Membrane reconstitution approach for the investigation of lipid peroxidation mechanisms and its pathological effects

用于研究脂质过氧化机制及其病理效应的膜重建方法

• 批准号: 10707400
• 负责人: Jean Chung
• 金额: $ 37.24万
• 依托单位: COLORADO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-21 至 2027-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10707400
• 关键词: Aging; Alzheimer' s Disease; Apoptosis; Biological; Biological Assay; Cell Death; Cells; Cellular Membrane; Cessation of life; Chemicals; Disease; Enzymes; Fluorescence; Fluorescence Microscopy; Generations; Health; Heart Diseases; Impairment; Investigation; Iron; Kinetics; Lipid Bilayers; Lipid Binding; Lipid Peroxidation; Lipid Peroxides; Lipids; Lipoxygenase; Malignant Neoplasms; Measurement; Measures; Membrane; Membrane Lipids; Membrane Proteins; Nature; Organic solvent product; Outcome; Pathologic; Pathway interactions; Play; Process; Role; Signaling Protein; Stroke; Surface; Testing; Therapeutic; age related; cell type; chemical reaction; combat; membrane reconstitution; oxidative damage; peroxidation; senescence

Project Summary This project aims to determine the chemical mechanisms of lipid peroxidation in membranes. The accumulation of lipid peroxides on cellular membranes is a process that accompanies aging, as well as a host of age-related diseases, such as cancer, Alzheimer’s disease, stroke, and heart diseases. More recently, it was discovered that lipid peroxidation leads to a programmed cell death, called ferroptosis, which may provide the connection between lipid peroxidation and diseases. Despite their fundamental role in aging and health, the mechanistic nature of lipid peroxidation and its connection to the diseased state in cells remain elusive. This may be because lipid peroxidation studies are usually carried out in live cell context, where quantitative measurements are difficult, or with lipids in organic solvents, where it lacks biological relevance. There are two important questions with respect to lipid peroxidation and ferroptosis: one is which of the two fundamentally different pathways, enzymatic peroxidation or non-enzymatic autoxidation involving free iron, is responsible for the membrane lipid peroxidation in cells. The other is how lipid peroxidation leads to ferroptosis and other adverse cellular outcomes. To investigate these problems, we will develop a membrane-based assay for quantitative measurement of lipid peroxidation. By using surface-selective fluorescence microscopy to detect the generation of lipid peroxides, we will elucidate chemical mechanism of lipid peroxidation in the membrane context. We hypothesize that both the enzymatic and non-enzymatic pathways are important in efficient lipid peroxidation, as they play different roles in the process: the enzyme, lipoxygenase, initiates peroxidation with the selective binding for lipid substrates. Then, after a critical amount of lipids have been oxidized, membrane structural disintegration allows iron- catalyzed propagation of further peroxidation. We will test this hypothesis by directly measuring the rate of lipid peroxidation in membranes under enzyme-driven and autoxidative conditions. We further hypothesize that lipid peroxidation inflicts a chemical damage to membrane proteins, and the impairment of their function leads to cellular death. To test this hypothesis, we will evaluate the structural and functional impact of lipid peroxidation on Ras, an important signaling protein that is known to be sensitive to oxidative damage. The results of this study will provide a better understanding of lipid peroxidation, which underlies many age-related health concerns and therapeutic strategies to combat them.

项目摘要 本项目旨在确定膜中脂质过氧化的化学机制。积累 细胞膜上的过氧化脂质是一个伴随着衰老的过程，也是一系列与年龄相关的 疾病，如癌症、阿尔茨海默病、中风和心脏病。最近，人们发现 脂质过氧化导致细胞程序性死亡，称为铁下垂，这可能提供了联系 脂质过氧化与疾病之间的关系。尽管它们在衰老和健康方面起着基础性作用，但机械论 脂质过氧化的本质及其与细胞内疾病状态的联系仍然难以捉摸。这可能是因为 脂质过氧化研究通常在活细胞环境中进行，其中定量测量是 困难，或与有机溶剂中的脂类，在那里它缺乏生物相关性。有两个重要的问题 关于脂质过氧化和铁下垂：一是两条根本不同的途径中的哪一条， 涉及游离铁的酶促过氧化或非酶自氧化是造成膜脂的原因。 细胞内的过氧化作用。另一个是脂质过氧化如何导致铁下垂和其他不利的细胞后果。 为了研究这些问题，我们将开发一种基于膜的脂肪定量测定方法。 过氧化。通过使用表面选择性荧光显微镜来检测过氧化脂质的产生，我们 将从膜的角度阐明脂质过氧化的化学机制。我们假设这两个 酶途径和非酶途径在有效的脂质过氧化中是重要的，因为它们扮演着不同的角色。 在这个过程中：脂氧合酶通过选择性结合脂质底物来启动过氧化反应。 然后，在一定量的脂类被氧化后，膜结构的解体允许铁- 催化了进一步的过氧化反应。我们将通过直接测量血脂的比率来检验这一假设 在酶驱动和自氧化条件下膜的过氧化作用。我们进一步假设脂类 过氧化对膜蛋白造成化学损伤，其功能的损害导致 细胞死亡。为了验证这一假设，我们将评估脂质过氧化的结构和功能影响。 RAS上的一种重要的信号蛋白，已知对氧化损伤敏感。这项研究的结果 将提供对脂质过氧化的更好的理解，这是许多与年龄相关的健康问题和 与之抗争的治疗策略。