RESUMO
BACKGROUND: We have previously described an approach to predicting the substrate specificity of serine-threonine protein kinases. The method, named Predikin, identifies key conserved substrate-determining residues in the kinase catalytic domain that contact the substrate in the region of the phosphorylation site and so determine the sequence surrounding the phosphorylation site. Predikin was implemented originally as a web application written in Javascript. RESULTS: Here, we describe a new version of Predikin, completely revised and rewritten as a modular framework that provides multiple enhancements compared with the original. Predikin now consists of two components: (i) PredikinDB, a database of phosphorylation sites that links substrates to kinase sequences and (ii) a Perl module, which provides methods to classify protein kinases, reliably identify substrate-determining residues, generate scoring matrices and score putative phosphorylation sites in query sequences. The performance of Predikin as measured using receiver operator characteristic (ROC) graph analysis equals or surpasses that of existing comparable methods. The Predikin website has been redesigned to incorporate the new features. CONCLUSION: New features in Predikin include the use of SQL queries to PredikinDB to generate predictions, scoring of predictions, more reliable identification of substrate-determining residues and putative phosphorylation sites, extended options to handle protein kinase and substrate data and an improved web interface. The new features significantly enhance the ability of Predikin to analyse protein kinases and their substrates. Predikin is available at http://predikin.biosci.uq.edu.au.
Assuntos
Domínio Catalítico , Proteínas Serina-Treonina Quinases/classificação , Proteínas Serina-Treonina Quinases/ultraestrutura , Software , Sequência de Aminoácidos , Animais , Sítios de Ligação , Domínio Catalítico/genética , Bases de Dados de Proteínas , Camundongos , Fosforilação , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Análise de Sequência de Proteína , Especificidade por Substrato/genéticaRESUMO
Classification of rare missense variants as neutral or disease causing is a challenge and has important implications for genetic counseling. A multifactorial likelihood model for classification of unclassified variants in BRCA1 and BRCA2 has previously been developed, which uses data on co-occurrence of the unclassified variant with pathogenic mutations in the same gene, cosegregation of the unclassified variant with affected status, and Grantham analysis of the fit between the missense substitution and the evolutionary range of variation observed at its position in the protein. We have further developed this model to take into account relevant features of BRCA1- and BRCA2-associated tumors, such as the characteristic histopathology and immunochemical profiles associated with pathogenic mutations in BRCA1, and the fact that approximately 80% of tumors from BRCA1 and BRCA2 carriers undergo inactivation of the wild-type allele by loss of heterozygosity. We examined 10 BRCA1 and 15 BRCA2 unclassified variants identified in Australian, multiple-case breast cancer families. By a combination of genetic, in silico, and histopathologic analyses, we were able to classify one BRCA1 variant as pathogenic and six BRCA1 and seven BRCA2 variants as neutral. Five of these neutral variants were also found in at least 1 of 180 healthy controls, suggesting that screening a large number of appropriate controls might be a useful adjunct to other methods for evaluation of unclassified variants.
Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/patologia , DNA de Neoplasias/genética , Genes BRCA1 , Genes BRCA2 , Alelos , Sequência de Bases , Feminino , Humanos , Imuno-Histoquímica , Perda de Heterozigosidade , Pessoa de Meia-Idade , Modelos Genéticos , Mutação , Mutação de Sentido IncorretoRESUMO
BACKGROUND: Protein phosphorylation is an extremely important mechanism of cellular regulation. A large-scale study of phosphoproteins in a whole-cell lysate of Saccharomyces cerevisiae has previously identified 383 phosphorylation sites in 216 peptide sequences. However, the protein kinases responsible for the phosphorylation of the identified proteins have not previously been assigned. RESULTS: We used Predikin in combination with other bioinformatic tools, to predict which of 116 unique protein kinases in yeast phosphorylates each experimentally determined site in the phosphoproteome. The prediction was based on the match between the phosphorylated 7-residue sequence and the predicted substrate specificity of each kinase, with the highest weight applied to the residues or positions that contribute most to the substrate specificity. We estimated the reliability of the predictions by performing a parallel prediction on phosphopeptides for which the kinase has been experimentally determined. CONCLUSION: The results reveal that the functions of the protein kinases and their predicted phosphoprotein substrates are often correlated, for example in endocytosis, cytokinesis, transcription, replication, carbohydrate metabolism and stress response. The predictions link phosphoproteins of unknown function with protein kinases with known functions and vice versa, suggesting functions for the uncharacterized proteins. The study indicates that the phosphoproteins and the associated protein kinases represented in our dataset have housekeeping cellular roles; certain kinases are not represented because they may only be activated during specific cellular responses. Our results demonstrate the utility of our previously reported protein kinase substrate prediction approach (Predikin) as a tool for establishing links between kinases and phosphoproteins that can subsequently be tested experimentally.
