Dynamics of auxin sensing by an SCF-E3 type ubiquitin ligase and its degradation targets

泛素依赖的蛋白分解(UPS)对生长素的感知和信号传递是必不可少的，因为UPS机制催化激素依赖的转录调节因子的周转，介导该途径的上调或下调。生长素通过促进AUX/IAA转录抑制物的降解来激活生长素反应基因的转录。我们以前的研究表明，生长素是由F-box蛋白转运蛋白抑制反应1(TIR1)或其类似的生长素信号F-box 1-5(AFB1-5)及其降解靶标AUX/IAAs组成的共同受体系统。确切地说，生长素通过增加疏水接触和作为分子胶来增强TIR1/AFB-AUX/IAA的相互作用。因此，我们用生化方法确定了不同的TIR1/AFB-AUX/IAA共受体对生长素具有不同的感知能力，并可能在体内构成不同的生长素感受器。TIR1/AFBs作为SCF型E3连接酶的底物受体，与AUX/IAA的降解结合，从而导致蛋白酶体的降解。退化区以外的区域是否决定生长素感知和目标识别仍然是一个谜。拟南芥中AUX/IAA和TIR1/AFB蛋白家族的多样性导致了一系列可能的共受体组合。由于不同的生长素和不同的生长素浓度调节植物的可塑性，植物细胞可能通过组装具有不同生长素感知特性的特定辅助受体来感知这些差异。泛素介导的蛋白质降解对激素的反应是动力学的典范，因为激素浓度的轻微变化不仅意味着靶-受体复合体的组装和拆解，而且决定了基因激活发生的时间和地点。这个动态系统控制着植物的生长和发育。植物细胞只有对内部信号和外部信号作出反应，不断地改变联结，才能生存、适应和茁壮成长。由于SCFTIR1/AFBs对AUX/IAA靶标的识别，生长素结合和靶标降解是高度特异的、及时调节的和动态的过程；我们的研究计划旨在获得对生长素依赖的蛋白质降解的生化和结构方面的见解，以预测基因的激活。这项拟议的工作还旨在确定生长素传感过程中是否发生构象转换和竞争相互作用，并表征SCFTIR1/AFB底物(AUX/IAA)加工的决定因素，从而确定26S蛋白酶体的决定因素。通过这个项目，我们想要证实在体外观察到的激素感应的调节机制，并研究小分子增强的SCF活性的结构决定因素。

Ubiquitin-dependent proteolysis (UPS) is essential to auxin perception and signaling, as the UPS machinery catalyzes hormone-dependent turnover of transcriptional regulators, mediating up- or down-regulation of the pathway. Auxin activates transcription of auxin response genes by promoting the degradation of AUX/IAA transcriptional repressors. We showed previously auxin is perceived by a co-receptor system formed by the F-box protein TRANSPORT INHIBITOR RESPONSE 1 (TIR1) or one of its paralogs AUXIN SIGNALING F-BOX 1-5 (AFB1-5) and its degradation targets, AUX/IAAs. Precisely, auxin enhances TIR1/AFB-AUX/IAA interaction by increasing hydrophobic contacts and acting as molecular glue. Thus, we biochemically determined that distinct TIR1/AFB-AUX/IAA co-receptor pairs are differentially perceptive to auxin and might constitute various auxin sensors in vivo. TIR1/AFBs act as substrate receptors of SCF-type E3 ligases, which bind to the degron of AUX/IAAs targeting them to proteasomal degradation. Whether regions besides the degron dictate auxin sensing and target recognition is still a puzzle. The diversity of AUX/IAA, as well as TIR1/AFB protein families in Arabidopsis gives rise to a repertoire of possible co-receptor combinations. Since different auxins and various auxin concentrations modulate plant plasticity, plant cells might perceive these differences via assembly of specific co-receptors with distinct auxin sensing properties. Ubiquitin-mediated protein degradation in response to hormones is the paragon for dynamics, as slight changes in hormone concentration not only imply the assembly and disassembly of target-receptor complexes, but determine when and where gene activation takes place. This dynamic system governs plant growth and development. Only a constant turnover of associations in response to internal cues and external signals allow plant cells to survive, adapt and thrive. As AUX/IAA target recognition by SCFTIR1/AFBs, auxin binding and target degradation are highly specific, timely regulated and dynamic processes; our research plan aims to gain biochemical and structural insights into auxin-dependent protein degradation to predict gene activation. The proposed work aims also to determine whether conformational transitions and competitive interactions take place during auxin sensing, and to characterize the determinants of substrate (AUX/IAA) processing by SCFTIR1/AFBs, and thereby by the 26S proteasome. With this project, we want to substantiate the regulatory mechanism of hormone sensing observed in vitro, and to investigate the structural determinants of small molecule-potentiated SCF activity.