Analysis of the VHL-HIF hydroxylase pathway

描述（由申请人提供）：泛素修饰蛋白在包括肿瘤发生在内的多种生物学现象中起关键调节作用。几种癌蛋白和抗癌蛋白显示泛素连接酶活性，鉴定它们的底物是癌症研究的中心主题之一。本申请旨在了解肿瘤抑制泛素连接酶VHL的生物学作用。VHL目前被最好地理解为缺氧诱导因子（HIF）的负调节因子。在氧和铁的存在下，HIF中特定的脯氨酸残基被羟基化，这些羟基化的脯氨酸被VHL识别，这导致HIF的泛素化和降解。VHL突变与遗传性和散发性肾细胞癌、血管母细胞瘤和嗜铬细胞瘤有关。一些证据表明，除了HIF之外，VHL还存在未鉴定的底物，其在肿瘤抑制中起关键作用。理解VHL功能（以及其他泛素连接酶的功能）的一个主要障碍是难以确定其泛素化底物。采用一种新的定量蛋白质组学技术，ICAT（同位素编码的亲和标签），我们开发了一种方法来系统地确定泛素连接酶的底物，并使用这种方法，我们确定了一种新的底物VHL，它带有VHL功能的关键介质的特性。因此，本申请的目的是分析该底物的泛素化的机制和生物学后果，并进行进一步的蛋白质组学筛选以鉴定VHL的其他底物。这些研究应该提供一个更好的了解VHL的生物学功能，并推进基于蛋白质组学的生物学的新生领域。

DESCRIPTION (provided by applicant): Protein modification with ubiquitin plays key regulatory roles in a wide variety of biological phenomena including tumorigenesis. Several oncoproteins and anti-oncoproteins display ubiquitin ligase activity and identification of their substrates is one of the central themes in cancer research. This application is directed toward understanding the biological roles of a tumor-suppressing ubiquitin ligase, VHL. VHL is currently best understood as a negative regulator of hypoxia inducible factor (HIF). In the presence of oxygen and iron, specific proline residues in HIF are hydroxylated and these hydroxylated prolines are recognized by VHL, which results in ubiquitination and degradation of HIF. VHL mutation is associated with hereditary and sporadic renal cell carcinomas, hemangioblastomas, and pheochromocytomas. Several lines of evidence suggest that, in addition to HIF, there are unidentified substrates for VHL which play critical roles in tumor suppression. A major barrier to understanding the VHL functions (as well as the functions of other ubiquitin ligases) has been the difficulty in pinpointing its ubiquitination substrates. Employing a novel quantitative proteomics technology, ICAT (isotope-coded affinity tag), we developed an approach to systematically identify the substrates of ubiquitin ligases and using this method, we identified a novel substrate for VHL, which carries the character of a key mediator of VHL functions. Therefore the aims of this application are to analyze the mechanism and biological consequence of ubiquitination of this substrate and to conduct further proteomic screens to identify additional substrates for VHL. These studies should provide a better understanding of the biological functions of VHL and advance the nascent field of proteomics-based biology.