Mechanism of Auxin Action

描述(申请人提供)：植物激素生长素参与植物生长和发育的许多方面，也许是所有方面。生长素通过刺激称为AUX/IAA蛋白的转录抑制蛋白家族的降解来发挥作用，这一过程需要泛素蛋白连接酶(E3)SCFTIR1。在上一次授予期间，我们发现生长素直接与F-box蛋白TIR1结合，F-box蛋白TIR1是E3中称为SCFTIR1的底物结合亚基。生长素结合稳定了SCFTIR1和AUX/IAA蛋白之间的相互作用。这是激素感知和E3调节的一种新机制。此外，我们还发现TIR1与InsP6分子结合，提示InsP6可能是TIR1结构和/或功能所必需的。我们进一步证明TIR1是6个相关的生长素感受器F-box蛋白之一，统称为AFBs。目前的提案有五个具体目标。一是研究TIR1对生长素的分子调控机制。在前期工作的基础上，我们提出生长素起到分子胶的作用，稳定TIR1-AUX/IAA复合体。将进行生化研究，以测试该模型的各个方面。此外，我们还将确定InsP6对TIR1功能的重要性。第二个目的是研究黄曲霉毒素蛋白的生物化学多样性。将进行一系列体外研究，以确定生长素和AUX/IAA与该家族每个成员的结合。任何特定相互作用的意义都将在体内确定。第三个目的是鉴定和鉴定含有TIR1和/或AUX/IAA蛋白IAA28的蛋白质复合体，目的是获得关于TIR1调控的更多信息。此外，这一目标将提供关于TIR1和AUX/IAA相互作用的特异性以及TIR1-AUX/IAA复合体形成的细胞位置的更多信息。目的四是确定亲环素分子在生长素作用中的功能。第五个目标是描述生长素转录网络，重点是两个转录因子ARF5和ARF7。这些研究解决了细胞调节中的一些关键问题，并将对人类健康产生重要影响。泛素途径和干细胞因子E3尤其参与多种疾病过程，包括多种癌症。由于SCFTIR1是所有物种中特性最好的E3复合体之一，这项工作为我们提供了一个独特的机会来促进我们对这一与人类疾病公共卫生相关的关键方面的理解：泛素/蛋白酶体途径的蛋白质降解是细胞调控的中心方面。该通路的缺陷导致了包括癌症在内的许多疾病过程。这项研究将促进我们对泛素/蛋白酶体途径在细胞功能中的理解。

DESCRIPTION (provided by applicant): The plant hormone auxin is involved in many, perhaps all, aspects of plant growth and development. Auxin acts by stimulating the degradation of a family of transcriptional repressors called the Aux/IAA proteins, a process that requires the ubiquitin protein ligase (E3) SCFTIR1. During the last grant period we showed that auxin binds directly to the F-box protein TIR1, the substrate binding subunit in an E3 called SCFTIR1. Auxin binding stabilizes the interaction between SCFTIR1 and the Aux/IAA proteins. This is a novel mechanism of both hormone perception and E3 regulation. In addition we showed that TIR1 binds an InsP6 molecule and suggest that InsP6 may be required for TIR1 structure and/or function. Further we demonstrated that TIR1 is one of 6 related auxin sensor F-box proteins, collectively called the AFBs. The current proposal has five specific aims. The first is to investigate the molecular mechanism of auxin perception by TIR1. Based on our previous work, we propose that auxin acts like molecular glue to stabilize the TIR1- Aux/IAA complex. Biochemical studies will be performed to test various aspects of this model. In addition, we will determine the importance of InsP6 for TIR1 function. The second Aim is to characterize the biochemical diversity of the AFB proteins. A series of in vitro studies will be performed to characterize auxin and Aux/IAA binding to each member of the family. The significance of any specific interactions will be determined in vivo. The third Aim is to identify and characterize protein complexes containing TIR1 and/or the Aux/IAA proteins IAA28 with the goal of obtaining further information on the regulation of TIR1. In addition, this aim will provide additional information on the specificity of TIR1 and Aux/IAA interactions and the cellular location of TIR1-Aux/IAA complex formation. Aim four is to determine the function of cyclophilin molecules in auxin action. The fifth Aim is to characterize the auxin transcriptional network with a focus on two transcription factors called ARF5 and ARF7. These studies address a number of key issues in cellular regulation and will have important implications for human health. The ubiquitin pathway and SCF E3s in particular, are involved in diverse disease processes including numerous cancers. Because SCFTIR1 is one of the best-characterized E3 complexes in any species, this work provides a unique opportunity to advance our understanding of this critical aspect of human disease PUBLIC HEALTH RELEVANCE: Protein degradation by the ubiquitin/proteasome pathway is a central aspect of cellular regulation. Defects in the pathway contribute to many disease processes including cancers. This study will advance our understanding of the ubiquitin/proteasome pathway in cell function.