Functional Studies of Ubiquilin

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泛素-蛋白酶体系统中错误折叠蛋白质和缺陷的参与。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-蛋白酶体系统还参与蛋白质展开、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型蛋白质的创始成员，它似乎抑制了蛋白质的降解。泛素含有多个泛素相关基序，通常存在于泛素-蛋白酶体系统中的蛋白质中。我们通过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。细胞中泛素的过度表达增加了早老素蛋白水平，降低了早老素蛋白内溶N端和c端片段的水平，并降低了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，泛素的表达在细胞应激过程中被诱导，它可能作为分子伴侣、泛素受体或在细胞存活中起作用。

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.