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Merck
  • A novel persulfide detection method reveals protein persulfide- and polysulfide-reducing functions of thioredoxin and glutathione systems.

A novel persulfide detection method reveals protein persulfide- and polysulfide-reducing functions of thioredoxin and glutathione systems.

Science advances (2016-02-05)
Éva Dóka, Irina Pader, Adrienn Bíró, Katarina Johansson, Qing Cheng, Krisztina Ballagó, Justin R Prigge, Daniel Pastor-Flores, Tobias P Dick, Edward E Schmidt, Elias S J Arnér, Péter Nagy
摘要

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.

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Sigma-Aldrich
抗 兔 IgG(全分子)-碱性磷酸酶 山羊抗, affinity isolated antibody, buffered aqueous glycerol solution
Sigma-Aldrich
抗-白蛋白 兔抗, IgG fraction of antiserum