Modeling synaptic vesicles: how does alpha-Synuclein inhibit fusion?

描述(由申请人提供)：我们的目标是从机理上理解单体α-突触核蛋白(ALPHA)抑制突触小泡融合。这项研究将为帕金森病(PD)治疗的新治疗策略奠定基础。越来越多的人达成共识，认为单体α是突触囊泡运输的中心调节成分。虽然阿尔法小体的形成通常与帕金森病有关，但高水平的阿尔法小体也被证明可以干扰正常的囊泡运输，显著抑制神经递质的释放，而不是形成阿尔法小体聚集体。我们的方法将包括对突触囊泡膜的生物物理和力学性质的定量研究，为此，我们将结合粗粒度分子动力学模拟和一系列互补的生物物理实验。准确了解单体α蛋白和突触囊泡膜之间的天然相互作用将使我们能够评估该蛋白在囊泡运输缺陷中的作用，因为它们与帕金森病有关。有限的生物物理学数据对阿尔法蛋白的过度表达如何在没有纤维形成的情况下抑制囊泡运输和融合产生了相互矛盾的观点。在从酵母到啮齿动物的多种模型系统中，过多的α蛋白被证明阻碍了正常的突触小泡在质膜上的循环。一种观点认为，这种病理可能是由Alpas与其他突触或质膜蛋白(如SNARES)之间的相互作用所驱动的。我们根据我们实验室和其他实验室最近的工作提出了另一种观点。根据这一观点，阿尔法可以直接改变膜内脂类的物理性质--即膜的硬度和相。这是在没有与其他蛋白质的特定相互作用的情况下实现的。因此，我们推测Alpas可能具有控制突触囊泡融合的内在能力。这一假设是 我们的初步数据和发表的生物物理实验结果表明，阿尔法可以降低膜的刚性，改变膜的曲率，并可以抑制合成的(否则不含蛋白质的)脂泡的融合。我们提出的研究将计算模拟与实验X射线散射和原子力显微镜紧密结合起来，以弥合理解阿尔法如何为囊泡融合创造物理屏障的关键空白。拟议的研究途径将提供有关PD相关贩运缺陷的关键信息。最终，我们的工作将导致更好地了解阿尔法蛋白的正常和异常功能，并使社区能够开发利用蛋白质的天然状态(即恢复适当的囊泡运输)的新治疗策略。

DESCRIPTION (provided by applicant): Our goal is to develop a mechanistic understanding of the inhibition of synaptic vesicle fusion by monomeric alpha-Synuclein (alphaS). This research will establish the foundation for new therapeutic strategies in the treatment of Parkinson's disease (PD). There is growing consensus that monomeric alphaS is a central regulatory component of synaptic vesicle trafficking. Although the formation of Lewy bodies, mediated by the aggregation of alphaS into insoluble fibrils, is commonly associated with PD, high levels of alphaS have also been shown to disrupt normal vesicle trafficking and markedly inhibit neurotransmitter release without the formation of alphaS aggregates. Our approach will involve quantitative studies of the biophysical and mechanical properties of synaptic vesicle membranes for which we will combine coarse-grained molecular dynamics simulations with a panel of complementary biophysical experiments. A precise understanding of the native interactions between monomeric alphaS and synaptic vesicle membranes will position us to evaluate the protein's role in vesicle trafficking defects as they relate to PD. Limited biophysica data have yielded conflicting views on how alphaS over-expression inhibits vesicle trafficking and fusion in the absence of fibril formation. In multiple model systems from yeast to rodents, an overabundance of alphaS has been shown to stall proper synaptic vesicle cycling at the plasma membrane. One view is that this pathology may be driven by interactions between alphaS and other synaptic or plasma membrane proteins (e.g. SNARES). We propose an alternate view based on recent work both from our labs and others. According to this view, alphaS can directly alter the physical properties of lipids within membranes - namely membrane rigidity and phase. This is achieved in the absence of specific interactions with other proteins. We therefore reason that alphaS might have an intrinsic capacity to control synaptic vesicle fusion. This hypothesis is motivated by our preliminary data and published result from biophysical experiments, which show that alphaS reduces a membrane's rigidity, can alter membrane curvature, and can inhibit fusion of synthetic (otherwise protein-free) lipid vesicles. Our proposed research intimately combines computational modeling with experimental x-ray scattering and atomic force microscopy to bridge a critical gap in understanding how alphaS creates physical barriers to vesicle fusion. The proposed research avenue will provide critical information about PD associated trafficking defects. Ultimately, our work will lead to better understanding of normal and abnormal functions of alphaS and position the community to develop new therapeutic strategies that exploit the native state of the protein (i.e., restoring proper vesicle trafficking.