RESUMO
Large-scale two-hybrid screens have generated a wealth of information describing potential protein--protein interactions. When compiled with data from systematic localizations of proteins, mutant screens and other functional tests, a network of interactions among proteins and between proteins and other components of eukaryotic cells can be deduced. These networks can be viewed as maps of the cell, depicting potential signaling pathways and interactive complexes. Most importantly, they provide potential clues to the function of previously uncharacterized proteins. Focusing on recent experiments, we explore these protein-interaction studies and the maps derived from such efforts.
Assuntos
Proteínas/metabolismo , Proteoma/genética , Técnicas do Sistema de Duplo-Híbrido , Ciclo Celular/fisiologia , Células/metabolismo , Biologia Computacional , Modelos Biológicos , Mapeamento de Peptídeos , Proteínas/classificação , Proteínas/genética , Proteoma/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMO
A global analysis of 2,709 published interactions between proteins of the yeast Saccharomyces cerevisiae has been performed, enabling the establishment of a single large network of 2,358 interactions among 1,548 proteins. Proteins of known function and cellular location tend to cluster together, with 63% of the interactions occurring between proteins with a common functional assignment and 76% occurring between proteins found in the same subcellular compartment. Possible functions can be assigned to a protein based on the known functions of its interacting partners. This approach correctly predicts a functional category for 72% of the 1,393 characterized proteins with at least one partner of known function, and has been applied to predict functions for 364 previously uncharacterized proteins.
Assuntos
Biologia Computacional , Proteínas Fúngicas/metabolismo , Proteoma , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Algoritmos , Proteínas Fúngicas/genética , Genômica , Ligação Proteica , Técnicas do Sistema de Duplo-HíbridoRESUMO
Two-hybrid systems are one of the most popular, preferred, cost effective, and scalable in vivo genetic approaches for screening protein-protein interactions. A number of variants of yeast and bacterial two-hybrid systems exist, rendering them ideal for modern, flexible proteomics-driven studies. For mapping protein interactions at genome scales (that is, constructing an interactome), the yeast two-hybrid system has been extensively tested and is preferred over bacterial two-hybrid systems, given that users have created more resources such as a variety of vectors and other modifications. Each system has its own advantages and limitations and thus needs to be compared directly. For instance, the bacterial two-hybrid method seems a better fit than the yeast two-hybrid system to screen membrane-associated proteins. In this chapter, we provide detailed protocols for yeast and bacterial two-hybrid systems as well as a comparison of outcomes for each approach using our own and published data.
Assuntos
Técnicas do Sistema de Duplo-Híbrido , Escherichia coli , Ligação Proteica , Mapas de Interação de Proteínas , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae , Transformação BacterianaRESUMO
The functions of roughly a third of all proteins in Streptococcus pneumoniae, a significant human-pathogenic bacterium, are unknown. Using a yeast two-hybrid approach, we have determined more than 2,000 novel protein interactions in this organism. We augmented this network with meta-interactome data that we defined as the pool of all interactions between evolutionarily conserved proteins in other bacteria. We found that such interactions significantly improved our ability to predict a protein's function, allowing us to provide functional predictions for 299 S. pneumoniae proteins with previously unknown functions. IMPORTANCE Identification of protein interactions in bacterial species can help define the individual roles that proteins play in cellular pathways and pathogenesis. Very few protein interactions have been identified for the important human pathogen S. pneumoniae. We used an experimental approach to identify over 2,000 new protein interactions for S. pneumoniae, the most extensive interactome data for this bacterium to date. To predict protein function, we used our interactome data augmented with interactions from other closely related bacteria. The combination of the experimental data and meta-interactome data significantly improved the prediction results, allowing us to assign possible functions to a large number of poorly characterized proteins.
RESUMO
The increasing rate at which complete genome sequences become available necessitates rapid and robust methods for investigating the functions of their encoded proteins. Efforts have been made to study protein function by systematically screening large sets of proteins using the two-hybrid method. Analyses of the complete proteomes of baceriophage T7, the mammalian viruses hepatitis C and vaccinia, as well as of several protein complexes including RNA splicing proteins and RNA polymerase III from yeast, have been undertaken. Saccharomyces cerevisiae has been studied extensively by two-hybrid methods, with more than 2500 protein-protein interactions described. Systematic studies on metazoan proteomes are, however, still in their infancy.
