The Dithiol Glutaredoxins of African Trypanosomes Have Distinct Roles and Are Closely Linked to the Unique Trypanothione Metabolism
The Dithiol Glutaredoxins of African Trypanosomes Have Distinct Roles and Are Closely Linked to the Unique Trypanothione Metabolism
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DOI：
10.1074/jbc.m110.165860
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发表时间：
2010-11-05
影响因子：
4.8
通讯作者：
Krauth-Siegel, R. Luise
中科院分区：
文献类型：
--
作者：
Ceylan, Sevgi;Seidel, Vera;Krauth-Siegel, R. Luise
布氏锥虫是非洲昏睡病的病原体，拥有两种二硫醇谷氧还蛋白（Grx1 和 Grx2）。 Grx1 存在于细胞质中，催化蛋白质去谷胱甘肽化，k(cat)/K-m 值高达 2 x 10(5) M-1 s(-1)。它加速了锥硫酮对核糖核苷酸还原酶的还原，尽管效率低于寄生虫锥虫氧还蛋白，并且二硫化胰岛素还原酶活性较低。尽管具有经典的 CPYC 活性位点，Grx1 仍与 GSH、谷胱甘肽亚精胺或锥硫酮作为非蛋白质配体形成二聚体铁硫复合物。因此，与普遍接受的假设相反，更换 Pro 并不是集群形成的先决条件。 T. brucei Grx2 显示出一个不寻常的 CQFC 活性位点，并且直向同源物仅出现在锥虫中。 Grx2 在线粒体中富集，分级分离的洋地黄皂苷裂解导致与细胞色素 c 共洗脱，表明定位于线粒体膜间隙。 Grx2 催化二硫化胰岛素的还原，但不催化核糖核苷酸还原酶的还原，并且其去谷胱甘肽活性比 Grx1 低 10 倍。 RNA 干扰 Grx2 导致原循环细胞生长迟缓，这与重要作用一致。 Grx1和Grx2在哺乳动物血流和昆虫原循环形式中分别以约2μM和200nM的细胞浓度组成型表达。锥硫酮还原两种蛋白质的二硫键形式，其表观速率常数比谷胱甘肽高 3 个数量级。 Grx1 以及效率较低的 Grx2 也催化锥硫酮对 GSSG 的还原。因此，Grxs 在非洲锥虫基于锥硫酮的硫醇氧化还原代谢中发挥独特的作用。
Trypanosoma brucei, the causative agent of African sleeping sickness, possesses two dithiol glutaredoxins (Grx1 and Grx2). Grx1 occurs in the cytosol and catalyzes protein deglutathionylations with k(cat)/K-m-values of up to 2 x 10(5) M-1 s(-1). It accelerates the reduction of ribonucleotide reductase by trypanothione although less efficiently than the parasite tryparedoxin and has low insulin disulfide reductase activity. Despite its classical CPYC active site, Grx1 forms dimeric iron-sulfur complexes with GSH, glutathionylspermidine, or trypanothione as nonprotein ligands. Thus, contrary to the generally accepted assumption, replacement of the Pro is not a prerequisite for cluster formation. T. brucei Grx2 shows an unusual CQFC active site, and orthologues occur exclusively in trypanosomatids. Grx2 is enriched in mitoplasts, and fractionated digitonin lysis resulted in a co-elution with cytochrome c, suggesting localization in the mitochondrial intermembrane space. Grx2 catalyzes the reduction of insulin disulfide but not of ribonucleotide reductase and exerts deglutathionylation activity 10-fold lower than that of Grx1. RNA interference against Grx2 caused a growth retardation of procyclic cells consistent with an essential role. Grx1 and Grx2 are constitutively expressed with cellular concentrations of about 2 mu M and 200 nM, respectively, in both the mammalian bloodstream and insect procyclic forms. Trypanothione reduces the disulfide form of both proteins with apparent rate constants that are 3 orders of magnitude higher than those with glutathione. Grx1 and, less efficiently, also Grx2 catalyze the reduction of GSSG by trypanothione. Thus, the Grxs play exclusive roles in the trypanothione-based thiol redox metabolism of African trypanosomes.