title: Glutathione-mediated reversibility of covalent modification of ubiquitin carboxyl-terminal hydrolase L1 by 1,2-naphthoquinone through Cys152, but not Lys4.
abstract: Covalent modification of cellular proteins by electrophiles affects electrophilic signal transduction and the dysfunction of enzymes that is involved in cytotoxicity. We have recently found a unique reaction which restores glyceraldehyde-3-phosphate dehydrogenase (GAPDH) that has been modified by 1,2-naphthoquinone (1,2-NQ) through a glutathione (GSH)-dependent S-transarylation reaction. We report here that ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) undergoes the same reaction. Exposure of human neuroblastoma SH-SY5Y cells to 1,2-NQ after pretreatment with buthionine sulfoximine (BSO) to deplete GSH resulted in an enhancement of covalent modification of UCH-L1 by 1,2-NQ. With recombinant human UCH-L1, we demonstrated that UCH-L1 underwent arylation by 1,2-NQ through Cys152 and Lys4, thereby decreasing its catalytic activity. Addition of GSH to an incubation mixture of 1,2-NQ-UCH-L1 adduct partially restored this decline in enzyme activity which was accompanied by decreased covalent attachment of 1,2-NQ, together with production of 1,2-NQ-GSH adduct. UCH-L1 in which Lys4 was mutated exhibited a lower level of covalent modification and enzyme inhibition, but completely recovered after addition of GSH. Taken together, these results suggest that Cys152 modification in UCH-L1 by 1,2-NQ is reversible via GSH-mediated S-transarylation reaction whereas Lys4 modification by 1,2-NQ is irreversible by GSH. Because UCH-L1 dysfunction has been associated with neurodegeneration, the electrophilic modification of Lys rather than Cys in UCH-L1 may be implicated in such neurodegenerative diseases.
authors.affiliation: Doctoral Program in Biomedical Sciences, Graduate School of Comprehensive Human Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan; Research Fellow of the Japan Society for the Promotion of Science, 1-8 Chiyoda, Tokyo 102-8472, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Toyama
authors.affiliation: Doctoral Program in Biomedical Sciences, Graduate School of Comprehensive Human Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan; Master's Program in Medical Sciences, Graduate School of Comprehensive Human Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan.
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Shinkai
authors.affiliation: College of Biological Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan.
authors.forename: Aki
authors.initials: A
authors.lastname: Yazawa
authors.affiliation: Master's Program in Medical Sciences, Graduate School of Comprehensive Human Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan.
authors.forename: Hidenao
authors.initials: H
authors.lastname: Kakehashi
authors.affiliation: Department of Environmental Health, Faculty of Pharmaceutical Sciences, Tokyo University of Science, Noda, Chiba 278-8510, Japan.
authors.forename: Toshiyuki
authors.initials: T
authors.lastname: Kaji
authors.affiliation: Doctoral Program in Biomedical Sciences, Graduate School of Comprehensive Human Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan; Master's Program in Medical Sciences, Graduate School of Comprehensive Human Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan. Electronic address: yk-em-tu@md.tsukuba.ac.jp.
authors.forename: Yoshito
authors.initials: Y
authors.lastname: Kumagai
mesh_terms: D015153:Blotting, Western; D045744:Cell Line, Tumor; D003545:Cysteine / Q000737:chemistry*; D015180:Electrophoresis, Gel, Two-Dimensional; D005978:Glutathione / Q000502:physiology*; D006801:Humans; D008239:Lysine / Q000737:chemistry*; D009285:Naphthoquinones / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D043222:Ubiquitin Thiolesterase / Q000737:chemistry / Q000378:metabolism*; D054875:Ubiquitination
chemical_list: D009285:Naphthoquinones; D011994:Recombinant Proteins; C482860:UCHL1 protein, human; C040756:1,2-naphthoquinone; D043222:Ubiquitin Thiolesterase; D005978:Glutathione; D008239:Lysine; D003545:Cysteine
keywords: 1,2-Napthoquinone; Glutathione; Michael reaction; S-transarylation; UCH-L1