Regulation of the von Hippel-Lindau Protein

描述（由申请人提供）：在遗传性癌症综合征VHL患者中，von Hippel-Lindau （VHL）肿瘤抑制基因失活导致高血管性肿瘤的发展，包括肾细胞癌和半血管母细胞瘤。在大多数散发性肾癌患者中，VHL也灭活。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，靶向转录因子缺氧诱导因子-1 （HIF-1）进行蛋白酶体降解。缺乏pVHL的肿瘤在正常环境下不再降解HIF-1，导致下游基因如血管内皮生长因子（VEGF）的异常上调。虽然pVHL参与细胞对各种应激刺激的反应，但调控pVHL水平和细胞功能的因素尚不清楚。在我们的实验室，使用酵母-2杂交方法，我们已经确定Tat结合蛋白-1 （TBP-1）与pVHL相互作用。TBP-1是26S蛋白酶体的19S调节复合体的一个组成部分。

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.