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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. Protein sequence databases.

    PubMed

    Apweiler, Rolf; Bairoch, Amos; Wu, Cathy H

    2004-02-01

    A variety of protein sequence databases exist, ranging from simple sequence repositories, which store data with little or no manual intervention in the creation of the records, to expertly curated universal databases that cover all species and in which the original sequence data are enhanced by the manual addition of further information in each sequence record. As the focus of researchers moves from the genome to the proteins encoded by it, these databases will play an even more important role as central comprehensive resources of protein information. Several the leading protein sequence databases are discussed here, with special emphasis on the databases now provided by the Universal Protein Knowledgebase (UniProt) consortium. PMID:15036160

  4. Protein Model Database

    SciTech Connect

    Fidelis, K; Adzhubej, A; Kryshtafovych, A; Daniluk, P

    2005-02-23

    The phenomenal success of the genome sequencing projects reveals the power of completeness in revolutionizing biological science. Currently it is possible to sequence entire organisms at a time, allowing for a systemic rather than fractional view of their organization and the various genome-encoded functions. There is an international plan to move towards a similar goal in the area of protein structure. This will not be achieved by experiment alone, but rather by a combination of efforts in crystallography, NMR spectroscopy, and computational modeling. Only a small fraction of structures are expected to be identified experimentally, the remainder to be modeled. Presently there is no organized infrastructure to critically evaluate and present these data to the biological community. The goal of the Protein Model Database project is to create such infrastructure, including (1) public database of theoretically derived protein structures; (2) reliable annotation of protein model quality, (3) novel structure analysis tools, and (4) access to the highest quality modeling techniques available.

  5. 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

  6. 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. PMID:26387108

  7. 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)

  8. Human genome protein function database.

    PubMed Central

    Sorenson, D. K.

    1991-01-01

    A database which focuses on the normal functions of the currently-known protein products of the Human Genome was constructed. Information is stored as text, figures, tables, and diagrams. The program contains built-in functions to modify, update, categorize, hypertext, search, create reports, and establish links to other databases. The semi-automated categorization feature of the database program was used to classify these proteins in terms of biomedical functions. PMID:1807638

  9. Cellular senescence and protein degradation

    PubMed Central

    Deschênes-Simard, Xavier; Lessard, Frédéric; Gaumont-Leclerc, Marie-France; Bardeesy, Nabeel; Ferbeyre, Gerardo

    2014-01-01

    Autophagy and the ubiquitin–proteasome pathway (UPP) are the major protein degradation systems in eukaryotic cells. Whereas the former mediate a bulk nonspecific degradation, the UPP allows a rapid degradation of specific proteins. Both systems have been shown to play a role in tumorigenesis, and the interest in developing therapeutic agents inhibiting protein degradation is steadily growing. However, emerging data point to a critical role for autophagy in cellular senescence, an established tumor suppressor mechanism. Recently, a selective protein degradation process mediated by the UPP was also shown to contribute to the senescence phenotype. This process is tightly regulated by E3 ubiquitin ligases, deubiquitinases, and several post-translational modifications of target proteins. Illustrating the complexity of UPP, more than 600 human genes have been shown to encode E3 ubiquitin ligases, a number which exceeds that of the protein kinases. Nevertheless, our knowledge of proteasome-dependent protein degradation as a regulated process in cellular contexts such as cancer and senescence remains very limited. Here we discuss the implications of protein degradation in senescence and attempt to relate this function to the protein degradation pattern observed in cancer cells. PMID:24866342

  10. PINT: Protein-protein Interactions Thermodynamic Database.

    PubMed

    Kumar, M D Shaji; Gromiha, M Michael

    2006-01-01

    The first release of Protein-protein Interactions Thermodynamic Database (PINT) contains >1500 data of several thermodynamic parameters along with sequence and structural information, experimental conditions and literature information. Each entry contains numerical data for the free energy change, dissociation constant, association constant, enthalpy change, heat capacity change and so on of the interacting proteins upon binding, which are important for understanding the mechanism of protein-protein interactions. PINT also includes the name and source of the proteins involved in binding, their Protein Information Resource, SWISS-PROT and Protein Data Bank (PDB) codes, secondary structure and solvent accessibility of residues at mutant positions, measuring methods, experimental conditions, such as buffers, ions and additives, and literature information. A WWW interface facilitates users to search data based on various conditions, feasibility to select the terms for output and different sorting options. Further, PINT is cross-linked with other related databases, PIR, SWISS-PROT, PDB and NCBI PUBMED literature database. The database is freely available at http://www.bioinfodatabase.com/pint/index.html. PMID:16381844

  11. Protein accounting in the cellular economy

    PubMed Central

    Vázquez-Laslop, Nora; Mankin, Alexander S.

    2014-01-01

    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. (2014) gain insights into key aspects of translation regulation and fundamental principles of cellular strategies to adjust protein synthesis according to the needs. PMID:24766801

  12. 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. PMID:24766801

  13. TRIP Database: a manually curated database of protein–protein interactions for mammalian TRP channels

    PubMed Central

    Shin, Young-Cheul; Shin, Soo-Yong; So, Insuk; Kwon, Dongseop; Jeon, Ju-Hong

    2011-01-01

    Transient receptor potential (TRP) channels are a superfamily of Ca2+-permeable cation channels that translate cellular stimuli into electrochemical signals. Aberrant activity of TRP channels has been implicated in a variety of human diseases, such as neurological disorders, cardiovascular disease and cancer. To facilitate the understanding of the molecular network by which TRP channels are associated with biological and disease processes, we have developed the TRIP (TRansient receptor potential channel-Interacting Protein) Database (http://www.trpchannel.org), a manually curated database that aims to offer comprehensive information on protein–protein interactions (PPIs) of mammalian TRP channels. The TRIP Database was created by systematically curating 277 peer-reviewed literature; the current version documents 490 PPI pairs, 28 TRP channels and 297 cellular proteins. The TRIP Database provides a detailed summary of PPI data that fit into four categories: screening, validation, characterization and functional consequence. Users can find in-depth information specified in the literature on relevant analytical methods and experimental resources, such as gene constructs and cell/tissue types. The TRIP Database has user-friendly web interfaces with helpful features, including a search engine, an interaction map and a function for cross-referencing useful external databases. Our TRIP Database will provide a valuable tool to assist in understanding the molecular regulatory network of TRP channels. PMID:20851834

  14. The PIR-International Protein Sequence Database.

    PubMed Central

    George, D G; Barker, W C; Mewes, H W; Pfeiffer, F; Tsugita, A

    1994-01-01

    PIR-International is an association of macromolecular sequence data collection centers dedicated to fostering international cooperation as an essential element in the development of scientific databases. A major objective of PIR-International is to continue the development of the Protein Sequence Database as an essential public resource for protein sequence information. This paper briefly describes the architecture of the Protein Sequence Database and how it and associated data sets are distributed and can be accessed electronically. PMID:7937060

  15. 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

  16. Human protein reference database--2006 update.

    PubMed

    Mishra, Gopa R; Suresh, M; Kumaran, K; Kannabiran, N; Suresh, Shubha; Bala, P; Shivakumar, K; Anuradha, N; Reddy, Raghunath; Raghavan, T Madhan; Menon, Shalini; Hanumanthu, G; Gupta, Malvika; Upendran, Sapna; Gupta, Shweta; Mahesh, M; Jacob, Bincy; Mathew, Pinky; Chatterjee, Pritam; Arun, K S; Sharma, Salil; Chandrika, K N; Deshpande, Nandan; Palvankar, Kshitish; Raghavnath, R; Krishnakanth, R; Karathia, Hiren; Rekha, B; Nayak, Rashmi; Vishnupriya, G; Kumar, H G Mohan; Nagini, M; Kumar, G S Sameer; Jose, Rojan; Deepthi, P; Mohan, S Sujatha; Gandhi, T K B; Harsha, H C; Deshpande, Krishna S; Sarker, Malabika; Prasad, T S Keshava; Pandey, Akhilesh

    2006-01-01

    Human Protein Reference Database (HPRD) (http://www.hprd.org) was developed to serve as a comprehensive collection of protein features, post-translational modifications (PTMs) and protein-protein interactions. Since the original report, this database has increased to >20 000 proteins entries and has become the largest database for literature-derived protein-protein interactions (>30 000) and PTMs (>8000) for human proteins. We have also introduced several new features in HPRD including: (i) protein isoforms, (ii) enhanced search options, (iii) linking of pathway annotations and (iv) integration of a novel browser, GenProt Viewer (http://www.genprot.org), developed by us that allows integration of genomic and proteomic information. With the continued support and active participation by the biomedical community, we expect HPRD to become a unique source of curated information for the human proteome and spur biomedical discoveries based on integration of genomic, transcriptomic and proteomic data. PMID:16381900

  17. 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. PMID:23058924

  18. AMASS: a database for investigating protein structures

    PubMed Central

    Mielke, Clinton J.; Mandarino, Lawrence J.; Dinu, Valentin

    2014-01-01

    Motivation: Modern techniques have produced many sequence annotation databases and protein structure portals, but these Web resources are rarely integrated in ways that permit straightforward exploration of protein functional residues and their co-localization. Results: We have created the AMASS database, which maps 1D sequence annotation databases to 3D protein structures with an intuitive visualization interface. Our platform also provides an analysis service that screens mass spectrometry sequence data for post-translational modifications that reside in functionally relevant locations within protein structures. The system is built on the premise that functional residues such as active sites, cancer mutations and post-translational modifications within proteins may co-localize and share common functions. Availability and implementation: AMASS database is implemented with Biopython and Apache as a freely available Web server at amass-db.org. Contact: clinton.mielke@gmail.com PMID:24497503

  19. 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.

  20. Annotation and retrieval in protein interaction databases

    NASA Astrophysics Data System (ADS)

    Cannataro, Mario; Hiram Guzzi, Pietro; Veltri, Pierangelo

    2014-06-01

    Biological databases have been developed with a special focus on the efficient retrieval of single records or the efficient computation of specialized bioinformatics algorithms against the overall database, such as in sequence alignment. The continuos production of biological knowledge spread on several biological databases and ontologies, such as Gene Ontology, and the availability of efficient techniques to handle such knowledge, such as annotation and semantic similarity measures, enable the development on novel bioinformatics applications that explicitly use and integrate such knowledge. After introducing the annotation process and the main semantic similarity measures, this paper shows how annotations and semantic similarity can be exploited to improve the extraction and analysis of biologically relevant data from protein interaction databases. As case studies, the paper presents two novel software tools, OntoPIN and CytoSeVis, both based on the use of Gene Ontology annotations, for the advanced querying of protein interaction databases and for the enhanced visualization of protein interaction networks.

  1. The Protein-DNA Interface database

    PubMed Central

    2010-01-01

    The Protein-DNA Interface database (PDIdb) is a repository containing relevant structural information of Protein-DNA complexes solved by X-ray crystallography and available at the Protein Data Bank. The database includes a simple functional classification of the protein-DNA complexes that consists of three hierarchical levels: Class, Type and Subtype. This classification has been defined and manually curated by humans based on the information gathered from several sources that include PDB, PubMed, CATH, SCOP and COPS. The current version of the database contains only structures with resolution of 2.5 Å or higher, accounting for a total of 922 entries. The major aim of this database is to contribute to the understanding of the main rules that underlie the molecular recognition process between DNA and proteins. To this end, the database is focused on each specific atomic interface rather than on the separated binding partners. Therefore, each entry in this database consists of a single and independent protein-DNA interface. We hope that PDIdb will be useful to many researchers working in fields such as the prediction of transcription factor binding sites in DNA, the study of specificity determinants that mediate enzyme recognition events, engineering and design of new DNA binding proteins with distinct binding specificity and affinity, among others. Finally, due to its friendly and easy-to-use web interface, we hope that PDIdb will also serve educational and teaching purposes. PMID:20482798

  2. BISC: binary subcomplexes in proteins database.

    PubMed

    Juettemann, Thomas; Gerloff, Dietlind L

    2011-01-01

    Binary subcomplexes in proteins database (BISC) is a new protein-protein interaction (PPI) database linking up the two communities most active in their characterization: structural biology and functional genomics researchers. The BISC resource offers users (i) a structural perspective and related information about binary subcomplexes (i.e. physical direct interactions between proteins) that are either structurally characterized or modellable entries in the main functional genomics PPI databases BioGRID, IntAct and HPRD; (ii) selected web services to further investigate the validity of postulated PPI by inspection of their hypothetical modelled interfaces. Among other uses we envision that this resource can help identify possible false positive PPI in current database records. BISC is freely available at http://bisc.cse.ucsc.edu. PMID:21081561

  3. 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

  4. 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

  5. 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…

  6. 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.

  7. Recognition of cisplatin adducts by cellular proteins.

    PubMed

    Kartalou, M; Essigmann, J M

    2001-07-01

    Cisplatin is a widely used chemotherapeutic agent. It reacts with nucleophilic bases in DNA and forms 1,2-d(ApG), 1,2-d(GpG) and 1,3-d(GpTpG) intrastrand crosslinks, interstrand crosslinks and monofunctional adducts. The presence of these adducts in DNA is through to be responsible for the therapeutic efficacy of cisplatin. The exact signal transduction pathway that leads to cell cycle arrest and cell death following treatment with the drug is not known but cell death is believed to be mediated by the recognition of the adducts by cellular proteins. Here we describe the structural information available for cisplatin and related platinum adducts, the interactions of the adducts with cellular proteins and the implications of these interactions for cell survival. PMID:11406166

  8. GWIDD: Genome-wide protein docking database

    PubMed Central

    Kundrotas, Petras J.; Zhu, Zhengwei; Vakser, Ilya A.

    2010-01-01

    Structural information on interacting proteins is important for understanding life processes at the molecular level. Genome-wide docking database is an integrated resource for structural studies of protein–protein interactions on the genome scale, which combines the available experimental data with models obtained by docking techniques. Current database version (August 2009) contains 25 559 experimental and modeled 3D structures for 771 organisms spanned over the entire universe of life from viruses to humans. Data are organized in a relational database with user-friendly search interface allowing exploration of the database content by a number of parameters. Search results can be interactively previewed and downloaded as PDB-formatted files, along with the information relevant to the specified interactions. The resource is freely available at http://gwidd.bioinformatics.ku.edu. PMID:19900970

  9. The PIR-International Protein Sequence Database.

    PubMed

    George, D G; Barker, W C; Mewes, H W; Pfeiffer, F; Tsugita, A

    1996-01-01

    From its origin the Protein Sequence Database has been designed to support research and has focused on comprehensive coverage, quality control and organization of the data in accordance with biological principles. Since 1988 the database has been maintained collaboratively within the framework of PIR-International, an association of macromolecular sequence data collection centers dedicated to fostering international cooperation as an essential element in the development of scientific databases. The database is widely distributed and is available on the World Wide Web, via ftp, email server, on CD-ROM and magnetic media. It is widely redistributed and incorporated into many other protein sequence data compilations, including SWISS-PROT and the Entrez system of the NCBI. PMID:8594572

  10. SCOP: a structural classification of proteins database.

    PubMed Central

    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/ PMID:9016544

  11. 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.

  12. PIPs: human protein–protein interaction prediction database

    PubMed Central

    McDowall, Mark D.; Scott, Michelle S.; Barton, Geoffrey J.

    2009-01-01

    The PIPs database (http://www.compbio.dundee.ac.uk/www-pips) is a resource for studying protein–protein interactions in human. It contains predictions of >37 000 high probability interactions of which >34 000 are not reported in the interaction databases HPRD, BIND, DIP or OPHID. The interactions in PIPs were calculated by a Bayesian method that combines information from expression, orthology, domain co-occurrence, post-translational modifications and sub-cellular location. The predictions also take account of the topology of the predicted interaction network. The web interface to PIPs ranks predictions according to their likelihood of interaction broken down by the contribution from each information source and with easy access to the evidence that supports each prediction. Where data exists in OPHID, HPRD, DIP or BIND for a protein pair this is also reported in the output tables returned by a search. A network browser is included to allow convenient browsing of the interaction network for any protein in the database. The PIPs database provides a new resource on protein–protein interactions in human that is straightforward to browse, or can be exploited completely, for interaction network modelling. PMID:18988626

  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. Protein structure database search and evolutionary classification.

    PubMed

    Yang, Jinn-Moon; Tung, Chi-Hua

    2006-01-01

    As more protein structures become available and structural genomics efforts provide structural models in a genome-wide strategy, there is a growing need for fast and accurate methods for discovering homologous proteins and evolutionary classifications of newly determined structures. We have developed 3D-BLAST, in part, to address these issues. 3D-BLAST is as fast as BLAST and calculates the statistical significance (E-value) of an alignment to indicate the reliability of the prediction. Using this method, we first identified 23 states of the structural alphabet that represent pattern profiles of the backbone fragments and then used them to represent protein structure databases as structural alphabet sequence databases (SADB). Our method enhanced BLAST as a search method, using a new structural alphabet substitution matrix (SASM) to find the longest common substructures with high-scoring structured segment pairs from an SADB database. Using personal computers with Intel Pentium4 (2.8 GHz) processors, our method searched more than 10 000 protein structures in 1.3 s and achieved a good agreement with search results from detailed structure alignment methods. [3D-BLAST is available at http://3d-blast.life.nctu.edu.tw]. PMID:16885238

  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. PPIM: A Protein-Protein Interaction Database for Maize.

    PubMed

    Zhu, Guanghui; Wu, Aibo; Xu, Xin-Jian; Xiao, Pei-Pei; Lu, Le; Liu, Jingdong; Cao, Yongwei; Chen, Luonan; Wu, Jun; Zhao, Xing-Ming

    2016-02-01

    Maize (Zea mays) is one of the most important crops worldwide. To understand the biological processes underlying various traits of the crop (e.g. yield and response to stress), a detailed protein-protein interaction (PPI) network is highly demanded. Unfortunately, there are very few such PPIs available in the literature. Therefore, in this work, we present the Protein-Protein Interaction Database for Maize (PPIM), which covers 2,762,560 interactions among 14,000 proteins. The PPIM contains not only accurately predicted PPIs but also those molecular interactions collected from the literature. The database is freely available at http://comp-sysbio.org/ppim with a user-friendly powerful interface. We believe that the PPIM resource can help biologists better understand the maize crop. PMID:26620522

  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. PepBind: a comprehensive database and computational tool for analysis of protein-peptide interactions.

    PubMed

    Das, Arindam Atanu; Sharma, Om Prakash; Kumar, Muthuvel Suresh; Krishna, Ramadas; Mathur, Premendu P

    2013-08-01

    Protein-peptide interactions, where one partner is a globular protein (domain) and the other is a flexible linear peptide, are key components of cellular processes predominantly in signaling and regulatory networks, hence are prime targets for drug design. To derive the details of the protein-peptide interaction mechanism is often a cumbersome task, though it can be made easier with the availability of specific databases and tools. The Peptide Binding Protein Database (PepBind) is a curated and searchable repository of the structures, sequences and experimental observations of 3100 protein-peptide complexes. The web interface contains a computational tool, protein inter-chain interaction (PICI), for computing several types of weak or strong interactions at the protein-peptide interaction interface and visualizing the identified interactions between residues in Jmol viewer. This initial database release focuses on providing protein-peptide interface information along with structure and sequence information for protein-peptide complexes deposited in the Protein Data Bank (PDB). Structures in PepBind are classified based on their cellular activity. More than 40% of the structures in the database are found to be involved in different regulatory pathways and nearly 20% in the immune system. These data indicate the importance of protein-peptide complexes in the regulation of cellular processes. PMID:23896518

  20. α-Cleavage of cellular prion protein

    PubMed Central

    Liang, Jingjing; Kong, Qingzhong

    2012-01-01

    The cellular prion protein (PrPC) is subjected to various processing under physiological and pathological conditions, of which the α-cleavage within the central hydrophobic domain not only disrupts a region critical for both PrP toxicity and PrPC to PrPSc conversion but also produces the N1 fragment that is neuroprotective and the C1 fragment that enhances the pro-apoptotic effect of staurosporine in one report and inhibits prion in another. The proteases responsible for the α-cleavage of PrPC are controversial. The effect of ADAM10, ADAM17, and ADAM9 on N1 secretion clearly indicates their involvement in the α-cleavage of PrPC, but there has been no report of direct PrPC α-cleavage activity with any of the three ADAMs in a purified protein form. We demonstrated that, in muscle cells, ADAM8 is the primary protease for the α-cleavage of PrPC, but another unidentified protease(s) must also play a minor role. We also found that PrPC regulates ADAM8 expression, suggesting that a close examination on the relationships between PrPC and its processing enzymes may reveal novel roles and underlying mechanisms for PrPC in non-prion diseases such as asthma and cancer. PMID:23052041

  1. Thermodynamic database for proteins: features and applications.

    PubMed

    Gromiha, M Michael; Sarai, Akinori

    2010-01-01

    We have developed a thermodynamic database for proteins and mutants, ProTherm, which is a collection of a large number of thermodynamic data on protein stability along with the sequence and structure information, experimental methods and conditions, and literature information. This is a valuable resource for understanding/predicting the stability of proteins, and it can be accessible at http://www.gibk26.bse.kyutech.ac.jp/jouhou/Protherm/protherm.html . ProTherm has several features including various search, display, and sorting options and visualization tools. We have analyzed the data in ProTherm to examine the relationship among thermodynamics, structure, and function of proteins. We describe the progress on the development of methods for understanding/predicting protein stability, such as (i) relationship between the stability of protein mutants and amino acid properties, (ii) average assignment method, (iii) empirical energy functions, (iv) torsion, distance, and contact potentials, and (v) machine learning techniques. The list of online resources for predicting protein stability has also been provided. PMID:20221915

  2. 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

  3. 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/

  4. An object-oriented database for protein structure analysis.

    PubMed

    Gray, P M; Paton, N W; Kemp, G J; Fothergill, J E

    1990-03-01

    An object-oriented database system has been developed which is being used to store protein structure data. The database can be queried using the logic programming language Prolog or the query language Daplex. Queries retrieve information by navigating through a network of objects which represent the primary, secondary and tertiary structures of proteins. Routines written in both Prolog and Daplex can integrate complex calculations with the retrieval of data from the database, and can also be stored in the database for sharing among users. Thus object-oriented databases are better suited to prototyping applications and answering complex queries about protein structure than relational databases. This system has been used to find loops of varying length and anchor positions when modelling homologous protein structures. PMID:2188261

  5. A family of cellular proteins related to snake venom disintegrins.

    PubMed Central

    Weskamp, G; Blobel, C P

    1994-01-01

    Disintegrins are short soluble integrin ligands that were initially identified in snake venom. A previously recognized cellular protein with a disintegrin domain was the guinea pig sperm protein PH-30, a protein implicated in sperm-egg membrane binding and fusion. Here we present peptide sequences that are characteristic for several cellular disintegrin-domain proteins. These peptide sequences were deduced from cDNA sequence tags that were generated by polymerase chain reaction from various mouse tissue and a mouse muscle cell line. Northern blot analysis with four sequence tags revealed distinct mRNA expression patterns. Evidently, cellular proteins containing a disintegrin domain define a superfamily of potential integrin ligands that are likely to function in important cell-cell and cell-matrix interactions. Images PMID:8146185

  6. PSSARD: protein sequence-structure analysis relational database.

    PubMed

    Guruprasad, Kunchur; Srikanth, K; Babu, A V N

    2005-09-15

    We have implemented a relational database comprising a representative dataset of amino acid sequences and their associated secondary structure. The representative amino acid sequences were selected according to the PDB_SELECT program by choosing proteins corresponding to protein crystal structure data deposited in the protein data bank that share less than 25% overall pair-wise sequence identity. The secondary structure was extracted from the protein data bank website. The information content in the database includes the protein description, PDB code, crystal structure resolution, total number of amino acid residues in the protein chain, amino acid sequence, secondary structure conformation and its summary. The database is freely accessible from the website mentioned below and is useful to query on any of the above fields. The database is particularly useful to quickly retrieve amino acid sequences that are compatible to any super-secondary structure conformation from several proteins simultaneously. PMID:16054209

  7. Piezo Proteins: Regulators of Mechanosensation and Other Cellular Processes*

    PubMed Central

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

    2014-01-01

    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. PMID:25305018

  8. MIPS: a database for genomes and protein sequences

    PubMed Central

    Mewes, H. W.; Frishman, D.; Gruber, C.; Geier, B.; Haase, D.; Kaps, A.; Lemcke, K.; Mannhaupt, G.; Pfeiffer, F.; Schüller, C.; Stocker, S.; Weil, B.

    2000-01-01

    The Munich Information Center for Protein Sequences (MIPS-GSF), Martinsried, near Munich, Germany, continues its longstanding tradition to develop and maintain high quality curated genome databases. In addition, efforts have been intensified to cover the wealth of complete genome sequences in a systematic, comprehensive form. Bioinformatics, supporting national as well as European sequencing and functional analysis projects, has resulted in several up-to-date genome-oriented databases. This report describes growing databases reflecting the progress of sequencing the Arabidopsis thaliana (MATDB) and Neurospora crassa genomes (MNCDB), the yeast genome database (MYGD) extended by functional analysis data, the database of annotated human EST-clusters (HIB) and the database of the complete cDNA sequences from the DHGP (German Human Genome Project). It also contains information on the up-to-date database of complete genomes (PEDANT), the classification of protein sequences (ProtFam) and the collection of protein sequence data within the framework of the PIR-International Protein Sequence Database. These databases can be accessed through the MIPS WWW server (http://www. mips.biochem.mpg.de ). PMID:10592176

  9. 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.

  10. Heat Shock Proteins: Cellular and molecular mechanisms in the CNS

    PubMed Central

    Stetler, R. Anne; Gan, Yu; Zhang, Wenting; Liou, Anthony K.; Gao, Yanqin; Cao, Guodong; Chen, Jun

    2010-01-01

    Emerging evidence describe heat shock proteins (HSPs) as critical regulators in normal neural physiological function as well as in cell stress responses. The functions of HSPs represent an enormous and diverse range of cellular activities, far beyond the originally identified role in protein folding and chaperoning. Now understood to be involved in processes such as synaptic transmission, autophagy, ER stress response, protein kinase and cell death signaling as well as protein chaperone and folding, manipulation of HSPs have robust effects on the fate of cells in neurological injury and disease states. The ongoing exploration of multiple HSP superfamilies has underscored the pluripotent nature of HSPs in the cellular context, and demanded the recent restructuring of the nomenclature referring to these families to reflect a re-organization based on structure and function. In keeping with this re-organization, we have first discussed the HSP superfamilies in terms of protein structure, regulation and expression and distribution in the brain. We then explore major cellular functions of HSPs that are relevant to neural physiological states, and from there discuss known and proposed HSP impact on major neurological disease states. This review article presents a three-part discussion on the array of HSPs families relevant to neuronal tissue, their cellular functions, and the exploration of therapeutic targets of these proteins in the context of neurological diseases. PMID:20685377

  11. 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

  12. 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

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

    PubMed

    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 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

  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. PMID:27055826

  16. Characterization of Protein Complexes and Subcomplexes in Protein-Protein Interaction Databases

    PubMed Central

    Zaki, Nazar; Mohamed, Elfadil A.; Mora, Antonio

    2015-01-01

    The identification and characterization of protein complexes implicated in protein-protein interaction data are crucial to the understanding of the molecular events under normal and abnormal physiological conditions. This paper provides a novel characterization of subcomplexes in protein interaction databases, stressing definition and representation issues, quantification, biological validation, network metrics, motifs, modularity, and gene ontology (GO) terms. The paper introduces the concept of “nested group” as a way to represent subcomplexes and estimates that around 15% of those nested group with the higher Jaccard index may be a result of data artifacts in protein interaction databases, while a number of them can be found in biologically important modular structures or dynamic structures. We also found that network centralities, enrichment in essential proteins, GO terms related to regulation, imperfect 5-clique motifs, and higher GO homogeneity can be used to identify proteins in nested complexes. PMID:25722891

  17. SWISS-PROT: connecting biomolecular knowledge via a protein database.

    PubMed

    Gasteiger, E; Jung, E; Bairoch, A

    2001-07-01

    With the explosive growth of biological data, the development of new means of data storage was needed. More and more often biological information is no longer published in the conventional way via a publication in a scientific journal, but only deposited into a database. In the last two decades these databases have become essential tools for researchers in biological sciences. Biological databases can be classified according to the type of information they contain. There are basically three types of sequence-related databases (nucleic acid sequences, protein sequences and protein tertiary structures) as well as various specialized data collections. It is important to provide the users of biomolecular databases with a degree of integration between these databases as by nature all of these databases are connected in a scientific sense and each one of them is an important piece to biological complexity. In this review we will highlight our effort in connecting biological information as demonstrated in the SWISS-PROT protein database. PMID:11488411

  18. EXProt: a database for proteins with an experimentally verified function.

    PubMed

    Ursing, Björn M; van Enckevort, Frank H J; Leunissen, Jack A M; Siezen, Roland J

    2002-01-01

    EXProt is a non-redundant protein database containing a selection of entries from genome annotation projects and public databases, aimed at including only proteins with an experimentally verified function. In EXProt release 2.0 we have collected entries from the Pseudomonas aeruginosa community annotation project (PseudoCAP), the Escherichia coli genome and proteome database (GenProtEC) and the translated coding sequences from the Prokaryotes division of EMBL nucleotide sequence database, which are described as having an experimentally verified function. Each entry in EXProt has a unique ID number and contains information about the species, amino acid sequence, functional annotation and, in most cases, links to references in MEDLINE/PubMed and to the entry in the original database. EXProt is indexed in SRS at CMBI (http://www.cmbi.kun.nl/srs/) and can be searched with BLAST and FASTA through the EXProt web page (http://www.cmbi.kun.nl/EXProt/). PMID:11752251

  19. PCDq: human protein complex database with quality index which summarizes different levels of evidences of protein complexes predicted from H-Invitational protein-protein interactions integrative dataset

    PubMed Central

    2012-01-01

    Background Proteins interact with other proteins or biomolecules in complexes to perform cellular functions. Existing protein-protein interaction (PPI) databases and protein complex databases for human proteins are not organized to provide protein complex information or facilitate the discovery of novel subunits. Data integration of PPIs focused specifically on protein complexes, subunits, and their functions. Predicted candidate complexes or subunits are also important for experimental biologists. Description Based on integrated PPI data and literature, we have developed a human protein complex database with a complex quality index (PCDq), which includes both known and predicted complexes and subunits. We integrated six PPI data (BIND, DIP, MINT, HPRD, IntAct, and GNP_Y2H), and predicted human protein complexes by finding densely connected regions in the PPI networks. They were curated with the literature so that missing proteins were complemented and some complexes were merged, resulting in 1,264 complexes comprising 9,268 proteins with 32,198 PPIs. The evidence level of each subunit was assigned as a categorical variable. This indicated whether it was a known subunit, and a specific function was inferable from sequence or network analysis. To summarize the categories of all the subunits in a complex, we devised a complex quality index (CQI) and assigned it to each complex. We examined the proportion of consistency of Gene Ontology (GO) terms among protein subunits of a complex. Next, we compared the expression profiles of the corresponding genes and found that many proteins in larger complexes tend to be expressed cooperatively at the transcript level. The proportion of duplicated genes in a complex was evaluated. Finally, we identified 78 hypothetical proteins that were annotated as subunits of 82 complexes, which included known complexes. Of these hypothetical proteins, after our prediction had been made, four were reported to be actual subunits of the

  20. [Construction of nervous system relative protein and gene secondary database].

    PubMed

    Wang, Pan; Chen, Xinhao; Liu, Xiangming

    2007-10-01

    Along with the rapid research of neural molecular biology, abundant data are produced so that the collection and coordination of high-throughout data about nervous system relative proteins and genes are imperative. Through analyzing the biological primary databases maintained by NCBI and RCSB as the main data source and designing a new data model, a local specialized secondary database is constructed, which mainly includes nucleotide sequences, protein sequences and protein structures, and is established on Sun Blade 2000 System and Oracle 9i. All programs are developed by Java technology. A method of web information automatic retrieval with XML is proposed for sequence data collection and submission to the database. JSP + JavaBean technology is used to support data promulgation on Internet. The establishment of this database provides an excellent platform for the research of neural molecular biology and the pathogenesis of related diseases. PMID:18027688

  1. 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

  2. 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

  3. Identification of Protein Interactions Involved in Cellular Signaling

    PubMed Central

    Westermarck, Jukka; Ivaska, Johanna; Corthals, Garry L.

    2013-01-01

    Protein-protein interactions drive biological processes. They are critical for all intra- and extracellular functions, and the technologies to analyze them are widely applied throughout the various fields of biological sciences. This study takes an in-depth view of some common principles of cellular regulation and provides a detailed account of approaches required to comprehensively map signaling protein-protein interactions in any particular cellular system or condition. We provide a critical review of the benefits and disadvantages of the yeast two-hybrid method and affinity purification coupled with mass spectrometric procedures for identification of signaling protein-protein interactions. In particular, we emphasize the quantitative and qualitative differences between tandem affinity and one-step purification (such as FLAG and Strep tag) methods. Although applicable to all types of interaction studies, a special section is devoted in this review to aspects that should be considered when attempting to identify signaling protein interactions that often are transient and weak by nature. Finally, we discuss shotgun and quantitative information that can be gleaned by MS-coupled methods for analysis of multiprotein complexes. PMID:23481661

  4. 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.

  5. 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

  6. 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

  7. ProTherm: Thermodynamic Database for Proteins and Mutants.

    PubMed

    Gromiha, M M; An, J; Kono, H; Oobatake, M; Uedaira, H; Sarai, A

    1999-01-01

    The first release of the Thermodynamic Database for Proteins and Mutants (ProTherm) contains more than 3300 data of several thermodynamic parameters for wild type and mutant proteins. Each entry includes numerical data for unfolding Gibbs free energy change, enthalpy change, heat capacity change, transition temperature, activity etc., which are important for understanding the mechanism of protein stability. ProTherm also includes structural information such as secondary structure and solvent accessibility of wild type residues, and experimental methods and other conditions. A WWW interface enables users to search data based on various conditions with different sorting options for outputs. Further, ProTherm is cross-linked with NCBI PUBMED literature database, Protein Mutant Database, Enzyme Code and Protein Data Bank structural database. Moreover, all the mutation sites associated with each PDB structure are automatically mapped and can be directly viewed through 3DinSight developed in our laboratory. The database is available at the URL, http://www.rtc.riken.go.jp/protherm.htm l PMID:9847203

  8. BCL2DB: database of BCL-2 family members and BH3-only proteins.

    PubMed

    Rech de Laval, Valentine; Deléage, Gilbert; Aouacheria, Abdel; Combet, Christophe

    2014-01-01

    BCL2DB (http://bcl2db.ibcp.fr) is a database designed to integrate data on BCL-2 family members and BH3-only proteins. These proteins control the mitochondrial apoptotic pathway and probably many other cellular processes as well. This large protein group is formed by a family of pro-apoptotic and anti-apoptotic homologs that have phylogenetic relationships with BCL-2, and by a collection of evolutionarily and structurally unrelated proteins characterized by the presence of a region of local sequence similarity with BCL-2, termed the BH3 motif. BCL2DB is monthly built, thanks to an automated procedure relying on a set of homemade profile HMMs computed from seed reference sequences representative of the various BCL-2 homologs and BH3-only proteins. The BCL2DB entries integrate data from the Ensembl, Ensembl Genomes, European Nucleotide Archive and Protein Data Bank databases and are enriched with specific information like protein classification into orthology groups and distribution of BH motifs along the sequences. The Web interface allows for easy browsing of the site and fast access to data, as well as sequence analysis with generic and specific tools. BCL2DB provides a helpful and powerful tool to both 'BCL-2-ologists' and researchers working in the various fields of physiopathology. Database URL: http://bcl2db.ibcp.fr. PMID:24608034

  9. HMG Nuclear Proteins: Linking Chromatin Structure to Cellular Phenotype

    PubMed Central

    Reeves, Raymond

    2009-01-01

    I. Summary Although the three families of mammalian HMG proteins (HMGA, HMGB and HMGN) participate in many of the same nuclear processes, each family plays its own unique role in modulating chromatin structure and regulating genomic function. This review focuses on the similarities and differences in the mechanisms by which the different HMG families impact chromatin structure and influence cellular phenotype. The biological implications of having three architectural transcription factor families with complementary, but partially overlapping, nuclear functions are discussed. PMID:19748605

  10. 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

  11. 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

  12. 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

  13. Thermal proteome profiling monitors ligand interactions with cellular membrane proteins.

    PubMed

    Reinhard, Friedrich B M; Eberhard, Dirk; Werner, Thilo; Franken, Holger; Childs, Dorothee; Doce, Carola; Savitski, Maria Fälth; Huber, Wolfgang; Bantscheff, Marcus; Savitski, Mikhail M; Drewes, Gerard

    2015-12-01

    We extended thermal proteome profiling to detect transmembrane protein-small molecule interactions in cultured human cells. When we assessed the effects of detergents on ATP-binding profiles, we observed shifts in denaturation temperature for ATP-binding transmembrane proteins. We also observed cellular thermal shifts in pervanadate-induced T cell-receptor signaling, delineating the membrane target CD45 and components of the downstream pathway, and with drugs affecting the transmembrane transporters ATP1A1 and MDR1. PMID:26524241

  14. 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.

