izvor podataka: poirot

Rab10 u biogenezi endosomalnog reciklirajućeg odjeljka

Rab10 in the biogenesis of endosomal recycling compartment


Endoplazmatski reciklirajući odjeljak (ERC) je složena stanična organela u koju se slijeva membranski promet iz ranog endosoma (EE) i predstavlja čvorište endosomalnog sortiranja nužno za održavanje stanične homeostaze. Usprkos višegodišnjim istraživanjima, biogeneza i organizacija ERCa tek se nazire, između ostalog zbog nedostatka prikladnog modela za istraživanje u fiziološkim uvjetima. U ovom projektu će se ispitivati uloga Rab10 proteina u biogenezi ERC na modelu stanica inficiranih mišjim citomegalovirusom (MCMV) jer rano tijekom infekcije dolazi do ekspanzije Rab10-pozitivnog odjeljka. Dosadašnje spoznaje o funkciji Rab10 uglavnom se temelje na istraživanjima polariziranih stanica, ne obuhvaćaju biogenezu ERC, i pribavljene su istraživanjima u nefiziološkim uvjetima (npr. nakon transfekcije) . Virusna infekcija ekspandira Rab10-pozitivne odjeljke, što ukazuje da je Rab10-međuodjeljak dio biogeneze ERC u nepolariziranim stanicama i omogućuje istraživanje njegove uloge pri fiziološkim koncentracijama regulatornih molekula. Dosadašnje spoznaje upućuju na tri moguća puta aktivacije Rab10 u biogenezi ERC: putem kaskadne aktivacije Rab10 nakon Rab5, Rab35 i Rab11. Naša istraživanja fokusirati će se na Rab5 put aktivacije u ranoj fazi infekcije MCMVom na ekspandiranom Rab10-odjeljku. Cilj nam je mapirati regulacijsku mrežu oko Rab10 utvrđivanjem izražaja njegovih regulatora (GEF i GAP) i efektora te potvrditi njihov interakciju u pull-down eksperimentima. Analizom izražaja i blokiranjem fosfoinozitida (PI(3)P i PI(4,5)P2) te utišavanjem ključnih regulatora utvrdit ćemo da li Rab10 djeluje nizvodno od Rab5 i testirati hipotezu da je Rab10 ključan u ranoj fazi biogeneze ERC. Konačnu sliku interaktoma Rab10 rekonstruirat ćemo koristeći bioinformatičkim metodama, a dobivene rezultate provjeriti na neinficiranim stanicama. U konačnici ćemo dobivenu mrežu Rab10 regulatora i efektora iskoristiti za ispitivanje njihovog izražaja na stanicama karcinoma endometrija. Time bismo doprinijeli razumijevanju fiziologije recikliranja, a moguće i u dijagnostici karcinoma.

Endoplasmic recycling compartment (ERC) is a complex cellular organelle where membrane traffic is targeted from the early endosome (EE). It also represents an endocytic sorting hub and is necessary for the maintenance of the cell homeostasis. Although it has been intensively investigated, biogenesis and organization of ERC are still poorly clarified. The lack of the suitable model for testing in the physiological conditions is undoubtedly one of the reasons. This project will investigate the role of Rab10 protein during the process of ERC biogenesis, and the model of murine cytomegalovirus (MCMV) infected cells will be applied. Namely, Rab10-positive compartment expands during the early phase of the infection. Current knowledge about the Rab10 function is mostly achieved in polarized cells, do not consider ERC biogenesis, and were performed in nonphysiological conditions (e.g., after transfection). The MCMV induced expanding of Rab10-positive compartments indicates that Rab10-subcompartment could play a significant role during the ERC biogenesis. Therefore, the investigation of Rab10 is now possible to perform under the physiological concentrations of regulatory molecules. Our present knowledge indicates three possible routes of Rab10 cascade activation: following Rab5, Rab35, and Rab11. Our investigations will be focused on Rab5 activation pathway on expanded Rab10 compartment during the early phase of infection. The goal is a mapping of the Rab10 regulatory network by establishing the expression of their regulators (GAPs and GEFs) and effectors. The possible interactions should be confirmed by pull-down experiments. Furthermore, analysis of phosphoinositides (PI(3)P, and PI(4,5)P2) expression and silencing of key regulators will help us to determine if Rab10 acts downstream of Rab5 – then Rab10 would be essential during the ERC biogenesis. The final picture will be reconstructed by using bioinformatics methods, and the results will be verified on noninfected cells. The reconstructed network of Rab10 regulators and effectors will be tested in endometrial carcinoma. Therefore, we would contribute to the understanding the physiology of recycling, and possibly to the carcinoma diagnostics.


nije evidentirano

Znanstveno-istraživački projekti

nije evidentirano

uniri-biomed-18-180

07.03.2019

31.12.2021

nije evidentirano

HRK 79.292,53