Regulation of the von Hippel-Lindau Protein

描述(申请人提供)：在遗传性癌症综合征VHL疾病患者中，von Hippel-Lindau(VHL)肿瘤抑制基因的失活会导致高血管肿瘤的发展，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也是失活的。最近的研究表明，VHL蛋白(PVHL)是以转录因子缺氧诱导因子-1(HIF-1)为靶标的多蛋白E3泛素连接酶的一部分，用于蛋白酶体的降解。缺乏pVHL的肿瘤在常氧条件下不再降解HIF-1，导致下游基因异常上调，如血管内皮生长因子(VEGF)。虽然pVHL参与了细胞对各种应激刺激的反应，但调节细胞内pVHL水平和功能的因素却知之甚少。在我们的实验室，使用酵母-2杂交法，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19S调控复合体的一个组成部分。 初步研究表明，TBP-1通过保护pVHL免受蛋白酶体降解，在体内结合并稳定pVHL。此外，TBP-1似乎增强了pVHL介导的HIF1α的降解。我们将通过检验以下假设来探讨TBP-1在VHL功能调节中的作用：(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的特异性突变会干扰与TBP-1的结合，导致pVHL的稳定性降低；(3)泛素介导的蛋白降解在控制pVHL的细胞内水平方面起着核心作用。本研究的具体目标如下：(1)分析TBP-1在细胞对低氧和其他应激刺激的反应中对pVHL功能的影响。(2)利用酵母-2杂交系统中的缺失结构和自然发生的VHL突变，以及体内外免疫共沉淀实验，确定pVHL与TBP-1相互作用的蛋白质结构域；(3)研究pVHL在细胞应激条件下的表达水平，并探讨pVHL在体内的降解机制。

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.