Cysteine 343 in the substrate binding domain is the primary S-Nitrosylated site in protein disulfide isomerase
Cysteine 343 in the substrate binding domain is the primary S-Nitrosylated site in protein disulfide isomerase
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DOI：
10.1016/j.freeradbiomed.2020.07.029
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发表时间：
2020-11-20
影响因子：
7.4
通讯作者：
Mano, Nariyasu
中科院分区：
文献类型：
--
作者：
Ogura, Jiro;Ruddock, Lloyd W.;Mano, Nariyasu
蛋白质异常积聚是神经退行性疾病的典型病理特征。蛋白质二硫键异构酶（PDI）是蛋白质氧化折叠的关键酶。在神经退行性疾病患者的死后脑中已经检测到S-亚硝基化PDI，但是S-亚硝基化对PDI功能和发展神经退行性疾病的影响还没有详细阐明。在这项研究中，我们确定了在体外和体内发生的C343的B'结构域的PDI的亚硝基化。还原重组人PDI（hPDI）与S-亚硝基化合物反应迅速，观察到预期的S-亚硝基化物质增加，活性位点出现二硫键状态。从hPDI的亚硝基化中观察到单亚硝基化和二亚硝基化。二亚硝基化的物种是在活性位点的两个半胱氨酸S-亚硝基化。但是，至少部分单亚硝基化物质在底物结合B'结构域的半胱氨酸343上被S-亚硝基化。虽然活性位点S-亚硝基化是可逆的还原型谷胱甘肽，S-亚硝基化的C343是比较稳定的。S-亚硝基半胱氨酸（SNOC）处理SH-SY 5 Y细胞后，可观察到细胞内PDI的S-亚硝基化，去除SNOC后24 h仍能检测到S-亚硝基化的PDI。虽然野生型PDI被S-亚硝基化，但过表达细胞中C343 S突变体的S-亚硝基化水平大幅降低，并且在去除过表达细胞中的SNOC后24小时，仅保留S-亚硝基化的野生型PDI。体内外实验结果表明，PDI中C343的S-亚硝基化可能是神经退行性疾病患者生理变化的原因，并可能有助于神经退行性疾病药物的开发。
Abnormal protein accumulations are typical pathological features for neurodegenerative diseases. Protein disulfide isomerase (PDI) is a critical enzyme in oxidative protein folding. S-nitrosylated PDI has been detected in the postmortem brain in neurodegenerative disease patients, but the effect of S-nitrosylation on PDI function and developing neurodegeneration was not clarified in detail. In this study, we identified that in vitro and in vivo Snitrosylation of C343 in the b' domain of PDI occurs. Reduced recombinant human PDI (hPDI) reacted quickly with S-nitrosocompounds, with an observed increase in the expected S-nitrosylated species and the appearance of the disulfide state of the active sites. Both Mononitrosylated and dinitrosylated were observed from the Snitrosylation of hPDI. Dinitrosylated species were S-nitrosylated both cysteines at active site. But, at least in part, mononitrosylated species were S-nitrosylated on cysteine 343 in the substrate binding b' domain. Although active site S-nitrosylation is reversible by reduced glutathione, S-nitrosylation of C343 is comparative stable. Snitrosylation of PDI in SH-SY5Y cells was observed after the S-nitrosocysteine (SNOC) treatment and S-nitrosylated PDI was still detected 24 h after removing SNOC. While wild-type PDI was S-nitrosylated, the level of S-nitrosylation of the C343S mutant in over-expressed cells was substantially lower and only wild-type PDI of Snitrosylation remained 24 h after removing SNOC in over-expressed cells. Both of in vitro and in vivo results suggested that S-nitrosylation of C343 in PDI may be the causative effect on physiological changes in neurodegerenative disease patients, and may be useful for the drug development for neurodegenerative diseases.