Assuntos
Fosfoproteínas/química , Proteínas Quinases/química , Proteoma/química , Saccharomyces cerevisiae/enzimologia , Análise de Sequência de Proteína/métodos , Sequência de Aminoácidos , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Dados de Sequência Molecular , Fosfoproteínas/metabolismo , Fosforilação , Mapeamento de Interação de Proteínas/métodos , Proteínas Quinases/classificação , Proteínas Quinases/metabolismo , Proteoma/metabolismo , Saccharomyces cerevisiae/química , Alinhamento de Sequência/métodos , Especificidade por SubstratoRESUMO
To ensure signalling fidelity, kinases must act only on a defined subset of cellular targets. Appreciating the basis for this substrate specificity is essential for understanding the role of an individual protein kinase in a particular cellular process. The specificity in the cell is determined by a combination of "peptide specificity" of the kinase (the molecular recognition of the sequence surrounding the phosphorylation site), substrate recruitment and phosphatase activity. Peptide specificity plays a crucial role and depends on the complementarity between the kinase and the substrate and therefore on their three-dimensional structures. Methods for experimental identification of kinase substrates and characterization of specificity are expensive and laborious, therefore, computational approaches are being developed to reduce the amount of experimental work required in substrate identification. We discuss the structural basis of substrate specificity of protein kinases and review the experimental and computational methods used to obtain specificity information.
Assuntos
Biologia Computacional/métodos , Proteínas Quinases/química , Especificidade por Substrato , Sítios de Ligação , Modelos Moleculares , Peptídeos/química , Peptídeos/metabolismo , Fosfoproteínas Fosfatases/química , Fosfoproteínas Fosfatases/metabolismo , Fosforilação , Proteínas Quinases/metabolismo , Estrutura Secundária de ProteínaRESUMO
Two bovine MHC class II alleles, BoLA-DRB3*0201 and BoLA-DRB3*3301, contain a three base pair deletion which results in the deletion of a lysine (K beta 65) in the antigen recognition site (ARS). Modelling of BoLA-DRB3*0201 with the conserved lysine K beta 65 and BoLA-DRB3*0201 without K beta 65 indicated that this deletion altered the peptide specificity of the ARS, and may impact on the immune response. To test this hypothesis, the presence of K beta 65 was analysed in a sample of cattle vaccinated with the commercial cattle tick vaccine (TickGARD). Homozygous deletion of K beta 65 was significantly associated with high response to TickGARD (P<0.05). Screening of the TickGARD antigen identified a potential T cell epitope that is recognised better by animals that are homozygous for the K beta 65 deletion. This study provides evidence that changes in the ARS of MHC class II molecules may be associated with the well recognised animal to animal variation in magnitude of vaccine response.
Assuntos
Antígenos/imunologia , Doenças dos Bovinos/imunologia , Genes MHC da Classe II/imunologia , Carrapatos/imunologia , Vacinas/imunologia , Animais , Anticorpos/sangue , Antígenos/metabolismo , Sítios de Ligação , Bovinos , Doenças dos Bovinos/parasitologia , Epitopos de Linfócito T/imunologia , Epitopos de Linfócito T/metabolismo , Genes MHC da Classe II/genética , Modelos Moleculares , Mutação/imunologia , Mutação/fisiologia , Ligação Proteica , Conformação Proteica , Infestações por Carrapato/prevenção & controle , Vacinas/normasRESUMO
PURPOSE: Rare missense substitutions and in-frame deletions of BRCA1 and BRCA2 genes present a challenge for genetic counseling of individuals carrying such unclassified variants. We assessed the value of tumor immunohistochemical markers in conjunction with genetic and evolutionary approaches for investigating the clinical significance of unclassified variants. PATIENTS AND METHODS: We studied 10 BRCA1 and 12 BRCA2 variants identified in Australian families with breast cancer. Analyses assumed a prior probability based on revised cross-species sequence alignment methods assessing amino acid evolutionary conservation and position, combined with likelihoods from data on co-occurrence with pathogenic mutations in the same gene, segregation analysis, and immunohistochemistry. We specifically explored the value of estrogen receptor, cytokeratin 5/6, and cytokeratin 14 as tumor markers of BRCA1 mutation status. RESULTS: Posterior probabilities classified 72% of variants. BRCA1 variants IVS18+1 G>T (del exon 18) and 5632 T >A (V1838E) were classified as pathogenic, with >99% posterior probability of being deleterious, and tumor histopathology was particularly important for their classification. BRCA2 variant classification was improved over previous studies, largely by incorporating the prior probability of pathogenicity based on amino acid cross-species sequence alignments. CONCLUSION: Variant classification was considerably improved by analysis of estrogen receptor, cytokeratin 5/6, and cytokeratin 14 tumor expression, and use of updated methods estimating the clinical relevance of amino acid evolutionary conservation and position. These methodologies may assist genetic counseling of individuals with unclassified sequence variants.
