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: Takashi Toyama, Yasuhiro Shinkai, Aki Yazawa, Hidenao Kakehashi, Toshiyuki Kaji, Yoshito Kumagai
• Journal: Chemico-biological interactions (Chem Biol Interact) Vol. 214 Pg. 41-8 (May 05 2014) ISSN: 1872-7786 [Electronic] Ireland
• PMID: 24582816 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
• Copyright: Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Chemical References

• Naphthoquinones
• Recombinant Proteins
• UCHL1 protein, human
• 1,2-naphthoquinone
• Ubiquitin Thiolesterase
• Glutathione
• Lysine
• Cysteine

Topics

• Blotting, Western
• Cell Line, Tumor
• Cysteine (chemistry)
• Electrophoresis, Gel, Two-Dimensional
• Glutathione (physiology)
• Humans
• Lysine (chemistry)
• Naphthoquinones (chemistry)
• Recombinant Proteins (chemistry, metabolism)
• Ubiquitin Thiolesterase (chemistry, metabolism)
• Ubiquitination