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The predicted protein product of open reading frame slr0328 from Synechocystis sp. PCC 6803, SynPTP, possesses significant amino acid sequence similarity with known low molecular weight protein tyrosine phosphatases (PTPs). To determine the gross functional properties of this hypothetical protein, open reading frame slr0328 was cloned, and its predicted protein product was expressed in E. coli. The recombinant protein, SynPTP, was purified by metal ion column chromatography. The catalytic activity of SynPTP was examined toward several exogenous protein substrates that had been phosphorylated on either tyrosine residues or serine residues. SynPTP exhibited phosphatase activity toward tyrosine phosphorylated protein substrates (Vmax toward phosphotyrosyl 32P-casein was 1.5 nmol/min/mg). However, no phosphatase activity was detected toward serine phosphorylated protein substrates. SynPTP displayed phosphohydrolase activity toward several organophosphoesters including para-nitrophenyl phosphate (p-NPP), beta-napthyl phosphate and phosphotyrosine but not toward alpha-napthyl phosphate, phosphoserine, or phosphothreonine. Kinetic analysis indicated that the Km (0.6 mM) and Vmax (3.2 mmole/min/mg) values for SynPTP toward pNPP are similar to those of other known bacterial low molecular weight PTPs. The protein phosphatase activity of SynPTP was inhibited by sodium orthovanadate, a known inhibitor for tyrosine phosphatases, but not by okadaic acid, an inhibitor for many serine/threonine phosphatases. Mutagenic alteration of the predicted catalytic cysteine, Cys7, to serine abolished enzyme activity. Several phosphotyrosine containing proteins were detected from the whole cell extracts of Synechocystis sp. PCC 6803 through immunoreactions using anti-phosphotyrosine antibody. SynPTP was observed to dephosphorylate three of these proteins in vitro. Two of these proteins were identified by peptide-mass fingerprinting analysis, as PsaD (photosystem I subunit II) and CpcD (phycocyanin rod linker protein). In addition, several phosphotyrosine proteins were detected from the soluble and membrane fractions of Synechocystis sp. PCC 6803 cell extracts by in vitro substrate trapping as potential endogenous substrates of SynPTP. Two of these proteins were identified as the alpha and beta subunits of phycocyanin. We therefore speculate that SynPTP might be involved in the regulation of photosynthesis in Synechocystis sp. PCC 6803. / Ph. D.

Diss. (sammanfattning) Umeå : Umeå universitet, 2010. / Härtill 5 uppsatser.

Tyrosyl phosphorylation plays an important role in many fundamental cellular processes, including cell growth, differentiation and proliferation. The levels of phosphotyrosine (pY) are regulated by the opposing actions of protein-tyrosine kinases (PTKs) and protein-tyrosine phosphatases (PTPs). A limitation to understanding the roles of PTPs in physiological and pathological cell signaling has been the absence of global proteomic approaches that enable the systematic and comprehensive analysis of PTP expression, regulation and function. This dissertation describes the development and application of novel proteomic methodologies that permit the global analysis of PTP expression (qPTPome), regulation (by oxidation and nitrosylation; q-oxPTPome) and substrates/binding proteins. These methods provide a workflow to begin assessing PTP function at a systems level, rather than its current targeted format. Application of these techniques will provide invaluable information to begin bridging the gap in our understanding of PTP and PTK function in normal and malignant cell signaling.

Tyrosyl phosphorylation plays an important role in many fundamental cellular processes, including cell growth, differentiation and proliferation. The levels of phosphotyrosine (pY) are regulated by the opposing actions of protein-tyrosine kinases (PTKs) and protein-tyrosine phosphatases (PTPs). A limitation to understanding the roles of PTPs in physiological and pathological cell signaling has been the absence of global proteomic approaches that enable the systematic and comprehensive analysis of PTP expression, regulation and function. This dissertation describes the development and application of novel proteomic methodologies that permit the global analysis of PTP expression (qPTPome), regulation (by oxidation and nitrosylation; q-oxPTPome) and substrates/binding proteins. These methods provide a workflow to begin assessing PTP function at a systems level, rather than its current targeted format. Application of these techniques will provide invaluable information to begin bridging the gap in our understanding of PTP and PTK function in normal and malignant cell signaling.

