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

蛋白S-谷胱甘肽化是谷胱甘肽和蛋白质上可接近的游离巯基之间的混合二硫键的可逆翻译后形成，已成为参与激酶信号通路、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调节的重要机制。因此，将特定蛋白质的S-谷胱甘肽化与癌症、心血管和肺部疾病、糖尿病和神经退行性疾病结合起来讨论并不奇怪。谷氧还蛋白（GRX）和可能的ω-类谷胱甘肽S-转移酶（GSTO）在这些（去）谷胱甘肽化过程中起着重要作用。该提案是我们对谷胱甘肽依赖性酶的全面调查的一部分，其长期目标是有助于理解它们在（i）氧化还原稳态，（ii）环境压力的传感和信号传导以及（iii）适应环境变化中的作用。C.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、定位和天然表达动力学的极好模型系统，所述氧化还原蛋白和抗氧化解毒系统通常是多细胞生物，特别是动物所特有的。我们计划在正常和氧化应激条件下确定S-谷胱甘肽化的靶蛋白。在这里，我们将重点确定，验证和分析的三个胞质二巯基GRX和GSTO-1的具体目标。一种新的“突变蛋白捕获策略”将被用来识别靶蛋白。这种体外方法基于CXXS突变的GRX或GSTO还原谷胱甘肽混合二硫键，对其进行标记，然后进行富集和蛋白质组学鉴定。我们对传统方法进行了升级和微调，（i）使用重组C。线虫CXXS突变的GRX和GSTO在脱谷胱甘肽化步骤中，从而选择性地捕获它们的特异性靶蛋白，（ii）使用蠕虫突变体，其中相应的内源性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中过表达氧化还原酶来直接识别氧化还原酶的靶标。这使得能够原位捕获靶蛋白。在验证选定靶蛋白的S-谷胱甘肽化后，将进行体外下拉试验以验证靶相互作用。最后，一种新的GFP募集试验将在C. elegans，其使得能够在体内分析选定的GRX（GSTO）-靶蛋白相互作用。这些相互作用的功能后果将进行分析，我们预计，新的目标的S-谷胱甘肽的识别将导致这一基本的细胞过程的知识的进步，也许建立新的治疗策略的基础。

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.