, Arthur S.

    2014-01-01

    Expression of recombinant proteins in bacterial or eukaryotic systems often results in aggregation rendering them unavailable for biochemical or structural studies. Protein aggregation is a costly problem for biomedical research. It forces research laboratories and the biomedical industry to search for alternative, more soluble, non-human proteins and limits the number of potential “druggable” targets. In this study we present a highly reproducible protocol that introduces the systematic use of an extensive number of detergents to solubilize aggregated proteins expressed in bacterial and eukaryotic systems. We validate the usefulness of this protocol by solubilizing traditionally difficult human protein targets to milligram quantities and confirm their biological activity. We use this method to solubilize monomeric or multimeric components of multi-protein complexes and demonstrate its efficacy to reconstitute large cellular machines. This protocol works equally well on cytosolic, nuclear and membrane proteins and can be easily adapted to a high throughput format. PMID:23137940

  19. Nuclear protein accumulation in cellular senescence and organismal aging revealed with a novel single-cell resolution fluorescence microscopy assay

    PubMed Central

    De Cecco, Marco; Jeyapalan, Jessie; Zhao, Xiaoai; Tamamori-Adachi, Mimi; Sedivy, John M.

    2011-01-01

    Replicative cellular senescence was discovered some 50 years ago. The phenotypes of senescent cells have been investigated extensively in cell culture, and found to affect essentially all aspects of cellular physiology. The relevance of cellular senescence in the context of age-associated pathologies as well as normal aging is a topic of active and ongoing interest. Considerable effort has been devoted to biomarker discovery to enable the microscopic detection of single senescent cells in tissues. One characteristic of senescent cells documented very early in cell culture studies was an increase in cell size and total protein content, but whether this occurs in vivo is not known. A limiting factor for studies of protein content and localization has been the lack of suitable fluorescence microscopy tools. We have developed an easy and flexible method, based on the merocyanine dye known as NanoOrange, to visualize and quantitatively measure total protein levels by high resolution fluorescence microscopy. NanoOrange staining can be combined with antibody-based immunofluorescence, thus providing both specific target and total protein information in the same specimen. These methods are optimally combined with automated image analysis platforms for high throughput analysis. We document here increasing protein content and density in nuclei of senescent human and mouse fibroblasts in vitro, and in liver nuclei of aged mice in vivo. Additionally, in aged liver nuclei NanoOrange revealed protein-dense foci that colocalize with centromeric heterochromatin. PMID:22006542

  20. CancerResource—updated database of cancer-relevant proteins, mutations and interacting drugs

    PubMed Central

    Gohlke, Bjoern-Oliver; Nickel, Janette; Otto, Raik; Dunkel, Mathias; Preissner, Robert

    2016-01-01

    Here, we present an updated version of CancerResource, freely available without registration at http://bioinformatics.charite.de/care. With upcoming information on target expression and mutations in patients’ tumors, the need for systems supporting decisions on individual therapy is growing. This knowledge is based on numerous, experimentally validated drug-target interactions and supporting analyses such as measuring changes in gene expression using microarrays and HTS-efforts on cell lines. To enable a better overview about similar drug-target data and supporting information, a series of novel information connections are established and made available as described in the following. CancerResource contains about 91 000 drug-target relations, more than 2000 cancer cell lines and drug sensitivity data for about 50 000 drugs. CancerResource enables the capability of uploading external expression and mutation data and comparing them to the database's cell lines. Target genes and compounds are projected onto cancer-related pathways to get a better overview about how drug-target interactions benefit the treatment of cancer. Features like cellular fingerprints comprising of mutations, expression values and drug-sensitivity data can promote the understanding of genotype to drug sensitivity associations. Ultimately, these profiles can also be used to determine the most effective drug treatment for a cancer cell line most similar to a patient's tumor cells. PMID:26590406

  1. deconSTRUCT: general purpose protein database search on the substructure level.

    PubMed

    Zhang, Zong Hong; Bharatham, Kavitha; Sherman, Westley A; Mihalek, Ivana

    2010-07-01

    deconSTRUCT webserver offers an interface to a protein database search engine, usable for a general purpose detection of similar protein (sub)structures. Initially, it deconstructs the query structure into its secondary structure elements (SSEs) and reassembles the match to the target by requiring a (tunable) degree of similarity in the direction and sequential order of SSEs. Hierarchical organization and judicious use of the information about protein structure enables deconSTRUCT to achieve the sensitivity and specificity of the established search engines at orders of magnitude increased speed, without tying up irretrievably the substructure information in the form of a hash. In a post-processing step, a match on the level of the backbone atoms is constructed. The results presented to the user consist of the list of the matched SSEs, the transformation matrix for rigid superposition of the structures and several ways of visualization, both downloadable and implemented as a web-browser plug-in. The server is available at http://epsf.bmad.bii.a-star.edu.sg/struct_server.html.

  2. Cellular localization of the Ca2+ binding TCH3 protein of Arabidopsis

    NASA Technical Reports Server (NTRS)

    Antosiewicz, D. M.; Polisensky, D. H.; Braam, J.

