Protein-Lipid Bilayer Interactions in Aggregation

聚集中的蛋白质-脂质双层相互作用

• 批准号: 6776826
• 负责人: ANNETA RAZATOS
• 金额: $ 6.54万
• 依托单位: ARIZONA STATE UNIVERSITY-TEMPE CAMPUS
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-05-01 至 2006-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6776826
• 关键词: Lewy body; Parkinson' s disease; alpha synuclein; anions; atomic force microscopy; intermolecular interaction; ionic bond; lipid bilayer membrane; lipid structure; molecular assembly /self assembly; neurofilament; neurogenesis; phospholipids; protein binding; protein protein interaction

Role of Protein-Lipid Bilayer Interactions in alpha-Synuclein Aggregation This proposal is in response to the NIA's Pilot Research Grant Program on Research Objective #7--Protein Modification, Aggregation and Degradation in Aging and Age-related Diseases. Parkinson's Disease (PD) is an age-related, neurodegenerative disease characterized by resting tremor, muscular rigidity, postural instability, and bradykinesia. Although the molecular mechanism responsible for PD is currently unknown, the soluble neural protein, alpha-synuclein (aS), is believed to be involved in the pathological cascade, alphaS is the major constituent of Lewy bodies, intracellular fibrillar aggregates that are a pathological hallmark of PD. AlphaS is known to self-aggregate in vitro into multimers and fibrils that resemble those found in Lewy bodies. Numerous in vivo and in vitro studies have also demonstrated that alphaS co-exists with lipids in Lewy bodies, and that aS directly interacts with phospholipids in vesicle or cell membranes. At the present time, there are two proposed mechanisms for aS aggregation and fibril formation in neural cells. The first mechanisms is that soluble aS protein adheres to the cell surface via lipid-protein interactions, and serves a seed or nucleus for aggregation and fibril formation. The second mechanism is that the natural state of alphaS is to be associated or bound to lipids such that the protein is not free to aggregate with other soluble proteins. The objective of this research is to distinguish between the two proposed mechanisms. Our hypothesis is that the interactions between alphaS and lipids play a role in protein aggregation. The atomic force microscope (AFM) will be used in this study to characterize the initial interactions between alphaS and supported lipid bilayers, and to determine how these interactions affect protein aggregation. The objective of this research will be achieved through the following specific aims: 1) Characterize the strength of the interactions between alphaS and supported lipid bilayers containing various mixtures of anionic and neutral phospholipids representative of neural cell membranes. 2) Determine if electrostatic interactions mediate protein-lipid binding. 3) Evaluate aS aggregation on lipid bilayers as a function of phospholipid composition. A correlation between the strength of protein-lipid interactions and protein aggregation will directly support our hypothesis of lipid-induced self-aggregation of alphaS. The AFM, which can provide unique quantitative information on the magnitude of biological interactions, will be used in this study to evaluate protein-lipid bilayer interactions. Protein will be immobilized directly on the tips of AFM cantilevers and used to probe supported lipid bilayers in buffer solutions. The AFM will also be used to observe the formation of alphaS aggregates on lipid bilayers. Ultimately, results from this study will be used to develop therapies that prevent alphaS aggregation.

蛋白质-脂质双层相互作用在 α-突触核蛋白聚集中的作用 该提案是为了响应 NIA 的研究目标 #7 试点研究资助计划——衰老和年龄相关疾病中的蛋白质修饰、聚集和降解。帕金森病 (PD) 是一种与年龄相关的神经退行性疾病，其特征为静止性震颤、肌肉强直、姿势不稳和运动迟缓。尽管PD的分子机制目前尚不清楚，但可溶性神经蛋白， α-突触核蛋白 (aS) 被认为参与病理级联反应，αS 是路易体的主要成分，路易体是细胞内纤维聚集体，是 PD 的病理标志。众所周知，AlphaS 在体外会自我聚集成多聚体和原纤维，类似于路易体中的那些。许多体内和体外研究也表明 证明 αS 与路易体中的脂质共存，并且 aS 直接与囊泡或细胞膜中的磷脂相互作用。 目前，神经细胞中的aS聚集和原纤维形成有两种机制。第一种机制是可溶性 aS 蛋白通过脂质-蛋白质相互作用粘附到细胞表面，并作为种子或细胞核用于聚集和原纤维形成。第二种机制是，αS 的天然状态是与脂质结合或结合，使得该蛋白质不会自由地与其他可溶性蛋白质聚集。本研究的目的是区分两种提议的机制。我们的假设是 alphaS 和脂质之间的相互作用在蛋白质聚集中发挥作用。 本研究将使用原子力显微镜 (AFM) 来表征 alphaS 和支持的脂质双层之间的初始相互作用，并确定这些相互作用如何影响蛋白质聚集。本研究的目的将通过以下具体目标来实现： 1) 表征 alphaS 和支持的脂质双层之间相互作用的强度，该脂质双层含有代表神经细胞膜的阴离子和中性磷脂的各种混合物。 2) 确定静电相互作用是否介导蛋白质-脂质结合。 3) 评估脂质双层上的 aS 聚集作为磷脂组成的函数。 蛋白质-脂质相互作用的强度与蛋白质聚集之间的相关性将直接支持我们的脂质诱导 αS 自我聚集的假设。 AFM 可以提供有关生物相互作用程度的独特定量信息，将在本研究中用于评估蛋白质-脂质双层相互作用。蛋白质将直接固定在 AFM 悬臂的尖端上，并用于探测缓冲液中支持的脂质双层 解决方案。 AFM 还将用于观察脂质双层上 alphaS 聚集体的形成。最终，这项研究的结果将用于开发防止 alphaS 聚集的疗法。