The dynamic function of the cytosolic redox regulatory network and the sensory role of the type II peroxiredoxins B/C/D.

硫醇氧化还原调节网络的存在是包括胞质溶胶在内的所有浆细胞区室的共同特征。它共同控制许多过程，如新陈代谢和信号传导。在植物中，叶绿体的氧化还原网络是最好的理解。然而，它的具体特点和功能在胞质溶胶仍然主要是要探讨，特别是相对于II型过氧化物酶（PRXIIB/C/D）的氧化还原传感器相比，谷胱甘肽过氧化物酶样蛋白（GPXL）的作用。对A. thaliana将讨论电子竞争、相互作用的特异性和氧化还原传递的动力学。氧化刺激的传播将被监测在一个新的重组氧化还原系统组成的各种网络组件。使用高分辨率定量质谱（MS）为基础的蛋白质组学，这种设置将区分硫醇，肽之间的二硫化物，和S-谷胱甘肽化，并剖析系统中每种类型的氧化还原元素的作用。将使用CRISPR/Cas在敲除系中探索三种PRXIIB/C/D同种型的功能，并与缺乏GPXL 2/8的突变体（例如，在光转移或RBOH激活后。PRXIIB的相互作用物组将使用基于亲和色谱的相互作用物的下拉（pull-down）在体外测定，然后进行MS鉴定，并使用Turbo-ID-PRXII构建体在体内测定。该网络的意义将在两个案例研究中进一步研究，即细胞溶质甘油醛-3-磷酸脱氢酶（GAPC）和液泡H+-ATP酶的氧化还原调节。总体而言，该项目计划测试“氧化还原体”作为监管单位的概念。先进的定量理解的胞质氧化还原子网络将尝试通过数学建模和动态模拟，以提供新的洞察网络组件之间的动力学耦合。

The presence of a thiol redox-regulatory network is a common feature of all plasmatic cell compartments including the cytosol. It co-controls many processes such as metabolism and signaling. In plants, the redox network is best understood for chloroplasts. However, its specific features and functions in the cytosol remain mostly to be explored, in particular with respect to the role of type II peroxiredoxins (PRXIIB/C/D) as redox sensors in comparison to glutathione-peroxidase-like proteins (GPXL). The proposed work on A. thaliana will address the competition for electrons, specificity of interaction, and kinetics of redox transmission. The propagation of an oxidative stimulus will be monitored in a novel reconstituted redox system consisting of various network components. Using high resolution quantitative mass spectrometry (MS)-based proteomics, this setup will distinguish thiols, disulphides between peptides, and S-glutathionylation and dissect the roles of each type of redox element in the system. The function of the three PRXIIB/C/D isoforms will be explored in knock out lines using CRISPR/Cas and compared with mutants lacking GPXL2/8, e.g., after light shifts or RBOH activation. The interactome of PRXIIB will be determined in vitro using affinity chromatography-based pull-down of interactors followed by MS identification and in vivo using Turbo-ID-PRXII constructs. The significance of the network will be further scrutinized in two case studies namely the redox regulation of cytosolic glyceraldehyde-3-phosphate dehydrogenase (GAPC) and vacuolar H+-ATPase. Overall, the project is planned to test the concept of “redoxosomes” as regulatory units. Advanced quantitative understanding of the cytosolic redox subnetworks will be attempted by mathematical modelling and dynamic simulation in order to deliver novel insight into the kinetic coupling between the network components.