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）：出现的酵母（S. cerevisiae）已成为理解许多人类疾病分子方面的强大模型系统。与某些神经退行性疾病（NDD）相关的蛋白质错误折叠，例如亨廷顿疾病，Lou Gehrig病和prion病，在酿酒酵母中已成功概括，并导致鉴定出与误后折叠的治疗相关调节剂的鉴定。尚未据报道帕金森氏病（PD）或牙齿牙脂脂脂萎缩症（DRPLA）的酿酒酵母模型。 PD是最常见的NDD之一，而Drpla是三胞胎重复疾病家族的罕见遗传NDD。在两种疾病中，都认为特定蛋白质（PD的α-突触核蛋白和Drpla的脂蛋白）的错误折叠被认为会导致选择性的神经元死亡。与亨廷顿疾病中特征良好的亨廷顿蛋白（与Drpla具有许多相似之处）不同，对Drpla中突变型脂蛋白的折叠折箱的了解较少。最近在我们的实验室中开发了用于研究α-核蛋白的酿酒酵母表达系统。初步证据支持野生型和与疾病相关的突变体在酵母细胞中汇总并在纯化后聚集。正在进行类似的努力，以在酵母中建立Atrophin-1表达。为了扩展酵母中α-核蛋白的初始观察结果，并完全开发了脂蛋白的酵母模型，提出了三个目标。 1）将研究两种蛋白质的野生型和突变版本之间的错误折叠特性，并在体内进行研究（免疫荧光和基于GFP的定位以及蛋白质半寿命的评估）和体外（通过测量蛋白酶敏感性和差异可溶性）。 2）伴侣蛋白和泛素 - 蛋白酶体途径蛋白对这些蛋白质的折叠和降解的影响将以伴侣/蛋白酶体功能损害的菌株进行评估，或者通过过表达伴侣蛋白的功能，以及通过共免疫收集评估评估。 3）将开发出α-突触核蛋白和脂蛋白特性的裂变酵母（S. pombe）表达模型（如AIM 1中），并与酿酒酵母模型进行比较； NDD模型尚未在S. pombe中报告。这些研究可能会进一步阐明PD和DRPLA连接蛋白的错误折叠和降解的分子碱基，并扩展酵母模型的有用性。重要的是，将支持许多本科生的科学训练，从而增强其细胞生物学和分子遗传学技能以及对模型生物的欣赏。