去泛素化酶UBP12/13通过调控ESCRTs关键组分VPS23参与ABA信号通路的研究

Phytohormone ABA regulates many processes that are necessary for plant development and growth and environmental stresses adaption. Analyzing the regulatory mechanism in ABA signaling pathway is helpful for gene mining for crop improvement in the aspect of stress resistance. In our previous work, the E2-like protein VPS23 was found to function as a key component of ESCRTs to regulate ABA receptors stability through endomembrane trafficking pathway. We found VPS23 can be degraded through 26S proteasome pathway, similar to its homolog TSG101 in mammals. To further understand the crosstalk between endomembrane system related vacuolar degradation pathway and the 26S proteasome degradation pathway, and the upstream regulatory mechanism of VPS23 in ABA signaling, yeast two-hybrid assay was carried out to screen the interacting proteins of VPS23. UBP12 and UBP13 was found to interact with VPS23. As we know, UBP12 and UBP13 are two deubiquitinases, which probably function in removing or editing specific forms of ubiquitin chains to modulate protein stability. Preliminary data showed that both UBP12 and UBP13 accelerate VPS23 degradation, and ubp12 and ubp13 knockout mutants both present ABA insensitive phenotype, which is contrary to the ABA sensitive phenotype of vps23. This project will focus on investigating the biochemical and genetic relationship between VPS23 and UBP12/13, and uncover the regulatory role of the ESCRTs component VPS23 by UBP12/13 in plant response to ABA signal.

脱落酸（Abscisic acid，ABA）参与植物的生长发育和各种生物和非生物胁迫应对过程。通过对ABA信号通路分子机理的深入探索和研究，有望进一步发掘相关功能基因，培育出抗旱耐盐等优良性状的作物。在前期探索蛋白质翻译后修饰泛素化对ABA信号通路调控的研究中，发现E2-like蛋白VPS23作为内膜运输系统中ESCRTs复合体的一个关键组分参与调控ABA信号，但是VPS23的上游调控机制仍不清楚。本研究利用酵母双杂交的方法筛选其互作候选蛋白，初步结果显示，去泛素化酶UBP12和UBP13与VPS23相互作用，并促进VPS23蛋白的降解；敲除单突变体ubp12和ubp13均表现出ABA不敏感的表型，与vps23突变体的表型相反。本项目拟通过深入探究VPS23与UBP12/13的功能关系，解析ESCRTs组分VPS23在植物中受调控的分子机制及其互作蛋白在植物响应ABA信号过程中的功能机制。

泛素化修饰调控脱落酸(ABA)信号途径在精细调控植物水分利用及帮助植物响应干旱中发挥着极其重要的作用。过去二十年在该领域的研究主要集中在解析E3泛素连接酶与底物之间的关系上，然而，去泛素化酶如何去除ABA信号途径相关底物上的泛素分子的机制并不是很清楚。实验室前期工作证明VPS23A作为内吞分选复合体I中的关键组分，发挥着识别、分选以及促进ABA受体进入内膜运输途径降解的作用。鉴于VPS23A在ABA信号途径中发挥重要作用，我们试图进一步揭示VPS23A自身被调控的分子机制。通过酵母双杂交筛库实验筛选VPS23A的互作蛋白，得到VPS23A可能的互作蛋白去泛素化酶UBP13。由于UBP12是UBP13的同源蛋白，因此我们集中解析了UBP12/13与VPS23A之间的关系。..通过各种蛋白互作实验证明了UBP12/13确实均与VPS23A互作。分子以及生化实验数据证明UBP12/13均可去除VPS23A上的泛素化修饰以促进VPS23A的稳定性。ubp12和ubp13均表现出相对野生型Col-0对ABA敏感及更加抗旱的表型，并且积累更高水平的ABA受体。遗传学实验证明UBP12/13通过调控ABA受体的积累参与ABA信号途径。进一步遗传学分析表明VPS23A过表达可部分恢复ubp12-2w和ubp13-1的ABA敏感表型。以上实验数据说明UBP12/13为ABA信号途径的负调控因子，通过去除VPS23A的泛素化修饰进而稳定VPS23A以调控ABA受体的积累。然而，我们发现ABA促进VPS23A的去泛素化酶UBP12/13的积累的同时却促进VPS23A的降解。为了揭示其中机制，通过实验发现UBP12/13不仅可以去泛素化VPS23A，还可以在ABA的处理下去泛素化并稳定XBAT35.2，形成一个正向反馈调控机制拮抗调控VPS23A，最终导致VPS23A蛋白水平降低从而增强植物对ABA的响应。.因此，本项目证明了去泛素化酶UBP12和UBP13是ABA信号途径新的负向调控因子，并揭示了去泛素化酶在ABA响应过程中精细调控XBAT35.2/VPS23A这一对泛素化调控模块，进一步加深了领域内对于植物中泛素化以及去泛素化机器之间精细调控的理解。