?-SYNUCLEIN FIBRIL

?-突触核蛋白原纤维

• 批准号: 7953806
• 负责人: Jeffrey A Kelly
• 金额: $ 0.87万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-12-01 至 2009-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7953806
• 关键词: Adopted; Amyloid Fibrils; Area; Brain; Caliber; Computer Retrieval of Information on Scientific Projects Database; Degenerative Disorder; Electron Microscopy; Fiber; Funding; Grant; Guanidinium Chloride; High Pressure Liquid Chromatography; Human; In Vitro; Incubated; Institution; Length; Nerve Degeneration; Parkinson Disease; Patients; Phase; Property; Proteins; Recombinant Proteins; Research; Research Personnel; Resources; Sampling; Seeds; Solutions; Source; Structure; Ultracentrifugation; United States National Institutes of Health; dopaminergic neuron; macromolecule; monomer; protein aggregate; synuclein

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. ?-Synuclein is a 14.5 kDa protein that aggregates into amyloid fibrils within dopaminergic neurons comprising the brains of Parkinson disease patients, putatively resulting in neurodegeneration. Amyloid fibrils are an intermolecular cross ?-sheet quaternary structure adopted by numerous proteins associated with human aggregation-associated degenerative diseases. Amyloid fibrils prepared in vitro by incubating recombinant proteins are typically long, straight, unbranched fibers 8-12 nm in diameter. These can grow to several microns in length. ?-Synuclein aggregates into amyloid fibrils when shaken in PBS in the presence of 1% seed (sonicated pre-formed fibrils to which monomers can add to elongate into fibrils). We have found that when these fibrils are diluted into high concentrations (5M) of guanidine hydrochloride (GdnHCl) they denature to monomer . The amount of monomer and aggregate in solution can be determined by ultracentrifugation of the sample at 60,000 rpm, collecting the supernatant and pellet, dissolving the pellet in 8M GdnHCl, and injecting both the supernatant and pellet samples on a reverse phase HPLC column. The area of the peak of ?-synuclein can be compared to a standard curve to determine the amount of material in each fraction and, assuming only monomer remains in the supernatant, the amount of protein aggregated. Interestingly, when ?-synuclein fibrils are diluted into 3M GdnHCl they denature to mostly monomer but upon further incubation, re-aggregate to roughly 50% aggregated fibers. Additionally, ?-synuclein aggregated from monomer will also aggregate to the extent of about 50% in 3M GdnHCl, either in the presence or absence of seed. Because the fibrils formed in PBS do not seem to be stable in 3M GdnHCl, as they at first disaggregate to monomer in the presence of 3M GdnHCl, the aggregates that later form in 3M GdnHCl ought to have different properties and likely a different structure than the fibrils formed in PBS. We are trying to compare the two types of aggregates to investigate those differences.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目和 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 β-突触核蛋白是一种 14.5 kDa 的蛋白质，在构成帕金森病患者大脑的多巴胺能神经元内聚集成淀粉样原纤维，推测导致神经变性。淀粉样原纤维是一种分子间跨β片层四级结构，被许多与人类聚集相关退行性疾病相关的蛋白质所采用。通过孵育重组蛋白在体外制备的淀粉样原纤维通常是直径为 8-12 nm 的长、直、无分支纤维。它们的长度可以长到几微米。 当在 1% 种子存在的 PBS 中摇动时，α-突触核蛋白聚集成淀粉样原纤维（超声处理的预形成原纤维，单体可以添加到其中以伸长成原纤维）。我们发现，当这些原纤维被稀释到高浓度 (5M) 盐酸胍 (GdnHCl) 中时，它们会变性为单体。溶液中单体和聚集体的量可以通过以下方式测定：以 60,000 rpm 超速离心样品，收集上清液和沉淀，将沉淀溶解在 8M GdnHCl 中，并将上清液和沉淀样品注射到反相 HPLC 柱上。可以将β-突触核蛋白的峰面积与标准曲线进行比较，以确定每个级分中的材料量，并且假设上清液中仅保留单体，则确定聚集的蛋白质的量。 有趣的是，当 β-突触核蛋白原纤维被稀释到 3M GdnHCl 中时，它们大部分变性为单体，但在进一步孵育后，重新聚集成大约 50% 的聚集纤维。另外，无论有或没有种子，从单体聚集的β-突触核蛋白在3M GdnHCl中也将聚集至约50％的程度。由于 PBS 中形成的原纤维在 3M GdnHCl 中似乎不稳定，因为它们首先在 3M GdnHCl 存在下分解为单体，因此后来在 3M GdnHCl 中形成的聚集体应该具有与 PBS 中形成的原纤维不同的特性，并且可能具有不同的结构。我们正在尝试比较这两种类型的聚合以调查这些差异。