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Molecular mechanism of regulation and activation of membrane proteins in native membrane milieu

天然膜环境中膜蛋白调节和激活的分子机制

基本信息

批准号：
10501008
负责人：
Moitrayee Bhattacharyya
金额：
$ 26.03万
依托单位：
YALE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-09-01 至 2027-06-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10501008
关键词：
Address Binding Biochemical Biological Biological Assay Biological Process Biophysics Cell membrane Cellular Membrane Chemicals Dimensions Disease Encapsulated Environment Family Frequencies Goals Guanosine Triphosphate Phosphohydrolases Heterogeneity Immune System Diseases Impairment Lipids Malignant Neoplasms Malignant neoplasm of pancreas Mammalian Cell Mass Spectrum Analysis Membrane Membrane Proteins Molecular Monomeric GTP-Binding Proteins Mutation Nerve Degeneration Oncogenes Play Property Proteins Proteome Proteomics Receptor Protein-Tyrosine Kinases Regulation Resolution Role Signal Pathway Signal Transduction Signaling Protein Styrenes Total Internal Reflection Fluorescent analog clinically relevant copolymer interest lipidome lipidomics maleic acid membrane activity nanodisk neuron development neuronal survival neurotrophic factor pain perception preservation protein function single molecule spatiotemporal

项目摘要

Project Summary/Abstract Heterogeneity in spatial and temporal dimensions is a hallmark of mammalian cell membranes. This partitions cellular membranes into distinct nanodomains distinguished by specific lipid-lipid or lipid-protein interactions. These nanodomains provide a dynamic, spatiotemporally organized platform for membrane-localized regulation of critical signaling pathways, imparting unique organization and bioactivity to the signaling proteins (and their effectors/substrates) that are associated with or embedded in them. This regulation of membrane proteins by their endogenous microenvironment is fundamental to critical biological functions and is often impaired in diseases such as cancer, neurodegeneration, and immune disorders. Despite the significance of the native membrane milieu in modulating membrane protein functions, there is a scarcity of experimental approaches for studying membrane proteins in their native lipid environment with simultaneous spatial, temporal, and molecular resolution. Motivated by this inherent challenge, my goal is to develop an experimental platform to understand the hierarchical and functional organization of membrane proteins in an endogenous cell membrane environment with precise spatial and molecular resolution. I will use an amphipathic styrene-maleic acid copolymer (SMA and its chemical analogs) to excise circular patches of the cell membrane, generating ~10-15 nm-sized “native nanodiscs”. SMA-encapsulated membrane proteins of interest are surrounded by an annular ring of endogenous lipids and interacting proteins, preserving their local membrane microenvironments and offering unprecedented spatial resolution. Following further enrichment and purification, I will use single-molecule TIRF microscopy, native mass spectrometry, lipidomics and proteomics, functional assays, and structural studies to investigate the biophysical and biochemical properties of these SMA- encapsulated membrane proteins within their endogenous environment. I will study two clinically relevant membrane/membrane-associated proteins using this approach – (1) KRas, a small GTPase that is a prominent oncogene with >95% mutation frequency in pancreatic cancers, and (2) Trk family receptor tyrosine kinases that bind neurotrophins and are central to neuronal development, differentiation, and survival and are implicated in pain perception. I will address outstanding questions about the structural and functional organization of these proteins on the native membrane, how the spatially enriched proteome and lipidome around these proteins regulate their organization, how that influences the biological activity of these membrane proteins, and ultimately how that impacts downstream signaling. The direct involvement of protein- lipid interaction and dynamics in several diseases necessitates a quantitative understanding of membrane proteins in their native membrane milieu. My proposal outlines a general experimental pipeline with a broad and transformative impact on understanding the regulation and activation of membrane proteins in the context of their endogenous environment with precise spatial and molecular resolution.

项目总结/文摘