Assuntos
Proteínas de Bactérias/genética , Proteínas Fúngicas/genética , Técnicas do Sistema de Duplo-Híbrido , Proteínas Virais/genética , Animais , Testes Genéticos/métodosRESUMO
A two-hybrid screen against an activation domain array of Saccharomyces cerevisiae proteins was carried out for 31 yeast proteasome proteins. Fifty-five putative interactions were identified: 21 between components of the proteasome complex and 34 between proteasome proteins and other proteins. Many of these latter interactions involved either proteins of the ubiquitin pathway, cell cycle proteins, protein kinases or a translation initiation factor subunit. The role of eleven proteins associated with proteasome function by these screens was analyzed by examining the corresponding deletion strains for temperature sensitivity and canavanine sensitivity and for the stability of a ubiquitin-beta-galactosidase fusion protein. These assays additionally implicated three proteins, Bim1, Ump1, and YKL171W, in proteasome function. This study demonstrates the utility of genome-wide two-hybrid assays as an entry point for the further analysis of a large protein complex.
Assuntos
Peptídeo Hidrolases/química , Peptídeo Hidrolases/metabolismo , Complexo de Endopeptidases do Proteassoma , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Técnicas do Sistema de Duplo-Híbrido , Catálise , Deleção de Genes , Biblioteca Gênica , Substâncias Macromoleculares , Peptídeo Hidrolases/genética , Fenótipo , Ligação Proteica , Subunidades Proteicas , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Temperatura , Termodinâmica , Ubiquitina/metabolismoRESUMO
We have isolated the murine gene encoding the 5-HT3 receptor (5-HT3R), a member of the ligand-gated ion channels, that mediates a variety of physiological effects in central and peripheral neurons. DNA sequence analysis of the 5-HT3R gene revealed its organisation in 9 exons distributed over approximately 12 kbp of DNA. Alternative use of exon 9 splice acceptor sites generated two 5-HT3R variants. The 5-HT3R gene, whose structure is closely related to neuronal and muscle AChR alpha genes, as demonstrated by four common splice junctions, was localised on human chromosome 11.
Assuntos
Mapeamento Cromossômico , Cromossomos Humanos Par 11 , Receptores de Serotonina/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Química Encefálica , DNA Complementar/isolamento & purificação , Feminino , Expressão Gênica , Técnicas de Transferência de Genes , Humanos , Camundongos , Dados de Sequência Molecular , Músculos/química , Neurônios/química , Oócitos/metabolismo , Splicing de RNA , Receptores de Serotonina/química , Análise de Sequência de DNA , Homologia de Sequência , Xenopus laevisAssuntos
Proteínas Recombinantes de Fusão/biossíntese , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Sequência de Bases , Clonagem Molecular/métodos , Primers do DNA , Proteínas de Ligação a DNA , Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/genética , Dados de Sequência Molecular , Fases de Leitura Aberta , Plasmídeos , Proteínas Recombinantes de Fusão/química , Mapeamento por Restrição , Robótica , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética , Técnicas do Sistema de Duplo-HíbridoRESUMO
Ld proteins (formins) are encoded by the limb deformity (ld) gene and define a family of related gene products regulating establishment of embryonic polarity. In this study we establish that chicken and murine Ld proteins interact directly with Src family kinases (c-Src and c-Fyn). Specific binding is mediated by the proline-rich domain present in Ld proteins and the ligand binding surface of the Src SH3 domain. Co-immunoprecipitation of Ld and c-Src proteins from transfected cells shows that these proteins associate in vivo. Immunolocalization and biochemical fractionation of fibroblasts confirms the predominant nuclear localization of Ld proteins, but unexpectedly identifies a population of Ld proteins associated to cellular membranes. This population of Ld proteins co-localizes with membrane-associated c-Src proteins at both plasma and perinuclear membranes. These studies indicate that the morphoregulatory Ld proteins interact with signal transduction cascades by association to membrane-bound Src family kinases.
Assuntos
Proteínas Fetais/metabolismo , Proteínas Nucleares/metabolismo , Quinases da Família src/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Células COS , Galinhas , Forminas , Camundongos , Proteínas dos Microfilamentos , Dados de Sequência Molecular , Alinhamento de Sequência , Distribuição Tecidual , TransfecçãoRESUMO
Two large-scale yeast two-hybrid screens were undertaken to identify protein-protein interactions between full-length open reading frames predicted from the Saccharomyces cerevisiae genome sequence. In one approach, we constructed a protein array of about 6,000 yeast transformants, with each transformant expressing one of the open reading frames as a fusion to an activation domain. This array was screened by a simple and automated procedure for 192 yeast proteins, with positive responses identified by their positions in the array. In a second approach, we pooled cells expressing one of about 6,000 activation domain fusions to generate a library. We used a high-throughput screening procedure to screen nearly all of the 6,000 predicted yeast proteins, expressed as Gal4 DNA-binding domain fusion proteins, against the library, and characterized positives by sequence analysis. These approaches resulted in the detection of 957 putative interactions involving 1,004 S. cerevisiae proteins. These data reveal interactions that place functionally unclassified proteins in a biological context, interactions between proteins involved in the same biological function, and interactions that link biological functions together into larger cellular processes. The results of these screens are shown here.