Endogenous NO upon estradiol-17β stimulation and NO donor differentially regulate mitochondrial S-nitrosylation in endothelial cells.

Adduction of a nitric oxide (NO) moiety (NO(•)) to cysteines termed as S-nitrosylation (SNO) has emerged as a crucial mechanism for NO signaling crucial for mediating the vascular effects of estrogens. Mitochondrion is a known vascular risk factor; however, the effects of estrogens on mitochondrial SNO are incompletely understood. In this study we determined the effects of estradiol-17β (E2β) on mitochondrial protein SNO in primary human umbilical vein endothelial cells and compared the mitochondrial nitroso-proteomes in E2β- and a NO donor S-nitrosoglutathione (GSNO)-treated cells using a proteomics approach. Treatment with 10 nM E2β and 1 mM GSNO for 30 minutes significantly increased the levels of mitochondrial SNO-proteins. Subcellular localization of SNO-proteins showed mitochondria as the major cellular organelle for protein SNO in response to E2β and GSNO. E2β stimulated mitochondrial endothelial nitric oxide synthase (eNOS) phosphorylation and mitochondrial protein SNO that was enhanced by overexpression of mitochondrion or Golgi, but not membrane targeting eNOS constructs. We identified 11, 32, and 54 SNO-proteins in the mitochondria from the untreated, E2β-, and GSNO-treated human umbilical vein endothelial cells, respectively. Comparisons of the nitroso-proteomes revealed that common and different mitochondrial SNO-proteins were affected by endogenous NO on E2β stimulation and exogenous NO from donor. These SNO-proteins were associated with various mitochondrial functions, including energy and redox regulation, transport, iron homeostasis, translation, mitochondrial morphology, and apoptosis, etc. Collectively, we conclude that estrogens rapidly stimulate protein SNO in endothelial mitochondria via mitochondrial eNOS, providing a mechanism for mediating the vascular effects of estrogens.