Role of Protein-Lipid Bilayer Interactions in alpha-Syn*

蛋白质-脂质双层相互作用在 α-Syn* 中的作用

• 批准号: 6892320
• 负责人: ANNETA RAZATOS
• 金额: $ 6.61万
• 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 2007-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6892320
• 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 - 脂质双层相互作用在α-核蛋白聚集中的作用 该提案是对NIA的研究目标＃7-蛋白质修饰，聚集和降解和与年龄相关疾病的质量修饰，聚集和降解的回应。帕金森氏病（PD）是一种与年龄相关的神经退行性疾病，其特征是静止震颤，肌肉僵硬，姿势不稳定和胸肌。尽管目前尚不清楚负责PD的分子机制，但可溶性神经蛋白， 据信，α-突触核蛋白（AS）与病理级联有关，Alpha是Lewy体的主要组成部分，细胞内的原纤维骨料是PD的病理标志。已知Alpha在体外自我聚集成类似于Lewy身体的多聚体和原纤维。许多体内和体外研究也 证明Alpha与Lewy体中的脂质共存，并且直接与囊泡或细胞膜中的磷脂相互作用。 目前，在神经细胞中有两种提出的作为聚集和原纤维形成的机制。第一种机制是，作为蛋白质可溶性，通过脂质 - 蛋白质相互作用粘附在细胞表面上，并提供种子或细胞核，用于聚集和原纤维形成。第二种机制是，自然状态应与脂质相关或与脂质结合，以使该蛋白质不能与其他可溶性蛋白质聚集。这项研究的目的是区分两种提出的机制。我们的假设是，alpha和脂质之间的相互作用在蛋白质聚集中起作用。 本研究将使用原子力显微镜（AFM）来表征alpha和受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。这项研究的目的将通过以下特定目的实现： 1）表征了alpha和受支持的脂质双层之间相互作用的强度，这些脂质双层包含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）评估作为脂质双层的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的强度之间的相关性将直接支持我们对脂质诱导的alpha自我聚集的假设。在本研究中将使用AFM提供有关生物相互作用幅度的独特定量信息，以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的脂质双层 解决方案。 AFM还将用于观察脂质双层的Alphas聚集体的形成。最终，这项研究的结果将用于开发防止alpha聚集的疗法。