    1995-01-01

    TCH3 is an Arabidopsis touch (TCH) gene isolated as a result of its strong and rapid upregulation in response to mechanical stimuli, such as touch and wind. TCH3 encodes an unusual calcium ion-binding protein that is closely related to calmodulin but has the potential to bind six calcium ions. Here it is shown that TCH3 shows a restricted pattern of accumulation during Arabidopsis vegetative development. These data provide insight into the endogenous signals that may regulate TCH3 expression and the sites of TCH3 action. TCH3 is abundant in the shoot apical meristem, vascular tissue, the root columella and pericycle cells that give rise to lateral roots. In addition, TCH3 accumulation in cells of developing shoots and roots closely correlates with the process of cellular expansion. Following wind stimulation, TCH3 becomes more abundant in specific regions including the branchpoints of leaf primordia and stipules, pith parenchyma, and the vascular tissue. The consequences of TCH3 upregulation by wind are therefore spatially restricted and TCH3 may function at these sites to modify cell or tissue characteristics following mechanical stimulation. Because TCH3 accumulates specifically in cells and tissues that are thought to be under the influence of auxin, auxin levels may regulate TCH3 expression during development. TCH3 is upregulated in response to low levels of exogenous indole-3-acetic acid (IAA), but not by inactive auxin-related compounds. These results suggest that TCH3 protein may play roles in mediating physiological responses to auxin and mechanical environmental stimuli.

  3. Transglutaminase type 2-dependent selective recruitment of proteins into exosomes under stressful cellular conditions.

    PubMed

    Diaz-Hidalgo, Laura; Altuntas, Sara; Rossin, Federica; D'Eletto, Manuela; Marsella, Claudia; Farrace, Maria Grazia; Falasca, Laura; Antonioli, Manuela; Fimia, Gian Maria; Piacentini, Mauro

    2016-08-01

    Numerous studies are revealing a role of exosomes in intercellular communication, and growing evidence indicates an important function for these vesicles in the progression and pathogenesis of cancer and neurodegenerative diseases. However, the biogenesis process of exosomes is still unclear. Tissue transglutaminase (TG2) is a multifunctional enzyme with different subcellular localizations. Particularly, under stressful conditions, the enzyme has been also detected in the extracellular matrix, but the mechanism(s) by which TG2 is released outside the cells requires further investigation. Therefore, the goal of the present study was to determine whether exosomes might be a vehicle for TG2 to reach the extracellular space, and whether TG2 could be involved in exosomes biogenesis. To address this issue, we isolated and characterized exosomes derived from cells either expressing or not TG2, under stressful conditions (i.e. proteasome impairment or expressing a mutated form of huntingtin (mHtt) containing 84 polyglutamine repeats). Our results show that TG2 is present in the exosomes only upon proteasome blockade, a condition in which TG2 interacts with TSG101 and ALIX, two key proteins involved in exosome biogenesis. Interestingly, we found that TG2 favours the assembly of a protein complex including mHtt, ALIX, TSG101 and BAG3, a co-chaperone involved in the clearance of mHtt. The formation of this complex is paralleled by the selective recruitment of mHtt and BAG3 in the exosomes derived from TG2 proficient cells only. Overall, our data indicate that TG2 is an important player in the biogenesis of exosomes controlling the selectivity of their cargo under stressful cellular conditions. In addition, these vesicles represent the way by which cells can release TG2 into the extracellular space under proteostasis impairment.

  4. Secretory proteins characteristic of environmental changes in cellular signal transduction: Expression in oral fluid

    NASA Astrophysics Data System (ADS)

    Mednieks, M. I.; Burke, J. C.; Sivakumar, T. P.; Hand, A. R.; Grindeland, R. E.

    2000-01-01

    Past studies have shown that both hypo- and hyper-gravity have significant consequences on a variety of tissues and organ systems. It is not known if the effects of environmental stimuli such as altered gravity are beneficial or detrimental, and if the effects can be prevented or reversed. Animal experiments from the Space Lab and Cosmos missions indicate that events that are mediated by cyclic AMP, such as cellular responses to catecholamine and peptide hormone action, are significantly altered in a number of tissues as a consequence of space flight. A secretory cyclic AMP-receptor protein (cARP), is present in saliva, and can serve as an indicator of individual responses to physiologic and environmental stress. Animal experiments have shown that the hypergravity component of space flight is a significant stress factor. In humans, cARP levels in each individual are constant under normal conditions, but elevated after acute stress. Additionally, the levels of cARP in secreted saliva can be compared to those in gingival crevicular fluid (GCF), which reflects the protein composition of serum. The ratio of cARP in saliva to that in GCF can be used as a measure of basal compared to hyper-or hypo-gravity values. An ultimate goal is to test hyper and zero G responses in human saliva to determine if cARP is a suitable index of acute and chronic stress. A miniaturized test kit for saliva collection has been designed. Samples can be collected and stored till analyses are carried out that will distinguish the effects of increased gravity from those of one and zero G. Such tests can serve as an individualized monitoring system for physiologic responses either in space or on earth. .

