Superoxide Induces Endothelial Nitric-oxide Synthase Protein Thiyl Radical Formation, a Novel Mechanism Regulating eNOS Function and Coupling

Abstract

An increase in production of reactive oxygen species resulting in a decrease in nitric oxide bioavailability in the endothelium contributes to many cardiovascular diseases, and these reactive oxygen species can oxidize cellular macromolecules. Protein thiols are critical reducing equivalents that maintain cellular redox state and are primary targets for oxidative modification. We demonstrate endothelial NOS (eNOS) oxidant-induced protein thiyl radical formation from tetrahydrobiopterin-free enzyme or following exposure to exogenous superoxide using immunoblotting, immunostaining, and mass spectrometry. Spin trapping with 5,5-dimethyl-1-pyrroline N-oxide (DMPO) followed by immunoblotting using an anti-DMPO antibody demonstrated the formation of eNOS protein radicals, which were abolished by superoxide dismutase and L-NAME, indicating that protein radical formation was due to superoxide generation from the eNOS heme. With tetrahydrobiopterin-reconstituted eNOS, eNOS protein radical formation was completely inhibited. Using mass spectrometric and mutagenesis analysis, we identified Cys-908 as the residue involved in protein radical formation. Mutagenesis of this key cysteine to alanine abolished eNOS thiyl radical formation and uncoupled eNOS, leading to increased superoxide generation. Protein thiyl radical formation leads to oxidation or modification of cysteine with either disulfide bond formation or S-glutathionylation, which induces eNOS uncoupling. Furthermore, in endothelial cells treated with menadione to trigger cellular superoxide generation, eNOS protein radical formation, as visualized with confocal microscopy, was increased, and these results were confirmed by immunoprecipitation with anti-eNOS antibody, followed by immunoblotting with an anti-DMPO antibody. Thus, eNOS protein radical formation provides the basis for a mechanism of superoxide-directed regulation of eNOS, involving thiol oxidation, defining a unique pathway for the redox regulation of cardiovascular function.

Subcellular Localization of Oxidants and Redox Modulation of Endothelial Nitric Oxide Synthase

“…Molecular modeling suggests that these cysteines are likely to be surrounded by amino acid residues that permit nucleophilic attack by the thiolate anion. 83 S-glutathionylation has been demonstrated to induce eNOS uncoupling (Figure 6). In turn, site-directed mutagenesis substituting cysteine 908 for alanine abolished eNOS thiyl radical formation.…”

“…In turn, site-directed mutagenesis substituting cysteine 908 for alanine abolished eNOS thiyl radical formation. 83 Oxidant stress generated through the S-glutathionylation reaction is not inhibited by competitive inhibitors of the NOS substrate L-arginine. 84 The relevance of this pathway has been studied in Wistar rats, in which S-glutathionylation is associated with impaired endothelium-dependent vasodilation.…”

Luteolinidin Protects the Postischemic Heart through CD38 Inhibition with Preservation of NAD(P)(H)

“…Important contributors to reactive oxygen species formation in the heart include the electron transport chain of mitochondria, NADPH oxidase, xanthine oxidase, and uncoupled endothelial nitric oxide synthase (eNOS) within endothelial cells (Zweier et al, 1988;Dumitrescu et al, 2007;Loukogeorgakis et al, 2010;De Pascali et al, 2014). eNOS dysfunction occurs secondary to oxidative depletion of its cofactor tetrahydrobiopterin (BH 4 ) and oxidation and glutathionylation of critical enzyme cysteines that cause eNOS to switch from production of nitric oxide (NO) to superoxide (Dumitrescu et al, 2007;Chen et al, 2010Chen et al, , 2011.…”

Nos Uncoupling Is Accompanied With Induction of the Oxidative Stress and the Cardiohemodynamics Disturbances in Hypertension

“…• О 2 -неспряженою eNOS [11][12][13][14][15], а не лише, як раніше вважалося, його надходженням з таких джерел, як мітохондрії [16] , НАДФН-оксидаза [17][18][19], ксантиноксидаза [20] чи ліпо-та циклооксигенази [21].…”

“…Він може утворюватися за допомогою de novo синтезу індуцибельною NO-синтазою (іNOS), внас-лідок реутилізації стабільних метаболітів NO нітрат-і нітрит-аніонів відповідними редуктазами чи декомпозиції нітрозотіолів, що є депо NO, в судинній стінці [7][8][9][10]. Серед механізмів виникнення оксидативного стресу крім зниження активності ферментів антиоксидантної системи та сполук, що ней-тралізують вільні радикали, перевага остан-нім часом надається збільшенню генерації• О 2 -неспряженою eNOS [11][12][13][14][15] …”