Identifying the targets of oncogenic/tumor-suppressive F box proteins

 描述(申请人提供)：泛素生物学代表一种主要的细胞调节模式。泛素化的最后一步涉及“泛素连接酶”对底物的识别。人类编码了700多种这样的连接酶，大大超过了基因组编码的激酶数量。在这些泛素连接酶基因中，有许多癌基因和肿瘤抑制基因。在许多情况下，这些连接酶的底物没有得到很好的定义，部分原因是缺乏可靠的技术来识别这些底物。我们开发了一种方法，我们称之为“连接酶捕获”，它捕获泛素连接酶上新生的泛素化底物，并允许对其进行共纯化和鉴定。在这里，我们将把这项技术应用于一组被称为F盒蛋白的泛素连接酶特异性亚基。人类编码大约80个这样的蛋白质，我们将重点关注9个已被证明在肿瘤中突变或错误表达，或具有与肿瘤发生相关的功能的靶点(如细胞周期调节蛋白和DNA损伤反应蛋白)。此外，我们将使用这项技术来表征这些基板粘结剂的结合表面。总而言之，这些数据将有助于识别潜在的药物靶点，更重要的是，将允许产生F盒蛋白本身的抑制剂，从而影响 最相关底物的周转。

 DESCRIPTION (provided by applicant): Ubiquitin biology represents a major mode of cellular regulation. The final step of ubiquitination involves the recognition of a substrate by a "ubiquiti ligase". Humans encode over 700 such ligases, significantly exceeding the number of kinases encoded by the genome. Among these ubiquitin ligase genes are many oncogenes and tumor suppressors. In many cases the substrates of these ligases have not been well defined, in part due to a lack of robust technologies for identifying these substrates. We have developed a method, which we call "Ligase Trapping" which traps the nascent ubiquitinated substrate on the ubiquitin ligase and allows for its co-purification and identification. Here, we will apply this technology to a group of ubiquitin ligase specificity subunits called F box proteins. Human's encode about 80 such proteins, and we will focus on nine that have either been shown to be mutated or mis-expressed in tumors, or have targets whose functions have been linked to tumorigenesis (such as cell cycle regulators and DNA damage response proteins). In addition, we will use this technology to characterize the binding surfaces of these substrate binders. Together, these data will allow the identification of potential drug targets, and, more importantly will allow the generation of inhibitors of the F box proteins themselves in such a way as to affect the turnover of the most relevant substrates.