Regulation of eNOS Enzyme Activity by Posttranslational Modification

Abstract

The regulation of endothelial NO synthase (eNOS) employs multiple different cellular control mechanisms impinging on level and activity of the enzyme. This review aims at summarizing the current knowledge on the posttranslational modifications of eNOS, including acylation, nitrosylation, phosphorylation, acetylation, glycosylation and glutathionylation. Sites, mediators and impact on enzyme localization and activity of the single modifications will be discussed. Moreover, interdependence, cooperativity and competition between the different posttranslational modifications will be elaborated with special emphasis on the susceptibility of eNOS to metabolic cues.

Thirty Years of Saying NO

“…Image derived from Vanhoutte et al 14 2+ or by PKA, whereas the PP2A subtype dephosphorylates Ser1177 on activation by various stimuli (eg, aldosterone, C-reactive proteins, and the bioactive lipid metabolite ceramide). 25,27,28,50,51 Furthermore, phosphorylation at tyrosine residues of eNOS also modifies enzyme activity; the best characterized is Tyr657 phosphorylation by proline-rich tyrosine kinase (eg, in response to stimulation by angiotensin II, hydrogen peroxide, insulin, or shear stress) leading to reduction of eNOS activity and this process exerts a negative feedback to moderate NO-output thus preventing BH 4 depletion and eNOS uncoupl ing. 25,27,28,50,51 Phosphorylation of eNOS at Tyr81 by Src kinase occurs in response to various stimuli (eg, acetylcholine, bradykinin, estrogens, and hydrogen peroxide); while it facilitates NO production, it does not affect the maximal activity of the enzyme (Figures 1, bottom, and 2).…”

“…25,27,28,50,51 Furthermore, phosphorylation at tyrosine residues of eNOS also modifies enzyme activity; the best characterized is Tyr657 phosphorylation by proline-rich tyrosine kinase (eg, in response to stimulation by angiotensin II, hydrogen peroxide, insulin, or shear stress) leading to reduction of eNOS activity and this process exerts a negative feedback to moderate NO-output thus preventing BH 4 depletion and eNOS uncoupl ing. 25,27,28,50,51 Phosphorylation of eNOS at Tyr81 by Src kinase occurs in response to various stimuli (eg, acetylcholine, bradykinin, estrogens, and hydrogen peroxide); while it facilitates NO production, it does not affect the maximal activity of the enzyme (Figures 1, bottom, and 2). 25,27,28,50,51 In addition to phosphorylation, the activity of eNOS can also be regulated post-translationally by (1) thiopalmitoylation (addition of palmitoyl groups to cysteine residues), a process that is mediated by Asp-His-His-Cys-motif palmitoyl-acyltransferases (of which subtypes 2, 3, 7, 8, and 21 are present in the Golgi apparatus of endothelial cells) and depends on the N-myristoylation of eNOS (irreversible addition of a myristoyl group to Gly2 of the enzyme during protein translation that targets it to biological membranes to permit thiopalmitoylation).…”

“…25,27,28,50,51 Phosphorylation of eNOS at Tyr81 by Src kinase occurs in response to various stimuli (eg, acetylcholine, bradykinin, estrogens, and hydrogen peroxide); while it facilitates NO production, it does not affect the maximal activity of the enzyme (Figures 1, bottom, and 2). 25,27,28,50,51 In addition to phosphorylation, the activity of eNOS can also be regulated post-translationally by (1) thiopalmitoylation (addition of palmitoyl groups to cysteine residues), a process that is mediated by Asp-His-His-Cys-motif palmitoyl-acyltransferases (of which subtypes 2, 3, 7, 8, and 21 are present in the Golgi apparatus of endothelial cells) and depends on the N-myristoylation of eNOS (irreversible addition of a myristoyl group to Gly2 of the enzyme during protein translation that targets it to biological membranes to permit thiopalmitoylation). In particular, thiopalmitoylations at Cys15 and Cys26 facilitate the hydrophobic interactions of eNOS with the caveolae, where, as mentioned above, it comes in close contact with regulatory molecules including G-protein-coupled receptors and Akt.…”

