Identification of the ubiquitination sites in mitochondrial protein TOMM20 modified by the E3 ubiquitin-ligase SCF(Fbxo7)

• Grant number: 17/22153-4
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: February 01, 2018
• End date: July 31, 2019
• Field of knowledge: Biological Sciences - Biochemistry
• Principal Investigator: Felipe Roberti Teixeira
• Grantee: Natália Borges Simaroli
• Host Institution: Centro de Ciências Biológicas e da Saúde (CCBS). Universidade Federal de São Carlos (UFSCAR). São Carlos , SP, Brazil

Abstract

The Translocase of Outer Mitochondrial Membrane 20 (TOMM20) recognizes location signals of proteins that will be imported to the mitochondrial matrix, contributing to mitochondrial homeostasis. TOMM20 has also a major role in mitophagy pathway triggered by mitochondrial depolarization, blocking PINK1 importation and promoting its stabilization in mitochondrial outer membrane. Recently, our group in collaboration with Dr. Heike Laman (University of Cambridge/UK) has demonstrated that TOMM20 is a substrate for the E3-ubiquitin ligase complex SCF (Fbxo7) being either monoubiquitinated or polyubiquitinatedin vitro and in vivo via lysine 63 (K63), which is a signal of function regulation instead of degradation via proteasome. In fact, we have verified that the reduction or increasing of the Fbxo7 levels led to the reduction or increasing of the TOMM20protein content, respectively. These results indicate a functional dependence relationship that is not associated with the change of TOMM20 stability. In an ubiquitinome study of HEK293T cells, it was described that TOMM20 has ubiquitin chains in three internal lysine residues (K56, K61 and K68), however, it was not described which E3 ligase was responsible for these modifications. Therefore, this proposal aims to identify which lysine residue of TOMM20 is modified by SCF(Fbxo7). We will construct the triple mutant TOMM20-K56R,K61R, K68R -HA and develop ubiquitination in vivo assays with this mutant in comparison with the wild type TOMM20-HA in HEK293T cells. This study will provide information to the investigation of the function of this post translational modification in mitochondrial protein importation and mitophagy, which are aims of the ongoing Regular Project "Functional characterization of the E3 ubiquitin-ligase SCF(Fbxo7/PARK15) in the oncogenic Wnt pathway and mitochondrial homeostasis"(Processo FAPESP: 2016/00792-2). (AU)

Scientific publications

(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)

SPAGNOL, VALENTINE; OLIVEIRA, CAIO A. B.; RANDLE, SUZANNE J.; PASSOS, PATRICIA M. S.; CORREIA, CAMILA R. S. T. B.; SIMAROLI, NATALIA B.; OLIVEIRA, JOICE S.; MEVISSEN, TYCHO E. T.; MEDEIROS, ANA CARLA; GOMES, MARCELO D.; et al. The E3 ubiquitin ligase SCF(Fbxo7) mediates proteasomal degradation of UXT isoform 2 (UXT-V2) to inhibit the NF-kappa B signaling pathway. BIOCHIMICA ET BIOPHYSICA ACTA-GENERAL SUBJECTS, v. 1865, n. 1, JAN 2021. (16/25798-3, 16/21310-6, 16/00792-2, 17/22153-4, 17/07879-9, 18/09204-1, 19/03943-0, 18/01308-2)