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.

Thesis (Ph.D. in Neuroscience) -- University of Colorado Denver, 2007. / Typescript. Includes bibliographical references (leaves 200-233). Free to UCD affiliates. Online version available via ProQuest Digital Dissertations;

Thesis (Ph.D. in Pharmacology) -- University of Colorado Denver, 2007. / Typescript. Includes bibliographical references (leaves 82-92; 128-134). 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.

Coordination of mitosis and cytokinesis is crucial to generate healthy daughter cells with equal amounts of genetic and cytoplasmic materials. In the fission yeast Schizosaccharomyces pombe, an evolutionarily conserved Cdc14-like phosphatase (Clp1) functions to couple mitosis and cytokinesis by antagonizing CDK activity. The activity of Clp1 is thought to be regulated in part by its subcellular localization. It is sequestered in the nucleolus and the spindle pole body (SPB) during interphase. Upon mitotic entry, it is released into the cytoplasm and localized to the kinetochores, the actomyosin ring, and the mitotic spindle to carry out distinct functions. It is not clear how Clp1 is released from the nucleolus, however, once released, a conserved signaling pathway termed Septation Initiation Network (SIN) functions to retain Clp1 in the cytoplasm until completion of cytokinesis. The SIN and Clp1 function together in a positive feedback loop to promote each other’s activity. That is, the SIN promotes cytoplasmic retention of Clp1, and cytoplasmic Clp1 antagonizes CDK activity and reverses CDK inhibition on the SIN pathway to promote its function and activity. However, at the start of this thesis, the mechanism by which the SIN regulated Clp1 was unknown. The SIN pathway is also required to promote constriction of the actomyosin ring, and the septum formation. However, its downstream targets were still uncharacterized. In two separate studies, we studied how Clp1 is released from the nucleolus at mitotic entry and how the SIN kinase Sid2 acts to retain Clp1 in the cytoplasm. We identified several Sid2 candidate substrates, and revealed other functions of the SIN pathway in coordinating mitotic events.

No description available.

<p>The pathological hallmarks of sickle cell disease originate from a single mutation of the beta hemoglobin gene resulting in a valine at position 6 instead of the canonical glutamic acid. This small change perpetuates many factors, manifesting into chronic embolic processes in the microvasculature, causing painful vaso-occlusive episodes and eventual organ failure. There have been numerous therapies developed to reduce the mortality of sickle cell ranging from agents to induce production of fetal hemoglobin to chronic blood transfusions. Although each of these options are effective at improving the quality of life for sickle cell patients, they only treat one aspect of the disease and, for some, become ineffective over time. In the hope of producing a better therapy, a better understanding of the pathogenesis of vaso-occlusive episodes is needed. While many models have been offered to account for these vaso-occlusive events, one recently proposed mechanism stems from the elevated tyrosine phosphorylation of the cytoplasmic domain of the major erythrocyte membrane protein, Band 3. Band 3 serves as a hub for many critical proteins in the red cell. It binds ankyrin, which associates the spectrin cortical cytoskeleton to the red cell membrane, deoxygenated hemoglobin, the kinases Wnk1 and OSR1, which regulate cation transport, and a glycolytic enzyme metabolon that regulates the production of ATP and glutathione. When Band 3 is tyrosine phosphorylated, each of these proteins dissociate, causing significant changes to red cell homeostasis. These changes include an accumulation of reactive oxygen species, vesiculation and release of prothrombotic microvesicles, leakage of cell free hemoglobin, and a decrease in cell volume. Normally, Band 3 exists in a predominantly unphosphorylated state, however, in sickle cell disease, Band 3 is abundantly tyrosine phosphorylated. Reduction in the tyrosine phosphorylation of Band 3 has been documented to prevent the release of microvesicles and hemoglobin from sickle cell red blood cells. Because these microvesicles and cell free hemoglobin contribute to the vaso-occlusive episodes in sickle cell patients, inhibiting the mechanism for their release offers a potential therapeutic option. But to accomplish this, the molecular cause for the elevated tyrosine phosphorylation in sickle cell disease must be identified. Since tyrosine phosphorylation is performed by a tyrosine kinase and removed by a tyrosine phosphatase, the elevation in phosphorylation must be due to changes in both of these processes. Unfortunately, the identity and nature of these kinases and phosphatases are poorly understood. In this dissertation, I identified the tyrosine kinases Syk, Lyn, and Src attributed to Band 3</p> <p>15</p> <p>phosphorylation that facilitates the release of microvesicles and hemoglobin in sickle cell red blood cells. Inhibition of Syk or one of the two Src family kinases is sufficient to prevent the destabilization of the red blood cell membrane. These kinases function in a hierarchy, where one of the three Src family kinase, Lyn phosphorylates Syk, activating it, and promoting the phosphorylation of Band 3 at tyrosines 8 and 21. Prevention of either phosphorylation event prevents the release of microvesicles and cell free hemoglobin. I also report the identification of PTP1B as the tyrosine phosphatase responsible for maintaining Band 3 in an unphosphorylated state. Interestingly, in sickle cell disease, this tyrosine phosphatase is proteolytically cleaved, resulting in a reduction in dephosphorylating potential. It has been reported previously that PTP1B is a substrate of the calcium dependent protease, calpain and that calpain inhibitors improve the cell morphology of sickle erythrocytes. Inhibition of this proteolytic process may offer an additional therapeutic option for the treatment of sickle cell disease.</p>