Mechanism of Auxin Action

描述（由申请人提供）：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。生长素通过刺激被称为Aux/IAA蛋白的转录抑制因子家族的降解而起作用，这一过程需要泛素蛋白连接酶（E3） SCFTIR1。在上一个资助期间，我们发现生长素直接与F-box蛋白TIR1结合，这是E3中称为SCFTIR1的底物结合亚基。生长素结合稳定了SCFTIR1与Aux/IAA蛋白之间的相互作用。这是一种激素感知和E3调控的新机制。此外，我们发现TIR1结合了一个InsP6分子，并表明InsP6可能是TIR1结构和/或功能所必需的。进一步，我们证明了TIR1是6个相关的生长素传感器F-box蛋白之一，统称为afb。目前的提议有五个具体目标。首先是研究TIR1感知生长素的分子机制。基于我们之前的工作，我们提出生长素像分子胶一样稳定TIR1- Aux/IAA复合物。将进行生化研究以测试该模型的各个方面。此外，我们将确定InsP6对于TIR1功能的重要性。第二个目的是表征AFB蛋白的生化多样性。将进行一系列体外研究，以表征生长素和Aux/IAA与每个家族成员的结合。任何特定相互作用的重要性将在体内确定。第三个目标是鉴定和表征含有TIR1和/或Aux/IAA蛋白IAA28的蛋白复合物，以获得有关TIR1调控的进一步信息。此外，这一目的将提供关于TIR1和Aux/IAA相互作用的特异性以及TIR1-Aux/IAA复合物形成的细胞位置的额外信息。目的四是确定亲环素分子在生长素作用中的作用。第五个目标是表征生长素转录网络，重点关注两个转录因子ARF5和ARF7。这些研究解决了细胞调控中的一些关键问题，并将对人类健康产生重要影响。泛素通路，特别是scfe3，参与多种疾病过程，包括许多癌症。因为SCFTIR1是任何物种中最具特征的E3复合物之一，这项工作提供了一个独特的机会来推进我们对人类疾病公共卫生相关性的这一关键方面的理解：泛素/蛋白酶体途径的蛋白质降解是细胞调节的一个核心方面。该通路中的缺陷导致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.