title: Cross-linking protein glutathionylation mediated by O2-arylated bis-diazeniumdiolate "Double JS-K".
abstract: Attachment of glutathione (GSH) to cysteine residues in proteins (S-glutathionylation) is a reversible post-translational modification that can profoundly alter protein structure and function. Often serving in a protective role, for example, by temporarily saving protein thiols from irreversible oxidation and inactivation, glutathionylation can be identified and semiquantitatively assessed using anti-GSH antibodies, thought to be specific for recognition of the S-glutathionylation modification. Here, we describe an alternate mechanism of protein glutathionylation in which the sulfur atoms of the GSH and the protein's thiol group are covalently bound via a cross-linking agent, rather than through a disulfide bond. This form of thiol cross-linking has been shown to occur and has been confirmed by mass spectrometry at the solution chemistry level, as well as in experiments documenting the potent antiproliferative activity of the bis-diazeniumdiolate Double JS-K in H1703 cells in vitro and in vivo. The modification is recognized by the anti-GSH antibody as if it were authentic S-glutathionylation, requiring mass spectrometry to distinguish between them.
authors.affiliation: Frederick National Laboratory for Cancer Research, Frederick, Maryland 21702, United States. hollandrj@mail.nih.gov
authors.forename: Ryan J
authors.initials: RJ
authors.lastname: Holland
authors.forename: Anna E
authors.initials: AE
authors.lastname: Maciag
authors.forename: Varun
authors.initials: V
authors.lastname: Kumar
authors.forename: Lei
authors.initials: L
authors.lastname: Shi
authors.forename: Joseph E
authors.initials: JE
authors.lastname: Saavedra
authors.forename: Robert K
authors.initials: RK
authors.lastname: Prud'homme
authors.forename: Harinath
authors.initials: H
authors.lastname: Chakrapani
authors.forename: Larry K
authors.initials: LK
authors.lastname: Keefer
mesh_terms: D000111:Acetylcysteine / Q000737:chemistry; D000199:Actins / Q000378:metabolism; D000818:Animals; D000970:Antineoplastic Agents / Q000737:chemistry / Q000494:pharmacology*; D001391:Azo Compounds / Q000737:chemistry / Q000494:pharmacology*; D045744:Cell Line, Tumor; D004139:Dinitrofluorobenzene / Q000031:analogs & derivatives / Q000737:chemistry; D005260:Female; D005978:Glutathione / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D051379:Mice; D008819:Mice, Nude; D053758:Nanoparticles / Q000737:chemistry; D009569:Nitric Oxide / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism; D010879:Piperazines / Q000737:chemistry / Q000494:pharmacology*
chemical_list: D000199:Actins; D000970:Antineoplastic Agents; D001391:Azo Compounds; C474958:O(2)-(2,4-dinitrophenyl) 1-((4-ethoxycarbonyl)piperazin-1-yl)diazen-1-ium-1,2-diolate; D010879:Piperazines; D009569:Nitric Oxide; D004139:Dinitrofluorobenzene; C002041:1,5-difluoro-2,4-dinitrobenzene; D005978:Glutathione; D010100:Oxygen; D000111:Acetylcysteine