  15. 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.

  16. 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

  17. 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

  18. Knockdown of the cellular protein LRPPRC attenuates HIV-1 infection.

    PubMed

    Schweitzer, Cameron J; Matthews, John M; Madson, Christian J; Donnellan, Meghan R; Cerny, Ronald L; Belshan, Michael

    2012-01-01

    HIV-1 exploits numerous host cellular pathways for productive infection. To identify novel factors involved in HIV-1 replication, HIV-1 integrase and matrix protein complexes were captured at 4 hours post infection for proteomic analysis using an affinity purification system. Leucine-rich PPR-motif containing (LRPPRC) protein, a cellular protein involved in mitochondrial function, cell metabolism, and cell-cycle progression was identified as one of the candidate HIV-1 factors. Co-immunoprecipitation RT-PCR experiments confirmed that LRPPRC associated with HIV-1 nucleic acids during the early steps of virus infection. To establish if LRPPRC was critical for HIV-1 infection, three independent LRPPRC knockdown cell lines were constructed (2.7, 3.6, and 4.1). Subcellular fractionation of these cell lines revealed differential knockdown of LRPPRC in subcellular compartments. LRPPRC was knocked down in the insoluble/cytoskeletal fractions of all three cell lines, but the 3.6 and 4.1 cells also showed a reduction in nuclear LRPPRC. Additionally, several cellular factors were downregulated and/or disrupted by loss of LRPPRC. HIV-1 infection was reduced in all three cell lines, but virus production and RNA encapsidation were unaffected, suggesting that LRPPRC was critical for the afferent stage of virus replication. Two of the three cell lines (3.6, 4.1) were refractory for murine leukemia virus infection, a virus dependent on cellular proliferation for productive infection. Consistent with this, these two cell lines exhibited reduced cellular growth with no loss of cellular viability or change in cell cycle phenotype. The early steps of virus infection were also differentially affected among the cell lines. A reduced level of preintegration complex formation was observed in all three cell lines, but viral DNA nuclear import was reduced only in the 3.6 and 4.1 cells. Combined, these data identify LRPPRC as a HIV-1 factor that is involved in HIV-1 replication through more

  19. The ProDom database of protein domain families.

    PubMed Central

    Corpet, F; Gouzy, J; Kahn, D

    1998-01-01

    The ProDom database contains protein domain families generated from the SWISS-PROT database by automated sequence comparisons. It can be searched on the World Wide Web (http://protein.toulouse.inra. fr/prodom.html ) or by E-mail (prodom@toulouse.inra.fr) to study domain arrangements within known families or new proteins. Strong emphasis has been put on the graphical user interface which allows for interactive analysis of protein homology relationships. Recent improvements to the server include: ProDom search by keyword; links to PROSITE and PDB entries; more sensitive ProDom similarity search with BLAST or WU-BLAST; alignments of query sequences with homologous ProDom domain families; and links to the SWISS-MODEL server (http: //www.expasy.ch/swissmod/SWISS-MODEL.html ) for homology based 3-D domain modelling where possible. PMID:9399865

  20. 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

  1. 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

  2. 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

  3. Cellular Actions of Insulin-Like Growth Factor Binding Proteins

    PubMed Central

    Ferry, R. J.; Katz, L. E. L.; Grimberg, Adda; Cohen, P.; Weinzimer, S. A.

    2014-01-01

    The insulin-like growth factors (IGFs), insulin-like growth factor binding proteins (IGFBPs), and the IGFBP proteases are involved in the regulation of somatic growth and cellular proliferation both in vivo and in vitro. IGFs are potent mitogenic agents whose actions are determined by the availability of free IGFs to interact with the IGF receptors. IGFBPs comprise a family of proteins that bind IGFs with high affinity and specificity and thereby regulate IGF-dependent actions. IGFBPs have recently emerged as IGF-independent regulators of cell growth. Various IGFBP association proteins as well as cleavage of IGFBPs by specific proteases modulate levels of free IGFs and IGFBPs. The ubiquity and complexity of the IGF axis promise exciting discoveries and applications for the future. PMID:10226802

  4. Method for Rapid Protein Identification in a Large Database

    PubMed Central

    Zhang, Wenli; Zhao, Xiaofang

    2013-01-01

    Protein identification is an integral part of proteomics research. The available tools to identify proteins in tandem mass spectrometry experiments are not optimized to face current challenges in terms of identification scale and speed owing to the exponential growth of the protein database and the accelerated generation of mass spectrometry data, as well as the demand for nonspecific digestion and post-modifications in complex-sample identification. As a result, a rapid method is required to mitigate such complexity and computation challenges. This paper thus aims to present an open method to prevent enzyme and modification specificity on a large database. This paper designed and developed a distributed program to facilitate application to computer resources. With this optimization, nearly linear speedup and real-time support are achieved on a large database with nonspecific digestion, thus enabling testing with two classical large protein databases in a 20-blade cluster. This work aids in the discovery of more significant biological results, such as modification sites, and enables the identification of more complex samples, such as metaproteomics samples. PMID:24000323

  5. InterPro: the integrative protein signature database.

    PubMed

    Hunter, Sarah; Apweiler, Rolf; Attwood, Teresa K; Bairoch, Amos; Bateman, Alex; Binns, David; Bork, Peer; Das, Ujjwal; Daugherty, Louise; Duquenne, Lauranne; Finn, Robert D; Gough, Julian; Haft, Daniel; Hulo, Nicolas; Kahn, Daniel; Kelly, Elizabeth; Laugraud, Aurélie; Letunic, Ivica; Lonsdale, David; Lopez, Rodrigo; Madera, Martin; Maslen, John; McAnulla, Craig; McDowall, Jennifer; Mistry, Jaina; Mitchell, Alex; Mulder, Nicola; Natale, Darren; Orengo, Christine; Quinn, Antony F; Selengut, Jeremy D; Sigrist, Christian J A; Thimma, Manjula; Thomas, Paul D; Valentin, Franck; Wilson, Derek; Wu, Cathy H; Yeats, Corin

    2009-01-01

    The InterPro database (http://www.ebi.ac.uk/interpro/) integrates together predictive models or 'signatures' representing protein domains, families and functional sites from multiple, diverse source databases: Gene3D, PANTHER, Pfam, PIRSF, PRINTS, ProDom, PROSITE, SMART, SUPERFAMILY and TIGRFAMs. Integration is performed manually and approximately half of the total approximately 58,000 signatures available in the source databases belong to an InterPro entry. Recently, we have started to also display the remaining un-integrated signatures via our web interface. Other developments include the provision of non-signature data, such as structural data, in new XML files on our FTP site, as well as the inclusion of matchless UniProtKB proteins in the existing match XML files. The web interface has been extended and now links out to the ADAN predicted protein-protein interaction database and the SPICE and Dasty viewers. The latest public release (v18.0) covers 79.8% of UniProtKB (v14.1) and consists of 16 549 entries. InterPro data may be accessed either via the web address above, via web services, by downloading files by anonymous FTP or by using the InterProScan search software (http://www.ebi.ac.uk/Tools/InterProScan/). PMID:18940856

  6. The Protein Identifier Cross-Referencing (PICR) service: reconciling protein identifiers across multiple source databases

    PubMed Central

    Côté, Richard G; Jones, Philip; Martens, Lennart; Kerrien, Samuel; Reisinger, Florian; Lin, Quan; Leinonen, Rasko; Apweiler, Rolf; Hermjakob, Henning

    2007-01-01

    Background Each major protein database uses its own conventions when assigning protein identifiers. Resolving the various, potentially unstable, identifiers that refer to identical proteins is a major challenge. This is a common problem when attempting to unify datasets that have been annotated with proteins from multiple data sources or querying data providers with one flavour of protein identifiers when the source database uses another. Partial solutions for protein identifier mapping exist but they are limited to specific species or techniques and to a very small number of databases. As a result, we have not found a solution that is generic enough and broad enough in mapping scope to suit our needs. Results We have created the Protein Identifier Cross-Reference (PICR) service, a web application that provides interactive and programmatic (SOAP and REST) access to a mapping algorithm that uses the UniProt Archive (UniParc) as a data warehouse to offer protein cross-references based on 100% sequence identity to proteins from over 70 distinct source databases loaded into UniParc. Mappings can be limited by source database, taxonomic ID and activity status in the source database. Users can copy/paste or upload files containing protein identifiers or sequences in FASTA format to obtain mappings using the interactive interface. Search results can be viewed in simple or detailed HTML tables or downloaded as comma-separated values (CSV) or Microsoft Excel (XLS) files suitable for use in a local database or a spreadsheet. Alternatively, a SOAP interface is available to integrate PICR functionality in other applications, as is a lightweight REST interface. Conclusion We offer a publicly available service that can interactively map protein identifiers and protein sequences to the majority of commonly used protein databases. Programmatic access is available through a standards-compliant SOAP interface or a lightweight REST interface. The PICR interface, documentation and

  7. CELLULAR AND MOLECULAR INTERACTIONS OF PHOSPHOINOSITIDES AND PERIPHERAL PROTEINS

    PubMed Central

    Stahelin, Robert V.; Scott, Jordan L.; Frick, Cary T.

    2015-01-01

    Anionic lipids act as signals for the recruitment of proteins containing cationic clusters to biological membranes. A family of anionic lipids known as the phosphoinositides (PIPs) are low in abundance, yet play a critical role in recruitment of peripheral proteins to the membrane interface. PIPs are mono-, bis-, or trisphosphorylated derivatives of phosphatidylinositol (PI) yielding seven species with different structure and anionic charge. The differential spatial distribution and temporal appearance of PIPs is key to their role in communicating information to target proteins. Selective recognition of PIPs came into play with the discovery that the substrate of protein kinase C termed pleckstrin possessed the first PIP binding region termed the pleckstrin homology (PH) domain. Since the discovery of the PH domain, more than ten PIP binding domains have been identified including PH, ENTH, FYVE, PX, and C2 domains. Representative examples of each of these domains have been thoroughly characterized to understand how they coordinate PIP headgroups in membranes, translocate to specific membrane docking sites in the cell, and function to regulate the activity of their full-length proteins. In addition, a number of novel mechanisms of PIP-mediated membrane association have emerged, such as coincidence detection – specificity for two distinct lipid headgroups. Other PIP-binding domains may also harbor selectivity for a membrane physical property such as charge or membrane curvature. This review summarizes the current understanding of the cellular distribution of PIPs and their molecular interaction with peripheral proteins. PMID:24556335

  8. Cellular unfolded protein response against viruses used in gene therapy

    PubMed Central

    Sen, Dwaipayan; Balakrishnan, Balaji; Jayandharan, Giridhara R.

    2014-01-01

    Viruses are excellent vehicles for gene therapy due to their natural ability to infect and deliver the cargo to specific tissues with high efficiency. Although such vectors are usually “gutted” and are replication defective, they are subjected to clearance by the host cells by immune recognition and destruction. Unfolded protein response (UPR) is a naturally evolved cyto-protective signaling pathway which is triggered due to endoplasmic reticulum (ER) stress caused by accumulation of unfolded/misfolded proteins in its lumen. The UPR signaling consists of three signaling pathways, namely PKR-like ER kinase, activating transcription factor 6, and inositol-requiring protein-1. Once activated, UPR triggers the production of ER molecular chaperones and stress response proteins to help reduce the protein load within the ER. This occurs by degradation of the misfolded proteins and ensues in the arrest of protein translation machinery. If the burden of protein load in ER is beyond its processing capacity, UPR can activate pro-apoptotic pathways or autophagy leading to cell death. Viruses are naturally evolved in hijacking the host cellular translation machinery to generate a large amount of proteins. This phenomenon disrupts ER homeostasis and leads to ER stress. Alternatively, in the case of gutted vectors used in gene therapy, the excess load of recombinant vectors administered and encountered by the cell can trigger UPR. Thus, in the context of gene therapy, UPR becomes a major roadblock that can potentially trigger inflammatory responses against the vectors and reduce the efficiency of gene transfer. PMID:24904562

  9. 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

  10. CentrosomeDB: a human centrosomal proteins database

    PubMed Central

    Nogales-Cadenas, Rubén; Abascal, Federico; Díez-Pérez, Javier; Carazo, José María; Pascual-Montano, Alberto

    2009-01-01

    Active research on the biology of the centrosome during the past decades has allowed the identification and characterization of many centrosomal proteins. Unfortunately, the accumulated data is still dispersed among heterogeneous sources of information. Here we present centrosome:db, which intends to compile and integrate relevant information related to the human centrosome. We have compiled a set of 383 likely human centrosomal genes and recorded the associated supporting evidences. Centrosome:db offers several perspectives to study the human centrosome including evolution, function and structure. The database contains information on the orthology relationships with other species, including fungi, nematodes, arthropods, urochordates and vertebrates. Predictions of the domain organization of centrosome:db proteins are graphically represented at different sections of the database, including sets of alternative protein isoforms, interacting proteins, groups of orthologs and the homologs identified with blast. Centrosome:db also contains information related to function, gene–disease associations, SNPs and the 3D structure of proteins. Apart from important differences in the coverage of the set of centrosomal genes, our database differentiates from other similar initiatives in the way information is treated and analyzed. Centrosome:db is publicly available at http://centrosome.dacya.ucm.es. PMID:18971254

  11. Cellular factors modulating the mechanism of tau protein aggregation.

    PubMed

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

    2015-05-01

    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

  12. Fluorescent Proteins in Cellular Organelles: Serious Pitfalls and Some Solutions

    PubMed Central

    Costantini, Lindsey M.

    2013-01-01

    Fluorescent proteins (FPs) have been powerful tools for cell biologists for over 15 years. The large variety of FPs available rarely comes with an instruction manual or a warning label. The potential pitfalls of the use of FPs in cellular organelles represent a significant concern for investigators. FPs generally did not evolve in the often distinctive physicochemical environments of subcellular organelles. In organelles, FPs can misfold, go dark, and even distort organelle morphology. In this minireview, we describe the issues associated with FPs in organelles and provide solutions to enable investigators to better exploit FP technology in cells. PMID:23971632

  13. 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

  14. Protein engineering strategies with potential applications for altering clinically relevant cellular pathways at the protein level.

    PubMed

    Regan, Lynne; Hinrichsen, Michael R; Oi, Curran

    2016-05-01

    All diseases can be fundamentally viewed as the result of malfunctioning cellular pathways. Protein engineering offers the potential to develop new tools that will allow these dysfunctional pathways to be better understood, in addition to potentially providing new routes to restore proper function. Here we discuss different approaches that can be used to change the intracellular activity of a protein by intervening at the protein level: targeted protein sequestration, protein recruitment, protein degradation, and selective inhibition of binding interfaces. The potential of each of these tools to be developed into effective therapeutic treatments will also be discussed, along with any major barriers that currently block their translation into the clinic. PMID:27031866

  15. InterPro: the integrative protein signature database

    PubMed Central

    Hunter, Sarah; Apweiler, Rolf; Attwood, Teresa K.; Bairoch, Amos; Bateman, Alex; Binns, David; Bork, Peer; Das, Ujjwal; Daugherty, Louise; Duquenne, Lauranne; Finn, Robert D.; Gough, Julian; Haft, Daniel; Hulo, Nicolas; Kahn, Daniel; Kelly, Elizabeth; Laugraud, Aurélie; Letunic, Ivica; Lonsdale, David; Lopez, Rodrigo; Madera, Martin; Maslen, John; McAnulla, Craig; McDowall, Jennifer; Mistry, Jaina; Mitchell, Alex; Mulder, Nicola; Natale, Darren; Orengo, Christine; Quinn, Antony F.; Selengut, Jeremy D.; Sigrist, Christian J. A.; Thimma, Manjula; Thomas, Paul D.; Valentin, Franck; Wilson, Derek; Wu, Cathy H.; Yeats, Corin

    2009-01-01

    The InterPro database (http://www.ebi.ac.uk/interpro/) integrates together predictive models or ‘signatures’ representing protein domains, families and functional sites from multiple, diverse source databases: Gene3D, PANTHER, Pfam, PIRSF, PRINTS, ProDom, PROSITE, SMART, SUPERFAMILY and TIGRFAMs. Integration is performed manually and approximately half of the total ∼58 000 signatures available in the source databases belong to an InterPro entry. Recently, we have started to also display the remaining un-integrated signatures via our web interface. Other developments include the provision of non-signature data, such as structural data, in new XML files on our FTP site, as well as the inclusion of matchless UniProtKB proteins in the existing match XML files. The web interface has been extended and now links out to the ADAN predicted protein–protein interaction database and the SPICE and Dasty viewers. The latest public release (v18.0) covers 79.8% of UniProtKB (v14.1) and consists of 16 549 entries. InterPro data may be accessed either via the web address above, via web services, by downloading files by anonymous FTP or by using the InterProScan search software (http://www.ebi.ac.uk/Tools/InterProScan/). PMID:18940856

  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. Efficient cellular solid-state NMR of membrane proteins by targeted protein labeling.

    PubMed

    Baker, Lindsay A; Daniëls, Mark; van der Cruijsen, Elwin A W; Folkers, Gert E; Baldus, Marc

    2015-06-01

    Solid-state NMR spectroscopy (ssNMR) has made significant progress towards the study of membrane proteins in their native cellular membranes. However, reduced spectroscopic sensitivity and high background signal levels can complicate these experiments. Here, we describe a method for ssNMR to specifically label a single protein by repressing endogenous protein expression with rifampicin. Our results demonstrate that treatment of E. coli with rifampicin during induction of recombinant membrane protein expression reduces background signals for different expression levels and improves sensitivity in cellular membrane samples. Further, the method reduces the amount of time and resources needed to produce membrane protein samples, enabling new strategies for studying challenging membrane proteins by ssNMR. PMID:25956570

  18. 3-D lookup: Fast protein structure database searches

    SciTech Connect

    Holm. L.; Sander, C.

    1995-12-31

    There are far fewer classes of three-dimensional protein folds than sequence families but the problem of detecting three-dimensional similarities is NP-complete. We present a novel heuristic for identifying 3-D similarities between a query structure and the database of known protein structures. Many methods for structure alignment use a bottom-up approach, identifying first local matches and then solving a combinatorial problem in building up larger clusters of matching substructures. Here the top-down approach is to start with the global comparison and select a rough superimposition using a fast 3-D lookup of secondary structure motifs. The superimposition is then extended to an alignment of C{sup {alpha}} atoms by an iterative dynamic programming step. An all-against-all comparison of 385-representative proteins (150,000 pair comparisons) took 1 day of computer time on a single R8000 processor. In other words, one query structure is scanned against the database in a matter of minutes. The method is rated at 90% reliability at capturing statistically significant similarities. It is useful as a rapid preprocessor to a comprehensive protein structure database search system.

  19. 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.

  20. 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

  1. 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

  2. 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

  3. Distinctive Behaviors of Druggable Proteins in Cellular Networks

    PubMed Central

    Workman, Paul; Al-Lazikani, Bissan

    2015-01-01

    The interaction environment of a protein in a cellular network is important in defining the role that the protein plays in the system as a whole, and thus its potential suitability as a drug target. Despite the importance of the network environment, it is neglected during target selection for drug discovery. Here, we present the first systematic, comprehensive computational analysis of topological, community and graphical network parameters of the human interactome and identify discriminatory network patterns that strongly distinguish drug targets from the interactome as a whole. Importantly, we identify striking differences in the network behavior of targets of cancer drugs versus targets from other therapeutic areas and explore how they may relate to successful drug combinations to overcome acquired resistance to cancer drugs. We develop, computationally validate and provide the first public domain predictive algorithm for identifying druggable neighborhoods based on network parameters. We also make available full predictions for 13,345 proteins to aid target selection for drug discovery. All target predictions are available through canSAR.icr.ac.uk. Underlying data and tools are available at https://cansar.icr.ac.uk/cansar/publications/druggable_network_neighbourhoods/. PMID:26699810

  4. WDSPdb: a database for WD40-repeat proteins

    PubMed Central

    Wang, Yang; Hu, Xue-Jia; Zou, Xu-Dong; Wu, Xian-Hui; Ye, Zhi-Qiang; Wu, Yun-Dong

    2015-01-01

    WD40-repeat proteins, as one of the largest protein families, often serve as platforms to assemble functional complexes through the hotspot residues on their domain surfaces, and thus play vital roles in many biological processes. Consequently, it is highly required for researchers who study WD40 proteins and protein–protein interactions to obtain structural information of WD40 domains. Systematic identification of WD40-repeat proteins, including prediction of their secondary structures, tertiary structures and potential hotspot residues responsible for protein–protein interactions, may constitute a valuable resource upon this request. To achieve this goal, we developed a specialized database WDSPdb (http://wu.scbb.pkusz.edu.cn/wdsp/) to provide these details of WD40-repeat proteins based on our recently published method WDSP. The WDSPdb contains 63 211 WD40-repeat proteins identified from 3383 species, including most well-known model organisms. To better serve the community, we implemented a user-friendly interactive web interface to browse, search and download the secondary structures, 3D structure models and potential hotspot residues provided by WDSPdb. PMID:25348404

  5. 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

  6. 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

  7. Cellular Biochemistry Methods for Investigating Protein Tyrosine Phosphatases

    PubMed Central

    Stanford, Stephanie M.; Ahmed, Vanessa

    2014-01-01

    Abstract Significance: The protein tyrosine phosphatases (PTPs) are a family of proteins that play critical roles in cellular signaling and influence many aspects of human health and disease. Although a wealth of information has been collected about PTPs since their discovery, many questions regarding their regulation and function still remain. Critical Issues: Of particular importance are the elucidation of the biological substrates of individual PTPs and understanding of the chemical and biological basis for temporal and spatial resolution of PTP activity within a cell. Recent Advances: Drawing from recent advances in both biology and chemistry, innovative approaches have been developed to study the intracellular biochemistry and physiology of PTPs. We provide a summary of PTP-tailored techniques and approaches, emphasizing methodologies to study PTP activity within a cellular context. We first provide a discussion of methods for identifying PTP substrates, including substrate-trapping mutants and synthetic peptide libraries for substrate selectivity profiling. We next provide an overview of approaches for monitoring intracellular PTP activity, including a discussion of mechanistic-based probes, gel-based assays, substrates that can be used intracellularly, and assays tied to cell growth. Finally, we review approaches used for monitoring PTP oxidation, a key regulatory pathway for these enzymes, discussing the biotin switch method and variants of this approach, along with affinity trapping techniques and probes designed to detect PTP oxidation. Future Directions: Further development of approaches to investigate the intracellular PTP activity and functions will provide specific insight into their mechanisms of action and control of diverse signaling pathways. Antioxid. Redox Signal. 20, 2160–2178. PMID:24294920

  8. Fluorescent, bioactive protein nanoparticles (prodots) for rapid, improved cellular uptake.

    PubMed

    Deshapriya, Inoka K; Stromer, Bobbi S; Pattammattel, Ajith; Kim, Christina S; Iglesias-Bartolome, Ramiro; Gonzalez-Fajardo, Laura; Patel, Vyomesh; Gutkind, J Silvio; Lu, Xiuling; Kumar, Challa V

    2015-03-18

    A simple and effective method for synthesizing highly fluorescent, protein-based nanoparticles (Prodots) and their facile uptake into the cytoplasm of cells is described here. Prodots made from bovine serum albumin (nBSA), glucose oxidase (nGO), horseradish peroxidase (nHRP), catalase (nCatalase), and lipase (nLipase) were found to be 15-50 nm wide and have been characterized by gel electrophoresis, transmission electron microscopy (TEM), circular dichroism (CD), fluorescence spectroscopy, dynamic light scattering (DLS), and optical microscopic methods. Data showed that the secondary structure of the protein in Prodots is retained to a significant extent and specific activities of nGO, nHRP, nCatalase, and nLipase were 80%, 70%, 65%, and 50% of their respective unmodified enzyme activities. Calorimetric studies indicated that the denaturation temperatures of nGO and nBSA increased while those of other Prodots remained nearly unchanged, and accelerated storage half-lives of Prodots at 60 °C increased by 4- to 8-fold. Exposure of nGO and nBSA+ nGO to cells indicated rapid uptake within 1-3 h, accompanied by significant blebbing of the plasma membrane, but no uptake has been noted in the absence of nGO. The presence of nGO/glucose in the media facilitated the uptake, and hydrogen peroxide induced membrane permeability could be responsible for this rapid uptake of Prodots. In control studies, FITC alone did not enter the cell, BSA-FITC was not internalized even in the presence of nGO, and there has been no uptake of nBSA-FITC in the absence of nGO. These are the very first examples of very rapid cellular uptake of fluorescent nanoparticles into cells, particularly nanoparticles made from pure proteins. The current approach is a simple and efficient method for the preparation of bioactive, fluorescent protein nanoparticles of controllable size for cellular imaging, and cell uptake is under the control of two separate chemical triggers. PMID:25642999

  9. 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

  10. Ebolavirus Database: Gene and Protein Information Resource for Ebolaviruses

    PubMed Central

    Sekar, Kanagaraj

    2016-01-01

    Ebola Virus Disease (EVD) is a life-threatening haemorrhagic fever in humans. Even though there are many reports on EVD, the protein precursor functions and virulent factors of ebolaviruses remain poorly understood. Comparative analyses of Ebolavirus genomes will help in the identification of these important features. This prompted us to develop the Ebolavirus Database (EDB) and we have provided links to various tools that will aid researchers to locate important regions in both the genomes and proteomes of Ebolavirus. The genomic analyses of ebolaviruses will provide important clues for locating the essential and core functional genes. The aim of EDB is to act as an integrated resource for ebolaviruses and we strongly believe that the database will be a useful tool for clinicians, microbiologists, health care workers, and bioscience researchers. PMID:27190508

  11. 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

  12. 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

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

    PubMed Central

    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. PMID:18940865

  14. 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

  15. SAFE Software and FED Database to Uncover Protein-Protein Interactions using Gene Fusion Analysis.

    PubMed

    Tsagrasoulis, Dimosthenis; Danos, Vasilis; Kissa, Maria; Trimpalis, Philip; Koumandou, V Lila; Karagouni, Amalia D; Tsakalidis, Athanasios; Kossida, Sophia

    2012-01-01

    Domain Fusion Analysis takes advantage of the fact that certain proteins in a given proteome A, are found to have statistically significant similarity with two separate proteins in another proteome B. In other words, the result of a fusion event between two separate proteins in proteome B is a specific full-length protein in proteome A. In such a case, it can be safely concluded that the protein pair has a common biological function or even interacts physically. In this paper, we present the Fusion Events Database (FED), a database for the maintenance and retrieval of fusion data both in prokaryotic and eukaryotic organisms and the Software for the Analysis of Fusion Events (SAFE), a computational platform implemented for the automated detection, filtering and visualization of fusion events (both available at: http://www.bioacademy.gr/bioinformatics/projects/ProteinFusion/index.htm). Finally, we analyze the proteomes of three microorganisms using these tools in order to demonstrate their functionality. PMID:22267904

  16. Cellular Prion Protein Promotes Brucella Infection into Macrophages

    PubMed Central

    Watarai, Masahisa; Kim, Suk; Erdenebaatar, Janchivdorj; Makino, Sou-ichi; Horiuchi, Motohiro; Shirahata, Toshikazu; Sakaguchi, Suehiro; Katamine, Shigeru

    2003-01-01

    The products of the Brucella abortus virB gene locus, which are highly similar to conjugative DNA transfer system, enable the bacterium to replicate within macrophage vacuoles. The replicative phagosome is thought to be established by the interaction of a substrate of the VirB complex with macrophages, although the substrate and its host cellular target have not yet been identified. We report here that Hsp60, a member of the GroEL family of chaperonins, of B. abortus is capable of interacting directly or indirectly with cellular prion protein (PrPC) on host cells. Aggregation of PrPC tail-like formation was observed during bacterial swimming internalization into macrophages and PrPC was selectively incorporated into macropinosomes containing B. abortus. Hsp60 reacted strongly with serum from human brucellosis patients and was exposed on the bacterial surface via a VirB complex–associated process. Under in vitro and in vivo conditions, Hsp60 of B. abortus bound to PrPC. Hsp60 of B. abortus, expressed on the surface of Lactococcus lactis, promoted the aggregation of PrPC but not PrPC tail formation on macrophages. The PrPC deficiency prevented swimming internalization and intracellular replication of B. abortus, with the result that phagosomes bearing the bacteria were targeted into the endocytic network. These results indicate that signal transduction induced by the interaction between bacterial Hsp60 and PrPC on macrophages contributes to the establishment of B. abortus infection. PMID:12847134

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

    PubMed Central

    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. PMID:23193284

  18. 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

  19. TuberQ: a Mycobacterium tuberculosis protein druggability database

    PubMed Central

    Radusky, Leandro; Lanzarotti, Esteban; Luque, Javier; Barril, Xavier; Marti, Marcelo A.; Turjanski, Adrián G.

    2014-01-01

    In 2012 an estimated 8.6 million people developed tuberculosis (TB) and 1.3 million died from the disease [including 320 000 deaths among human immunodeficiency virus (HIV)-positive people]. There is an urgent need for new anti-TB drugs owing to the following: the fact that current treatments have severe side effects, the increasing emergence of multidrug-resistant strains of Mycobacterium tuberculosis (Mtb), the negative drug–drug interactions with certain HIV (or other disease) treatments and the ineffectiveness against dormant Mtb. In this context we present here the TuberQ database, a novel resource for all researchers working in the field of drug development in TB. The main feature of TuberQ is to provide a druggability analysis of Mtb proteins in a consistent and effective manner, contributing to a better selection of potential drug targets for screening campaigns and the analysis of targets for structure-based drug design projects. The structural druggability analysis is combined with features related to the characteristics of putative inhibitor binding pockets and with functional and biological data of proteins. The structural analysis is performed on all available unique Mtb structures and high-quality structural homology-based models. This information is shown in an interactive manner, depicting the protein structure, the pockets and the associated characteristics for each protein. TuberQ also provides information about gene essentiality information, as determined from whole cell–based knockout experiments, and expression information obtained from microarray experiments done in different stress-related conditions. We hope that TuberQ will be a powerful tool for researchers working in TB and eventually will lead to the identification of novel putative targets and progresses in therapeutic activities. Database URL: http://tuberq.proteinq.com.ar/ PMID:24816183

  20. 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

  1. 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

  2. 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

  3. 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

  4. Identification of Cellular Proteins that Interact with Human Cytomegalovirus Immediate-Early Protein 1 by Protein Array Assay

    PubMed Central

    Puerta Martínez, Francisco; Tang, Qiyi

    2013-01-01

    Human cytomegalovirus (HCMV) gene expression during infection is characterized as a sequential process including immediate-early (IE), early (E), and late (L)-stage gene expression. The most abundantly expressed gene at the IE stage of infection is the major IE (MIE) gene that produces IE1 and IE2. IE1 has been the focus of study because it is an important protein, not only for viral gene expression but also for viral replication. It is believed that IE1 plays important roles in viral gene regulation by interacting with cellular proteins. In the current study, we performed protein array assays and identified 83 cellular proteins that interact with IE1. Among them, seven are RNA-binding proteins that are important in RNA processing; more than half are nuclear proteins that are involved in gene regulations. Tumorigenesis-related proteins are also found to interact with IE1, implying that the role of IE1 in tumorigenesis might need to be reevaluated. Unexpectedly, cytoplasmic proteins, such as Golgi autoantigen and GGA1 (both related to the Golgi trafficking protein), are also found to be associated with IE1. We also employed a coimmunoprecipitation assay to test the interactions of IE1 and some of the proteins identified in the protein array assays and confirmed that the results from the protein array assays are reliable. Many of the proteins identified by the protein array assay have not been previously reported. Therefore, the functions of the IE1-protein interactions need to be further explored in the future. PMID:24385082

  5. 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

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

    PubMed

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

    2016-03-14

    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. PMID:26804616

  7. The Protein Information Resource (PIR) and the PIR-International Protein Sequence Database.

    PubMed Central

    George, D G; Dodson, R J; Garavelli, J S; Haft, D H; Hunt, L T; Marzec, C R; Orcutt, B C; Sidman, K E; Srinivasarao, G Y; Yeh, L S; Arminski, L M; Ledley, R S; Tsugita, A; Barker, W C

    1997-01-01

    From its origin, the PIR has aspired to support research in computational biology and genomics through the compilation of a comprehensive, quality controlled and well-organized protein sequence information resource. The resource originated with the pioneering work of the late Margaret O. Dayhoff in the early 1960s. Since 1988, the Protein Sequence Database has been maintained collaboratively by PIR-International, an association of macromolecular sequence data collection centers dedicated to fostering international cooperation as an essential element in the development of scientific databases. The work of the resource is widely distributed and is available on the World Wide Web, via FTP, E-mail server, CD-ROM and magnetic media. It is widely redistributed and incorporated into many other protein sequence data compilations including SWISS-PROT and theEntrezsystem of the NCBI. PMID:9016497

  8. CancerPPD: a database of anticancer peptides and proteins

    PubMed Central

    Tyagi, Atul; Tuknait, Abhishek; Anand, Priya; Gupta, Sudheer; Sharma, Minakshi; Mathur, Deepika; Joshi, Anshika; Singh, Sandeep; Gautam, Ankur; Raghava, Gajendra P.S.

