Endothelial Nitric Oxide Synthase–Derived Nitric Oxide Prevents Dihydrofolate Reductase Degradation via Promoting S-Nitrosylation
Objective—Dihydrofolate reductase (DHFR) is a key protein involved in tetrahydrobiopterin (BH4) regeneration from 7,8-dihydrobiopterin (BH2). Dysfunctional DHFR may induce endothelial nitric oxide (NO) synthase (eNOS) uncoupling resulting in enzyme production of superoxide anions instead of NO. The mechanism by which DHFR is regulated is unknown. Here, we investigate whether eNOS-derived NO maintains DHFR stability.
Approach and Results—DHFR activity, BH4 content, eNOS activity, and S-nitrosylation were assessed in human umbilical vein endothelial cells and in aortas isolated from wild-type and eNOS knockout mice. In human umbilical vein endothelial cells, depletion of intracellular NO by transfection with eNOS-specific siRNA or by the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide—both of which had no effect on DHFR mRNA levels—markedly reduced DHFR protein levels in parallel with increased DHFR polyubiquitination. Supplementation of S-nitroso-l-glutathione, a NO donor, or MG132, a potent inhibitor of the 26S proteasome, prevented eNOS silencing and 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide–induced DHFR reduction in human umbilical vein endothelial cells. 2-(4-Carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide suppressed S-nitrosylation of DHFR, whereas S-nitroso-l-glutathione promoted DHFR S-nitrosylation. Mutational analysis confirmed that cysteine 7 of DHFR was S-nitrosylated. Cysteine 7 S-nitrosylation stabilized DHFR from ubiquitination and degradation. Experiments performed in aortas confirmed that 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide or eNOS deficiency reduces endothelial DHFR, which can be abolished by MG132 supplementation.
Conclusions—We conclude that S-nitrosylation of DHFR at cysteine 7 by eNOS-derived NO is crucial for DHFR stability. We also conclude that NO-induced stabilization of DHFR prevents eNOS uncoupling via regeneration of tetrahydrobiopterin, an essential eNOS cofactor.
- human umbilical vein endothelial cells
- nitric oxide
- Nos3 protein, mouse
- tetrahydrofolate dehydrogenase
- Received April 22, 2015.
- Accepted September 4, 2015.
- © 2015 American Heart Association, Inc.