Thesis (Ph.D. in Cell Biology, Stem Cells, & Development) -- University of Colorado Denver, 2008. / Typescript. Includes bibliographical references (leaves 138-155). Free to UCD Anschutz Medical Campus. Online version available via ProQuest Digital Dissertations;

A Análise de Acoplamentos Estatísticos é uma técnica computacional capaz de identificar resíduos importantes para a estrutura e função de proteínas em uma família por meio da quantificação de conservação posicional, correlação entre posições e identificação de grupos de resíduos correlacionados entre si. Neste trabalho, a análise de acoplamentos estatísticos foi implementada e aplicada ao estudo das proteínas tirosina fosfatases. Em conjunto com as proteínas tirosina quinases (PTKs), que adicionam um grupo fosforil a um resíduo de tirosina em uma proteína, as proteínas tirosina fosfatases (PTPs), que o removem, são responsáveis por diversos processos de sinalização celular. Elas são um caso de evolução convergente, onde um subgrupo (as proteínas tirosina fosfatases de baixo peso molecular) não apresenta homologia às chamadas PTPs \"clássicas\", capazes de defosforilar apenas resíduos de tirosina, e às fosfatases de especifidicade dupla, capazes de defosforilar também resíduos de serina e treonina, além de substratos não-protéicos. Em comum, as três subfamílias apresentam apenas o motivo CX5R, característico para todas as PTPs. Através do estudo das três subfamílias utilizando a análise de acoplamentos estatísticos, foi possível obter uma descrição detalhada de suas características conservadas e correlacionadas, relacionando-as ao conhecimento acumulado sobre proteínas tirosina fosfatases e a questões em aberto como a regulação por dimerização, a especificidade e mutações relacionadas a patologias. Foi possível também apresentar um método capaz de distinguir proteínas tirosina fosfatases de baixo peso molecular das arsenato redutases, derivadas das primeiras por evolução divergente. Adicionalmente, a técnica foi aplicada ao estudo das hexoquinases, às superóxido dismutases e às peroxidases. A tese descreve também estudos desenvolvidos pelo autor na área de cristalografia de proteínas a determinação das estruturas da Transtirretina humana em complexo com genisteína, da holo-Hexoquinase PI de S. cerevisae, do complexo IL-22/IL-22R1 e da Laminarinase de R. marinus. / The statistical coupling analysis is a computational technique which can identify important residues for the structure and function of proteins in a family by quantifying positional conservation, correlation between positions and identifying groups of self-correlating residues. Its implementation in this research group was applied to the study of the protein tyrosine phosphatases. Together with the protein tyrosine kinases (PTKs), which add a phosphoryl group to a tyrosine residue in proteins, the protein tyrosine phosphatases (PTPs), which remove it, are responsible for a variety of cell signaling processes. They are a case of convergent evolution, since one subgroup (the low molecular weight protein tyrosine phosphatases) are not homologous to the classical phosphatases, which can only dephosphorilate tyrosine residues, and the dual-specificity phosphatases, which can also dephosphorilate serine and threonine residues, and also non-proteinaceous substrates. All three sub-families have, in common, the CX5R motif, a characteristic of all PTPs. By applying the statistical coupling analysis to the study of the three sub-families, it was possible to obtain a detailed depiction of their conserved and correlated characteristics, relating them to the accumulated knowledge on protein tyrosine phosphatases and open questions such as protein regulation by dimerization, specificity and disease-related mutations. It was also possible to present a method to distinguish between low molecular weight phosphatases and arsenate reductases, which are derived by the former by divergent evolution. In addition, the technique was applied to the study of hexokinases, superoxide dismutases and peroxidases. The thesis also describe studies developed by the author in the field of protein crystallography the structure determination of human transthyretin in complex with genistein, holo-hexokinase PI from S. cerevisae, the IL-22/IL-22R1 complex and the laminarinase from R. marinus.