    2015-01-01

    CancerPPD (http://crdd.osdd.net/raghava/cancerppd/) is a repository of experimentally verified anticancer peptides (ACPs) and anticancer proteins. Data were manually collected from published research articles, patents and from other databases. The current release of CancerPPD consists of 3491 ACP and 121 anticancer protein entries. Each entry provides comprehensive information related to a peptide like its source of origin, nature of the peptide, anticancer activity, N- and C-terminal modifications, conformation, etc. Additionally, CancerPPD provides the information of around 249 types of cancer cell lines and 16 different assays used for testing the ACPs. In addition to natural peptides, CancerPPD contains peptides having non-natural, chemically modified residues and D-amino acids. Besides this primary information, CancerPPD stores predicted tertiary structures as well as peptide sequences in SMILES format. Tertiary structures of peptides were predicted using the state-of-art method, PEPstr and secondary structural states were assigned using DSSP. In order to assist users, a number of web-based tools have been integrated, these include keyword search, data browsing, sequence and structural similarity search. We believe that CancerPPD will be very useful in designing peptide-based anticancer therapeutics. PMID:25270878

  9. Characterizing protein interactions employing a genome-wide siRNA cellular phenotyping screen.

    PubMed

    Suratanee, Apichat; Schaefer, Martin H; Betts, Matthew J; Soons, Zita; Mannsperger, Heiko; Harder, Nathalie; Oswald, Marcus; Gipp, Markus; Ramminger, Ellen; Marcus, Guillermo; Männer, Reinhard; Rohr, Karl; Wanker, Erich; Russell, Robert B; Andrade-Navarro, Miguel A; Eils, Roland; König, Rainer

    2014-09-01

    Characterizing the activating and inhibiting effect of protein-protein interactions (PPI) is fundamental to gain insight into the complex signaling system of a human cell. A plethora of methods has been suggested to infer PPI from data on a large scale, but none of them is able to characterize the effect of this interaction. Here, we present a novel computational development that employs mitotic phenotypes of a genome-wide RNAi knockdown screen and enables identifying the activating and inhibiting effects of PPIs. Exemplarily, we applied our technique to a knockdown screen of HeLa cells cultivated at standard conditions. Using a machine learning approach, we obtained high accuracy (82% AUC of the receiver operating characteristics) by cross-validation using 6,870 known activating and inhibiting PPIs as gold standard. We predicted de novo unknown activating and inhibiting effects for 1,954 PPIs in HeLa cells covering the ten major signaling pathways of the Kyoto Encyclopedia of Genes and Genomes, and made these predictions publicly available in a database. We finally demonstrate that the predicted effects can be used to cluster knockdown genes of similar biological processes in coherent subgroups. The characterization of the activating or inhibiting effect of individual PPIs opens up new perspectives for the interpretation of large datasets of PPIs and thus considerably increases the value of PPIs as an integrated resource for studying the detailed function of signaling pathways of the cellular system of interest. PMID:25255318

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

    PubMed

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

    2014-01-01

    The analyses of protein-protein interactions are 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

  11. Methods for the Analysis of Protein Phosphorylation-Mediated Cellular Signaling Networks.

    PubMed

    White, Forest M; Wolf-Yadlin, Alejandro

    2016-06-12

    Protein phosphorylation-mediated cellular signaling networks regulate almost all aspects of cell biology, including the responses to cellular stimulation and environmental alterations. These networks are highly complex and comprise hundreds of proteins and potentially thousands of phosphorylation sites. Multiple analytical methods have been developed over the past several decades to identify proteins and protein phosphorylation sites regulating cellular signaling, and to quantify the dynamic response of these sites to different cellular stimulation. Here we provide an overview of these methods, including the fundamental principles governing each method, their relative strengths and weaknesses, and some examples of how each method has been applied to the analysis of complex signaling networks. When applied correctly, each of these techniques can provide insight into the topology, dynamics, and regulation of protein phosphorylation signaling networks. PMID:27049636

  12. Methods for the Analysis of Protein Phosphorylation–Mediated Cellular Signaling Networks

    NASA Astrophysics Data System (ADS)

    White, Forest M.; Wolf-Yadlin, Alejandro

    2016-06-01

    Protein phosphorylation–mediated cellular signaling networks regulate almost all aspects of cell biology, including the responses to cellular stimulation and environmental alterations. These networks are highly complex and comprise hundreds of proteins and potentially thousands of phosphorylation sites. Multiple analytical methods have been developed over the past several decades to identify proteins and protein phosphorylation sites regulating cellular signaling, and to quantify the dynamic response of these sites to different cellular stimulation. Here we provide an overview of these methods, including the fundamental principles governing each method, their relative strengths and weaknesses, and some examples of how each method has been applied to the analysis of complex signaling networks. When applied correctly, each of these techniques can provide insight into the topology, dynamics, and regulation of protein phosphorylation signaling networks.

  13. 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

  14. 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.

  15. The Crystalline Structure of Escherichia Coli Derived, - and Holo-Rat Cellular Retinol Binding Protein II

    NASA Astrophysics Data System (ADS)

    Winter, Nathan Shoup

    1993-01-01

    Crystal of apo- and holo-rat cellular retinol binding protein II from the recombinant protein isolated from E. coli were grown. X-ray data to about 2A resolution for both crystal forms were collected. The phases for both data sets were determined by the molecular replacement technique using cellular retinol binding protein. The structures were then refined. The electron density from bound retinol was observed in the holo-form. Other than the presence or absence of bound retinol, little difference was noted in the structures of the apo- and holo-protein. The retinol was bound in a interior cavity with the hydroxyl group in the center of the protein, and the ionone ring near the surface. The hydroxyl group of the retinol made a hydrogen bond to glutamine 108, and the amine group of lysine 40 came into Van der Waals contact with the isoprene chain. The structure of cellular retinol binding protein II was then compared with the structures of five other intracellular lipid binding proteins: adipocyte lipid binding protein, cellular retinol binding protein, intestinal fatty acid binding protein, p2 protein from myelin sheaths, and a midgut fatty acid binding protein.

  16. PhIN: A Protein Pharmacology Interaction Network Database

    PubMed Central

    Wang, Z; Li, J; Dang, R; Liang, L; Lin, J

    2015-01-01

    Network pharmacology is a new and hot concept in drug discovery for its ability to investigate the complexity of polypharmacology, and becomes more and more important in drug development. Here we report a protein pharmacology interaction network database (PhIN), aiming to assist multitarget drug discovery by providing comprehensive and flexible network pharmacology analysis. Overall, PhIN contains 1,126,060 target–target interaction pairs in terms of shared compounds and 3,428,020 pairs in terms of shared scaffolds, which involve 12,419,700 activity data, 9,414 targets, 314 viral targets, 652 pathways, 1,359,400 compounds, and 309,556 scaffolds. Using PhIN, users can obtain interacting target networks within or across human pathways, between human and virus, by defining the number of shared compounds or scaffolds under an activity cutoff. We expect PhIN to be a useful tool for multitarget drug development. PhIN is freely available at http://cadd.pharmacy.nankai.edu.cn/phin/. PMID:26225242

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

    PubMed

    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

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

    PubMed

    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. PMID:25792605

  19. ERM proteins: from cellular architecture to cell signaling.

    PubMed

    Louvet-Vallée, S

    2000-08-01

    ERM (ezrin/radixin/moesin) proteins, concentrated in actin rich cell-surface structures, cross-link actin filaments with the plasma membrane. They are involved in the formation of microvilli, cell-cell adhesion, maintenance of cell shape, cell motility and membrane trafficking. Recent analyses reveal that they are not only involved in cytoskeleton organization but also in signaling pathway. They play an important role in the activation of members of the Rho family by recruiting their regulators. The functions of ERM proteins are regulated by their conformational charges: the intramolecular interaction between the N- and C-terminal domains of ERM proteins charges masks several binding sites, leading to a dormant protein. Different activation signals regulate ERM proteins functions by modulating these intramolecular interactions. The involvement of ERM proteins in many signaling pathways has led to study their role during development of different species. PMID:11071040

  20. 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.

  1. The REF52 protein database. Methods of database construction and analysis using the QUEST system and characterizations of protein patterns from proliferating and quiescent REF52 cells.

    PubMed

    Garrels, J I; Franza, B R

    1989-03-25

    The construction and analysis of protein databases using the QUEST system is described, and the REF52 protein database is presented. A protein database provides the means to store and compare quantitative and descriptive data for up to 2000 proteins from many experiments that employ computer-analyzed two-dimensional gel electrophoresis. The QUEST system provides the tools to manage, analyze, and communicate these data. The REF52 database contains experiments with normal and transformed rat cell lines. In this report, many of the proteins on the REF52 map are identified by name, by subcellular localization, and by mode of post-translational modification. The quantitative experiments analyzed and compared here include 1) a study of the quantitative reproducibility of the analysis system, 2) a study of the clonal reproducibility of REF52 cells, 3) a study of growth-related changes in REF52 cells, and 4) a study of the effects of labeling cells for varying lengths of time. Of the proteins analyzed from REF52 cells, 10% are nuclear, 6% are phosphoproteins, and 4% are mannose-labeled glycoproteins. The mannose-labeled proteins are more prominent in patterns from quiescent cells, while the synthesis of cytoskeletal proteins is generally repressed at quiescence. A small set of proteins, selected for elevated rates of synthesis is generally repressed at quiescence. A small set of proteins, selected for elevated rates of synthesis in quiescent versus proliferating cells includes one of the tropomyosin isoforms, a myosin light chain isoform, and several prominent glycoproteins. These proteins are thought to be characteristic of the differentiated state of untransformed REF52 cells. Proteins induced early versus late after refeeding quiescent cells show very different patterns of growth regulation. These studies lay the foundations of the REF52 database and provide information needed to interpret the experiments with transformed REF52 cells, which are reported in the

  2. 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. PMID:26277093

  3. Cellular retinol-binding protein and retinoic acid-binding protein in rat testes: effect of retinol depletion.

    PubMed

    Ong, D E; Tsai, C H; Chytil, F

    1976-02-01

    Testes of rats contain two cellular binding proteins of interest in vitamin A metabolism. One protein binds retinoic acid with high specificity; the other binds retinol with high specificity. When the cellular retinol-binding protein was partially purified from rat testes, it exhibited fluorescence excitation and emission spectra similar to that of all-trans-retinol in hexane. Exposure of this preparation to UV light destroyed this fluorescence but spectra identical to the original were obtained after addition of retinol. Hexane extracts of the binding protein had fluorescence spectra identical to all-trans-retinol, suggesting that this compound is bound to the protein in vivo. Extracts of testes from retinol depleted rats were submitted to gel filtration but failed to show a retinol-like fluorescence at the elution position of retinol binding protein. This fluorescence was observed in the preparations from pair fed control animals. However, after addition of all-trans-retinol to the extracts from the depleted rats, fluorescence at that elution position was observed. This indicates that in testes of retinol depleted rats the cellular retinol binding protein is present but without bound retinol, in contrast to the non-depleted rats where 30-43% of the binding protein had bound retinol. The amounts of cellular retinol binding protein and retinoic acid binding protein in testes, as determined by sucrose gradient centrifugation, were found to be similar for retinol depleted and pair fed control rats. PMID:942996

  4. 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. PMID:26667279

  5. 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. PMID:26980515

  6. 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). PMID:17825976

  7. 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

  8. Determining the Sub-Cellular Localization of Proteins within Caenorhabditis elegans Body Wall Muscle

    PubMed Central

    Viveiros, Ryan; Warner, Adam; Plastino, Lorena; Lorch, Adam; Granger, Laure; Segalat, Laurent; Moerman, Donald G.

    2011-01-01

    Determining the sub-cellular localization of a protein within a cell is often an essential step towards understanding its function. In Caenorhabditis elegans, the relatively large size of the body wall muscle cells and the exquisite organization of their sarcomeres offer an opportunity to identify the precise position of proteins within cell substructures. Our goal in this study is to generate a comprehensive “localizome” for C. elegans body wall muscle by GFP-tagging proteins expressed in muscle and determining their location within the cell. For this project, we focused on proteins that we know are expressed in muscle and are orthologs or at least homologs of human proteins. To date we have analyzed the expression of about 227 GFP-tagged proteins that show localized expression in the body wall muscle of this nematode (e.g. dense bodies, M-lines, myofilaments, mitochondria, cell membrane, nucleus or nucleolus). For most proteins analyzed in this study no prior data on sub-cellular localization was available. In addition to discrete sub-cellular localization we observe overlapping patterns of localization including the presence of a protein in the dense body and the nucleus, or the dense body and the M-lines. In total we discern more than 14 sub-cellular localization patterns within nematode body wall muscle. The localization of this large set of proteins within a muscle cell will serve as an invaluable resource in our investigation of muscle sarcomere assembly and function. PMID:21611156

  9. Nanoparticle-cell interactions: molecular structure of the protein corona and cellular outcomes.

    PubMed

    Fleischer, Candace C; Payne, Christine K

    2014-08-19

    The use of nanoparticles (NPs) in biology and medicine requires a molecular-level understanding of how NPs interact with cells in a physiological environment. A critical difference between well-controlled in vitro experiments and in vivo applications is the presence of a complex mixture of extracellular proteins. It has been established that extracellular serum proteins present in blood will adsorb onto the surface of NPs, forming a "protein corona". Our goal was to understand how this protein layer affected cellular-level events, including NP binding, internalization, and transport. A combination of microscopy, which provides spatial resolution, and spectroscopy, which provides molecular information, is necessary to probe protein-NP-cell interactions. Initial experiments used a model system composed of polystyrene NPs functionalized with either amine or carboxylate groups to provide a cationic or anionic surface, respectively. Serum proteins adsorb onto the surface of both cationic and anionic NPs, forming a net anionic protein-NP complex. Although these protein-NP complexes have similar diameters and effective surface charges, they show the exact opposite behavior in terms of cellular binding. In the presence of bovine serum albumin (BSA), the cellular binding of BSA-NP complexes formed from cationic NPs is enhanced, whereas the cellular binding of BSA-NP complexes formed from anionic NPs is inhibited. These trends are independent of NP diameter or cell type. Similar results were obtained for anionic quantum dots and colloidal gold nanospheres. Using competition assays, we determined that BSA-NP complexes formed from anionic NPs bind to albumin receptors on the cell surface. BSA-NP complexes formed from cationic NPs are redirected to scavenger receptors. The observation that similar NPs with identical protein corona compositions bind to different cellular receptors suggested that a difference in the structure of the adsorbed protein may be responsible for the

  10. 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

  11. 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.

  12. Cellular functions of phosphatidylinositol 3-phosphate and FYVE domain proteins.

    PubMed Central

    Gillooly, D J; Simonsen, A; Stenmark, H

    2001-01-01

    PtdIns3P is a phosphoinositide 3-kinase product that has been strongly implicated in regulating membrane trafficking in both mammalian and yeast cells. PtdIns3P has been shown to be specifically located on membranes associated with the endocytic pathway. Proteins that contain FYVE zinc-finger domains are recruited to PtdIns3P-containing membranes. Structural information is now available concerning the interaction between FYVE domains and PtdIns3P. A number of proteins have been identified which contain a FYVE domain, and in this review we discuss the functions of PtdIns3P and its FYVE-domain-containing effector proteins in membrane trafficking, cytoskeletal regulation and receptor signalling. PMID:11284710

  13. 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.

  14. 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...

  15. SNAPPI-DB: a database and API of Structures, iNterfaces and Alignments for Protein-Protein Interactions.

    PubMed

    Jefferson, Emily R; Walsh, Thomas P; Roberts, Timothy J; Barton, Geoffrey J

    2007-01-01

    SNAPPI-DB, a high performance database of Structures, iNterfaces and Alignments of Protein-Protein Interactions, and its associated Java Application Programming Interface (API) is described. SNAPPI-DB contains structural data, down to the level of atom co-ordinates, for each structure in the Protein Data Bank (PDB) together with associated data including SCOP, CATH, Pfam, SWISSPROT, InterPro, GO terms, Protein Quaternary Structures (PQS) and secondary structure information. Domain-domain interactions are stored for multiple domain definitions and are classified by their Superfamily/Family pair and interaction interface. Each set of classified domain-domain interactions has an associated multiple structure alignment for each partner. The API facilitates data access via PDB entries, domains and domain-domain interactions. Rapid development, fast database access and the ability to perform advanced queries without the requirement for complex SQL statements are provided via an object oriented database and the Java Data Objects (JDO) API. SNAPPI-DB contains many features which are not available in other databases of structural protein-protein interactions. It has been applied in three studies on the properties of protein-protein interactions and is currently being employed to train a protein-protein interaction predictor and a functional residue predictor. The database, API and manual are available for download at: http://www.compbio.dundee.ac.uk/SNAPPI/downloads.jsp. PMID:17202171

  16. 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

  17. 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

  18. 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

  19. 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

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

    PubMed Central

    Stokes-Rees, Ian; Sliz, Piotr

    2010-01-01

    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. PMID:21098306

  1. 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. PMID:21098306

  2. Adaptations of proteins to cellular and subcellular pH

    PubMed Central

    2009-01-01

    Bioinformatics-based searches for correlations between subcellular localization and pI or charge distribution of proteins have failed to detect meaningful correlations. Recent work published in BMC Biology finds that a physicochemical metric of charge distribution correlates better with subcellular pH than does pI. See research article http://www.biomedcentral.com/1741-7007/7/69 PMID:20017887

  3. Adaptations of proteins to cellular and subcellular pH.

    PubMed

    Garcia-Moreno, Bertrand

    2009-01-01

    Bioinformatics-based searches for correlations between subcellular localization and pI or charge distribution of proteins have failed to detect meaningful correlations. Recent work published in BMC Biology finds that a physicochemical metric of charge distribution correlates better with subcellular pH than does pI. See research article http://www.biomedcentral.com/1741-7007/7/69. PMID:20017887

  4. 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

  5. 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

  6. 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

  7. A relational database of protein structures designed for flexible enquiries about conformation.

    PubMed

    Islam, S A; Sternberg, M J

    1989-03-01

    A relational database of protein structure has been developed to enable rapid and flexible enquiries about the occurrence of many aspects of protein architecture. The coordinates of 294 proteins from the Brookhaven Data Bank have been processed by standard computer programs to generate many additional terms that quantify aspects of protein structure. These terms include solvent accessibility, main-chain and side-chain dihedral angles, and secondary structure. In a relational database, the information is stored in tables with columns holding the different terms and rows holding the different entries for the terms. The different relational base tables store the information about the protein coordinate set, the different chains in the protein, the amino acid residues and ligands, the atomic coordinates, the salt bridges, the hydrogen bonds, the disulphide bridges and the close tertiary contacts. The database was established under ORACLE management system. Enquiries are constructed in ORACLE using SQL (structured query language) which is simple to use and alleviates the need for extensive computer programs. A single table can be searched for entries that meet various criteria, e.g. all protein solved to better than a given resolution. The power of the database occurs when several tables, or the entries in a single table, are cross-correlated. For example the dihedral angles of proline in the fourth position in an alpha-helix in high resolution structures can be rapidly obtained. The structural database provides a powerful tool to obtain empirical rules about protein conformation. This database of protein structures is part of a joint project between Birkbeck College and Leeds University to establish an integrated data resource of protein sequences and structures (ISIS) that encodes the complex patterns of residues and coordinates that define protein conformation. The entire data resource (ISIS) will provide a system to guide all areas of protein modelling including

  8. Redox modulation of cellular metabolism through targeted degradation of signaling proteins by the proteasome

    SciTech Connect

    Squier, Thomas C.

    2006-02-01

    Under conditions of oxidative stress, the 20S proteasome plays a critical role in maintaining cellular homeostasis through the selective degradation of oxidized and damaged proteins. This adaptive stress response is distinct from ubiquitin-dependent pathways in that oxidized proteins are recognized and degraded in an ATP-independent mechanism, which can involve the molecular chaperone Hsp90. Like the regulatory complexes 19S and 11S REG, Hsp90 tightly associates with the 20S proteasome to mediate the recognition of aberrant proteins for degradation. In the case of the calcium signaling protein calmodulin, proteasomal degradation results from the oxidation of a single surface exposed methionine (i.e., Met145); oxidation of the other eight methionines has a minimal effect on the recognition and degradation of calmodulin by the proteasome. Since cellular concentrations of calmodulin are limiting, the targeted degradation of this critical signaling protein under conditions of oxidative stress will result in the downregulation of cellular metabolism, serving as a feedback regulation to diminish the generation of reactive oxygen species. The targeted degradation of critical signaling proteins, such as calmodulin, can function as sensors of oxidative stress to downregulate global rates of metabolism and enhance cellular survival.

  9. 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

  10. Development of Human Protein Reference Database as an Initial Platform for Approaching Systems Biology in Humans

    PubMed Central

    Peri, Suraj; Navarro, J. Daniel; Amanchy, Ramars; Kristiansen, Troels Z.; Jonnalagadda, Chandra Kiran; Surendranath, Vineeth; Niranjan, Vidya; Muthusamy, Babylakshmi; Gandhi, T.K.B.; Gronborg, Mads; Ibarrola, Nieves; Deshpande, Nandan; Shanker, K.; Shivashankar, H.N.; Rashmi, B.P.; Ramya, M.A.; Zhao, Zhixing; Chandrika, K.N.; Padma, N.; Harsha, H.C.; Yatish, A.J.; Kavitha, M.P.; Menezes, Minal; Choudhury, Dipanwita Roy; Suresh, Shubha; Ghosh, Neelanjana; Saravana, R.; Chandran, Sreenath; Krishna, Subhalakshmi; Joy, Mary; Anand, Sanjeev K.; Madavan, V.; Joseph, Ansamma; Wong, Guang W.; Schiemann, William P.; Constantinescu, Stefan N.; Huang, Lily; Khosravi-Far, Roya; Steen, Hanno; Tewari, Muneesh; Ghaffari, Saghi; Blobe, Gerard C.; Dang, Chi V.; Garcia, Joe G.N.; Pevsner, Jonathan; Jensen, Ole N.; Roepstorff, Peter; Deshpande, Krishna S.; Chinnaiyan, Arul M.; Hamosh, Ada; Chakravarti, Aravinda; Pandey, Akhilesh

    2003-01-01

    Human Protein Reference Database (HPRD) is an object database that integrates a wealth of information relevant to the function of human proteins in health and disease. Data pertaining to thousands of protein-protein interactions, posttranslational modifications, enzyme/substrate relationships, disease associations, tissue expression, and subcellular localization were extracted from the literature for a nonredundant set of 2750 human proteins. Almost all the information was obtained manually by biologists who read and interpreted >300,000 published articles during the annotation process. This database, which has an intuitive query interface allowing easy access to all the features of proteins, was built by using open source technologies and will be freely available at http://www.hprd.org to the academic community. This unified bioinformatics platform will be useful in cataloging and mining the large number of proteomic interactions and alterations that will be discovered in the postgenomic era. PMID:14525934

  11. 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.

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

    NASA Astrophysics Data System (ADS)

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

    2004-05-01

    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.

  13. 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. PMID:23121485

  14. Cellular deficiency in the RGS10 protein facilitates chemoresistant ovarian cancer

    PubMed Central

    Hooks, Shelley B; Murph, Mandi M

    2015-01-01

    More than 30 regulators of G protein signaling (RGS) proteins encompass the RGS protein superfamily of critical regulators essential to cellular homeostasis. There is enormous structural and functional diversity among the RGS superfamily, and as such they serve a wide range of functions in regulating cell biology and physiology. Recent evidence has suggested roles for multiple RGS proteins in cancer initiation and progression, which has prompted research toward the potential modulation of these proteins as a new approach in cancer therapy. This article will discuss basic RGS molecular pharmacology, summarize the cellular functions and epigenetic regulation of RGS10, review ovarian cancer chemotherapy and describe the role of RGS10 in ovarian cancer survival signaling. PMID:26293348

  15. Nrf2 Protein Up-regulates Antiapoptotic Protein Bcl-2 and Prevents Cellular Apoptosis*

    PubMed Central

    Niture, Suryakant K.; Jaiswal, Anil K.

    2012-01-01

    Nuclear transcription factor Nrf2 regulates the expression and coordinated induction of a battery of genes encoding cytoprotective and drug transporter proteins in response to chemical and radiation stress. This leads to reduced apoptosis, enhanced cell survival, and increased drug resistance. In this study, we investigated the role of Nrf2 in up-regulation of antiapoptotic protein Bcl-2 and its contribution to stress-induced apoptosis and cell survival. Exposure of mouse hepatoma (Hepa-1) and human hepatoblastoma (HepG2) cells to antioxidant tert-butylhydroquinone led to induction of Bcl-2. Mutagenesis and transfection assays identified an antioxidant response element between nucleotides −3148 and −3140 on the reverse strand of the Bcl-2 gene promoter that was essential for activation of Bcl-2 gene expression. Band/supershift and ChIP assays demonstrated binding of Nrf2 to Bcl-2 antioxidant response element. Alterations in Nrf2 led to altered Bcl-2 induction and cellular apoptosis. Moreover, dysfunctional/mutant inhibitor of Nrf2 (INrf2) in human lung cancer cells failed to degrade Nrf2, resulting in an increased Bcl-2 level and decreased etoposide- and UV/γ radiation-mediated DNA fragmentation. In addition, siRNA-mediated down-regulation of Nrf2 also led to decreased apoptosis and increased cell survival. Furthermore, the specific knockdown of Bcl-2 in Nrf2-activated tumor cells led to increased etoposide-induced apoptosis and decreased cell survival and growth/proliferation. These data provide the first evidence of Nrf2 in control of Bcl-2 expression and apoptotic cell death with implications in antioxidant protection, survival of cancer cells, and drug resistance. PMID:22275372

  16. 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

  17. Ubiquitin ligase ITCH recruitment suppresses the aggregation and cellular toxicity of cytoplasmic misfolded proteins

    PubMed Central

    Chhangani, Deepak; Upadhyay, Arun; Amanullah, Ayeman; Joshi, Vibhuti; Mishra, Amit

    2014-01-01

    The protein quality control (QC) system protects cells against cellular toxicity induced by misfolded proteins and maintains overall cellular fitness. Inefficient clearance of or failure to degrade damaged proteins causes several diseases, especially age-linked neurodegenerative disorders. Attenuation of misfolded protein degradation under severe stress conditions leads to the rapid over-accumulation of toxic proteinaceous aggregates in the cytoplasmic compartment. However, the precise cytoplasmic quality control degradation mechanism is unknown. In the present study, we demonstrate that the Nedd4-like E3 ubiquitin ligase ITCH specifically interacts with mutant bona fide misfolded proteins and colocalizes with their perinuclear aggregates. In a cell culture model, we demonstrate ITCH recruitment by cytoplasmic inclusions containing polyglutamine-expanded huntingtin or ataxin-3 proteins. Transient overexpression of ITCH dramatically induced the degradation of thermally denatured misfolded luciferase protein. Partial depletion of ITCH increased the rate of aggregate formation and cell death generated by expanded polyglutamine proteins. Finally, we demonstrate that overexpression of ITCH alleviates the cytotoxic potential of expanded polyglutamine proteins and reduces aggregation. These observations indicate that ITCH is involved in the cytosolic quality control pathway and may help to explain how abnormal proteins are targeted by QC ubiquitin-protein ligases. PMID:24865853

  18. 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

  19. Identification of protein functions from a molecular surface database, eF-site.

    PubMed

    Kinoshita, Kengo; Furui, Jun'ichi; Nakamura, Haruki

    2002-01-01

    A bioinformatics method was developed to identify the protein surface around the functional site and to estimate the biochemical function, using a newly constructed molecular surface database named the eF-site (electrostatic surface of Functional site. Molecular surfaces of protein molecules were computed based on the atom coordinates, and the eF-site database was prepared by adding the physical properties on the constructed molecular surfaces. The electrostatic potential on each molecular surface was individually calculated solving the Poisson-Boltzmann equation numerically for the precise continuum model, and the hydrophobicity information of each residue was also included. The eF-site database is accessed by the internet (http://pi.protein.osaka-u.ac.jp/eF-site/). We have prepared four different databases, eF-site/antibody, eF-site/prosite, eF-site/P-site, and eF-site/ActiveSite, corresponding to the antigen binding sites of antibodies with the same orientations, the molecular surfaces for the individual motifs in PROSITE database, the phosphate binding sites, and the active site surfaces for the representatives of the individual protein family, respectively. An algorithm using the clique detection method as an applied graph theory was developed to search of the eF-site database, so as to recognize and discriminate the characteristic molecular surfaces of the proteins. The method identifies the active site having the similar function to those of the known proteins. PMID:12836670

  20. 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

  1. 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

  2. A cellular system that degrades misfolded proteins and protects against neurodegeneration.

    PubMed

    Guo, Lili; Giasson, Benoit I; Glavis-Bloom, Alex; Brewer, Michael D; Shorter, James; Gitler, Aaron D; Yang, Xiaolu

    2014-07-01

    Misfolded proteins compromise cellular function and cause disease. How these proteins are detected and degraded is not well understood. Here we show that PML/TRIM19 and the SUMO-dependent ubiquitin ligase RNF4 act together to promote the degradation of misfolded proteins in the mammalian cell nucleus. PML selectively interacts with misfolded proteins through distinct substrate recognition sites and conjugates these proteins with the small ubiquitin-like modifiers (SUMOs) through its SUMO ligase activity. SUMOylated misfolded proteins are then recognized and ubiquitinated by RNF4 and are subsequently targeted for proteasomal degradation. We further show that PML deficiency exacerbates polyglutamine (polyQ) disease in a mouse model of spinocerebellar ataxia 1 (SCA1). These findings reveal a mammalian system that removes misfolded proteins through sequential SUMOylation and ubiquitination and define its role in protection against protein-misfolding diseases. PMID:24882209

  3. 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

  4. Efficiency of database search for identification of mutated and modified proteins via mass spectrometry.

    PubMed

    Pevzner, P A; Mulyukov, Z; Dancik, V; Tang, C L

    2001-02-01

    Although protein identification by matching tandem mass spectra (MS/MS) against protein databases is a widespread tool in mass spectrometry, the question about reliability of such searches remains open. Absence of rigorous significance scores in MS/MS database search makes it difficult to discard random database hits and may lead to erroneous protein identification, particularly in the case of mutated or post-translationally modified peptides. This problem is especially important for high-throughput MS/MS projects when the possibility of expert analysis is limited. Thus, algorithms that sort out reliable database hits from unreliable ones and identify mutated and modified peptides are sought. Most MS/MS database search algorithms rely on variations of the Shared Peaks Count approach that scores pairs of spectra by the peaks (masses) they have in common. Although this approach proved to be useful, it has a high error rate in identification of mutated and modified peptides. We describe new MS/MS database search tools, MS-CONVOLUTION and MS-ALIGNMENT, which implement the spectral convolution and spectral alignment approaches to peptide identification. We further analyze these approaches to identification of modified peptides and demonstrate their advantages over the Shared Peaks Count. We also use the spectral alignment approach as a filter in a new database search algorithm that reliably identifies peptides differing by up to two mutations/modifications from a peptide in a database. PMID:11157792

  5. Monitoring protein phosphatase 1 isoform levels as a marker for cellular stress.

    PubMed

    Amador, Fátima Camões; Henriques, Ana Gabriela; da Cruz E Silva, Odete A B; da Cruz E Silva, Edgar F

    2004-01-01

    Reversible protein phosphorylation is a central mechanism regulating many biological functions, and abnormal protein phosphorylation can have a devastating impact on cellular control mechanisms, including a contributing role in neurodegenerative processes. Hence, many promising novel drug development strategies involve targeting protein phosphorylation systems. In this study, we demonstrate that various cellular stresses relevant to neurodegeneration can specifically affect the protein expression levels of protein phosphatase 1 (PP1). PP1 levels were altered upon exposure of PC12 and COS-1 cells to aluminium, Abeta peptides, sodium azide, and even heat shock. Particularly interesting, given PP1's involvement in aging and neurodegeneration, was the consistent decrease in PP1gamma(1) levels in response to stress agents. In fact, alterations in the expression levels of PP1 appear to correspond to an early response of stress induction, that is, before alterations in heat shock proteins can be detected. Our data suggest that monitoring PP1 isoform expression could constitute a useful diagnostic tool for cellular stress, possibly even neurodegeneration. Additionally, our results strengthen the rationale for signal transduction therapeutics and indicate that altering the specific activity of PP1 either directly or by targeting its regulatory proteins may be a useful therapeutic development strategy for the future. PMID:15113600

  6. 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

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

    PubMed

    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

  8. DBBP: database of binding pairs in protein-nucleic acid interactions

    PubMed Central

    2014-01-01

    Background Interaction of proteins with other molecules plays an important role in many biological activities. As many structures of protein-DNA complexes and protein-RNA complexes have been determined in the past years, several databases have been constructed to provide structure data of the complexes. However, the information on the binding sites between proteins and nucleic acids is not readily available from the structure data since the data consists mostly of the three-dimensional coordinates of the atoms in the complexes. Results We analyzed the huge amount of structure data for the hydrogen bonding interactions between proteins and nucleic acids and developed a database called DBBP (DataBase of Binding Pairs in protein-nucleic acid interactions, http://bclab.inha.ac.kr/dbbp). DBBP contains 44,955 hydrogen bonds (H-bonds) of protein-DNA interactions and 77,947 H-bonds of protein-RNA interactions. Conclusions Analysis of the huge amount of structure data of protein-nucleic acid complexes is labor-intensive, yet provides useful information for studying protein-nucleic acid interactions. DBBP provides the detailed information of hydrogen-bonding interactions between proteins and nucleic acids at various levels from the atomic level to the residue level. The binding information can be used as a valuable resource for developing a computational method aiming at predicting new binding sites in proteins or nucleic acids. PMID:25474259

  9. 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

  10. 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

  11. 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

  12. Finding the right RNA: identification of cellular mRNA substrates for RNA-binding proteins.

    PubMed Central

    Trifillis, P; Day, N; Kiledjian, M

    1999-01-01

    Defects in RNA-binding proteins have been implicated in human genetic disorders. However, efforts in understanding the functions of these proteins have been hampered by the inability to obtain their mRNA substrates. To identify cognate cellular mRNAs associated with an RNA-binding protein, we devised a strategy termed isolation of specific nucleic acids associated with proteins (SNAAP). The SNAAP technique allows isolation and subsequent identification of these mRNAs. To assess the validity of this approach, we utilized cellular mRNA and protein from K562 cells and alphaCP1, a protein implicated in a-globin mRNA stability, as a model system. Immobilization of an RNA-binding protein with the glutathione-S-transferase (GST) domain enables isolation of mRNA within an mRNP context and the identity of the bound mRNAs is determined by the differential display assay. The specificity of protein-RNA interactions was considerably enhanced when the interactions were carried out in the presence of cellular extract rather than purified components. Two of the mRNAs specifically bound by alphaCP1 were mRNAs encoding the transmembrane receptor protein, TAPA-1, and the mitochondrial cytochrome c oxidase subunit II enzyme, coxII. A specific poly(C)-sensitive complex formed on the TAPA-1 and coxII 3' UTRs consistent with the binding of aCP1. Furthermore, direct binding of purified alphaCP proteins to these 3' UTRs was demonstrated and the binding sites determined. These results support the feasibility of the SNAAP technique and suggest a broad applicability for the approach in identifying mRNA targets for clinically relevant RNA-binding proteins that will provide insights into their possible functions. PMID:10445881

  13. 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.

  14. Protein corona fingerprinting predicts the cellular interaction of gold and silver nanoparticles.

    PubMed

    Walkey, Carl D; Olsen, Jonathan B; Song, Fayi; Liu, Rong; Guo, Hongbo; Olsen, D Wesley H; Cohen, Yoram; Emili, Andrew; Chan, Warren C W

    2014-03-25

    Using quantitative models to predict the biological interactions of nanoparticles will accelerate the translation of nanotechnology. Here, we characterized the serum protein corona 'fingerprint' formed around a library of 105 surface-modified gold nanoparticles. Applying a bioinformatics-inspired approach, we developed a multivariate model that uses the protein corona fingerprint to predict cell association 50% more accurately than a model that uses parameters describing nanoparticle size, aggregation state, and surface charge. Our model implicates a set of hyaluronan-binding proteins as mediators of nanoparticle-cell interactions. This study establishes a framework for developing a comprehensive database of protein corona fingerprints and biological responses for multiple nanoparticle types. Such a database can be used to develop quantitative relationships that predict the biological responses to nanoparticles and will aid in uncovering the fundamental mechanisms of nano-bio interactions. PMID:24517450

  15. 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

  16. 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

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

    PubMed Central

    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. PMID:26673716

  18. 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. PMID:26673716

  19. 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

  20. 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.

