CHARACTERIZATION OF POLYGLUTAMINE AGGREGATES BY MASS SPECTROMETRY

通过质谱法表征聚谷氨酰胺聚集体

• 批准号: 8170904
• 负责人: Michael Y Sherman
• 金额: $ 3.37万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-06-01 至 2011-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8170904
• 关键词: Affect; Cell Death; Cells; Complex; Computer Retrieval of Information on Scientific Projects Database; Data; Disease; Doctor of Philosophy; Funding; Grant; Human; Huntington protein; Inherited; Institution; Journals; Lead; Manuscripts; Mass Spectrum Analysis; Methods; Modeling; Neurodegenerative Disorders; Neurons; Phase; Procedures; Protein Isoforms; Proteins; Proteomics; Publishing; Research; Research Personnel; Resources; Source; United States National Institutes of Health; Yeasts; crosslink; mutant; novel; polyglutamine; protein aggregate; protein protein interaction; research study; spatial relationship

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. Polyglutamine diseases are inherited neurodegenerative diseases caused by the expansion of polyglutamine tract in the disease-causing mutant proteins. A unifying pathological feature of polyglutamine disorders is the presence of microscopically discernible intracellular inclusions in the affected neurons. It has been proposed that expanded polyglutamine tract formed-aggregates participate in inappropriate protein-protein interactions that lead to cell death. To date, there is still no reliable method to isolate highly purified aggregates which are usually morphologically heterogeneous. We have developed a new purification method and the proteomics procedure was carried out to analyze the aggregate-interacting proteins by mass spectrometry. Mass spectrometry provides a powerful and direct analysis of aggregate proteins in tiny quantities and will help us understand the nativity of aggregates in the polyglutamine disorders. We have obtained mass spectral data that indicates the presence of several expected proteins in the aggregate and others that would not have been easily predicted. Immunochemical approaches have confirmed these aggregate interacting proteins. A manuscript describing our novel purification method was recently published. Recent experiments have provided evidence that the yeast cells form aggresomes from some isoforms of the Huntington proteins; this is the first indication that aggresomes can form in yeast. The manuscript describing the identification of proteins that are required for aggresome formation and presenting our new model for aggresome formation has been published in FASEB journal. Extensive details are included in the PhD thesis of Y. Wang, completed in June 2008. In the current phase of the research, human cells are being employed. The components of the complex are again being identified using proteomic approaches. In addition, their spatial relationships to one another will be probed using the top-down crosslinking methods being developed within a TRD project.

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。子项目及 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。 多聚谷氨酰胺疾病是遗传性神经退行性疾病，由致病突变蛋白中多聚谷氨酰胺链的扩张引起。多聚谷氨酰胺疾病的一个统一的病理特征是受影响的神经元中存在显微镜可辨别的细胞内包涵体。 有人提出，扩展的聚谷氨酰胺束形成的聚集体参与不适当的蛋白质-蛋白质相互作用，从而导致细胞死亡。迄今为止，仍然没有可靠的方法来分离高度纯化的聚集体，这些聚集体通常在形态上是异质的。我们开发了一种新的纯化方法，并进行蛋白质组学程序，通过质谱分析聚集体相互作用的蛋白质。质谱法可以对微量聚集蛋白进行强大而直接的分析，并将帮助我们了解多聚谷氨酰胺疾病中聚集体的本质。 我们获得的质谱数据表明聚集体中存在几种预期蛋白质以及其他不易预测的蛋白质。免疫化学方法已经证实了这些聚集的相互作用蛋白。最近发表了一篇描述我们新颖的纯化方法的手稿。最近的实验提供了证据，表明酵母细胞从亨廷顿蛋白的某些亚型形成聚集体。这是酵母中可以形成聚集体的第一个迹象。描述攻击体形成所需蛋白质的鉴定并展示我们的攻击体形成新模型的手稿已发表在 FASEB 杂志上。 Y. Wang 于 2008 年 6 月完成的博士论文包含了大量细节。在当前阶段的研究中，正在使用人体细胞。使用蛋白质组学方法再次鉴定了复合物的成分。此外，将使用 TRD 项目中开发的自上而下的交联方法来探讨它们之间的空间关系。