Navegando por Palavras-chave "Mutantes"

Agora exibindo 1 - 2 de 2
  • Imagem de Miniatura
    Item
    Acesso aberto (Open Access)
    Mutantes do receptor B2 de cininas : análise da interação droga-receptor e sua aplicabilidade no estudo do angioedema hereditário
    (Universidade Federal de São Paulo (UNIFESP), 2017-12-13) Silva, Rafael Filippelli da [UNIFESP]; Pesquero, João Bosco [UNIFESP]; Costa Neto, Cláudio Miguel da [UNIFESP]; http://lattes.cnpq.br/8887116099313093; http://lattes.cnpq.br/0856630824759511; http://lattes.cnpq.br/6408852011134966; Universidade Federal de São Paulo (UNIFESP)
    Introduction: bradykinin (8K) is an important pressure regulating peptide arterial, nociception, inflammation and is associated with the pathophysiology of angioedema (HAE). The main clinical aspects of HAE are mediated by 8K, which exercise by the activation of receptor 82 (82R), a member of the family of G protein coupled receptors (GPCR). The objective of the present study was to evaluate if 82R mutations found in HAE patients could alter the profile pharmacological effect of 8K signal transduction, leading to relevant for the pathophysiology of the disease. Materials and methods: we use G-and-3-arrestin biosensors to perform 8RET analyzes and evaluate functional profiles of mutant 82Rs. All the experiments were carried out in transiently transfected HEK 293T cells, either with the wild-type or with the human 82R mutants. Competitive binding assays using 8K radiolabeled (3H-8K) were performed to estimate the 8K affinity for mutant receptors. The 8RET assays, used to evaluate the activation of G protein and the recruitment of β-arrestin 1 and 2, were performed using a method adapted from that described by Quoyer et al. (2013). Results: the binding assays showed that 8K retains a similar affinity at mutant receptors R14C, W344C, G354E and V376M, when compared to the wild-type receptor. In analysis, 8K triggered the coupling of G protein and mobilization intracellular calcium with similar potency and efficacy at R14C and G354E compared to WT. On the other hand, these receptors were not able to to recruit! 3-arrestinas. In the W344C and V376M mutants, the potency and efficacy of 8K was reduced on activation of all of the signaling pathways tested. Conclusions: These findings provide strong evidence for a biased agonist profile endogenous 8K G protein by activating mutants R14C and G354E 82R identified in patients with HAE. On the other hand, mutants W344C and V376M showed a decrease in the pharmacological responses induced by 8K. We believe that the current data contribute to revealing the mechanisms molecules involved in HAE and thus opening up new perspectives and approaches to study and treat this disease.
  • Nenhuma Miniatura disponível
    Item
    Somente Metadadados
    Variantes no gene GLA e o perfil enzimático da alfagalactosidase a em pacientes com suspeita de Doença de Fabry
    (Universidade Federal de São Paulo (UNIFESP), 2019-03-28) Teixeira, Patricia Varela Lima [UNIFESP]; Pesquero, Joao Bosco [UNIFESP]; http://lattes.cnpq.br/0856630824759511; http://lattes.cnpq.br/1816388063402796; Universidade Federal de São Paulo (UNIFESP)
    Objective: To analyze a database with molecular and biochemical results of patients with suspected Fabry disease. From these results, to evaluate the pathogenicity of the variants, as well as to carry out the in vitro characterization of novel mutations, and finally to trace the enzymatic profile of the genotypes in order to evaluate the impact of these variants on the enzyme. Methods: Bioinformatics tools were used to analyze the variants found in the GLA gene of patients with Fabry disease suspected. Patients were divided according to the genotype they carried in order to trace the enzymatic profile and correlation with the pathogenicity. For in vitrocharacterization, experiments were conducted on HeLa cells transfected with wild-type GLA or with mutants plasmids, finally, gene expression as well as the enzymatic activity of α-Gal A were evaluated. Results: 215 men and 48 women presented 103 variants in GLA exons; 57 variants were previously described as pathogenic, eleven described as unknown effect and the other 35 are family-specific mutations. The other patients presented variants in non-coding regions or had no alterations inGLA. In vitro analysis confirmed pathogenicity in mutations classified as pathogenic and possibly pathogenic; in the new mutations classified as with unknown effect, pathogenicity was confirmed in three mutations, the other four did not cause DF. The enzymatic profile revealed a possible non-pathogenicity of variants of unknown effect, as well as variants found in non-coding regions. Finally, the study of haplotypes formed by variants in non-coding regions revealed a high frequency in the control population, similar to that found in patients. Conclusion: The DBS enzymatic activity was markedly reduced in patients with mutations described as pathogenic, as well as in novel variants classified as pathogenic or possibly pathogenic (with in vitro confirmation) when compared with individuals presenting VUS, variants in non-coding regions or without variants, indicating non-pathogenic potential of these latter genotypes.