  5. High expression of cellular retinol binding protein-1 in lung adenocarcinoma is associated with poor prognosis

    PubMed Central

    Doldo, Elena; Costanza, Gaetana; Ferlosio, Amedeo; Pompeo, Eugenio; Agostinelli, Sara; Bellezza, Guido; Mazzaglia, Donatella; Giunta, Alessandro; Sidoni, Angelo; Orlandi, Augusto

    2015-01-01

    Purpose Adenocarcinoma, the most common non-small cell lung cancer is a leading cause of death worldwide, with a low overall survival (OS) despite increasing attempts to achieve an early diagnosis and accomplish surgical and multimodality treatment strategies. Cellular retinol binding protein-1 (CRBP-1) regulates retinol bioavailability and cell differentiation, but its role in lung cancerogenesis remains uncertain. Experimental design CRBP-1 expression, clinical outcome and other prognostic factors were investigated in 167 lung adenocarcinoma patients. CRBP-1 expression was evaluated by immunohistochemistry of tissue microarray sections, gene copy number analysis and tumor methylation specific PCR. Effects of CRBP-1 expression on proliferation/apoptosis gene array, protein and transcripts were investigated in transfected A549 lung adenocarcinoma cells. Results CRBP-1High expression was observed in 62.3% of adenocarcinomas and correlated with increased tumor grade and reduced OS as an independent prognostic factor. CRBP-1 gene copy gain also associated with tumor CRBP-1High status and dedifferentiation. CRBP-1-transfected (CRBP-1+) A549 grew more than CRBP-1− A549 cells. At >1μM concentrations, all trans-retinoic acid and retinol reduced viability more in CRBP-1+ than in CRBP-1− A549 cells. CRBP-1+ A549 cells showed up-regulated RARα/ RXRα and proliferative and transcriptional genes including pAkt, pEGFR, pErk1/2, creb1 and c-jun, whereas RARβ and p53 were strongly down-regulated; pAkt/pErk/ pEGFR inhibitors counteracted proliferative advantage and increased RARα/RXRα, c-jun and CD44 expression in CRBP-1+ A549 cells. Conclusion CRBP-1High expression in lung adenocarcinoma correlated with increased tumor grade and reduced OS, likely through increased Akt/Erk/EGFR-mediated cell proliferation and differentiation. CRBP-1High expression can be considered an additional marker of poor prognosis in lung adenocarcinoma patients. PMID:26807202

  6. Transglutaminase type 2-dependent selective recruitment of proteins into exosomes under stressful cellular conditions.

    PubMed

    Diaz-Hidalgo, Laura; Altuntas, Sara; Rossin, Federica; D'Eletto, Manuela; Marsella, Claudia; Farrace, Maria Grazia; Falasca, Laura; Antonioli, Manuela; Fimia, Gian Maria; Piacentini, Mauro

    2016-08-01

    Numerous studies are revealing a role of exosomes in intercellular communication, and growing evidence indicates an important function for these vesicles in the progression and pathogenesis of cancer and neurodegenerative diseases. However, the biogenesis process of exosomes is still unclear. Tissue transglutaminase (TG2) is a multifunctional enzyme with different subcellular localizations. Particularly, under stressful conditions, the enzyme has been also detected in the extracellular matrix, but the mechanism(s) by which TG2 is released outside the cells requires further investigation. Therefore, the goal of the present study was to determine whether exosomes might be a vehicle for TG2 to reach the extracellular space, and whether TG2 could be involved in exosomes biogenesis. To address this issue, we isolated and characterized exosomes derived from cells either expressing or not TG2, under stressful conditions (i.e. proteasome impairment or expressing a mutated form of huntingtin (mHtt) containing 84 polyglutamine repeats). Our results show that TG2 is present in the exosomes only upon proteasome blockade, a condition in which TG2 interacts with TSG101 and ALIX, two key proteins involved in exosome biogenesis. Interestingly, we found that TG2 favours the assembly of a protein complex including mHtt, ALIX, TSG101 and BAG3, a co-chaperone involved in the clearance of mHtt. The formation of this complex is paralleled by the selective recruitment of mHtt and BAG3 in the exosomes derived from TG2 proficient cells only. Overall, our data indicate that TG2 is an important player in the biogenesis of exosomes controlling the selectivity of their cargo under stressful cellular conditions. In addition, these vesicles represent the way by which cells can release TG2 into the extracellular space under proteostasis impairment. PMID:27169926

  7. LepChorionDB, a database of Lepidopteran chorion proteins and a set of tools useful for the identification of chorion proteins in Lepidopteran proteomes.

    PubMed

    Giannopoulos, Nikolaos G; Michalopoulos, Ioannis; Papandreou, Nikos C; Malatras, Apostolos; Iconomidou, Vassiliki A; Hamodrakas, Stavros J

    2013-02-01

    Chorion proteins of Lepidoptera have a tripartite structure, which consists of a central domain and two, more variable, flanking arms. The central domain is highly conserved and it is used for the classification of chorion proteins into two major classes, A and B. Annotated and unreviewed Lepidopteran chorion protein sequences are available in various databases. A database, named LepChorionDB, was constructed by searching 5 different protein databases using class A and B central domain-specific profile Hidden Markov Models (pHMMs), developed in this work. A total of 413 Lepidopteran chorion proteins from 9 moths and 1 butterfly species were retrieved. These data were enriched and organised in order to populate LepChorionDB, the first relational database, available on the web, containing Lepidopteran chorion proteins grouped in A and B classes. LepChorionDB may provide insights in future functional and evolutionary studies of Lepidopteran chorion proteins and thus, it will be a useful tool for the Lepidopteran scientific community and Lepidopteran genome annotators, since it also provides access to the two pHMMs developed in this work, which may be used to discriminate A and B class chorion proteins. LepChorionDB is freely available at http://bioinformatics.biol.uoa.gr/LepChorionDB.

