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

项目概要 该项目旨在确定膜中脂质过氧化的化学机制。积累 细胞膜上脂质过氧化物的生成是伴随衰老以及许多与年龄相关的过程。 疾病，例如癌症、阿尔茨海默病、中风和心脏病。最近，人们发现 脂质过氧化导致程序性细胞死亡，称为铁死亡，这可能提供这种联系 脂质过氧化与疾病之间的关系。尽管它们在衰老和健康中发挥着重要作用，但其机制 脂质过氧化的性质及其与细胞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，或有机溶剂中的脂质，缺乏生物相关性。有两个重要问题 关于脂质过氧化和铁死亡：其一是两种根本不同的途径中的哪一种， 涉及游离铁的酶促过氧化或非酶促自氧化，负责膜脂 细胞内的过氧化。另一个是脂质过氧化如何导致铁死亡和其他不良细胞结果。 为了研究这些问题，我们将开发一种基于膜的脂质定量测量方法 过氧化。通过使用表面选择性荧光显微镜来检测脂质过氧化物的产生，我们 将阐明膜背景下脂质过氧化的化学机制。我们假设两者 酶促和非酶促途径对于有效的脂质过氧化很重要，因为它们发挥着不同的作用 在此过程中：脂氧合酶通过选择性结合脂质底物引发过氧化反应。 然后，在临界量的脂质被氧化后，膜结构崩解使得铁- 催化进一步过氧化反应的传播。我们将通过直接测量血脂率来检验这一假设 在酶驱动和自氧化条件下膜中的过氧化。我们进一步假设脂质 过氧化会对膜蛋白造成化学损伤，其功能受损会导致 细胞死亡。为了检验这一假设，我们将评估脂质过氧化的结构和功能影响 Ras 是一种重要的信号蛋白，已知对氧化损伤敏感。本研究结果 将提供对脂质过氧化的更好理解，脂质过氧化是许多与年龄相关的健康问题的基础 对抗它们的治疗策略。

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.