Ca2+-dependent lipid scrambling and ion transport by TMEM16 proteins

TMEM16 蛋白的 Ca2 依赖性脂质扰乱和离子传输

基本信息

批准号：
10798983
负责人：
Alessio Accardi
金额：
$ 10.77万
依托单位：
WEILL MEDICAL COLL OF CORNELL UNIV
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-06-04 至 2024-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10798983
关键词：
Address Affect Apoptotic Binding Binding Sites Biological Assay Blood Cells Blood coagulation Cell membrane Cells Cellular Membrane Cellular Structures Ceramides Characteristics Charge Complex Coupling Cryoelectron Microscopy Data Elements Endothelial Cells Environment Face Family Genetic Diseases Goals Grant Human Impairment In Vitro Integral Membrane Protein Intervention Ion Transport Ions Ligand Binding Ligands Lipid Bilayers Lipids Mediating Membrane Membrane Fusion Membrane Proteins Modeling Molecular Molecular Conformation Movement Pathway interactions Phosphatidylserines Phospholipid Interaction Phospholipids Physiological Physiology Process Property Proteins Protocols documentation Regulation Resolution Role Signal Transduction Signal Transduction Pathway Site Stroke Structure Testing Thinness Visualization design experimental study extracellular in vitro activity in vivo insight lipid transport mechanical properties member molecular dynamics mutant nanodisk novel novel strategies novel therapeutics particle pharmacologic phospholipid scramblase reconstitution repaired response vesicular release

项目摘要

ABSTRACT Members of the TMEM16 family of integral membrane proteins are Ca2+-dependent phospholipid scramblases. Because mechanisms of lipid scrambling by the TMEM16s remain poorly understood, the ability to interpret their function in human physiology and to design targeted pharmacological interventions that would selectively manipulate the activity of these proteins, is limited. Our goal is to overcome these limitations by determining how the TMEM16 scramblases are activated in response to Ca2+ binding, how and for what purpose they remodel cellular membranes, and how they are affected by specific components of these membranes. We address this mechanistic goal with an integrated strategy combining experimentation with structural, functional, and computational approaches. To understand how these proteins are modulated in vivo we will focus on ceramides as the first class of molecules found to inhibit the function of TMEM16 scramblases and to be associated in vivo with excessive exposure of PS in endothelial cells. Our 1st aim is to determine the Ca2+-dependent gating mechanism of the TMEM16 scramblases using a combination of cryo-electron microscopy (cryoEM), molecular dynamics (MD) simulations and functional assays. These experiments will reveal the allosteric coupling mechanism between the Ca2+ binding site and the structural elements gating the lipid pathway. Our 2nd aim is to determine how the TMEM16 scramblases interact with, and alter the structure of, their surrounding membrane environment in support of their function. Using structure determination with cryoEM we will visualize afTMEM16 complexes with membranes with a variety of physicochemical properties and compositions, in different functional states. In combination with MD simulations and functional assays we will identify the energetic and molecular determinants for membrane-protein interactions and membrane remodeling, and their role in scrambling. Our 3rd aim is to determine the mechanism and in vivo role of ceramide regulation of TMEM16 scramblases using functional assays to identify the molecular determinants of ceramide inhibition, and structural and computational experiments to determine their mechanism of action, and the role of specific ceramides in the in vivo regulation of TMEM16F.

抽象的 TMEM16 整合膜蛋白家族的成员是 Ca2+ 依赖性磷脂扰乱酶。由于 TMEM16 的脂质扰乱机制仍知之甚少，因此无法解释其人类生理学中的功能并设计有针对性的药理学干预措施，选择性地操纵这些蛋白质的活性是有限的。我们的目标是通过确定如何克服这些限制 TMEM16 扰乱酶响应 Ca2+ 结合而被激活，它们重塑的方式和目的细胞膜，以及它们如何受到这些膜的特定成分的影响。我们解决这个问题机械目标与综合策略相结合的实验与结构、功能和计算方法。为了了解这些蛋白质在体内如何调节，我们将重点关注神经酰胺作为第一类被发现抑制 TMEM16 扰乱酶功能并在体内相关的分子内皮细胞中 PS 过度暴露。我们的第一个目标是确定 Ca2+ 依赖性门控使用冷冻电子显微镜 (cryoEM)、分子动力学（MD）模拟和功能分析。这些实验将揭示变构耦合 Ca2+ 结合位点与脂质通路门控结构元件之间的机制。我们的第二个目标是确定 TMEM16 扰乱酶如何与其周围膜相互作用并改变其结构支持其功能的环境。使用冷冻电镜进行结构测定，我们将可视化 afTMEM16 具有多种理化性质和成分的膜复合物，具有不同的功能州。结合MD模拟和功能测定，我们将识别能量和分子膜蛋白相互作用和膜重塑的决定因素及其在扰乱中的作用。我们的第三个目标是确定神经酰胺调节 TMEM16 扰乱酶的机制和体内作用功能测定，以确定神经酰胺抑制的分子决定因素，以及结构和计算实验以确定其作用机制以及特定神经酰胺在体内调节中的作用 TMEM16F。