Regulation of the von Hippel-Lindau Protein

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性肿瘤的发展，包括肾细胞癌和半血管母细胞瘤。 VHL在大多数散发性肾癌患者中也是失活的。 最近的研究表明，VHL蛋白（pVHL）的功能作为一个多蛋白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）的蛋白酶体降解。 缺乏pVHL的肿瘤在常氧条件下不再降解HIF-1，导致下游基因如血管内皮生长因子（VEGF）的异常上调。 虽然pVHL参与了对各种应激刺激的细胞反应，但对调节pVHL水平和细胞功能的因素知之甚少。 在我们的实验室中，使用酵母双杂交方法，我们已经确定达特结合蛋白-1（TBP-1）与pVHL相互作用。 TBP-1是26 S蛋白酶体的19 S调节复合物的组分。 初步研究表明，TBP-1结合并稳定pVHL在体内保护它免受蛋白酶体降解。 此外，TBP-1似乎增强pVHL介导的HIF 1 α降解。 我们将通过以下假设来探讨TBP-1在调节VHL功能中的作用：（1）TBP-1是pVHL功能的重要调节因子。 (2)在人类肿瘤中发现的pVHL的特定突变将干扰与TBP-1的结合，导致pVHL稳定性的降低，和（3）泛素介导的蛋白水解在控制pVHL的细胞内水平中起核心作用。 本研究的具体目的如下：（1）分析TBP-1对pVHL在缺氧等应激反应中的作用。 (2)利用酵母双杂交系统中的缺失构建体和天然存在的VHL突变体，并利用体外和体内免疫共沉淀实验，鉴定负责pVHL和TBP-1之间相互作用的蛋白结构域。（3）表征pVHL在细胞应激下的表达水平，并研究pVHL体内降解的机制。

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.