A New E3 Ligase Implicated in Protein Quality Control and Neurodegeneration

描述(由申请人提供)：背景和相关性：这项基础研究提案的目标是在分子水平上更好地了解神经退行性疾病的原因。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)。神经退行性疾病是一种无法治愈的疾病，随着患病率的增加而变得虚弱，如果没有足够详细的致病机制被阐明，我们产生纠正治疗干预的理论基础的能力就会受到极大的限制。我们之前描述了一种新的由Lister基因突变引起的神经退行性变的小鼠模型。与重要的生物学功能一致，该基因在从酵母到人类的所有生物中都是保守的。因此，我们一直在利用各种模型来发现基因功能以及该功能的缺陷如何导致疾病。Lister基因编码一种蛋白质，它充当E3泛素连接酶，名为Listerin。最近，我们使用酵母发现了Listerin的一个功能，这与哺乳动物神经退化中的一个角色是一致的：它在一个被称为蛋白质质量控制的过程中发挥作用，通过这个过程，异常蛋白质被定位为降解的目标。具体地说，它的靶标是由缺失终止密码子的有缺陷的信使核糖核酸(“非停止蛋白”)编码的蛋白质。Listerin通过用泛素(泛素化)分子标记这些异常蛋白来发挥作用， 这通常是一个毁灭的信号。李斯特林的突变会导致这些有毒蛋白质的积累。通过这项拟议的研究，我们计划明确李斯特林介导的泛素化的特征，并将研究结果扩展到疾病。目的/假设：我们的主要假设是：(A)Listerin通过泛化停滞在核糖体中的不间断多肽而在蛋白质质量控制中发挥作用，以及(B)Listerin介导的缺陷降解导致蛋白质聚集体和包涵体的形成，这是神经退行性变的标志。其具体目的是表征Listerin在蛋白质不间断降解中的功能，研究Listerin在蛋白质质量控制中功能的保守程度，以及该功能的缺陷是如何导致神经退化的。我们的长期目标是帮助阐明涉及人类神经退行性疾病发病机制的分子机制。研究设计：我们将利用生物化学和酵母分子遗传学来确定E3如何识别其特定的靶底物；利用哺乳动物组织培养和生物化学来研究哺乳动物Listerin对不间断蛋白质降解的调节；以及生物化学和细胞生物学来研究不间断蛋白质在细胞中积累的后果。 公共卫生相关性：这项基础研究计划的目标是确定一种与神经退行性变有关的新基因的功能及其在疾病中的作用。在分子水平上更好地了解神经退行性疾病的原因有望为开发新的治疗原理和方法(例如，通过发现新的药物靶点)开辟道路。

DESCRIPTION (provided by applicant): 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 therapeutc 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, it 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. PUBLIC HEALTH RELEVANCE: The goal of this basic research proposal is to characterize the function of a new gene implicated in neurodegeneration, and its role in disease. The better understanding of the causes of neurodegenerative disorders at the molecular level is expected to open the way to the development of new therapeutic rationale and approaches (e.g., by discovering new drug targets).