In vivo targeting of unfolded protein to a phase-separated Huntingtin inclusion

体内将未折叠蛋白靶向相分离的亨廷顿蛋白包合物

• 批准号: 10377389
• 负责人: Lesley Rachel Emtage
• 金额: $ 12.43万
• 依托单位: YORK COLLEGE
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-04-09 至 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10377389
• 关键词: Address; Adult; Amyloid fibers; Cell Death; Cells; Complex; Cytoplasm; Data; Dependence; Development; Diffuse; Disease; Electrophoresis; Etiology; Excision; Family; Funding; Gel; Gene Deletion; Genes; Genetic; Goals; Growth; Human; Huntington Disease; Huntington gene; Huntington protein; Image; Impaired cognition; In Vitro; Inclusion Bodies; Individual; Lead; Length; Life; Mass Spectrum Analysis; Modeling; Molecular; Molecular Chaperones; Mutation; Neuraxis; Neurodegenerative Disorders; Pathway interactions; Phase; Play; Process; Proteins; Quarantine; Research; Research Personnel; Role; Saccharomyces cerevisiae; Saccharomycetales; Sepharose; System; Testing; Tissues; Training; Ubiquitin; Western Blotting; Work; Yeasts; college; effective therapy; heat-shock proteins 110; imaging modality; in vivo; insight; member; molecular targeted therapies; multicatalytic endopeptidase complex; mutant; particle; polyglutamine; protein aggregation; therapeutic target; tool; undergraduate student

All major neurodegenerative disorders are characterized by the accumulation of large aggregates of unfolded protein in the central nervous system. Among neurodegenerative disorders, the etiology of Huntington's Disease (HD) is uniquely simple: the sole cause of HD is a class of dominant mutations in the huntingtin gene, which lead to the expansion of a polyglutamine repeat region in the huntingtin protein (Htt). The length of the polyglutamine tract correlates with increased instability and unfolding of mutant Htt protein, causing aggregation. Although Htt protein is widely expressed in all tissues throughout life, only a subset of cells degenerate; most cells are able to tolerate mutant Htt without significant ill effect. How do cells cope with large quantities of unfolded protein? Many studies have found that the presence of large inclusions of aggregated protein correlates poorly with cell death; in contrast, the concentration of small oligomeric aggregates is more predictive of cell death. Similarly, in S. cerevisiae, cells that form a single ovoid mutant Htt inclusion grow normally, whereas numerous widely distributed Htt aggregates are toxic to the cell. These observations have led many researchers to conclude that inclusions are cytoprotective, a cellular mechanism for collecting and quarantining harmful small aggregative species. Our studies have shown that the mHtt inclusion is a mobile phase-separated compartment that grows through the coalescence of the inclusion with small aggregates that diffuse through the cytoplasm. Evidence suggests that the formation of an inclusion body (IB) is nucleated and occurs even when the ubiquitin-proteasome system has excess capacity. Our goal is to elucidate the mechanisms by which inclusions are initiated and material is incorporated into them. Typically, a yeast cell develops a single ovoid mutant Htt IB, but IBs fail to form in a number of strains carrying single-gene deletions. Two genes that are absolutely required to form single ovoid inclusions are chaperone proteins; our evidence suggests that chaperones cooperate with other proteins in vivo to target material to IBs. The proposed work will help us better understand the interactions and additional functions of chaperone systems in the complex cellular milieu. Importantly, this project will also facilitate the continued development of the PI, as well as maintaining a highly active research lab that has provided training for 17 York College undergraduates in the past three years.

所有主要的神经退行性疾病的特征是大量聚集的 中枢神经系统中未折叠的蛋白质。在神经退行性疾病中， 亨廷顿病(HD)非常简单：HD的唯一原因是一类显性突变 Huntingtin基因，导致亨廷顿蛋白(Huntingtin Protein，Htt)中一个多谷氨酰胺重复区域的扩张。 多聚谷氨酰胺束的长度与突变型Htt的不稳定性增加和展开有关 蛋白质，引起聚集。尽管Htt蛋白在整个生命过程中在所有组织中广泛表达，但只有 细胞亚群退化；大多数细胞能够耐受突变的Htt而没有明显的不良影响。做什么 细胞应对大量未折叠的蛋白质？许多研究发现，存在大量的 聚集蛋白的包涵体与细胞死亡的相关性较差；相比之下，小分子蛋白的浓度 寡聚体更能预测细胞死亡。同样，在酿酒酵母中，形成单个 卵球形突变体Htt包裹体正常生长，而大量广泛分布的Htt聚集体对 牢房。这些观察使许多研究人员得出结论，包涵体具有细胞保护作用 收集和检疫有害小聚集性物种的细胞机制。我们的研究已经 表明mHTT包裹体是一个移动的相分离的隔室，它通过 包涵体与弥漫在细胞质中的小聚集体结合。有证据表明 包涵体(IB)的形成是有核的，即使当泛素-蛋白酶体 系统容量过剩。我们的目标是阐明包裹体的启动机制和 材料被融入其中。通常，酵母细胞产生一个卵形突变体Htt IB，但IBS失败 形成多个携带单基因缺失的菌株。两个绝对需要的基因 形式单一的卵形包涵体是伴侣蛋白；我们的证据表明伴侣蛋白与 体内的其他蛋白质以IBS为靶向物质。拟议的工作将有助于我们更好地了解 复杂细胞环境中伴侣系统的相互作用和附加功能。重要的是，这 项目还将促进PI的继续发展，以及保持高度活跃 该研究实验室在过去三年中为17名约克学院本科生提供了培训。