Lipid droplets and the regulation of ferroptosis

脂滴与铁死亡的调节

• 批准号: 10248497
• 负责人: Siti Nur Sarah Morris
• 金额: --
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-07-09 至 2021-09-25
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10248497
• 关键词: Acyl Coenzyme A; Address; Alcohols; Alternative Splicing; Biochemistry; Cell Death; Cell Line; Cell membrane; Cell physiology; Cells; Cellular Membrane; Cellular biology; Cessation of life; Chemicals; Chimeric Proteins; Coenzyme A Ligases; Cysteine; Defect; Degenerative Disorder; Disease; Disease model; Drug Sensitization; Drug resistance; Enzymes; Esters; Etiology; Extravasation; Fatty Acids; Generations; Genes; Glutamates; Glutathione; Goals; Health; Hydrogen; Iron; Isoenzymes; Isotopes; Knock-out; Lead; Length; Lipid Peroxidation; Lipid Peroxides; Lipids; Location; Malignant Neoplasms; Membrane; Methods; Neurodegenerative Disorders; Organelles; Oxygen; Pathway interactions; Peroxidases; Peroxides; Pharmacology; Phospholipids; Play; Polyunsaturated Fatty Acids; Protein Isoforms; RNA Splicing; Raman Spectrum Analysis; Reactive Oxygen Species; Regulation; Regulatory Pathway; Research; Resistance; Role; Series; Structure; Tail; Testing; Traction; Travel; age related; cancer cell; cell injury; fatty acid metabolism; in vitro Model; in vivo; lipid metabolism; lipidomics; long chain fatty acid; metabolomics; negative affect; nervous system disorder; new therapeutic target; novel anticancer drug; novel strategies; oxidative damage; peroxidation; phospholipid-hydroperoxide glutathione peroxidase; prevent; refractory cancer; response; success; targeted treatment

PROJECT SUMMARY Ferroptosis is a form of regulated cell death that generates reactive oxygen species (ROS) in an iron- dependent fashion. The most important of the ROS are lipid peroxides, which self-propogate along the plasma membrane and result in the accumulation of oxidatively damaged lipids. These lipid peroxides lead to a loss of membrane integrity, leakage of cellular and subcellular content and, ultimately, cell death. The main protective mechanism against ferroptosis relies on a gluathione-dependent peroxidase (GPX4); this enzyme converts lipid peroxides to lipid alcohols, thus blocking cell death. Defects in this protective pathway have been implicated in the etiology of a number of neurodegenerative disorders. Conversely, abrogation of GPX4 has sensitized drug-resistant cancer cells to chemotherapeutics. Unfortunately, our understanding of the mechanisms underlying ferroptosis remain limited, but one possibility of ferroptotic regulation may be found in the enzyme acyl-CoA synthetase long-chain 4 (ACSL4). This enzyme converts free long-chain fatty acids into fatty acyl-CoA esters, the building blocks of the phospholipids that are fated to propagate ferroptosis. Knockout of ACSL4 has been shown to stave off ferroptosis in multiple in vivo and in vitro models. Interestingly, the ACSL4 gene can produce two isoenzymes via alternative splicing; these isoforms differ in length and in localization, where the long form co-localizes to lipid droplets (LDs) and the short form co- localizes to the plasma membrane (PM). We hypothesize that this differential localization modulates ferroptotic sensitivity as the sequestration of lipid peroxides to the LDs may prevent death by ferroptosis. In order to address this question, cell lines will be created in which endogenous ACSL4 has been eliminated and replaced with a different expression construct for the two major splice forms of ACSL4. Moreover, these splice forms will be targeted to various subcellular localizations to investigate the role of these compartments in the execution of ferroptotic cell death. Additionally, since ACSL4 is integral to lipid metabolism, we will investigate the composition of fatty acids via Raman spectroscopy and characterize fatty acid flux through lipidomics. This project addresses an important step in the regulation of ferroptosis, namely how sequestration of damaged lipids away from critical membrane regions may negatively affect ferroptotic cell death. The enzyme that governs this sequestration, ACSL4, thus has great pharmacologic potential, not only as a drug target for novel anti-cancer drugs, but also in age-related neurological diseases.

项目摘要 铁凋亡是一种调节性细胞死亡的形式，其在铁-蛋白质结合中产生活性氧（ROS）。 依赖的方式。最重要的活性氧是脂质过氧化物，它沿着血浆沿着自我繁殖 膜，并导致氧化损伤脂质的积累。这些过氧化脂质会导致 膜完整性、细胞和亚细胞内容物的泄漏以及最终的细胞死亡。主要保护性 抗铁凋亡的机制依赖于谷胱甘肽依赖性过氧化物酶（GPX 4）;这种酶将 脂质过氧化物转化为脂质醇，从而阻止细胞死亡。这种保护途径的缺陷已经被 与许多神经退行性疾病的病因学有关。相反，GPX 4的废除 使耐药癌细胞对化疗药物敏感。不幸的是，我们对 铁凋亡的潜在机制仍然有限，但铁凋亡调节的一种可能性可能被发现， 酰基辅酶A合成酶长链4（ACSL 4）。这种酶将游离的长链脂肪酸转化为 脂肪酰辅酶A酯，磷脂的结构单元，注定要传播铁凋亡。 ACSL 4的敲除已显示在多种体内和体外模型中避免铁凋亡。 有趣的是，ACSL 4基因可以通过选择性剪接产生两种同工酶;这些同工酶在以下方面不同： 长度和定位，其中长形式共定位于脂滴（LD），短形式共定位于脂滴（LD）。 定位于质膜（PM）。我们假设，这种差异定位调节铁电位 由于脂质过氧化物对LD的螯合作用可以防止铁凋亡引起的死亡，因此可以提高敏感性。为了 为了解决这一问题，将建立内源性ACSL 4已被消除和取代的细胞系 ACSL 4的两种主要剪接形式具有不同的表达构建体。此外，这些剪接形式将 以各种亚细胞定位为目标，以研究这些隔室在执行 铁凋亡性细胞死亡此外，由于ACSL 4是脂质代谢不可或缺的，我们将研究ACSL 4在脂质代谢中的作用。 通过拉曼光谱分析脂肪酸组成，并通过脂质组学表征脂肪酸通量。这 该项目解决了铁凋亡调节的重要一步，即如何螯合受损的铁， 远离关键膜区域的脂质可负面地影响铁凋亡细胞死亡。的酶 ACSL 4控制这种隔离，因此具有巨大的药理学潜力，不仅作为新的药物靶点， 抗癌药物，而且在与年龄有关的神经系统疾病。