Mechanisms of Curvature Sensing and Generation by Peripheral Membrane Proteins

外周膜蛋白曲率传感和产生的机制

• 批准号: 8194640
• 负责人: Tobias Baumgart
• 金额: $ 28.64万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-01 至 2016-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8194640
• 关键词: Affect; Alzheimer' s Disease; Area; Arthritis; Aspirate substance; Basic Science; Binding; Binding Proteins; Cell physiology; Cells; Cellular Membrane; Centronuclear myopathy; Collaborations; Coupled; Couples; Coupling; Data; Diabetes Mellitus; Disease; Down-Regulation; Electron Microscopy; Epilepsy; Equilibrium; Evaluation; Fluorescence; GTP-Binding Proteins; Generations; Goals; Guanosine Triphosphate Phosphohydrolases; Huntington Disease; Image; Intracellular Membranes; Label; Lead; Letters; Life; Ligand Binding; Ligands; Link; Malignant Neoplasms; Mammalian Cell; Measurement; Mechanics; Membrane; Membrane Lipids; Membrane Proteins; Mental Retardation; Methodology; Molecular; Monitor; Mutate; Mutation; N-terminal; Organ; Parkinson Disease; Peripheral; Plasmids; Play; Point Mutation; Protein Binding Domain; Proteins; RNA Splicing; Regulation; Role; SH3 Domains; Shapes; Sorting - Cell Movement; Stiff-Person Syndrome; Structure; Tertiary Protein Structure; Testing; Thermodynamics; Tube; Tubular formation; Ursidae Family; Vesicle; amphiphysin; amphiphysin 2; cancer cell; cancer type; density; epsin; fluorescence imaging; improved; insight; interest; member; membrane model; mutant; optical traps; research study; scaffold; simulation; synuclein; theories

DESCRIPTION (provided by applicant): It has very recently been recognized that numerous proteins involved in membrane curvature (MC) sensing and regulation are also implicated in a variety of diseases. Thus, the need to understand how MC couples with cellular function has emerged as a focus area both from a basic research as well as a biomedical perspective. Several membrane-binding protein domains have been shown to be capable of generating MC. Some of the most prominent members of these protein domains are the Bin/amphiphysin/Rvs (BAR) and Epsin N-terminal homology (ENTH) domains. These domains have been found in numerous disease-related proteins. To date, studies of these proteins rely heavily on electron microscopy imaging. The mechanisms by which these domains couple to MC and in particular a quantitative understanding of the hypothesized mechanisms have remained elusive. Membrane curvature sensing and generating proteins can be distinguished by several different types of disease involvement mechanisms. Firstly, altered expression levels are often found in malignant cells. For example, down-regulation or mis-splicing of BAR domain-containing proteins is found in cancers of various organs. Secondly, specific point mutations are encountered in proteins related to MC. For example, several point mutations in the BAR domain of the protein BIN1 (amphiphysin-2) are found to be linked to centronuclear myopathy. These mutations have been connected with membrane tubulation (i.e. curvature generation) activity. Furthermore, mutations within SH3 domains of BAR proteins, or mutations in SH3 domain ligands, are linked to several diseases. Our goal is to understand how protein concentration affects MC sensing and MC generation, and by which mechanisms these phenomena are effected by wild type and strategically mutated proteins. We will also investigate how ligand/SH3 domain interactions couple to membrane curvature. In order to achieve this goal, we have developed a biophysical approach that allows us to separately quantify MC sensing and generation. Curvature sensing will be determined through fluorescence measurements. These will monitor curvature-dependent re-localization of membrane-binding proteins and their mutants on membranes with precisely adjustable curvature. MC generation will be quantified through probing mechanical aspects of bent membranes. We will focus on using micropipette-aspiration and optical trapping to manipulate cylindrical membranes pulled from vesicles that are either self-assembled or purified from cells. We will provide improved mechanistic understanding of our quantitative data by comparing them to statistical mechanical, thermodynamic and mechanical theories. Our studies will provide valuable insight into MC regulation and sensing by proteins which may aid in understanding the role that these proteins play in numerous diseases. PUBLIC HEALTH RELEVANCE: Membrane proteins coupling with cellular membrane curvature are involved in numerous diseases, including Alzheimer's, Huntington's, and Parkinson's disease, arthritis, centronuclear myopathy, diabetes, epilepsy, mental retardation, stiff person syndrome, and several types of cancer. The field of experimental research into curvature regulation and sensing by peripheral proteins is currently dominated by qualitative observations and insufficient mechanistic understanding. This project aims at providing a quantitative understanding of membrane curvature sensing and generation of three important classes of proteins, including mutants that occur in diseases such as centronuclear myopathy and Parkinson's disease.

描述（由申请人提供）：最近人们认识到许多参与膜曲率（MC）传感和调节的蛋白质也与多种疾病有关。因此，从基础研究和生物医学角度来看，了解 MC 如何与细胞功能结合的需求已成为一个重点领域。一些膜结合蛋白结构域已被证明能够产生 MC。这些蛋白质结构域中最重要的一些成员是 Bin/amphiphyn/Rvs (BAR) 和 Epsin N 末端同源性 (ENTH) 结构域。这些结构域已在许多疾病相关蛋白质中发现。迄今为止，这些蛋白质的研究很大程度上依赖于电子显微镜成像。这些域与 MC 耦合的机制，特别是对假设机制的定量理解仍然难以捉摸。 膜曲率传感和生成蛋白质可以通过几种不同类型的疾病参与机制来区分。首先，在恶性细胞中经常发现表达水平改变。例如，在多种器官的癌症中发现了含有 BAR 结构域的蛋白质的下调或错误剪接。其次，与MC相关的蛋白质会遇到特定的点突变。例如，发现蛋白质 BIN1（amphiphyn-2）的 BAR 结构域中的几个点突变与中心核肌病有关。这些突变与膜管（即曲率生成）活性有关。此外，BAR 蛋白的 SH3 结构域内的突变或 SH3 结构域配体的突变与多种疾病有关。我们的目标是了解蛋白质浓度如何影响 MC 传感和 MC 生成，以及野生型和策略性突变蛋白质通过何种机制影响这些现象。我们还将研究配体/SH3 结构域相互作用如何与膜曲率耦合。 为了实现这一目标，我们开发了一种生物物理方法，使我们能够单独量化 MC 传感和生成。曲率传感将通过荧光测量来确定。这些将监测膜结合蛋白及其突变体在具有精确可调节曲率的膜上的曲率依赖性重新定位。 MC 的产生将通过探测弯曲膜的机械方面进行量化。我们将重点关注使用微移液器抽吸和光学捕获来操纵从自组装或从细胞中纯化的囊泡中拉出的圆柱形膜。我们将通过将定量数据与统计力学、热力学和机械理论进行比较，提供对定量数据更好的机械理解。 我们的研究将为蛋白质的 MC 调节和传感提供有价值的见解，这可能有助于了解这些蛋白质在许多疾病中发挥的作用。 公共健康相关性：与细胞膜曲率耦合的膜蛋白与多种疾病有关，包括阿尔茨海默病、亨廷顿病和帕金森病、关节炎、中心核肌病、糖尿病、癫痫、智力低下、僵人综合症和几种类型的癌症。目前，外周蛋白曲率调节和传感的实验研究领域主要以定性观察和机制理解不足为主。该项目旨在定量了解膜曲率传感和三类重要蛋白质的生成，包括中心核肌病和帕金森病等疾病中出现的突变体。