Mei-Hui Hsu

Structures of human microsomal cytochrome P450 2A6 complexed with coumarin and methoxsalen

Human microsomal cytochrome P450 2A6 (CYP2A6) contributes extensively to nicotine detoxication but also activates tobacco-specific procarcinogens to mutagenic products. The CYP2A6 structure shows a compact, hydrophobic active site with one hydrogen bond donor, Asn297, that orients coumarin for regioselective oxidation. The inhibitor methoxsalen effectively fills the active site cavity without substantially perturbing the structure. The structure should aid the design of inhibitors to reduce smoking and tobacco-related cancers.

A Carboxyl-terminal Extension of the Zinc Finger Domain Contributes to the Specificity and Polarity of Peroxisome Proliferator-activated Receptor DNA Binding*

Heterodimers of the peroxisome proliferator-activated receptors (PPAR) and the retinoid X receptors (RXR) recognize response elements (PPREs) that exhibit the consensus sequence 5′-A(A/T)CT(A/G)GGNCAAAG(G/T)TCA-3′. The consensus PPRE includes both a 5′-extension and a direct repeat (DR1) comprised of two canonical core recognition sequences (underlined) for nuclear receptor zinc fingers separated by a single nucleotide spacer. The extended binding site recognized by PPARs is very similar to sites that bind monomers of the nuclear receptors Rev-ErbA and ROR suggesting that the latter could bind to PPREs and affect gene transcription. However, Rev-ErbA and ROR bind weakly to naturally occurring PPREs relative to the consensus binding site, and significant effects on PPARα transactivation of a CYP4A6-Z reporter were not observed. In contrast, PPAR/RXR heterodimers bind to a DR2 element containing the conserved 5′-extended sequence that is recognized by dimers of RORα or Rev-ErbA. PPARα/RXRα positively regulate transcription from this element, and co-expression of Rev-ErbA blocks this effect. The nuclear receptors NGFI-B and ROR utilize a carboxyl-terminal extension (CTE) of the zinc finger DNA binding domain in their interactions with the 5′-extension of a single zinc finger-binding site. DNA binding domains (DBD) of PPARs α, δ, and γ that contain the zinc finger motif and a CTE display binding to core recognition sequences that is dependent on the 5′-extended sequence found in PPREs. Unlike DBDs of other nuclear receptors that form heterodimers with RXR, the PPAR-DBDs did not exhibit cooperative binding with the DBD of RXR and exhibit the opposite polarity for binding to the direct repeat motif. In contrast to the corresponding DBD of RXR, the PPAR-DBDs bind as monomers to a single extended binding site as well as to the consensus PPRE. A chimera linking the zinc finger domain of RXRα to the CTE from PPARα bound to a single extended binding site indicating a functional role for the CTE of PPARs in extended binding site recognition.

Human cytochrome P450 family 4 enzymes : Function, genetic variation and regulation

The microsomal cytochrome P450 (CYP) family 4 monooxygenases are the major fatty acid ω-hydroxylases. These enzymes remove excess free fatty acids to prevent lipotoxicity, catabolize leukotrienes and prostanoids, and also produce bioactive metabolites from arachidonic acid ω-hydroxylation. In addition to endogenous substrates, recent evidence indicates that CYP4 monooxygenases can also metabolize xenobiotics, including therapeutic drugs. This review focuses on human CYP4 enzymes and updates current knowledge concerning catalytic activity profiles, genetic variation and regulation of expression. Comparative differences between the human and rodent CYP4 enzymes regarding catalytic function and conditional expression are also discussed.

Characterization of a cDNA encoding a human kidney, cytochrome P-450 4A fatty acid ω-hydroxylase and the cognate enzyme expressed in Escherichia coli

A cDNA encoding a cytochrome P-450 4A (CYP4AII) was cloned from a human kidney cDNA library. Northern blot analysis and RNase protection assays indicate that related mRNAs occur in kidney and liver with the highest abundance found in kidney. The enzyme was expressed from its cDNA in Escherichia coli. A solubilized preparation of the enzyme reconstituted with cytochrome P-450 reductase catalyzed the omega-hydroxylation of lauric acid, palmitic acid, and arachidonic acid with turnover numbers of 9.8, 2.2 and 0.55 min-1, respectively. Little or no activity was detected toward prostaglandins A1 and E1.

