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Nitric oxide-regulated proteolysis of human CYP2B6 via the ubiquitin-proteasome system.

ABSTRACT: We showed previously that rat cytochrome P450 CYP2B1 undergoes NO-dependent proteasomal degradation in response to inflammatory stimuli, and that the related human enzyme CYP2B6 is also down-regulated by NO in primary human hepatocytes. To investigate the mechanism of CYP2B6 down-regulation, we made several cell lines (HeLa and HuH7 cells) in which native CYP2B6 or CYP2B6 with a C-terminal V5 tag (CYP2B6V5) are expressed from a lentiviral vector with a cytomegalovirus promoter. Native CYP2B6 protein was rapidly down-regulated in HeLa cells within 3h of treatment with the NO donor (Z)-1-[2-(2-Aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate, while its mRNA level was not down-regulated. Treatment of the cells with the NO donor (Z)-1-[N-(3-aminopropyl)-N-(3-ammoniopropyl)amino]diazen-1-ium-1,2-diolate also resulted in rapid down-regulation of CYP2B6 activity, measured as the formation of 7-hydroxy-4-trifluoromethylcoumarin, as well as 2B6 protein in the CYP2B6 HeLa cell line. CYP2B6V5 was also down-regulated by NO donors in HuH7 cells. Down-regulation was observed in the presence of cycloheximide, demonstrating that this occurs via a post-translational mechanism. We generated a HeLa cell line expressing both CYP2B6V5 and human nitric oxide synthase 2 (NOS2), the latter under positive control by tetracycline. The cellular NO produced by doxycycline treatment also effectively down-regulated CYP2B6 protein, which was blocked by the co-treatment with the NOS2 competitive inhibitor L-NG-nitroarginine methyl ester (L-NAME). We next investigated the proteolytic enzymes responsible for NO-dependent CYP2B6 degradation. Neither calpain inhibitors (N-Acetyl-L-leucyl-L-leucyl-L-norleucinal, carbobenzoxy-valinyl-phenylalaninal), nor lysosomal protease inhibitors (3-methyladenine and chloroquine) inhibited the NO dependent CYP2B6V5 down-regulation. The proteasome inhibitors MG132 and bortezomib attenuated, but did not completely block the NO-induced down-regulation in the HuH7 cell line. However, when cells were co-treated with NO donor and proteasome inhibitors, high molecular mass species could be detected on native CYP2B6 as well as CYP2B6V5 Western blots. Further investigation demonstrated that CYP2B6 protein was polyubiquitinated and this was dramatically enhanced by co-treatment with NO donor and bortezomib. Taken together, our data demonstrate that CYP2B6 is down-regulated in an NO-dependent manner via ubiquitination and proteasomal degradation.

SUBMITTER: Lee CM

PROVIDER: S-EPMC5507215 | biostudies-literature | 2017 Jul

REPOSITORIES: biostudies-literature

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Nitric oxide-regulated proteolysis of human CYP2B6 via the ubiquitin-proteasome system.

Lee Choon-Myung CM Tripathi Shweta S Morgan Edward T ET

Free radical biology & medicine 20170417

We showed previously that rat cytochrome P450 CYP2B1 undergoes NO-dependent proteasomal degradation in response to inflammatory stimuli, and that the related human enzyme CYP2B6 is also down-regulated by NO in primary human hepatocytes. To investigate the mechanism of CYP2B6 down-regulation, we made several cell lines (HeLa and HuH7 cells) in which native CYP2B6 or CYP2B6 with a C-terminal V5 tag (CYP2B6V5) are expressed from a lentiviral vector with a cytomegalovirus promoter. Native CYP2B6 pro ...[more]

PMID: 28427998

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Project description:UNLABELLED:The molecular mechanisms of targeted proteolysis in archaea are poorly understood, yet they may have deep evolutionary roots shared with the ubiquitin-proteasome system of eukaryotic cells. Here, we demonstrate in archaea that TBP2, a TATA-binding protein (TBP) modified by ubiquitin-like isopeptide bonds, is phosphorylated and targeted for degradation by proteasomes. Rapid turnover of TBP2 required the functions of UbaA (the E1/MoeB/ThiF homolog of archaea), AAA ATPases (Cdc48/p97 and Rpt types), a type 2 JAB1/MPN/MOV34 metalloenzyme (JAMM/MPN+) homolog (JAMM2), and 20S proteasomes. The ubiquitin-like protein modifier small archaeal modifier protein 2 (SAMP2) stimulated the degradation of TBP2, but SAMP2 itself was not degraded. Analysis of the TBP2 fractions that were not modified by ubiquitin-like linkages revealed that TBP2 had multiple N termini, including Met1-Ser2, Ser2, and Met1-Ser2(p) [where (p) represents phosphorylation]. The evidence suggested that the Met1-Ser2(p) form accumulated in cells that were unable to degrade TBP2. We propose a model in archaea in which the attachment of ubiquitin-like tags can target proteins for degradation by proteasomes and be controlled by N-terminal degrons. In support of a proteolytic mechanism that is energy dependent and recycles the ubiquitin-like protein tags, we find that a network of AAA ATPases and a JAMM/MPN+ metalloprotease are required, in addition to 20S proteasomes, for controlled intracellular proteolysis. IMPORTANCE:This study advances the fundamental knowledge of signal-guided proteolysis in archaea and sheds light on components that are related to the ubiquitin-proteasome system of eukaryotes. In archaea, the ubiquitin-like proteasome system is found to require function of an E1/MoeB/ThiF homolog, a type 2 JAMM/MPN+ metalloprotease, and a network of AAA ATPases for the targeted destruction of proteins. We provide evidence that the attachment of the ubiquitin-like protein is controlled by an N-terminal degron and stimulates proteasome-mediated proteolysis.

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.

Dataset Information

Nitric oxide-regulated proteolysis of human CYP2B6 via the ubiquitin-proteasome system.

Publications

Nitric oxide-regulated proteolysis of human CYP2B6 via the ubiquitin-proteasome system.

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