Assuntos
Biomarcadores Tumorais/genética , Neoplasias da Mama/genética , Genes BRCA1 , Genes BRCA2 , Variação Genética , Queratinas/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Evolução Biológica , Cromatografia Líquida de Alta Pressão , Análise Mutacional de DNA , Feminino , Humanos , Imuno-Histoquímica , Funções Verossimilhança , Dados de Sequência Molecular , Mutação , Linhagem , Receptores de Estrogênio/biossíntese , Alinhamento de Sequência , Análise de Sequência de DNARESUMO
In budding yeast, partitioning of the cytoplasm during cytokinesis can proceed via a pathway dependent on the contractile actomyosin ring, as in other eukaryotes, or alternatively via a septum deposition pathway dependent on an SH3 domain protein, Hof1/Cyk2 (the yeast PSTPIP1 ortholog). In dividing yeast cells, Hof1 forms a ring at the bud neck distinct from the actomyosin ring, and this zone is active in septum deposition. We previously showed the yeast Wiskott-Aldrich syndrome protein (WASP)-interacting protein (WIP) ortholog, verprolin/Vrp1/End5, interacts with Hof1 and facilitates Hof1 recruitment to the bud neck. A Vrp1 fragment unable to interact with yeast WASP (Las17/Bee1), localize to the actin cytoskeleton or function in polarization of the cortical actin cytoskeleton nevertheless retains function in Hof1 recruitment and cytokinesis. Here, we show the ability of this Vrp1 fragment to bind the Hof1 SH3 domain via its Hof one trap (HOT) domain is critical for cytokinesis. The Vrp1 HOT domain consists of three tandem proline-rich motifs flanked by serines. Unexpectedly, the Hof1 SH3 domain itself is not required for cytokinesis and indeed appears to negatively regulate cytokinesis. The Vrp1 HOT domain promotes cytokinesis by binding to the Hof1 SH3 domain and counteracting its inhibitory effect.
Assuntos
Ciclo Celular/fisiologia , Proteínas dos Microfilamentos/fisiologia , Proteínas Associadas aos Microtúbulos/fisiologia , Proteínas de Saccharomyces cerevisiae/fisiologia , Motivos de Aminoácidos , Proteínas dos Microfilamentos/metabolismo , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/metabolismo , Modelos Moleculares , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiologia , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Serina/metabolismo , Domínios de Homologia de srcRESUMO
The large number of protein kinases makes it impractical to determine their specificities and substrates experimentally. Using the available crystal structures, molecular modeling, and sequence analyses of kinases and substrates, we developed a set of rules governing the binding of a heptapeptide substrate motif (surrounding the phosphorylation site) to the kinase and implemented these rules in a web-interfaced program for automated prediction of optimal substrate peptides, taking only the amino acid sequence of a protein kinase as input. We show the utility of the method by analyzing yeast cell cycle control and DNA damage checkpoint pathways. Our method is the only available predictive method generally applicable for identifying possible substrate proteins for protein serinethreonine kinases and helps in silico construction of signaling pathways. The accuracy of prediction is comparable to the accuracy of data from systematic large-scale experimental approaches.
Assuntos
Oligopeptídeos/metabolismo , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Ciclo Celular , Sequência Consenso , Proteínas Quinases Dependentes de AMP Cíclico/química , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Dano ao DNA , Bases de Dados de Proteínas , Modelos Biológicos , Modelos Moleculares , Dados de Sequência Molecular , Oligopeptídeos/química , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Transdução de Sinais , Software , Especificidade por SubstratoRESUMO
Protein kinases exhibit various degrees of substrate specificity. The large number of different protein kinases in the eukaryotic proteomes makes it impractical to determine the specificity of each enzyme experimentally. To test if it were possible to discriminate potential substrates from non-substrates by simple computational techniques, we analysed the binding enthalpies of modelled enzyme-substrate complexes and attempted to correlate it with experimental enzyme kinetics measurements. The crystal structures of phosphorylase kinase and cAMP-dependent protein kinase were used to generate models of the enzyme with a series of known peptide substrates and non-substrates, and the approximate enthalpy of binding assessed following energy minimization. We show that the computed enthalpies do not correlate closely with kinetic measurements, but the method can distinguish good substrates from weak substrates and non-substrates.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/química , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Fosforilase Quinase/química , Sítios de Ligação , Biologia Computacional , AMP Cíclico/farmacologia , Cinética , Modelos Químicos , Modelos Moleculares , Fosforilase Quinase/metabolismo , Conformação Proteica , Especificidade por Substrato , Fatores de TempoRESUMO
Importin-alpha is the nuclear import receptor that recognizes cargo proteins carrying conventional basic monopartite and bipartite nuclear localization sequences (NLSs) and facilitates their transport into the nucleus. Bipartite NLSs contain two clusters of basic residues, connected by linkers of variable lengths. To determine the structural basis of the recognition of diverse bipartite NLSs by mammalian importin-alpha, we co-crystallized a non-autoinhibited mouse receptor protein with peptides corresponding to the NLSs from human retinoblastoma protein and Xenopus laevis phosphoprotein N1N2, containing diverse sequences and lengths of the linker. We show that the basic clusters interact analogously in both NLSs, but the linker sequences adopt different conformations, whereas both make specific contacts with the receptor. The available data allow us to draw general conclusions about the specificity of NLS binding by importin-alpha and facilitate an improved definition of the consensus sequence of a conventional basic/bipartite NLS (KRX10-12KRRK) that can be used to identify novel nuclear proteins.