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

巯基氧化还原调节网络的存在是包括细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，如代谢和信号传导。在植物中，对叶绿体的氧化还原网络了解最多。然而，与谷胱甘肽过氧化物酶样蛋白（GPXL）相比，其在细胞质中的具体特征和功能仍有待探索，特别是II型过氧化物还毒素（PRXIIB/C/D）作为氧化还原传感器的作用。拟开展的拟南芥研究将涉及电子竞争、相互作用的特异性和氧化还原传输动力学。氧化刺激的传播将在一个由各种网络组成的新型重组氧化还原系统中进行监测。使用高分辨率定量质谱（MS）为基础的蛋白质组学，该设置将区分硫醇，肽之间的二硫化物和s -谷胱甘肽化，并剖析每种氧化还原元素在系统中的作用。我们将利用CRISPR/Cas在敲除系中探索这三种PRXIIB/C/D异构体的功能，并将其与缺乏GPXL2/8的突变体进行比较，例如在光移或RBOH激活后。PRXIIB的相互作用组将在体外使用基于亲和层析的下拉相互作用体，然后进行质谱鉴定，在体内使用Turbo-ID-PRXII构建体。该网络的意义将在两个案例研究中进一步研究，即胞质甘油醛-3-磷酸脱氢酶（GAPC）和液泡H+- atp酶的氧化还原调节。总的来说，该项目计划测试“氧化质体”作为调节单位的概念。将通过数学建模和动态模拟来尝试对细胞质氧化还原子网络进行高级定量理解，以便对网络组件之间的动力学耦合提供新的见解。

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.