Role of CSN in the activity and dynamic cycling of cullin-RING ubiquitin ligases

描述（由申请人提供）：cullin-RING泛素连接酶（CRL）家族包含超过240个泛素连接酶，可调节广泛的细胞和有机体过程。与其在调控生物学中的关键作用一致，CRL与人类疾病的发病机制和治疗密切相关：CRL亚基的突变是几种人类癌症发展的基础，CRL4CRBN被认为是抗骨髓瘤药物Thalomid的靶标。CRL通路受多种机制调控，包括CRL组装/拆卸的动态循环和泛素样蛋白Nedd8对cullin亚基的翻译后修饰。CSN是一种Nedd8异肽酶，通过去除Nedd8修饰来调节crl。从CRL中去除Nedd8会导致其失活，并使其底物受体容易被交换因子Cand1分解。虽然CSN的异肽酶活性抑制CRL活性，但遗传学研究表明CSN是CRL的正调节因子，这表明需要周期性的CRL失活和拆卸来维持CRL活性。最近，Nedd8偶联通路已成为癌症化疗的药物靶点，Csn5的活性位点亚基在myc驱动的癌症模型中参与维持致瘤性。总之，这些数据表明，crl的动态循环对癌细胞增殖至关重要。推动这个项目的首要假设是，CSN在调节CRL酶的活性和CRL网络的结构中起关键作用，并且CSN活性的扰动对癌细胞是有毒的。我们计划在三个具体目标中探讨这些主题。目标1

DESCRIPTION (provided by applicant): The cullin-RING ubiquitin ligase (CRL) family contains upwards of 240 ubiquitin ligases that regulate a broad range of cellular and organismal processes. Consistent with their critical roles in regulatory biology, CRLs have been intimately linked to human disease pathogenesis and therapy: mutations in CRL subunits underlie the development of several human cancers and CRL4CRBN has been implicated as the target of the anti- myeloma agent Thalomid. CRL pathways are regulated by multiple mechanisms, including dynamic cycles of CRL assembly/disassembly and post-translational modification of the cullin subunit by the ubiquitin-like protein Nedd8. CSN is a Nedd8 isopeptidase that regulates CRLs by removing the Nedd8 modification. Removal of Nedd8 from a CRL results in its deactivation and renders its substrate receptor susceptible to disassembly by the exchange factor, Cand1. Although the isopeptidase activity of CSN inhibits CRL activity, genetic studies indicate that CSN is a positive regulator of CRLs, which suggests that periodic deactivation and disassembly of CRLs is required to sustain CRL activity. Recently, the Nedd8 conjugation pathway has emerged as a drug target for cancer chemotherapy and the active site subunit of Csn5 has been implicated in sustaining tumorigenicity in a Myc-driven cancer model. Together, these data point to dynamic cycling of CRLs as being critical for cancer cell proliferation. The over-arching hypotheses that animate this project are that CSN plays a critical role in regulating the activity of CRL enzymes and architecture of the CRL network, and that perturbation of CSN activity is toxic to cancer cells. We plan to explore these topics in three Specific Aims. In Aim 1 we will use quantitative multidimensional mass spectrometry to characterize the architecture and dynamics of the CRL proteome. In Aim 2, we will characterize a novel non-enzymatic CRL-inhibitory activity of CSN. In Aim 3, we will generate a CSN isopeptidase inhibitor to use as a research tool and to evaluate whether CSN is a promising target for chemotherapy of cancer.