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Content archived on 2024-05-29

Comparative imaging study of the trafficking and processing of gamma-secretase substrates

Objective

Regulated intramembrane proteolysis (RIP) is of emerging importance in a broad range of physiological processes by abrogating or initiating downstream signalling events.

beta-Secretase is by far the most diverse RIP mechanism if not only for the many substrates it targets. It consists of a catalytic component, presenilin that requires nicastrin, pen-2 and aph-1 for activity. The targets of beta-secretase are type I transmembrane proteins with the prerequisite however of ectodomain shedding.

Since most of these substrates somehow function as cell surface receptors, we hypothesize that regulation of their dual processing essentially occurs at the surface or en route to the endosomal system. In most cases, little is known about the co-distribution of different substrates or their spatial organization between the different microdomains including their individual routes of entry in the cell.

Moreover, as evidenced from the analysis of PS deficient cells, we postulate that a crosstalk exists between presenilin and/or presenilin-associated beta-secretase activity and sorting/internalization of substrates.

To address these questions, we aim for a comparative imaging analysis of three known substrates, i.e. the amyloid precursor protein (APP), Notch and the more recent p75 neurotrophin receptor, p75NTR. For these substrates, their incorporation or exclusion from lipid rafts as well as their routes of entry will be under focus.

To study events at the cell surface, we will establish a cell-free assay of isolated plasma membranes heets derived from wild-type, beta-secretase deficient fibroblasts or neuronal cell lines. Live and confocal imaging of single and double fluorescently tagged substrates will allow us to explore the routes and delivery to the endosomal system.

Finally, it is of increasing importance to establish the trafficking route of these substrates as well as the exact site of their proteolysis to understand their individual vital biological functions in health and disease.

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Call for proposal

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FP6-2005-MOBILITY-5
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Funding Scheme

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EIF - Marie Curie actions-Intra-European Fellowships

Coordinator

VIB VZW
EU contribution
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Total cost

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