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: 8323294
• 负责人: Tobias Baumgart
• 金额: $ 28.61万
• 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/8323294
• 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; unilamellar vesicle

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.

描述(由申请人提供)：最近人们认识到，与膜曲率(MC)感知和调节有关的许多蛋白质也与各种疾病有关。因此，从基础研究和生物医学的角度来看，了解MC如何与细胞功能结合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一些最重要的成员是Bin/AmiPhyin/RVS(BAR)和Epsin N-末端同源(Ten)结构域。这些结构域在许多与疾病相关的蛋白质中都被发现。迄今为止，对这些蛋白质的研究在很大程度上依赖于电子显微镜成像。这些结构域与MC偶联的机制，特别是对假设机制的定量理解仍然难以捉摸。膜曲率感知和生成蛋白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，在各种器官的癌症中发现含有bar结构域的蛋白质下调或错误剪接。其次，在与MC相关的蛋白质中遇到特定的点突变。例如，蛋白质BIN1(两面体蛋白-2)的bar结构域中的几个点突变被发现与中心核肌病有关。这些突变与膜管形成(即曲率生成)活性有关。此外，BAR蛋白SH3区域内的突变或SH3区域配体的突变与几种疾病有关。我们的目标是了解蛋白质浓度如何影响MC的感知和MC的产生，以及野生型和策略性突变的蛋白质通过哪些机制影响这些现象。我们还将研究配体/SH3结构域相互作用如何耦合到膜曲率。为了实现这一目标，我们开发了一种生物物理方法，允许我们分别量化MC传感和生成。曲率传感将通过荧光测量来确定。这些将监测与曲率相关的膜结合蛋白及其突变体在曲率精确可调的膜上的重新定位。MC的产生将通过探测弯曲的膜的机械方面来量化。我们将专注于使用微吸管吸入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薄膜。我们将通过将我们的定量数据与统计力学、热力学和力学理论进行比较，提供更好的机械理解。我们的研究将对MC的调节和蛋白质的感知提供有价值的见解，这可能有助于理解这些蛋白质在许多疾病中所起的作用。