Internalização e tráfego da Stress Inducible Protein-1 (STI-1), um ligante da proteína prion celular

AUTOR(ES)

Fabiana Andrade Caetano

DATA DE PUBLICAÇÃO

2006

RESUMO

The cellular prion protein (PrPc) is a glycosylphosphatidylinositol (GPI)-anchored plasma membrane glycoprotein. The PrPc abnormal isoform, PrPsc, is an infectious form involved in the prion disease pathogenesis. Identification of ligands that interact with PrPc can help in the understanding the physiological function of this protein. The STI-1 (stress inducible protein 1) is a specific PrPc ligand that promotes neuroprotection and neuritogenesis by distinct signaling pathways. STI-1 is a co-chaperone that can act as a soluble neurotrophic factor. In order to understand possible physiological functions of STI-1 and PrPc we investigated the cellular interactions and intracellular trafficking of STI-1. In this work, we characterized the interaction of recombinant STI-1 with SN56 cells and showed that this interaction is mediated by a specific and saturable binding site in SN56 cells. We observed that the interaction between STI-1 and SN56 cells promotes STI-1 internalization. Double labeling experiments using fluorescent STI-1 and clathrin-GFP indicated that the major endocytic pathway to the STI-1 internalization is clathrin independent. Next, our aim was to evaluate wether the STI-1 was targeted to early endosomes after endocytosis. To test this hypothesis, we used two markers of early endosomes: Transferrin and Rab5-GFP. The experiment of co-localization between STI-1 and Alexa Fluor 488-Transferrin or Rab5-GFP suggested that STI-1 is not targeted to early endosomes after endocytosis. As STI-1 was not found in early endosomes, we decided to evaluate if STI-1 is found in acidic vesicles. To this purpose we used Lysosensor Green labelling. The result indicated strong co-localization between STI-1 and Lysosensor, suggesting that STI-1 is target to acidic vesicles. To identify these vesicles, we used the constitutively active mutant Rab7Q67L, a late endosome/lysosome marker. The co-localization studies showed that the most STI-1 positive vesicles were positives to Rab7Q67L-positives suggesting that STI-1 is targeted to late endosomes/lysosomes. Taken together, our results indicate that STI-1 binds to cells in a saturable and specific way, it is internalized by a clathrin independent pathway and is targeted to late endosomes/lysosomes bypassing therefore early endosomes

ASSUNTO(S)

farmacologia teses. proteína de ligação teses.

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