Made available in DSpace on 2015-12-06T23:47:07Z (GMT). No. of bitstreams: 0 Previous issue date: 2007 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O controle traducional mediado pela fosforilacao da subunidade alfa do fator de inicio de traducao 2 (eIF2a) e um ponto central para programas de expressao genica induzidos por estresse. Tripanossomatideos, importantes patogenos humanos, apresentam processos de diferenciacao desencadeados pelo contato com os distintos ambientes encontrados em seus insetos vetores e hospedeiros mamiferos, provavelmente representando situacoes de estresse. Trypanosoma brucei, o agente causador da tripanossomiase africana, codifica tres potenciais quinases de eIF2α (TbeIF2Kl-K3). Neste trabalho, nos mostramos que TbeIF2K2 e uma glicoproteina associada a membrana, expressa tanto na forma prociclica quanta na sanguinea. 0 dominio catalitico de TbeIF2K2 fosforila eIF2α de levedura e de mamiferos na Ser51. A quinase tambem fosforila a incomum forma de eIF2α encontrada em tripanossomatideos, especificamente no residuo Thr169, que corresponde a Ser51 em outros eucariotos. 0 eIF2α de T brucei, no entanto, nao e um substrato para GCN2 ou PKR in vitro. 0 dominio regulatorio putativo de TbeIF2K2 nao apresenta nenhuma similaridade de sequencia com as quinases de eIF2α conhecidas. Tanto na forma sanguinea quanto na prociclica, TbeIF2K2 esta localizada principalmente na bolsa flagelar, organela que e o local exclusivo de exo e endocitose nesses parasitas. Ela tambem pode ser detectada em compartimentos endociticos, mas nao em lisossomos, sugerindo que a quinase e reciclada entre os endossomos e a bolsa flagelar. A localizacao de TbeIF2K2 sugere que ela possa funcionar como um sensor do transporte de nutrientes ou proteinas em T brucei, um organismo que depende de mecanismos regulatorios pos-transcricionais para controlar a expressao genica em diferentes situacoes. Essa e a primeira quinase de eIF2α associada a membrana descrita em eucariotos unicelulares / Translational control mediated by phosphorylation of the alpha subunit of the eukaryotic initiation factor 2 (eIF2α) is central to stress-induced programs of gene expression. Trypanosomatids, important human pathogens, display differentiation processes elicited by contact with the distinct physiological milieu found in their insect vectors and mammalian hosts, likely representing stress situations. Trypanosoma brucei, the agent of African trypanosomiasis, encodes three potential eIF2α kinases (TbeIF2K1-K3). We show here that TbeIF2K2 is a transmembrane glycoprotein expressed both in procyclic and bloodstream forms. The catalytic domain of TbeIF2K2 phosphorylates yeast and mammalian eIF2α at Ser51. It also phosphorylates the highly unusual form of eIF2α found in trypanosomatids specifically at residue Thr169, that corresponds to Ser51 in other eukaryotes. T. brucei eIF2α, however, is not a substrate for GCN2 or PKR in vitro. The putative regulatory domain of TbeIF2K2 does not share any sequence similarity with known eIF2α kinases. In both procyclic and bloodstream forms TbeIF2K2 is mainly localized in the membrane of the flagellar pocket, an organelle that is the exclusive site of exo- and endocytosis in these parasites. It can also be detected in endocytic compartments but not in lysosomes, suggesting it is recycled between endosomes and the flagellar pocket. TbeIF2K2 location suggests a relevance in sensing protein or nutrient transport in T. brucei, an organism that relies heavily on posttranscriptional regulatory mechanisms to control gene expression in different environmental conditions. This is the first membrane-associated eIF2α kinase described in unicellular eukaryotes. / FAPESP: 02/13783-9 / BV UNIFESP: Teses e dissertações