Identifying and characterizing protein quality control mechanisms in the nucleus

描述（由申请人提供）：受损或错误折叠蛋白质的积累会对细胞生理和活力产生破坏性影响。为了保护自己，细胞拥有许多蛋白质质量控制（PQC）系统，以尽量减少这些异常蛋白质的持久性和有害影响。虽然各种PQC系统在细胞质、内质网和线粒体中已经被表征，但细胞核如何管理异常蛋白却知之甚少。但是，了解核生物学的这个基本方面是很重要的，因为许多与年龄相关的神经肌肉疾病（亨廷顿氏病，肯尼迪氏病，几种脊髓-小脑共济失调）是由细胞核中异常蛋白质的毒性积累引起的。因此，我们的长期目标是通过识别在细胞核中运作的PQC系统，描述这些系统如何识别和靶向异常蛋白，检查核PQC系统中的病变是否需要这些病理发展，以及确定被异常核蛋白破坏导致毒性的特定核过程，从而了解细胞核通常如何保护自己免受有毒异常蛋白的侵害。从我们对酿酒酵母的初步研究中，我们已经确定了第一个已知的在细胞核中起作用的PQC降解系统。这个核PQC系统的核心参与者是San1，这是一种核定位的泛素蛋白连接酶，针对异常核蛋白进行泛素化和蛋白酶体降解。随着San1的发现，我们现在可以很好地探索核PQC降解系统如何识别其底物，以及它识别这些底物中的异常。这笔拨款的具体目的是：(1)确定San1中负责底物靶向的区域。San1的突变将用于识别对底物识别重要的顺式区域。(2)确定被San1识别的异常蛋白的特征。San1底物的诱变将用于确定San1靶向所需的区域。(3)识别和表征San1通路的其他成分。遗传和生化方法的结合将用于鉴定额外的San1途径成分。公共卫生相关性：许多与年龄相关的神经肌肉疾病（亨廷顿氏病、肯尼迪氏病、几种脊髓-小脑共济失调和眼咽肌营养不良等）是由细胞核中易聚集蛋白的毒性积累引起的。我们的目标是了解细胞核如何在正常情况下保护自己免受有毒聚集蛋白的侵害，以及为什么它在这些疾病和其他疾病中无法做到这一点。

DESCRIPTION (provided by applicant): The accumulation of damaged or misfolded proteins can have devastating effects on cellular physiology and viability. To protect itself, the cell possesses numerous Protein Quality Control (PQC) systems that minimize the persistence and detrimental effects of these aberrant proteins. While a variety of PQC systems have been characterized in the cytoplasm, ER, and mitochondria, how the nucleus manages aberrant proteins is poorly understood. But, understanding this fundamental aspect of nuclear biology is important because many age-correlated neuromuscular disorders (Huntington's, Kennedy's, several spinal-cerebellar ataxias) result from toxic accumulation of aberrant proteins in the nucleus. Therefore, our long-term goals are to understand how the nucleus normally protects itself from toxic aberrant proteins by identifying PQC systems that operate in the nucleus, characterizing how these systems recognize and target aberrant proteins, examining if lesions in nuclear PQC systems are required for these pathologies to develop, and determining the specific nuclear processes that are disrupted by aberrant nuclear proteins to cause toxicity. From our initial studies in S. cerevisiae, we've identified the first-known PQC degradation system that acts in the nucleus. The central player of this nuclear PQC system is San1, a nuclear-localized ubiquitin- protein ligase that targets aberrant nuclear proteins for ubiquitination and proteasome degradation. With the discovery of San1, we're now well positioned to explore how a nuclear PQC degradation system recognizes its substrates, and what it recognizes as aberrant within those substrates. The specific aims for this grant are to: (1) Determine the regions within San1 responsible for substrate targeting. Mutagenesis of San1 will be used to identify in cis regions important for substrate recognition. (2) Determine the features of an aberrant protein recognized by San1. Mutagenesis of San1 substrates will be used to identify regions required for targeting by San1. (3) Identify & characterize additional components of the San1 pathway. A combination of genetic and biochemical approaches will be used to identify additional San1 pathway components. PUBLIC HEALTH RELEVANCE: Many age-correlated neuromuscular disorders (Huntington's, Kennedy's, several spinal-cerebellar ataxias, and oculopharyngeal muscular dystrophy to name a few) result from toxic accumulation of aggregation-prone proteins in the cell's nucleus. Our goals are to understand how the nucleus normally protects itself from toxic aggregation-prone proteins, and why it fails to do so in these and other diseases.