A carregar...
Publicação
Estudo dos mecanismos moleculares regulados pelo H2O2 nas células endoteliais num contexto tumoral
| Nome: | Descrição: | Tamanho: | Formato: | |
|---|---|---|---|---|
| 1.75 MB | Adobe PDF |
Autores
Orientador(es)
Resumo(s)
In vivo, o H2O2 está envolvido em várias vias de sinalização e na regulação de inúmeros processos biológicos, incluindo a angiogénese. Esta molécula é produzida em maiores quantidades em tumores, não só pelas células tumorais como também por componentes do seu microambiente. Um dos objectivos desta tese foi compreender o papel do H2O2 na regulação da função do receptor da laminina 37/67kDa (37LRP/67LR) e na sua interacção com a laminina-1. Este receptor é sobrexpresso em tumores e é considerado como um marcador de mau prognóstico. Analisando a sequência e estrutura secundária do 37LRP/67LR humano, verificou-se que este possui dois resíduos de cisteína conservados, os quais são considerados alvos preferenciais para oxidação pelo H2O2. Inicialmente, observou-se que a exposição celular a concentrações sub-letais de H2O2 aumenta a migração das células endoteliais (CEs) à laminina-1 e diminuem a adesão de CEs e tumorais a esse substrato. Seguidamente, através de ensaios in vitro, foi observado que o 37LRP/67LR está envolvido na adesão de células tumorais à laminina-1 apenas na presença de H2O2, e que os resíduos de cisteína deste receptor desempenham um papel fundamental neste fenómeno. Estes resultados mostram que, apesar da diminuição da adesão celular na presença de H2O2, as células que apresentam sobrexpressão do 37LRP/67LR esta não é tão acentuada. Este processo é dependente da oxidação da proteína. Por outro lado, pretendeu-se estudar a função do H2O2na sinalização pelo VEGF, uma vez que a activação deste factor de crescimento leva à produção de H2O2. Assim, analisou-se o proteoma de cisteínas sensíveis ao H2O2 após tratamento com VEGF. Com este estudo foi possível encontrar proteínas com alteração do seu estado de oxidação dependente da sinalização do VEGF. Os resultados obtidos permitiram demonstrar que o H2O2 regula directamente a função de proteínas específicas relacionadas com a adesão e migração celulares e poderá desta forma ter um papel fundamental para a progressão de determinadas doenças com caracter oxidativo, como é o caso do cancro.
In vivo, H2O2 is involved in multiple signaling pathways and in the regulation of several biological processes, including angiogenesis. This molecule is produced in large quantities in case of diseases such as cancer and inflammation. In this thesis, we aimed at understanding the role of H2O2 in the regulation of laminin receptor 37/67kDa (37LRP/67LR) function and its interaction with laminin-1. This receptor is overexpressed in tumors, both in endothelial cells and tumor cells, and is a marker for poor prognostic. Despite the numerous functions and locations described for this protein, its molecular mechanism is still unclear. The identification of H2O2-dependent alterations in structure/function of 37LRP/67LR, can elucidate the role of this protein and show the importance of H2O2 in tumor development. Sequence and structure analysis of 37LRP/67LR have shown that this protein possesses two cysteines, which are common oxidation targets for H2O2. These aminoacids are conserved which indicates a possible role for these two amino acids in the regulation of 37LRP/67LR function. First, we have observed that cellular exposure to sub-lethal concentrations of H2O2 increases endothelial cell migration on Laminin-1 and decreases adhesion of endothelial and tumor cells to this substrate. Secondly, we have demonstrated that the 37LRP/67LR increases tumor cell adhesion to laminin-1, only in presence of H2O2, and that the molecular mechanism is dependent on the cysteines present in the protein. These results show that, even though H2O2 decreases cell adhesion to Laminin-1, 37LRP/67LR overexpression prevents partially this effect by a mechanism dependent on cysteine oxidation. We also aimed at unravel the role of H2O2 in VEGF signaling, which induces H2O2 production. Therefore, we analyzed the proteome of cysteines sensitive to H2O2 after treatment with VEGF. In this study it was possible to find four proteins that are oxidized and three that are reduced in a VEGF-dependent manner. We have demonstrated that H2O2 direct regulation of specific proteins might have a key role in the progression of diseases with oxidative properties, such as cancer.
In vivo, H2O2 is involved in multiple signaling pathways and in the regulation of several biological processes, including angiogenesis. This molecule is produced in large quantities in case of diseases such as cancer and inflammation. In this thesis, we aimed at understanding the role of H2O2 in the regulation of laminin receptor 37/67kDa (37LRP/67LR) function and its interaction with laminin-1. This receptor is overexpressed in tumors, both in endothelial cells and tumor cells, and is a marker for poor prognostic. Despite the numerous functions and locations described for this protein, its molecular mechanism is still unclear. The identification of H2O2-dependent alterations in structure/function of 37LRP/67LR, can elucidate the role of this protein and show the importance of H2O2 in tumor development. Sequence and structure analysis of 37LRP/67LR have shown that this protein possesses two cysteines, which are common oxidation targets for H2O2. These aminoacids are conserved which indicates a possible role for these two amino acids in the regulation of 37LRP/67LR function. First, we have observed that cellular exposure to sub-lethal concentrations of H2O2 increases endothelial cell migration on Laminin-1 and decreases adhesion of endothelial and tumor cells to this substrate. Secondly, we have demonstrated that the 37LRP/67LR increases tumor cell adhesion to laminin-1, only in presence of H2O2, and that the molecular mechanism is dependent on the cysteines present in the protein. These results show that, even though H2O2 decreases cell adhesion to Laminin-1, 37LRP/67LR overexpression prevents partially this effect by a mechanism dependent on cysteine oxidation. We also aimed at unravel the role of H2O2 in VEGF signaling, which induces H2O2 production. Therefore, we analyzed the proteome of cysteines sensitive to H2O2 after treatment with VEGF. In this study it was possible to find four proteins that are oxidized and three that are reduced in a VEGF-dependent manner. We have demonstrated that H2O2 direct regulation of specific proteins might have a key role in the progression of diseases with oxidative properties, such as cancer.
Descrição
Tese de mestrado em Bioquímica (Bioquímica Médica), apresentada à Universidade de Lisboa, através da Faculdade de Ciências, 2012
Palavras-chave
H2O2 Lamina-1 Receptor da lamina 37/67kDa Angiogénese VEGF Cancro Teses de mestrado - 2012