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

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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.