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Tyrosine Nitration Contributes to Nitric Oxide-Stimulated Degradation of CYP2B6
Choon-Myung Lee1, P Ross Wilderman1, Ji Won Park1
1Department of Pharmacology and Chemical Biology, Emory University, Atlanta, Georgia (C.-m.L., J.W.P., T.J.M., E.T.M.) and University of Connecticut School of Pharmacy, Storrs, Connecticut (P.R.W.).
Abstract:
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.
Insights
Nitric oxide (NO) causes the degradation of human cytochrome P450 CYP2B6 by targeting specific tyrosine residues for nitration, leading to enzyme destabilization and proteasomal breakdown. This process is crucial for understanding CYP2B6 regulation in response to inflammation.
Area of Science:
- Biochemistry
- Molecular Biology
- Pharmacology
Background:
- Human cytochrome P450 (P450) enzymes, particularly CYP2B6, play critical roles in drug metabolism.
- Nitric oxide (NO) is implicated in cellular signaling and can influence protein stability and degradation.
- Understanding the regulation of CYP2B6 is essential for predicting drug efficacy and toxicity.
Purpose of the Study:
- To investigate the mechanism by which nitric oxide (NO) induces the degradation of human CYP2B6.
- To identify specific residues in CYP2B6 that are targeted by NO-mediated post-translational modifications.
- To elucidate the role of tyrosine nitration and heme interaction in CYP2B6 downregulation.
Main Methods:
- Site-directed mutagenesis of predicted tyrosine residues (Y190, Y317, Y380) in CYP2B6.
- Expression of wild-type and mutant CYP2B6 proteins in HuH7 cells.
- Treatment with NO donors (DPTA), proteasome inhibitors (bortezomib), and CO-releasing molecules.
- Analysis of protein degradation, high molecular mass (HMM) species formation, and ubiquitination.
- Inhibition studies using a specific CYP2B6 inhibitor.
- Molecular dynamics simulations to predict structural changes upon tyrosine nitration.
Main Results:
- Mutant CYP2B6 proteins with Y317A, Y380A, or Y190A substitutions showed reduced sensitivity to NO-dependent degradation.
- Y317A and Y380A mutants did not form high molecular mass (HMM) species upon treatment with NO donors and proteasome inhibitors.
- Carbon monoxide-releasing molecule 2 treatment suppressed CYP2B6 activity and induced ubiquitination but not degradation.
- A CYP2B6 inhibitor blocked NO-dependent degradation, indicating the importance of NO access to the active site.
- Molecular dynamics simulations predicted that tyrosine nitration destabilizes CYP2B6 structure.
Conclusions:
- Nitric oxide (NO) triggers the proteasomal degradation of CYP2B6 through nitration of specific tyrosine residues (Y190, Y317, Y380).
- Tyrosine nitration destabilizes the CYP2B6 protein structure, potentially in conjunction with heme nitrosylation, targeting it for ubiquitination and degradation.
- These findings reveal a novel mechanism for CYP2B6 regulation by NO, relevant to inflammatory conditions affecting hepatocytes.
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