Abstract
Endothelial nitric oxide synthase (eNOS) is responsible for maintaining systemic blood pressure, vascular remodeling and angiogenesis. Previous studies showed that bovine eNOS serine 1179 (Serine 1177 for human eNOS) phosphorylation enhanced NO synthesis. Meanwhile, heat shock protein 90 (Hsp90) plays a critical role in maintenance of eNOS structure and function. However, the regulatory difference and importance between Serine 1179 phosphorylation and Hsp90 on eNOS activity have not been evaluated. In current studies, S1179D eNOS was employed to mimic phospho-eNOS and exhibited markedly increased enzyme activity than wild type eNOS (WT eNOS). Hsp90 showed a dose-dependent increase for both WT eNOS and S1179D eNOS activity at the presence of all eNOS cofactors, such as Calcium/Calmodulin (Ca2+ /CaM), BH4, and NADPH etc. The enhancement effects were abolished by dominant-negative mutant Hsp 90 protein. ENOS-cofactors dynamic assay showed that Hsp90 enhanced WT eNOS affinity to NADPH, L-arginine, and CaM but not to Ca2+ and BH4. The impact of eNOS Serine 1179 phosphorylation and Hsp90 on eNOS affinity to cofactors has also been compared. Different from the effect of Hsp90 on eNOS affinity to specific cofactors, Serine 1179 phosphorylation significantly increased eNOS affinity to all cofactors. Moreover, VEGF-induced eNOS phosphorylation in bovine aortic endothelial cells (BAECs) and more NO generation from eNOS compared to control. Inhibition of Hsp90 by geldanamycin decreased eNOS activity and decreased endothelial viability. In conclusion, by changing eNOS structure, Hsp90 profoundly affected eNOS functions, including change of affinity of eNOS to cofactors like Ca2+, L-arginine, BH4 and further affecting NO generation capability. These specific cofactors regulated by Hsp 90 could become potential therapeutic targets of the eNOS-related diseases in future.
Highlights
Endothelial nitric oxide synthase exists in various organs and tissue endothelium
Serine 1179 aspartic acid mutation (S1179D) Endothelial nitric oxide synthase (eNOS) exhibited a two times higher conversion rate of L-[14C]-citrulline compared with wild type eNOS, which is consistent with our previous report and other group’s report (Data not shown)[4, 17]
ENOS activity was significantly blocked by dominant negative heat shock protein 90 (Hsp90) (DNHsp90), but was not affected by BSA(Fig 1A); Compared with wild type eNOS (WT eNOS), S1179D eNOS has significantly higher activity as same dosage of Hsp90 incubated with S1179D eNOS (2.5 times higher than control; Fig 1B S1179D eNOS)
Summary
Endothelial nitric oxide synthase (eNOS) exists in various organs and tissue endothelium. ENOS converts L-arginine into L-citrulline and produces a high active molecule, nitric oxide (NO) which is a potent cell signaling and vasodilator molecule that plays important and diverse roles in biological processes, including control of vascular tone, vascular remodeling and angiogenesis [1]. ENOS activity is regulated by its cofactors, such as Ca2+, CaM, L-arginine and BH4 [2], and by its ammonia acid modifications, such as Serine 1177 and Threonine 495 phosphorylation (serine 1179 and Threonine 497 for bovine eNOS, respectively) [3,4,5]. VEGF and shear-stress mediated eNOS serine/threonine phosphorylation accompanied activation of eNOS and increase of NO production [6]. Mimicking the phosphorylation of Ser 1177 by mutation of Ser1177 to aspartic acid enhances enzyme activity and alters the sensitivity of the enzyme to Ca2+ [7]
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