  8. Sialic Acid within the Glycosylphosphatidylinositol Anchor Targets the Cellular Prion Protein to Synapses.

    PubMed

    Bate, Clive; Nolan, William; McHale-Owen, Harriet; Williams, Alun

    2016-08-12

    Although the cellular prion protein (PrP(C)) is concentrated at synapses, the factors that target PrP(C) to synapses are not understood. Here we demonstrate that exogenous PrP(C) was rapidly targeted to synapses in recipient neurons derived from Prnp knock-out((0/0)) mice. The targeting of PrP(C) to synapses was dependent upon both neuronal cholesterol concentrations and the lipid and glycan composition of its glycosylphosphatidylinositol (GPI) anchor. Thus, the removal of either an acyl chain or sialic acid from the GPI anchor reduced the targeting of PrP(C) to synapses. Isolated GPIs (derived from PrP(C)) were also targeted to synapses, as was IgG conjugated to these GPIs. The removal of sialic acid from GPIs prevented the targeting of either the isolated GPIs or the IgG-GPI conjugate to synapses. Competition studies showed that pretreatment with sialylated GPIs prevented the targeting of PrP(C) to synapses. These results are consistent with the hypothesis that the sialylated GPI anchor attached to PrP(C) acts as a synapse homing signal. PMID:27325697

  9. A cationic tetrapyrrole inhibits toxic activities of the cellular prion protein

    PubMed Central

    Massignan, Tania; Cimini, Sara; Stincardini, Claudia; Cerovic, Milica; Vanni, Ilaria; Elezgarai, Saioa R.; Moreno, Jorge; Stravalaci, Matteo; Negro, Alessandro; Sangiovanni, Valeria; Restelli, Elena; Riccardi, Geraldina; Gobbi, Marco; Castilla, Joaquín; Borsello, Tiziana; Nonno, Romolo; Biasini, Emiliano

    2016-01-01

    Prion diseases are rare neurodegenerative conditions associated with the conformational conversion of the cellular prion protein (PrPC) into PrPSc, a self-replicating isoform (prion) that accumulates in the central nervous system of affected individuals. The structure of PrPSc is poorly defined, and likely to be heterogeneous, as suggested by the existence of different prion strains. The latter represents a relevant problem for therapy in prion diseases, as some potent anti-prion compounds have shown strain-specificity. Designing therapeutics that target PrPC may provide an opportunity to overcome these problems. PrPC ligands may theoretically inhibit the replication of multiple prion strains, by acting on the common substrate of any prion replication reaction. Here, we characterized the properties of a cationic tetrapyrrole [Fe(III)-TMPyP], which was previously shown to bind PrPC, and inhibit the replication of a mouse prion strain. We report that the compound is active against multiple prion strains in vitro and in cells. Interestingly, we also find that Fe(III)-TMPyP inhibits several PrPC-related toxic activities, including the channel-forming ability of a PrP mutant, and the PrPC-dependent synaptotoxicity of amyloid-β (Aβ) oligomers, which are associated with Alzheimer’s Disease. These results demonstrate that molecules binding to PrPC may produce a dual effect of blocking prion replication and inhibiting PrPC-mediated toxicity. PMID:26976106

  10. Protein ADP-ribosylation and the cellular response to DNA strand breaks.

    PubMed

    Caldecott, K W

    2014-07-01

    DNA strand breaks arise continuously in cells and can lead to chromosome rearrangements and genome instability or cell death. The commonest DNA breaks are DNA single-strand breaks, which arise at a frequency of tens-of-thousands per cell each day and which can block the progression of RNA/DNA polymerases and disrupt gene transcription and genome duplication. If not rapidly repaired, SSBs can be converted into DNA double-strand breaks (DSBs) during genome duplication, eliciting a complex series of DNA damage responses that attempt to protect cells from irreversible replication fork collapse. DSBs are the most cytotoxic and clastogenic type of DNA breaks, and can also arise independently of DNA replication, albeit at a frequency several orders of magnitude lower than SSBs. Here, I discuss the evidence that DNA single- and double -strand break repair pathways, and cellular tolerance mechanisms for protecting replication forks during genome duplication, utilize signalling by protein ADP-ribosyltransferases to protect cells from the harmful impact of DNA strand breakage.

  11. Promoter analysis of the membrane protein gp64 gene of the cellular slime mold Polysphondylium pallidum.

    PubMed

    Takaoka, N; Fukuzawa, M; Saito, T; Sakaitani, T; Ochiai, H

    1999-10-28

    We cloned a genomic fragment of the membrane protein gp64 gene of the cellular slime mold Polysphondylium pallidum by inverse PCR. Primer extension analysis identified a major transcription start site 65 bp upstream of the translation start codon. The promoter region of the gp64 gene contains sequences homologous to a TATA box at position -47 to -37 and to an initiator (Inr, PyPyCAPyPyPyPy) at position -3 to +5 from the transcription start site. Successively truncated segments of the promoter were tested for their ability to drive expression of the beta-galactosidase reporter gene in transformed cells; also the difference in activity between growth conditions was compared. The results indicated that there are two positive vegetative regulatory elements extending between -187 and -62 bp from the transcription start site of the gp64 promoter; also their activity was two to three times higher in the cells grown with bacteria in shaken suspension than in the cells grown in an axenic medium. PMID:10542319

  12. The NS1 Protein of Influenza A Virus Interacts with Cellular Processing Bodies and Stress Granules through RNA-Associated Protein 55 (RAP55) during Virus Infection

    PubMed Central

    Mok, Bobo Wing-Yee; Song, Wenjun; Wang, Pui; Tai, Hung; Chen, Yixin; Zheng, Min; Wen, Xi; Lau, Siu-Ying; Wu, Wai Lan; Matsumoto, Ken