  1. 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

  2. Impact of Protein Stability, Cellular Localization, and Abundance on Proteomic Detection of Tumor-Derived Proteins in Plasma

    PubMed Central

    Faca, Vitor M.; Zhang, Wenxuan; Zhang, Qing; Jain, Anjali; Hanash, Sam; Agus, David B.; McIntosh, Martin W.; Mallick, Parag

    2011-01-01

    Tumor-derived, circulating proteins are potentially useful as biomarkers for detection of cancer, for monitoring of disease progression, regression and recurrence, and for assessment of therapeutic response. Here we interrogated how a protein's stability, cellular localization, and abundance affect its observability in blood by mass-spectrometry-based proteomics techniques. We performed proteomic profiling on tumors and plasma from two different xenograft mouse models. A statistical analysis of this data revealed protein properties indicative of the detection level in plasma. Though 20% of the proteins identified in plasma were tumor-derived, only 5% of the proteins observed in the tumor tissue were found in plasma. Both intracellular and extracellular tumor proteins were observed in plasma; however, after normalizing for tumor abundance, extracellular proteins were seven times more likely to be detected. Although proteins that were more abundant in the tumor were also more likely to be observed in plasma, the relationship was nonlinear: Doubling the spectral count increased detection rate by only 50%. Many secreted proteins, even those with relatively low spectral count, were observed in plasma, but few low abundance intracellular proteins were observed. Proteins predicted to be stable by dipeptide composition were significantly more likely to be identified in plasma than less stable proteins. The number of tryptic peptides in a protein was not significantly related to the chance of a protein being observed in plasma. Quantitative comparison of large versus small tumors revealed that the abundance of proteins in plasma as measured by spectral count was associated with the tumor size, but the relationship was not one-to-one; a 3-fold decrease in tumor size resulted in a 16-fold decrease in protein abundance in plasma. This study provides quantitative support for a tumor-derived marker prioritization strategy that favors secreted and stable proteins over all but the

  3. Impact of protein stability, cellular localization, and abundance on proteomic detection of tumor-derived proteins in plasma.

    PubMed

    Fang, Qiaojun; Kani, Kian; Faca, Vitor M; Zhang, Wenxuan; Zhang, Qing; Jain, Anjali; Hanash, Sam; Agus, David B; McIntosh, Martin W; Mallick, Parag

    2011-01-01

    Tumor-derived, circulating proteins are potentially useful as biomarkers for detection of cancer, for monitoring of disease progression, regression and recurrence, and for assessment of therapeutic response. Here we interrogated how a protein's stability, cellular localization, and abundance affect its observability in blood by mass-spectrometry-based proteomics techniques. We performed proteomic profiling on tumors and plasma from two different xenograft mouse models. A statistical analysis of this data revealed protein properties indicative of the detection level in plasma. Though 20% of the proteins identified in plasma were tumor-derived, only 5% of the proteins observed in the tumor tissue were found in plasma. Both intracellular and extracellular tumor proteins were observed in plasma; however, after normalizing for tumor abundance, extracellular proteins were seven times more likely to be detected. Although proteins that were more abundant in the tumor were also more likely to be observed in plasma, the relationship was nonlinear: Doubling the spectral count increased detection rate by only 50%. Many secreted proteins, even those with relatively low spectral count, were observed in plasma, but few low abundance intracellular proteins were observed. Proteins predicted to be stable by dipeptide composition were significantly more likely to be identified in plasma than less stable proteins. The number of tryptic peptides in a protein was not significantly related to the chance of a protein being observed in plasma. Quantitative comparison of large versus small tumors revealed that the abundance of proteins in plasma as measured by spectral count was associated with the tumor size, but the relationship was not one-to-one; a 3-fold decrease in tumor size resulted in a 16-fold decrease in protein abundance in plasma. This study provides quantitative support for a tumor-derived marker prioritization strategy that favors secreted and stable proteins over all but the

  4. MitoNuc: a database of nuclear genes coding for mitochondrial proteins. Update 2002.

    PubMed

    Attimonelli, Marcella; Catalano, Domenico; Gissi, Carmela; Grillo, Giorgio; Licciulli, Flavio; Liuni, Sabino; Santamaria, Monica; Pesole, Graziano; Saccone, Cecilia

    2002-01-01

    Mitochondria, besides their central role in energy metabolism, have recently been found to be involved in a number of basic processes of cell life and to contribute to the pathogenesis of many degenerative diseases. All functions of mitochondria depend on the interaction of nuclear and organelle genomes. Mitochondrial genomes have been extensively sequenced and analysed and data have been collected in several specialised databases. In order to collect information on nuclear coded mitochondrial proteins we developed MitoNuc, a database containing detailed information on sequenced nuclear genes coding for mitochondrial proteins in Metazoa. The MitoNuc database can be retrieved through SRS and is available via the web site http://bighost.area.ba.cnr.it/mitochondriome where other mitochondrial databases developed by our group, the complete list of the sequenced mitochondrial genomes, links to other mitochondrial sites and related information, are available. The MitoAln database, related to MitoNuc in the previous release, reporting the multiple alignments of the relevant homologous protein coding regions, is no longer supported in the present release. In order to keep the links among entries in MitoNuc from homologous proteins, a new field in the database has been defined: the cluster identifier, an alpha numeric code used to identify each cluster of homologous proteins. A comment field derived from the corresponding SWISS-PROT entry has been introduced; this reports clinical data related to dysfunction of the protein. The logic scheme of MitoNuc database has been implemented in the ORACLE DBMS. This will allow the end-users to retrieve data through a friendly interface that will be soon implemented. PMID:11752284

  5. MitoNuc: a database of nuclear genes coding for mitochondrial proteins. Update 2002

    PubMed Central

    Attimonelli, Marcella; Catalano, Domenico; Gissi, Carmela; Grillo, Giorgio; Licciulli, Flavio; Liuni, Sabino; Santamaria, Monica; Pesole, Graziano; Saccone, Cecilia

    2002-01-01

    Mitochondria, besides their central role in energy metabolism, have recently been found to be involved in a number of basic processes of cell life and to contribute to the pathogenesis of many degenerative diseases. All functions of mitochondria depend on the interaction of nuclear and organelle genomes. Mitochondrial genomes have been extensively sequenced and analysed and data have been collected in several specialised databases. In order to collect information on nuclear coded mitochondrial proteins we developed MitoNuc, a database containing detailed information on sequenced nuclear genes coding for mitochondrial proteins in Metazoa. The MitoNuc database can be retrieved through SRS and is available via the web site http://bighost.area.ba.cnr.it/mitochondriome where other mitochondrial databases developed by our group, the complete list of the sequenced mitochondrial genomes, links to other mitochondrial sites and related information, are available. The MitoAln database, related to MitoNuc in the previous release, reporting the multiple alignments of the relevant homologous protein coding regions, is no longer supported in the present release. In order to keep the links among entries in MitoNuc from homologous proteins, a new field in the database has been defined: the cluster identifier, an alpha numeric code used to identify each cluster of homologous proteins. A comment field derived from the corresponding SWISS-PROT entry has been introduced; this reports clinical data related to dysfunction of the protein. The logic scheme of MitoNuc database has been implemented in the ORACLE DBMS. This will allow the end-users to retrieve data through a friendly interface that will be soon implemented. PMID:11752284

  6. 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

  7. 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

  8. 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

  9. 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

  10. 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

  11. GALT protein database: querying structural and functional features of GALT enzyme.

    PubMed

    d'Acierno, Antonio; Facchiano, Angelo; Marabotti, Anna

    2014-09-01

    Knowledge of the impact of variations on protein structure can enhance the comprehension of the mechanisms of genetic diseases related to that protein. Here, we present a new version of GALT Protein Database, a Web-accessible data repository for the storage and interrogation of structural effects of variations of the enzyme galactose-1-phosphate uridylyltransferase (GALT), the impairment of which leads to classic Galactosemia, a rare genetic disease. This new version of this database now contains the models of 201 missense variants of GALT enzyme, including heterozygous variants, and it allows users not only to retrieve information about the missense variations affecting this protein, but also to investigate their impact on substrate binding, intersubunit interactions, stability, and other structural features. In addition, it allows the interactive visualization of the models of variants collected into the database. We have developed additional tools to improve the use of the database by nonspecialized users. This Web-accessible database (http://bioinformatica.isa.cnr.it/GALT/GALT2.0) represents a model of tools potentially suitable for application to other proteins that are involved in human pathologies and that are subjected to genetic variations. PMID:24990533

  12. Novel Mechanism of Regulation of Tomato Bushy Stunt Virus Replication by Cellular WW-Domain Proteins

    PubMed Central

    Barajas, Daniel; Kovalev, Nikolay; Qin, Jun

    2014-01-01

    ABSTRACT Replication of (+)RNA viruses depends on several co-opted host proteins but is also under the control of cell-intrinsic restriction factors (CIRFs). By using tombusviruses, small model viruses of plants, we dissect the mechanism of inhibition of viral replication by cellular WW-domain-containing proteins, which act as CIRFs. By using fusion proteins between the WW domain and the p33 replication protein, we show that the WW domain inhibits the ability of p33 to bind to the viral RNA and to other p33 and p92 replication proteins leading to inhibition of viral replication in yeast and in a cell extract. Overexpression of WW-domain protein in yeast also leads to reduction of several co-opted host factors in the viral replicase complex (VRC). These host proteins, such as eEF1A, Cdc34 E2 ubiquitin-conjugating enzyme, and ESCRT proteins (Bro1p and Vps4p), are known to be involved in VRC assembly. Simultaneous coexpression of proviral cellular factors with WW-domain protein partly neutralizes the inhibitory effect of the WW-domain protein. We propose that cellular WW-domain proteins act as CIRFs and also as regulators of tombusvirus replication by inhibiting the assembly of new membrane-bound VRCs at the late stage of infection. We suggest that tombusviruses could sense the status of the infected cells via the availability of cellular susceptibility factors versus WW-domain proteins for binding to p33 replication protein that ultimately controls the formation of new VRCs. This regulatory mechanism might explain how tombusviruses could adjust the efficiency of RNA replication to the limiting resources of the host cells during infections. IMPORTANCE Replication of positive-stranded RNA viruses, which are major pathogens of plants, animals, and humans, is inhibited by several cell-intrinsic restriction factors (CIRFs) in infected cells. We define here the inhibitory roles of the cellular Rsp5 ubiquitin ligase and its WW domain in plant-infecting tombusvirus

  13. 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.

  14. 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

  15. 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.

  16. Correlation between Protein Sequence Similarity and Crystallization Reagents in the Biological Macromolecule Crystallization Database

    PubMed Central

    Lu, Hui-Meng; Yin, Da-Chuan; Liu, Yong-Ming; Guo, Wei-Hong; Zhou, Ren-Bin

    2012-01-01

    The protein structural entries grew far slower than the sequence entries. This is partly due to the bottleneck in obtaining diffraction quality protein crystals for structural determination using X-ray crystallography. The first step to achieve protein crystallization is to find out suitable chemical reagents. However, it is not an easy task. Exhausting trial and error tests of numerous combinations of different reagents mixed with the protein solution are usually necessary to screen out the pursuing crystallization conditions. Therefore, any attempts to help find suitable reagents for protein crystallization are helpful. In this paper, an analysis of the relationship between the protein sequence similarity and the crystallization reagents according to the information from the existing databases is presented. We extracted information of reagents and sequences from the Biological Macromolecule Crystallization Database (BMCD) and the Protein Data Bank (PDB) database, classified the proteins into different clusters according to the sequence similarity, and statistically analyzed the relationship between the sequence similarity and the crystallization reagents. The results showed that there is a pronounced positive correlation between them. Therefore, according to the correlation, prediction of feasible chemical reagents that are suitable to be used in crystallization screens for a specific protein is possible. PMID:22949812

  17. Influenza A virus non-structural protein 1 (NS1) interacts with cellular multifunctional protein nucleolin during infection.

    PubMed

    Murayama, Rikinori; Harada, Yuichi; Shibata, Toshikatsu; Kuroda, Kazumichi; Hayakawa, Satoshi; Shimizu, Kazufumi; Tanaka, Torahiko

    2007-11-01

    Influenza A virus non-structural protein 1 (NS1) is the most important viral regulatory factor that controls cellular processes to facilitate viral replication. To gain further insight into the role of NS1, we tried to find novel cellular factors that interact with NS1. The complexes of NS1 and target proteins were pulled down from an infected cell lysate using anti-NS1 (A/Udorn/72) single-chain Fv and identified by peptide mass fingerprinting analysis. We identified nucleolin, a multifunctional major nucleolar protein, as a novel NS1-binding protein. The RNA-binding domain of NS1 was responsible for this binding, as judged by a GST (glutathione S-transferase) pull-down assay with the GST-fused functional domains of NS1. By laser confocal microscopy, we observed the co-localization of NS1 with nucleolin most clearly in the nucleoli, indicating that NS1 is interacting with nucleolin during infection. Our results suggest a novel function of NS1, namely, affecting cellular events via interaction with nucleolin. PMID:17767916

  18. 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

  19. 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

  20. PTM-SD: a database of structurally resolved and annotated posttranslational modifications in proteins

    PubMed Central

    Craveur, Pierrick; Rebehmed, Joseph; de Brevern, Alexandre G.

    2014-01-01

    Posttranslational modifications (PTMs) define covalent and chemical modifications of protein residues. They play important roles in modulating various biological functions. Current PTM databases contain important sequence annotations but do not provide informative 3D structural resource about these modifications. Posttranslational modification structural database (PTM-SD) provides access to structurally solved modified residues, which are experimentally annotated as PTMs. It combines different PTM information and annotation gathered from other databases, e.g. Protein DataBank for the protein structures and dbPTM and PTMCuration for fine sequence annotation. PTM-SD gives an accurate detection of PTMs in structural data. PTM-SD can be browsed by PDB id, UniProt accession number, organism and classic PTM annotation. Advanced queries can also be performed, i.e. detailed PTM annotations, amino acid type, secondary structure, SCOP class classification, PDB chain length and number of PTMs by chain. Statistics and analyses can be computed on a selected dataset of PTMs. Each PTM entry is detailed in a dedicated page with information on the protein sequence, local conformation with secondary structure and Protein Blocks. PTM-SD gives valuable information on observed PTMs in protein 3D structure, which is of great interest for studying sequence–structure– function relationships at the light of PTMs, and could provide insights for comparative modeling and PTM predictions protocols. Database URL: PTM-SD can be accessed at http://www.dsimb.inserm.fr/dsimb_tools/PTM-SD/. PMID:24857970

  1. 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

  2. Aggresomes do not represent a general cellular response to protein misfolding in mammalian cells

    PubMed Central

    Beaudoin, Simon; Goggin, Kevin; Bissonnette, Cyntia; Grenier, Catherine; Roucou, Xavier

    2008-01-01

    Background Aggresomes are juxtanuclear inclusion bodies that have been proposed to represent a general cellular response to misfolded proteins in mammalian cells. Yet, why aggresomes are not a pathological characteristic of protein misfolding diseases is unclear. Here, we investigate if a misfolded protein inevitably forms aggresomes in mammalian cells. Results We show that a cytoplasmic form of the prion protein may form aggresomes or dispersed aggregates in different cell lines. In contrast to aggresomes, the formation of dispersed aggregates is insensitive to histone deacetylase 6 inhibitors and does not result in cytoskeleton rearrangements. Modulation of expression levels or proteasome inhibitors does not alter the formation of dispersed aggregates. Conclusion Our results establish that aggresomes are not obligatory products of protein misfolding in vivo. PMID:18937858

  3. A comprehensive manually curated protein–protein interaction database for the Death Domain superfamily

    PubMed Central

    Kwon, Dongseop; Yoon, Jong Hwan; Shin, Soo-Yong; Jang, Tae-Ho; Kim, Hong-Gee; So, Insuk; Jeon, Ju-Hong; Park, Hyun Ho

    2012-01-01

    The Death Domain (DD) superfamily, which is one of the largest classes of protein interaction modules, plays a pivotal role in apoptosis, inflammation, necrosis and immune cell signaling pathways. Because aberrant or inappropriate DD superfamily-mediated signaling events are associated with various human diseases, such as cancers, neurodegenerative diseases and immunological disorders, the studies in these fields are of great biological and clinical importance. To facilitate the understanding of the molecular mechanisms by which the DD superfamily is associated with biological and disease processes, we have developed the DD database (http://www.deathdomain.org), a manually curated database that aims to offer comprehensive information on protein–protein interactions (PPIs) of the DD superfamily. The DD database was created by manually curating 295 peer-reviewed studies that were published in the literature; the current version documents 175 PPI pairs among the 99 DD superfamily proteins. The DD database provides a detailed summary of the DD superfamily proteins and their PPI data. Users can find in-depth information that is specified in the literature on relevant analytical methods, experimental resources and domain structures. Our database provides a definitive and valuable tool that assists researchers in understanding the signaling network that is mediated by the DD superfamily. PMID:22135292

  4. 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

  5. Cell-Specific Establishment of Poliovirus Resistance to an Inhibitor Targeting a Cellular Protein

    PubMed Central

    Viktorova, Ekaterina G.; Nchoutmboube, Jules; Ford-Siltz, Lauren A.

    2015-01-01

    ABSTRACT It is hypothesized that targeting stable cellular factors involved in viral replication instead of virus-specific proteins may raise the barrier for development of resistant mutants, which is especially important for highly adaptable small (+)RNA viruses. However, contrary to this assumption, the accumulated evidence shows that these viruses easily generate mutants resistant to the inhibitors of cellular proteins at least in some systems. We investigated here the development of poliovirus resistance to brefeldin A (BFA), an inhibitor of the cellular protein GBF1, a guanine nucleotide exchange factor for the small cellular GTPase Arf1. We found that while resistant viruses can be easily selected in HeLa cells, they do not emerge in Vero cells, in spite that in the absence of the drug both cultures support robust virus replication. Our data show that the viral replication is much more resilient to BFA than functioning of the cellular secretory pathway, suggesting that the role of GBF1 in the viral replication is independent of its Arf activating function. We demonstrate that the level of recruitment of GBF1 to the replication complexes limits the establishment and expression of a BFA resistance phenotype in both HeLa and Vero cells. Moreover, the BFA resistance phenotype of poliovirus mutants is also cell type dependent in different cells of human origin and results in a fitness loss in the form of reduced efficiency of RNA replication in the absence of the drug. Thus, a rational approach to the development of host-targeting antivirals may overcome the superior adaptability of (+)RNA viruses. IMPORTANCE Compared to the number of viral diseases, the number of available vaccines is miniscule. For some viruses vaccine development has not been successful after multiple attempts, and for many others vaccination is not a viable option. Antiviral drugs are needed for clinical practice and public health emergencies. However, viruses are highly adaptable and can

  6. 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

  7. Improving the quality of NMR and crystallographic protein structures by means of a conformational database potential derived from structure databases.

    PubMed

    Kuszewski, J; Gronenborn, A M; Clore, G M

    1996-06-01

    A new conformational database potential involving dihedral angle relationships in databases of high-resolution highly refined protein crystal structures is presented as a method for improving the quality of structures generated from NMR data. The rationale for this procedure is based on the observation that uncertainties in the description of the nonbonded contacts present a key limiting factor in the attainable accuracy of protein NMR structures and that the nonbonded interaction terms presently used have poor discriminatory power between high- and low-probability local conformations. The idea behind the conformational database potential is to restrict sampling during simulated annealing refinement to conformations that are likely to be energetically possible by effectively limiting the choices of dihedral angles to those that are known to be physically realizable. In this manner, the variability in the structures produced by this method is primarily a function of the experimental restraints, rather than an artifact of a poor nonbonded interaction model. We tested this approach with the experimental NMR data (comprising an average of about 30 restraints per residue and consisting of interproton distances, torsion angles, 3JHN alpha coupling constants, and 13C chemical shifts) used previously to calculate the solution structure of reduced human thioredoxin (Qin J, Clore GM, Gronenborn AM, 1994, Structure 2:503-522). Incorporation of the conformational database potential into the target function used for refinement (which also includes terms for the experimental restraints, covalent geometry, and nonbonded interactions in the form of either a repulsive, repulsive-attractive, or 6-12 Lennard-Jones potential) results in a significant improvement in various quantitative measures of quality (Ramachandran plot, side-chain torsion angles, overall packing). This is achieved without compromising the agreement with the experimental restraints and the deviations from

  8. 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

  9. 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

  10. The adenovirus E1A protein overrides the requirement for cellular ras in initiating DNA synthesis.

    PubMed Central

    Stacey, D W; Dobrowolski, S F; Piotrkowski, A; Harter, M L

    1994-01-01

    The adenovirus E1A protein can induce cellular DNA synthesis in growth-arrested cells by interacting with the cellular protein p300 or pRb. In addition, serum- and growth factor-dependent cells require ras activity to initiate DNA synthesis and recently we have shown that Balb/c 3T3 cells can be blocked in either early or late G1 following microinjection of an anti-ras antibody. In this study, the E1A 243 amino acid protein is shown through microinjection not only to shorten the G0 to S phase interval but, what is more important, to override the inhibitory effects exerted by the anti-ras antibody in either early or late G1. Specifically, whether E1A is co-injected with anti-ras into quiescent cells or injected 18 h following a separate injection of anti-ras after serum stimulation, it efficiently induces cellular DNA synthesis in cells that would otherwise be blocked in G0/G1. Moreover, injection of a mutant form of E1A that can no longer associate with p300 is just as efficient as wild-type E1A in stimulating DNA synthesis in cells whose ras activity has been neutralized by anti-ras. The results presented here show that E1A is capable of overriding the requirement of cellular ras activity in promoting the entry of cells into S phase. Moreover, the results suggest the possibility that pRb and/or pRb-related proteins may function in a ras-dependent pathway that enables E1A to achieve this activity. Images PMID:7813447

  11. 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

  12. 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

  13. 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

  14. 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.

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

    PubMed Central

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

    2013-01-01

    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 HIV-1 infection of myeloid-lineage cells 1,2 as well as resting CD4+ T cells 3,4 by reducing the cellular dNTP concentration to a level where the viral reverse transcriptase cannot function 5,6. In other lentiviruses, including HIV-2 and related 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 7,8. 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. We present here the crystal structure of a ternary complex of Vpx with the host cell’s E3 ligase substrate adaptor DCAF1 and the C-terminal region of SAMHD1. Vpx is made up of a three-helical bundle, stabilised 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 provides the first 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. PMID:24336198

  16. 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

  17. 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

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

    PubMed

    Li, Ming; 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

    2016-08-01

    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

  19. Cellular accumulation of Cys326-OGG1 protein complexes under conditions of oxidative stress

    PubMed Central

    Kaur, M.P.; Guggenheim, E.J.; Pulisciano, C.; Akbar, S.; Kershaw, R.M.; Hodges, N.J.

    2014-01-01

    The common Ser326Cys polymorphism in the base excision repair protein 8-oxoguanine glycosylase 1 is associated with a reduced capacity to repair oxidative DNA damage particularly under conditions of intracellular oxidative stress and there is evidence that Cys326-OGG1 homozygous individuals have increased susceptibility to specific cancer types. Indirect biochemical studies have shown that reduced repair capacity is related to OGG1 redox modification and also possibly OGG1 dimer formation. In the current study we have used bimolecular fluorescence complementation to study for the first time a component of the base excision repair pathway and applied it to visualise accumulation of Cys326-OGG1 protein complexes in the native cellular environment. Fluorescence was observed both within and around the cell nucleus, was shown to be specific to cells expressing Cys326-OGG1 and only occurred in cells under conditions of cellular oxidative stress following depletion of intracellular glutathione levels by treatment with buthionine sulphoximine. Furthermore, OGG1 complex formation was inhibited by incubation of cells with the thiol reducing agents β-mercaptoethanol and dithiothreitol and the antioxidant dimethylsulfoxide indicating a causative role for oxidative stress in the formation of OGG1 cellular complexes. In conclusion, this study has provided for the first time evidence of redox sensitive Cys326-OGG1 protein accumulation in cells under conditions of intracellular oxidative stress that may be related to the previously reported reduced repair capacity of Cys326-OGG1 specifically under conditions of oxidative stress. PMID:24680828

  20. Cellular content of p53 protein in rat skin after exposure to the space environment.

    PubMed

    Ohnishi, T; Inoue, N; Matsumoto, H; Omatsu, T; Ohira, Y; Nagaoka, S

    1996-07-01

    The effects of stress in space, microgravity and space radiation, on living organisms are still unknown. We have examined the cellular content of p53 protein, a tumor-suppressor gene product, in skin from rats by the Western blot method using a blotting-amplification system. Three groups of rats were used. The first group was kept on Earth normally and showed hardly any detectable p53 protein. The second group made a 14-day flight into space on the second Spacelab Life Sciences-2 mission (F). The last group was subjected to the same kinds of stress as the rats in the second group except for spaceflight (SC). The F and the SC rats were killed on day zero (F-0 and SC-0 groups) and day nine (F-9 and SC-9 groups) after return. F-0 rats showed marked accumulation of p53 protein, whereas SC-0 rats showed a slight decrease. F-9 and SC-9 rats showed almost the same amount of p53 protein, but F-9 rats showed a slightly higher expression. From these results, it is suggested that the accumulation of cellular p53 protein is induced in rat skin cells by exposure to the space environment. PMID:8828662

  1. 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

  2. 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.

  3. Proteome-wide cellular protein concentrations of the human pathogen Leptospira interrogans

    PubMed Central

    Malmström, Johan; Beck, Martin; Schmidt, Alexander; Lange, Vinzenz; Deutsch, Eric W.; Aebersold, Ruedi

    2009-01-01

    Mass spectrometry based methods for relative proteome quantification have broadly impacted life science research. However, important research directions, particularly those involving mathematical modeling and simulation of biological processes, also critically depend on absolutely quantitative data, i.e. knowledge of the concentration of the expressed proteins as a function of cellular state. Until now, absolute protein concentration measurements of a significant fraction of the proteome (73%) have only been derived from genetically altered S. cerevisiae cells 1, a technique that is not directly portable from yeast to other species. In this study we developed and applied a mass spectrometry based strategy to determine the absolute quantity i.e. the average number of protein copies per cell in a cell population, for a significant fraction of the proteome in genetically unperturbed cells. Applying the technology to the human pathogen Leptospira interrogans, a spirochete responsible for Leptospirosis 4, we generated an absolute protein abundance scale for 83% of the mass spectrometry detectable proteome, from cells at different states. Taking advantage of the unique cellular dimensions of L. interrogans, we used cryo electron tomography (cryoET) morphological measurements to verify at the single cell level the average absolute abundance values of selected proteins determined by mass spectrometry on a population of cells. As the strategy is relatively fast and applicable to any cell type we expect that it will become a cornerstone of quantitative biology and systems biology. PMID:19606093

  4. The InterPro database, an integrated documentation resource for protein families, domains and functional sites.

    PubMed

    Apweiler, R; Attwood, T K; Bairoch, A; Bateman, A; Birney, E; Biswas, M; Bucher, P; Cerutti, L; Corpet, F; Croning, M D; Durbin, R; Falquet, L; Fleischmann, W; Gouzy, J; Hermjakob, H; Hulo, N; Jonassen, I; Kahn, D; Kanapin, A; Karavidopoulou, Y; Lopez, R; Marx, B; Mulder, N J; Oinn, T M; Pagni, M; Servant, F; Sigrist, C J; Zdobnov, E M

    2001-01-01

    Signature databases are vital tools for identifying distant relationships in novel sequences and hence for inferring protein function. InterPro is an integrated documentation resource for protein families, domains and functional sites, which amalgamates the efforts of the PROSITE, PRINTS, Pfam and ProDom database projects. Each InterPro entry includes a functional description, annotation, literature references and links back to the relevant member database(s). Release 2.0 of InterPro (October 2000) contains over 3000 entries, representing families, domains, repeats and sites of post-translational modification encoded by a total of 6804 different regular expressions, profiles, fingerprints and Hidden Markov Models. Each InterPro entry lists all the matches against SWISS-PROT and TrEMBL (more than 1,000,000 hits from 462,500 proteins in SWISS-PROT and TrEMBL). The database is accessible for text- and sequence-based searches at http://www.ebi.ac.uk/interpro/. Questions can be emailed to interhelp@ebi.ac.uk. PMID:11125043

  5. The InterPro database, an integrated documentation resource for protein families, domains and functional sites

    PubMed Central

    Apweiler, R.; Attwood, T. K.; Bairoch, A.; Bateman, A.; Birney, E.; Biswas, M.; Bucher, P.; Cerutti, L.; Corpet, F.; Croning, M. D. R.; Durbin, R.; Falquet, L.; Fleischmann, W.; Gouzy, J.; Hermjakob, H.; Hulo, N.; Jonassen, I.; Kahn, D.; Kanapin, A.; Karavidopoulou, Y.; Lopez, R.; Marx, B.; Mulder, N. J.; Oinn, T. M.; Pagni, M.; Servant, F.; Sigrist, C. J. A.; Zdobnov, E. M.

    2001-01-01

    Signature databases are vital tools for identifying distant relationships in novel sequences and hence for inferring protein function. InterPro is an integrated documentation resource for protein families, domains and functional sites, which amalgamates the efforts of the PROSITE, PRINTS, Pfam and ProDom database projects. Each InterPro entry includes a functional description, annotation, literature references and links back to the relevant member database(s). Release 2.0 of InterPro (October 2000) contains over 3000 entries, representing families, domains, repeats and sites of post-translational modification encoded by a total of 6804 different regular expressions, profiles, fingerprints and Hidden Markov Models. Each InterPro entry lists all the matches against SWISS-PROT and TrEMBL (more than 1 000 000 hits from 462 500 proteins in SWISS-PROT and TrEMBL). The database is accessible for text- and sequence-based searches at http://www.ebi.ac.uk/interpro/. Questions can be emailed to interhelp@ebi.ac.uk. PMID:11125043

  6. PROSITE, a protein domain database for functional characterization and annotation.

    PubMed

    Sigrist, Christian J A; Cerutti, Lorenzo; de Castro, Edouard; Langendijk-Genevaux, Petra S; Bulliard, Virginie; Bairoch, Amos; Hulo, Nicolas

    2010-01-01

    PROSITE consists of documentation entries describing protein domains, families and functional sites, as well as associated patterns and profiles to identify them. It is complemented by ProRule, a collection of rules based on profiles and patterns, which increases the discriminatory power of these profiles and patterns by providing additional information about functionally and/or structurally critical amino acids. PROSITE is largely used for the annotation of domain features of UniProtKB/Swiss-Prot entries. Among the 983 (DNA-binding) domains, repeats and zinc fingers present in Swiss-Prot (release 57.8 of 22 September 2009), 696 ( approximately 70%) are annotated with PROSITE descriptors using information from ProRule. In order to allow better functional characterization of domains, PROSITE developments focus on subfamily specific profiles and a new profile building method giving more weight to functionally important residues. Here, we describe AMSA, an annotated multiple sequence alignment format used to build a new generation of generalized profiles, the migration of ScanProsite to Vital-IT, a cluster of 633 CPUs, and the adoption of the Distributed Annotation System (DAS) to facilitate PROSITE data integration and interchange with other sources. The latest version of PROSITE (release 20.54, of 22 September 2009) contains 1308 patterns, 863 profiles and 869 ProRules. PROSITE is accessible at: http://www.expasy.org/prosite/. PMID:19858104

  7. PROSITE, a protein domain database for functional characterization and annotation

    PubMed Central

    Sigrist, Christian J. A.; Cerutti, Lorenzo; de Castro, Edouard; Langendijk-Genevaux, Petra S.; Bulliard, Virginie; Bairoch, Amos; Hulo, Nicolas

    2010-01-01

    PROSITE consists of documentation entries describing protein domains, families and functional sites, as well as associated patterns and profiles to identify them. It is complemented by ProRule, a collection of rules based on profiles and patterns, which increases the discriminatory power of these profiles and patterns by providing additional information about functionally and/or structurally critical amino acids. PROSITE is largely used for the annotation of domain features of UniProtKB/Swiss-Prot entries. Among the 983 (DNA-binding) domains, repeats and zinc fingers present in Swiss-Prot (release 57.8 of 22 September 2009), 696 (∼70%) are annotated with PROSITE descriptors using information from ProRule. In order to allow better functional characterization of domains, PROSITE developments focus on subfamily specific profiles and a new profile building method giving more weight to functionally important residues. Here, we describe AMSA, an annotated multiple sequence alignment format used to build a new generation of generalized profiles, the migration of ScanProsite to Vital-IT, a cluster of 633 CPUs, and the adoption of the Distributed Annotation System (DAS) to facilitate PROSITE data integration and interchange with other sources. The latest version of PROSITE (release 20.54, of 22 September 2009) contains 1308 patterns, 863 profiles and 869 ProRules. PROSITE is accessible at: http://www.expasy.org/prosite/. PMID:19858104

  8. The cellular response to DNA damage: A focus on MDC1 and its interacting proteins

    PubMed Central

    Coster, Gideon

    2010-01-01

    The DNA damage response (DDR) is comprised of a network of proteins that respond to DNA damage. Mediator of DNA Damage Checkpoint 1 (MDC1) plays an early and important role in the DDR. Recent data show that MDC1 binds multiple proteins that participate in various aspects of the DDR, positioning it at the core of the DDR. Furthermore, interactions with non-DDR proteins were also revealed, suggesting novel roles for MDC1. In this review we provide a comprehensive overview of all known MDC1-binding proteins and discuss their role. We present these binding partners according to their function, thereby providing the reader with a detailed and updated overview of the cellular response to DNA damage. We discuss more recent findings in detail and conclude by presenting the challenges the field faces in the future. PMID:21326949

  9. 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.

  10. Global analysis of cellular protein flux quantifies the selectivity of basal autophagy.

    PubMed

    Zhang, Tian; Ghaemmaghami, Sina

    2016-08-01

    In eukaryotic cells, the macroautophagy pathway has been implicated in the degradation of long-lived proteins and damaged organelles. Although it has been demonstrated that macroautophagy can selectively degrade specific targets, its contribution to the basal turnover of cellular proteins had previously not been quantified on proteome-wide scales. In a recent study, we utilized dynamic proteomics to provide a global comparison of protein half-lives between wild-type and autophagy-deficient cells. Our results indicated that in quiescent fibroblasts, macroautophagy contributes to the basal turnover of a substantial fraction of the proteome. However, the contribution of macroautophagy to constitutive protein turnover is variable within the proteome. The methodology outlined in the study provides a global strategy for quantifying the selectivity of basal macroautophagy. PMID:27248575

  11. 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

  12. Structural and functional characterization of recombinant human cellular retinaldehyde-binding protein.

    PubMed Central

    Crabb, J. W.; Carlson, A.; Chen, Y.; Goldflam, S.; Intres, R.; West, K. A.; Hulmes, J. D.; Kapron, J. T.; Luck, L. A.; Horwitz, J.; Bok, D.

