Nuclear Localization and Function of the Transmembrane Doa10 Ubiquitin Ligase

跨膜 Doa10 泛素连接酶的核定位和功能

• 批准号: 7943917
• 负责人: Eric Meyer Rubenstein
• 金额: $ 5.05万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-01 至 2012-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7943917
• 关键词: Biochemical; Biological; Cell Nucleus; Cells; Characteristics; Chimeric Proteins; Cystic Fibrosis; DNA Binding; Development; Diabetes Mellitus; Disease; Endoplasmic Reticulum; Enzymes; Foundations; Genomics; Human; Malignant Neoplasms; Mediating; Medical; Membrane Proteins; Methodology; Mission; Monitor; National Institute of General Medical Sciences; Neurodegenerative Disorders; Nuclear; Nuclear Envelope; Nuclear Inner Membrane; Nuclear Localization Signal; Nuclear Protein; Nuclear Proteins; Patients; Proteins; Research; Site; Specificity; System; Therapeutic; Therapeutic Intervention; Ubiquitin; Ubiquitination; Work; Yeasts; human disease; improved; multicatalytic endopeptidase complex; novel; nucleocytoplasmic transport; public health relevance; research study; ubiquitin ligase

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.

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完善。人类中保守的酵母跨膜泛素连接酶 Doa10 催化 ERAD 底物和调节性核蛋白的泛素化 (2)。 Doa10 通过定位于内核膜 (INM) 和 ER 来访问其底物 (3)。拟议研究的主要目标是调查控制 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。该提案的具体目的是描述 Doa10 定位到 INM 的决定因素和后果，并研究 Doa10 和 Hex3-Slx8 对 DNA 结合底物的作用。将采用生物化学和细胞生物学方法来实现这些目标。核定位信号 (NLS)（定位另一种 INM 蛋白 (4) 所需）将附加到野生型 Doa10 和大体积 Doa10 融合蛋白上，该蛋白通常无法进入细胞核，可能是由于无辅助入核的大小限制 (3)。将监测融合蛋白的定位，并评估 Doa10 活性对其核和 ER 相关底物定位改变的后果。将确定 NLS 修饰蛋白是否需要活性核转运系统才能进入核，就像可溶性 NLS 承载蛋白一样。其次，新的 ChlP-seq 方法将用于确定 Doa10 和 Hex3-Slx8 相互作用的染色体位点。这些相互作用的要求和后果将得到表征。该策略还可能有助于发现新型 Doa10 和 Hex3-Slx8 底物。这些实验很可能为 UPS 相关疾病的治疗进展奠定基础，这与国家普通医学科学研究所的使命一致。公共健康相关性：许多人类疾病，例如特定形式的癌症、神经退行性疾病、囊性纤维化和糖尿病，都与破坏人体细胞中蛋白质所需的细胞系统功能障碍有关 (1)。这里提出的工作将增进对酵母中一种名为 Doa10 的重要酶（与具有类似功能的人类酶密切相关）的理解，该酶在蛋白质破坏中发挥着至关重要的作用。这些研究可能有助于开发针对蛋白质破坏异常疾病患者的新疗法。