Nuclear Localization and Function of the Transmembrane Doa10 Ubiquitin Ligase

描述(由申请人提供)：泛素-蛋白酶体系统(UPS)和内质网相关降解(ERAD)的失调是许多人类疾病的特征，包括癌症、神经退行性疾病、囊性纤维化和糖尿病(1)。针对UPS的治疗干预措施仍然不发达。酵母跨膜泛素连接酶Doa10在人类中保守，催化ERAD底物和调节核蛋白的泛素化(2)。DoA10通过定位于内核膜(INM)和内质网(3)而进入底物。这项研究的主要目标是研究控制Doa10定位的因素，以及Doa10和Hex3-SLx8(一种具有重叠特异性的泛素连接酶)与其核底物相互作用的方式。这项建议的具体目的是表征Doa10定位到INM的决定因素和后果，并研究Doa10和Hex3-slx8在DNA结合底物上的作用。将采用生物化学和细胞生物学方法来实现这些目标。一个核定位信号(NLS)(定位另一个INM蛋白(4)所需)将被附加到野生型Doa10和一个笨重的Doa10融合蛋白上，该融合蛋白通常无法进入细胞核，可能是由于对非辅助核进入的大小限制(3)。将监测融合蛋白的定位，并将评估Doa10活性对其核和ER相关底物的定位改变的后果。将确定NLS修饰的蛋白质是否需要一个活跃的核运输系统才能进入核，携带NLS的可溶性蛋白质也是如此。第二，新的ChlP-seq方法将被用来确定Doa10和Hex3-slx8相互作用的染色体位点。将描述这些交互的要求和结果。这一策略还可以发现新的Doa10和Hex3-SLx8底物。这些实验极有可能为UPS相关疾病的治疗进展提供基础，这与国家普通医学研究所的使命是一致的。公共卫生相关性：许多人类疾病，如特定形式的癌症、神经退行性疾病、囊性纤维化和糖尿病，都与破坏人类细胞中蛋白质所需的细胞系统功能障碍有关(1)。这里提出的工作将提高对酵母中一种名为Doa10的重要酶的理解(它与一种功能相似的人类酶密切相关)，它与蛋白质破坏密切相关。这些研究可能有助于为蛋白质异常破坏疾病患者开发新的医学治疗方法。

DESCRIPTION (provided by applicant): Misregulation of the ubiquitin-proteasome system (UPS) and Endoplasmic Reticulum-Associated Degradation (ERAD) is characteristic of many human diseases, including cancer, neurodegenerative disorders, cystic fibrosis, and diabetes (1). Therapeutic interventions targeting the UPS remain underdeveloped. The yeast transmembrane ubiquitin ligase Doa10, conserved in humans, catalyzes ubiquitination of ERAD substrates and regulatory nuclear proteins (2). Doa10 accesses its substrates by localizing to both the inner nuclear membrane (INM) and ER (3). The broad objectives of the proposed research are to investigate factors governing the localization of Doa10 and the manner by which Doa10 and Hex3-Slx8, a ubiquitin ligase with overlapping specificity, interact with their nuclear substrates. The specific aims of this proposal are to characterize the determinants and consequences of Doa10 localization to the INM and investigate the action of Doa10 and Hex3-Slx8 on DNA-bound substrates. Biochemical and cell- biological approaches will be employed to accomplish these aims. A nuclear localization signal (NLS) (required for the localization of another INM protein (4)) will be appended to wild-type Doa10 and a bulky Doa10 fusion protein that cannot normally gain access to the nucleus, presumably due to a size restriction on unassisted nuclear entry (3). Localization of the fusion proteins will be monitored, and the consequences of altered localization for Doa10 activity toward its nuclear and ER-associated substrates will be assessed. It will be determined if the NLS-modified proteins require an active nuclear transport system for nuclear entry, as do soluble NLS-bearing proteins. Second, novel ChlP-seq methodology will be used to determine the chromosomal loci with which Doa10 and Hex3-Slx8 interact. The requirements and consequences for these interactions will be characterized. This strategy may also enable discovery of novel Doa10 and Hex3-Slx8 substrates. These experiments are highly likely to provide a foundation for therapeutic advances for UPS- associated conditions, consistent with the mission of the National Institute of General Medical Sciences. Public Health Relevance: Many human diseases, such as specific forms of cancer, neurodegenerative disorders, cystic fibrosis, and diabetes, are associated with malfunctioning of the cellular system required for the destruction of proteins in human cells (1). The work proposed here will improve the understanding of an important enzyme in yeast called Doa10 (which is closely related to a human enzyme of similar function) that is critically involved in protein destruction. These studies are likely to assist in the development of new medical treatments for patients with diseases of aberrant protein destruction.