Peroxynitrite-Dependent Zinc Release and Inactivation of Guanosine 5′-Triphosphate Cyclohydrolase 1 Instigate Its Ubiquitination in Diabetes

  1. Ming-Hui Zou1,2,3
  1. 1Section of Molecular Medicine, Department of Medicine, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma
  2. 2Hubei Province Key Laboratory on Cardiovascular, Cerebrovascular, and Metabolic Disorders, Hubei University of Science and Technology, Xianning, Hubei, China
  3. 3Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma
  1. Corresponding author: Ming-Hui Zou, ming-hui-zou{at}ouhsc.edu.

Abstract

Aberrant degradation of guanosine 5′-triphosphate cyclohydrolase 1 (GTPCH1) with consequent deficiency of tetrahydrobiopterin is considered the primary cause for endothelial dysfunction in diabetes. How GTPCH1 becomes susceptible to the degradation remains unknown. We hypothesized that oxidation and release of the zinc ion by peroxynitrite (ONOO), a potent oxidant generated by nitric oxide and superoxide anions, instigates GTPCH1 ubiquitination and degradation. Zinc contents, GTPCH1 ubiquitination, and GTPCH1 activity were assayed in purified GTPCH1, endothelial cells, and hearts from diabetic mice. Exogenous ONOO dose-dependently released zinc, inhibited its activity, and increased the ubiquitin binding affinity of GTPCH1 in vitro and in endothelial cells. Consistently, high glucose (30 mmol/L) inhibited GTPCH1 activity with increased ubiquitination, which was inhibited by antioxidants. Furthermore, mutation of the zinc-binding cysteine (141) (C141R or C141A) significantly reduced GTPCH1 activity and reduced its half-life but increased GTPCH1 ubiquitination, indicating an essential role of the zinc ion in maintaining the catalytic activity and stability of GTPCH1. Finally, GTPCH1 ubiquitination and degradation markedly increased in parallel with decreased GTPCH1 activity in the aortas and hearts of diabetic mice, both of which were attenuated by the inhibitors of ONOO in mice in vivo. Taken together, we conclude that ONOO releases zinc and inhibits GTPCH1, resulting in its ubiquitination and degradation of the enzyme.

Footnotes

  • Received May 8, 2013.
  • Accepted August 14, 2013.

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  1. Diabetes vol. 62 no. 12 4247-4256
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