Targeting of metallothionein by L-homocysteine: a novel mechanism for disruption of zinc and redox homeostasis.
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.
Barbato
John C
JC
Department Cell Biology, Lerner Research Institute, The Cleveland Clinic, Cleveland, OH 44195, USA.
Catanescu
Otilia
O
Murray
Kelsey
K
DiBello
Patricia M
PM
Jacobsen
Donald W
DW
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*
C497325:EGR1 protein, human; D051766:Early Growth Response Protein 1; D006710:Homocysteine; D013481:Superoxides; D008668:Metallothionein; D015032:Zinc