Dóka Éva, Pader Irina, Bíró Adrienn, Johansson Katarina, Cheng Qing, Ballagó Krisztina, Prigge Justin R, Pastor-Flores Daniel, Dick Tobias P, Schmidt Edward E, Arnér Elias S J, Nagy Péter
Department of Molecular Immunology and Toxicology, National Institute of Oncology, Ráth György utca 7-9, Budapest 1122, Hungary.
Division of Biochemistry, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE-171 77 Stockholm, Sweden.
Sci Adv. 2016 Jan 22;2(1):e1500968. doi: 10.1126/sciadv.1500968. eCollection 2016 Jan.
Hydrogen sulfide signaling involves persulfide formation at specific protein Cys residues. However, overcoming current methodological challenges in persulfide detection and elucidation of Cys regeneration mechanisms from persulfides are prerequisites for constructing a bona fide signaling model. We here establish a novel, highly specific protein persulfide detection protocol, ProPerDP, with which we quantify 1.52 ± 0.6 and 11.6 ± 6.9 μg/mg protein steady-state protein persulfide concentrations in human embryonic kidney 293 (HEK293) cells and mouse liver, respectively. Upon treatment with polysulfides, HEK293 and A549 cells exhibited increased protein persulfidation. Deletion of the sulfide-producing cystathionine-γ-lyase or cystathionine-β-synthase enzymes in yeast diminished protein persulfide levels, thereby corroborating their involvement in protein persulfidation processes. We here establish that thioredoxin (Trx) and glutathione (GSH) systems can independently catalyze reductions of inorganic polysulfides and protein persulfides. Increased endogenous persulfide levels and protein persulfidation following polysulfide treatment in thioredoxin reductase-1 (TrxR1) or thioredoxin-related protein of 14 kDa (TRP14) knockdown HEK293 cells indicated that these enzymes constitute a potent regeneration system of Cys residues from persulfides in a cellular context. Furthermore, TrxR1-deficient cells were less viable upon treatment with toxic amounts of polysulfides compared to control cells. Emphasizing the dominant role of cytosolic disulfide reduction systems in maintaining sulfane sulfur homeostasis in vivo, protein persulfide levels were markedly elevated in mouse livers where hepatocytes lack both TrxR1 and glutathione reductase (TR/GR-null). The different persulfide patterns observed in wild-type, GR-null, and TR/GR-null livers suggest distinct roles for the Trx and GSH systems in regulating subsets of protein persulfides and thereby fine-tuning sulfide signaling pathways.
硫化氢信号传导涉及特定蛋白质半胱氨酸（Cys）残基处的过硫化物形成。然而，克服当前过硫化物检测方法的挑战以及阐明过硫化物中Cys再生机制是构建真正信号传导模型的先决条件。我们在此建立了一种新型、高度特异性的蛋白质过硫化物检测方案ProPerDP，利用该方案我们分别在人胚肾293（HEK293）细胞和小鼠肝脏中量化了稳态蛋白质过硫化物浓度，分别为1.52±0.6和11.6±6.9μg/mg蛋白质。用多硫化物处理后，HEK293和A549细胞的蛋白质过硫化作用增强。酵母中产生硫化物的胱硫醚-γ-裂解酶或胱硫醚-β-合酶的缺失降低了蛋白质过硫化物水平，从而证实了它们参与蛋白质过硫化过程。我们在此确定硫氧还蛋白（Trx）和谷胱甘肽（GSH）系统可以独立催化无机多硫化物和蛋白质过硫化物的还原。在硫氧还蛋白还原酶-1（TrxR1）或14 kDa硫氧还蛋白相关蛋白（TRP14）敲低的HEK293细胞中，多硫化物处理后内源性过硫化物水平和蛋白质过硫化作用增加，表明这些酶在细胞环境中构成了过硫化物中Cys残基的有效再生系统。此外，与对照细胞相比，用有毒量的多硫化物处理后，TrxR1缺陷细胞的活力较低。蛋白质过硫化物水平在肝细胞同时缺乏TrxR1和谷胱甘肽还原酶（TR/GR-null）的小鼠肝脏中显著升高，强调了胞质二硫键还原系统在体内维持次磺酸硫稳态中的主导作用。在野生型、GR-null和TR/GR-null肝脏中观察到的不同过硫化物模式表明Trx和GSH系统在调节蛋白质过硫化物亚群从而微调硫化物信号通路中具有不同作用。
