RESUMEN
Acyl-CoA conjugation of xenobiotic carboxylic acids is catalyzed by hepatic microsomal long-chain fatty acid CoA ligases (LCL, EC 6.2.1.3). Marmosets (Callithrix jacchus) are considered genetically closer to humans than rodents and are used in pharmacological and toxicological studies. We have demonstrated that marmoset liver microsomes catalyze nafenopin-, ciprofibroyl-, and palmitoyl-CoA conjugation and that only palmitoyl-CoA conjugation is significantly upregulated (1.7-fold, P < 0.02) by a high fat diet. Additionally, the apparent C(50) values for nafenopin-, ciprofibroyl-, and palmitoyl-CoA conjugation of 149.7, 413.4, and 3.4 microM were comparable to those reported for human liver microsomes viz, 213.7, 379.8, and 3.4 microM, respectively. Comparison with human data was enabled by the cloning of a full-length marmoset cDNA (MLCL1) that encoded a 698-amino-acid protein sharing 83% similarity with rat liver acyl-CoA synthetase (ACS1) and 93 and 90% similarity with human liver LCL1 and LCL2, respectively. MLCL1 transiently expressed in COS-7 cells activated nafenopin (C(50) 192.9 microM), ciprofibrate (C(50) 168.7 microM), and palmitic acid (C(50) 4.5 microM) to their respective CoA conjugates. This study also demonstrated that the sigmoidal kinetics observed for nafenopin- and ciprofibroyl-CoA conjugation were not unique to human liver microsomes but were also characteristic of marmoset liver microsomes and recombinant MLCL1. More extensive characterization of the substrate specificity of marmoset LCL isoforms will aid in determining further the suitability of marmosets as a model for human xenobiotic metabolism via acyl-CoA conjugation.
Asunto(s)
Callithrix/genética , Ácido Clofíbrico/análogos & derivados , Ácido Clofíbrico/metabolismo , Coenzima A Ligasas/genética , Coenzima A Ligasas/metabolismo , Microsomas Hepáticos/metabolismo , Nafenopina/metabolismo , Ácido Palmítico/metabolismo , Proteínas Represoras , Proteínas de Saccharomyces cerevisiae , Secuencia de Aminoácidos , Animales , Ácidos Fíbricos , Humanos , Masculino , Datos de Secuencia Molecular , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Especificidad de la Especie , Xenobióticos/metabolismoRESUMEN
Endogenous fatty acyl-CoAs play an important role in the acylation of proteins. A number of xenobiotic carboxylic acids are able to mimic fatty acids, forming CoA conjugates and acting as substrates in pathways of lipid metabolism. In this study nafenopin, a substrate for human hepatic fatty acid-CoA ligases, was chosen as a model compound to study xenobiotic acylation of human liver proteins. (3)H-nafenopin (+/- unlabeled palmitate) or (14)C-palmitate (+/- unlabeled nafenopin) were incubated for up to 120 min at 37 degrees C with ATP, CoA, and homogenate protein (1 mg/ml) from four individual human livers. Nafenopin covalently bound to proteins was detectable in all human livers and increased with time. Nafenopin adduct formation was directly proportional to nafenopin-CoA formation (r = 0.985, p < 0.05). Attachment of nafenopin to proteins involved both thioester and amide linkages with 76 and 24% of adducts formed with proteins > 100 and 50-100 kDa, respectively. Protein acylation by palmitate was also demonstrated. Palmitate significantly inhibited nafenopin-CoA formation by 29% but had no effect on nafenopin-CoA-mediated protein acylation. In contrast, nafenopin significantly inhibited protein palmitoylation by palmitoyl-CoA. This is the first study to demonstrate a direct relationship between xenobiotic-CoA formation, acylation of human liver proteins, and inhibition of endogenous palmitoylation. The ability of xenobiotics to acylate tissue proteins may have important biological consequences including perturbation of endogenous regulation of protein localization and function.