Regulation of P450 4A expression by peroxisome proliferator activated receptors

The induction of P450 4A enzymes by peroxisome proliferators (PPs) and fatty acids is mediated by the peroxisome proliferator activated receptor alpha (PPAR alpha) that binds to response elements in target genes as a heterodimer with the retinoid X receptor (RXR). The consensus sequence recognized by PPAR/RXR heterodimers, contains an imperfect direct repeat of two nuclear receptor binding motifs separated by a single nucleotide. This repeat is preceded by a conserved A/T rich sequence that is required for function. In mice, chronic exposure to PPs results in PPAR alpha mediated liver hypertrophy, hyperplasia and carcinogenesis accompanied by a proliferation of peroxisomes. In contrast, humans exhibit a reduced sensitivity to PP pathogenesis. This could reflect >10-fold lower PPAR alpha levels relative to mice as well as differences in targeted genes. In order to identify PPAR responsive human genes, the human hepatoma cell line, HepG2, was engineered to express increased levels of PPAR alpha. Several genes encoding rate-limiting enzymes and branch points in ketone body formation are regulated by PPAR alpha in these cells. In contrast, significant induction by PP is not evident for peroxisomal fatty acid oxidation that is associated with peroxisome proliferation in mice. Human P450 4A11 is not expressed in dividing cultures of cells with enhanced PPAR alpha levels, but it is expressed in confluent cultures expressing elevated amounts of PPAR alpha.

Crystal Structure of Human Cytochrome P450 2D6 with Prinomastat Bound

Background: P450 2D6 contributes significantly to the metabolic clearance of many drugs. Results: Binding of prinomastat to P450 2D6 reveals a distinctive active site topology. Conclusion: P450 2D6 structural flexibility contributes to its catalytic versatility. Significance: This structure will aid efforts to minimize the impact of genetic variation and drug-drug interactions for new drugs. Human cytochrome P450 2D6 contributes to the metabolism of >15% of drugs used in clinical practice. This study determined the structure of P450 2D6 complexed with a substrate and potent inhibitor, prinomastat, to 2.85 Å resolution by x-ray crystallography. Prinomastat binding is well defined by electron density maps with its pyridyl nitrogen bound to the heme iron. The structure of ligand-bound P450 2D6 differs significantly from the ligand-free structure reported for the P450 2D6 Met-374 variant (Protein Data Bank code 2F9Q). Superposition of the structures reveals significant differences for β sheet 1, helices A, F, F′, G″, G, and H as well as the helix B-C loop. The structure of the ligand complex exhibits a closed active site cavity that conforms closely to the shape of prinomastat. The closure of the open cavity seen for the 2F9Q structure reflects a change in the direction and pitch of helix F and introduction of a turn at Gly-218, which is followed by a well defined helix F′ that was not observed in the 2F9Q structure. These differences reflect considerable structural flexibility that is likely to contribute to the catalytic versatility of P450 2D6, and this new structure provides an alternative model for in silico studies of substrate interactions with P450 2D6.

Regulation of Human Cytochrome P450 4F2 Expression by Sterol Regulatory Element-binding Protein and Lovastatin

This report provides the first evidence that human P450 4F2 (CYP4F2) is induced by statins, which are widely used to treat hypercholesterolemia. Real time PCR and immunoblots indicate that lovastatin treatment increases expression of the endogenous CYP4F2 gene in human primary hepatocytes and HepG2 cells. The effects of lovastatin on gene expression are often mediated through sterol regulatory element-binding proteins (SREBPs). Immunoblots indicate that lovastatin-treated human hepatocytes display increased proteolytic processing of SREBP-2. In HepG2 cells, co-administration of a potent suppressor of SREBP-2 activation, 25-hydroxycholesterol, inhibits CYP4F2 mRNA induction by lovastatin. HepG2 cells transfected with an expression vector for the active nuclear form of SREBP-1a (nSREBP-1a) also display elevated endogenous CYP4F2 expression. Luciferase reporters containing the CYP4F2 proximal promoter are transactivated by nSREBPs (-1a, -1c, and -2) or a dominant positive form of the SREBP cleavage-activating protein (SCAP), which facilitates activation of endogenous SREBPs. Lovastatin-induced reporter expression is inhibited by overexpressed Insig-1, which prevents proteolytic activation of endogenous SREBPs. Electrophoretic mobility shift assays with in vitro translated nSREBP-1a identified two SREBP binding sites at –169/–152 and –109/–92, relative to the CYP4F2 transcription start site. Mutations in each site abolish SREBP binding. Chromatin immunoprecipitation experiments indicate that more SREBP-1 is associated with the CYP4F2 promoter after overexpression of nSREBP-1a. Transfection studies and mutagenesis indicate that the –109/–92 region is the primary site responsible for the effects of statins. Collectively, these results demonstrate that SREBPs transactivate CYP4F2 transcription and that CYP4F2 induction by statins is mediated by SREBP-2.

Differential regulation of human CYP4A genes by peroxisome proliferators and dexamethasone.