    2012-01-01

    The nonstructural protein (NS1) of influenza A virus performs multiple functions in the virus life cycle. Proteomic screening for cellular proteins which interact with NS1 identified the cellular protein RAP55, which is one of the components of cellular processing bodies (P-bodies) and stress granules. To verify whether NS1 interacts with cellular P-bodies, interactions between NS1, RAP55, and other P-body-associated proteins (Ago1, Ago2, and DCP1a) were confirmed using coimmunoprecipitation and cellular colocalization assays. Overexpression of RAP55 induced RAP55-associated stress granule formation and suppressed virus replication. Knockdown of RAP55 with small interfering RNA (siRNA) or expression of a dominant-negative mutant RAP55 protein with defective interaction with P-bodies blocked NS1 colocalization to P-bodies in cells. Expression of NS1 inhibited RAP55 expression and formation of RAP55-associated P-bodies/stress granules. The viral nucleoprotein (NP) was found to be targeted to stress granules in the absence of NS1 but localized to P-bodies when NS1 was coexpressed. Restriction of virus replication via P-bodies occurred in the early phases of infection, as the number of RAP55-associated P-bodies in cells diminished over the course of virus infection. NS1 interaction with RAP55-associated P-bodies/stress granules was associated with RNA binding and mediated via a protein kinase R (PKR)-interacting viral element. Mutations introduced into either RNA binding sites (R38 and K41) or PKR interaction sites (I123, M124, K126, and N127) caused NS1 proteins to lose the ability to interact with RAP55 and to inhibit stress granules. These results reveal an interplay between virus and host during virus replication in which NP is targeted to P-bodies/stress granules while NS1 counteracts this host restriction mechanism. PMID:22973032

  13. Human Gene and Protein Database (HGPD): a novel database presenting a large quantity of experiment-based results in human proteomics.

    PubMed

    Maruyama, Yukio; Wakamatsu, Ai; Kawamura, Yoshifumi; Kimura, Kouichi; Yamamoto, Jun-ichi; Nishikawa, Tetsuo; Kisu, Yasutomo; Sugano, Sumio; Goshima, Naoki; Isogai, Takao; Nomura, Nobuo

    2009-01-01

    Completion of human genome sequencing has greatly accelerated functional genomic research. Full-length cDNA clones are essential experimental tools for functional analysis of human genes. In one of the projects of the New Energy and Industrial Technology Development Organization (NEDO) in Japan, the full-length human cDNA sequencing project (FLJ project), nucleotide sequences of approximately 30 000 human cDNA clones have been analyzed. The Gateway system is a versatile framework to construct a variety of expression clones for various experiments. We have constructed 33 275 human Gateway entry clones from full-length cDNAs, representing to our knowledge the largest collection in the world. Utilizing these clones with a highly efficient cell-free protein synthesis system based on wheat germ extract, we have systematically and comprehensively produced and analyzed human proteins in vitro. Sequence information for both amino acids and nucleotides of open reading frames of cDNAs cloned into Gateway entry clones and in vitro expression data using those clones can be retrieved from the Human Gene and Protein Database (HGPD, http://www.HGPD.jp). HGPD is a unique database that stores the information of a set of human Gateway entry clones and protein expression data and helps the user to search the Gateway entry clones.

  14. Induction of Cellular Senescence by Insulin-like Growth Factor Binding Protein-5 through a p53-dependent Mechanism

    PubMed Central

    Kim, Kwang Seok; Seu, Young Bae; Baek, Suk-Hwan; Kim, Mi Jin; Kim, Keuk Jun; Kim, Jung Hye

    2007-01-01

    The insulin-like growth factor (IGF) signaling pathway plays a crucial role in the regulation of cell growth, differentiation, apoptosis, and aging. IGF-binding proteins (IGFBPs) are important members of the IGF axis. IGFBP-5 is up-regulated during cellular senescence in human dermal fibroblasts and endothelial cells, but the function of IGFBP-5 in cellular senescence is unknown. Here we show that IGFBP-5 plays important roles in the regulation of cellular senescence. Knockdown of IGFBP-5 in old human umbilical endothelial cells (HUVECs) with IGFBP-5 micro-RNA lentivirus caused partial reduction of a variety of senescent phenotypes, such as changes in cell morphology, increases in cell proliferation, and decreases in senescence-associated β-galactosidase (SA-β-gal) staining. In addition, treatment with IGFBP-5 protein or up-regulation of IGFBP-5 in young cells accelerates cellular senescence, as confirmed by cell proliferation and SA-β-gal staining. Premature senescence induced by IGFBP-5 up-regulation in young cells was rescued by knockdown of p53, but not by knockdown of p16. Furthermore, atherosclerotic arteries exhibited strong IGFBP-5–positive staining along intimal plaques. These results suggest that IGFBP-5 plays a role in the regulation of cellular senescence via a p53-dependent pathway and in aging-associated vascular diseases. PMID:17804819

  15. PANTHER: a browsable database of gene products organized by biological function, using curated protein family and subfamily classification.

