Navegando por Palavras-chave "Ácido glutâmico"
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- ItemAcesso aberto (Open Access)Caracterização do mecanismo catalítico da SGP, enzima protótipo das glutâmico peptidases(Universidade Federal de São Paulo (UNIFESP), 2012-02-01) Kondo, Marcia Yuri [UNIFESP]; Juliano, Luiz [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)Proteolytic enzymes are widespread in all organisms and are involved in a wide range of biological roles. There are seven distinct classes of proteases (serine, cysteine, aspartate, metalloproteases, threonine, glutamate and the newly identified asparagine peptide lyases), grouped according to their catalytic mechanism. The glutamic peptidases are carboxylic (acidic) proteases insensitive to pepstatin inhibitor and were annotated as a new group in 2004 (Family G1 Merops). These proteases have a unique catalytic dyad of residues Glu and Gln and a previously undescribed structure fold. Scytalidoglutamic peptidase (SGP), isolated from the wood-degrading fungus Scytalidium lignicolum on the 1970’s, is the forming member of glutamic peptidases. The three dimensional structures of scytalidoglutamic peptidase (SGP) and aspergilloglutamic peptidase (AGP) from Aspergillus niger revealed that the overall structure of these enzymes are almost identical, however two different catalytic mechanisms were proposed. Aiming at understanding the reaction mechanism of glutamic peptidases, in this thesis the following topics are highlighted, (1) biochemical characterization of the catalytic mechanism of SGP by use of pH rates profiles and solvent kinetic isotopic effects (SKIE) on the enzyme hydrolysis of FRET peptides and (2) specificity of the protease for P1 and P1’ positions describing the interactions of S1 subsite during catalysis. The paper showing results of these studies is attached to this thesis (Kondo, MY et al, JBC 2010).
- ItemAcesso aberto (Open Access)Estudo da sinalização glutamatérgica, estresse oxidativo e morte celular em cérebros de ratos durante o envelhecimento(Universidade Federal de São Paulo (UNIFESP), 2008-06-25) Ureshino, Rodrigo Portes [UNIFESP]; Smaili, Soraya Soubhi [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)O envelhecimento é um processo multi-fatorial associado a déficits funcionais, sendo que o cérebro é um dos órgãos com maior susceptibilidade a doenças crônico-degenerativas. Dentre essas, as doenças de Alzheimer e de Parkinson apresentam maior prevalência na população global e levam à incapacitação severa do indivíduo. Assim, o entendimento dos mecanismos dessas doenças que estão relacionados com o envelhecimento é importante para a busca de alternativas de tratamento. Há evidências de que, em doenças neurodegenerativas, ocorrem alterações na homeostase do cálcio (Ca2+), o que pode contribuir para a morte celular por apoptose. No presente trabalho, buscamos investigar fenômenos envolvidos com a tríade Ca2+ -mitocondria- EROs (espécies reativas do oxigênio) (TOESCU, 2005) e a apoptose em corpo estriado de ratos no envelhecimento. Foram avaliadas a sinalização intracelular dinâmica (em tempo real) e estática (biologia molecular), a morfologia celular e ultraestrutural, a morfometria e bioenergética. Utilizando fatias cerebrais de ratos, observamos que os anmais senescentes apresentaram um aumento de Ca2+ citosólico maior que os animais jovens, após a estimulação glutamatérgica. Em seguida, utilizamos antagonistas parciais das duas classes de receptores, os metabotrópicos do grupo I e os ionotrópicos (NMDAR), para estudar os componentes desse aumento de Ca2+. Avaliamos também o aumento de Ca2+ citosólico mediado por agentes que mobilizam esse íon do retículo endoplasmático e da mitocôndria, mostrando que esses estoques de Ca2+ podem estar aumentados no envelhecimento. As medidas do ∆ψm basal mostraram que hei urna diminuição deste parâmetro no envelhecimento, sendo estas alterações condizentes com a inibição mais acentuada do complexo I da cadeia transportadora de elétrons e do aumento na produção de EROs. As alterações funcionais não implicaram em mudanças ultraestruturais da mitocôndria. Foram investigados a expressão gênica e o conteúdo proteíco de Bax e Bcl-2, mostrando um aumento da expressão de bax e uma redução de proteínas Bcl-2, o que pode ter uma relação com o aumento de apoptose encontrado no estriado dos animais senescentes. Desse modo, os resultados indicam que, no envelhecimento, existem alterações no controle intracelular de sinalização de Ca2+ e na bioenergética, que podem contribuir para o aumento de apoptose.
