Characterization of TMEM164 as novel multi-pass transmembrane enzyme and its role in ferroptosis

点击翻译按钮获取中文摘要

Project Summary/Abstract The goal of this proposal is to understand how transmembrane protein 164 (TMEM164) attenuates polyunsaturated fatty acid (PUFA) metabolism and ferroptotic signaling. Ferroptosis is a novel iron-dependent form of cell death implicated in a broad range of human diseases, including cancer and type 2 diabetes mellitus (T2DM). In T2DM, the severity of diabetic state increases risk for renal tubular injury related to iron overload and lipid peroxidation, two key features of ferroptosis. Characterizing enzymes that regulate the PUFA content of human cells would not only deepen our understanding of ferroptosis, but also identify therapeutic targets for treating diseases like T2DM where this form of cell death is dysregulated. Recent genome editing screens have identified TMEM164, a multi-pass transmembrane protein of uncharacterized function, as a key regulator of ferroptosis. We hypothesize, based on its Alphafold-predicted structure, that TMEM164 is a novel type of cysteine-dependent transmembrane enzyme that regulates cellular PUFA content. Here we seek to test this hypothesis and identify the specific enzymatic functions performed by TMEM164 in PUFA lipid metabolism, as well as their contribution to conferring ferroptosis sensitivity to human cells. In Aim 1, I will determine the lipid profiles of cells genetically deleted for TMEM164 and test whether wild type, but not a putative catalytic cysteine mutant form of TMEM164 can rescue these lipid perturbations. From these data, I will define candidate physiological substrates for TMEM164, which will be tested in recombinant protein systems. In Aim 2, I will leverage our lab’s longstanding expertise in covalent inhibitor discovery to identify electrophilic compounds that block TMEM164 activity through modifying the putative catalytic cysteine of the protein. Successful completion of this project will broaden our fundamental understanding of the lipid metabolic pathways involved in ferroptosis and identify new candidate drug targets for suppressing the contribution of ferroptosis to human degenerative diseases like T2DM.