Genetics of Cell Cycle Regulators in C. elegans

泛素连接酶调节大多数细胞蛋白质的降解。最大的 泛素连接酶的一个类别是Cullin-RING泛素连接酶（CRL）家族。 CRL调节许多动态细胞过程，包括细胞周期，转录， 信号转导和发育。CRL是多亚基复合物， cullin，其形成用于组装复合物的刚性支架。CRL要求 泛素样肽Nedd 8与cullin的缀合以获得完全活性。Cullins说， 缺乏Nedd 8的细胞可以被抑制剂CAND 1结合和隔离。Neddylation和 去卷曲化事件与cullin螯合和CRL复合物的循环有关 在建议的CRL激活周期中组装。CRL激活的许多方面都不是 很好理解，包括cullin与CAND 1的关联是如何调节的。在 C. CAND-1是cullin neddylation的重要负调控因子， cand-1突变体中neddylation水平显著增加。CAND-1不是 对主要CRL功能至关重要，但在体内确实促进CRL活性。这项建议 采用遗传学方法来揭示CAND-1与cullin的抑制性结合 并确定新的调节CRL激活周期。全基因组 RNAi筛选将用于鉴定cand-1增强子和cand-1抑制子， 将克隆先前从化学诱变筛选中分离的基因。增强子 和抑制基因将进行分子和遗传分析，以确定它们如何 调节CAND-1功能和CRL活性。初步筛查显示 介导蛋白质翻译后修饰的cand-1突变体的增强子。 我们将检验这一假设，即cullin蛋白的翻译后修饰调节其 与CAND-1的相互作用。我们用串联质谱法来鉴定后- CUL-2和CUL-4的翻译修饰。功能 翻译后修饰的意义将通过体内 在修饰位点中具有位点定向变化的cullin的分析。拟议 遗传和生物化学实验将进一步加深我们对调控的理解。 控制最重要的一类泛素连接酶的途径。异常 CRL的调节与癌症进展有关，因此， 从这项研究中获得的信息将有助于了解这种疾病的起源。 摘要章节变更说明： 我们已经确定了cand-1的抑制因子，摘要已经被修改以反映 这是通过消除对新的cand-1抑制基因的筛选。我们现在也有 cullin CUL-2和CUL-4的已鉴定翻译后修饰（PTM） 使用亲和纯化/串联质谱法。摘要已被 修改以消除先前提出的筛选以识别剔除PTM。

Ubiquitin ligases regulate the degradation of the majority of cellular proteins. The largest category of ubiquitin ligases is the family of Cullin-RING ubiquitin ligases (CRLs). CRLs regulate many dynamic cellular processes including the cell cycle, transcription, signal transduction, and development. CRLs are multisubunit complexes that contain a cullin, which forms a rigid scaffold for the assembly of the complex. CRLs require the conjugation of a ubiquitin-like peptide, Nedd8, to the cullin for full activity. Cullins that lack Nedd8 can be bound and sequestered by the inhibitor CAND1. Neddylation and deneddylation events are linked to a cycle of cullin sequestration and CRL complex assembly in a proposed CRL activation cycle. Many aspects of CRL activation are not well understood, including how the association of cullins with CAND1 is regulated. In C. elegans, CAND-1 is an important negative regulator of cullin neddylation, and cullin neddylation levels are significantly increased in cand-1 mutants. CAND-1 is not essential for major CRL functions, but does promote CRL activity in vivo. This proposal employs genetic approaches to uncover how the inhibitory binding of CAND-1 to cullins is regulated and to identify novel regulators of the CRL activation cycle. A genome-wide RNAi screen will be used to identify cand-1 enhancers; and cand-1 suppressors that have previously been isolated from chemical mutagenesis screens will be cloned. Enhancer and suppressor genes will be analyzed molecularly and genetically to determine how they regulate CAND-1 function and CRL activity. Preliminary screens have identified enhancers of the cand-1 mutant that mediate post-translational modification of proteins. We will test the hypothesis that post-translational modifications of cullins regulate their interaction with CAND-1. We have used tandem mass spectrometry to identify post- translational modifications for the cullins CUL-2 and CUL-4. The functional significance of the post-translational modifications will be assessed through the in vivo analysis of cullins with site-directed changes in the modification sites. The proposed genetic and biochemical experiments will further our understanding of the regulatory pathways that control one of the most important classes of ubiquitin ligases. Aberrant regulation of CRLs is implicated in cancer progression, and therefore the insights obtained from this study will have relevance for understanding the genesis of this disease. Notes on changes to Abstract Section: We have already identified cand-1 suppressors, and the abstract has been altered to reflect this by eliminating the screen for new cand-1 suppressor genes. We have also now identified post-translational modifications (PTMs) for the cullins CUL-2 and CUL-4 using an affinity purification/tandem mass spectrometry approach. The abstract has been modified to eliminate the previously proposed screen to identify cullin PTMs.