Regulation of the von Hippel-Lindau Protein

von Hippel-Lindau 蛋白的调节

• 批准号: 7128117
• 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/7128117
• 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）肿瘤抑制基因失活会导致高度血管肿瘤的发展，包括肾脏细胞癌和半肌母细胞瘤。 在大多数零星肾癌患者中，VHL也被灭活。 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，该连接酶靶向蛋白酶体降解的转录因子低氧诱导因子1（HIF-1）。 缺乏PVHL的肿瘤在常氧化条件下不再降解HIF-1，导致下游基因的异常上调，例如血管内皮生长因子（VEGF）。 虽然PVHL参与了对各种应激刺激的细胞反应，但对细胞中PVHL水平和功能的因素的了解很少。 在我们的实验室中，使用酵母2杂交方法，我们确定TAT结合蛋白-1（TBP-1）与PVHL相互作用。 TBP-1是26S蛋白酶体19s调节复合物的组成部分。 初步研究表明，TBP-1通过保护其免受蛋白酶体降解而与体内结合并稳定PVHL。 另外，TBP-1似乎可以增强PVHL介导的HIF1ALPHA降解。 我们将通过测试以下假设来探讨TBP-1在调节VHL功能中的作用：（1）TBP-1充当PVHL功能的重要调节剂。 （2）在人类肿瘤中发现的PVHL的特异性突变将干扰与TBP-1的结合，从而导致PVHL稳定性降低，（3）泛素介导的蛋白水解在控制PVHL的细胞内水平方面起着核心作用。 将追求以下特定目标：（1）分析在细胞对缺氧和其他压力刺激的细胞反应期间TBP-1对PVHL功能的影响。 （2）使用缺失构建体和酵母2-杂交系统中的自然存在的VHL突变，并使用体外和体内免疫沉淀实验（3）在蜂窝压力下表征PVHL的表达水平，以确定PVHL和TBP-1之间相互作用的蛋白质结构域，并使用共释放实验来表征PVHL的表达水平。