Spelling suggestions: "subject:"phosphotyrosinephosphatase"" "subject:"phosphoproteinphosphatase""
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High-throughput profiling of sequence recognition by phosphotyrosine signaling proteinsLi, Allyson January 2023 (has links)
Protein tyrosine kinase and phosphatase domains have binding specificities that depend on the amino acid sequence surrounding the target (phospho)tyrosine residue on their substrates. Although the preferred recognition motifs of many kinase and phosphatase domains have been characterized, we lack a quantitative description of sequence specificity that could guide predictions about signaling pathways or be used to design sequences for biomedical applications.
Here, we present a platform that combines genetically-encoded peptide libraries and deep sequencing to profile sequence recognition by tyrosine kinases. We screened several tyrosine kinases against a million-peptide random library and used the resulting profiles to design high-activity sequences and predict phosphorylation efficiencies of substrates. We then screened several kinases against a library containing thousands of human proteome-derived peptides and their naturally-occurring variants. These screens recapitulated independently measured phosphorylation rates and revealed hundreds of phosphosite-proximal mutations that impact phosphosite recognition by tyrosine kinases.
Finally, we have made progress towards extending this platform to the analysis of tyrosine phosphatase domains, by optimizing methods to produce tyrosine-phosphorylated bacterial display libraries and implementing methods to detect peptide dephosphorylation on the cell surface. Collectively, these experiments demonstrate the utility of our platform for rapid profiling of sequence specificity by tyrosine kinases and will shed new light on phosphotyrosine signaling.
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Characterization of the Function and Interaction of Proteins Involved in Exopolysaccharide Synthesis in Streptococcus thermophilus, Streptococcus iniae, and Lactococcus lactis subsp. cremorisCefalo, Angela D. 01 May 2012 (has links)
Amino acid residues that are important for metal binding and catalysis in Grampositive phosphotyrosine phosphatases were identified in Streptococcus thermophilus Wzh/EpsB proteins. The Wzh protein from S. thermophilus MR-1C was purified after heterologous expression and tested for phosphatase activity against synthetic phosphotyrosine and phosphoserine/threonine peptides. The purified Wzh protein was able to remove phosphate from both phosphotyrosine peptides tested and the phosphatase activity of Wzh was dramatically reduced by the presence of the phosphotyrosine phosphatase inhibitor sodium vanadate at concentrations of 1, 5, and 10 mM. Purified Wzh had no activity against the synthetic phosphoserine/threonine peptide. These results established that Wzh functions as a phosphotyrosine phosphatase. By using the yeast two-hybrid system, strong intraspecific protein interactions were detected in S. thermophilus MR-1C, Streptococcus iniae 9066, and Lactococcus lactis subsp. cremoris JRF1 between the putative transmembrane activation protein (Wzd, CpsC, and EpsA, respectively) and the putative protein tyrosine kinase (Wze, CpsD, and EpsB, respectively). Weaker protein interactions take place forming a dimer between two identical protein tyrosine kinases and between the protein tyrosine kinase and phosphotyrosine phosphatase (Wzh, CpsB, and EpsC, respectively) in these species. Protein-protein interactions involving a S. thermophilus MR-1C Wzd/Wze fusion protein and Wzd and Wze indicated that these proteins may form multi-protein complexes. All combinations of the S. thermophilus Wzh, Wzd, Wze, Wzg (regulation), CpsE (glycosyl-1-phosphate transferase), CpsS (polymerization), CpsL (unknown), CpsW (regulation), and CpsU (membrane translocation) proteins were analyzed for protein-protein interactions but no additional interactions were discovered. For each of the intraspecific interactions detected, interspecific interactions were also detected when one protein was from S. iniae and the other was from S. thermophilus. Interactions were also observed between two protein tyrosine kinases when one protein was from either of the Streptococcus species and the other from L. lactis subsp. cremoris. These results and sequence comparisons performed in this study support the conclusion that interactions among the components of the tyrosine kinase/phosphatase regulatory system are conserved in the family Streptococcaceae. Interspecific protein-protein interactions suggest that functional regulatory complexes can be formed in naturally occurring and genetically engineered recombinant strains.
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PROFILING THE INTRINSIC SEQUENCE SPECIFICITY OF PROTEIN TYROSINE PHOSPHATASESSelner, Nicholas January 2013 (has links)
No description available.
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Études biochimiques et cellulaires de tyrosine-kinases bactériennes / Biochemical and cellular studies of bacterial tyrosine-kinasesNourikyan, Julien 19 December 2014 (has links)
Les bactéries possèdent une famille particulière de tyrosine-autokinases, les BY-kinases. Ces enzymes sont impliquées dans la régulation de plusieurs processus cellulaires dont la synthèse et l'export des polysaccharides extracellulaires qui jouent un rôle crucial dans la virulence de certaines bactéries pathogènes. Cependant, les mécanismes de régulation sous-jacents sont inconnus. L'objectif de ma thèse a été de caractériser d'un point de vue structural et fonctionnel le rôle des BY-kinases. Pour cela, j'ai réalisé trois études indépendantes dans trois modèles bactériens différents. Chez Escherichia coli, j'ai identifié et étudié la surface d'interaction entre la BY-kinase Wzc et sa phosphatase associée Wzb afin de comprendre comment Wzb déphosphoryle Wzc. Chez Staphylococcus aureus, j'ai participé à la caractérisation structurale de la pseudo-BY-kinase CapB1. Par comparaison avec la structure de son homologue actif CapB2, mes études ont permis de suggérer l'existence d'un mécanisme de régulation de la synthèse des polysaccharides extracellulaires impliquant CapB2 et CapB1. Enfin, chez Streptococcus pneumoniae, j'ai mis en évidence que si l'autophosphorylation de la BY-kinase CpsD était indispensable à la synthèse de la capsule polysaccharidique, elle était également indispensable à la division de la cellule. Ainsi, mes travaux ont permis de proposer l'existence d'un mécanisme de coordination de la production de la capsule et du cycle cellulaire du pneumocoque. D'un point de vue appliqué, l'ensemble de mes travaux représente une étape préalable et prometteuse au développement de nouvelles molécules, ciblant les BY-kinases de manière spécifique, afin de lutter contre la virulence de certaines bactéries pathogènes / Bacteria possess a particular family of tyrosine-autokinases named BY-kinases. These enzymes are involved in the regulation of numerous cellular functions including the synthesis and export of extracellular polysaccharides that play a critical role in the virulence of different bacterial pathogens. However, the mechanisms of these processes remain unknown. The aim of my thesis was to characterize the role of these BY-kinases by structural and functional approaches. For that, I have realized three independent studies in three bacterial models. In Escherichia coli, I have characterized the interaction surface between the BY-kinase Wzc and its cognate phosphatase Wzb to understand how Wzb dephosphorylates Wzc. In Staphylococcus aureus, I have studied structurally the pseudo-BY-kinase CapB1. By comparison with the structure of its active homologue CapB2, my studies have allowed to suggest the existence of a mechanism controlling capsular polysaccharides synthesis involving both CapB1 and CapB2. Last, in Streptococcus pneumoniae, I have showed that while the autophosphorylation of the BY-kinase CpsD is necessary for proper synthesis of capsular polysaccharides, it is also involved in cell division. Thus, my work shows that a mechanism coordinating capsule production and cellular cycle exists in the pneumococcus. These works constitute a preliminary and promising step toward the development of new molecules, targeting specifically BY-kinases and aim to combat the virulence of bacterial pathogens
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