    1998-01-01

    Cellular retinaldehyde-binding protein (CRALBP) is abundant in the retinal pigment epithelium (RPE) and Müller cells of the retina where it is thought to function in retinoid metabolism and visual pigment regeneration. The protein carries 11-cis-retinal and/or 11-cis-retinol as endogenous ligands in the RPE and retina and mutations in human CRALBP that destroy retinoid binding functionality have been linked to autosomal recessive retinitis pigmentosa. CRALBP is also present in brain without endogenous retinoids, suggesting other ligands and physiological roles exist for the protein. Human recombinant cellular retinaldehyde-binding protein (rCRALBP) has been over expressed as non-fusion and fusion proteins in Escherichia coli from pET3a and pET19b vectors, respectively. The recombinant proteins typically constitute 15-20% of the soluble bacterial lysate protein and after purification, yield about 3-8 mg per liter of bacterial culture. Liquid chromatography electrospray mass spectrometry, amino acid analysis, and Edman degradation were used to demonstrate that rCRALBP exhibits the correct primary structure and mass. Circular dichroism, retinoid HPLC, UV-visible absorption spectroscopy, and solution state 19F-NMR were used to characterize the secondary structure and retinoid binding properties of rCRALBP. Human rCRALBP appears virtually identical to bovine retinal CRALBP in terms of secondary structure, thermal stability, and stereoselective retinoid-binding properties. Ligand-dependent conformational changes appear to influence a newly detected difference in the bathochromic shift exhibited by bovine and human CRALBP when complexed with 9-cis-retinal. These recombinant preparations provide valid models for human CRALBP structure-function studies. PMID:9541407

  13. 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.

  14. Fluorophore targeting to cellular proteins via enzyme-mediated azide ligation and strain-promoted cycloaddition

    PubMed Central

    Yao, Jennifer Z.; Uttamapinant, Chayasith; Poloukhtine, Andrei; Baskin, Jeremy M.; Codelli, Julian A.; Sletten, Ellen M.; Bertozzi, Carolyn R.; Popik, Vladimir V.; Ting, Alice Y.

    2012-01-01

    Methods for fluorophore targeting to cellular proteins can allow imaging with dyes that are smaller, brighter, and more photostable than fluorescent proteins. Previously, we reported targeting of the blue fluorophore coumarin to cellular proteins fused to a 13-amino acid recognition sequence (LAP), catalyzed by a mutant of the E. coli enzyme lipoic acid ligase (LplA). Here, we extend LplA-based labeling to green- and red-emitting fluorophores by employing a two-step targeting scheme. First, we found that the W37I mutant of LplA catalyzes site-specific ligation of 10-azidodecanoic acid to LAP in cells, in nearly quantitative yield after 30 minutes. Second, we evaluated a panel of five different cyclooctyne structures, and found that fluorophore conjugates to aza-dibenzocyclooctyne (ADIBO) gave the highest and most specific derivatization of azide-conjugated LAP in cells. However, for targeting of hydrophobic fluorophores such as ATTO 647N, the hydrophobicity of ADIBO was detrimental, and superior targeting was achieved by conjugation to the less hydrophobic monofluorinated cyclooctyne (MOFO). Our optimized two-step enzymatic/chemical labeling scheme was used to tag and image a variety of LAP fusion proteins in multiple mammalian cell lines with diverse fluorophores including fluorescein, rhodamine, Alexa Fluor 568, ATTO 647N, and ATTO 655. PMID:22239252

  15. The HSV-1 tegument protein pUL46 associates with cellular membranes and viral capsids

    SciTech Connect

    Murphy, Michael A.; Bucks, Michelle A.; O'Regan, Kevin J.; Courtney, Richard J.

    2008-07-05

    The molecular mechanisms responsible for the addition of tegument proteins into nascent herpesvirus particles are poorly understood. To better understand the tegumentation process of herpes simplex virus type 1 (HSV-1) virions, we initiated studies that showed the tegument protein pUL46 (VP11/12) has a similar cellular localization to the membrane-associated tegument protein VP22. Using membrane flotation analysis we found that pUL46 associates with membranes in both the presence and absence of other HSV-1 proteins. However, when purified virions were stripped of their envelope, the majority of pUL46 was found to associate with the capsid fraction. This strong affinity of pUL46 for capsids was confirmed by an in vitro capsid pull-down assay in which purified pUL46-GST was able to interact specifically with capsids purified from the nuclear fraction of HSV-1 infected cells. These results suggest that pUL46 displays a dynamic interaction between cellular membranes and capsids.

  16. The TATA-binding protein as a regulator of cellular transformation.

    PubMed

    Johnson, Sandra A S; Dubeau, Louis; White, Robert J; Johnson, Deborah L

    2003-01-01

    The TATA-binding protein, TBP, is used by all three RNA polymerases and is therefore central to the process of gene expression. TBP associates with several subsets of proteins, called TATA-binding protein-associated factors (TAFs). This results in the formation of at least three distinct complexes, SL1, TFIID, and TFIIIB, which dictates whether TBP functions in RNA polymerase (pol) I, pol II, or pol III transcription, respectively. The regulation of gene expression has focused largely on proteins that serve to modulate the efficiency by which the general transcription components, such as TBP, interact with promoters. The possibility of a basal transcription factor, itself, being regulated, and influencing cellular homeostasis, has not been extensively considered. However, recent studies have indicated that TBP is indeed regulated, and that modulation of its cellular concentration has a profound, and surprisingly selective, impact on gene expression that can mediate the normal proliferative responses of cells to growth stimuli as well as the transformation potential of cells. PMID:12963838

  17. DPY30 regulates pathways in cellular senescence through ID protein expression

    PubMed Central

    Simboeck, Elisabeth; Gutierrez, Arantxa; Cozzuto, Luca; Beringer, Malte; Caizzi, Livia; M Keyes, William; Di Croce, Luciano

    2013-01-01

    Cellular senescence is an intrinsic defense mechanism to various cellular stresses: while still metabolically active, senescent cells stop dividing and enter a proliferation arrest. Here, we identify DPY30, a member of all mammalian histone H3K4 histone methyltransferases (HMTases), as a key regulator of the proliferation potential of human primary cells. Following depletion of DPY30, cells show a severe proliferation defect and display a senescent phenotype, including a flattened and enlarged morphology, elevated level of reactive oxygen species (ROS), increased SA-β-galactosidase activity, and formation of senescence-associated heterochromatin foci (SAHFs). While DPY30 depletion leads to a reduced level of H3K4me3-marked active chromatin, we observed a concomitant activation of CDK inhibitors, including p16INK4a, independent of H3K4me3. ChIP experiments show that key regulators of cell-cycle progression, including ID proteins, are under direct control of DPY30. Because ID proteins are negative regulators of the transcription factors ETS1/2, depletion of DPY30 leads to the transcriptional activation of p16INK4a by ETS1/2 and thus to a senescent-like phenotype. Ectoptic re-introduction of ID protein expression can partially rescue the senescence-like phenotype induced by DPY30 depletion. Thus, our data indicate that DPY30 controls proliferation by regulating ID proteins expression, which in turn lead to senescence bypass. PMID:23872946

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

    PubMed Central

    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

  19. 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

  20. 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.

  1. Characterization of a Fasciola gigantica protein carrying two DM9 domains reveals cellular relocalization property.

    PubMed

    Phadungsil, Wansika; Smooker, Peter M; Vichasri-Grams, Suksiri; Grams, Rudi

    2016-01-01

    Even at the present age of whole-organism analysis, e.g., genomics, transcriptomics, and proteomics, the biological roles of many proteins remain unresolved. Classified among the proteins of unknown function is a family of proteins harboring repeats of the DM9 domain, a 60-75 amino acids motif first described in a small number of Drosophila melanogaster proteins. Proteins may carry two or more DM9 domains either in combination with other domains or as their sole constituent. Here we have characterized a 16.8kDa Fasciola gigantica protein comprising two tandem repeated DM9 domains (FgDM9-1). The protein was located in the parenchyma of the immature and mature parasite and consequently it was not detected in the ES product of the parasite but only in the whole worm extract. Interestingly, extraction with SDS yielded a substantially higher amount of the protein suggesting association with insoluble cell components. In Sf9 insect cells a heterologously expressed EGFP-FgDM9-1 chimera showed cell-wide distribution but relocated to vesicle-like structures in the cytoplasm after stimulating cellular stress by bacteria, heat shock or chloroquine. These structures did not colocalize with the markers of endocytosis/phagocytosis ubiquitin, RAB7, GABARAP. The same behavior was noted for Aedes aegypti PRS1, a homologous mosquito DM9 protein as a positive control while EGFP did not exhibit such relocation in the insect cells. Cross-linking experiments on soluble recombinant FgDM9-1 indicated that the protein can undergo specific oligomerization. It is speculated that proteins carrying the DM9 domain have a role in vesicular transport in flatworms and insects. PMID:26946400

  2. Differential protein-protein interactions of LRRK1 and LRRK2 indicate roles in distinct cellular signaling pathways

    PubMed Central

    Reyniers, Lauran; Del Giudice, Maria Grazia; Civiero, Laura; Belluzzi, Elisa; Lobbestael, Evy; Beilina, Alexandra; Arrigoni, Giorgio; Derua, Rita; Waelkens, Etienne; Li, Yan; Crosio, Claudia; Iaccarino, Ciro; Cookson, Mark R.; Baekelandt, Veerle; Greggio, Elisa; Taymans, Jean-Marc

    2014-01-01

    Genetic studies show that LRRK2, and not its closest paralogue LRRK1, is linked to Parkinson’s disease. To gain insight into the molecular and cellular basis of this discrepancy, we searched for LRRK1- and LRRK2-specific cellular processes by identifying their distinct interacting proteins. A protein microarray-based interaction screen was performed with recombinant 3xFlag-LRRK1 and 3xFlag-LRRK2 and, in parallel, co-immunoprecipitation followed by mass spectrometry was performed from SH-SY5Y neuroblastoma cell lines stably expressing 3xFlag-LRRK1 or 3xFlag-LRRK2. We identified a set of LRRK1- and LRRK2-specific as well as common interactors. One of our most prominent findings was that both screens pointed to epidermal growth factor receptor (EGF-R) as a LRRK1-specific interactor, while 14-3-3 proteins were LRRK2-specific. This is consistent with phosphosite mapping of LRRK1, revealing phosphosites outside of 14-3-3 consensus binding motifs. To assess the functional relevance of these interactions, SH-SY5Y-LRRK1 and -LRRK2 cell lines were treated with LRRK2 kinase inhibitors that disrupt 14-3-3 binding, or with EGF, an EGF-R agonist. Redistribution of LRRK2, not LRRK1, from diffuse cytoplasmic to filamentous aggregates was observed after inhibitor treatment. Similarly, EGF induced translocation of LRRK1, but not of LRRK2, to endosomes. Our study confirms that LRRK1 and LRRK2 can carry out distinct functions by interacting with different cellular proteins. PMID:24947832

  3. 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

  4. 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

  5. Autotransporters: The Cellular Environment Reshapes a Folding Mechanism to Promote Protein Transport

    PubMed Central

    Braselmann, Esther; Clark, Patricia L.

    2012-01-01

    We know very little about how the cellular environment affects protein folding mechanisms. Here, we focus on one unique aspect of that environment that is difficult to recapitulate in the test tube: the effect of a folding vector. When protein folding is initiated at one end of the polypeptide chain, folding starts from a much smaller ensemble of conformations than during refolding of a full-length polypeptide chain. But to what extent can vectorial folding affect protein folding kinetics and the conformations of folding intermediates? We focus on recent studies of autotransporter proteins, the largest class of virulence proteins from pathogenic Gram-negative bacteria. Autotransporter proteins are secreted across the bacterial inner membrane from N→C-terminus, which, like refolding in vitro, retards folding. But in contrast, upon C→N-terminal secretion across the outer membrane autotransporter folding proceeds orders of magnitude faster. The potential impact of vectorial folding on the folding mechanisms of other proteins is also discussed. PMID:23687560

  6. Identification of Dynamic Changes in Proteins Associated with the Cellular Cytoskeleton after Exposure to Okadaic Acid

    PubMed Central

    Opsahl, Jill A.; Ljostveit, Sonja; Solstad, Therese; Risa, Kristin; Roepstorff, Peter; Fladmark, Kari E.

    2013-01-01

    Exposure of cells to the diarrhetic shellfish poison, okadaic acid, leads to a dramatic reorganization of cytoskeletal architecture and loss of cell-cell contact. When cells are exposed to high concentrations of okadaic acid (100–500 nM), the morphological rearrangement is followed by apoptotic cell death. Okadaic acid inhibits the broad acting Ser/Thr protein phosphatases 1 and 2A, which results in hyperphosphorylation of a large number of proteins. Some of these hyperphosphorylated proteins are most likely key players in the reorganization of the cell morphology induced by okadaic acid. We wanted to identify these phosphoproteins and searched for them in the cellular lipid rafts, which have been found to contain proteins that regulate cytoskeletal dynamics and cell adhesion. By using stable isotope labeling by amino acids in cell culture cells treated with okadaic acid (400 nM) could be combined with control cells before the isolation of lipid rafts. Protein phosphorylation events and translocations induced by okadaic acid were identified by mass spectrometry. Okadaic acid was shown to regulate the phosphorylation status and location of proteins associated with the actin cytoskeleton, microtubules and cell adhesion structures. A large number of these okadaic acid-regulated proteins have previously also been shown to be similarly regulated prior to cell proliferation and migration. Our results suggest that okadaic acid activates general cell signaling pathways that induce breakdown of the cortical actin cytoskeleton and cell detachment. PMID:23708184

  7. 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. PMID:26513421

  8. 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

  9. Enterovirus 71 inhibits cellular type I interferon signaling by downregulating JAK1 protein expression.

    PubMed

    Liu, Ying; Zhang, Zhe; Zhao, Xinghui; Yu, Rui; Zhang, Xiaopeng; Wu, Shipo; Liu, Ju; Chi, Xiangyang; Song, Xiaohong; Fu, Ling; Yu, Yingqun; Hou, Lihua; Chen, Wei

    2014-08-01

    Enterovirus 71 (EV71) infection can cause severe disease and lead to death in children. Recurring outbreaks of EV71 have been reported in several countries. Interferons (IFNs) have been used for decades to treat several types of viral infection, but have a limited ability to inhibit EV71 replication. Herein, we intend to investigate the mechanisms by which EV71 inhibits the cellular type I IFN response. In this study, MRC-5 (human embryonic lung fibroblast) or RD (human rhabdomyosarcoma) cells were infected with EV71, and then treated with or without IFN-α2b. Cells were harvested and analyzed by flow cytometry to determine the level of IFNAR1. Cell lysis were prepared to detect the levels of STAT1, STAT2, phosphorylated STAT1, phosphorylated STAT2, IFNAR1, JAK1, and TYK2 by Western blotting. The phosphorylation of STAT1 and STAT2 induced by IFN were inhibited without significant downregulation of IFNAR1 in EV71-infected cells. The EV71-induced suppression of STAT1 and STAT2 phosphorylation was not rescued by the protein tyrosine phosphatases inhibitor, and was independent of suppressor of cytokine signaling protein 1/3 levels. The phosphorylation of JAK1 and TYK2 were inhibited accompanied by EV71-induced downregulation of JAK1, which occurred at a post-transcriptional level and was proteasome independent. JAK1 expression did not decrease, and IFN-α-stimulated STAT1 and STAT2 phosphorylation were not blocked in HEK293T cells overexpressing the EV71 viral protein 2A or 3C. This study demonstrates that EV71 inhibits the cellular type I IFN antiviral pathway by downregulating JAK1, while the expression of IFNAR1 does not significantly alter in EV71-infected cells. Additionally, the EV71 viral proteins 2A and 3C do not act as antagonists of cellular type I IFN signaling. PMID:24905060

  10. Plin2 Inhibits Cellular Glucose Uptake through Interactions with SNAP23, a SNARE Complex Protein

    PubMed Central

    Senthivinayagam, Subramanian; McIntosh, Avery L.; Moon, Kenneth C.; Atshaves, Barbara P.

    2013-01-01

    Although a link between excess lipid storage and aberrant glucose metabolism has been recognized for many years, little is known what role lipid storage droplets and associated proteins such as Plin2 play in managing cellular glucose levels. To address this issue, the influence of Plin2 on glucose uptake was examined using 2-NBD-Glucose and [3H]-2-deoxyglucose to show that insulin-mediated glucose uptake was decreased 1.7- and 1.8-fold, respectively in L cell fibroblasts overexpressing Plin2. Conversely, suppression of Plin2 levels by RNAi-mediated knockdown increased 2-NBD-Glucose uptake several fold in transfected L cells and differentiated 3T3-L1 cells. The effect of Plin2 expression on proteins involved in glucose uptake and transport was also examined. Expression of the SNARE protein SNAP23 was increased 1.6-fold while levels of syntaxin-5 were decreased 1.7-fold in Plin2 overexpression cells with no significant changes observed in lipid droplet associated proteins Plin1 or FSP27 or with the insulin receptor, GLUT1, or VAMP4. FRET experiments revealed a close proximity of Plin2 to SNAP23 on lipid droplets to within an intramolecular distance of 51 Å. The extent of targeting of SNAP23 to lipid droplets was determined by co-localization and co-immunoprecipitation experiments to show increased partitioning of SNAP23 to lipid droplets when Plin2 was overexpressed. Taken together, these results suggest that Plin2 inhibits glucose uptake by interacting with, and regulating cellular targeting of SNAP23 to lipid droplets. In summary, the current study for the first time provides direct evidence for the role of Plin2 in mediating cellular glucose uptake. PMID:24040030

  11. Changes in cellular microRNA expression induced by porcine circovirus type 2-encoded proteins.

    PubMed

    Hong, Jae-Sang; Kim, Nam-Hoon; Choi, Chang-Yong; Lee, Jun-Seong; Na, Dokyun; Chun, Taehoon; Lee, Young Sik

    2015-01-01

    Porcine circovirus type 2 (PCV2) is the primary causative agent of postweaning multisystemic wasting syndrome, which leads to serious economic losses in the pig industry worldwide. While the molecular basis of PCV2 replication and pathogenicity remains elusive, it is increasingly apparent that the microRNA (miRNA) pathway plays a key role in controlling virus-host interactions, in addition to a wide range of cellular processes. Here, we employed Solexa deep sequencing technology to determine which cellular miRNAs were differentially regulated after expression of each of three PCV2-encoded open reading frames (ORFs) in porcine kidney epithelial (PK15) cells. We identified 51 ORF1-regulated miRNAs, 74 ORF2-regulated miRNAs, and 32 ORF3-regulated miRNAs that differed in abundance compared to the control. Gene ontology analysis of the putative targets of these miRNAs identified transcriptional regulation as the most significantly enriched biological process, while KEGG pathway analysis revealed significant enrichment for several pathways including MAPK signaling, which is activated during PCV2 infection. Among the potential target genes of ORF-regulated miRNAs, two genes encoding proteins that are known to interact with PCV2-encoded proteins, zinc finger protein 265 (ZNF265) and regulator of G protein signaling 16 (RGS16), were selected for further analysis. We provide evidence that ZNF265 and RGS16 are direct targets of miR-139-5p and let-7e, respectively, which are both down-regulated by ORF2. Our data will initiate further studies to elucidate the roles of ORF-regulated cellular miRNAs in PCV2-host interactions. PMID:25885539

  12. The SWISS-PROT protein sequence database and its supplement TrEMBL in 2000

    PubMed Central

    Bairoch, Amos; Apweiler, Rolf

    2000-01-01

    SWISS-PROT is a curated protein sequence database which strives to provide a high level of annotation (such as the description of the function of a protein, its domains structure, post-translational modifications, variants, etc.), a minimal level of redundancy and high level of integration with other databases. Recent developments of the database include format and content enhancements, cross-references to additional databases, new documentation files and improvements to TrEMBL, a computer-annotated supplement to SWISS-PROT. TrEMBL consists of entries in SWISS-PROT-like format derived from the translation of all coding sequences (CDSs) in the EMBL Nucleotide Sequence Database, except the CDSs already included in SWISS-PROT. We also describe the Human Proteomics Initiative (HPI), a major project to annotate all known human sequences according to the quality standards of SWISS-PROT. SWISS-PROT is available at: http://www.expasy.ch/sprot/ and http://www.ebi.ac.uk/swissprot/ PMID:10592178

  13. The SWISS-PROT protein sequence database and its supplement TrEMBL in 2000.

    PubMed

    Bairoch, A; Apweiler, R

    2000-01-01

    SWISS-PROT is a curated protein sequence database which strives to provide a high level of annotation (such as the description of the function of a protein, its domains structure, post-translational modifications, variants, etc.), a minimal level of redundancy and high level of integration with other databases. Recent developments of the database include format and content enhancements, cross-references to additional databases, new documentation files and improvements to TrEMBL, a computer-annotated supplement to SWISS-PROT. TrEMBL consists of entries in SWISS-PROT-like format derived from the translation of all coding sequences (CDSs) in the EMBL Nucleotide Sequence Database, except the CDSs already included in SWISS-PROT. We also describe the Human Proteomics Initiative (HPI), a major project to annotate all known human sequences according to the quality standards of SWISS-PROT. SWISS-PROT is available at: http://www.expasy.ch/sprot/ and http://www.ebi.ac.uk/swissprot/ PMID:10592178

  14. 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

  15. MitoDrome: a database of Drosophila melanogaster nuclear genes encoding proteins targeted to the mitochondrion

    PubMed Central

    Sardiello, Marco; Licciulli, Flavio; Catalano, Domenico; Attimonelli, Marcella; Caggese, Corrado

    2003-01-01

    Mitochondria are organelles present in the cytoplasm of most eukaryotic cells; although they have their own DNA, the majority of the proteins necessary for a functional mitochondrion are coded by the nuclear DNA and only after transcription and translation they are imported in the mitochondrion as proteins. The primary role of the mitochondrion is electron transport and oxidative phosphorylation. Although it has been studied for a long time, the interest of researchers in mitochondria is still alive thanks to the discovery of mitochondrial role in apoptosis, aging and cancer. Aim of the MitoDrome database is to annotate the Drosophila melanogaster nuclear genes coding for mitochondrial proteins in order to contribute to the functional characterization of nuclear genes coding for mitochondrial proteins and to knowledge of gene diseases related to mitochondrial dysfunctions. Indeed D. melanogaster is one of the most studied organisms and a model for the Human genome. Data are derived from the comparison of Human mitochondrial proteins versus the Drosophila genome, ESTs and cDNA sequence data available in the FlyBase database. Links from the MitoDrome entries to the related homologous entries available in MitoNuC will be soon imple-mented. The MitoDrome database is available at http://bighost.area.ba.cnr.it/BIG/MitoDrome. Data are organised in a flat-file format and can be retrieved using the SRS system. PMID:12520013

  16. SynDB: a Synapse protein DataBase based on synapse ontology.

    PubMed

    Zhang, Wuxue; Zhang, Yong; Zheng, Hui; Zhang, Chen; Xiong, Wei; Olyarchuk, John G; Walker, Michael; Xu, Weifeng; Zhao, Min; Zhao, Shuqi; Zhou, Zhuan; Wei, Liping

    2007-01-01

    A synapse is the junction across which a nerve impulse passes from an axon terminal to a neuron, muscle cell or gland cell. The functions and building molecules of the synapse are essential to almost all neurobiological processes. To describe synaptic structures and functions, we have developed Synapse Ontology (SynO), a hierarchical representation that includes 177 terms with hundreds of synonyms and branches up to eight levels deep. associated 125 additional protein keywords and 109 InterPro domains with these SynO terms. Using a combination of automated keyword searches, domain searches and manual curation, we collected 14,000 non-redundant synapse-related proteins, including 3000 in human. We extensively annotated the proteins with information about sequence, structure, function, expression, pathways, interactions and disease associations and with hyperlinks to external databases. The data are stored and presented in the Synapse protein DataBase (SynDB, http://syndb.cbi.pku.edu.cn). SynDB can be interactively browsed by SynO, Gene Ontology (GO), domain families, species, chromosomal locations or Tribe-MCL clusters. It can also be searched by text (including Boolean operators) or by sequence similarity. SynDB is the most comprehensive database to date for synaptic proteins. PMID:17098931

  17. Exploring Cellular Interactions of Liposomes Using Protein Corona Fingerprints and Physicochemical Properties.

    PubMed

    Bigdeli, Arafeh; Palchetti, Sara; Pozzi, Daniela; Hormozi-Nezhad, Mohammad Reza; Baldelli Bombelli, Francesca; Caracciolo, Giulio; Mahmoudi, Morteza

    2016-03-22

    To control liposomes fate and transport upon contact with biofluids, it is essential to consider several parameters affecting the synthetic and biological identity of liposomes, as well as liposome-protein corona (PC) aspects. As a powerful tool in this data mining adventure, quantitative structure-activity relationship (QSAR) approach is used to correlate physicochemical properties of liposomes and their PC fingerprints to multiple quantified biological responses. In the present study, the relationship between cellular interactions of a set of structurally diverse liposomal formulations and their physicochemical and PC properties has been investigated via linear and nonlinear QSAR models. Significant parameters affecting cellular uptake and cell viability of liposomes in two important cancer cell lines (PC3 and HeLa) have been identified. The developed QSARs have the capacity to be implemented in advanced targeted delivery of liposomal drugs. PMID:26882007

  18. 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

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

    PubMed

    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

    2015-05-01

    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

  20. 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...

  1. 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...

  2. MANGANESE UPREGULATES CELLULAR PRION PROTEINS AND INHIBITS THE RATE OF PROTEINASE-K DEPENDENT LIMITED PROTEOLYSIS IN NEURONAL CELLS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The key 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 cations such as copper to th...

  3. 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

  4. 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

  5. A Critical Role of a Cellular Membrane Traffic Protein in Poliovirus RNA Replication

    PubMed Central

    Belov, George A.; Feng, Qian; Nikovics, Krisztina; Jackson, Catherine L.; Ehrenfeld, Ellie

    2008-01-01

    Replication of many RNA viruses is accompanied by extensive remodeling of intracellular membranes. In poliovirus-infected cells, ER and Golgi stacks disappear, while new clusters of vesicle-like structures form sites for viral RNA synthesis. Virus replication is inhibited by brefeldin A (BFA), implicating some components(s) of the cellular secretory pathway in virus growth. Formation of characteristic vesicles induced by expression of viral proteins was not inhibited by BFA, but they were functionally deficient. GBF1, a guanine nucleotide exchange factor for the small cellular GTPases, Arf, is responsible for the sensitivity of virus infection to BFA, and is required for virus replication. Knockdown of GBF1 expression inhibited virus replication, which was rescued by catalytically active protein with an intact N-terminal sequence. We identified a mutation in GBF1 that allows growth of poliovirus in the presence of BFA. Interaction between GBF1 and viral protein 3A determined the outcome of infection in the presence of BFA. PMID:19023417

  6. Cryo-Microscopic Analysis of the Effects of Extra Cellular Proteins on Polycrystalline Ice Structure

    NASA Astrophysics Data System (ADS)

    Brox, T.; Skidmore, M. L.; Christner, B. C.; Achberger, A.

    2010-12-01

    Recent work has demonstrated that microorganisms can occupy the liquid filled inter-crystalline veins in ice and maintain their metabolic activity under these conditions. While these discoveries have increased the extent of the biosphere to include the large continental ice sheets of Antarctica and Greenland as biomes, the habitat of the microorganisms within the inter-crystalline liquid veins is poorly understood. Certain cold tolerant organisms produce extra cellular proteins (i.e., ice-binding proteins) that have the ability to bind to the prism face of an ice crystal and inhibit recrystallization of ice. This phenotype affects the physical ice structure and the liquid vein network, potentially providing ice-inhabiting species a protective mechanism with which to control their habitat. One such microorganism is Chryseobacterium sp. V3519-10, a bacterium isolated from a depth of 3519 m in the Vostok Ice Core. Our investigation is examining the impact of extra cellular proteins from this ice-adapted bacterium on the formation of ice crystals and characterizing the inter-crystalline liquid filled vein network using cryo-microscopy.

  7. 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. PMID:26890218

  8. 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

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

    PubMed Central

    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

  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. 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/. PMID:26590254

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

    PubMed Central

    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/. PMID:26590254

  13. 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

  14. Cellular prion protein (PrPC) and its role in stress responses

    PubMed Central

    Zeng, Liang; Zou, Wenquan; Wang, Gongxian

    2015-01-01

    Investigation of the physiological function of cellular prion protein (PrPC) has been developed by the generation of transgenic mice, however, the pathological mechanisms related to PrPC in prion diseases such as transmissible spongiform encephalopathies (TSEs) are still abstruse. Regardless of some differences, most studies describe the neuroprotective role of PrPC in environmental stresses. In this review, we will update the current knowledge on the responses of PrPC to various stresses, especially those correlated with cell signaling and neural degeneration, including ischemia, oxidative stress, inflammation and autophagy. PMID:26221369

  15. 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

  16. AMP-activated protein kinase regulates L-arginine mediated cellular responses

    PubMed Central

    2013-01-01

    Background Our prior study revealed the loss in short-term L-Arginine (ARG) therapeutic efficacy after continuous exposure; resulting in tolerance development, mediated by endothelial nitric oxide synthase (eNOS) down-regulation, secondary to oxidative stress and induced glucose accumulation. However, the potential factor regulating ARG cellular response is presently unknown. Method Human umbilical vein endothelial cells were incubated with 100 μM ARG for 2 h in buffer (short-term or acute), or for 7 days in culture medium and challenged for 2 h in buffer (continuous or chronic), in the presence or absence of other agents. eNOS activity was determined by analyzing cellular nitrite/nitrate (NO2–/NO3–), and AMP-activated protein kinase (AMPK) activity was assayed using SAMS peptide. 13C6 glucose was added to medium to measure glucose uptake during cellular treatments, which were determined by LC-MS/MS. Cellular glucose was identified by o-toluidine method. Superoxide (O2•–) was identified by EPR-spin-trap, and peroxynitrite (ONOO–) was measured by flow-cytometer using aminophenyl fluorescein dye. Results Short-term incubation of cells with 100 μM ARG in the presence or absence of 30 μM L-NG-Nitroarginine methyl ester (L-NAME) or 30 μM AMPK inhibitor (compound C, CMP-C) increased cellular oxidative stress and overall glucose accumulation with no variation in glucose transporter-1 (GLUT-1), or AMPK activity from control. The increase in total NO2–/NO3– after 2 h 100 μM ARG exposure, was suppressed in cells co-incubated with 30 μM CMP-C or L-NAME. Long-term exposure of ARG with or without CMP-C or L-NAME suppressed NO2–/NO3–, glucose uptake, GLUT-1, AMPK expression and activity below control, and increased overall cellular glucose, O2•– and ONOO–. Gluconeogenesis inhibition with 30 μM 5-Chloro-2-N-2,5-dichlorobenzenesulfonamido-benzoxazole (CDB) during ARG exposure for 2 h maintained overall cellular glucose to control, but increased

  17. HUGE: a database for human large proteins identified in the Kazusa cDNA sequencing project.

    PubMed

    Kikuno, R; Nagase, T; Suyama, M; Waki, M; Hirosawa, M; Ohara, O

    2000-01-01

    HUGE is a database for human large proteins newly identified in the Kazusa cDNA project, the aim of which is to predict the primary structure of proteins from the sequences of human large cDNAs (>4 kb). In particular, cDNA clones capable of coding for large proteins (>50 kDa) are the current targets of the project. HUGE contains >1100 cDNA sequences and detailed information obtained through analysis of the sequences of cDNAs and the predicted proteins. Besides an increase in the number of cDNA entries, the amount of experimental data for expression profiling has been largely increased and data on chromosomal locations have been newly added. All of the protein-coding regions were examined by GeneMark analysis, and the results of a motif/domain search of each predicted protein sequence against the Pfam database have been newly added. HUGE is available through the WWW at http://www.kazusa.or.jp/huge PMID:10592264

  18. Protein structure databases with new web services for structural biology and biomedical research.

    PubMed

    Standley, Daron M; Kinjo, Akira R; Kinoshita, Kengo; Nakamura, Haruki

    2008-07-01

    The Protein Data Bank Japan (PDBj) curates, edits and distributes protein structural data as a member of the worldwide Protein Data Bank (wwPDB) and currently processes approximately 25-30% of all deposited data in the world. Structural information is enhanced by the addition of biological and biochemical functional data as well as experimental details extracted from the literature and other databases. Several applications have been developed at PDBj for structural biology and biomedical studies: (i) a Java-based molecular graphics viewer, jV; (ii) display of electron density maps for the evaluation of structure quality; (iii) an extensive database of molecular surfaces for functional sites, eF-site, as well as a search service for similar molecular surfaces, eF-seek; (iv) identification of sequence and structural neighbors; (v) a graphical user interface to all known protein folds with links to the above applications, Protein Globe. Recent examples are shown that highlight the utility of these tools in recognizing remote homologies between pairs of protein structures and in assigning putative biochemical functions to newly determined targets from structural genomics projects. PMID:18430752

  19. 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

  20. 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.

  1. 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. PMID:26245978

  2. 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.

  3. 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.

  4. A quantitative chaperone interaction network reveals the architecture of cellular protein homeostasis pathways.

    PubMed

    Taipale, Mikko; Tucker, George; Peng, Jian; Krykbaeva, Irina; Lin, Zhen-Yuan; Larsen, Brett; Choi, Hyungwon; Berger, Bonnie; Gingras, Anne-Claude; Lindquist, Susan

    2014-07-17

    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 cofactors (cochaperones) that regulate their specificity and function. However, how these cochaperones regulate protein folding and whether they have chaperone-independent biological functions is largely unknown. We combined mass spectrometry and quantitative high-throughput LUMIER assays to systematically characterize the chaperone-cochaperone-client interaction network in human cells. We uncover hundreds of chaperone clients, delineate their participation in specific cochaperone complexes, and establish a surprisingly distinct network of protein-protein interactions for cochaperones. As a salient example of the power of such analysis, we establish that NUDC family cochaperones specifically associate with structurally related but evolutionarily distinct β-propeller folds. We provide a framework for deciphering the proteostasis network and its regulation in development and disease and expand the use of chaperones as sensors for drug-target engagement. PMID:25036637

  5. Accumulation of soluble and nucleolar-associated p53 proteins following cellular stress.

    PubMed

    Klibanov, S A; O'Hagan, H M; Ljungman, M

    2001-05-01

    The tumor suppressor p53 is a nucleocytoplasmic shuttling protein that accumulates in the nucleus of cells exposed to various cellular stresses. One important role of nuclear p53 is to mobilize a stress response by transactivating target genes such as the p21(Waf1) gene. In this study, we investigated more closely the localization of p53 in cells following various stresses. Immunocytochemistry of fixed human fibroblasts treated with either UV light, the kinase and transcription inhibitor DRB or the proteasome inhibitor MG132 revealed abundant p53 localized to the nucleus. When cells treated with UV or DRB were permeabilized prior to fixation to allow soluble proteins to diffuse, the nuclear p53 signal was abolished. However, in cells treated with MG132, residual p53 localized to distinct large foci. Furthermore, nucleolin co-localized with p53 to these foci, suggesting that these foci were nucleolar structures. Interestingly, the MDM2 protein was found to co-localize with p53 to nucleolar structures following proteasome inhibition. Our results suggest that the p53 proteins accumulating in the nucleus following UV-irradiation or blockage of transcription are freely soluble and, thus, should be able to roam the nucleus to ensure high occupancy of p53 binding sites. However, inhibition of proteasome activity may be a unique stress in that it leads to the sequestering of p53 proteins to the nucleolus, thereby blunting the p53-mediated transactivation of target genes. PMID:11329373

  6. 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

  7. Functional diversity of protein phosphatase-1, a cellular economizer and reset button.

    PubMed

    Ceulemans, Hugo; Bollen, Mathieu

    2004-01-01

    The protein serine/threonine phosphatase protein phosphatase-1 (PP1) is a ubiquitous eukaryotic enzyme that regulates a variety of cellular processes through the dephosphorylation of dozens of substrates. This multifunctionality of PP1 relies on its association with a host of function-specific targetting and substrate-specifying proteins. In this review we discuss how PP1 affects the biochemistry and physiology of eukaryotic cells. The picture of PP1 that emerges from this analysis is that of a "green" enzyme that promotes the rational use of energy, the recycling of protein factors, and a reversal of the cell to a basal and/or energy-conserving state. Thus PP1 promotes a shift to the more energy-efficient fuels when nutrients are abundant and stimulates the storage of energy in the form of glycogen. PP1 also enables the relaxation of actomyosin fibers, the return to basal patterns of protein synthesis, and the recycling of transcription and splicing factors. In addition, PP1 plays a key role in the recovery from stress but promotes apoptosis when cells are damaged beyond repair. Furthermore, PP1 downregulates ion pumps and transporters in various tissues and ion channels that are involved in the excitation of neurons. Finally, PP1 promotes the exit from mitosis and maintains cells in the G1 or G2 phases of the cell cycle. PMID:14715909

  8. 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

  9. dbPAF: an integrative database of protein phosphorylation in animals and fungi.