HepG2 cells that stably overexpress PPARalpha were used to examine the regulation of the two known human CYP4A genes by Wy14643. Specific PCR amplification across intron 5 and restriction endonuclease analysis indicated that HepG2 cells possess genes corresponding to both the CYP4A11 cDNA and a more recently characterized gene, CYP4A22, that exhibits 95% identity to CYP4A11 in the coding region. These are unlikely to represent alleles because both genes were present in DNA samples from 100 of 100 individuals. Quantitative real-time PCR determined that CYP4A22 mRNA is expressed at significantly lower levels than CYP4A11 mRNA in human liver samples. The PPARalpha agonist Wy14643 induced CYP4A11 mRNA in confluent cultures of HepG2 cells stably expressing the murine PPARalpha-E282G mutant. This mutant exhibits a significantly decreased ligand-independent trans-activation and can be activated by Wy14643 to a level similar to that of wild-type PPARalpha. Dexamethasone induced CYP4A11 mRNA in both control and PPARalpha- E282G-expressing HepG2 cells, indicating that the induction of CYP4A11 by dexamethasone is independent of elevated PPARalpha expression. Wy14643 or dexamethasone induction of CYP4A22 mRNA was not evident in either control or PPARalpha -E282G-expressing HepG2 cells. The results indicate that CYP4A11 expression can be induced by glucocorticoids and peroxisome proliferators.

Genistein, Resveratrol, and 5-Aminoimidazole-4-carboxamide-1-β-d-ribofuranoside Induce Cytochrome P450 4F2 Expression through an AMP-Activated Protein Kinase-Dependent Pathway

Activators of AMP-activated protein kinase (AMPK) increase the expression of the human microsomal fatty acid ω-hydroxylase CYP4F2. A 24-h treatment of either primary human hepatocytes or the human hepatoma cell line HepG2 with 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR), which is converted to 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranosyl 5′-monophosphate, an activator of AMPK, caused an average 2.5- or 7-fold increase, respectively, of CYP4F2 mRNA expression but not of CYP4A11 or CYP4F3, CYP4F11, and CYP4F12 mRNA. Activation of CYP4F2 expression by AICAR was significantly reduced in HepG2 cells by an AMPK inhibitor, 6-[4-(2-piperidin-1-yl-ethoxy)-phenyl)]-3-pyridin-4-yl-pyrrazolo[1,5-a]-pyrimidine (compound C) or by transfection with small interfering RNAs for AMPKα isoforms α1 and α2. A 2.5-fold increase in CYP4F2 mRNA expression was observed upon treatment of HepG2 cells with 6,7-dihydro-4-hydroxy-3-(2′-hydroxy[1,1′-biphenyl]-4-yl)-6-oxo-thieno[2,3-b]pyridine-5-carbonitrile (A-769662), a direct activator for AMPK. In addition, the indirect activators of AMPK, genistein and resveratrol increased CYP4F2 mRNA expression in HepG2 cells. Pretreatment with compound C or 1,2-dihydro-3H-naphtho[2,1-b]pyran-3-one (splitomicin), an inhibitor of the NAD+ activated deacetylase SIRT1, only partially blocked activation of CYP4F2 expression by resveratrol, suggesting that a SIRT1/AMPK-independent pathway also contributes to increased CYP4F2 expression. Compound C greatly diminished genistein activation of CYP4F2 expression. 7H-benz[de]benzimidazo[2,1-a]isoquinoline-7-one-3-carboxylic acid acetate (STO-609), a calmodulin kinase kinase (CaMKK) inhibitor, reduced the level of expression of CYP4F2 elicited by genistein, suggesting that CaMKK activation contributed to AMPK activation by genistein. Transient transfection studies in HepG2 cells with reporter constructs containing the CYP4F2 proximal promoter demonstrated that AICAR, genistein, and resveratrol stimulated transcription of the reporter gene. These results suggest that activation of AMPK by cellular stress and endocrine or pharmacologic stimulation is likely to activate CYP4F2 gene expression.

Opposing Roles of Peroxisome Proliferator-activated Receptor α and Growth Hormone in the Regulation of CYP4A11 Expression in a Transgenic Mouse Model

CYP4A11 transgenic mice (CYP4A11 Tg) were generated to examine in vivo regulation of the human CYP4A11 gene. Expression of CYP4A11 in mice yields liver and kidney P450 4A11 levels similar to those found in the corresponding human tissues and leads to an increased microsomal capacity for ω-hydroxylation of lauric acid. Fasted CYP4A11 Tg mice exhibit 2–3-fold increases in hepatic CYP4A11 mRNA and protein, and this response is absent in peroxisome proliferator-activated receptor α (PPARα) null mice. Dietary administration of either of the PPARα agonists, fenofibrate or clofibric acid, increases hepatic and renal CYP4A11 levels by 2–3-fold, and these responses were also abrogated in PPARα null mice. Basal liver CYP4A11 levels are reduced differentially in PPARα−/− females (>95%) and males (<50%) compared with PPARα−/+ mice. Quantitative and temporal differences in growth hormone secretion are known to alter hepatic lipid metabolism and to underlie sexually dimorphic gene expression, respectively. Continuous infusion of low levels of growth hormone reduced CYP4A11 expression by 50% in PPARα-proficient male and female transgenic mice. A larger decrease was observed for the expression of CYP4A11 in PPARα−/− CYP4A11 Tg male mice to levels similar to that of female PPARα-deficient mice. These results suggest that PPARα contributes to the maintenance of basal CYP4A11 expression and mediates CYP4A11 induction in response to fibrates or fasting. In contrast, increased exposure to growth hormone down-regulates CYP4A11 expression in liver.