Yeast Model for Two Neurodegeneration-Linked Proteins

两种神经变性相关蛋白的酵母模型

• 批准号: 6756139
• 负责人: SHUBHIK KUMAR DEBBURMAN
• 金额: $ 19.58万
• 依托单位: LAKE FOREST COLLEGE
• 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/6756139
• 关键词: Saccharomyces cerevisiae; Schizosaccharomyces pombe; alpha synuclein; chemical models; chemical stability; conformation; fluorescence microscopy; fungal proteins; gene mutation; green fluorescent proteins; immunofluorescence technique; immunoprecipitation; model design /development; molecular chaperones; nerve /myelin protein; neural degeneration; proteasome; protein degradation; protein folding; protein localization; radiotracer; transfection /expression vector; ubiquitin

DESCRIPTION (provided by applicant): Budding Yeast (S. cerevisiae) has emerged as a powerful model system for understanding molecular aspects of many human diseases. Protein misfolding linked to certain neurodegenerative diseases (NDDs) like Huntington Disease, Lou Gehrig's disease, and prion diseases have been successfully recapitulated in S. cerevisiae and led to identification of therapeutically relevant regulators of misfolding. No S. cerevisiae models for Parkinson's Disease (PD) or dentatorubral pallidoluysian atrophy (DRPLA) have been reported. PD is one of the most common NDDs, while DRPLA is a rare inherited NDD of the triplet repeat disease family. In both diseases, misfolding of a specific protein (alpha-synuclein for PD and atrophin for DRPLA) is thought to cause selective neuronal death. Unlike the well-characterized huntingtin protein in Huntington Disease (which shares many similarities to DRPLA), less is known about the misfolding of mutant atrophin in DRPLA. A S. cerevisiae expression system for studying alpha-synuclein has recently been developed in our lab. Preliminary evidence supports that both wildtype and disease-associated mutants are aggregating within yeast cells and upon purification. A similar effort to establish atrophin-1 expression in yeast is underway. To extend initial observations with alpha-synuclein in yeast and fully develop a yeast model for atrophin, three goals are proposed. 1) Misfolding properties between wildtype and mutant versions of both proteins will be investigated in vivo (immunofluorescence and GFP-based localization and assessment of protein half-life) and in vitro (by measuring protease sensitivity and differential solubility). 2) Influences of chaperones and ubiquitin-proteasomal pathway proteins on folding and degradation of these proteins will be assessed in strains compromised for chaperone/proteasomal function, or those that overexpress chaperones, and by co-immunoprecipitation assessment. 3) A fission yeast (S. pombe) expression model for alpha-synuclein and atrophin properties (as in Aim 1) will be developed and compared with the S. cerevisiae model; NDD models have not been reported in S. pombe. These studies may further clarify the molecular bases for misfolding and degradation of PD- and DRPLA-linked proteins and extend the usefulness of yeast models. Importantly, the scientific training of many undergraduates will be supported, strengthening their cell biology and molecular genetics skills and appreciation for model organisms.

描述（由申请人提供）：芽殖酵母（S. cerevisiae）已成为了解许多人类疾病分子方面的强大模型系统。与亨廷顿病、卢伽雷氏病和朊病毒病等某些神经退行性疾病 (NDD) 相关的蛋白质错误折叠已在酿酒酵母中成功重现，并导致鉴定出与治疗相关的错误折叠调节因子。尚未报道帕金森病 (PD) 或齿状红核苍白球路易体萎缩症 (DRPLA) 的酿酒酵母模型。 PD是最常见的NDD之一，而DRPLA是三联体重复疾病家族中罕见的遗传性NDD。在这两种疾病中，特定蛋白质（PD 的α-突触核蛋白和 DRPLA 的萎缩蛋白）的错误折叠被认为会导致选择性神经元死亡。与亨廷顿病中众所周知的亨廷顿蛋白（与 DRPLA 有许多相似之处）不同，人们对 DRPLA 中突变型萎缩蛋白的错误折叠知之甚少。我们的实验室最近开发了用于研究α-突触核蛋白的酿酒酵母表达系统。初步证据支持野生型和疾病相关突变体在酵母细胞内和纯化后聚集。一项类似的努力也在酵母中建立 atropin-1 的表达。为了扩展酵母中α-突触核蛋白的初步观察并充分开发萎缩蛋白的酵母模型，提出了三个目标。 1) 将在体内（免疫荧光和基于 GFP 的蛋白质半衰期的定位和评估）和体外（通过测量蛋白酶敏感性和差异溶解度）研究两种蛋白质的野生型和突变体版本之间的错误折叠特性。 2) 伴侣和泛素-蛋白酶体途径蛋白对这些蛋白质折叠和降解的影响将在伴侣/蛋白酶体功能受损的菌株或过度表达伴侣的菌株中进行评估，并通过免疫共沉淀评估进行评估。 3) 将开发α-突触核蛋白和萎缩蛋白特性（如目标 1）的裂殖酵母（S. pombe）表达模型，并与酿酒酵母模型进行比较； NDD 模型尚未在粟酒裂殖酵母中报道。这些研究可能会进一步阐明 PD 和 DRPLA 连接蛋白错误折叠和降解的分子基础，并扩展酵母模型的用途。重要的是，许多本科生的科学培训将得到支持，加强他们的细胞生物学和分子遗传学技能以及对模式生物的欣赏。