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EMC translocase is Required for membrane targeting of RAS

Grant number: 22/09519-8
Support type:Scholarships abroad - Research Internship - Doctorate
Effective date (Start): October 01, 2022
Effective date (End): September 30, 2023
Field of knowledge:Biological Sciences - Genetics - Human and Medical Genetics
Principal researcher:Enilza Maria Espreafico
Grantee:Airton de Carvalho Junior
Supervisor abroad: Mark Reid Philips
Home Institution: Faculdade de Medicina de Ribeirão Preto (FMRP). Universidade de São Paulo (USP). Ribeirão Preto , SP, Brazil
Research place: NYU Langone Medical Center, United States  
Associated to the scholarship:19/01285-5 - Post-translational regulation of RAS by the EMC complex: a potential target for anti-oncogenic therapy, BP.DR

Abstract

The RAS proteins are small GTPases that regulate a number of signaling pathways including the mitogen-activated protein kinase (MAPK) pathway. To perform this function, RAS proteins must associate with the plasma membrane (PM). All RAS isoforms (HRAS, NRAS and, KRAS) are post-translational modified at a C-terminal CaaX motif to gain affinity for cellular membranes. Prenylation (lipidation) of RAS occurs in the cytosol and directs RAS proteins to the cytosolic face of the endoplasmic reticulum (ER) for further processing. The endoplasmic reticulum membrane protein complex (EMC) plays roles in a wide range of cellular functions, including folding of membrane proteins and lipid biosynthesis. We discovered that knockdown of either EMC1 or EMC3 results in the suppression of the rough eye phenotype of Drosophila melanogaster, well established to be a consequence of expression of constitutively active RasV12. Consistent with this finding, silencing EMC1 or EMC3 in HEK293T cells inhibited MAPK signaling. Strikingly, knockdown of these EMC subunits in the HEK293T cells impaired RAS farnesylation and blocked delivery to the PM, suggesting that its effects on signaling relate to membrane targeting. We conclude that EMC is required for HRAS farnesylation, trafficking and activity, although the molecular mechanisms remain unknown. In this BEPE project, we will focus on elucidating the mechanisms underlying the requirement of EMC protein complex for membrane targeting of RAS and oncogenic transformation, by pursuing the following specific aims: 1) define the spectrum of CaaX proteins that require EMC for proper trafficking and if all 10 subunits of the EMC translocase and their clients/interactors are involved; 2) determine which enzymes involved in the post-translational modification of RAS require EMC for stability and proper localization; (3) determine the effect of loss of EMC function on RAS driven transformation in vitro and in vivo. We will use advanced molecular and cellular techniques, such as biosensors of RAS localization and activity, high resolution confocal microscopy, metabolic labeling, and in vivo genetic editing to modulate KRAS-induced tumorigenesis in the airway epithelium of LSL-KRAS12D; p53”/” transgenically engineered oncomouse, available at NYU, in the laboratory of Mark Philips. This study has the potential to lead to new drug development for cancer treatment. (AU)

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