ITP-GAMMAS · Comparative imaging study of the trafficking and processing of gamma-secretase substrates
6РП — Действия „Мария Кюри“
- Период
- 2007-03-01 → 2009-02-28
- Финансиране от ЕС
- 151 618 €
- Участници
- 1
- Схема
- EIF
Линиите свързват координатора с партньорите.
Накратко на български
Разпределението на ензимите гама-секретази и техните мишени, като протеина APP, се проследява чрез специално изображения в клетката. Разбирането на тези разлики помага при търсенето на начини за избирателно спиране на конкретни процеси в организма.
Кратко обяснение, генерирано от езиков модел по текста на CORDIS. Оригиналът е по-долу.
Резултати накратко
Final Activity Report Summary - ITP-GAMMAS (Comparative imaging study of the trafficking and processing of gamma-secretase substrates)
Regulated intramembrane proteolysis (RIP) is an unusual proteolytic event that can abrogate or initiate downstream signalling by cleaving substrate proteins within their transmembrane domains. One such mechanism is governed by gamma-secretase that catalyses intramembrane cleavage of over 30 type I transmembrane proteins, including the amyloid precursor protein (APP), Notch, E-Cadherin, p75NTR and syndecans. Its activity is confined to a complex consisting of presenilin (PS), nicastrin (NCT), Aph-1 and Pen-2. The existence of two PS homologues (PS1 and PS2) and several Aph-1 isoforms suggest the presence of distinct complexes and this heterogeneity opens new avenues for selective inhibition. The spatial and temporal localisation of substrates versus gamma-secretase is of major importance for the regulation of intramembrane proteolysis. Therefore, we proposed an imaging approach to compare the trafficking route of diverse substrates versus the gamma-secrestase complexes. Confocal analysis of PS1 or PS2 knockout MEFs stably rescued with their respective fluorescently tagged human orthologues (EGFP-PS1 and EGFP-PS2) demonstrated significant differences on the relative distribution of PS1 vs PS2. PS1 is broadly distributed along the secretory and endocytic pathway whereas PS2 is essentially restricted to late endosomal/lysosomal compartments. Only limited levels of PS2, relative to PS1 and NCT, are present at the plasma membrane based on cell surface biotinylation and analysis of isolated plasma membrane sheets. Furthermore, PS2 when present at the plasma membrane does not colocalise with PS1 supporting the idea that they indeed associate with different microdomains. Together, our findings strengthen the hypothesis that the distinct subcellular distribution of gamma-secretase complexes can provide a basis for substrate and/or cleavage specificity. Secondly, we identified independent internalization routes for APP versus its processing enzymes BACE1 and gamma-secretase. As they likely meet downstream in early to late endosomes, we provide a mechanism to interfere with APP processing through modulating the specific entry routes of the secretases. Both research lines are currently further explored using superresolution nanoscopy with the purpose of obtaining stoichiometric information on the composition and nanodomain association of gamma-secretase complexes.
Текст от CORDIS, на английски · Данни: CORDIS, © Европейски съюз
Цел на проекта
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.
Оригинален текст от CORDIS (на английски).
Участници
- VIB VZW · ZWIJNAARDEКоординаторБелгия
Връзки
Данни: CORDIS, © Европейски съюз