Functional Studies of Ubiquilin

从神经退行性疾病研究中出现的一个共同主题是 蛋白质的错误折叠和泛素-蛋白酶体系统的缺陷。泛素-蛋白酶体系统是 最初被认为是标记和销毁不需要的蛋白质的机器。然而，最近 有证据表明，泛素-蛋白酶体系统也参与了蛋白质在细胞内的展开 I蛋白靶向、细胞信号和转录。我们的实验室确认了泛比奎林，它是 一类令人兴奋的新蛋白质，似乎能抑制蛋白质的降解。泛素含有多个 泛素相关基序通常存在于泛素-蛋白酶体系统中涉及的蛋白质中。我们确认了 泛素通过与早老素蛋白的相互作用，其突变与早发性相关 家族性阿尔茨海默病。泛素在细胞中的过表达增加了早老素的蛋白水平， 降低内源性蛋白水解性N-末端和C-末端早老素片段的水平，并减少泛素化 早老素蛋白。包括我们实验室的结果在内的几条证据表明，泛素 表达是在细胞应激过程中诱导的，它可能作为一种分子伴侣发挥作用， 泛素受体，或在细胞生存中。 我们建议使用多管齐下的方法来确定泛素蛋白在细胞和 有机体。利用细胞、分子和免疫学技术，我们将确定其表达 不同泛素蛋白在组织和组织培养细胞以及细胞内的分布 不同泛素亚型的定位特性。我们将确定泛素的不同域如何 多肽都参与了蛋白质的功能。我们将使用两种转染法以及一种 体外细胞自由翻译实验，以确定泛素在泛素-蛋白酶体系统中的作用。我们会 用免疫共沉淀法和酵母双杂交试验鉴定泛素相互作用蛋白。 最后，我们建议在小鼠中使用基因敲除，在线虫和人类组织中使用反义抑制 细胞培养和抗体中和，以确定泛素缺失对细胞和 有机体。拟议研究的结果将有助于更好地了解 泛素在细胞和整个生物体中的功能作用，最终，它在健康和疾病中的作用。

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 Iprotein 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 familia_ 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 celt-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 have 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.