    PubMed

    Ullah, Shahid; Lin, Shaofeng; Xu, Yang; Deng, Wankun; Ma, Lili; Zhang, Ying; Liu, Zexian; Xue, Yu

    2016-01-01

    Protein phosphorylation is one of the most important post-translational modifications (PTMs) and regulates a broad spectrum of biological processes. Recent progresses in phosphoproteomic identifications have generated a flood of phosphorylation sites, while the integration of these sites is an urgent need. In this work, we developed a curated database of dbPAF, containing known phosphorylation sites in H. sapiens, M. musculus, R. norvegicus, D. melanogaster, C. elegans, S. pombe and S. cerevisiae. From the scientific literature and public databases, we totally collected and integrated 54,148 phosphoproteins with 483,001 phosphorylation sites. Multiple options were provided for accessing the data, while original references and other annotations were also present for each phosphoprotein. Based on the new data set, we computationally detected significantly over-represented sequence motifs around phosphorylation sites, predicted potential kinases that are responsible for the modification of collected phospho-sites, and evolutionarily analyzed phosphorylation conservation states across different species. Besides to be largely consistent with previous reports, our results also proposed new features of phospho-regulation. Taken together, our database can be useful for further analyses of protein phosphorylation in human and other model organisms. The dbPAF database was implemented in PHP + MySQL and freely available at http://dbpaf.biocuckoo.org. PMID:27010073

  10. dbPAF: an integrative database of protein phosphorylation in animals and fungi

    PubMed Central

    Ullah, Shahid; Lin, Shaofeng; Xu, Yang; Deng, Wankun; Ma, Lili; Zhang, Ying; Liu, Zexian; Xue, Yu

    2016-01-01

    Protein phosphorylation is one of the most important post-translational modifications (PTMs) and regulates a broad spectrum of biological processes. Recent progresses in phosphoproteomic identifications have generated a flood of phosphorylation sites, while the integration of these sites is an urgent need. In this work, we developed a curated database of dbPAF, containing known phosphorylation sites in H. sapiens, M. musculus, R. norvegicus, D. melanogaster, C. elegans, S. pombe and S. cerevisiae. From the scientific literature and public databases, we totally collected and integrated 54,148 phosphoproteins with 483,001 phosphorylation sites. Multiple options were provided for accessing the data, while original references and other annotations were also present for each phosphoprotein. Based on the new data set, we computationally detected significantly over-represented sequence motifs around phosphorylation sites, predicted potential kinases that are responsible for the modification of collected phospho-sites, and evolutionarily analyzed phosphorylation conservation states across different species. Besides to be largely consistent with previous reports, our results also proposed new features of phospho-regulation. Taken together, our database can be useful for further analyses of protein phosphorylation in human and other model organisms. The dbPAF database was implemented in PHP + MySQL and freely available at http://dbpaf.biocuckoo.org. PMID:27010073

  11. 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

  12. Manipulation of cellular light from green fluorescent protein by a femtosecond laser

    NASA Astrophysics Data System (ADS)

    He, Hao; Li, Shiyang; Wang, Shaoyang; Hu, Minglie; Cao, Youjia; Wang, Chingyue

    2012-10-01

    Green fluorescent protein (GFP) is one of the most widely studied and exploited proteins in biochemistry and cell biology. It emits fluorescence following optical excitation, which is usually provided by a laser. Here, we report that fluorescence from enhanced GFP can be `turned off' by exposing cells to laser light. A short flash of femtosecond laser light is shown to deplete calcium in the endoplasmic reticulum of cells. Calcium-release-activated calcium channels are then activated by stromal interaction molecule 1 (STIM1). The rise in intracellular Ca2+ depolarizes mitochondria and increases the leakage of reactive oxygen species, which then permanently bleach the GFP. This controllable optical scheme for reactive oxygen species generation can also be used to modulate the photoconversion of GFP fluorescence from green to red emission and provide a mechanism for influencing cellular molecular dynamics.

  13. 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

  14. 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.

  15. Redox Modulation of Cellular Signaling and Metabolism Through Reversible Oxidation of Methionine Sensors in Calcium Regulatory Proteins

    SciTech Connect

    Bigelow, Diana J.; Squier, Thomas C.

    2005-01-17

    Adaptive responses associated with environmental stressors are critical to cell survival. These involve the modulation of central signaling protein functions through site-specific and enzymatically reversible oxidative modifications of methionines to coordinate cellular metabolism, energy utilization, and calcium signaling. Under conditions when cellular redox and antioxidant defenses are overwhelmed, the selective oxidation of critical methionines within selected protein sensors functions to down-regulate energy metabolism and the further generation of reactive oxygen species (ROS). Mechanistically, these functional changes within protein sensors take advantage of the helix-breaking character of methionine sulfoxide. Thus, depending on either the ecological niche of the organism or the cellular milieu of different organ systems, cellular metabolism can be fine-tuned to maintain optimal function in the face of variable amounts of collateral oxidative damage. The sensitivity of several calcium regulatory proteins to oxidative modification provides cellular sensors that link oxidative stress to cellular response and recovery. Calmodulin (CaM) is one such critical calcium regulatory protein, which is functionally sensitive to methionine oxidation. Helix destabilization resulting from the oxidation of either Met{sup 144} or Met{sup 145} results in the nonproductive association between CaM and target proteins. The ability of oxidized CaM to stabilize its target proteins in an inhibited state with an affinity similar to that of native (unoxidized) CaM permits this central regulatory protein to function as a cellular rheostat that down-regulates energy metabolism in response to oxidative stress. Likewise, oxidation of a methionine within a critical switch region of the regulatory protein phospholamban is expected to destabilize the phosphorylationdependent helix formation necessary for the release of enzyme inhibition, resulting in a down-regulation of the Ca-ATPase in

  16. MannDB – A microbial database of automated protein sequence analyses and evidence integration for protein characterization

    PubMed Central

    Zhou, Carol L Ecale; Lam, Marisa W; Smith, Jason R; Zemla, Adam T; Dyer, Matthew D; Kuczmarski, Thomas A; Vitalis, Elizabeth A; Slezak, Thomas R

    2006-01-01

    Background 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. Description 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. Conclusion MannDB comprises a large number of genomes and comprehensive protein sequence analyses

  17. 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). PMID:24922051

  18. 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

  19. Interaction of Gamma-Herpesvirus Genome Maintenance Proteins with Cellular Chromatin

    PubMed Central

    Callegari, Simone; Gastaldello, Stefano; Masucci, Maria G.

    2013-01-01

    The capacity of gamma-herpesviruses to establish lifelong infections is dependent on the expression of genome maintenance proteins (GMPs) that tether the viral episomes to cellular chromatin and allow their persistence in latently infected proliferating cells. Here we have characterized the chromatin interaction of GMPs encoded by viruses belonging to the genera Lymphocryptovirus (LCV) and Rhadinovirus (RHV). We found that, in addition to a similar diffuse nuclear localization and comparable detergent resistant interaction with chromatin in transfected cells, all GMPs shared the capacity to promote the decondensation of heterochromatin in the A03-1 reporter cell line. They differed, however, in their mobility measured by fluorescence recovery after photobleaching (FRAP), and in the capacity to recruit accessory molecules required for the chromatin remodeling function. While the AT-hook containing GMPs of LCVs were highly mobile, a great variability was observed among GMPs encoded by RHV, ranging from virtually immobile to significantly reduced mobility compared to LCV GMPs. Only the RHV GMPs recruited the bromo- and extra terminal domain (BET) proteins BRD2 and BRD4 to the site of chromatin remodeling. These findings suggest that differences in the mode of interaction with cellular chromatin may underlie different strategies adopted by these viruses for reprogramming of the host cells during latency. PMID:23667520

  20. 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

  1. Hexokinase 2 controls cellular stress response through localization of an RNA-binding protein

    PubMed Central

    Courteau, L; Crasto, J; Hassanzadeh, G; Baird, S D; Hodgins, J; Liwak-Muir, U; Fung, G; Luo, H; Stojdl, D F; Screaton, R A; Holcik, M

    2015-01-01

    Subcellular localization of RNA-binding proteins is a key determinant of their ability to control RNA metabolism and cellular stress response. Using an RNAi-based kinome-wide screen, we identified hexokinase 2 (HK2) as a regulator of the cytoplasmic accumulation of hnRNP A1 in response to hypertonic stress and human rhinovirus infection (HRV). We show that inhibition of HK2 expression or pharmacological inhibition of HK2 activity blocks the cytoplasmic accumulation of heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1), restores expression of B-cell lymphoma-extra large (Bcl-xL), and protects cells against hypertonic stress-induced apoptosis. Reduction of HK2 protein levels by knockdown results in decreased HRV replication, a delay in HRV-induced cell death, and a reduced number of infected cells, all of which can be rescued by forced expression of a cytoplasm-restricted hnRNP A1. Our data elucidate a novel role for HK2 in cellular stress response and viral infection that could be exploited for therapeutic intervention. PMID:26247723

  2. 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

  3. The cellular bromodomain protein Brd4 has multiple functions in E2-mediated papillomavirus transcription activation.

    PubMed

    Helfer, Christine M; Yan, Junpeng; You, Jianxin

    2014-08-01

    The cellular bromodomain protein Brd4 functions in multiple processes of the papillomavirus life cycle, including viral replication, genome maintenance, and gene transcription through its interaction with the viral protein, E2. However, the mechanisms by which E2 and Brd4 activate viral transcription are still not completely understood. In this study, we show that recruitment of positive transcription elongation factor b (P-TEFb), a functional interaction partner of Brd4 in transcription activation, is important for E2's transcription activation activity. Furthermore, chromatin immunoprecipitation (ChIP) analyses demonstrate that P-TEFb is recruited to the actual papillomavirus episomes. We also show that E2's interaction with cellular chromatin through Brd4 correlates with its papillomavirus transcription activation function since JQ1(+), a bromodomain inhibitor that efficiently dissociates E2-Brd4 complexes from chromatin, potently reduces papillomavirus transcription. Our study identifies a specific function of Brd4 in papillomavirus gene transcription and highlights the potential use of bromodomain inhibitors as a method to disrupt the human papillomavirus (HPV) life cycle. PMID:25140737

  4. 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

  5. Looking for a needle in a haystack: Cellular proteins that may interact with the tyrosine-based sorting signal of the TGEV S protein.

    PubMed

    Trincone, Anna; Schwegmann-Weßels, Christel

    2015-04-16

    The spike protein S of transmissible gastroenteritis virus, an Alphacoronavirus, contains a tyrosine-based sorting signal that is responsible for ERGIC retention and may be important for a correct viral assembly process. To find out whether the S protein interacts with cellular proteins via this sorting signal, a pulldown assay with GST fusion proteins was performed. Filamin A has been identified as a putative interaction candidate. Immunofluorescence assays confirmed a co-localization between the TGEV S protein and filamin A. Further experiments have to be performed to prove a significant impact of filamin A on TGEV infection. Different approaches of several researchers for the identification of cellular interaction candidates relevant for coronavirus replication are summarized. These results may help in the future to identify the role of cellular proteins during coronavirus assembly at the ER-Golgi intermediate compartment. PMID:25481285

  6. 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.

  7. LMPID: A manually curated database of linear motifs mediating protein–protein interactions

    PubMed Central

    Sarkar, Debasree; Jana, Tanmoy; Saha, Sudipto

    2015-01-01

    Linear motifs (LMs), used by a subset of all protein–protein interactions (PPIs), bind to globular receptors or domains and play an important role in signaling networks. LMPID (Linear Motif mediated Protein Interaction Database) is a manually curated database which provides comprehensive experimentally validated information about the LMs mediating PPIs from all organisms on a single platform. About 2200 entries have been compiled by detailed manual curation of PubMed abstracts, of which about 1000 LM entries were being annotated for the first time, as compared with the Eukaryotic LM resource. The users can submit their query through a user-friendly search page and browse the data in the alphabetical order of the bait gene names and according to the domains interacting with the LM. LMPID is freely accessible at http://bicresources.jcbose. ac.in/ssaha4/lmpid and contains 1750 unique LM instances found within 1181 baits interacting with 552 prey proteins. In summary, LMPID is an attempt to enrich the existing repertoire of resources available for studying the LMs implicated in PPIs and may help in understanding the patterns of LMs binding to a specific domain and develop prediction model to identify novel LMs specific to a domain and further able to predict inhibitors/modulators of PPI of interest. Database URL: http://bicresources.jcbose.ac.in/ssaha4/lmpid PMID:25776024

  8. 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. PMID:24174539

  9. 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

  10. 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

  11. 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

  12. Methylated arsenic metabolites bind to PML protein but do not induce cellular differentiation and PML-RARα protein degradation

    PubMed Central

    Zhang, Yan Fang; Bu, Na; Zhou, Jin; Cao, Feng Lin; Naranmandura, Hua

    2015-01-01

    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-RARa degradation. However, after injection, As2O3 is rapidly methylated in body to different intermediate metabolites such as trivalent monomethylarsonous acid (MMAIII) and dimethylarsinous acid (DMAIII), 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 iAsIII) and its intermediate metabolites (i.e., MMAIII/DMAIII) in NB4 cells. Inorganic iAsIII predominantly showed induction of cell differentiation, while MMAIII and DMAIII specifically showed to induce mitochondria and endoplasmic reticulum-mediated apoptosis, respectively. On the other hand, in contrast to iAsIII, MMAIII 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-RARa fusion protein. Moreover, methylated arsenicals can efficiently lead to cellular apoptosis, however, they are incapable of inducing NB4 cell differentiation. PMID:26213848

  13. The role of ORMDL proteins, guardians of cellular sphingolipids, in asthma.

    PubMed

    Paulenda, T; Draber, P

    2016-07-01

    A family of widely expressed ORM-like (ORMDL) proteins has been recently linked to asthma in genomewide association studies in humans and extensively explored in in vivo studies in mice. ORMDL proteins are key regulators of serine palmitoyltransferase, an enzyme catalyzing the initial step of sphingolipid biosynthesis. Sphingolipids play prominent roles in cell signaling and response to stress, and they affect the mechanistic properties of cellular membranes. Deregulation of sphingolipid biosynthesis and their recycling has been proven to support and even cause several diseases including allergy, inflammation, and asthma. ORMDL3, the most extensively studied member of the ORMDL family, has been shown to be important for endoplasmic reticulum homeostasis by regulating the unfolded protein response and calcium response. In immune cells, ORMDL3 is involved in migration and in the production of proinflammatory cytokines. Furthermore, changes in the expression level of ORMDL3 are important in allergen-induced asthma pathologies. This review focuses on functional aspects of the ORMDL family proteins, which may serve as new therapeutic targets for the treatment of asthma and some other life-threatening diseases. PMID:26969910

  14. Cellular stress induces Bax-regulated nuclear bubble budding and rupture followed by nuclear protein release.

    PubMed

    Lindenboim, Liora; Sasson, Tiki; Worman, Howard J; Borner, Christoph; Stein, Reuven

    2014-01-01

    Cellular stress triggers many pathways including nuclear protein redistribution. We previously discovered that this process is regulated by Bax but the underlying mechanism has not yet been studied. Here we define this mechanism by showing that apoptotic stimuli cause Bax-regulated disturbances in lamin A/C and nuclear envelope (NE)-associated proteins which results in the generation and subsequent rupture of nuclear protein-containing bubbles. The bubbles do not contain DNA and are encapsulated by impaired nuclear pore-depleted NE. Stress-induced generation and rupture of nuclear bubbles ultimately leads to the discharge of nuclear proteins into the cytoplasm. This process precedes morphological changes of apoptosis and occurs independently of caspases. Rescue experiments revealed that this Bax effect is non-canonical, i.e. it requires the BH3 domain and α-helices 5 and 6 but it is not inhibited by Bcl(-)xL. Targeting Bax to the NE by the Klarsicht/ANC-1/Syne-1 homology (KASH) domain effectively triggers the generation and rupture of nuclear bubbles. Overall, our findings provide evidence for a novel stress-response, which is regulated by a non-canonical action of Bax on the NE. PMID:25482068

  15. Induction of cellular prion protein (PrPc) under hypoxia inhibits apoptosis caused by TRAIL treatment

    PubMed Central

    Lee, Ju-Hee; Moon, Ji-Hong; Kim, Sung-Wook; Lee, You-Jin; Park, Sang-Youel

    2015-01-01

    Hypoxia decreases cytotoxic responses to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) protein. Cellular prion protein (PrPc) is regulated by HIF-1α in neurons. We hypothesized that PrPc is involved in hypoxia-mediated resistance to TRAIL-induced apoptosis. We found that hypoxia induced PrPc protein and inhibited TRAIL-induced apoptosis. Thus silencing of PrPc increased TRAIL-induced apoptosis under hypoxia. Overexpression of PrPc protein using an adenoviral vector inhibited TRAIL-induced apoptosis. In xenograft model in vivo, shPrPc transfected cells were more sensitive to TRAIL-induced apoptosis than in shMock transfected cells. Molecular chemo-therapy approaches based on the regulation of PrPc expression need to address anti-tumor function of TRAIL under hypoxia. Molecular chemo-therapy approaches based on the regulation of PrPc expression need to address anti-tumor function of TRAIL under hypoxia. PMID:25742790

  16. 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

  17. 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.

  18. 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

  19. Addressing statistical biases in nucleotide-derived protein databases for proteogenomic search strategies.

    PubMed

    Blakeley, Paul; Overton, Ian M; Hubbard, Simon J

    2012-11-01

    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

  20. Identification of Cellular Calcium Binding Protein Calmodulin as a Regulator of Rotavirus A Infection during Comparative Proteomic Study

    PubMed Central

    Chattopadhyay, Shiladitya; Basak, Trayambak; Nayak, Mukti Kant; Bhardwaj, Gourav; Mukherjee, Anupam; Bhowmick, Rahul; Sengupta, Shantanu; Chakrabarti, Oishee; Chatterjee, Nabendu S.; Chawla-Sarkar, Mamta

    2013-01-01

    Rotavirus (RV) being the major diarrhoegenic virus causes around 527000 children death (<5years age) worldwide. In cellular environment, viruses constantly adapt and modulate to survive and replicate while the host cell also responds to combat the situation and this results in the differential regulation of cellular proteins. To identify the virus induced differential expression of proteins, 2D-DIGE (Two-dimensional Difference Gel Electrophoresis) based proteomics was used. For this, HT-29 cells were infected with RV strain SA11 for 0 hours, 3 hours and 9 hours post infection (hpi), differentially expressed spots were excised from the gel and identified using MALDI-TOF/TOF mass spectrometry. 2D-DIGE based proteomics study identified 32 differentially modulated proteins, of which 22 were unique. Some of these were validated in HT-29 cell line and in BALB/c mice model. One of the modulated cellular proteins, calmodulin (CaM) was found to directly interact with RV protein VP6 in the presence of Ca2+. Ca2+-CaM/VP6 interaction positively regulates RV propagation since both CaM inhibitor (W-7) and Ca2+ chelator (BAPTA-AM) resulted in decreased viral titers. This study not only identifies differentially modulated cellular proteins upon infection with rotavirus in 2D-DIGE but also confirmed positive engagement of cellular Ca2+/CaM during viral pathogenesis. PMID:23437200

  1. 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...

  2. 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

  3. Role of the cellular prion protein in the neuron adaptation strategy to copper deficiency.

    PubMed

    Urso, Emanuela; Manno, Daniela; Serra, Antonio; Buccolieri, Alessandro; Rizzello, Antonia; Danieli, Antonio; Acierno, Raffaele; Salvato, Benedetto; Maffia, Michele

    2012-08-01

    Copper transporter 1 (CTR1), cellular prion protein (PrP(C)), natural resistance-associated macrophage protein 2 (NRAMP2) and ATP7A proteins control the cell absorption and efflux of copper (Cu) ions in nervous tissues upon physiological conditions. Little is known about their regulation under reduced Cu availability, a condition underlying the onset of diffused neurodegenerative disorders. In this study, rat neuron-like cells were exposed to Cu starvation for 48 h. The activation of Caspase-3 enzymes and the impairment of Cu,Zn superoxide dismutase (Cu,Zn SOD) activity depicted the initiation of a pro-apoptotic program, preliminary to the appearance of the morphological signs of apoptosis. The transcriptional response related to Cu transport proteins has been investigated. Notably, PrP(C) transcript and protein levels were consistently elevated upon Cu deficiency. The CTR1 protein amount was stable, despite a two-fold increase in the transcript amount, meaning the activation of post-translational regulatory mechanisms. NRAMP2 and ATP7A expressions were unvaried. The up-regulated PrP(C) has been demonstrated to enhance the cell Cu uptake ability by about 50% with respect to the basal transport, and so sustain the Cu delivery to the Cu,Zn SOD cuproenzymes. Conclusively, the study suggests a pivotal role for PrP(C) in the cell adaptation to Cu limitation through a direct activity of ion uptake. In this view, the PrP(C) accumulation observed in several cancer cell lines could be interpreted as a molecular marker of cell Cu deficiency and a potential target of therapeutic interventions against disorders caused by metal imbalances. PMID:22362149

  4. TSQ, a Common Fluorescent Sensor for Cellular Zinc, Images Zinc Proteins

    PubMed Central

    Meeusen, Jeffrey W.; Tomasiewicz, Henry; Nowakowski, Andrew; Petering, David H.

    2011-01-01

    Zn2+ is a necessary cofactor for thousands of mammalian proteins. Research has suggested that transient fluxes of cellular Zn2+ are also involved in processes such as apoptosis. Observations of Zn2+ trafficking have been collected using Zn2+ responsive fluorescent dyes. A commonly used Zn2+ fluorophore is TSQ. The chemical species responsible for TSQ's observed fluorescence in resting or activated cells have not been characterized. Parallel fluorescence microscopy and spectrofluorometry of LLC-PK1 cells incubated with TSQ demonstrated punctate staining that concentrated around the nucleus and was characterized by an emission maximum near 470 nm. Addition of cell permeable Zn-pyrithione resulted in greatly increased, diffuse fluorescence that shifted the emission peak to 490 nm, indicative of the formation of Zn(TSQ)2. TPEN, a cell permeant Zn2+ chelator, largely quenched TSQ fluorescence returning the residual fluorescence to the 470 nm emission maximum. Gel filtration chromatography of cell supernatant from LLC-PK1 cells treated with TSQ revealed that TSQ fluorescence (470 nm emission) eluted with the proteome fractions. Similarly, addition of TSQ to proteome prior to chromatography resulted in 470 nm fluorescence emission that was not observed in smaller molecular weight fractions. It is hypothesized that Zn-TSQ fluorescence, blue-shifted from the 490 nm emission maximum of Zn(TSQ)2, results from ternary complex, TSQ-Zn-protein formation. As an example, Zn-carbonic anhydrase formed a ternary adduct with TSQ characterized by a fluorescence emission maximum of 470 nm and a dissociation constant of 1.55 × 10-7 M. Quantification of TSQ-Zn-proteome fluorescence indicated that approximately 8% of cellular Zn2+ was imaged by TSQ. These results were generalized to other cell types and model Zn-proteins. PMID:21774459

  5. Cellular IAP proteins and LUBAC differentially regulate necrosome-associated RIP1 ubiquitination.

    PubMed

    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

  6. 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

  7. 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.

  8. 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.

  9. 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. PMID:26237451

  10. 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 Central

    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

    ABSTRACT Prion diseases involve the conversion of the endogenous cellular prion protein, PrPC, into a misfolded infectious isoform, PrPSc. Several functions have been attributed to PrPC, and its role has also been investigated in the olfactory system. PrPC 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 PrPC expression in OE and its putative role in olfaction, we screened a mouse OE cDNA library to identify novel PrPC-binding partners. Ten different putative PrPC 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 PrPC 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 PrPC-Stub1 interaction are under investigation. The PrPC-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 PrPC 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. PMID:26237451

  11. 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

  12. 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

  13. 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

  14. 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

  15. NCBI Reference Sequence (RefSeq): a curated non-redundant sequence database of genomes, transcripts and proteins.

    PubMed

    Pruitt, Kim D; Tatusova, Tatiana; Maglott, Donna R

    2005-01-01

    The National Center for Biotechnology Information (NCBI) Reference Sequence (RefSeq) database (http://www.ncbi.nlm.nih.gov/RefSeq/) provides a non-redundant collection of sequences representing genomic data, transcripts and proteins. Although the goal is to provide a comprehensive dataset representing the complete sequence information for any given species, the database pragmatically includes sequence data that are currently publicly available in the archival databases. The database incorporates data from over 2400 organisms and includes over one million proteins representing significant taxonomic diversity spanning prokaryotes, eukaryotes and viruses. Nucleotide and protein sequences are explicitly linked, and the sequences are linked to other resources including the NCBI Map Viewer and Gene. Sequences are annotated to include coding regions, conserved domains, variation, references, names, database cross-references, and other features using a combined approach of collaboration and other input from the scientific community, automated annotation, propagation from GenBank and curation by NCBI staff. PMID:15608248

  16. HNdb: an integrated database of gene and protein information on head and neck squamous cell carcinoma

    PubMed Central

    Henrique, Tiago; José Freitas da Silveira, Nelson; Henrique Cunha Volpato, Arthur; Mioto, Mayra Mataruco; Carolina Buzzo Stefanini, Ana; Bachir Fares, Adil; Gustavo da Silva Castro Andrade, João; Masson, Carolina; Verónica Mendoza López, Rossana; Daumas Nunes, Fabio; Paulo Kowalski, Luis; Severino, Patricia; Tajara, Eloiza Helena

    2016-01-01

    The total amount of scientific literature has grown rapidly in recent years. Specifically, there are several million citations in the field of cancer. This makes it difficult, if not impossible, to manually retrieve relevant information on the mechanisms that govern tumor behavior or the neoplastic process. Furthermore, cancer is a complex disease or, more accurately, a set of diseases. The heterogeneity that permeates many tumors is particularly evident in head and neck (HN) cancer, one of the most common types of cancer worldwide. In this study, we present HNdb, a free database that aims to provide a unified and comprehensive resource of information on genes and proteins involved in HN squamous cell carcinoma, covering data on genomics, transcriptomics, proteomics, literature citations and also cross-references of external databases. Different literature searches of MEDLINE abstracts were performed using specific Medical Subject Headings (MeSH terms) for oral, oropharyngeal, hypopharyngeal and laryngeal squamous cell carcinomas. A curated gene-to-publication assignment yielded a total of 1370 genes related to HN cancer. The diversity of results allowed identifying novel and mostly unexplored gene associations, revealing, for example, that processes linked to response to steroid hormone stimulus are significantly enriched in genes related to HN carcinomas. Thus, our database expands the possibilities for gene networks investigation, providing potential hypothesis to be tested. Database URL: http://www.gencapo.famerp.br/hndb PMID:27013077

  17. Kin-Driver: a database of driver mutations in protein kinases.

    PubMed

    Simonetti, Franco L; Tornador, Cristian; Nabau-Moretó, Nuria; Molina-Vila, Miguel A; Marino-Buslje, Cristina

    2014-01-01

    Somatic mutations in protein kinases (PKs) are frequent driver events in many human tumors, while germ-line mutations are associated with hereditary diseases. Here we present Kin-driver, the first database that compiles driver mutations in PKs with experimental evidence demonstrating their functional role. Kin-driver is a manual expert-curated database that pays special attention to activating mutations (AMs) and can serve as a validation set to develop new generation tools focused on the prediction of gain-of-function driver mutations. It also offers an easy and intuitive environment to facilitate the visualization and analysis of mutations in PKs. Because all mutations are mapped onto a multiple sequence alignment, analogue positions between kinases can be identified and tentative new mutations can be proposed for studying by transferring annotation. Finally, our database can also be of use to clinical and translational laboratories, helping them to identify uncommon AMs that can correlate with response to new antitumor drugs. The website was developed using PHP and JavaScript, which are supported by all major browsers; the database was built using MySQL server. Kin-driver is available at: http://kin-driver.leloir.org.ar/ PMID:25414382

  18. IUPHAR-DB: the IUPHAR database of G protein-coupled receptors and ion channels.

    PubMed

    Harmar, Anthony J; Hills, Rebecca A; Rosser, Edward M; Jones, Martin; Buneman, O Peter; Dunbar, Donald R; Greenhill, Stuart D; Hale, Valerie A; Sharman, Joanna L; Bonner, Tom I; Catterall, William A; Davenport, Anthony P; Delagrange, Philippe; Dollery, Colin T; Foord, Steven M; Gutman, George A; Laudet, Vincent; Neubig, Richard R; Ohlstein, Eliot H; Olsen, Richard W; Peters, John; Pin, Jean-Philippe; Ruffolo, Robert R; Searls, David B; Wright, Mathew W; Spedding, Michael

    2009-01-01

    The IUPHAR database (IUPHAR-DB) integrates peer-reviewed pharmacological, chemical, genetic, functional and anatomical information on the 354 nonsensory G protein-coupled receptors (GPCRs), 71 ligand-gated ion channel subunits and 141 voltage-gated-like ion channel subunits encoded by the human, rat and mouse genomes. These genes represent the targets of approximately one-third of currently approved drugs and are a major focus of drug discovery and development programs in the pharmaceutical industry. IUPHAR-DB provides a comprehensive description of the genes and their functions, with information on protein structure and interactions, ligands, expression patterns, signaling mechanisms, functional assays and biologically important receptor variants (e.g. single nucleotide polymorphisms and splice variants). In addition, the phenotypes resulting from altered gene expression (e.g. in genetically altered animals or in human genetic disorders) are described. The content of the database is peer reviewed by members of the International Union of Basic and Clinical Pharmacology Committee on Receptor Nomenclature and Drug Classification (NC-IUPHAR); the data are provided through manual curation of the primary literature by a network of over 60 subcommittees of NC-IUPHAR. Links to other bioinformatics resources, such as NCBI, Uniprot, HGNC and the rat and mouse genome databases are provided. IUPHAR-DB is freely available at http://www.iuphar-db.org. PMID:18948278

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

    PubMed Central

    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

  20. 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...

  1. 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. PMID:24758495

  2. Combinatorial effects of continuous protein synthesis, ERK-signaling, and reactive oxygen species on induction of cellular senescence.

    PubMed

    Takauji, Yuki; En, Atsuki; Miki, Kensuke; Ayusawa, Dai; Fujii, Michihiko

    2016-07-15

    Mammalian cells, when treated with sub-lethal doses of genotoxic stresses, slow down DNA synthesis but continue protein synthesis. Thus, these cells show an accumulation of proteins and undergo unbalanced growth. In the previous studies, we have shown that HeLa cells treated with excess thymidine or camptothecin undergo unbalanced growth, and prolonged unbalanced growth causes induction of cellular senescence, which is suppressed by restriction of protein synthesis or inhibition of ERK-signaling. In this study, we found that restriction of protein synthesis, inhibition of ERK-signaling, and elimination of reactive oxygen species showed a combinatorial effect on suppression of cellular senescence induced by excess thymidine or camptothecin. Of these, restriction of protein synthesis most effectively suppressed cellular senescence. Importantly, a similar combinatorial effect was observed in replicative senescence in normal human diploid fibroblasts. Our findings suggested that various stresses were cumulatively involved in cellular senescence, and suppression of cellular senescence was improved by combining the treatments that reduce the stresses. PMID:27339653

  3. Protein partners of the calcium channel β subunit highlight new cellular functions.

    PubMed

    Rima, Mohamad; Daghsni, Marwa; Fajloun, Ziad; M'rad, Ridha; Brusés, Juan L; Ronjat, Michel; De Waard, Michel

    2016-07-01

    Calcium plays a key role in cell signalling by its intervention in a wide range of physiological processes. Its entry into cells occurs mainly via voltage-gated calcium channels (VGCC), which are found not only in the plasma membrane of excitable cells but also in cells insensitive to electrical signals. VGCC are composed of different subunits, α1, β, α2δ and γ, among which the cytosolic β subunit (Cavβ) controls the trafficking of the channel to the plasma membrane, its regulation and its gating properties. For many years, these were the main functions associated with Cavβ. However, a growing number of proteins have been found to interact with Cavβ, emphasizing the multifunctional role of this versatile protein. Interestingly, some of the newly assigned functions of Cavβ are independent of its role in the regulation of VGCC, and thus further increase its functional roles. Based on the identity of Cavβ protein partners, this review emphasizes the diverse cellular functions of Cavβ and summarizes both past findings as well as recent progress in the understanding of VGCC. PMID:27354560

  4. 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

  5. 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

  6. HMGB1 and HMGB2 proteins up-regulate cellular expression of human topoisomerase IIα

    PubMed Central

    Štros, Michal; Polanská, Eva; Štruncová, Soňa; Pospíšilová, Šárka

    2009-01-01

    Topoisomerase IIα (topo IIα) is a nuclear enzyme involved in several critical processes, including chromosome replication, segregation and recombination. Previously we have shown that chromosomal protein HMGB1 interacts with topo IIα, and stimulates its catalytic activity. Here we show the effect of HMGB1 on the activity of the human topo IIα gene promoter in different cell lines. We demonstrate that HMGB1, but not a mutant of HMGB1 incapable of DNA bending, up-regulates the activity of the topo IIα promoter in human cells that lack functional retinoblastoma protein pRb. Transient over-expression of pRb in pRb-negative Saos-2 cells inhibits the ability of HMGB1 to activate the topo IIα promoter. The involvement of HMGB1 and its close relative, HMGB2, in modulation of activity of the topo IIα gene is further supported by knock-down of HMGB1/2, as evidenced by significantly decreased levels of topo IIα mRNA and protein. Our experiments suggest a mechanism of up-regulation of cellular expression of topo IIα by HMGB1/2 in pRb-negative cells by modulation of binding of transcription factor NF-Y to the topo IIα promoter, and the results are discussed in the framework of previously observed pRb-inactivation, and increased levels of HMGB1/2 and topo IIα in tumors. PMID:19223331

  7. 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

    2015-01-01

    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

  8. Comparative study of the expression of cellular cycle proteins in cervical intraepithelial lesions.

    PubMed

    Queiroz, Conceição; Silva, Tânia Correia; Alves, Venâncio A F; Villa, Luisa L; Costa, Maria Cecília; Travassos, Ana Gabriela; Filho, José Bouzas Araújo; Studart, Eduardo; Cheto, Tatiana; de Freitas, Luiz Antonio R

    2006-01-01

    Interaction of human papilloma virus oncoproteins E6 and E7 with cell cycle proteins leads to disturbances of the cell cycle mechanism and subsequent alteration in the expression of some proteins, such as p16INK4a, cyclin D1, p53 and KI67. In this study, we compared alterations in the expression of these proteins during several stages of intraepithelial cervical carcinogenesis. Accordingly, an immunohistochemical study was performed on 50 cervical biopsies, including negative cases and intraepithelial neoplasias. The expression patterns of these markers were correlated with the histopathological diagnosis and infection with HPV. The p16INK4a, followed by Ki67, showed better correlation with cancer progression than p53 and cyclin D1, which recommends their use in the evaluation of cervical carcinogenesis. These monoclonal antibodies can be applied to cervical biopsy specimens to identify lesions transformed by oncogenic HPV, separating CIN 1 (p16INK4a positive) and identifying high-grade lesions by an increase in the cellular proliferation index (Ki67). In this way, we propose immunomarkers that can be applied in clinical practice to separate patients who need a conservative therapeutic approach from those who require a more aggressive treatment. PMID:16979303

  9. 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

  10. 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

  11. Cellular Promyelocytic Leukemia Protein Is an Important Dengue Virus Restriction Factor

    PubMed Central

    Giovannoni, Federico; Damonte, Elsa B.; García, Cybele C.

