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Abstract(s)
O centrossoma é o principal centro organizador de microtúbulos das células animais, desempenhando funções celulares essenciais no processo de divisão celular, uma vez que regula a nucleação e organização espacial dos microtúbulos, estando também implicado no posicionamento de organelos na célula, como o complexo de Golgi, no estabelecimento da polaridade celular, na migração e adesão celulares e na ciliogénese. Nas células animais o centrossoma encontra-se posicionado no centro da célula em estreita associação com o núcleo. Os cofactores da tubulina (TBCA-E) são proteínas que participam na via de folding da tubulina e possuem funções relacionadas com o citoesqueleto, desempenhando papéis essenciais nas células eucariotas. A proteína TBCCD1 (TBCC-domain containing protein 1) é uma proteína centrossomal relacionada com o TBCC e com a proteína RP2, uma vez que possui os domínios funcionais TBCC e CARP, porém não parece possuir actividade de GAP para a tubulina. O TBCCD1 é um potencial regulador do posicionamento do centrossoma em estreita interacção com o núcleo e da organização citoplasmática. Neste trabalho descrevemos a identificação do domínio responsável pela localização centrossomal da proteína TBCCD1 humana, constituído pelos primeiros 20 resíduos de aminoácidos da sua região N-terminal. Em células humanas observámos que a expressão da proteína TBCCD1 com uma mutação pontual no resíduo de prolina na posição 24 leva à deslocalização da γ-tubulina do centrossoma. Verificámos também que três mutações pontuais distintas nos motivos VxPX e KRAK da proteína causam uma menor eficiência na formação de cílios primários. Concluindo, a proteína centrossomal TBCCD1 humana parece ter uma ligação à γ-tubulina, contudo ainda não está esclarecido se esta interacção é directa ou indirecta, podendo a γ-tubulina ser um parceiro molecular do TBCCD1. O TBCCD1 deverá também ter um papel essencial in vivo resultante do seu envolvimento na manutenção da ligação do centrossoma ao núcleo e no processo de ciliogénese.
The centrosome is the major microtubule organizing center in animal cells, playing an essential role in cellular processes such cell division, since it regulates the nucleation and spatial organization of microtubules, and is also implicated in organelle positioning in the cell, such as the Golgi apparatus, cell polarity establishment, cell migration and adhesion and ciliogenesis. In animal cells, the centrosome is positioned in the center of the cell in close association with the nucleus. The tubulin cofactors (TBCA-E) are proteins involved in tubulin folding pathway that have emerged as proteins with crucial roles in eukaryotic cells related to the cytoskeleton. The TBCCD1 protein (TBCC domain-containing protein 1) is a centrossomal protein related to TBCC and RP2 protein, since it contains the TBCC and CARP domains. However, TBCCD1 probably doesn’t have a GAP activity towards tubulin. The TBCCD1 is a potential regulator of the positioning of the centrosome and cytoplasmic organization. In this work we described the identification of the centrosome targeting motif of the human TBCCD1, corresponding to the first 20 amino acid residues of its N-terminus region. Our studies performed in mammalian cell lines revealed that the expression of TBCCD1 with a point mutation in the proline residue at position 24 leads to the mis-localization of γ-tubulin from the centrosome. Furthermore, we also found that three distinct point mutations in the motifs VxPX and KRAK lead to a lower efficiency of transfected cells to assemble primary cilia. Also, the obtained results clearly show that the human centrossomal TBCCD1 protein seems to have an interaction with γ-tubulin, but whether this is direct or indirect is still not clear. However, our results support the idea that γ-tubulin will probably be a molecular partner of TBCCD1. The TBCCD1 should also have an essential role in vivo resulting from their involvement in maintaining the nucleus-centrosome association and its involvement in ciliogenesis.
The centrosome is the major microtubule organizing center in animal cells, playing an essential role in cellular processes such cell division, since it regulates the nucleation and spatial organization of microtubules, and is also implicated in organelle positioning in the cell, such as the Golgi apparatus, cell polarity establishment, cell migration and adhesion and ciliogenesis. In animal cells, the centrosome is positioned in the center of the cell in close association with the nucleus. The tubulin cofactors (TBCA-E) are proteins involved in tubulin folding pathway that have emerged as proteins with crucial roles in eukaryotic cells related to the cytoskeleton. The TBCCD1 protein (TBCC domain-containing protein 1) is a centrossomal protein related to TBCC and RP2 protein, since it contains the TBCC and CARP domains. However, TBCCD1 probably doesn’t have a GAP activity towards tubulin. The TBCCD1 is a potential regulator of the positioning of the centrosome and cytoplasmic organization. In this work we described the identification of the centrosome targeting motif of the human TBCCD1, corresponding to the first 20 amino acid residues of its N-terminus region. Our studies performed in mammalian cell lines revealed that the expression of TBCCD1 with a point mutation in the proline residue at position 24 leads to the mis-localization of γ-tubulin from the centrosome. Furthermore, we also found that three distinct point mutations in the motifs VxPX and KRAK lead to a lower efficiency of transfected cells to assemble primary cilia. Also, the obtained results clearly show that the human centrossomal TBCCD1 protein seems to have an interaction with γ-tubulin, but whether this is direct or indirect is still not clear. However, our results support the idea that γ-tubulin will probably be a molecular partner of TBCCD1. The TBCCD1 should also have an essential role in vivo resulting from their involvement in maintaining the nucleus-centrosome association and its involvement in ciliogenesis.
Description
Tese de mestrado em Bioquímica, apresentada à Universidade de Lisboa, através da Faculdade de Ciências, 2011
Keywords
TBCCD1 Centrossoma y-tubulina Cílios Teses de mestrado - 2011