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

摘要 TMEM16家族的膜整合蛋白的成员是Ca2+依赖性磷脂乱序酶。 由于对TMEM16的脂质扰乱机制仍然知之甚少，因此解释其机制的能力还有待进一步研究。 功能，并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如何克服这些限制 TMEM 16扰乱酶响应于Ca2+结合而被激活，它们如何以及为了什么目的重塑 细胞膜，以及它们如何受到这些膜的特定成分的影响。我们解决这个 机械目标与综合战略相结合的实验与结构，功能， 计算方法。为了了解这些蛋白质是如何在体内调节，我们将重点放在神经酰胺 作为第一类被发现抑制TMEM 16乱序酶功能并在体内结合的分子 内皮细胞中PS的过度暴露。我们的第一个目标是确定钙依赖性门控 使用低温电子显微镜（cryoEM）、分子生物学和分子生物学的组合， 动力学（MD）模拟和功能测定。这些实验将揭示 Ca2+结合位点和脂质通路的结构元件之间的机制。我们的第二个目标是 以确定TMEM16乱序酶如何与其周围膜相互作用并改变其结构， 环境，以支持其功能。使用cryoEM的结构测定，我们将可视化afTMEM 16 复合物与膜具有各种物理化学性质和组成，在不同的功能 states.结合MD模拟和功能测定，我们将确定能量和分子 膜蛋白相互作用和膜重塑的决定因素，以及它们在混乱中的作用。我们 第三个目的是确定神经酰胺调节TMEM 16乱序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.