    2015-01-01

    The intrinsic antiviral defense is based on cellular restriction factors that are constitutively expressed and, thus, active even before a pathogen enters the cell. The promyelocytic leukemia (PML) nuclear bodies (NBs) are discrete nuclear foci that contain several cellular proteins involved in intrinsic antiviral responses against a number of viruses. Accumulating reports have shown the importance of PML as a DNA virus restriction factor and how these pathogens evade this antiviral activity. However, very little information is available regarding the antiviral role of PML against RNA viruses. Dengue virus (DENV) is an RNA emerging mosquito-borne human pathogen affecting millions of individuals each year by causing severe and potentially fatal syndromes. Since no licensed antiviral drug against DENV infection is currently available, it is of great importance to understand the factors mediating intrinsic immunity that may lead to the development of new pharmacological agents that can boost their potency and thereby lead to treatments for this viral disease. In the present study, we investigated the in vitro antiviral role of PML in DENV-2 A549 infected cells. PMID:25962098

  12. An efficient bit string implementation of a database cross-field association system (with an application to protein sequence patterns)

    SciTech Connect

    Guigo, R.; Vazquez, I.; Smith, T.F.

    1992-08-01

    We present a fast implementation of an algorithm to infer correlation between database queries. The implementation has been primarily designed to automatically obtain the best description for the function of a given protein sequence pattern. We assume that such a description is the query on the functional annotation of a protein sequence database having the closet extension in the database to the extension of the pattern. The functional annotation of a protein sequence database can be described as a set-valued attribute whose domain is a set of one-place predicates with biological meaning. The query language is then a first order language and the query space can be mapped into a set algebra in which a measure of set similarity is introduced. We have previously developed an algorithm to search such an algebra when negation is not considered. Here, we present an efficient implementation of such and algorithm and we develop a method to search exhaustively a protein sequence database for biologically relevant protein sequence patterns, incorporating such an implementation. The method relies on the initial generation of an extensive collection of amino acid sequence motifs that correspond to high information dense regions in long consensus patterns derived from homologous protein families -and their automatic evaluation using above implementation. We have used this method to automatically search the SWISSPROT protein sequence database. The results obtained show that potentially meaningful amino acid sequence patterns may have been discovered.

  13. An efficient bit string implementation of a database cross-field association system (with an application to protein sequence patterns)

    SciTech Connect

    Guigo, R. ); Vazquez, I.; Smith, T.F. )

    1992-01-01

    We present a fast implementation of an algorithm to infer correlation between database queries. The implementation has been primarily designed to automatically obtain the best description for the function of a given protein sequence pattern. We assume that such a description is the query on the functional annotation of a protein sequence database having the closet extension in the database to the extension of the pattern. The functional annotation of a protein sequence database can be described as a set-valued attribute whose domain is a set of one-place predicates with biological meaning. The query language is then a first order language and the query space can be mapped into a set algebra in which a measure of set similarity is introduced. We have previously developed an algorithm to search such an algebra when negation is not considered. Here, we present an efficient implementation of such and algorithm and we develop a method to search exhaustively a protein sequence database for biologically relevant protein sequence patterns, incorporating such an implementation. The method relies on the initial generation of an extensive collection of amino acid sequence motifs that correspond to high information dense regions in long consensus patterns derived from homologous protein families -and their automatic evaluation using above implementation. We have used this method to automatically search the SWISSPROT protein sequence database. The results obtained show that potentially meaningful amino acid sequence patterns may have been discovered.

  14. 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

  15. Cellular defense against latent colonization foiled by human cytomegalovirus UL138 protein

    PubMed Central

    Lee, Song Hee; Albright, Emily R.; Lee, Jeong-Hee; Jacobs, Derek; Kalejta, Robert F.

    2015-01-01

    Intrinsic immune defenses mediated by restriction factors inhibit productive viral infections. Select viruses rapidly establish latent infections and, with gene expression profiles that imply cell-autonomous intrinsic defenses, may be the most effective immune control measure against latent reservoirs. We illustrate that lysine-specific demethylases (KDMs) are restriction factors that prevent human cytomegalovirus from establishing latency by removing repressive epigenetic modifications from histones associated with the viral major immediate early promoter (MIEP), stimulating the expression of a viral lytic phase target of cell-mediated adaptive immunity. The viral UL138 protein negates this defense by preventing KDM association with the MIEP. The presence of an intrinsic defense against latency and the emergence of a cognate neutralizing viral factor indicate that “arms races” between hosts and viruses over lifelong colonization exist at the cellular level. PMID:26702450

  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. 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.

  18. Gene Composer: database software for protein construct design, codon engineering, and gene synthesis

    PubMed Central

    Lorimer, Don; Raymond, Amy; Walchli, John; Mixon, Mark; Barrow, Adrienne; Wallace, Ellen; Grice, Rena; Burgin, Alex; Stewart, Lance

    2009-01-01

    Background To improve efficiency in high throughput protein structure determination, we have developed a database software package, Gene Composer, which facilitates the information-rich design of protein constructs and their codon engineered synthetic gene sequences. With its modular workflow design and numerous graphical user interfaces, Gene Composer enables researchers to perform all common bio-informatics steps used in modern structure guided protein engineering and synthetic gene engineering. Results An interactive Alignment Viewer allows the researcher to simultaneously visualize sequence conservation in the context of known protein secondary structure, ligand contacts, water contacts, crystal contacts, B-factors, solvent accessible area, residue property type and several other useful property views. The Construct Design Module enables the facile design of novel protein constructs with altered N- and C-termini, internal insertions or deletions, point mutations, and desired affinity tags. The modifications can be combined and permuted into multiple protein constructs, and then virtually cloned in silico into defined expression vectors. The Gene Design Module uses a protein-to-gene algorithm that automates the back-translation of a protein amino acid sequence into a codon engineered nucleic acid gene sequence according to a selected codon usage table with minimal codon usage threshold, defined G:C% content, and desired sequence features achieved through synonymous codon selection that is optimized for the intended expression system. The gene-to-oligo algorithm of the Gene Design Module plans out all of the required overlapping oligonucleotides and mutagenic primers needed to synthesize the desired gene constructs by PCR, and for physically cloning them into selected vectors by the most popular subcloning strategies. Conclusion We present a complete description of Gene Composer functionality, and an efficient PCR-based synthetic gene assembly procedure with mis

  19. The alpha/beta fold family of proteins database and the cholinesterase gene server ESTHER.

    PubMed Central

    Cousin, X; Hotelier, T; Giles, K; Lievin, P; Toutant, J P; Chatonnet, A

    1997-01-01

    ESTHER (for esterases, alpha/betahydrolase enzyme and relatives) is a database of sequences phylogenetically related to cholinesterases. These sequences define a homogeneous group of enzymes (carboxylesterases, lipases and hormone-sensitive lipases) sharing a similar structure of a central beta-sheet surrounded by alpha-helices. Among these proteins a wide range of functions can be found (hydrolases, adhesion molecules, hormone precursors). The purpose of ESTHER is to help comparison of structures and functions of members of the family. Since the last release, new features have been added to the server. A BLAST comparison tool allows sequence homology searches within the database sequences. New sections are available: kinetics and inhibitors of cholinesterases, fasciculin-acetylcholinesterase interaction and a gene structure review. The mutation analysis compilation has been improved with three-dimensional images. A mailing list has been created. PMID:9016525

  20. Trichinella spiralis: genome database searches for the presence and immunolocalization of protein disulphide isomerase family members.

    PubMed

    Freitas, C P; Clemente, I; Mendes, T; Novo, C

    2016-01-01

    The formation of nurse cells in host muscle cells during Trichinella spiralis infection is a key step in the infective mechanism. Collagen trimerization is set up via disulphide bond formation, catalysed by protein disulphide isomerase (PDI). In T. spiralis, some PDI family members have been identified but no localization is described and no antibodies specific for T. spiralis PDIs are available. In this work, computational approaches were used to search for non-described PDIs in the T. spiralis genome database and to check the cross-reactivity of commercial anti-human antibodies with T. spiralis orthologues. In addition to a previously described PDI (PDIA2), endoplasmic reticulum protein (ERp57/PDIA3), ERp72/PDIA4, and the molecular chaperones calreticulin (CRT), calnexin (CNX) and immunoglobulin-binding protein/glucose-regulated protein (BIP/GRP78), we identified orthologues of the human thioredoxin-related-transmembrane proteins (TMX1, TMX2 and TMX3) in the genome protein database, as well as ERp44 (PDIA10) and endoplasmic reticulum disulphide reductase (ERdj5/PDIA19). Immunocytochemical staining of paraffin sections of muscle infected by T. spiralis enabled us to localize some orthologues of the human PDIs (PDIA3 and TMX1) and the chaperone GRP78. A theoretical three-dimensional model for T. spiralis PDIA3 was constructed. The localization and characteristics of the predicted linear B-cell epitopes and amino acid sequence of the immunogens used for commercial production of anti-human PDIA3 antibodies validated the use of these antibodies for the immunolocalization of T. spiralis PDIA3 orthologues. These results suggest that further study of the role of the PDIs and chaperones during nurse cell formation is desirable. PMID:25475092

  1. 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

  2. Formation of a Protein Corona on Silver Nanoparticles Mediates Cellular Toxicity via Scavenger Receptors

    PubMed Central

    Shannahan, Jonathan H.; Podila, Ramakrishna; Aldossari, Abdullah A.; Emerson, Hilary; Powell, Brian A.; Ke, Pu Chun; Rao, Apparao M.; Brown, Jared M.

    2015-01-01

    Addition of a protein corona (PC) or protein adsorption layer on the surface of nanomaterials following their introduction into physiological environments may modify their activity, bio-distribution, cellular uptake, clearance, and toxicity. We hypothesize that silver nanoparticles (AgNPs) will associate with proteins common to human serum and cell culture media forming a PC that will impact cell activation and cytotoxicity. Furthermore, the role of scavenger receptor BI (SR-BI) in mediating this toxicity was evaluated. Citrate-suspended 20 nm AgNPs were incubated with human serum albumin (HSA), bovine serum albumin (BSA), high-density lipoprotein (HDL), or water (control) to form a PC. AgNPs associated with each protein (HSA, BSA, and HDL) forming PCs as assessed by electron microscopy, hyperspectral analysis, ζ-potential, and hydrodynamic size. Addition of the PC decreased uptake of AgNPs by rat lung epithelial and rat aortic endothelial cells. Hyperspectral analysis demonstrated a loss of the AgNP PC following internalization. Cells demonstrated concentration-dependent cytotoxicity following exposure to AgNPs with or without PCs (0, 6.25, 12.5, 25 or 50 μg/ml). All PC-coated AgNPs were found to activate cells by inducing IL-6 mRNA expression. A small molecule SR-BI inhibitor was utilized to determine the role of SR-BI in the observed effects. Pretreatment with the SR-BI inhibitor decreased internalization of AgNPs with or without PCs, and reduced both cytotoxicity and IL-6 mRNA expression. This study characterizes the formation of a PC on AgNPs and demonstrates its influence on cytotoxicity and cell activation through a cell surface receptor. PMID:25326241

  3. 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

  4. PolyQ 2.0: an improved version of PolyQ, a database of human polyglutamine proteins

    PubMed Central

    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

  5. 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

  6. 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

  7. A versatile transreplication-based system to identify cellular proteins involved in geminivirus replication.

    PubMed

    Morilla, Gabriel; Castillo, Araceli G; Preiss, Werner; Jeske, Holger; Bejarano, Eduardo R

    2006-04-01

    A versatile green fluorescent protein (GFP) expression cassette containing the replication origins of the monopartite begomovirus Tomato yellow leaf curl Sardinia virus (TYLCSV) is described. Transgenic Nicotiana benthamiana plants containing one copy of the cassette stably integrated into their genome were superinfected with TYLCSV, which mobilized and replicated the cassette as an episomal replicon. The expression of the reporter gene (the GFP gene) was thereby modified. Whereas GFP fluorescence was dimmed in the intercostal areas, an increase of green fluorescence in veins of all leaves placed above the inoculation site, as well as in transport tissues of roots and stems, was observed. The release of episomal trans replicons from the transgene and the increase in GFP expression were dependent on the cognate geminiviral replication-associated protein (Rep) and required interaction between Rep and the intergenic region of TYLCSV. This expression system is able to monitor the replication status of TYLCSV in plants, as induction of GFP expression is only produced in those tissues where Rep is present. To further confirm this notion, the expression of a host factor required for geminivirus replication, the proliferating cellular nuclear antigen (PCNA) was transiently silenced. Inhibition of PCNA prevented GFP induction in veins and reduced viral DNA. We propose that these plants could be widely used to easily identify host factors required for geminivirus replication by virus-induced gene silencing. PMID:16537630

  8. A Versatile Transreplication-Based System To Identify Cellular Proteins Involved in Geminivirus Replication

    PubMed Central

    Morilla, Gabriel; Castillo, Araceli G.; Preiss, Werner; Jeske, Holger; Bejarano, Eduardo R.

    2006-01-01

    A versatile green fluorescent protein (GFP) expression cassette containing the replication origins of the monopartite begomovirus Tomato yellow leaf curl Sardinia virus (TYLCSV) is described. Transgenic Nicotiana benthamiana plants containing one copy of the cassette stably integrated into their genome were superinfected with TYLCSV, which mobilized and replicated the cassette as an episomal replicon. The expression of the reporter gene (the GFP gene) was thereby modified. Whereas GFP fluorescence was dimmed in the intercostal areas, an increase of green fluorescence in veins of all leaves placed above the inoculation site, as well as in transport tissues of roots and stems, was observed. The release of episomal trans replicons from the transgene and the increase in GFP expression were dependent on the cognate geminiviral replication-associated protein (Rep) and required interaction between Rep and the intergenic region of TYLCSV. This expression system is able to monitor the replication status of TYLCSV in plants, as induction of GFP expression is only produced in those tissues where Rep is present. To further confirm this notion, the expression of a host factor required for geminivirus replication, the proliferating cellular nuclear antigen (PCNA) was transiently silenced. Inhibition of PCNA prevented GFP induction in veins and reduced viral DNA. We propose that these plants could be widely used to easily identify host factors required for geminivirus replication by virus-induced gene silencing. PMID:16537630

  9. 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

  10. 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

  11. 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

  12. Identification and characterization of cellular proteins interacting with Hepatitis E virus untranslated regions.

    PubMed

    Paingankar, Mandar S; Arankalle, Vidya A

    2015-10-01

    Lack of robust cell culture systems for Hepatitis E virus (HEV) infection has hampered understanding of HEV biology. We attempted to identify the host cellular factors that interact with HEV 5' and 3' untranslated regions (UTRs) by RNA affinity chromatography followed by mass spectrometry analysis. Hepatitis E virus genotype-1 (HEV-1) and Hepatitis E virus genotype-4 (HEV-4) and three cell lines (HepG2/C3A, A549 and Caco2) were employed to understand the UTR-host protein interaction. RNA pull-down and matrix-assisted laser desorption/ionization time-of-flight/time-of-flight (MALDI TOF/TOF) analysis revealed that DHX9, PTK-7, DIS3 and TCR E chain (CD3ɛ) of all the three cell lines interacted with HEV 3'UTR while RAD50 and TLE-4 interacted with HEV 5'UTR. RNA immuno-precipitation studies further confirmed the interaction of DHX9, DIS3 and TCR E chain. The expression changes in genes associated with the identified proteins were quantitated in the peripheral blood mononuclear cells (PBMCs) of Hepatitis E patients during acute and recovery phases. The data revealed that HEV infection influences the exosomes, T cell receptor signalling and Wnt signalling pathways. Interactions of DIS3 with HEV UTRs suggest that exosomes might have important implication in HEV life cycle. PMID:26087402

  13. 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.

  14. A Highly Toxic Cellular Prion Protein Induces a Novel, Nonapoptotic Form of Neuronal Death

    PubMed Central

    Christensen, Heather M.; Dikranian, Krikor; Li, Aimin; Baysac, Kathleen C.; Walls, Ken C.; Olney, John W.; Roth, Kevin A.; Harris, David A.

    2010-01-01

    Several different deletions within the N-terminal tail of the prion protein (PrP) induce massive neuronal death when expressed in transgenic mice. This toxicity is dose-dependently suppressed by coexpression of full-length PrP, suggesting that it results from subversion of a normal physiological activity of cellular PrP. We performed a combined biochemical and morphological analysis of Tg(ΔCR) mice, which express PrP carrying a 21-aa deletion (residues 105-125) within a highly conserved region of the protein. Death of cerebellar granule neurons in Tg(ΔCR) mice is not accompanied by activation of either caspase-3 or caspase-8 or by increased levels of the autophagy marker, LC3-II. In electron micrographs, degenerating granule neurons displayed a unique morphology characterized by heterogeneous condensation of the nuclear matrix without formation of discrete chromatin masses typical of neuronal apoptosis. Our data demonstrate that perturbations in PrP functional activity induce a novel, nonapoptotic, nonautophagic form of neuronal death whose morphological features are reminiscent of those associated with excitotoxic stress. PMID:20472884

  15. SED1/MFG-E8: a bi-motif protein that orchestrates diverse cellular interactions

    PubMed Central

    Raymond, Adam; Ensslin, Michael A.; Shur, Barry D.

    2009-01-01

    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

  16. 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

  17. The electric dipole moment of DNA-binding HU protein calculated by the use of an NMR database.

    PubMed

    Takashima, S; Yamaoka, K

    1999-08-30

    Electric birefringence measurements indicated the presence of a large permanent dipole moment in HU protein-DNA complex. In order to substantiate this observation, numerical computation of the dipole moment of HU protein homodimer was carried out by using NMR protein databases. The dipole moments of globular proteins have hitherto been calculated with X-ray databases and NMR data have never been used before. The advantages of NMR databases are: (a) NMR data are obtained, unlike X-ray databases, using protein solutions. Accordingly, this method eliminates the bothersome question as to the possible alteration of the protein structure due to the transition from the crystalline state to the solution state. This question is particularly important for proteins such as HU protein which has some degree of internal flexibility; (b) the three-dimensional coordinates of hydrogen atoms in protein molecules can be determined with a sufficient resolution and this enables the N-H as well as C = O bond moments to be calculated. Since the NMR database of HU protein from Bacillus stearothermophilus consists of 25 models, the surface charge as well as the core dipole moments were computed for each of these structures. The results of these calculations show that the net permanent dipole moments of HU protein homodimer is approximately 500-530 D (1 D = 3.33 x 10(-30) Cm) at pH 7.5 and 600-630 D at the isoelectric point (pH 10.5). These permanent dipole moments are unusually large for a small protein of the size of 19.5 kDa. Nevertheless, the result of numerical calculations is compatible with the electro-optical observation, confirming a very large dipole moment in this protein. PMID:10483709

  18. Use of protein database for the computation of the dipole moments of normal and abnormal hemoglobins.

    PubMed Central

    Takashima, S

    1993-01-01

    Previously, we discussed the calculation of the dipole moments of small proteins using the three-dimensional protein data-base. Our results demonstrate that the calculated dipole moments are in acceptable agreement with measured values. We, however, noted the difficulty of the calculation with larger proteins, in particular those consisting of several subunits. Hemoglobin (Hb) is a protein having a molecular weight of 64,000 that consists of four subunits, a typical case where the computation was found to be difficult. To circumvent the difficulties, we calculated the dipole moment of each subunit separately. The dipole moment of the whole protein was calculated by the vectorial summation of subunit moments. With this method, the calculated net dipole moment is in good agreement with the experimental value. Our calculation shows that the dipole moment vectors of subunits are, by and large, antiparallel in tetramers causing partial cancellation of the net dipole moment. In addition to normal HbA, the dipole moment of abnormal HbS was calculated using an approximate computational technique. Because of the loss of two negative changes as a result of the replacement of glutamic acid with valine in beta-chains, the dipole moment of HbS was found, experimentally and theoretically, to be significantly smaller than that of HbA. PMID:8324190

  19. 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

  20. Exploring the Ligand-Protein Networks in Traditional Chinese Medicine: Current Databases, Methods, and Applications

    PubMed Central

    Zhao, Mingzhu; Wei, Dong-Qing

    2013-01-01

    The traditional Chinese medicine (TCM), which has thousands of years of clinical application among China and other Asian countries, is the pioneer of the “multicomponent-multitarget” and network pharmacology. Although there is no doubt of the efficacy, it is difficult to elucidate convincing underlying mechanism of TCM due to its complex composition and unclear pharmacology. The use of ligand-protein networks has been gaining significant value in the history of drug discovery while its application in TCM is still in its early stage. This paper firstly surveys TCM databases for virtual screening that have been greatly expanded in size and data diversity in recent years. On that basis, different screening methods and strategies for identifying active ingredients and targets of TCM are outlined based on the amount of network information available, both on sides of ligand bioactivity and the protein structures. Furthermore, applications of successful in silico target identification attempts are discussed in detail along with experiments in exploring the ligand-protein networks of TCM. Finally, it will be concluded that the prospective application of ligand-protein networks can be used not only to predict protein targets of a small molecule, but also to explore the mode of action of TCM. PMID:23818932

  1. KinMutBase, a database of human disease-causing protein kinase mutations.

    PubMed

    Stenberg, K A; Riikonen, P T; Vihinen, M

    1999-01-01

    KinMutBase (http://www.uta.fi/laitokset/imt/KinMut Base.html) is a registry of mutations in human protein kinases related to disorders. Kinases are essential cellular signalling molecules, in which mutations can lead into diseases including, e.g., immunodeficiencies, cancers and endocrine disorders. The first release of KinMutBase contains information for nine protein tyrosine kinases. There are altogether 170 entries representing 273 families and 403 patients. Mutations appear both in conserved hallmark residues of the kinases as well as in non-homologous sites. The KinMutBase WWW pages provide plenty of information, namely mutation statistics and display, clickable sequences with mutations, restriction enzyme patterns and online submission. PMID:9847229

  2. SIMAP—the database of all-against-all protein sequence similarities and annotations with new interfaces and increased coverage

    PubMed Central

    Arnold, Roland; Goldenberg, Florian; Mewes, Hans-Werner; Rattei, Thomas

    2014-01-01

    The Similarity Matrix of Proteins (SIMAP, http://mips.gsf.de/simap/) database has been designed to massively accelerate computationally expensive protein sequence analysis tasks in bioinformatics. It provides pre-calculated sequence similarities interconnecting the entire known protein sequence universe, complemented by pre-calculated protein features and domains, similarity clusters and functional annotations. SIMAP covers all major public protein databases as well as many consistently re-annotated metagenomes from different repositories. As of September 2013, SIMAP contains >163 million proteins corresponding to ∼70 million non-redundant sequences. SIMAP uses the sensitive FASTA search heuristics, the Smith–Waterman alignment algorithm, the InterPro database of protein domain models and the BLAST2GO functional annotation algorithm. SIMAP assists biologists by facilitating the interactive exploration of the protein sequence universe. Web-Service and DAS interfaces allow connecting SIMAP with any other bioinformatic tool and resource. All-against-all protein sequence similarity matrices of project-specific protein collections are generated on request. Recent improvements allow SIMAP to cover the rapidly growing sequenced protein sequence universe. New Web-Service interfaces enhance the connectivity of SIMAP. Novel tools for interactive extraction of protein similarity networks have been added. Open access to SIMAP is provided through the web portal; the portal also contains instructions and links for software access and flat file downloads. PMID:24165881

  3. Osmotic stress stimulates phosphorylation and cellular expression of heat shock proteins in rhesus macaque sperm.

    PubMed

    Cole, Julie A; Meyers, Stuart A

    2011-01-01

    The cryosurvival of sperm requires cell signaling mechanisms to adapt to anisotonic conditions during the freezing and thawing process. Chaperone proteins heat shock protein 70 (HSP 70) and heat shock protein 90 (HSP 90; recently renamed HSPA and HSPC, respectively) facilitate some of these cell signaling events in somatic cells. Sperm were evaluated for their cellular expression and levels of phosphorylation of both HSP 70 and HSP 90 under anisotonic conditions as a potential model for cell signaling during the cryopreservation of macaque spermatozoa. In order to monitor the level of stress, the motility and viability parameters were evaluated at various time points. Cells were then either prepared for phosphoprotein enrichment or indirect immunocytochemistry. As controls, the phosphoserine, phosphothreonine, and phosphotyrosine levels were measured under capacitation and cryopreservation conditions and were compared with the phosphoprotein levels expressed under osmotic conditions. As expected, there was an increase in the level of tyrosine phosphorylation under capacitation and cryopreservation conditions. There was also a significant increase in the level of all phosphoproteins under hyperosmotic conditions. There was no change in the level of expression of HSP 70 or 90 under osmotic stress conditions as measured by Western blot. The enrichment of phosphoproteins followed by Western immunoblotting revealed an increase in the phosphorylation of HSP 70 but not HSP 90 under osmotic stress conditions. Indirect immunofluorescence localized HSP 70 to the postacrosomal region of sperm, and the level of membrane expression of HSP 70 was significantly affected by anisotonic conditions, as measured by flow cytometry. Taken together, these results suggest a differential role for HSP 70 and HSP 90 during osmotic stress conditions in rhesus macaque sperm. PMID:21088232

  4. 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

  5. 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

  6. 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

  7. 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

  8. Heat induction of heat shock protein 25 requires cellular glutamine in intestinal epithelial cells.

    PubMed

    Phanvijhitsiri, Kittiporn; Musch, Mark W; Ropeleski, Mark J; Chang, Eugene B

    2006-08-01

    Glutamine is considered a nonessential amino acid; however, it becomes conditionally essential during critical illness when consumption exceeds production. Glutamine may modulate the heat shock/stress response, an important adaptive cellular response for survival. Glutamine increases heat induction of heat shock protein (Hsp) 25 in both intestinal epithelial cells (IEC-18) and mesenchymal NIH/3T3 cells, an effect that is neither glucose nor serum dependent. Neither arginine, histidine, proline, leucine, asparagine, nor tyrosine acts as physiological substitutes for glutamine for heat induction of Hsp25. The lack of effect of these amino acids was not caused by deficient transport, although some amino acids, including glutamate (a major direct metabolite of glutamine), were transported poorly by IEC-18 cells. Glutamate uptake could be augmented in a concentration- and time-dependent manner by increasing either media concentration and/or duration of exposure. Under these conditions, glutamate promoted heat induction of Hsp25, albeit not as efficiently as glutamine. Further evidence for the role of glutamine conversion to glutamate was obtained with the glutaminase inhibitor 6-diazo-5-oxo-l-norleucine (DON), which inhibited the effect of glutamine on heat-induced Hsp25. DON inhibited phosphate-dependent glutaminase by 75% after 3 h, decreasing cell glutamate. Increased glutamine/glutamate conversion to glutathione was not involved, since the glutathione synthesis inhibitor, buthionine sulfoximine, did not block glutamine's effect on heat induction of Hsp25. A large drop in ATP levels did not appear to account for the diminished Hsp25 induction during glutamine deficiency. In summary, glutamine is an important amino acid, and its requirement for heat-induced Hsp25 supports a role for glutamine supplementation to optimize cellular responses to pathophysiological stress. PMID:16554407

  9. The CATH database: an extended protein family resource for structural and functional genomics

    PubMed Central

    Pearl, F. M. G.; Bennett, C. F.; Bray, J. E.; Harrison, A. P.; Martin, N.; Shepherd, A.; Sillitoe, I.; Thornton, J.; Orengo, C. A.

    2003-01-01

    The CATH database of protein domain structures (http://www.biochem.ucl.ac.uk/bsm/cath_new) currently contains 34 287 domain structures classified into 1383 superfamilies and 3285 sequence families. Each structural family is expanded with domain sequence relatives recruited from GenBank using a variety of efficient sequence search protocols and reliable thresholds. This extended resource, known as the CATH-protein family database (CATH-PFDB) contains a total of 310 000 domain sequences classified into 26 812 sequence families. New sequence search protocols have been designed, based on these intermediate sequence libraries, to allow more regular updating of the classification. Further developments include the adaptation of a recently developed method for rapid structure comparison, based on secondary structure matching, for domain boundary assignment. The philosophy behind CATHEDRAL is the recognition of recurrent folds already classified in CATH. Benchmarking of CATHEDRAL, using manually validated domain assignments, demonstrated that 43% of domains boundaries could be completely automatically assigned. This is an improvement on a previous consensus approach for which only 10–20% of domains could be reliably processed in a completely automated fashion. Since domain boundary assignment is a significant bottleneck in the classification of new structures, CATHEDRAL will also help to increase the frequency of CATH updates. PMID:12520050

  10. 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. PMID:24185697

  11. Coverage of whole proteome by structural genomics observed through protein homology modeling database

    PubMed Central

    Yamaguchi, Akihiro; Go, Mitiko

    2006-01-01

    We have been developing FAMSBASE, a protein homology-modeling database of whole ORFs predicted from genome sequences. The latest update of FAMSBASE (http://daisy.nagahama-i-bio.ac.jp/Famsbase/), which is based on the protein three-dimensional (3D) structures released by November 2003, contains modeled 3D structures for 368,724 open reading frames (ORFs) derived from genomes of 276 species, namely 17 archaebacterial, 130 eubacterial, 18 eukaryotic and 111 phage genomes. Those 276 genomes are predicted to have 734,193 ORFs in total and the current FAMSBASE contains protein 3D structure of approximately 50% of the ORF products. However, cases that a modeled 3D structure covers the whole part of an ORF product are rare. When portion of an ORF with 3D structure is compared in three kingdoms of life, in archaebacteria and eubacteria, approximately 60% of the ORFs have modeled 3D structures covering almost the entire amino acid sequences, however, the percentage falls to about 30% in eukaryotes. When annual differences in the number of ORFs with modeled 3D structure are calculated, the fraction of modeled 3D structures of soluble protein for archaebacteria is increased by 5%, and that for eubacteria by 7% in the last 3 years. Assuming that this rate would be maintained and that determination of 3D structures for predicted disordered regions is unattainable, whole soluble protein model structures of prokaryotes without the putative disordered regions will be in hand within 15 years. For eukaryotic proteins, they will be in hand within 25 years. The 3D structures we will have at those times are not the 3D structure of the entire proteins encoded in single ORFs, but the 3D structures of separate structural domains. Measuring or predicting spatial arrangements of structural domains in an ORF will then be a coming issue of structural genomics. PMID:17146617

  12. Protein binding modulates the cellular uptake of silver nanoparticles into human cells: Implications for in vitro to in vivo extrapolations?

    PubMed Central

    Monteiro-Riviere, Nancy A.; Samberg, Meghan E.; Oldenburg, Steven J.; Riviere, Jim E.

    2013-01-01

    Nanoparticles (NP) absorbed in the body will come in contact with blood proteins and form NP/protein complexes termed protein coronas, which may modulate NP cellular uptake. This study quantitated human epidermal keratinocyte (HEK) uptake of silver (Ag) NP complexed to different human serum proteins. Prior to HEK dosing, AgNP (20 nm and 110 nm citrate BioPure™; 40 nm and 120 nm silica-coated) were preincubated for 2 h at 37 °C without (control) or with physiological levels of albumin (44 mg/ml), IgG (14.5 mg/ml) or transferrin (3 mg/ml) to form protein-complexed NP. HEK were exposed to the protein incubated AgNP for 3 h, rinsed and incubated for 24 h, rinsed in buffer and lysed. Ag was assayed by inductively-coupled plasma optical emission spectrometry. Uptake of Ag in HEK was <4.1% of applied dose with proteins suppressing citrate, but not silica coated Ag uptake. IgG exposure dramatically reduced 110 nm citrate AgNP uptake. In contrast, greatest uptake of 20 nm silica AgNP was seen with IgG, while 110 nm silica AgNP showed minimal protein effects. Electron microscopy confirmed cellular uptake of all NP but showed differences in the appearance and agglomeration state of the NP within HEK vacuoles. This work suggests that NP association with different serum proteins, purportedly forming different protein coronas, significantly modulates Ag uptake into HEK compared to native NP uptake, suggesting caution in extrapolating in vitro uptake data to predict behavior in vivo where the nature of the protein corona may determine patterns of cellular uptake, and thus biodistribution, biological activity and toxicity. PMID:23660336

  13. 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. PMID:25614100

  14. 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

  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. 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

  17. 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

  18. Using the SUBcellular database for Arabidopsis proteins to localize the Deg protease family.

    PubMed

    Tanz, Sandra K; Castleden, Ian; Hooper, Cornelia M; Small, Ian; Millar, A Harvey

    2014-01-01

    Sub-functionalization during the expansion of gene families in eukaryotes has occurred in part through specific subcellular localization of different family members. To better understand this process in plants, compiled records of large-scale proteomic and fluorescent protein localization datasets can be explored and bioinformatic predictions for protein localization can be used to predict the gaps in experimental data. This process can be followed by targeted experiments to test predictions. The SUBA3 database is a free web-service at http://suba.plantenergy.uwa.edu.au that helps users to explore reported experimental data and predictions concerning proteins encoded by gene families and to define the experiments required to locate these homologous sets of proteins. Here we show how SUBA3 can be used to explore the subcellular location of the Deg protease family of ATP-independent serine endopeptidases (Deg1-Deg16). Combined data integration and new experiments refined location information for Deg1 and Deg9, confirmed Deg2, Deg5, and Deg8 in plastids and Deg 15 in peroxisomes and provide substantial experimental evidence for mitochondrial localized Deg proteases. Two of these, Deg3 and Deg10, additionally localized to the plastid, revealing novel dual-targeted Deg proteases in the plastid and the mitochondrion. SUBA3 is continually updated to ensure that researchers can use the latest published data when planning the experimental steps remaining to localize gene family functions. PMID:25161662

  19. Using the SUBcellular database for Arabidopsis proteins to localize the Deg protease family

    PubMed Central

    Tanz, Sandra K.; Castleden, Ian; Hooper, Cornelia M.; Small, Ian; Millar, A. Harvey

    2014-01-01

    Sub-functionalization during the expansion of gene families in eukaryotes has occurred in part through specific subcellular localization of different family members. To better understand this process in plants, compiled records of large-scale proteomic and fluorescent protein localization datasets can be explored and bioinformatic predictions for protein localization can be used to predict the gaps in experimental data. This process can be followed by targeted experiments to test predictions. The SUBA3 database is a free web-service at http://suba.plantenergy.uwa.edu.au that helps users to explore reported experimental data and predictions concerning proteins encoded by gene families and to define the experiments required to locate these homologous sets of proteins. Here we show how SUBA3 can be used to explore the subcellular location of the Deg protease family of ATP-independent serine endopeptidases (Deg1–Deg16). Combined data integration and new experiments refined location information for Deg1 and Deg9, confirmed Deg2, Deg5, and Deg8 in plastids and Deg 15 in peroxisomes and provide substantial experimental evidence for mitochondrial localized Deg proteases. Two of these, Deg3 and Deg10, additionally localized to the plastid, revealing novel dual-targeted Deg proteases in the plastid and the mitochondrion. SUBA3 is continually updated to ensure that researchers can use the latest published data when planning the experimental steps remaining to localize gene family functions. PMID:25161662

  20. Relationship between binding affinities to cellular retinoic acid-binding protein and biological potency of a new series of retinoids.

