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

摘要 TMEM16完整膜蛋白家族的成员是钙依赖的磷脂扰乱酶。 由于TMEM16的脂质扰乱机制仍然知之甚少，因此解释其 在人体生理中的作用，并设计有针对性的药物干预措施，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扰乱酶是响应于钙结合而被激活的，它们如何以及为了什么目的重塑 细胞膜，以及这些膜的特定成分是如何影响它们的。我们解决了这个问题 机械目标，综合策略，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是如何调节的，我们将重点研究神经酰胺。 作为第一类被发现抑制TMEM16扰乱酶功能并在体内结合的分子 血管内皮细胞过度暴露PS。我们的第一个目标是确定钙依赖的门控 冷冻电子显微镜结合分子生物学方法研究TMEM16扰乱酶的机制 动力学(MD)模拟和功能分析。这些实验将揭示变构耦合 钙结合部位与控制脂质途径的结构元件之间的机制。我们的第二个目标是 以确定TMEM16扰乱酶如何与其周围的膜相互作用并改变其结构 支持其职能的环境。使用冷冻电子显微镜的结构确定，我们将可视化AFTMEM16 具有不同物理化学性质和组成的膜的络合物，具有不同的功能 各州。结合MD模拟和功能分析，我们将确定能量和分子 膜-蛋白质相互作用和膜重塑的决定因素，以及它们在争抢中的作用。我们的 第三个目的是确定神经酰胺对TMEM16扰乱酶的调节机制和体内作用。 鉴定神经酰胺抑制的分子决定因素的功能分析，以及结构和计算 实验确定它们的作用机制，以及特定神经酰胺在体内调节中的作用 TMEM16F。

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.