Scientific Report 2007

Molecular and Experimental Medicine

Division Of Biochemistry

Cytochrome P450: Regulation, Structure, and Function

E.F. Johnson, K.J. Griffin, M.-H. Hsu, R.L. Reynald, S. Sansen, Ü. Savas

Enzymes in the cytochrome P450 superfamily primarily serve 2 purposes in human physiology. Some P450s catalyze specific biotransformations in autocrine, paracrine, and endocrine signal transduction pathways. A second, relatively large group of P450 monooxygenases play defensive roles by converting toxic compounds to less toxic forms that are more soluble and more easily excreted than are the parent compounds. Each xenobiotic-metabolizing P450 generally oxidizes structurally diverse substrates, leading to a wide-ranging protective capacity for elimination of toxic chemicals. Often the expression levels of these enzymes are increased in response to exposure to xenobiotics or altered physiologic states. We wish to understand how the structural diversity and genetic regulation of P450s that metabolize xenobiotics contribute to a person's ability to avoid the adverse effects of environmental chemicals or alter the clearance and bioavailability of therapeutic drugs.

Although extensive information on the conditional expression of P450 genes in experimental animal species is available, in humans the transcriptional responses of P450 genes to environmental stimuli and to physiologic changes are poorly understood. To address this problem, we use human cell lines, primary cultures of human cells, and transgenic mice to study mechanisms that regulate human family 4 P450 genes. These genes encode enzymes that are involved in both signal transduction and the metabolism of endogenous lipids and xenobiotics. Studies with cell lines are providing new information about endocrine and autocrine signal transduction pathways that govern the conditional expression of these genes in response to nutritional, hormonal, and xenobiotic signals.

Research is in progress to test whether more complex physiologic conditions such as pregnancy or caloric restriction alter the expression of the human enzymes in transgenic mice. We produced 2 independent transgenic mouse strains containing the human gene for CYP4A11 and extensive flanking intergenic regions. We found that the gene is expressed predominantly in kidney and liver at concentrations observed in human tissue samples. The expression of the transgene is elevated in response to fasting or exposure to agonists of the peroxisome proliferator-activated receptor α. Interestingly, the basal level of CYP4A11 expression is lowered in mice that do not express this receptor.

We also discovered that the human long-chain fatty acid ω-hydroxylase, CYP4F2, is induced in primary cultures of human hepatocytes and in cell lines by several statins, drugs used to lower serum levels of cholesterol. The induction of CYP4F2 could contribute to the reported reduction by statins of long-chain fatty acids that accumulate in X-linked adrenoleukodystrophy. The induction of CYP4F2 by statins could also aid in the treatment of patients with Refsum's disease, a congenital deficiency in the oxidation of branched-chain fatty acids that is exacerbated by dietary phytanic acid. The ω-hydroxylation of phytanic acid by CYP4F2 enables further metabolism by β-oxidation.

In collaboration with C.D. Stout, Department of Molecular Biology, we are defining the atomic structures of individual human P450s to understand the structural basis for the broad yet unique catalytic selectivity of each enzyme. This information can be used to better understand the adverse effects of oxidation of drugs and toxins and the potential for metabolic drug-drug interactions. These consequences of multidrug therapies can be life threatening and contribute extensively to the attrition of promising new candidate drugs. Toxicity and poor metabolic properties are significant barriers to the development of new drugs.

Mammalian P450s are tethered to the endoplasmic reticulum by a transmembrane segment at the amino terminus and by additional interactions of the catalytic domain with the cytoplasmic side of the membrane. Although membrane proteins are difficult to crystallize, we developed methods to express, purify, and crystallize genetically modified mammalian P450s that retain a native catalytic domain. Using this approach, we have determined the atomic structures of several of the most important human drug-metabolizing P450s: 1A2, 2A6, 2C8, 2C9, and 3A4. Through these studies, we determined how the flexibility of the P450s and the diversity of their amino acid sequence shape catalytic specificity. Our recent publication of the structure of P450 1A2 provides the first structure of a family 1 P450. This structure indicates that family 1 P450s are highly adapted for the oxidation of large aromatic hydrocarbons, which are often produced by combustion and are generally carcinogenic (Fig. 1). The structure of the enzyme's active site complements the active sites of family 2 and 3 P450s.

Fig. 1. The substrate-binding cavity (mesh surface) of human cytochrome P450 1A2 is narrow and well suited for large planar molecules like 7,8-benzoflavone (stick figure with yellow carbons), which was cocrystallized with the protein. Molecular oxygen is reduced by the heme prosthetic group (stick figure with pink carbons) to form a reactive intermediate that oxygenates the substrate. Parts of the protein backbone are shown as a cyan ribbon.

The P450 2A6 is the principal nicotine-detoxication enzyme and can also activate the tobacco smoke–specific carcinogen nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone to its carcinogenic form. Several reports indicate that because of the increased side effects of nicotine, persons who are genetically deficient in 2A6 activity are less likely to smoke than are persons not genetically deficient in this activity. In collaboration with J. Cashman, Human Biomolecular Research Institute, La Jolla, California, we are developing inhibitors of P450 2A6 that could reduce smoking behavior and diminish the likelihood of tobacco-related lung cancers.

Publications

Hsu, M.-H., Savas, Ü., Griffin, K.J., Johnson, E.F. Human cytochrome P450 family 4 enzymes: function and regulation. Drug Metab. Rev., in press.

Hsu, M.-H., Savas, Ü., Griffin, K.J., Johnson, E.F. Regulation of human cytochrome P450 4F2 expression by sterol regulatory element-binding protein and lovastatin. J. Biol. Chem. 282:5225, 2007.

Sansen, S., Hsu, M.-H., Stout, C.D., Johnson, E.F. Structural insight into the altered substrate specificity of human cytochrome P450 2A6 mutants. Arch. Biochem. Biophys., in press.

Sansen, S., Yano, J.K., Reynald, R.L., Schoch, G.A., Griffin, K.J., Stout, C.D., Johnson, E.F. Adaptations for the oxidation of polycyclic aromatic hydrocarbons exhibited by the structure of human P450 1A2. J. Biol. Chem. 282:14348, 2007.

Yano, J.K., Denton, T.T., Cerny, M.A., Zhang, X., Johnson, E.F., Cashman, J.R. Synthetic inhibitors of cytochrome P-450 2A6: inhibitory activity, difference spectra, mechanism of inhibition, and protein cocrystallization. J. Med. Chem. 49:6987, 2006.