- ItemAcesso aberto (Open Access)Estudos da competição entre laminina e ollgômeros Aβ pela ligação à PrPc e seus efeitos no ritmo circadiano(Universidade Federal de São Paulo (UNIFESP), 2017-05-30) Luz, Marcio Henrique Mello da [UNIFESP]; Lee, Kil Sun [UNIFESP]; http://lattes.cnpq.br/7705881286363327; http://lattes.cnpq.br/7724620775199307; Universidade Federal de São Paulo (UNIFESP)Interaction between PrPC and Laminin (LN) promotes neuritogenesis via ERK1/2, while the interaction between PrPC and amyloid-beta oligomer (oAβ) triggers synaptic dysfunction via Fyn kinase, but both complexes use mGluR1 as co-receptor. The involvement of PrPC in these opposite functions suggests that PrPC-mGluR1 acts as a key complex in the regulation of synaptic activity. To better understand how this complex works, we evaluated the interference of oAβ on LN binding to PrPC. We also verified levels of these molecules in different circadian periods and after sleep deprivation (SD), as the synaptic activity oscillates along the sleep-wake cycle. Our results showed that the addition of oAβ reduced LN-PrPC binding, suggesting that these ligands can compete with each other. Regarding the expression levels, we observed a reduction of PrPC in sleep deprived animals, independent of circadian period. mGluR1 levels were increased during the activity period in control animals (CTa) compared to the rest period (CTr), however this variation was attenuated with SD. These results indicate that the signaling triggered by PrPC-mGluR1 might undergo circadian regulation through the alteration of mGluR1 levels, and that SD might impair this regulation. Moreover, the levels of amyloid-beta peptides (pAβ) were increased by SD in rest period (SDr) when compared with CTr group. This increase correlated with hyperphosphorylation of SRC kinases in SDr group and might have contributed to a higher deviation in NMDAR phosphorylation of the same group. Total NMDAR levels in CTr and CTa showed the same profile of mGluR1. Laminin levels did not varied between groups, however ERK1/2 phosphorylation presented subtle decrease in CTa group compared to CTr group, which was not observed in SD groups. These results suggest that ERK1/2 and NMDAR tend to function in an opposite way during the activity time in control animals. However, SD seems to attenuate the variation of ERK/12 activity and hyperactivate SRC kinases leading to a NMDAR deregulation.
- ItemAcesso aberto (Open Access)Fisiopatologia da cefaléia crônica diária: estudo do líquido cefalorraquidiano(Universidade Federal de São Paulo (UNIFESP), 2008-03-26) Vieira, Domingos Sávio de Souza [UNIFESP]; Peres, Mario Fernando Prieto [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)Introduction: Chronic daily headaches consist of a group of headaches, among them chronic migraine, that is comorbid with depression, overuse of medication, obesity and recently, cases of idiopathic intracranial hypertension without papilloedema. Objectives: To establish idiopathic intracranial hypertension without papilloedema prevalence and glutamate and gamma-aminobutyric acid levels in cerebrospinal fluid from patients with chronic migraine compared to other groups of patients. Methods: We studied patients with chronic migraine, who underwent lumbar puncture to rule out idiopathic intracranial hypertension without papilloedema. Amino acids glutamate and gamma-aminobutyric acid levels were measured by high performance liquid chromatography in cerebrospinal fluid. Results: Six patients, among sixty, had CSF open pressure higher than 200 mm H20 without papilloedema on fundoscopy. Patients who overused triptans had glutamate levels lower than those with abuse of other analgesic types and nonoverusers. The gamma-aminobutyric acid levels in cerebrospinal fluid were lower in depressed patients when compared to patients without depression and controls. Conclusions: The study of the cerebrospinal fluid was important in patients with chronic migraine for the exclusion of idiopathic intracranial hypertension without papilloedema, opening perspectives for the understanding of the physiopathology and development of new drug therapies for migraine and its comorbidities.