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

项目摘要/摘要 这项提案的目标是了解跨膜蛋白164(TMEM164)是如何衰减的 多不饱和脂肪酸(PUFA)代谢与铁链信号转导。铁性下垂是一种新的铁依赖性疾病 一种与多种人类疾病有关的细胞死亡形式，包括癌症和2型糖尿病 (T2 DM)。在T2 DM中，糖尿病状态的严重程度增加了与铁超载和 脂质过氧化是铁性上睑下垂的两个主要特征。调节多不饱和脂肪酸含量的酶的特性 人类细胞不仅可以加深我们对铁性下垂的了解，还可以确定治疗的靶点。 治疗像T2 DM这样的疾病，这种形式的细胞死亡是不受调控的。最近的基因组编辑屏幕已经 TMEM164是一种功能未知的多通道跨膜蛋白，是一种关键的调节蛋白。 铁性下垂。我们根据其Alpha折叠式预测结构推测，TMEM164是一种新型的 调节细胞多不饱和脂肪酸含量的半胱氨酸依赖的跨膜酶。在这里，我们试图测试这一点 假设和确定TMEM164在PUFA脂代谢中执行的特定酶功能，AS 以及它们在赋予人类细胞对铁性下垂敏感性方面的贡献。在目标1中，我将测定脂质 TMEM164基因缺失细胞的图谱，并检测野生型，但不是假定的催化半胱氨酸 突变形式的TMEM164可以挽救这些血脂紊乱。从这些数据中，我将确定候选人 TMEM164的生理底物，将在重组蛋白系统中进行测试。在《目标2》中，我会 利用我们实验室在共价抑制剂发现方面的长期专业知识来识别亲电化合物 通过修饰蛋白的催化半胱氨酸来阻断TMEM164的活性。成功完成 这个项目将扩大我们对铁下垂所涉及的脂代谢途径的基本了解。 并确定新的候选药物靶点，以抑制铁下垂对人类退行性病变的贡献 像T2 DM这样的疾病。

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.