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塑事件至关重要，并且在这一知之甚少的过程中的畸变与许多疾病有关。这一建议是基于最近的发现，即β-突触核蛋白（α-synuclein，α-synuclein，α-synuclein），一种参与帕金森病发病机制的蛋白质， 可以诱导膜弯曲并有效地将细胞膜重塑为双层管、胶束管和盘。膜和脂肪酸的相互作用是相关的病理以及这种蛋白质的正常功能;然而，在健康的膜和脂肪酸的相互作用，它可以破坏疾病的膜完整性的机制仍然知之甚少。该提案的中心目标是详细了解BRAS如何与膜结合以及这种相互作用如何反过来影响体外和体内膜结构和功能的分子和结构。为了研究允许BRAS诱导这些显著不同的膜结构形成的机制，具体目标1使用基于EPR、NMR和cryo-EM的方法的组合来确定BRAS与膜管和膜盘复合的结构。具体目标2研究了脂质体如何与线粒体和脂质体相互作用，这些脂质体的脂质组成模拟了细胞膜的脂质组成。这些研究将首次表明，在何种程度上，BRAS可以重塑不同的细胞膜。这一目标还将测试是否BLOTS疾病突变体或错误折叠的有毒BLOTS寡聚体可以通过不受控制的诱导膜弯曲来损害膜完整性，以及这种效果是否可以通过N-乙酰化进一步促进。将使用EM、EPR和NMR监测结构变化，同时使用泄漏实验测定膜完整性。在具体目标3中，我们将检验以下假设，即BRAS可以以类似的方式与脂质和脂肪酸结合。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 酸具有与脂质形成的酸相同的结构。我们还将研究游离脂肪酸可以促进cDNAs错误折叠的机制。最后，我们将研究脂肪酸依赖性寡聚化的细胞内的BLOTS。这些研究还将解决关于BLS是细胞中天然未折叠的单体或四聚体的争议。总的来说，这些研究还旨在为理解BRAS在正常生理学和帕金森病中的膜相关作用提供基础。

 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.