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Sci Adv. 2016 Jan 22;2(1):e1500968. doi: 10.1126/sciadv.1500968. eCollection 2016 Jan.

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

Author information

1
Department of Molecular Immunology and Toxicology, National Institute of Oncology, Ráth György utca 7-9, Budapest 1122, Hungary.
2
Division of Biochemistry, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE-171 77 Stockholm, Sweden.
3
Department of Microbiology and Immunology, Montana State University, Cooley Hall, PO Box 173520, Bozeman, MT 59717, USA.
4
Division of Redox Regulation, German Cancer Research Center (DKFZ), DKFZ-ZMBH Alliance, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany.

Abstract

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.

KEYWORDS:

Glutathione system; Life sciences; ProPerDP method; Proteins; Thioredoxins; biochemistry; cell signaling; hydrogen sulfide; persulfide

PMID:
26844296
PMCID:
PMC4737208
DOI:
10.1126/sciadv.1500968
[Indexed for MEDLINE]
Free PMC Article

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