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

项目摘要/摘要 空间和时间维度的异质性是哺乳动物细胞膜的一个标志。此分区 通过特定的脂-脂或脂-蛋白相互作用将细胞膜分解成不同的纳米结构域。 这些纳米结构域为膜局部调控提供了动态的、时空组织的平台 关键的信号通路，赋予信号蛋白独特的组织和生物活性(及其 效应器/衬底)与其相关联或嵌入其中。这种对膜蛋白的调节是通过它们的 内源性微环境是重要生物功能的基础，在疾病中经常受损 如癌症、神经退行性变和免疫紊乱。 尽管天然的膜环境在调节膜蛋白功能方面具有重要意义，但还有 缺乏在天然脂环境中研究膜蛋白的实验方法 同时的空间、时间和分子分辨率。在这种内在挑战的驱使下，我的目标是 开发一个了解膜的分层和功能组织的实验平台 具有精确空间和分子分辨率的内源细胞膜环境中的蛋白质。我会用 一种两亲性的苯乙烯-马来酸共聚物(SMA及其化学类似物)，用于切除皮肤的圆形斑块 细胞膜，产生约10-15纳米大小的“天然纳米盘”。SMA包被的膜蛋白 兴趣被一个由内源性脂质和相互作用的蛋白质组成的环形环所包围，保持它们的局部 膜微环境，并提供前所未有的空间分辨率。在进一步丰富和 纯化，我将使用单分子TIRF显微镜，天然质谱学，脂质组学和蛋白质组学， 功能分析和结构研究，以研究这些SMA-的生物物理和生化特性- 在其内源环境中被包裹的膜蛋白。 我将用这种方法研究两种临床上相关的膜/膜相关蛋白--(1)KRAS， 一种小的GTP酶，在胰腺癌中是一种突出的癌基因，突变频率为95%，以及(2)Trk 家族受体酪氨酸激酶与神经营养因子结合，对神经元的发育、分化、 和生存有关，并与疼痛感知有关。我将解决有关结构和 这些蛋白质在天然膜上的功能组织，空间丰富的蛋白质组和 这些蛋白质周围的脂体调节它们的组织，这如何影响这些蛋白质的生物活性 膜蛋白，以及最终如何影响下游信号转导。蛋白质的直接参与- 几种疾病中的脂质相互作用和动力学需要对膜蛋白进行定量的了解 在它们的原生膜环境中。我的提案勾勒出了一条具有广泛和广泛的 在其背景下对理解膜蛋白的调节和激活的变革性影响 具有精确空间和分子分辨率的内生环境。

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.