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：
10624809
负责人：
Alessio Accardi
金额：
$ 41.53万
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/10624809
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

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.

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，因此解释的能力它们在人体生理学中的功能，并设计有针对性的药理干预措施，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来克服这些限制 TMEM16 扰乱酶如何响应 Ca2+ 结合而被激活，它们如何以及用于什么目的重塑细胞膜，以及它们如何受到这些膜的特定成分的影响。我们通过将实验与结构、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是第一类被发现能抑制 TMEM16 扰乱酶功能的分子，体内与内皮细胞中 PS 的过度暴露有关。我们的第一个目标是确定 Ca2+- 结合冷冻电子显微镜观察 TMEM16 扰乱酶的依赖门控机制（冷冻电镜）、分子动力学 (MD) 模拟和功能测定。这些实验将揭示 Ca2+ 结合位点与脂质门控结构元件之间的变构耦合机制途径。我们的第二个目标是确定 TMEM16 扰乱酶如何与以下物质相互作用并改变其结构：它们周围的膜环境支持它们的功能。使用结构测定通过冷冻电镜，我们将可视化 afTMEM16 复合物与具有各种理化特性的膜和组合物，处于不同的功能状态。结合 MD 模拟和功能分析，我们将确定膜-蛋白质相互作用和膜的能量和分子决定因素重塑，以及它们在扰乱中的作用。我们的第三个目标是确定其机制和体内作用使用功能测定来识别 TMEM16 扰乱酶的神经酰胺调节分子决定因素神经酰胺抑制，以及确定其作用机制的结构和计算实验，以及特定神经酰胺在 TMEM16F 体内调节中的作用。