S-glutathionylation uncouples eNOS and regulates its cellular and vascular function.

Area of Science:

• Biochemistry
• Cellular Biology
• Physiology

Background:

• Endothelial nitric oxide synthase (eNOS) regulates vascular function by producing nitric oxide (NO) and superoxide (O(2)(•-)).
• Tetrahydrobiopterin (BH(4)) is essential for NO synthesis; its absence leads to O(2)(•-) generation.
• While NOS dysfunction is linked to redox stress, BH(4) repletion offers only partial restoration of function, suggesting other regulatory mechanisms.

Purpose of the Study:

• To investigate the role of S-glutathionylation, a reversible protein modification, in the redox regulation of eNOS function.
• To identify specific cysteine residues in eNOS involved in redox-dependent regulation.
• To determine the impact of eNOS S-glutathionylation on endothelial function and vasodilation in physiological and pathological conditions.

Main Methods:

• Investigated the effect of oxidative stress on eNOS activity and O(2)(•-) generation in vitro and in endothelial cells.
• Identified and characterized S-glutathionylation sites on eNOS using biochemical and proteomic approaches.
• Assessed the functional consequences of eNOS S-glutathionylation on endothelium-dependent vasodilation in isolated vessels, including those from hypertensive models.

Main Results:

• S-glutathionylation of eNOS reversibly decreases its activity, shifting production from NO to O(2)(•-), primarily via the reductase domain.
• Two conserved cysteine residues were identified as critical sites for S-glutathionylation and redox regulation of eNOS.
• eNOS S-glutathionylation in endothelial cells correlated with impaired vasodilation, and this dysfunction was reversed by thiol-specific reducing agents in hypertensive vessels.

Conclusions:

• S-glutathionylation of eNOS acts as a critical redox-sensitive switch, modulating its activity and O(2)(•-) generation.
• This modification plays a significant role in regulating cellular signaling, endothelial function, and vascular tone, particularly under oxidative stress.
• Targeting eNOS S-glutathionylation with reducing agents may offer a therapeutic strategy for vascular dysfunction associated with redox imbalance.