In vivo targets of S-thiolation in Chlamydomonas reinhardtii
In vivo targets of S-thiolation in Chlamydomonas reinhardtii
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DOI：
10.1074/jbc.m802331200
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发表时间：
2008-08-01
影响因子：
4.8
通讯作者：
Decottignies, Paulette
中科院分区：
文献类型：
--
作者：
Michelet, Laure;Zaffagnini, Mirko;Decottignies, Paulette
谷胱甘肽基化是细胞内S硫基化的主要形式。这种可逆的氧化还原翻译后修饰包括在蛋白质上的游离硫醇和谷胱甘肽分子之间形成混合的二硫键。这种最近被描述的修饰被认为是在氧化应激下发生的，可以保护半胱氨酸残基免受不可逆氧化，并对不同蛋白质的活性产生正向或负向的改变。到目前为止，这种修饰及其靶标主要是在非光合作用生物中进行的研究。我们报道了第一个在体内对具有光合作用能力的细胞进行蛋白质组学的方法，使用真核单细胞绿藻衣藻用放射性标记的半胱氨酸[S-35]标记谷胱甘肽库和二胺作为氧化剂。这种方法可以识别25个参与各种代谢过程的靶标，主要是氯化塑料。有几个靶标与光合作用有关，如卡尔文循环酶磷酸甘油酸激酶和核糖-5-磷酸异构酶。许多靶标，如伴侣和过氧化还蛋白，都与应激反应有关。体外实验证实HSP70B、叶绿体2-半胱氨酸过氧化还蛋白和异柠檬酸裂解酶在纯化蛋白上发生谷胱甘肽基化，并鉴定了目标残基。
Glutathionylation is the major form of S-thiolation in cells. This reversible redox post-translational modification consists of the formation of a mixed disulfide between a free thiol on a protein and a molecule of glutathione. This recently described modification, which is considered to occur under oxidative stress, can protect cysteine residues from irreversible oxidation, and alter positively or negatively the activity of diverse proteins. This modification and its targets have been mainly studied in non-photosynthetic organisms so far. We report here the first proteomic approach performed in vivo on photosynthetically competent cells, using the eukaryotic unicellular green alga Chlamydomonas reinhardtii with radiolabeled [S-35] cysteine to label the glutathione pool and diamide as oxidant. This method allowed the identification of 25 targets, mainly chloroplastic, involved in various metabolic processes. Several targets are related to photosynthesis, such as the Calvin cycle enzymes phosphoglycerate kinase and ribose-5-phosphate isomerase. A number of targets, such as chaperones and peroxiredoxins, are related to stress responses. The glutathionylation of HSP70B, chloroplastic 2-Cys peroxiredoxin and isocitrate lyase was confirmed in vitro on purified proteins and the targeted residues were identified.