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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2007;27:49.)
© 2007 American Heart Association, Inc.

Vascular Biology

Targeting of Metallothionein by L-Homocysteine

A Novel Mechanism for Disruption of Zinc and Redox Homeostasis

John C. Barbato; Otilia Catanescu; Kelsey Murray; Patricia M. DiBello; Donald W. Jacobsen

From Department of Cell Biology (J.C.B., O.C., K.M., P.M.D., D.W.J.), Lerner Research Institute, The Cleveland Clinic, Department of Molecular Medicine (D.W.J.), Cleveland Clinic Lerner College of Medicine of Case Western Reserve University, Cleveland, Ohio.

Correspondence to Donald W. Jacobsen, Department Cell Biology, NC-10, Lerner Research Institute, The Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195. E-mail jacobsd{at}ccf.org

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.

Methods and 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.

Conclusion— 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.

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. 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.

Key Words: early growth response protein-1 • endothelial dysfunction • hyperhomocysteinemia • metallothionein • superoxide anion radical scavenging • zinc homeostasis

Related Article:

Homocysteinylation of Metallothionein Impairs Intracellular Redox Homeostasis: The Enemy Within!

Stephen M. Colgan and Richard C. Austin
Arterioscler. Thromb. Vasc. Biol. 2007 27: 8-11. [Full Text] [PDF]

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				S. M. Colgan and R. C. Austin Homocysteinylation of Metallothionein Impairs Intracellular Redox Homeostasis: The Enemy Within! Arterioscler. Thromb. Vasc. Biol., January 1, 2007; 27(1): 8 - 11. [Full Text] [PDF]