Molecular mechanisms of auxin response in Arabidopsis

描述(申请人提供)：SCF和其他剔除环连合酶(CRL)与许多人类发育和疾病途径有关。这些酶在泛素蛋白分解途径中发挥作用，催化泛素转移到特定的蛋白质上，从而针对这些蛋白质进行降解。以往的研究表明，SCF的活性受到高度调控，包括CAND1、SGT1、COP9信号体(CSN)和RUB/NEDD8结合途径在内的许多蛋白质都参与了SCF活性的调节。虽然对这些因素的调控性质已经有了一些了解，但仍然存在许多问题，似乎还没有发现更多的SCF调控机制。植物激素生长素几乎调控植物生长发育的方方面面。SCFTIR1泛素连接酶通过靶向一类转录抑制因子家族来调节生长素的反应，泛素介导的蛋白分解是对生长素刺激的反应。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，已被证明是研究真核生物如何调控SCF泛素连接酶活性的一个极其强大的遗传系统。在生长素反应减弱的突变体的遗传筛选中，几乎每一个已知的SCF成分和调节因子的突变都被分离出来。该系统提供了几种新的遗传工具，包括CAND1和CSN亚基的活性突变等位基因，这是其他多细胞真核模型系统所没有的。该项目的长期目标是彻底阐明生长素介导的植物生长发育控制的分子机制。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性状。更广泛地说，SCF复合体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、分子和生化方法，以阐明控制SCFTIR1活性的调控机制。首先，将使用新的基因工具和简单的生化分析来检验通过组装和拆卸的调节周期来控制SCFTIR1的活性。其次，将表征CSN或包含生长素诱导基因表达所需的CSN3亚单位的独特复合体的新活性。第三，在设计用于识别SCF活性的负调控因子的遗传屏幕中分离的突变体集合将被表征，并被纳入当前生长素信号和SCF调节的模型中。拟议中的实验结果几乎肯定会与人类细胞用来调节SCF活性和其他信号过程的机制有直接的相似之处，从而增加对疾病发病机制的理解，并可能导致调节癌症和其他疾病中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.