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可能对细胞生理学和活力产生破坏性影响。为了保护自身，细胞拥有众多的蛋白质质量控​​制 (PQC) 系统，可以最大限度地减少这些异常蛋白质的持久性和有害影响。虽然细胞质、内质网和线粒体中的多种 PQC 系统已得到表征，但人们对细胞核如何管理异常蛋白质却知之甚少。但是，了解核生物学的这一基本方面非常重要，因为许多与年龄相关的神经肌肉疾病（亨廷顿舞蹈症、肯尼迪舞蹈症、几种脊髓小脑共济失调）都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行的 PQC 系统，了解细胞核通常如何保护自身免受有毒异常蛋白的侵害，描述这些系统如何识别和靶向异常蛋白，检查核 PQC 系统中的损伤是否是这些病理学发展所必需的，并确定被异常核蛋白破坏以引起毒性的特定核过程。通过对酿酒酵母的初步研究，我们发现了第一个已知的在细胞核中起作用的 PQC 降解系统。该核 PQC 系统的核心角色是 San1，它是一种核定位泛素蛋白连接酶，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内容。这笔资助的具体目标是： (1) 确定 San1 内负责底物靶向的区域。 San1 的诱变将用于识别对底物识别重要的顺式区域。 (2)确定San1识别的异常蛋白的特征。 San1 底物的诱变将用于识别 San1 靶向所需的区域。 (3) 识别并表征 San1 途径的其他成分。将结合遗传和生化方法来鉴定其他 San1 通路成分。公共健康相关性：许多与年龄相关的神经肌肉疾病（亨廷顿舞蹈症、肯尼迪舞蹈症、几种脊髓小脑共济失调和眼咽肌营养不良症等）都是由细胞核中易于聚集的蛋白质的毒性积累引起的。我们的目标是了解细胞核通常如何保护自身免受有毒聚集倾向蛋白质的侵害，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.