title: Nitric oxide-dependent proteasomal degradation of cytochrome P450 2B proteins.
abstract: Exposure to inflammatory agents or cytokines causes the suppression of cytochrome P450 (CYP) enzyme activities and expression in liver and primary hepatocyte cultures. We showed previously that phenobarbital-induced CYP2B protein is down-regulated in primary cultures of rat hepatocytes after exposure to bacterial endotoxin (lipopolysaccharide) in a nitric oxide (NO) -dependent manner. In this study, we found that CYP2B proteins in primary rat hepatocyte cultures were suppressed >60% after 6 h of treatment with interleukin-1beta (IL-1). This effect was NO-dependent, and treatment of cells with the NO donors (Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl) aminodiazen-1-ium-1,2-diolate (NOC-18), S-nitrosoglutathione, and S-nitroso-N-acetylpenicillamine also suppressed CYP2B proteins. However, the down-regulation by IL-1 was insensitive to inhibition of cGMP-dependent protein kinases. The down-regulation by IL-1 or NO donors was abolished by treatments with the proteasome inhibitors MG132 and lactacystin that did not affect NO production. The calpain inhibitor E64-d or the lysosomal protease inhibitors NH(4)Cl and chloroquine did not attenuate the down-regulation of CYP2B by IL-1. Treatment of HeLa cells expressing c-Myc-tagged CYP2B1 with NOC-18 down-regulated its expression and enhanced its ubiquitination. Treatment of rat liver microsomes with S-nitrosoglutathione caused S-nitrosylation of CYP2B protein and enhanced the ubiquitination pattern of CYP2B compared with unmodified CYP2B in an in vitro ubiquitination assay. These data are consistent with the hypothesis that NO-dependent CYP2B ubiquitination and proteasomal degradation are dependent on protein modification by reactive nitrogen species.
authors.affiliation: Department of Pharmacology, Emory University School of Medicine, Atlanta, Georgia 30322, USA.
authors.forename: Choon-Myung
authors.initials: CM
authors.lastname: Lee
authors.forename: Bong-Yoon
authors.initials: BY
authors.lastname: Kim
authors.forename: Lian
authors.initials: L
authors.lastname: Li
authors.forename: Edward T
authors.initials: ET
authors.lastname: Morgan
mesh_terms: D000818:Animals; D019362:Cytochrome P-450 CYP2B1 / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D022781:Hepatocytes / Q000201:enzymology*; D007375:Interleukin-1 / Q000494:pharmacology; D015850:Interleukin-6 / Q000494:pharmacology; D007700:Kinetics; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology*; D009569:Nitric Oxide / Q000494:pharmacology*; D046988:Proteasome Endopeptidase Complex / Q000187:drug effects / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D014158:Transcription, Genetic
chemical_list: D007375:Interleukin-1; D015850:Interleukin-6; D009569:Nitric Oxide; D003577:Cytochrome P-450 Enzyme System; D019362:Cytochrome P-450 CYP2B1; D046988:Proteasome Endopeptidase Complex