    PubMed

    Thomas, Paul D; Kejariwal, Anish; Campbell, Michael J; Mi, Huaiyu; Diemer, Karen; Guo, Nan; Ladunga, Istvan; Ulitsky-Lazareva, Betty; Muruganujan, Anushya; Rabkin, Steven; Vandergriff, Jody A; Doremieux, Olivier

    2003-01-01

    The PANTHER database was designed for high-throughput analysis of protein sequences. One of the key features is a simplified ontology of protein function, which allows browsing of the database by biological functions. Biologist curators have associated the ontology terms with groups of protein sequences rather than individual sequences. Statistical models (Hidden Markov Models, or HMMs) are built from each of these groups. The advantage of this approach is that new sequences can be automatically classified as they become available. To ensure accurate functional classification, HMMs are constructed not only for families, but also for functionally distinct subfamilies. Multiple sequence alignments and phylogenetic trees, including curator-assigned information, are available for each family. The current version of the PANTHER database includes training sequences from all organisms in the GenBank non-redundant protein database, and the HMMs have been used to classify gene products across the entire genomes of human, and Drosophila melanogaster. The ontology terms and protein families and subfamilies, as well as Drosophila gene c;assifications, can be browsed and searched for free. Due to outstanding contractual obligations, access to human gene classifications and to protein family trees and multiple sequence alignments will temporarily require a nominal registration fee. PANTHER is publicly available on the web at http://panther.celera.com.

  16. HSV-1 nucleocapsid egress mediated by UL31 in association with UL34 is impeded by cellular transmembrane protein 140

    SciTech Connect

    Guan, Ying; Guo, Lei; Yang, Erxia; Liao, Yun; Liu, Longding; Che, Yanchun; Zhang, Ying; Wang, Lichun; Wang, Jingjing; Li, Qihan

    2014-09-15

    During HSV-1 infection, the viral UL31 protein forms a complex with the UL34 protein at the cellular nuclear membrane, where both proteins play important roles in the envelopment of viral nucleocapsids and their egress into the cytoplasm. To characterize the mechanism of HSV-1 nucleocapsid egress, we screened host proteins to identify proteins that interacted with UL31 via yeast two-hybrid analysis. Transmembrane protein 140 (TMEM140), was identified and confirmed to bind to and co-localize with UL31 during viral infection. Further studies indicated that TMEM140 inhibits HSV-1 proliferation through selectively blocking viral nucleocapsid egress during the viral assembly process. The blockage function of TMEM140 is mediated by impeding the formation of the UL31–UL34 complex due to competitive binding to UL31. Collectively, these data suggest the essentiality of the UL31–UL34 interaction in the viral nucleocapsid egress process and provide a new anti-HSV-1 strategy in viral assembly process of nucleocapsid egress. - Highlights: • Cellular TMEM140 protein interacts with HSV-1 UL31 protein during viral infection. • Increasing expression of TMEM140 leads to inhibition of HSV-1 proliferation. • Increasing expression of TMEM140 blocks HSV-1 nucleocapsid egress process. • Binding to UL31 of TMEM140 impedes formation of HSV-1 UL31–UL34 complex.

  17. Differential rates of protein folding and cellular trafficking for the Hendra virus F and G proteins: implications for F-G complex formation.

    PubMed

    Whitman, Shannon D; Smith, Everett Clinton; Dutch, Rebecca Ellis

    2009-09-01

    Hendra virus F protein-promoted membrane fusion requires the presence of the viral attachment protein, G. However, events leading to the association of these glycoproteins remain unclear. Results presented here demonstrate that Hendra virus G undergoes slower secretory pathway trafficking than is observed for Hendra virus F. This slowed trafficking is not dependent on the G protein cytoplasmic tail, the presence of the G receptor ephrin B2, or interaction with other viral proteins. Instead, Hendra virus G was found to undergo intrinsically slow oligomerization within the endoplasmic reticulum. These results suggest that the critical F-G interactions occur only after the initial steps of synthesis and cellular transport.

  18. RADARS, a bioinformatics solution that automates proteome mass spectral analysis, optimises protein identification, and archives data in a relational database.

    PubMed

    Field, Helen I; Fenyö, David; Beavis, Ronald C

    2002-01-01

    RADARS, a rapid, automated, data archiving and retrieval software system for high-throughput proteomic mass spectral data processing and storage, is described. The majority of mass spectrometer data files are compatible with RADARS, for consistent processing. The system automatically takes unprocessed data files, identifies proteins via in silico database searching, then stores the processed data and search results in a relational database suitable for customized reporting. The system is robust, used in 24/7 operation, accessible to multiple users of an intranet through a web browser, may be monitored by Virtual Private Network, and is secure. RADARS is scalable for use on one or many computers, and is suited to multiple processor systems. It can incorporate any local database in FASTA format, and can search protein and DNA databases online. A key feature is a suite of visualisation tools (many available gratis), allowing facile manipulation of spectra, by hand annotation, reanalysis, and access to all procedures. We also described the use of Sonar MS/MS, a novel, rapid search engine requiring 40 MB RAM per process for searches against a genomic or EST database translated in all six reading frames. RADARS reduces the cost of analysis by its efficient algorithms: Sonar MS/MS can identifiy proteins without accurate knowledge of the parent ion mass and without protein tags. Statistical scoring methods provide close-to-expert accuracy and brings robust data analysis to the non-expert user.

  19. Exploiting the multiplexing capabilities of tandem mass tags for high-throughput estimation of cellular protein abundances by mass spectrometry.

