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

项目总结/摘要 空间和时间维度的异质性是哺乳动物细胞膜的标志。这个分区 细胞膜被分成不同的纳米结构域，通过特定的脂质-脂质或脂质-蛋白质相互作用来区分。 这些纳米结构域为膜定位调节提供了动态的时空组织平台 关键信号通路，赋予独特的组织和生物活性的信号蛋白（及其 效应物/底物）与它们相关联或嵌入其中。这种膜蛋白的调节， 内源性微环境是关键生物功能的基础，并且经常在疾病中受损 例如癌症、神经变性和免疫紊乱。 尽管天然膜环境在调节膜蛋白功能中具有重要意义， 缺乏研究膜蛋白在其天然脂质环境中的实验方法， 同时的空间、时间和分子分辨率。在这个固有挑战的激励下，我的目标是 开发一个实验平台，以了解膜的层次和功能组织 蛋白质在内源性细胞膜环境中具有精确的空间和分子分辨率。我会用 两亲性苯乙烯-马来酸共聚物（SMA及其化学类似物），以切除 细胞膜，产生约10-15 nm大小的“天然纳米盘”。SMA-囊膜蛋白 感兴趣的是被内源性脂质和相互作用蛋白质的环状环包围， 膜微环境，并提供前所未有的空间分辨率。经过进一步的浓缩和 纯化，我将使用单分子TIRF显微镜，天然质谱，脂质组学和蛋白质组学， 功能测定和结构研究，以研究这些SMA的生物物理和生物化学性质， 在其内源性环境中包封膜蛋白。 我将使用这种方法研究两种临床相关的膜/膜相关蛋白-（1）KRas， 一种小的GTk，它是胰腺癌中突变频率>95%的主要癌基因，和（2）Trk 家族受体酪氨酸激酶，其结合神经营养因子并且是神经元发育，分化， 和存活率之间的关系，并与疼痛感知有关。我将讨论有关结构和 这些蛋白质在天然膜上的功能组织，空间富集的蛋白质组和 这些蛋白质周围的脂质体调节它们的组织，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。蛋白质的直接参与- 在一些疾病中，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。我的建议概述了一个一般性的实验管道， 变革性的影响，了解调控和激活膜蛋白的背景下， 内源环境具有精确的空间和分子分辨率。

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.