Membrane remodeling by alpha-synuclein: implications for function and disease

 描述(由申请人提供)：膜形状和曲率的控制对于所有细胞膜重构事件和在这一鲜为人知的过程中的异常都是必不可少的，这一过程与许多疾病有关。这一建议是基于最近的一项发现，即参与帕金森病发病机制的蛋白质-突触核蛋白(S)， 可诱导细胞膜弯曲，并有效地将细胞膜重塑为双层管、胶束管和圆盘。S的膜和脂肪酸相互作用与该蛋白的病理和正常功能有关，但S在健康状态下与膜和脂肪酸相互作用以及在疾病中破坏膜完整性的机制尚不清楚。这项提议的中心目标是对S如何与膜结合以及这种相互作用在体外和体内如何影响膜结构和功能达成详细的分子和结构理解。为了研究S诱导这些显著不同的膜结构形成的机制，特定目标1使用了基于电子顺磁共振、核磁共振和冷冻EM的方法来确定S在膜管和膜盘的复合体中的结构。特定目的2研究S如何与线粒体和脂质体相互作用，这些线粒体和脂质体的脂组成与细胞膜上的相似。这些研究将首次表明S能够在多大程度上重塑不同的细胞膜。这一目的还将测试S病突变体或错误折叠、有毒的S寡聚体是否可以通过不受控制地诱导膜曲率而损害膜的完整性，以及N-乙酰化是否可以进一步促进这一效应。结构的变化将使用EM，EPR和核磁共振进行监测，而膜的完整性将通过泄漏实验进行分析。在具体目标3中，我们将检验S可以以类似的方式与脂质和脂肪酸结合的假设。作为这一目标的一部分，我们将进行结构研究，以测试圆盘和圆柱形胶束是否与脂肪形成 酸的结构与由脂形成的酸的结构相同。我们还将研究游离脂肪酸促进S错误折叠的机制。最后，我们将研究S在细胞内的脂肪酸依赖寡聚。这些研究还将解决关于S是细胞内天然展开的单体或四聚体的争议。总而言之，这些研究还旨在为理解S在正常生理和帕金森病中的膜相关角色提供基础。

 DESCRIPTION (provided by applicant): The control of membrane shape and curvature is essential for all cellular membrane remodeling events and aberrations in this poorly understood process have been linked to a number of diseases. This proposal is based upon the recent discovery that -synuclein (S), a protein involved in the pathogenesis of Parkinson's disease, can induce membrane curvature and potently remodel cellular membranes into bilayers tubes, micellar tubes and discs. Membrane and fatty acid interaction of S is of relevance for the pathological as well as the normal functions of this protein; however the mechanisms by which S interacts with membranes and fatty acids in health and by which it can disrupt membrane integrity in disease remain poorly understood. The central goal of this proposal is to arrive at a detailed molecular and structural understanding of how S binds to membranes and how this interaction, in turn, affects membrane structure and function in vitro and in vivo. In order to investigate the mechanisms that allow S to induce the formation of these remarkably diverse membrane structures, Specific Aim 1 uses a combination of EPR, NMR and cryo-EM based approaches to determine the structures of S in complex with membrane tubes and discs. Specific Aim 2 investigates how S can interact with mitochondria and liposomes whose lipid compositions mimic those of cellular membranes. These studies will give a first indication of the extent to which S can remodel different cellular membranes. This aim will also test whether S disease mutants or misfolded, toxic S oligomers can compromise membrane integrity by uncontrolled induction of membrane curvature and whether this effect can be further promoted by N-acetylation. Structural changes will be monitored using EM, EPR and NMR while membrane integrity will be assayed using leakages experiments. In Specific Aim 3, we will test the hypothesis that S can bind to lipids and fatty acids in similar manners. As part of this aim we will perform structural studies to test whether discs and cylindrical micelles formed with fatty acids have the same structure as those formed with lipids. We will also investigate the mechanism by which free fatty acids can promote the misfolding of S. Finally, we will investigate the fatty acid-dependent oligomerization of S within cells. These studies will also address the controversy with respect to S being a natively unfolded monomer or tetramer in the cell. Collectively, these studies also aim to provide a foundation for understanding S's membrane related roles in normal physiology and Parkinson's disease.