Proteínas tirosina-fosfatases (PTPs) catalisam a hidrólise de fosfotirosina de outras proteínas e, assim, regulam importantes processos bioquímicos. Dois representantes desta família são as fosfatases de dupla especificidade VHR e CDC25B. A primeira etapa de reação catalisada é um ataque nucleofílico da cadeia lateral de uma cisteína sobre o fósforo do substrato, com uma possível transferência de H+ de um ácido geral para o grupo de saída, formando uma PTP intermediária tiofosforilada e desfosforilando o substrato. Dúvidas ainda persistem sobre esta etapa, envolvendo os estados de protonação do substrato e do nucleófilo enzimático, a inatividade de certos mutantes e a identificação do ácido geral nas CDC25s. Procuramos solucionar estas questões por simulações computacionais das reações catalisadas pela VHR e pela CDC25B. Inicialmente, caminhos das reações de tiólise e alcoólise de ésteres de fosfato na fase gasosa foram determinados por cálculos de estrutura eletrônica ab initio e analisados como referências da reatividade intrínseca de ésteres de fosfato e como modelos da atividade enzimática. Um potencial híbrido de mecânica quântica e mecânica molecular foi amplamente testado e calibrado, empregando estes caminhos de reação e outros dados ab initio. A calibração permitiu que conclusões semiquantitativas pudessem ser obtidas a partir das simulações enzimáticas. Potenciais de força média foram determinados com o potencial híbrido para desfosforilação de diferentes substratos catalisada pelas PTPs selvagens e por suas mutantes. Os resultados mostram que o mecanismo da reação catalisada segue uma adição e eliminação simultânea, com um estado de transição dissociativo com caráter de metafosfato. As barreiras calculadas são bastante próximas às energias de ativação experimentais. O substrato enzimático é um diânion desprotonado e o nucleófilo está ionizado. As reações do substrato ou do nucleófilo protonados apresentam barreiras, no mínimo, 15 kcal/mol mais altas que os valores experimentais. A VHR mutante cisteína para serina no sítio ativo é inativa, porque a serina está protonada. As CDC25s não utilizam um ácido geral para catálise, ao contrário das outras PTPs. Propostas de que o ácido geral poderia ser o próprio substrato ou um dos ácidos glutâmicos presentes no sítio ativo são energeticamente inacessíveis. / Protein tyrosine phosphatases (PTPs) catalyse the hydrolysis of phosphotyrosine from other proteins and, hence, regulate important biochemical processes. Two members from this family are the dual specificity phosphatases VHR and CDC25B. The first step of the catalysed reaction is the nucleophilic attack from the side chain of a cystein towards the substrate, with a possible H+ transfer from a general acid to the leaving group, forming a PTP thiophosphorylated intermediate and dephosphorylating the substrate. There are still some doubts about this step, involving the protonation states of the substrate and the nzymatic nucleophile, the inactivity of certain mutants and the identification of the general acid in CDC25s. We tried to solve this questions by computer simulations of the reactions catalysed by VHR and by CDC25B. Initially, reaction pathways of phosphate esters thiolysis and alcoholysis in the gas-phase were determined by ab initio electronic structure calculations and analysed as benchmarks for the intrinsic reactivity of phosphate esters and as models of the enzymatic activity. A hybrid potential of quantum mechanics and molecular mechanics was fully tested and calibrated, employing these reaction pathways and other ab initio data. The calibration allowed that semiquantitative conclusions could be obtained from the enzymatic simulations. Potentials of mean force were determined with the hybrid potential for the dephosphorylation of different substrates catalysed by the wild-type PTPs and their mutants. The results show that the catalysed reaction mechanism follows a concerted addition and elimination, with a dissociative transition state with metaphosphate-like. The calculated barriers are very close to the experimental activation energies. The enzymatic substrate is a deprotonated dianion and the nucleophile is ionised. The reactions of the protonated substrate or nucleophile have barriers, at least, 15 kcal/mol higher than the experimental results. The active site cystein to serine VHR mutant is inactive because the serine is protonated. The CDC25s do not employ a general acid for catalysis, differently from the other PTPs. Proposals that the general acid is the substrate or one of the glutamic acids present in the active site are not energetically accessible.

The control of cellular tyrosine phosphorylation levels is of great importance in many biological systems. Among the kinases and phosphatases that modulate these levels, the LAR-RPTPs have been suggested to act in several key aspects of neural development, and in a dysfunctional manner in various pathologies from diabetes to cancer. The aim of this thesis is to describe the physiological functions of one of the members of this subfamily of RPTPs, namely RPTPsigma. First, we showed that glucose homeostasis is altered in RPTPsigma null mice. They are hypoglycemic and more sensitive to exogenous insulin and we proposed that the insulin hypersensitivity observed in RPTPsigma-null mice is likely secondary to their neuroendocrine dysplasia and GH/IGF-1 deficiency. In addition to regulating nervous system development, RPTPsigma was previously shown to regulate axonal regeneration after injury. In the absence of RPTPsigma, axonal regeneration in the sciatic, facial and optical nerves was enhanced following nerve crush. However, myelin-associated growth inhibitory proteins and components of the glial scar such as CSPGs (chondroitin sulfate proteoglycans) have long been known to inhibit axonal regeneration in the CNS, making spinal cord injury irreversible. In collaboration with Dr Samuel David, we unveiled that RPTPsigma null mice are able to regenerate their corticospinal tract following spinal cord hemisections as opposed to their WT littermates. We then isolated primary neurons from both sets of animals and found that the absence of RPTPsigma promotes the ability of the neurons to adhere to certain inhibitory substrates. Finally, in order to better understand the physiological role of RPTPsigma, we used a yeast substrate-trapping approach, to screen a murine embryonic library for new substrates. This screen identified the RhoGAP p250GAP as a new substrate, suggesting a downstream role for RPTPsigma in RhoGTPase signaling. We also identified p130Cas and Fyn as new binding partners. All these proteins have clear functional links to neurite extension. The characterization of RPTPsigma and its signaling partners is essential for understanding its role in neurological development and may one day translate into treatments of neural diseases and injuries.