“…Hence, thiopalmitoylation (which occurs mainly in the Golgi apparatus after eNOS synthesis and after its depalmitoylation and translocation to the cytosol [repalmitoylation]) contributes to the proper localization of the enzyme and permits its integration with upstream signaling initiated by neurohumoral (agonists and hormones) and physical (shear stress) stimuli. 25,27,28,50,51 This process is facilitated by the availability of palmitoyl coenzyme A (CoA), formed by fatty acid synthase (the expression of which is induced by insulin, possibly contributing to the vasoprotective role of the hormone) by increased levels of plasma glucose and fatty acids as occurs in obesity and during hypertension (see Reduced Protein Activity section of this article). Depalmitoylation, mediated by palmitoyl protein thioesterase and acyl-protein thioesterase 1 and facilitated by the Ca 2+ -calmodulin complex, occurs after prolonged agonist stimulation, resulting in the translocation of the enzyme to the cytosol and distancing it from exogenous stimuli 28,50,51 ; (2) S-nitrosylation (by NO produced by eNOS itself), which occurs when the enzyme is localized in the cell membrane/caveolae because the local hydrophobic environment favors the accumulation of NO and hence the formation of S-nitrosothiols.…”

“…Denitrosylation takes place when eNOS is translocated, on activation, to the cytosol, which contains a high concentration of reduced thiols thus facilitating reduction of S-nitrosothiols on eNOS, and permitting for maximal activity; this process is impaired under conditions of oxidative stress (eg, aging, hypertension, and diabetes mellitus) or by excessive intracellular levels of NO (eg, after iNOS induction during inflammation; see Reduced Protein Dimerization and Reduced NO Bioavailability sections of this article) 28,50,51 ; (3) both acetylation of eNOS at Lys610 by lysine acetyltransferases (depending on the availability of acetyl-CoA [major metabolite of the catabolism of sugars, fatty acids, and proteins], the breakdown of which is reduced by AMPK [inhibiting acetyl-CoA carboxylase to prevent metabolism of acetyl-CoA to melonyl-CoA] and enhanced by PP2A [dephosphorylating and hence activating acetyl-CoA carboxylase]) and its deacetylation at Lys497 and Lys507 (sites that are acetylated constitutively and prevent calmodulin binding) by silent mating-type information regulation 2 homologue 1 (SIRT1, the activity of which is reduced when the thiol group of its cysteine residues is oxidized and when NAD + is depleted by oxidative stress, as with aging and hyperglycemia; see Reduced NO Bioavailability section of this article), leading to increased enzyme activity 28,51 ; (4) glycosylation (attachment of N-acetylglucosamine by O-linked N-acetylglucosamine transferase) at Ser1177, a post-translational modification (characteristic of hyperglycemic conditions) that prevents phosphorylation and hence decreases eNOS activity; and (5) S-glutathionylation (addition of glutathione) at Cys689 and Cys908, determined by the cellular levels of oxidized versus reduced glutathione and prevented by thiol-reducing agents and enzymes (eg, thioredoxin reductase and glutathione reductase); such S-glutathionylation favors the uncoupling of eNOS (by promoting the reaction of the enzyme with oxygen and preventing the binding of the cofactors flavin adenine dinucleotide [FAD] and flavin mononucleotide [FMN] to it for NO synthesis; Figure 1) and hence its inactivation (see Reduced Protein Activity section of this article). 25,28,51 Other Endothelial Sources of NO Nitrite and nitrate not only are products of the metabolism of NO but also act as a reservoir for the endothelial mediator (Figure 2, left). Indeed, several enzymes (hemoglobin, myoglobin, xanthine oxidoreductase, mitochondrial complex IV [cytochrome c oxidase], aldehyde dehydrogenase 2, cytochrome P450 reductase, and cytochrome P450) can catalyze the reduction of nitrite or nitrate to generate NO.…”