Combinatorial effects of PTMs on a-Synuclein structure, function and aggregation

α-突触核蛋白是一种小的神经元蛋白，是帕金森氏病、路易体痴呆、多系统萎缩和其他突触核糖核病的标志蛋白聚集体的主要成分，并与阿尔茨海默病等相关神经退行性疾病有关。尽管进行了大量的研究，但对于导致αS在PD中聚集的环境因素以及在LBD和MSA中聚集的差异仍然缺乏了解。最近的证据支持这样一种观点，即α-突触核蛋白聚集体或菌株之间的结构差异是不同的突触核病的基础。虽然分子细节尚不清楚，但已有研究表明，α-突触核蛋白的翻译后修饰可能是不同品系之间构象差异的基础。然而，考虑到已报道的大量对α-突触核蛋白的翻译后修饰，以及它们在患者来源样本中的异质性分布，了解这些修饰如何影响聚集结构，并最终影响病理学，是极其具有挑战性的。从生化和生物物理的角度来看，许多这些修饰都是单独讨论的，并发现对α-Synuclein的性质有显著的影响，包括聚集动力学和细胞摄取和播种。然而，在我们对多个同时修饰如何协同作用来改变聚集结构或α-突触核蛋白功能的理解上存在着显著的差距。我们建议的研究将通过利用三个PI在蛋白质化学合成、细胞和分子生物物理学以及结构生物学方面的集体专业知识来解决这一缺陷。这将包括使用一种新的半合成策略--结合非天然氨基酸诱变、化学酶修饰、硫醇-烯反应和天然化学连接--产生在单个和多个位点特异修饰的α-突触核蛋白位点(目标1)；确定α-突触核蛋白修饰对与脂质双层的功能相互作用、对自结合动力学和聚集体结构特征的影响(目标2)；以及将这些结构效应与初级神经元对α-突触核蛋白的内化以及随后内源α-突触核蛋白的种子聚集(目标3)联系起来。我们在α-突触核蛋白上选择了七个不同的疾病相关位点，它们受到不同基团的修饰，包括磷酸化、乙酰化和泛素化，我们将比较和对比这些修饰的个体效果以及它们的串扰。我们的重点是在PD、LBD和MSA患者组织中发现的不同的修饰，现有的结构数据允许我们提出明确的机制假说。我们期望描述这些修饰对α-突触核蛋白功能相互作用以及纤维结构和扩散的影响。由此产生的影响将是提供一个透彻的了解在联核病中‘菌株’差异的分子基础，并指导针对翻译后修饰的治疗的发展，甚至是针对联核病和相关痴呆的全新治疗策略的发展。

α-Synuclein is a small neuronal protein that is the primary component of the proteinaceous aggregates that are the hallmark of Parkinson’s disease (PD), Lewy body dementia (LBD), multiple system atrophy (MSA) and other synucleinopathies, as well as being implicated in related neurodegenerative diseases such as Alzheimer’s disease. Despite intense study, an understanding of the environmental factors which lead to αS aggregation in PD and to differences in aggregation in LBD and MSA is still lacking. Recent evidence supports the idea that structural differences between α-Synuclein aggregates, or ‘strains’, underlie different synucleinopathies. While the molecular details are not yet well understood, it has been suggested that post-translational modifications to α-Synuclein may underlie conformational differences between ‘strains’. However, understanding how these modifications impact aggregate structure, and ultimately pathology, is extremely challenging, both given the large number of reported post-translational modifications to α-Synuclein, as well as their heterogeneous distribution in patient derived samples. From a biochemical and biophysical perspective, many of these modifications have been addressed individually and found to have striking impacts on α-Synuclein properties, including aggregation kinetics and cellular uptake and seeding. However, there is a significant gap in our understanding of how multiple simultaneous modifications may work cooperatively to alter aggregate structure or α-Synuclein function. Our proposed research will address this deficit by taking advantage of the collective expertise of the three PIs in protein chemical synthesis, cellular and molecular biophysics, and structural biology. This will include using a novel semi-synthesis strategy – combining unnatural amino acid mutagenesis, chemoenzymatic modification, thiol-ene reactions, and native chemical ligation – to produce α-Synuclein site specifically modified both at single and multiple sites (Aim 1); determining the impact of α-Synuclein modifications on functional interactions with lipid bilayers, on the kinetics of self-association and on the structural features of the aggregates (Aim 2); and relating these structural effects to internalization of α-Synuclein by primary neurons, and subsequent seeded aggregation of endogenous α-Synuclein (Aim 3). We have selected seven different disease-associated sites on α-Synuclein that are subject to modification with diverse groups, including phosphorylation, acetylation and ubiquitination, and we will compare and contrast the individual effects of these modifications as well as their cross-talk. Our focus is on modifications that are differentially found in PD, LBD, and MSA patient tissues and for which available structural data allow us to propose clear mechanistic hypotheses. We expect to characterize the impact of these modifications both on α-Synuclein functional interactions as well as fibrillar structure and spread. The resulting impact will be in providing a thorough understanding of the molecular basis of ‘strain’ differences in synucleinopathies and guiding the development of therapies targeted at post-translational modifications, or even entirely new therapeutic strategies for synucleinopathies and related dementias.