S-glutathionylation of metallothioneins by nitrosative/oxidative stress.
Cystein residues within metallothionein (MT) structure have been shown to be particularly prone to S-nitrosylation. The objective of this study was to examine the possibility that MTs undergo S-glutathionylation under nitrosative/oxidative stress. MT from rabbit liver was treated with different concentrations of GSNO, diamide plus GSH or H(2)O(2) plus GSH. Parallel sets of samples were treated with 10mM DTT for 30min at 37 degrees C to reduce mixed disulphides. Incubations were then processed for Western blot or dot-immunobinding assay. Western blot with anti-MT or anti-GSH were also performed on peripheral blood mononuclear cell extracts. Structural aspects of S-glutathionylation of MTs were also examined. Treatment with GSNO, diamide/GSH or H(2)O(2)/GSH induced a dose-dependent increase in the levels of MT S-glutathionylation. This effect was completely reversed by treatment with the reducing agent DTT, indicating that S-glutathionylation of MT protein was related to formation of protein-mixed disulphides. Structural analysis of rat MT indicated that Cys residues located in the N-terminal domain of the protein are the likely targets for S-glutathionylation, both for their solvent accessibility and electrostatics induced reactivity. S-Glutathionylation of MT, given its reversibility, would provide protection from irreversible oxidation of Cys residues, thus representing a mechanism of high potential biological relevance.
Casadei
Manuela
M
Dipartimento di Biologia, University ROMA TRE, Viale Marconi 446, 00146 Rome, Italy.
Persichini
Tiziana
T
Polticelli
Fabio
F
Musci
Giovanni
G
Colasanti
Marco
M
D000818:Animals; D015153:Blotting, Western; D003958:Diamide / Q000494:pharmacology; D005978:Glutathione / Q000031:analogs & derivatives* / Q000494:pharmacology; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007963:Leukocytes, Mononuclear; D008668:Metallothionein / Q000187:drug effects* / Q000378:metabolism*; D009574:Nitro Compounds / Q000494:pharmacology*; D016877:Oxidants / Q000494:pharmacology; D018384:Oxidative Stress / Q000502:physiology*; D011817:Rabbits; D013439:Sulfhydryl Reagents / Q000494:pharmacology
D009574:Nitro Compounds; D016877:Oxidants; C116027:S-nitroglutathione; D013439:Sulfhydryl Reagents; D003958:Diamide; D008668:Metallothionein; D006861:Hydrogen Peroxide; D005978:Glutathione