A New E3 Ligase Implicated in Protein Quality Control and Neurodegeneration

7.项目总结 背景和相关性：这项基础研究建议的目标是更好地了解 分子水平上的神经退行性疾病。其中最著名的例子是阿尔茨海默氏症， 帕金森氏症和亨廷顿氏症，以及肌萎缩侧索硬化症(ALS)。神经退行性疾病 是无法治愈和衰弱的疾病，患病率不断上升，并且没有其致病机制 在充分详细地阐明之后，我们产生纠正性治疗干预的理由的能力是 非常有限。我们之前已经描述了一种新的小鼠神经退行性变模型，该模型由 李斯特基因的突变。该基因与一种重要的生物学功能一致，在ALL中是保守的。 从酵母菌到人类。因此，我们一直在利用各种模型来发现这种基因 以及这一功能的缺陷如何导致疾病。LISTER基因编码一种蛋白质， 作为一种E3泛素连接酶，命名为Listerin。最近，我们使用酵母发现了一种用于列表的功能 这与哺乳动物神经退化中的一个作用是一致的：它在一个被称为蛋白质的过程中发挥作用 以异常蛋白质为目标进行降解的质量控制。具体地说，它的目标是蛋白质 由缺失终止密码子的有缺陷的信使核糖核酸(“非停止蛋白”)编码。Listerin函数通过标记 含有泛素(泛素化)分子的异常蛋白质，这通常起到破坏信号的作用。突变 会导致这些有毒蛋白质堆积。在拟议的研究中，我们计划具体说明 Listerin介导的泛素化的特征，并将发现扩展到疾病。 目标/假设：我们的主要假设是：(A)李斯特林通过以下途径参与蛋白质质量控制 核糖体中停滞的泛素化不间断多肽，以及(B)缺陷Listerin介导的降解 导致蛋白质聚集体和包涵体的形成，这是神经退化的标志。 其具体目的是表征Listerin在不间断蛋白质降解中的功能，研究 Listerin功能在蛋白质质量控制中的保守程度，并调查这种缺陷是如何 功能会导致神经退化。我们的长期目标是帮助阐明分子机制 参与人类神经退行性疾病的发病机制。 研究设计：我们将使用生物化学和酵母分子遗传学来确定E3是如何识别其 特异性靶标底物；哺乳动物组织培养和生化研究不间断调节 哺乳动物李斯特蛋白降解与生物化学和细胞生物学研究的后果 蛋白质在细胞内不间断地积累。

7. PROJECT SUMMARY Background and relevance: The goal of this basic research proposal is to better understand the causes of neurodegenerative disorders at the molecular level. Among the best known examples are Alzheimer's, Parkinson's and Huntington's diseases, and Amyotrophic Lateral Sclerosis (ALS). Neurodegenerative diseases are incurable and debilitating conditions with increasing prevalence, and without their pathogenic mechanisms being elucidated in sufficient detail, our ability to generate a rationale for corrective therapeutic interventions is greatly limited. We had previously characterized a novel mouse model of neurodegeneration caused by mutation of the LISTER gene. Consistent with an important biological function, this gene is conserved in all organisms from yeast to humans. Accordingly, we have been utilizing various models to discover the gene function and how defects in this function may lead to the disease. The LISTER gene encodes a protein that acts as an E3 ubiquitin ligase, named Listerin. More recently, using yeast we discovered a function for Listerin that is consistent with a role in neurodegeneration in mammals: it functions in a process known as protein quality control whereby aberrant proteins are targeted for degradation. Specifically, its targets are proteins encoded by defective mRNA lacking stop codons ("non-stop proteins"). Listerin functions by tagging those aberrant proteins with molecules of ubiquitin (ubiquitination), which often acts as a destruction signal. Mutation of Listerin causes those toxic proteins to accumulate. With the proposed research we plan to specify the features of Listerin-mediated ubiquitination and expand the findings towards disease. Objective/Hypothesis: Our main hypotheses are that (a) Listerin acts in protein quality control by ubiquitinating non-stop polypeptides stalled in ribosomes, and (b) defective Listerin-mediated degradation leads to the formation of protein aggregates and inclusions, which are hallmarks of neurodegeneration. The Specific Aims are to characterize Listerin's function in non-stop protein degradation, to investigate the extent of conservation of Listerin's function in protein quality control, and to investigate how defects in this function lead to neurodegeneration. Our long-term objective is to help elucidate molecular mechanisms involved in the pathogenesis of human neurodegenerative disease. Study design: We will use biochemistry and yeast molecular genetics to specify how the E3 recognizes its specific target substrates; mammalian tissue culture and biochemistry to investigate the regulation of non-stop protein degradation by mammalian Listerin and biochemistry and cell biology to study the consequences of non-stop protein accumulation in cells.