Functional Studies of Ubiquilin

泛素的功能研究

• 批准号: 6631110
• 负责人: Mervyn J Monteiro
• 金额: $ 29.4万
• 依托单位: UNIVERSITY OF MD BIOTECHNOLOGY INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-05-01 至 2007-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6631110
• 关键词: Caenorhabditis elegans; antibody neutralization test; cell free system; green fluorescent proteins; human tissue; immunoprecipitation; laboratory mouse; laboratory rat; molecular cloning; northern blottings; nucleic acid sequence; polymerase chain reaction; proteasome; protein degradation; protein isoforms; protein localization; protein structure function; site directed mutagenesis; tissue /cell culture; transfection; ubiquitin; western blottings; yeast two hybrid system

DESCRIPTION (provided by applicant): A common theme that is emerging from studies of neurodegenerative disorders is the involvement of misfolded proteins and defects in the ubiquitin-proteasome system. The ubiquitin-proteasome system was originally considered to be the machinery for tagging and destroying unwanted proteins. However, recent evidence indicates that the ubiquitin-proteasome system is also involved in protein unfolding, intracellular protein targeting, cell signaling and transcription. Our laboratory identified ubiquilin, the founding member of an exciting new class of proteins, which appears to inhibit degradation of proteins. Ubiquilin contains multiple ubiquitin-related motifs typically found in proteins involved in the ubiquitin-proteasome system. We identified ubiquilin through its interactions with presenilin proteins, mutations in which are associated with early-onset familial Alzheimer's disease. Overexpression of ubiquilin in cells increases presenilin protein levels, decreases levels of endoproteolytic N- and C-terminal presenilin fragments, and decreases ubiquitination of presenilin proteins. Several lines of evidence, including results from our laboratory, suggest that ubiquilin expression is induced during cell stress and that it may function as a molecular chaperone, a ubiquitin-receptor, or in cell survival. We propose to use a multi-pronged approach to determine the role ubiquilin proteins play in cells and organisms. Using cellular, molecular, and immunological techniques, we will determine the expression patterns of the different ubiquilin proteins in tissues and tissue culture cells as well as the intracellular localization properties of different ubiquilin isotypes. We will determine how different domains of the ubiquilin polypeptide are involved in the functions of the protein. We will use both transfection assays, as well as an in vitro cell-free translation assay, to identify the role ubiquilin plays in the ubiquitin-proteasome system. We will characterize ubiquilin-interacting proteins using co-immunoprecipitation and yeast two-hybrid assays. Finally, we propose to use gene knockout in mouse, anti-sense inhibition in C. elegans and human tissue culture cells, and antibody neutralization to identify the effects that loss of ubiquilin has in cells and organisms. The results obtained from the proposed research will lead to a better understanding of the functional role of ubiquilin in cells and in whole organisms, and ultimately, its role in health and in disease.

描述（由申请人提供）：神经退行性疾病研究中出现的一个共同主题是错误折叠蛋白和泛素-蛋白酶体系统缺陷的参与。泛素-蛋白酶体系统最初被认为是标记和破坏不需要的蛋白质的机器。然而，最近的证据表明，泛素-蛋白酶体系统也参与蛋白质解折叠，细胞内蛋白质靶向，细胞信号传导和转录。我们的实验室发现了泛素，这是一种令人兴奋的新蛋白质的创始成员，它似乎可以抑制蛋白质的降解。泛素包含多个泛素相关的基序，通常存在于参与泛素-蛋白酶体系统的蛋白质中。我们通过泛素与早老素蛋白的相互作用确定了泛素，早老素蛋白的突变与早发性家族性阿尔茨海默病有关。泛素在细胞中的过表达增加早老素蛋白水平，降低内蛋白水解的N-和C-末端早老素片段的水平，并降低早老素蛋白的泛素化。包括我们实验室的结果在内的几条证据表明，在细胞应激过程中，泛素的表达被诱导，并且它可能作为分子伴侣、泛素受体或在细胞存活中起作用。 我们建议使用多管齐下的方法来确定泛素蛋白在细胞和生物体中的作用。利用细胞、分子和免疫学技术，我们将确定不同泛素蛋白在组织和组织培养细胞中的表达模式以及不同泛素同种型的细胞内定位特性。我们将确定泛素多肽的不同结构域如何参与蛋白质的功能。我们将使用两种转染试验，以及在体外无细胞翻译试验，以确定在泛素蛋白酶体系统中的作用ubiquilin。我们将使用免疫共沉淀和酵母双杂交检测来表征泛素相互作用蛋白。最后，我们建议在小鼠中采用基因敲除，在C. elegans和人类组织培养细胞，以及抗体中和，以鉴定细胞和生物体中泛素缺失的影响。从拟议的研究中获得的结果将导致更好地了解泛素在细胞和整个生物体中的功能作用，并最终了解其在健康和疾病中的作用。