    PubMed

    Ahrné, Erik; Martinez-Segura, Amalia; Syed, Afzal Pasha; Vina-Vilaseca, Arnau; Gruber, Andreas J; Marguerat, Samuel; Schmidt, Alexander

    2015-09-01

    The generation of dynamic models of biological processes critically depends on the determination of precise cellular concentrations of biomolecules. Measurements of system-wide absolute protein levels are particularly valuable information in systems biology. Recently, mass spectrometry based proteomics approaches have been developed to estimate protein concentrations on a proteome-wide scale. However, for very complex proteomes, fractionation steps are required, increasing samples number and instrument analysis time. As a result, the number of full proteomes that can be routinely analyzed is limited. Here we combined absolute quantification strategies with the multiplexing capabilities of isobaric tandem mass tags to determine cellular protein abundances in a high throughput and proteome-wide scale even for highly complex biological systems, such as a whole human cell line. We generated two independent data sets to demonstrate the power of the approach regarding sample throughput, dynamic range, quantitative precision and accuracy as well as proteome coverage in comparison to existing mass spectrometry based strategies.

  20. Molecular Mechanism of Wide Photoabsorption Spectral Shifts of Color Variants of Human Cellular Retinol Binding Protein II.

    PubMed

    Cheng, Cheng; Kamiya, Motoshi; Uchida, Yoshihiro; Hayashi, Shigehiko

    2015-10-21

    Color variants of human cellular retinol binding protein II (hCRBPII) created by protein engineering were recently shown to exhibit anomalously wide photoabsorption spectral shifts over ∼200 nm across the visible region. The remarkable phenomenon provides a unique opportunity to gain insight into the molecular basis of the color tuning of retinal binding proteins for understanding of color vision as well as for engineering of novel color variants of retinal binding photoreceptor proteins employed in optogenetics. Here, we report a theoretical investigation of the molecular mechanism underlying the anomalously wide spectral shifts of the color variants of hCRBPII. Computational modeling of the color variants with hybrid molecular simulations of free energy geometry optimization succeeded in reproducing the experimentally observed wide spectral shifts, and revealed that protein flexibility, through which the active site structure of the protein and bound water molecules is altered by remote mutations, plays a significant role in inducing the large spectral shifts.

  1. hCLE/C14orf166, a cellular protein required for viral replication, is incorporated into influenza virus particles

    PubMed Central

    Rodriguez-Frandsen, Ariel; de Lucas, Susana; Pérez-González, Alicia; Pérez-Cidoncha, Maite; Roldan-Gomendio, Alejandro; Pazo, Alejandra; Marcos-Villar, Laura; Landeras-Bueno, Sara; Ortín, Juan; Nieto, Amelia

    2016-01-01

    The influenza A virus polymerase associates with a number of cellular transcription-related factors, including the RNA polymerase II (RNAP II). We previously described that the cellular protein hCLE/C14orf166 interacts with and stimulates influenza virus polymerase as well as RNAP II activities. Here we show that, despite the considerable cellular shut-off observed in infected cells, which includes RNAP II degradation, hCLE protein levels increase throughout infection in a virus replication-dependent manner. Human and avian influenza viruses of various subtypes increase hCLE levels, but other RNA or DNA viruses do not. hCLE colocalises and interacts with viral ribonucleoproteins (vRNP) in the nucleus, as well as in the cytoplasm late in infection. Furthermore, biochemical analysis of purified virus particles and immunoelectron microscopy of infected cells show hCLE in virions, in close association with viral vRNP. These findings indicate that hCLE, a cellular protein important for viral replication, is one of the very few examples of transcription factors that are incorporated into particles of an RNA-containing virus. PMID:26864902

  2. Chemical Genomics Identifies the PERK-Mediated Unfolded Protein Stress Response as a Cellular Target for Influenza Virus Inhibition

    PubMed Central

    Landeras-Bueno, Sara; Fernández, Yolanda; Falcón, Ana; Oliveros, Juan Carlos

    2016-01-01

    ABSTRACT Influenza A viruses generate annual epidemics and occasional pandemics of respiratory disease with important consequences for human health and the economy. Therefore, a large effort has been devoted to the development of new anti-influenza virus drugs directed to viral targets, as well as to the identification of cellular targets amenable to anti-influenza virus therapy. Here we have addressed the identification of such potential cellular targets by screening collections of drugs approved for human use. We reasoned that screening with a green fluorescent protein-based recombinant replicon system would identify cellular targets involved in virus transcription/replication and/or gene expression and hence address an early stage of virus infection. By using such a strategy, we identified Montelukast (MK) as an inhibitor of virus multiplication. MK inhibited virus gene expression but did not alter viral RNA synthesis in vitro or viral RNA accumulation in vivo. The low selectivity index of MK prevented its use as an antiviral, but it was sufficient to identify a new cellular pathway suitable for anti-influenza virus intervention. By deep sequencing of RNA isolated from mock- and virus-infected human cells, treated with MK or left untreated, we showed that it stimulates the PERK-mediated unfolded protein stress response. The phosphorylation of PERK was partly inhibited in virus-infected cells but stimulated in MK-treated cells. Accordingly, pharmacological inhibition of PERK phosphorylation led to increased viral gene expression, while inhibition of PERK phosphatase reduced viral protein synthesis. These results suggest the PERK-mediated unfolded protein response as a potential cellular target to modulate influenza virus infection. PMID:27094326

