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Molecular targeting of metallothionein by L‐homocysteine in human aortic endothelial cells
Author(s) -
Barbato John C.,
Catanescu Otilia,
DiBello Patricia M.,
Jacobsen Donald W.
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.4.a198-a
Subject(s) - metallothionein , homocysteine , chemistry , microbiology and biotechnology , biochemistry , biology , gene
Hyperhomocysteinemia is an independent risk factor for cardiovascular disease. Due to the reactive nature of the –SH group, homocysteine is likely to form mixed‐disulfide adducts with cysteine‐rich proteins and alter function of the targeted molecule. We hypothesized that metallothionein (MT), an essential intracellular zinc‐chaperone and superoxide anion scavenger, is an ideal target for homocysteinylation because of its high cysteine content. After incubation of human aortic endothelial cells (HAEC) with 35 S‐L‐homocysteine, 35 S‐L‐homocysteinylated‐MT was identified in lysates by phosphorimaging and Western blotting. Using zinc‐chelating Sepharose chromatography, we determined that homocysteine impairs the zinc‐binding capacity of MT. In addition, microscopic fluorometry on Zinquin‐AM loaded HAEC demonstrated that homocysteine caused a dose‐dependent rise in intracellular free zinc. This rise in free zinc was associated with expression of early growth response protein 1 (EGR1) 30 min after peak intracellular zinc levels were reached. Homocysteine was also shown to inhibit the superoxide anion scavenging ability of MT by 65%. For the first time, these studies provide evidence that homocysteine targets MT, an intracellular HAEC protein responsible for zinc homeostasis and superoxide scavenging and suggest a novel mechanism for homocysteine‐induced oxidative stress. This work was supported by NIH HL52234.