Molecular mechanisms of auxin response in Arabidopsis

描述（由申请人提供）：SCF和其他cullin-ring连接酶（CRLs）与许多人类发育和疾病途径有关。这些酶在泛素蛋白水解途径中起作用，催化泛素向特定蛋白质的转移，从而靶向这些蛋白质进行降解。先前的研究表明，SCF活性受到高度调控，包括CAND1、SGT1、COP9信号体（CSN）和RUB/NEDD8偶联途径在内的许多蛋白质都可调节SCF活性。虽然已经对这些因素的调控性质有了一些了解，但仍然存在许多问题，似乎还没有发现额外的SCF调控机制。植物激素生长素几乎调节着植物生长发育的各个方面。在生长素刺激下，SCFTIR1泛素连接酶通过靶向一个转录抑制家族进行泛素介导的蛋白水解来调节生长素反应。模式植物拟南芥中的生长素信号对SCFTIR1活性的扰动非常敏感，并且已被证明是研究真核生物如何调节SCF泛素连接酶活性的极其强大的遗传系统。几乎所有已知的SCF成分和调节因子的突变都已在基因筛选中分离出表现出生长素反应降低的突变体。该系统提供了几种新的遗传工具，包括CAND1和CSN亚基的活突变等位基因，这在其他多细胞真核模型系统中是不可用的。本项目的长期目标是彻底阐明生长素调控植物生长发育的分子机制。这些知识将有助于操纵植物的生长和发育，以改善粮食生产和其他有益于人类健康的植物特性。更广泛地说，SCF复合物和控制其活性的调控机制在高等真核生物中高度保守。建议的研究包括遗传学，分子和生化方法来阐明控制SCFTIR1活性的调控机制。首先，将使用新的遗传工具和简单的生化分析来检查SCFTIR1活性通过调节组装和拆卸周期的控制。其次，将描述生长素诱导基因表达所必需的CSN3亚基或CSN的新活性或独特复合物的特征。第三，从基因筛选中分离出一系列突变体，用于鉴定SCF活性的负调节因子，并将其特征化并纳入生长素信号传导和SCF调节的当前模型中。这些实验的发现几乎肯定与人类细胞调节SCF活性和其他信号过程的机制有直接的相似之处，从而增加了对疾病发病机制的理解，并有可能导致在癌症和其他疾病中调节SCF活性的新治疗策略。

DESCRIPTION (provided by applicant): SCF and other cullin-ring ligases (CRLs) have been implicated in numerous human developmental and disease pathways. These enzymes function in the ubiquitin proteolytic pathway, catalyzing the transfer of ubiquitin to specific proteins and thus targeting these proteins for degradation. Previous studies have revealed that SCF activity is highly regulated, with numerous proteins including CAND1, SGT1, the COP9 signalosome (CSN), and the RUB/NEDD8 conjugation pathway all acting to modulate SCF activity. While some understanding into the nature of the regulation by these factors has been achieved, many questions remain, and it seems likely that additional SCF regulatory mechanisms are yet to be discovered. The plant hormone auxin regulates virtually every aspect of plant growth and development. The SCFTIR1 ubiquitin-ligase regulates auxin response by targeting a family of transcriptional repressors for ubiquitin- mediated proteolysis in response to an auxin stimulus. Auxin signaling in the model plant Arabidopsis thaliana is exquisitely sensitive to perturbations in SCFTIR1 activity, and has proven to be an extremely powerful genetic system for studying how eukaryotic organisms regulate SCF ubiquitin-ligase activity. Mutations in virtually every known SCF component and regulator have been isolated in genetic screens for mutants exhibiting reduced auxin response. This system provides several novel genetic tools, including viable mutant alleles of CAND1 and CSN subunits, which are not available in other multicellular eukaryotic model systems. The long-term goal of this project is to thoroughly elucidate the molecular mechanisms underlying auxin- mediated control of plant growth and development. Such knowledge will facilitate the manipulation of plant growth and development to improve food production and other plant traits of benefit to human health. More broadly, SCF complexes and the regulatory mechanisms controlling their activity are highly conserved throughout higher eukaryotes. The proposed studies include genetic, molecular, and biochemical approaches to elucidate the regulatory mechanisms controlling SCFTIR1 activity. First, the control of SCFTIR1 activity by regulated cycles of assembly and disassembly will be examined using novel genetic tools and simple biochemical assays. Second, a novel activity of either the CSN or a unique complex containing the CSN3 subunit that is required for auxin-inducible gene expression will be characterized. Third, a collection of mutants isolated in a genetic screen designed to identify negative regulators of SCF activity will be characterized and incorporated into current models for auxin signaling and SCF regulation. The findings from the proposed experiments will almost certainly have direct parallels to the mechanisms human cells employ to regulate SCF activity and other signaling processes, thus increasing understanding of disease pathogenesis and potentially leading to novel therapeutic strategies for modulating SCF activity in cancer and other diseases.