  3. Automated clustering of ensembles of alternative models in protein structure databases.

    PubMed

    Domingues, Francisco S; Rahnenführer, Jörg; Lengauer, Thomas

    2004-06-01

    Experimentally determined protein structures have been classified in different public databases according to their structural and evolutionary relationships. Frequently, alternative structural models, determined using X-ray crystallography or NMR spectroscopy, are available for a protein. These models can present significant structural dissimilarity. Currently there is no classification available for these alternative structures. In order to classify them, we developed STRuster, an automated method for clustering ensembles of structural models according to their backbone structure. The method is based on the calculation of carbon alpha (Calpha) distance matrices. Two filters are applied in the calculation of the dissimilarity measure in order to identify both large and small (but significant) backbone conformational changes. The resulting dissimilarity value is used for hierarchical clustering and partitioning around medoids (PAM). Hierarchical clustering reflects the hierarchy of similarities between all pairs of models, while PAM groups the models into the 'optimal' number of clusters. The method has been applied to cluster the structures in each SCOP species level and can be easily applied to any other sets of conformers. The results are available at: http://bioinf.mpi-sb.mpg.de/projects/struster/. PMID:15319469

  4. Cellular immunity to the P6 outer membrane protein of nontypeable Haemophilus influenzae.

    PubMed Central

    Kodama, H; Faden, H

    1995-01-01

    Cellular immunity to nontypeable Haemophilus influenzae in a population of 10 healthy, immune adults was determined by measuring lymphocyte blast transformation and antibody secretion in response to the P6 outer membrane protein. P6 (200 microliters/ml) induced lymphocyte blast transformation that peaked on day 10 of incubation. The peak induction of antibody-secreting cells occurred on day 8 of incubation. In comparison with the response to tetanus toxoid stimulation, the peak lymphocyte blast transformation response to P6 was reduced (mean counts per minute +/- standard error of the mean [SEM], 3,457 +/- 503 versus 9,414 +/- 1,464; P = 0.0051) and delayed (mean days +/- SEM, 10.3 +/- 0.4 versus 8.4 +/- 0.5; P = 0.0169); however, P6 was a better stimulus of antibody secretion from lymphocytes, particularly antibody of the immunoglobulin M (IgM) class (mean peak numbers of antibody-secreting cells per 10(5) peripheral blood mononuclear cells +/- SEM: IgG, 85 +/- 29 versus 42 +/- 16 [P = 0.0469]; IgM, 81 +/- 20 versus 25 +/- 7 [P = 0.0125]; IgA, 24 +/- 8 versus 16 +/- 6 [P = 0.0526]). Thus, lymphocytes from immune individuals recognize P6 of nontypeable H. influenzae as an immunogen. These data provide a basis for future studies with otitis-prone children who fail to develop a normal antibody response to P6 antigen (N. Yamanaka and H. Faden, J. Pediatr. 122:212-218, 1993). PMID:7790058

  5. Cellular trafficking and degradation of erythropoietin and novel erythropoiesis stimulating protein (NESP).

    PubMed

    Gross, Alec W; Lodish, Harvey F

    2006-01-27

    Erythropoietin (Epo) is essential for the production of mature red blood cells, and recombinant Epo is commonly used to treat anemia, but how Epo is degraded and cleared from the body is not understood. Glycosylation of Epo is required for its in vivo bioactivity, although not for in vitro receptor binding or stimulation of Epo-dependent cell lines; Epo glycosylation actually reduces the affinity of Epo for the Epo receptor (EpoR). Interestingly, a hyperglycosylated analog of Epo, called novel erythropoiesis-stimulating protein (NESP), has a lower affinity than Epo for the EpoR but has greater in vivo activity and a longer serum half-life than Epo. We hypothesize that a major mechanism for degradation of Epo in the body occurs in cells expressing the Epo receptor, through receptor-mediated endocytosis of Epo followed by degradation in lysosomes, and therefore investigated the trafficking and degradation of Epo and NESP by EpoR-expressing cells. We show that Epo and NESP are degraded only by cultured cells that express the EpoR, and their receptor binding, dissociation, and trafficking properties determine their rates of intracellular degradation. Epo binds surface EpoR faster than NESP (k(on) = 5.0 x 10(8) m(-1) min(-1) versus 1.1 x 10(8) m(-1) min(-1)) but dissociates slower (k(off) = 0.029 min(-1) versus 0.042 min(-1)). Surface-bound Epo and NESP are internalized at the same rate (k(in) = 0.06 min(-1)), and after internalization 60% of each ligand is resecreted intact and 40% degraded. Our kinetic model of Epo and NESP receptor binding, intracellular trafficking, and degradation explains why Epo is degraded faster than NESP at the cellular level.

  6. Amyloid-β-induced Synapse Damage Is Mediated via Cross-linkage of Cellular Prion Proteins

    PubMed Central

    Bate, Clive; Williams, Alun

    2011-01-01

    The cellular prion protein (PrPC), which is highly expressed at synapses, was identified as a receptor for the amyloid-β (Aβ) oligomers that are associated with dementia in Alzheimer disease. Here, we report that Aβ oligomers secreted by 7PA2 cells caused synapse damage in cultured neurons via a PrPC-dependent process. Exogenous PrPC added to Prnp knock-out(0/0) neurons was targeted to synapses and significantly increased Aβ-induced synapse damage. In contrast, the synapse damage induced by a phospholipase A2-activating peptide was independent of PrPC. In Prnp wild-type(+/+) neurons Aβ oligomers activated synaptic cytoplasmic phospholipase A2 (cPLA2). In these cells, the addition of Aβ oligomers triggered the translocation of cPLA2 in synapses to cholesterol