Protein-S-glutathionylation in Caenorhabditis elegans - with focus on glutaredoxins and omega-class glutathione S-transferases

蛋白质S谷胱甘肽基化是蛋白质上谷胱甘肽和可获得的游离硫醇之间可逆的混合二硫键桥的翻译后形成，已成为调节蛋白激酶信号通路、糖酵解和能量代谢、抗氧化酶、钙稳态、蛋白质折叠、泛素-蛋白酶体和细胞凋亡的重要机制。因此，特定蛋白质的S谷胱甘肽基化与癌症、心血管和肺部疾病、糖尿病和神经变性疾病一起讨论也就不足为奇了。谷氧还蛋白(GRX)和可能的omega类谷胱甘肽S转移酶(GSTO)在这些(去)谷胱甘肽的过程中起着重要的作用。这项建议是我们对谷胱甘肽依赖酶的全面研究的一部分，长期目标是帮助理解它们在(I)氧化还原动态平衡、(Ii)环境压力的传感和信号以及(Iii)适应环境变化中的作用。线虫是研究氧化还原蛋白和抗氧化解毒系统的物理相互作用、定位和自然表达动力学的一个很好的模型系统，这些系统是多细胞生物所特有的，特别是动物。我们计划在正常和氧化应激条件下鉴定S谷胱甘肽基化的靶蛋白。在这里，我们将集中于鉴定、验证和分析三个胞质二硫醇GRX和GSTO-1的特异性靶标。一种新的“突变蛋白捕获策略”将被用来识别目标蛋白。这种体外方法是基于CXXS突变的GRX或GSTO还原谷胱甘肽混合二硫化物，然后标记它们，然后进行浓缩和蛋白质组学鉴定。我们通过以下方式对传统方法进行了升级和微调：(I)在去谷胱甘肽基化步骤中使用重组线虫CXXS突变的GRX和GSTO，从而选择性地捕获它们的特定目标蛋白；(Ii)使用蠕虫突变体，其中相应的内源GRX被敲除。这将大大增强外源GRX与靶蛋白之间的相互作用。在我们的体内方法中，我们希望通过在可用的GRX突变蠕虫的生理位置过度表达氧化还原酶来直接识别我们的氧化还原酶的靶标。这使得原位捕获目标蛋白成为可能。在验证了选定的目标蛋白的S-谷胱甘肽基化后，将进行体外下拉试验来验证目标相互作用。最后，一种新的GFP募集试验将用于线虫体内，从而能够在体内分析选定的GRX(GSTO)与目标蛋白的相互作用。我们将分析这些相互作用的功能后果，我们预计S谷胱甘肽基化的新靶点的确定将导致对这一基本细胞过程的了解的进步，并可能为新的治疗策略奠定基础。

Protein S-glutathionylation, the reversible post-translational formation of a mixed-disulfide bridge between glutathione and an accessible free thiol on a protein, has emerged as a crucial mechanism involved in the regulation of kinase signaling pathways, glycolysis and energy metabolism, antioxidant enzymes, calcium homeostasis, protein folding, ubiquitin-proteasome and apoptosis. It is therefore not surprising that S-glutathionylation of specific proteins is discussed in conjunction with cancer, cardiovascular and lung diseases, diabetes and neurodegerative diseases. Glutaredoxins (GRXs) and possibly the omega-class glutathione S-transferases (GSTOs) play a major role in these (de)glutathionylation processes. The proposal is part of our comprehensive investigation of glutathione-dependent enzymes with the long-term goal of contributing to the understanding of their role in (i) redox homeostasis, (ii) sensing and signaling of environmental stress and (iii) adaptation to environmental changes. C. elegans is an excellent model system to study the physical interaction, localization and native expression dynamics of redox proteins and antioxidant detoxification systems that are unique to multicellularity in general and animals in particular. We plan to identify target proteins of S-glutathionylation under normal and oxidative stress conditions. Here we will focus on identifying, verifying and analyzing the specific targets of three cytosolic dithiol GRXs and the GSTO-1. A novel 'mutant protein trapping strategy' will be used to identify target proteins. This in vitro method is based on the reduction of glutathione-mixed disulfides by CXXS-mutated GRX or GSTOs, their labeling followed by enrichment and proteomic identification. We have upgraded and fine-tuned the conventional method by (i) using the recombinant C. elegans CXXS-mutated GRXs and GSTOs in the deglutathionylation step, thereby selectively trapping their specific target proteins, (ii) using worm mutants where the corresponding endogenous GRX is knocked out. This will greatly enhance the interaction between the exogenous GRX and target proteins. In our in vivo approach we want to directly identify the targets of our redox enzymes by overexpressing them in their physiological location in the available grx-mutant worms. This enables the in situ trapping of the target proteins. Following verification of S-glutathionylation of selected target proteins, in vitro pull-down assays will be performed to validate target interactions. Finally, a novel GFP-recruitment assay will be used in C. elegans that enables the in vivo analysis of selected GRX (GSTO) - target protein interactions. Functional consequences of these interactions will be analysed and we expect that the identification of novel targets of S-glutathionylation will lead to an advancement in the knowledge of this fundamental cellular process and perhaps builds the basis for new therapeutic strategies.