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

PTM 对 a-Synuclein 结构、功能和聚集的组合效应

• 批准号: 10391709
• 负责人: Ernest James Petersson
• 金额: $ 170.61万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-04-15 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10391709
• 关键词: Acetylation; Address; Alzheimer' s Disease; Autopsy; Behavior; Binding; Biochemical; Biological Assay; Biology; Biophysics; Cellular biology; Chemicals; Collaborations; Complex; Data; Dementia; Dementia with Lewy Bodies; Deposition; Disease; Environmental Risk Factor; Exhibits; Fiber; Fluorescence; Genes; Heterogeneity; In Vitro; Individual; Inherited; Isotope Labeling; Joints; Kinetics; Label; Lead; Lewy Body Dementia; Ligation; Link; Lipid Bilayers; Literature; Mass Spectrum Analysis; Modification; Molecular; Molecular Conformation; Multiple System Atrophy; Mutagenesis; Mutation; N-terminal; Neurodegenerative Disorders; Neurons; Parkinson Disease; Pathologic; Pathology; Patients; Persons; Phosphorylation; Physiological; Play; Point Mutation; Polymorph; Post-Translational Protein Processing; Property; Proteins; Publications; Reaction; Recording of previous events; Reporting; Research; Role; Sampling; Seeds; Single Nucleotide Polymorphism; Site; Structure; Sulfhydryl Compounds; Testing; Therapeutic; Tissues; Ubiquitination; Variant; Work; alpha synuclein; base; biophysical analysis; biophysical properties; chemical group; combinatorial; design; experimental study; glycation; insight; member; monomer; novel; novel therapeutic intervention; peptide chemical synthesis; prion-like; single molecule; structural biology; success; synucleinopathy; targeted treatment; therapy development; unnatural amino acids; uptake

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.

α-突触核蛋白是一种小神经元蛋白，是蛋白质聚集体的主要成分，而蛋白质聚集体是帕金森病 (PD)、路易体痴呆 (LBD)、多系统萎缩 (MSA) 和其他突触核蛋白病的标志，并且与其他突触核蛋白病有关尽管进行了大量研究，但对导致帕金森病中αS聚集的环境因素以及导致帕金森病中聚集差异的环境因素的了解。目前仍缺乏 LBD 和 MSA 的证据支持这一观点，即 α-突触核蛋白聚集体或“菌株”之间的结构差异是不同突触核蛋白病的基础，虽然分子细节尚不清楚，但有人认为翻译后修饰是导致不同突触核蛋白病的原因。 α-突触核蛋白可能是“菌株”之间构象差异的基础。然而，考虑到大量报道的翻译后修饰，了解这些修饰如何影响聚集结构以及最终的病理学是极具挑战性的。从生物化学和生物物理学的角度来看，许多这些修饰已被单独解决，并发现对 α-突触核蛋白的特性（包括聚集动力学和细胞摄取）具有显着影响。然而，我们对多个同时修饰如何协同改变聚集结构或 α-突触核蛋白功能的理解存在重大差距，我们提出的研究将通过利用集体专业知识来解决这一缺陷。这将包括使用一种新颖的半合成策略——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——来生产蛋白质化学合成、细胞和分子生物物理学以及结构生物学中的三个 PI。 α-突触核蛋白位点在单个和多个位点进行了特异性修饰（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以及动力学的影响；自关联和聚集体的结构特征（目标 2）；并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性 α-突触核蛋白的种子聚集相关联（目标 3）。 α-突触核蛋白上与疾病相关的位点会受到不同基团的修饰，包括磷酸化、乙酰化和泛素化，我们将比较和对比个体效果我们的重点是在 PD、LBD 和 MSA 患者组织中发现的差异性修饰，并且可用的结构数据使我们能够提出明确的机制假设，我们希望能够描述这些修饰的影响。这些对 α-突触核蛋白功能相互作用以及纤维结构和扩散的修改所产生的影响将有助于全面了解突触核蛋白病“菌株”差异的分子基础，并指导针对该疾病的治疗的开发。翻译后修饰，甚至是针对突触核蛋白病和相关痴呆症的全新治疗策略。