title: Targeting of metallothionein by L-homocysteine: a novel mechanism for disruption of zinc and redox homeostasis.
abstract: OBJECTIVE
  L-homocysteine and/or L-homocystine interact in vivo with albumin and other extracellular proteins by forming mixed-disulfide conjugates. Because of its extremely rich cysteine content, we hypothesized that metallothionein, a ubiquitous intracellular zinc-chaperone and superoxide anion radical scavenger, reacts with L-homocysteine and that homocysteinylated-metallothionein suffers loss of function.
  
  
  RESULTS
  35S-homocysteinylated-metallothionein was resolved in lysates of cultured human aortic endothelial cells in the absence and presence of reduced glutathione by SDS-PAGE and identified by Western blotting and phosphorimaging. Using zinc-Sepharose chromatography, L-homocysteine was shown to impair the zinc-binding capacity of metallothionein even in the presence of reduced glutathione. L-Homocysteine induced a dose-dependent increase in intracellular free zinc in zinquin-loaded human aortic endothelial cells within 30 minutes, followed by the appearance of early growth response protein-1 within 60 minutes. In addition, intracellular reactive oxygen species dramatically increased 6 hours after L-homocysteine treatment. In vitro studies demonstrated that L-homocysteine is a potent inhibitor of the superoxide anion radical scavenging ability of metallothionein.
  
  
  CONCLUSIONS
  These studies provide the first evidence that L-homocysteine targets intracellular metallothionein by forming a mixed-disulfide conjugate and that loss of function occurs after homocysteinylation. The data support a novel mechanism for disruption of zinc and redox homeostasis.
authors.affiliation: Department Cell Biology, Lerner Research Institute, The Cleveland Clinic, Cleveland, OH 44195, USA.
authors.forename: John C
authors.initials: JC
authors.lastname: Barbato
authors.forename: Otilia
authors.initials: O
authors.lastname: Catanescu
authors.forename: Kelsey
authors.initials: K
authors.lastname: Murray
authors.forename: Patricia M
authors.initials: PM
authors.lastname: DiBello
authors.forename: Donald W
authors.initials: DW
authors.lastname: Jacobsen
mesh_terms: D001013:Aorta, Thoracic / Q000166:cytology; D002478:Cells, Cultured; D002847:Chromatography, Agarose; D004305:Dose-Response Relationship, Drug; D051766:Early Growth Response Protein 1 / Q000235:genetics / Q000378:metabolism; D004730:Endothelium, Vascular / Q000378:metabolism* / Q000473:pathology / Q000503:physiopathology; D005786:Gene Expression Regulation; D006706:Homeostasis / Q000502:physiology*; D006710:Homocysteine / Q000378:metabolism / Q000494:pharmacology*; D006801:Humans; D008668:Metallothionein / Q000378:metabolism*; D010084:Oxidation-Reduction; D013481:Superoxides / Q000378:metabolism; D013997:Time Factors; D015032:Zinc / Q000378:metabolism*
chemical_list: C497325:EGR1 protein, human; D051766:Early Growth Response Protein 1; D006710:Homocysteine; D013481:Superoxides; D008668:Metallothionein; D015032:Zinc