title: Protein polysulfidation-dependent persulfide dioxygenase activity of ethylmalonic encephalopathy protein 1.
abstract: Reactive persulfide species such as glutathione persulfide (GSSH) are highly abundant biomolecules. Persulfide dioxygenase (also called ethylmalonic encephalopathy protein 1, ETHE1) reportedly metabolizes GSSH to GSH with simultaneous oxygen consumption. How ETHE1 activity is regulated is still unclear, however. In this study, we describe the possible role of protein polysulfidation in the catalytic activity of ETHE1. We first found that ETHE1 catalyzed the persulfide dioxygenase reaction mostly for glutathione polysulfides, GS-(S)n-H, as well as for GSSH, but not for other endogenous persulfides such as cysteine and homocysteine persulfides/polysulfides. We then developed a novel method to detect protein polysulfidation and named it the polyethylene glycol-conjugated maleimide-labeling gel shift assay (PMSA). PMSA analysis indicated that most cysteine residues in ETHE1 were polysulfidated. Site-directed mutagenesis of cysteine residues in ETHE1 combined with liquid chromatography tandem mass spectrometry for polysulfidation determination surprisingly indicated that the Cys247 residue was important for polysulfidation of other Cys residues and that the C247S mutant possessed no persulfide dioxygenase activity. These results suggested that ETHE1 is a major enzyme regulating endogenous GSSH/GS-(S)n-H and that its activity is controlled by polysulfidation of the Cys247 residue.
authors.affiliation: Department of Environmental Health Sciences and Molecular Toxicology, Tohoku University Graduate School of Medicine, Sendai, 980-8575, Japan.
authors.forename: Minkyung
authors.initials: M
authors.lastname: Jung
authors.affiliation: Department of Environmental Health Sciences and Molecular Toxicology, Tohoku University Graduate School of Medicine, Sendai, 980-8575, Japan.
authors.forename: Shingo
authors.initials: S
authors.lastname: Kasamatsu
authors.affiliation: Department of Environmental Health Sciences and Molecular Toxicology, Tohoku University Graduate School of Medicine, Sendai, 980-8575, Japan.
authors.forename: Tetsuro
authors.initials: T
authors.lastname: Matsunaga
authors.affiliation: Department of Environmental Health Sciences and Molecular Toxicology, Tohoku University Graduate School of Medicine, Sendai, 980-8575, Japan.
authors.forename: Soichiro
authors.initials: S
authors.lastname: Akashi
authors.affiliation: Department of Microbiology, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, 860-8556, Japan.
authors.forename: Katsuhiko
authors.initials: K
authors.lastname: Ono
authors.affiliation: Department of Environmental Health Sciences and Molecular Toxicology, Tohoku University Graduate School of Medicine, Sendai, 980-8575, Japan.
authors.forename: Akira
authors.initials: A
authors.lastname: Nishimura
authors.affiliation: Department of Environmental Health Sciences and Molecular Toxicology, Tohoku University Graduate School of Medicine, Sendai, 980-8575, Japan.
authors.forename: Masanobu
authors.initials: M
authors.lastname: Morita
authors.affiliation: Department of Environmental Health Sciences and Molecular Toxicology, Tohoku University Graduate School of Medicine, Sendai, 980-8575, Japan.
authors.forename: Hisyam
authors.initials: H
authors.lastname: Abdul Hamid
authors.affiliation: Department of Environmental Health Sciences and Molecular Toxicology, Tohoku University Graduate School of Medicine, Sendai, 980-8575, Japan.
authors.forename: Shigemoto
authors.initials: S
authors.lastname: Fujii
authors.affiliation: Department of Gene Expression Regulation, Institute of Development, Aging and Cancer, Tohoku University, Sendai, 980-8575, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Kitamura
authors.affiliation: Department of Microbiology, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, 860-8556, Japan.
authors.forename: Tomohiro
authors.initials: T
authors.lastname: Sawa
authors.affiliation: Department of Environmental Health Sciences and Molecular Toxicology, Tohoku University Graduate School of Medicine, Sendai, 980-8575, Japan.
authors.forename: Tomoaki
authors.initials: T
authors.lastname: Ida
authors.affiliation: Department of Gene Expression Regulation, Institute of Development, Aging and Cancer, Tohoku University, Sendai, 980-8575, Japan.
authors.forename: Hozumi
authors.initials: H
authors.lastname: Motohashi
authors.affiliation: Department of Environmental Health Sciences and Molecular Toxicology, Tohoku University Graduate School of Medicine, Sendai, 980-8575, Japan. Electronic address: takaike@med.tohoku.ac.jp.
authors.forename: Takaaki
authors.initials: T
authors.lastname: Akaike
mesh_terms: D000072283:A549 Cells; D003545:Cysteine / Q000737:chemistry; D049308:Dioxygenases / Q000235:genetics / Q000378:metabolism; D004220:Disulfides / Q000378:metabolism; D005978:Glutathione / Q000031:analogs & derivatives / Q000378:metabolism; D006801:Humans; D024101:Mitochondrial Proteins / Q000235:genetics / Q000378:metabolism*; D029543:Nucleocytoplasmic Transport Proteins / Q000235:genetics / Q000378:metabolism*; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity; D013440:Sulfides / Q000378:metabolism
chemical_list: D004220:Disulfides; C483228:ETHE1 protein, human; D024101:Mitochondrial Proteins; D029543:Nucleocytoplasmic Transport Proteins; D011506:Proteins; D013440:Sulfides; C000611742:glutathione persulfide; C051552:persulfides; D049308:Dioxygenases; D005978:Glutathione; D003545:Cysteine
keywords: Ethylmalonic encephalopathy protein 1; Persulfide dioxygenase; Protein polysulfidation; Reactive persulfide