Regulation of Cullin-RING Ligases by Nedd8

描述（由申请人提供）：本申请是对NOT-OD-09-058（NIH宣布恢复法案资金用于竞争性修订申请的可用性）的响应。我们寻求恢复法案基金的支持，以扩大GM 065997的具体目标、研究设计和方法的范围，该项目的标题是：Nedd 8对cullin-RING连接酶的调节。Nedd 8是一种与cullin-RING泛素连接酶（CRL）的cullin亚基可逆结合的泛素样蛋白。Nedd 8的缀合和解缀合对于维持人细胞中CRL的活性是必不可少的。在母案申请的第二个目的中，我们提出研究C 0 P9信号体（CSN）对cullin的Nedd 8去缀合如何通过将底物和连接酶复合物的其它亚基募集到cullin支架来调节。在本申请中，我们提出通过开发CSN的Nedd 8异肽酶活性的有效和特异性抑制剂来扩展该目标。我们将设计均相荧光偏振和荧光共振能量转移测定来监测Nedd 8-Cul 1的体外和体内切割，并将采用体外测定来进行化学文库的高通量筛选。将对命中物进行二次筛选，以评价其体内功效、效力和选择性。我们的目标是确定一个铅化合物的IC 50至少为1？M在体外和体内测定中。具有这些特征的引线将是非常有用的工具，用于研究体内CSN依赖性Nedd 8去缀合的生理作用，并评估该功能如何与CSN的底物特异性相关，这是母案申请的目的2的主题。这里提出的实验大大扩大了原来的同行评审的目的的母基金的范围，通过引入新的方法和途径，将产生新的工具来研究CSN的功能。对拟议实验的追求将加快我实验室对Nedd 8去结合对CRL调节的细胞后果的研究克里思，并将使新员工的招聘成为可能。 公共卫生相关性：cullin-RING泛素连接酶（CRL）的亚基在包括癌症在内的多种人类疾病中突变或过表达，并且Csn 5的过表达驱动与乳腺癌的不良预后紧密相关的基因表达程序。此外，Nedd 8活化酶与Csn 5一起介导Nedd 8修饰cullin的循环，是临床开发中有前途的抗癌药物的靶点。鉴定出一种能阻断Csn 5从cullin中切割Nedd 8的化学物质，将有助于更深入地了解Nedd 8去缀合的生理作用，并有助于更好地了解过度活跃的Csn 5是如何导致人类疾病的。除了作为一种重要的研究工具之外，Csn 5抑制剂还具有很大的潜力，可以作为开发新的治疗药物以调节CRL活性以获得临床益处的起点。

DESCRIPTION (provided by applicant): This application is responsive to NOT-OD-09-058 (NIH Announces the Availability of Recovery Act Funds for Competitive Revision Applications). We seek support from the Recovery Act Funds to expand the scope of the specific aims, research design, and methods of GM065997, which bears the title: Regulation of cullin-RING ligases by Nedd8. Nedd8 is a ubiquitin-like protein that is reversibly conjugated to the cullin subunit of cullin-RING ubiquitin ligases (CRLs). Both the conjugation and deconjugation of Nedd8 are essential to maintain the activity of CRLs in human cells. In the second Aim of the parent application, we proposed to investigate how Nedd8 deconjugation of cullins by the COP9 Signalosome (CSN) is regulated by the recruitment of substrates and other subunits of the ligase complex to the cullin scaffold. In the present application, we propose to expand upon this goal by developing a potent and specific inhibitor of the Nedd8 isopeptidase activity of CSN. We will devise homogeneous fluorescence polarization and fluorescence resonance energy transfer assays to monitor cleavage of Nedd8-Cul1 both in vitro and in vivo, and will employ the in vitro assay to conduct high- throughput screens of chemical libraries. Hits will be subjected to secondary screens designed to evaluate their in vivo efficacy, potency, and selectivity. Our goal is to identify a lead compound with an IC50 of at least 1 ?M in both in vitro and in vivo assays. A lead with these characteristics would be a very useful tool to study the physiological role of CSN-dependent Nedd8 deconjugation in vivo, and to evaluate how this function relates to the substrate specificity of CSN, which is the subject of Aim 2 of the parent application. The experiments proposed here substantially expand the scope of the original peer reviewed Aims of the parent grant by introducing new new methods and approaches that will yield new tools to study the function of CSN. Pursuit of the proposed experiments will accelerate the tempo of research in my lab on the cellular consequences of CRL regulation by Nedd8 deconjugation, and will enable the recruitment of new staff. PUBLIC HEALTH RELEVANCE: Subunits of cullin-RING ubiquitin ligases (CRLs) are mutated or overexpressed in a variety of human diseases including cancer and overexpression of Csn5 drives a gene expression program that is tightly linked to poor prognosis in breast cancer. In addition, the Nedd8 activating enzyme that together with Csn5 mediates the cycle of cullin modification by Nedd8 is the target of a promising anti-cancer drug that is in clinical development. Identification of a chemical that blocks cleavage of Nedd8 from cullins by Csn5 will lead to a deeper understanding of the physiological role of Nedd8 deconjugation and will lead to a better understanding of how overactive Csn5 underlies human disease. Besides being an important research tool, a Csn5 inhibitor has great potential to serve as a starting point for development of new therapeutic drugs to modulate CRL activity for clinical benefit.