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 3 h 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.

Nitric oxide stimulates cellular degradation of human CYP51A1, the highly conserved lanosterol 14α-demethylase

“…Like several rat P450 enzymes [9, 15, 26] and human CYP2B6 [21], CYP51A1 protein is targeted for degradation when exposed to nitric oxide generated under inflammatory conditions by NOS2 or released from NO donor compounds. Three pieces of evidence support this conclusion: 1) the magnitude of down-regulation of the protein in response to DPTA is greater than that of the mRNA; 2) down-regulation of the protein has similar magitude in the presence or absence of the protein synthesis inhibitor cycloheximide; 3) down-regulation of V5-tagged CYP51A1 expressed from a lentivirus controlled by a cytomegalovirus CMV promoter is almost identical to that of the native protein expressed in the same cells.…”

“…This leads us to conclude that CYP51A1 undergoes proteasome-dependent degradation in response to NO. In studying the effect of NO on V5-tagged CYP2B6 in Huh7 cells, we have also routinely observed this partial effect of bortezomib [21]. However, CYP51A1 degradation appears to be largely independent of ubiquitination, because increases in HMM complexes indicative of CYP51A1 ubiquitination were not observed in the presence of DPTA plus proteasome inhibition, and HA-Ub-tagged CYP51A1 was only detected at very low levels in cells transfected with HA-Ub.…”

“…Virus-containing supernatants were collected at 48h and 72h post-transfection, combined, passed through a 0.45 µm filter and stored at −80°C. We described previously the construction of the pLIX-hNOS2 virus expressing human NOS2 under control of tetracycline [21]. …”

Autophagy as a Physiological Response of the Body to Starvation

“…When cells are exposed to long-term nutrient deprivation and energy loss, an excess amount of lipolysis results in the production of ketone bodies, which are the end product of free fatty acid oxidation reaction and can be used as an energy source in muscle and neurons. Since not all cells have the ability to use ketone bodies as an energy source, the cells that cannot utilize these small molecules activate proteolysis as a response to starvation (Luo et al 2017;Lee et al 2017). Proteolysis can be activated in two different ways, depending on the tissue type and stress duration.…”

Anti‑inflammatory activity of Radix Angelicae biseratae in the treatment of osteoarthritis determined by systematic pharmacology and in vitro experiments

“…CYP2B6 is a member of the cytochrome P450 superfamily of enzymes that is closely related to drug metabolism (41). A recent study demonstrated that CYP2B6 may affect the metabolism of NO under inflammatory stimulation (42). The present study indicated that PTGS2, NOS3 and CYP2B6 are potential targets for Radix Angelicae biseratae in the treatment of OA, with high degree values in the 'target-target' network.…”