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Tyrosine nitration on calmodulin enhances calcium-dependent association and activation of nitric-oxide synthase.

ABSTRACT: Production of reactive oxygen species caused by dysregulated endothelial nitric-oxide synthase (eNOS) activity is linked to vascular dysfunction. eNOS is a major target protein of the primary calcium-sensing protein calmodulin. Calmodulin is often modified by the main biomarker of nitroxidative stress, 3-nitrotyrosine (nitroTyr). Despite nitroTyr being an abundant post-translational modification on calmodulin, the mechanistic role of this modification in altering calmodulin function and eNOS activation has not been investigated. Here, using genetic code expansion to site-specifically nitrate calmodulin at its two tyrosine residues, we assessed the effects of these alterations on calcium binding by calmodulin and on binding and activation of eNOS. We found that nitroTyr-calmodulin retains affinity for eNOS under resting physiological calcium concentrations. Results from in vitro eNOS assays with calmodulin nitrated at Tyr-99 revealed that this nitration reduces nitric-oxide production and increases eNOS decoupling compared with WT calmodulin. In contrast, calmodulin nitrated at Tyr-138 produced more nitric oxide and did so more efficiently than WT calmodulin. These results indicate that the nitroTyr post-translational modification, like tyrosine phosphorylation, can impact calmodulin sensitivity for calcium and reveal Tyr site-specific gain or loss of functions for calmodulin-induced eNOS activation.

SUBMITTER: Porter JJ

PROVIDER: S-EPMC7039552 | biostudies-literature | 2020 Feb

REPOSITORIES: biostudies-literature

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Project description:Human cytochrome P450 (P450) CYP2B6 undergoes nitric oxide (NO)-dependent proteasomal degradation in response to the NO donor dipropylenetriamine NONOate (DPTA) and biologic NO in HeLa and HuH7 cell lines. CYP2B6 is also downregulated by NO in primary human hepatocytes. We hypothesized that NO or derivative reactive nitrogen species may generate adducts of tyrosine and/or cysteine residues, causing CYP2B6 downregulation, and selected Tyr and Cys residues for mutation based on predicted solvent accessibility. CYP2B6V5-Y317A, -Y380A, and -Y190A mutant proteins expressed in HuH7 cells were less sensitive than wild-type (WT) enzyme to degradation evoked by DPTA, suggesting that these tyrosines are targets for NO-dependent downregulation. The Y317A or Y380A mutants did not show increases in high molecular mass (HMM) species after treatment with DPTA or bortezomib + DPTA, in contrast to the WT enzyme. Carbon monoxide-releasing molecule 2 treatment caused rapid suppression of 2B6 enzyme activity, significant HMM species generation, and ubiquitination of CYP2B6 protein but did not stimulate CYP2B6 degradation. The CYP2B6 inhibitor 4-(4-chlorophenyl)imidazole blocked NO-dependent CYP2B6 degradation, suggesting that NO access to the active site is important. Molecular dynamics simulations predicted that tyrosine nitrations of CYP2B6 would cause significant destabilizing perturbations of secondary structure and remove correlated motions likely required for enzyme function. We propose that cumulative nitrations of Y190, Y317, and Y380 by reactive nitrogen species cause destabilization of CYP2B6, which may act synergistically with heme nitrosylation to target the enzyme for degradation. SIGNIFICANCE STATEMENT: This work provides novel insight into the mechanisms by which nitric oxide, which is produced in hepatocytes in response to inflammation, triggers the ubiquitin-dependent proteasomal degradation of the cytochrome P450 (P450) enzyme CYP2B6. Our data demonstrate that both nitration of specific tyrosine residues and interaction of nitric oxide (NO) with the P450 heme are necessary for NO to trigger ubiquitination and protein degradation.

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Tyrosine nitration on calmodulin enhances calcium-dependent association and activation of nitric-oxide synthase.

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