Functional interaction of glutathione S-transferase pi and peroxiredoxin 6 in intact cells.
Functional interaction of glutathione S-transferase pi and peroxiredoxin 6 in intact cells.
复制标题
完整细胞中谷胱甘肽 S-转移酶 pi 和过氧化还原蛋白 6 的功能相互作用。
DOI：
10.1016/j.biocel.2012.11.005
复制
发表时间：
2013
期刊：
影响因子：
--
通讯作者：
Fisher,AronB
中科院分区：
文献类型：
--
作者：
Zhou,Suiping;Lien,Yu-Chin;Shuvaeva,Tea;DeBolt,Kristine;Feinstein,SheldonI;Fisher,AronB
过氧化氧还蛋白6 （Prdx6）是过氧化氧还蛋白超家族的1-Cys成员，在抗氧化防御中起重要作用。π谷胱甘肽s -转移酶（GST）介导重组Prdx6的谷胱甘肽化是体外氧化Cys还原和完成过氧化物催化循环所必需的。本研究考察了完整细胞对π - gst的需求。将表达π - gst的质粒转染到缺乏内源性π - gst的MCF7细胞系中，可以显著提高细胞裂解物中磷脂过氧化物酶的活性，并保护完整细胞免受过氧化应激。siRNA敲低表明这种增加的过氧化物酶活性依赖于Prdx6。在基础条件下，π - gst和Prdx6之间的相互作用很小，但在过氧化叔丁基氧化剂处理细胞后，π - gst和Prdx6之间的相互作用急剧增加。由于Prdx6过氧化物酶活性位点C47的突变，相互作用被终止。用丁硫氨酸亚砜处理细胞消耗GSH对Prdx6与πGST的相互作用没有影响。这些数据与Prdx6与π - gst相互作用需要催化半胱氨酸氧化的假设一致，并且这种相互作用在Prdx6的过氧化物酶活性再生中起重要作用。
Peroxiredoxin 6 (Prdx6) is a 1-Cys member of the peroxiredoxin superfamily that plays an important role in antioxidant defense. Glutathionylation of recombinant Prdx6 mediated by π glutathione S-transferase (GST) is required for reduction of the oxidized Cys and completion of the peroxidatic catalytic cycle in vitro. This study investigated the requirement for πGST in intact cells. Transfection with a plasmid construct expressing πGST into MCF7, a cell line that lacks endogenous πGST, significantly increased phospholipid peroxidase activity as measured in cell lysates and protected intact cells against a peroxidative stress. siRNA knockdown indicated that this increased peroxidase activity was Prdx6 dependent. Interaction between πGST and Prdx6, evaluated by the Duolink Proximity Ligation Assay, was minimal under basal conditions but increased dramatically following treatment of cells with the oxidant, tert-butyl hydroperoxide. Interaction was abolished by mutation of C47, the active site for Prdx6 peroxidase activity. Depletion of cellular GSH by treatment of cells with buthionine sulfoximine had no effect on the interaction of Prdx6 and πGST. These data are consistent with the hypothesis that oxidation of the catalytic cysteine in Prdx6 is required for its interaction with πGST and that the interaction plays an important role in regenerating the peroxidase activity of Prdx6.