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：
10406928
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/10406928
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 Pharmacology Phosphatidylserines Phospholipid Interaction Phospholipids Physiological Physiology Process Property Proteins Protocols documentation Regulation Resolution Role Signal Transduction Signal Transduction Pathway Site Stroke Structure Testing Thinness 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 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积分膜蛋白家族的成员是Ca2+依赖性的磷脂串联酶。因为TMEM16S的脂质机制仍然很少理解，所以解释的能力它们在人类生理学中的功能和设计有针对性的药理学干预措施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如何根据Ca2+结合，如何以及出于什么目的激活TMEM16串联酶重塑细胞膜，以及它们如何受这些膜特定成分的影响。我们通过联合策略与结构，功能，功能，和计算方法。要了解这些蛋白质如何在体内调节，我们将重点关注神经酰胺作为第一类分子，发现抑制TMEM16串联酶的功能并为在体内与内皮细胞中PS的过度暴露有关。我们的第一个目标是确定CA2+ - 使用冷冻电子显微镜的组合TMEM16串联酶的依赖门控机制（冷冻），分子动力学（MD）模拟和功能测定。这些实验将揭示 Ca2+结合位点与结构元件之间的变构耦合机制，结构元素门控脂质路径。我们的第二个目标是确定TMEM16串联酶如何与之相互作用，并改变他们周围的膜环境支持其功能。使用结构确定低温我们将可视化具有各种理化特性的膜的AftMem16复合物和组成，在不同的功能状态。结合MD模拟和功能测定，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及其在争夺中的作用。我们的第三个目标是确定使用功能测定法调节TMEM16串联酶的神经酰胺调节，以鉴定神经酰胺抑制作用以及结构和计算实验以确定其作用机理，并特定神经酰胺在TMEM16F体内调节中的作用。