拟南芥内质网氧化还原酶AtERO1/2的功能及植物内质网中二硫键传递的机制

The proper folding of the protein is critical for its function, the disulfide bond formation is one of the important types of protein folding. ER oxidoreductase1 (ERO1) and protein disulfide isomerase (PDI) synergistically catalyze the disulfide bond modification of the substrate protein in eukaryotes. The disulfide bond transfer pattern has been studied very extensive in animals, but little is known in plants. Our previous study found that, there are two homologues of ERO1 in Arabidopsis thaliana, AtERO1 and AtERO2, both of which can oxidize different PDIs, and the respective deletion mutants are sensitive to DTT. However, how the redox activity of AtERO1/2 is regulated? How the disulfide bond is delivered along the “AtERO1/2→PDI→substrate” during the formation of the disulfide bond? The understanding about these issues is still limited. We screened the substrates of AtPDIs and found a receptor kinase PDI5-interacting RLK1 (P5IR1) that interacts with PDI5, as predicted that many receptor kinases may have multiple disulfide bonds in the leucine-rich region (LRR). Therefore, this project will focus on the above academic issues to illuminate the transfer mechanism of the disulfide bond in plant endoplasmic reticulum, and explore the disulfide modification mechanism involved in regulating plant physiological activities. The implementation of this project will provide new ideas for studying the mechanism of redox regulation in plant signal transduction pathways.

蛋白质的正确折叠对其功能发挥至关重要，二硫键的形成对于蛋白质折叠非常关键。真核生物的内质网氧化还原酶ERO1和蛋白二硫键异构酶PDI协同催化底物蛋白的二硫键修饰，在动物中研究得非常广泛，在植物中却十分有限。本项目前期的研究发现，拟南芥中存在两个高度同源的ERO1(AtERO1和2)，可以氧化多个PDI，缺失突变体都对DTT敏感。然而AtERO1/2的氧化还原活性是如何调节的？在底物二硫键形成过程中，二硫键是如何沿着“AtERO1/2→PDI→底物”传递的？人们对这些问题的了解非常有限。我们筛选到一个与AtPDI5互作的受体激酶P5IR1，而许多受体激酶的亮氨酸富集区(LRR)可能存在多处二硫键修饰。因此，本项目将围绕以上学术问题，深入解析植物内质网中的二硫键传递机制，并对二硫键修饰参与调控植物生理活动的机理进行深入探讨。本项目的开展将为研究植物信号转导途径中的氧化还原调控机制提供新的思路。

蛋白质的正确折叠对其功能发挥至关重要。真核生物的质膜蛋白与分泌蛋白在内质网中形成二硫键从而实现其氧化折叠。内质网中的氧化还原酶ERO1和蛋白二硫键异构酶PDI协同催化底物蛋白二硫键的形成。蛋白质氧化折叠在动物中研究得非常广泛，在植物中却十分有限。本项目开展了项目书中计划的研究内容，主要包括：1） AtERO1 和 AtERO2 的蛋白特性分析 ；2） AtERO1 和 AtERO2 的功能解析 ；3）PDIs 底物蛋白的筛选、验证及功能分析。我们发现，AtERO2蛋白，而非AtERO1，特异地响应还原剂DTT处理后在根中引起的还原性胁迫。我们还发现，AtERO2介导的蛋白质氧化折叠参与了植物根的发育，AtERO2基因的突变体植物的根尖分生组织中细胞的分裂与分化能力存在缺陷。此外，我们还发现植物激素油菜素内酯的受体BRI1可能是PDI5的底物，而PDI11通过其D 结构域与内质网凝集素分子伴侣的CRT1/2相互作用。