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-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.尽管进行了深入的研究，但仍缺乏对PD的αS聚集以及LBD和MSA聚集的差异的理解。最近的证据支持了以下观点：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苷病。尽管分子细节尚未得到充分的了解，但已经提出，对α-突触核蛋白的翻译后修饰可能是“菌株”之间构象差异的基础。但是，鉴于报告了大量报告的翻译后修饰，以及对患者衍生样品中的异质分布，了解这些修饰如何影响骨料结构以及最终的病理是极为挑战的。从生化和生物物理的角度来看，许多这些修饰已被单独解决，并发现对α-突触核蛋白的特性产生了惊人的影响，包括聚集动力学以及细胞摄取和播种。但是，我们对多个简单修改如何合作改变聚集结构或α-突触核蛋白功能的理解存在很大的差距。我们提出的研究将通过利用蛋白质化学合成，细胞和分子生物物理学以及结构生物学的三个PI的集体专业知识来解决这种辩护。这将包括使用一种新型的半合成策略 - 结合非天然氨基酸诱变，化学酶修饰，硫醇 - 烯反应和天然化学结扎 - 以在单个和多个位点特异性修饰的α-突触核蛋白位点（AIM 1）；确定α-突触核蛋白修饰对与脂质双层功能相互作用的影响，对自相关的动力学以及聚集体的结构特征的影响（AIM 2）；并将这些结构效应与原发性神经元的α-突触核蛋白的内在化以及随后的内源性α-突触核蛋白的聚集（AIM 3）相关。我们在α-突触核蛋白上选择了七个不同的疾病相关位点，这些位点与潜水员组进行了修改，包括磷酸化，乙酰化和泛素化，我们将比较和对比这些修饰以及其串扰的个体效应。我们的重点是在PD，LBD和MSA患者组织中发现的修改以及可用的结构数据使我们提出明确的机械假设。我们期望表征这些修饰对α-突触核蛋白功能相互作用以及原纤维结构和扩散的影响。由此产生的影响将是对突触核苷的“应变”差异的分子基础的透彻理解，并指导针对翻译后修饰的疗法的发展，甚至针对突触核核酸和相关痴呆症的全新治疗策略。