Regulation of the von Hippel-Lindau Protein

von Hippel-Lindau 蛋白的调节

• 批准号: 6937249
• 负责人: Paul G. Corn
• 金额: $ 12.77万
• 依托单位: UNIVERSITY OF TX MD ANDERSON CAN CTR
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-09-10 至 2008-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6937249
• 关键词: SDS polyacrylamide gel electrophoresis; Von Hippel Lindau syndrome; binding proteins; binding sites; hypoxia; hypoxia inducible factor 1; immunofluorescence technique; immunoprecipitation; neoplasm /cancer genetics; oligonucleotides; proteasome; protein protein interaction; protein structure function; proteolysis; renal cell carcinoma; tumor suppressor genes; ubiquitin; vascular endothelial growth factors; yeast two hybrid system

DESCRIPTION (provided by applicant): In patients with the hereditary cancer syndrome VHL disease, inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene leads to the development of highly vascular tumors, including renal cell carcinomas and hemiangioblastomas. VHL is also inactivated in a majority of patients with sporadic renal carcinoma. Recent studies have suggested that the VHL protein (pVHL) functions as part of a multi-protein E3 ubiquitin ligase that targets the transcription factor Hypoxia-inducible factor-1 (HIF-1) for proteasomal degradation. Tumors lacking pVHL no longer degrade HIF-1 under normoxic conditions, leading to the aberrant upregulation of downstream genes such as vascular endothelial growth factor (VEGF). While pVHL is involved in the cellular response to various stress stimuli, factors that regulate pVHL levels and function in the cell are poorly understood. In our laboratory, using a yeast-2-hybrid approach, we have determined that Tat binding protein-1 (TBP-1) interacts with pVHL. TBP-1 is a component of the 19S regulatory complex of the 26S proteasome. Preliminary studies suggest that TBP-1 binds to and stabilizes pVHL in vivo by protecting it from proteasomal degradation. In addition TBP-1 appears to potentiate pVHL-mediated degradation of HIF1alpha. We will explore the role of TBP-1 in regulating VHL function by testing the following hypotheses: (1) TBP-1 acts as an important regulator of pVHL function. (2) Specific mutations of pVHL found in human tumors will interfere with binding to TBP-1, leading to a reduction in the stability of pVHL, and (3) Ubiquitin-mediated proteolysis plays a central role in controlling intracellular levels of pVHL. The following specific aims will be pursued: (1) To analyze the effects of TBP-1 on pVHL function during the cellular response to hypoxia and other stressful stimuli. (2) To identify the protein domains responsible for the interaction between pVHL and TBP-1 using deletion constructs and naturally occurring VHL mutations in a yeast-2-hybrid system, and using co-immunoprecipitation experiments in vitro and in vivo, (3) To characterize expression levels of pVHL under cellular stress and examine the mechanism for pVHL degradation in vivo.

描述（由申请人提供）：在患有遗传性癌症综合征 VHL 疾病的患者中，von Hippel-Lindau (VHL) 肿瘤抑制基因的失活导致高度血管肿瘤的发展，包括肾细胞癌和血管母细胞瘤。 大多数散发性肾癌患者的 VHL 也失活。 最近的研究表明，VHL 蛋白 (pVHL) 作为多蛋白 E3 泛素连接酶的一部分发挥作用，该连接酶以转录因子缺氧诱导因子-1 (HIF-1) 为目标进行蛋白酶体降解。 缺乏 pVHL 的肿瘤在常氧条件下不再降解 HIF-1，导致血管内皮生长因子 (VEGF) 等下游基因异常上调。 虽然 pVHL 参与细胞对各种应激刺激的反应，但调节 pVHL 水平和细胞功能的因素却知之甚少。 在我们的实验室中，使用酵母 2 杂交方法，我们确定了 Tat 结合蛋白 1 (TBP-1) 与 pVHL 相互作用。 TBP-1 是 26S 蛋白酶体 19S 调控复合物的组成部分。 初步研究表明，TBP-1 通过保护 pVHL 免受蛋白酶体降解而在体内结合并稳定 pVHL。 此外，TBP-1 似乎可以增强 pVHL 介导的 HIF1α 降解。 我们将通过检验以下假设来探讨TBP-1在调节VHL功能中的作用：（1）TBP-1作为pVHL功能的重要调节剂。 (2)人类肿瘤中发现的pVHL的特异性突变会干扰与TBP-1的结合，导致pVHL稳定性降低；(3)泛素介导的蛋白水解在控制pVHL的细胞内水平中发挥核心作用。 我们将追求以下具体目标：（1）分析TBP-1在细胞对缺氧和其他应激刺激的反应过程中对pVHL功能的影响。 (2) 使用酵母 2 杂交系统中的缺失构建体和天然发生的 VHL 突变，并使用体外和体内免疫共沉淀实验来鉴定负责 pVHL 和 TBP-1 之间相互作用的蛋白质结构域，(3)表征细胞应激下 pVHL 的表达水平并检查 pVHL 体内降解机制。