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Endoplasmic reticulum-plasma membrane contacts as hubs for Nox NADPH oxidase dependent cellular redox signaling (FAPESP-kcl)

Grant number: 16/50290-3
Support type:Regular Research Grants
Duration: June 01, 2019 - May 31, 2021
Field of knowledge:Health Sciences - Medicine - Medical Clinics
Cooperation agreement: King's College London
Principal Investigator:Francisco Rafael Martins Laurindo
Grantee:Francisco Rafael Martins Laurindo
Principal investigator abroad: Ajay M. Shah
Institution abroad: King's College London, England
Home Institution: Instituto do Coração Professor Euryclides de Jesus Zerbini (INCOR). Hospital das Clínicas da Faculdade de Medicina da USP (HCFMUSP). Secretaria da Saúde (São Paulo - Estado). São Paulo , SP, Brazil

Abstract

Redox processes strongly regulate (patho) physiological cell signaling, yet several fundamental underlying mechanisms remain unclear, including in particular the associated subcellular compartmentalization. The Shah group at KCI investigates endoplasmic reticulum (ER) signaling mechanisms and associated cardiovascular implications of Nox NAOPH oxidases, major cellular ROS enzyme sources. The Laurindo group at Incor (USP) investigates the convergence between vascular cell Noxes and the ER redox chaperone POI (protein disulfide isomerase). This proposal addresses a concerted effort of both groups to unravel mechanisms and functional implications of the interplay between Noxes and ER-plasma membrane (PM) contact sites, which emerged as subcellular organelle-like structures recently identified to display specific molecular signatures and functions. Several proteins belonging to ER-PM contacts undergo potential redox regulation. However, the involvement of Nox (es) in the formation of ER-PM contacts, the potential effect of their stabilization in sustaining Nox activity and the possible role of ER-PM contacts as platforms for POI-dependent Nox regulation are unknown. Moreover, our results suggest a convergence between POI and Rho family GTPases, which regulate the cytoskeleton and potentially ER structure dynamics. The central aim of our project focuses on the hypothesis of a mutual convergence - physical as well as functional - between Nox (es) and the formation/stabilization of dynamic ER-PM contacts. The specific aims are: 1) To map the extent of ER-PM contacts in wild-type vascular smooth muscle cells (VSMC) and in VSMC overexpressing E-Syt3, a protein that stabilizes ER-PM contacts; 2)To perform in those VSMC a combined approach to calcium/ROS imaging; 3) To explore in those VSMC effects of ER stress, calcium depletion, growth factor exposure and serum starvation on the extent of ER-PM contacts and calcium/ROS imaging; 4) To investigate through imaging tools and loss-of-function approaches the roles of Nox4, Nox1 and POIA 1 on the extent of ER-PM contacts and calcium/ROS imaging. This work may reveal novel important cellular subcompartments involved in redox modulation of cardiovascular physiology and pathology. (AU)