    PubMed

    Sani, B P; Dawson, M I; Hobbs, P D; Chan, R L; Schiff, L J

    1984-01-01

    Binding affinities of a new and unusual series of retinoic acid analogues to cellular retinoic acid-binding protein, a possible mediator of their biological function in the control of differentiation and tumorigenesis, and to serum albumin, their plasma transport protein, were determined. Also, biological activity of these retinoids in the reversal of keratinization in hamster tracheal organ cultures was assessed and compared with their binding affinities. Analogues that possessed high biological activity showed high binding efficiency to cellular retinoic acid-binding protein. Those that were biologically less active were poor binders to the binding protein. Three retinoids, 4657-57, 3920-59, and 4445-75, which showed 90 to 100% binding efficiency of that of retinoic acid for cellular retinoic acid-binding protein expressed high biological activity detectable in the range of 10(-10) M as against 10(-11) M for retinoic acid. The correlation noticed in these two activities not only enhances the confidence in the two assay procedures but also paves the way for design and development of potential chemopreventive agents. No apparent differences were observed in the binding affinities of the retinoids to binding proteins of a normal tissue or a tumor tissue. No correlation existed between the binding affinities of these retinoids to serum albumin and their biological activity. Structure-activity relationships of the retinoids in relation to their binding affinities and biological activities have been discussed. PMID:6317169

  1. 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.

  2. 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

  3. Cellular retinol binding protein 1 could be a tumor suppressor gene in cervical cancer

    PubMed Central

    Mendoza-Rodriguez, Mónica; Arreola, Hugo; Valdivia, Alejandra; Peralta, Raúl; Serna, Humberto; Villegas, Vanessa; Romero, Pablo; Alvarado-Hernández, Beatriz; Paniagua, Lucero; Marrero-Rodríguez, Daniel; Meraz, Marco A; Salcedo, Mauricio

    2013-01-01

    Aims: Cervical Cancer (CC) is one of the most important health problems in women. It frequently presents genetic changes at chromosome region 3q21. This region contains the Cellular Retinol Binding Protein 1 gene (CRBP1) which has been implicated as an important element in the development of other types of cancer. The main goal of the present work was to determine the molecular alterations of CRBP1 and its relationship to CC. Methods: To determine the molecular alterations of CRBP1 gene in CC; twenty-six CC and twenty-six healthy cervix samples were evaluated for: 1) Copy number gain by real-time PCR analysis, 2) expression levels by an immunohistochemistry assay on tissue microarray, and 3) the methylation status of the CRBP1 promoter region. Results: The increase in CRBP1 copy number was observed in 10 out of the 26 CC samples analyzed, while healthy cervices samples showed no changes in the copy number. In addition, there was a lack of expression of the CRBP1 gene in an important number of the CC samples (17/26), and the CRBP1 gene promoter was methylated in 15/26 of the CC samples. Interestingly, there was a significant association between the lack of expression of the CRBP1 gene and its methylation status. Conclusions: The data indicates that, both activating and inactivating changes in the CRBP1 gene could be significant events in the development and progression of CC, and the lack of expression of the CRBP1 protein could be related with to the development of CC. We believe that there is enough evidence to consider to CRBP1 gene as a tumor suppressor gene for CC. PMID:24040446

  4. 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

  5. Alzheimer's amyloid-β oligomers rescue cellular prion protein induced tau reduction via the Fyn pathway.

    PubMed

    Chen, Rong-Jie; Chang, Wei-Wei; Lin, Yu-Chun; Cheng, Pei-Lin; Chen, Yun-Ru

    2013-09-18

    Amyloid-β (Aβ) and tau are the pathogenic hallmarks in Alzheimer's disease (AD). Aβ oligomers are considered the actual toxic entities, and the toxicity relies on the presence of tau. Recently, Aβ oligomers have been shown to specifically interact with cellular prion protein (PrP(C)) where the role of PrP(C) in AD is still not fully understood. To investigate the downstream mechanism of PrP(C) and Aβ oligomer interaction and their possible relationships to tau, we examined tau expression in human neuroblastoma BE(2)-C cells transfected with murine PrP(C) and studied the effect under Aβ oligomer treatment. By Western blotting, we found that PrP(C) overexpression down-regulated tau protein and Aβ oligomer binding alleviated the tau reduction induced by wild type but not M128V PrP(C), the high AD risk polymorphic allele in human prion gene. PrP(C) lacking the Aβ oligomer binding site was incapable of rescuing the level of tau reduction. Quantitative RT-PCR showed the PrP(C) effect was attributed to tau reduction at the transcription level. Treatment with Fyn pathway inhibitors, Fyn kinase inhibitor PP2 and MEK inhibitor U0126, reversed the PrP(C)-induced tau reduction and Aβ oligomer treatment modulated Fyn kinase activity. The results suggested Fyn pathway regulated Aβ-PrP(C)-tau signaling. Overall, our results demonstrated that PrP(C) down-regulated tau via the Fyn pathway and the effect can be regulated by Aβ oligomers. Our study facilitated the understanding of molecular mechanisms among PrP(C), tau, and Aβ oligomers. PMID:23805846

  6. 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. .

  7. Photochemistry and stereoselectivity of cellular retinaldehyde-binding protein from bovine retina

    SciTech Connect

    Saari, J.C.; Bredberg, D.L.

    1987-06-05

    11-cis-Retinaldehyde bound to cellular retinaldehyde-binding protein (CRALBP) is unaffected in bovine eyecup preparations by illumination that bleaches approximately 70% of the rhodopsin. Illumination of retinal homogenates to which CRALBP X (/sup 3/H)11-cis-retinaldehyde had been added did not result in a reduction of the specific activity of recovered 11-cis-retinaldehyde, ruling out a bleaching regeneration cycle. The quantum efficiency of photoisomerization for CRALBP X 11-cis-retinaldehyde was determined by comparing the rate of photoisomerization of 11-cis-retinaldehyde bound to purified CRALBP and opsin. The low value obtained (0.07), coupled with a low molar extinction coefficient (15,400 M-1 cm-1), results in a photosensitivity only about 4% that of rhodopsin. CRALBP binds 9-cis- and 11-cis-retinaldehyde, producing complexes with absorption maxima at 405 and 425 nm, respectively. No complexes were detected with 13-cis- and all-trans-retinaldehyde. Following incubation of CRALBP X 11-cis-retinol with an equimolar mixture of 9-, 11-, 13-cis-, and all-trans-retinaldehydes, only 11-cis-retinaldehyde and residual 11-cis-retinol are present on the protein following separation from excess retinoids. A similar result is obtained following incubation of CRALBP X 11-cis-retinol with mixtures of 9- and 11-cis-retinaldehyde ranging in composition from 9:1 to 1:9 (9-cis-:11-cis-,mol/mol). The results indicate that CRALBP X 11-cis-retinol is sufficiently stereoselective in its binding properties to warrant consideration as a component of the mechanism for the generation of 11-cis-retinaldehyde in the dark.

  8. Photochemistry and stereoselectivity of cellular retinaldehyde-binding protein from bovine retina.

    PubMed

    Saari, J C; Bredberg, D L

    1987-06-01

    11-cis-Retinaldehyde bound to cellular retinaldehyde-binding protein (CRALBP) is unaffected in bovine eyecup preparations by illumination that bleaches approximately 70% of the rhodopsin. Illumination of retinal homogenates to which CRALBP X [3H]11-cis-retinaldehyde had been added did not result in a reduction of the specific activity of recovered 11-cis-retinaldehyde, ruling out a bleaching regeneration cycle. The quantum efficiency of photoisomerization for CRALBP X 11-cis-retinaldehyde was determined by comparing the rate of photoisomerization of 11-cis-retinaldehyde bound to purified CRALBP and opsin. The low value obtained (0.07), coupled with a low molar extinction coefficient (15,400 M-1 cm-1), results in a photosensitivity only about 4% that of rhodopsin. CRALBP binds 9-cis- and 11-cis-retinaldehyde, producing complexes with absorption maxima at 405 and 425 nm, respectively. No complexes were detected with 13-cis- and all-trans-retinaldehyde. Following incubation of CRALBP X 11-cis-retinol with an equimolar mixture of 9-, 11-, 13-cis-, and all-trans-retinaldehydes, only 11-cis-retinaldehyde and residual 11-cis-retinol are present on the protein following separation from excess retinoids. A similar result is obtained following incubation of CRALBP X 11-cis-retinol with mixtures of 9- and 11-cis-retinaldehyde ranging in composition from 9:1 to 1:9 (9-cis-:11-cis-,mol/mol). The results indicate that CRALBP X 11-cis-retinol is sufficiently stereoselective in its binding properties to warrant consideration as a component of the mechanism for the generation of 11-cis-retinaldehyde in the dark. PMID:3584132

  9. Biochemical and cellular analysis of human variants of the DYT1 dystonia protein, torsinA

    PubMed Central

    Hettich, Jasmin; Ryan, Scott D.; Norberto de Souza, Osmar; Timmers, Luís Fernando Saraiva Macedo; Tsai, Shelun; Atai, Nadia A.; da Hora, Cintia C.; Zhang, Xuan; Kothary, Rashmi; Snapp, Erik; Ericsson, Maria; Grundmann, Kathrin; Breakefield, Xandra O.; Nery, Flávia C.

    2014-01-01

    Early-onset dystonia is associated with the deletion of one of a pair of glutamic acid residues (c.904_906delGAG/c.907_909delGAG; p.Glu302del/Glu303del; ΔE 302/303) near the carboxyl-terminus of torsinA, a member of the AAA+ protein family that localizes to the endoplasmic reticulum (ER) lumen and nuclear envelope (NE). This deletion commonly underlies early-onset DYT1 dystonia. While the role of the disease-causing mutation, torsinAΔE, has been established through genetic association studies, it is much less clear whether other rare human variants of torsinA are pathogenic. Two missense variations have been described in single patients; R288Q (c.863G>A; p.Arg288Gln; R288Q) identified in a patient with onset of severe generalized dystonia and myoclonus since infancy, and F205I (c.613T>A, p.Phe205Ile; F205I) in a psychiatric patient with late-onset focal dystonia. In this study, we have undertaken a series of analyses comparing the biochemical and cellular effects of these rare variants to torsinAΔE and wild-type (wt) torsinA in order to reveal whether there are common dysfunctional features. The results revealed that the variants, R288Q and F205I, are more similar in their properties to torsinAΔE protein than to torsinAwt. These findings provide functional evidence for the potential pathogenic nature of these rare sequence variants in the TOR1A gene, thus implicating these pathologies in the development of dystonia. PMID:24930953

  10. 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

  11. Manganese Upregulates Cellular Prion Protein and Contributes to Altered Stabilization and Proteolysis: Relevance to Role of Metals in Pathogenesis of Prion Disease

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Prion diseases are fatal neurodegenerative diseases resulting from misfolding of normal cellular prion (PrP**C) into an abnormal form of scrapie prion (PrP**Sc). The cellular mechanisms underlying the misfolding of PrP**C are not well understood. Since cellular prion proteins harbor divalent metal b...

  12. Synthetic amyloid-beta oligomers impair long-term memory independently of cellular prion protein.

    PubMed

    Balducci, Claudia; Beeg, Marten; Stravalaci, Matteo; Bastone, Antonio; Sclip, Alessandra; Biasini, Emiliano; Tapella, Laura; Colombo, Laura; Manzoni, Claudia; Borsello, Tiziana; Chiesa, Roberto; Gobbi, Marco; Salmona, Mario; Forloni, Gianluigi

    2010-02-01

    Inability to form new memories is an early clinical sign of Alzheimer's disease (AD). There is ample evidence that the amyloid-beta (Abeta) peptide plays a key role in the pathogenesis of this disorder. Soluble, bio-derived oligomers of Abeta are proposed as the key mediators of synaptic and cognitive dysfunction, but more tractable models of Abeta-mediated cognitive impairment are needed. Here we report that, in mice, acute intracerebroventricular injections of synthetic Abeta(1-42) oligomers impaired consolidation of the long-term recognition memory, whereas mature Abeta(1-42) fibrils and freshly dissolved peptide did not. The deficit induced by oligomers was reversible and was prevented by an anti-Abeta antibody. It has been suggested that the cellular prion protein (PrP(C)) mediates the impairment of synaptic plasticity induced by Abeta. We confirmed that Abeta(1-42) oligomers interact with PrP(C), with nanomolar affinity. However, PrP-expressing and PrP knock-out mice were equally susceptible to this impairment. These data suggest that Abeta(1-42) oligomers are responsible for cognitive impairment in AD and that PrP(C) is not required. PMID:20133875

  13. Synthetic amyloid-β oligomers impair long-term memory independently of cellular prion protein

    PubMed Central

    Balducci, Claudia; Beeg, Marten; Stravalaci, Matteo; Bastone, Antonio; Sclip, Alessandra; Biasini, Emiliano; Tapella, Laura; Colombo, Laura; Manzoni, Claudia; Borsello, Tiziana; Chiesa, Roberto; Gobbi, Marco; Salmona, Mario; Forloni, Gianluigi

    2010-01-01

    Inability to form new memories is an early clinical sign of Alzheimer’s disease (AD). There is ample evidence that the amyloid-β (Aβ) peptide plays a key role in the pathogenesis of this disorder. Soluble, bio-derived oligomers of Aβ are proposed as the key mediators of synaptic and cognitive dysfunction, but more tractable models of Aβ−mediated cognitive impairment are needed. Here we report that, in mice, acute intracerebroventricular injections of synthetic Aβ1–42 oligomers impaired consolidation of the long-term recognition memory, whereas mature Aβ1–42 fibrils and freshly dissolved peptide did not. The deficit induced by oligomers was reversible and was prevented by an anti-Aβ antibody. It has been suggested that the cellular prion protein (PrPC) mediates the impairment of synaptic plasticity induced by Aβ. We confirmed that Aβ1–42 oligomers interact with PrPC, with nanomolar affinity. However, PrP-expressing and PrP knock-out mice were equally susceptible to this impairment. These data suggest that Aβ1–42 oligomers are responsible for cognitive impairment in AD and that PrPC is not required. PMID:20133875

  14. Characterization of a cellular immunostimulating peptide from a soybean protein fraction digested with peptidase R.

    PubMed

    Egusa, Shintaro; Otani, Hajime

    2009-01-01

    An immunostimulating glutamine-rich peptide was purified from a soybean protein fraction digested with Peptidase R produced by Rhizopus oryzae (Ro-digest) by a combination of SP-Sepharose column chromatography and reversed-phase high-performance liquid chromatography. The purified peptide was supposed to be located at or near the glutamine-rich region 202 to 222 of the glycinin G4 subunit. The peptide significantly increased the number of CD8(+), CD11b(+), and CD49b(+) cells in C3H/HeN mouse spleen cell cultures, while 2 chemically synthesized glutamine-rich peptides corresponding to residues 202 to 213 (QQQQQQKSHGGR) and residues 214 to 225 (KQGQHQQEEEEE) of the glycinin G4 subunit increased the number of interleukin (IL)-12(+)CD11b(+) cells. The peptide 202-213 also significantly increased the number of CD49b(+), IL-2(+)CD4(+), and interferon-gamma(+)CD4(+) cells and stimulated the cytotoxic activity of spleen cells toward the human erythroleukemia cell line K562. These results indicate that the glutamine-rich region of the soybean glycinin G4 subunit stimulates the cellular immune system in mouse spleen cell cultures. PMID:19926930

  15. 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

  16. 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

  17. Role of cellular prion proteins in the function of macrophages and dendritic cells.

    PubMed

    Nitta, Kayako; Sakudo, Akikazu; Masuyama, Jun; Xue, Guangai; Sugiura, Katsuaki; Onodera, Takashi

    2009-01-01

    The cellular isoform of prion proteins (PrPC) is expressed in hematopoietic stem cells, granulocytes, T and B lymphocyte natural killer cells, platelets, monocytes, dendritic cells, and follicular dendritic cells, which may act as carrier cells for the spread of its abnormal isoform (PrPSc) before manifesting transmissible spongiform encephalopathies (TSEs). In particular, macrophages and dendritic cells seem to be involved in the replication of PrPSc after ingestion. In addition, information on the role of PrPC during phagocytotic activity in these cells has been obtained. A recent study showed that resident macrophages from ZrchI PrP gene (Prnp)-deficient (Prnp-/-) mice show augmented phagocytotic activity compared to Prnp+/+ counterparts. In contrast, our study suggests that Rikn Prnp-/- peritoneal macrophages show pseudopodium extension arrest and up-regulation of phagocytotic activity compared to Prnp+/+ cells. Although reports regarding phagocytotic activity in resident and peritoneal macrophages are inconsistent between ZrchI and Rikn Prnp-/- mice, it seems plausible that PrPC in macrophages could contribute to maintain the immunological environment. This review will introduce the recent progress in understanding the functions of PrPC in macrophages and dendritic cells under physiological conditions and its involvement in the pathogenesis of prion diseases. PMID:19275736

  18. 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

  19. Human cellular retinaldehyde-binding protein has secondary thermal 9-cis-retinal isomerase activity.

    PubMed

    Bolze, Christin S; Helbling, Rachel E; Owen, Robin L; Pearson, Arwen R; Pompidor, Guillaume; Dworkowski, Florian; Fuchs, Martin R; Furrer, Julien; Golczak, Marcin; Palczewski, Krzysztof; Cascella, Michele; Stocker, Achim

    2014-01-01

    Cellular retinaldehyde-binding protein (CRALBP) chaperones 11-cis-retinal to convert opsin receptor molecules into photosensitive retinoid pigments of the eye. We report a thermal secondary isomerase activity of CRALBP when bound to 9-cis-retinal. UV/vis and (1)H NMR spectroscopy were used to characterize the product as 9,13-dicis-retinal. The X-ray structure of the CRALBP mutant R234W:9-cis-retinal complex at 1.9 Å resolution revealed a niche in the binding pocket for 9-cis-aldehyde different from that reported for 11-cis-retinal. Combined computational, kinetic, and structural data lead us to propose an isomerization mechanism catalyzed by a network of buried waters. Our findings highlight a specific role of water molecules in both CRALBP-assisted specificity toward 9-cis-retinal and its thermal isomerase activity yielding 9,13-dicis-retinal. Kinetic data from two point mutants of CRALBP support an essential role of Glu202 as the initial proton donor in this isomerization reaction. PMID:24328211

  20. CyanoPhyChe: A Database for Physico-Chemical Properties, Structure and Biochemical Pathway Information of Cyanobacterial Proteins

    PubMed Central

    Arun, P. V. Parvati Sai; Bakku, Ranjith Kumar; Subhashini, Mranu; Singh, Pankaj; Prabhu, N. Prakash; Suzuki, Iwane; Prakash, Jogadhenu S. S.

    2012-01-01

    CyanoPhyChe is a user friendly database that one can browse through for physico-chemical properties, structure and biochemical pathway information of cyanobacterial proteins. We downloaded all the protein sequences from the cyanobacterial genome database for calculating the physico-chemical properties, such as molecular weight, net charge of protein, isoelectric point, molar extinction coefficient, canonical variable for solubility, grand average hydropathy, aliphatic index, and number of charged residues. Based on the physico-chemical properties, we provide the polarity, structural stability and probability of a protein entering in to an inclusion body (PEPIB). We used the data generated on physico-chemical properties, structure and biochemical pathway information of all cyanobacterial proteins to construct CyanoPhyChe. The data can be used for optimizing methods of expression and characterization of cyanobacterial proteins. Moreover, the ‘Search’ and data export options provided will be useful for proteome analysis. Secondary structure was predicted for all the cyanobacterial proteins using PSIPRED tool and the data generated is made accessible to researchers working on cyanobacteria. In addition, external links are provided to biological databases such as PDB and KEGG for molecular structure and biochemical pathway information, respectively. External links are also provided to different cyanobacterial databases. CyanoPhyChe can be accessed from the following URL: http://bif.uohyd.ac.in/cpc. PMID:23185330

  1. 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

  2. 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.

  3. Modulation of cellular and viral promoters by mutant human p53 proteins found in tumor cells.

    PubMed Central

    Deb, S; Jackson, C T; Subler, M A; Martin, D W

    1992-01-01

    Wild-type p53 has recently been shown to repress transcription from several cellular and viral promoters. Since p53 mutations are the most frequently reported genetic defects in human cancers, it becomes important to study the effects of mutations of p53 on promoter functions. We, therefore, have studied the effects of wild-type and mutant human p53 on the human proliferating-cell nuclear antigen (PCNA) promoter and on several viral promoters, including the herpes simplex virus type 1 UL9 promoter, the human cytomegalovirus major immediate-early promoter-enhancer, and the long terminal repeat promoters of Rous sarcoma virus and human T-cell lymphotropic virus type I. HeLa cells were cotransfected with a wild-type or mutant p53 expression vector and a plasmid containing a chloramphenicol acetyltransferase reporter gene under viral (or cellular) promoter control. As expected, expression of the wild-type p53 inhibited promoter function. Expression of a p53 with a mutation at any one of the four amino acid positions 175, 248, 273, or 281, however, correlated with a significant increase of the PCNA promoter activity (2- to 11-fold). The viral promoters were also activated, although to a somewhat lesser extent. We also showed that activation by a mutant p53 requires a minimal promoter containing a lone TATA box. A more significant increase (25-fold) in activation occurs when the promoter contains a binding site for the activating transcription factor or cyclic AMP response element-binding protein. Using Saos-2 cells that do not express p53, we showed that activation by a mutant p53 was a direct enhancement. The mutant forms of p53 used in this study are found in various cancer cells. The activation of PCNA by mutant p53s may indicate a way to increase cell proliferation by the mutant p53s. Thus, our data indicate a possible functional role for the mutants of p53 found in cancer cells in activating several important loci, including PCNA. Images PMID:1356162

  4. Dynameomics: a multi-dimensional analysis-optimized database for dynamic protein data.

    PubMed

    Kehl, Catherine; Simms, Andrew M; Toofanny, Rudesh D; Daggett, Valerie

    2008-06-01

    The Dynameomics project is our effort to characterize the native-state dynamics and folding/unfolding pathways of representatives of all known protein folds by way of molecular dynamics simulations, as described by Beck et al. (in Protein Eng. Des. Select., the first paper in this series). The data produced by these simulations are highly multidimensional in structure and multi-terabytes in size. Both of these features present significant challenges for storage, retrieval and analysis. For optimal data modeling and flexibility, we needed a platform that supported both multidimensional indices and hierarchical relationships between related types of data and that could be integrated within our data warehouse, as described in the accompanying paper directly preceding this one. For these reasons, we have chosen On-line Analytical Processing (OLAP), a multi-dimensional analysis optimized database, as an analytical platform for these data. OLAP is a mature technology in the financial sector, but it has not been used extensively for scientific analysis. Our project is further more unusual for its focus on the multidimensional and analytical capabilities of OLAP rather than its aggregation capacities. The dimensional data model and hierarchies are very flexible. The query language is concise for complex analysis and rapid data retrieval. OLAP shows great promise for the dynamic protein analysis for bioengineering and biomedical applications. In addition, OLAP may have similar potential for other scientific and engineering applications involving large and complex datasets. PMID:18411222

  5. Sand fly salivary proteins induce strong cellular immunity in a natural reservoir of visceral leishmaniasis with adverse consequences for Leishmania.

    PubMed

    Collin, Nicolas; Gomes, Regis; Teixeira, Clarissa; Cheng, Lily; Laughinghouse, Andre; Ward, Jerrold M; Elnaiem, Dia-Eldin; Fischer, Laurent; Valenzuela, Jesus G; Kamhawi, Shaden

    2009-05-01

    Immunity to a sand fly salivary protein protects against visceral leishmaniasis (VL) in hamsters. This protection was associated with the development of cellular immunity in the form of a delayed-type hypersensitivity response and the presence of IFN-gamma at the site of sand fly bites. To date, there are no data available regarding the cellular immune response to sand fly saliva in dogs, the main reservoirs of VL in Latin America, and its role in protection from this fatal disease. Two of 35 salivary proteins from the vector sand fly Lutzomyia longipalpis, identified using a novel approach termed reverse antigen screening, elicited strong cellular immunity in dogs. Immunization with either molecule induced high IgG(2) antibody levels and significant IFN-gamma production following in vitro stimulation of PBMC with salivary gland homogenate (SGH). Upon challenge with uninfected or infected flies, immunized dogs developed a cellular response at the bite site characterized by lymphocytic infiltration and IFN-gamma and IL-12 expression. Additionally, SGH-stimulated lymphocytes from immunized dogs efficiently killed Leishmania infantum chagasi within autologous macrophages. Certain sand fly salivary proteins are potent immunogens obligatorily co-deposited with Leishmania parasites during transmission. Their inclusion in an anti-Leishmania vaccine would exploit anti-saliva immunity following an infective sand fly bite and set the stage for a protective anti-Leishmania immune response. PMID:19461875

  6. 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

  7. 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

  8. 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

  9. Measles Virus Infection Inactivates Cellular Protein Phosphatase 5 with Consequent Suppression of Sp1 and c-Myc Activities

    PubMed Central

    Sato, Hiroki; Yoneda, Misako; Honma, Reiko; Ikeda, Fusako; Watanabe, Shinya

    2015-01-01

    ABSTRACT Measles virus (MeV) causes several unique syndromes, including transient immunosuppression. To clarify the cellular responses to MeV infection, we previously analyzed a MeV-infected epithelial cell line and a lymphoid cell line by microarray and showed that the expression of numerous genes was up- or downregulated in the epithelial cells. In particular, there was a characteristic comprehensive downregulation of housekeeping genes during late stage infection. To identify the mechanism underlying this phenomenon, we examined the phosphorylation status of transcription factors and kinase/phosphatase activities in epithelial cells after infection. MeV infection inactivated cellular protein phosphatase 5 (PP5) that consequently inactivated DNA-dependent protein kinase, which reduced Sp1 phosphorylation levels, and c-Myc degradation, both of which downregulated the expression of many housekeeping genes. In addition, intracellular accumulation of viral nucleocapsid inactivated PP5 and subsequent downstream responses. These findings demonstrate a novel strategy of MeV during infection, which causes the collapse of host cellular functions. IMPORTANCE Measles virus (MeV) is one of the most important pathogens in humans. We previously showed that MeV infection induces the comprehensive downregulation of housekeeping genes in epithelial cells. By examining this phenomenon, we clarified the molecular mechanism underlying the constitutive expression of housekeeping genes in cells, which is maintained by cellular protein phosphatase 5 (PP5) and DNA-dependent protein kinase. We also demonstrated that MeV targets PP5 for downregulation in epithelial cells. This is the first report to show how MeV infection triggers a reduction in overall cellular functions of infected host cells. Our findings will help uncover unique pathogenicities caused by MeV. PMID:26157124

  10. Activation of the oncogenic potential of the avian cellular src protein by specific structural alteration of the carboxy terminus.

    PubMed Central

    Reynolds, A B; Vila, J; Lansing, T J; Potts, W M; Weber, M J; Parsons, J T

    1987-01-01

    The role of tyrosine phosphorylation in the regulation of tyrosine protein kinase activity was investigated using site-directed mutagenesis to alter the structure and environment of the three tyrosine residues present in the C terminus of avian pp60c-src. Mutations that change Tyr 527 to Phe or Ser activate in vivo tyrosine protein kinase activity and induce cellular transformation of chicken cells in culture. In contrast, alterations of tyrosine residues present at positions 511 or 519 in c-src do not induce transformation or in vivo tyrosine protein kinase activity. Amber mutations, which alter the structure of the pp60c-src C terminus by inducing premature termination of the c-src protein at either residue 518 or 523 also induce morphological transformation and increase in vivo tyrosine phosphorylation, whereas removal of the last four residues of c-src by chain termination at residue 530 does not alter the kinase activity or the biological activity of the resultant c-src protein. We conclude from these studies that C-terminal alterations which either remove or replace Tyr 527 serve to activate the c-src protein resulting in cellular transformation and increased in vivo tyrosine protein kinase activity. Images Fig. 2. Fig. 3. Fig. 4. PMID:2822389

  11. The unfolded protein response and cellular senescence. A review in the theme: cellular mechanisms of endoplasmic reticulum stress signaling in health and disease.

    PubMed

    Pluquet, Olivier; Pourtier, Albin; Abbadie, Corinne

    2015-03-15

    The endoplasmic reticulum (ER) is a multifunctional organelle critical for the proper folding and assembly of secreted and transmembrane proteins. Perturbations of ER functions cause ER stress, which activates a coordinated system of transcriptional and translational controls called the unfolded protein response (UPR), to cope with accumulation of misfolded proteins and proteotoxicity. It results in ER homeostasis restoration or in cell death. Senescence is a complex cell phenotype induced by several stresses such as telomere attrition, DNA damage, oxidative stress, and activation of some oncogenes. It is mainly characterized by a cell enlargement, a permanent cell-cycle arrest, and the production of a secretome enriched in proinflammatory cytokines and components of the extracellular matrix. Senescent cells accumulate with age in tissues and are suspected to play a role in age-associated diseases. Since senescence is a stress response, the question arises of whether an ER stress could occur concomitantly with senescence and participate in the onset or maintenance of the senescent features. Here, we described the interconnections between the UPR signaling and the different aspects of the cellular senescence programs and discuss the implication of UPR modulations in this context. PMID:25540175

  12. 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. PMID:11788990

  13. 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 dense membranes (lipid rafts) where it formed a complex also containing Aβ and PrPC. In contrast, the addition of Aβ to Prnp(0/0) neurons did not activate synaptic cPLA2, which remained in the cytoplasm and was not associated with Aβ. Filtration assays and non-denaturing gels demonstrated that Aβ oligomers cross-link PrPC. We propose that it is the cross-linkage of PrPC by Aβ oligomers that triggers abnormal activation of cPLA2 and synapse damage. This hypothesis was supported by our observation that monoclonal antibody mediated cross-linkage of PrPC also activated synaptic cPLA2 and caused synapse damage. PMID:21900234

  14. A cationic tetrapyrrole inhibits toxic activities of the cellular prion protein.

    PubMed

    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 (PrP(C)) into PrP(Sc), a self-replicating isoform (prion) that accumulates in the central nervous system of affected individuals. The structure of PrP(Sc) 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 PrP(C) may provide an opportunity to overcome these problems. PrP(C) 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 PrP(C), 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 PrP(C)-related toxic activities, including the channel-forming ability of a PrP mutant, and the PrP(C)-dependent synaptotoxicity of amyloid-β (Aβ) oligomers, which are associated with Alzheimer's Disease. These results demonstrate that molecules binding to PrP(C) may produce a dual effect of blocking prion replication and inhibiting PrP(C)-mediated toxicity. PMID:26976106

  15. Monoacylated Cellular Prion Protein Modifies Cell Membranes, Inhibits Cell Signaling, and Reduces Prion Formation*

    PubMed Central

    Bate, Clive; Williams, Alun

    2011-01-01

    Prion diseases occur following the conversion of the cellular prion protein (PrPC) into a disease related, protease-resistant isoform (PrPSc). In these studies, a cell painting technique was used to introduce PrPC to prion-infected neuronal cell lines (ScGT1, ScN2a, or SMB cells). The addition of PrPC resulted in increased PrPSc formation that was preceded by an increase in the cholesterol content of cell membranes and increased activation of cytoplasmic phospholipase A2 (cPLA2). In contrast, although PrPC lacking one of the two acyl chains from its glycosylphosphatidylinositol (GPI) anchor (PrPC-G-lyso-PI) bound readily to cells, it did not alter the amount of cholesterol in cell membranes, was not found within detergent-resistant membranes (lipid rafts), and did not activate cPLA2. It remained within cells for longer than PrPC with a conventional GPI anchor and was not converted to PrPSc. Moreover, the addition of high amounts of PrPC-G-lyso-PI displaced cPLA2 from PrPSc-containing lipid rafts, reduced the activation of cPLA2, and reduced PrPSc formation in all three cell lines. In addition, ScGT1 cells treated with PrPC-G-lyso-PI did not transmit infection following intracerebral injection to mice. We propose that that the chemical composition of the GPI anchor attached to PrPC modified the local membrane microenvironments that control cell signaling, the fate of PrPC, and hence PrPSc formation. In addition, our observations raise the possibility that pharmacological modification of GPI anchors might constitute a novel therapeutic approach to prion diseases. PMID:21212283

  16. Cellular inhibitor of apoptosis protein 2 controls human colonic epithelial restitution, migration, and Rac1 activation.

    PubMed

    Seidelin, Jakob Benedict; Larsen, Sylvester; Linnemann, Dorte; Vainer, Ben; Coskun, Mehmet; Troelsen, Jesper Thorvald; Nielsen, Ole Haagen

    2015-01-15

    Identification of pathways involved in wound healing is important for understanding the pathogenesis of various intestinal diseases. Cellular inhibitor of apoptosis protein 2 (cIAP2) regulates proliferation and migration in nonepithelial cells and is expressed in human colonocytes. The aim of the study was to investigate the role of cIAP2 for wound healing in the normal human colon. Wound tissue was generated by taking rectosigmoidal biopsies across an experimental ulcer in healthy subjects after 5, 24, and 48 h. In experimental ulcers, the expression of cIAP2 in regenerating intestinal epithelial cells (IECs) was increased at the wound edge after 24 h (P < 0.05), returned to normal after reepithelialization, and correlated with the inflammatory reaction in the experimental wounds (P < 0.001). cIAP2 was induced in vitro in regenerating Caco2 IECs after wound infliction (P < 0.01). Knockdown of cIAP2 caused a substantial impairment of the IEC regeneration through inhibition of migration (P < 0.005). cIAP2 overexpression lead to formation of migrating IECs and upregulation of expression of RhoA and Rac1 as well as GTP-activation of Rac1. Transforming growth factor-β1 enhanced the expression of cIAP2 but was not upregulated in wounds in vivo and in vitro. NF-κB and MAPK pathways did not affect cIAP2 expression. cIAP2 is in conclusion a regulator of human intestinal wound healing through enhanced migration along with activation of Rac1, and the findings suggest that cIAP2 could be a future therapeutic target to improve intestinal wound healing. PMID:25394657

  17. Intracellular Localization and Cellular Factors Interaction of HTLV-1 and HTLV-2 Tax Proteins: Similarities and Functional Differences

    PubMed Central

    Bertazzoni, Umberto; Turci, Marco; Avesani, Francesca; Di Gennaro, Gianfranco; Bidoia, Carlo; Romanelli, Maria Grazia

    2011-01-01

    Human T-lymphotropic viruses type 1 (HTLV-1) and type 2 (HTLV-2) present very similar genomic structures but HTLV-1 is more pathogenic than HTLV-2. Is this difference due to their transactivating Tax proteins, Tax-1 and Tax-2, which are responsible for viral and cellular gene activation? Do Tax-1 and Tax-2 differ in their cellular localization and in their interaction pattern with cellular factors? In this review, we summarize Tax-1 and Tax-2 structural and phenotypic properties, their interaction with factors involved in signal transduction and their localization-related behavior within the cell. Special attention will be given to the distinctions between Tax-1 and Tax-2 that likely play an important role in their transactivation activity. PMID:21994745