Molecular screens for pVHL-associated isopeptidase VDU1

描述(由申请人提供)： VDU1是最近发现的一种泛素异肽酶，它与vonHippel-Lindau蛋白(PVHL)的E3连接酶复合体结合并被其泛素化。VDU1已被发现是肿瘤抑制蛋白复合体VCB-CUL2(一种E3泛素连接酶复合体，由von Hippel-Lindau蛋白pVHL、细长蛋白C、细长蛋白B和cullin-2(Li，Na等人)组成的泛素化(和蛋白酶体降解)的靶标。2002年；Li，Wang等人。2002年)。PVHL在某些癌症中发生突变，并作为肿瘤抑制基因发挥作用。生化和遗传学证据表明，VDU1与pVHL E3连接酶的其他泛素化靶点一起，在建立或维持pVHL突变肿瘤的转化状态方面发挥了作用。E3连接酶确定的目标蛋白之一，HIFA，已知在特定条件下诱导血管生成因子，从而发挥癌基因产物的作用。PVHL的β结构域是VDU1相互作用的位点，是自然发生突变的部位，VDU1可以在VCB-CUL2复合体中免疫共沉淀。此外，通过依赖pVHL的途径泛素化和降解VDU1被VHL突变所废除，这些突变破坏了与VDU1的相互作用。因此，pVHL对VDU1的靶向降解在抑制肿瘤形成和/或维持方面可能是重要的，并且VDU1可能具有在缺乏功能连接酶的情况下未发现的致癌活性。在这项拟议的为期一年的第一阶段中，我们将为VDU1配置一种新的异肽酶分析方法，并将其配置为高通量筛选，目的是使用此方法来发现新的抗肿瘤药物，这些药物通过抑制通常由野生型pVHL阻止的细胞活性而发挥作用。我们还将根据其他肽底物筛选VDU1，以评估其切割模式。开发VDU1检测的最终商业目标是发现一种作为肾癌和其他癌症联合治疗的单一药剂或成分的药物。在第二阶段将开发一种相关的和高度同源的异肽酶VDU2的分析方法，并用该方法筛选VDU1分析的HITS。

DESCRIPTION (provided by applicant): VDU1 is a recently described ubiquitin isopeptidase that binds to and is ubiquitinated by the E3 ligase complex of the vonHippel-Lindau protein (pVHL). VDU1 has been found to be a target of ubiquitination (and proteasomal degradation) by the tumor suppressor protein complex VCB-CUL2 (an E3 ubiquitin ligase complex consisting of the von Hippel-Lindau protein pVHL, elongin C, elongin B, and cullin-2 (Li, Na et al. 2002; Li, Wang et al. 2002). pVHL is mutated in certain cancers and behaves as a tumor suppressor gene. Biochemical and genetic evidence suggests that VDU1, along with a limited number of other ubiquitination targets of the pVHL E3 ligase, has a role in establishing or maintaining the transformed state of pVHLmutant tumors. One of the E3 ligase's identified target proteins, HIFa, is known to induce angiogenic factors under certain conditions, and thus acts as an oncogene product. The beta-domain region of pVHL, which is the site of naturally occurring mutations, is the locus of VDU1 interaction, and VDU1 can be coimmunoprecipitated in the VCB-CUL2 complex. Moreover, the ubiquitination and degradation of VDU1 by a pVHL-dependent pathway is abrogated by VHL mutations that disrupt interactions with VDU1. Thus, targeted degradation of VDU1 by pVHL may be important in suppressing tumor formation and/or maintenance, and VDU1 may have oncogenic activity that is uncovered in the absence of the functional ligase. In this proposed one-year Phase I, we will configure a novel isopeptidase assay for VDU1 and configure it for high throughput screening, with the aim using this assay to discover novel antitumor drugs that act by inhibiting the cellular activity that would normally be prevented by wild type pVHL. We will also screen VDU1 against other peptide substrates to assess its cleavage patterns. The ultimate commercial goal of the development of an assay for VDU1 is to discover a drug with efficacy as a single agent or a component of conjoint therapy against renal and other cancers. An assay for the related and highly homologous isopeptidase VDU2 will be developed in Phase II and hits from the VDU1 assay screened with this assay as well.