Cytochrome P450 125A4, the Third Cholesterol C-26 Hydroxylase from Mycobacterium smegmatis.

Mycobacterium tuberculosis (Mtb) and Mycobacterium smegmatis (Msmeg) can grow on cholesterol as the sole carbon source. In Mtb the utilization of cholesterol can be initiated by CYP125A1 or CYP142A1 and in Msmeg by the orthologous CYP125A3 and CYP142A2. Double knockout of the two enzymes in Mtb prevents its growth on cholesterol, but the double knockout of Msmeg is still able to grow, albeit at a slower rate. We report here that Msmeg has a third enzyme, CYP125A4, that also oxidizes cholesterol, although it has a much higher activity for the oxidation of 7α-hydroxycholesterol. The ability of Msmeg CYP125A4 (and Mtb CYP125A1) to oxidize 7α-hydroxycholesterol is due, at least in part, to the presence of a smaller amino acid side chain facing C-7 of the sterol substrate than in CYP125A3. The ability to oxidize 7-substituted steroids broadens the range of sterol carbon sources for growth, but even more importantly in Mtb, additional biological effects are possible due to the potent immunomodulatory activity of 7α,26-dihydroxycholesterol.

Synergic hypocholesterolaemic effect of n-3 PUFA and oestrogen by modulation of hepatic cholesterol metabolism in female rats.

n-3 PUFA such as EPA and DHA as well as oestrogen have been reported to decrease blood levels of cholesterol, but their underlying mechanism is unclear. The purpose of this study was to determine the effects of the combination of n-3 PUFA supplementation and oestrogen injection on hepatic cholesterol metabolism. Rats were fed a modified AIN-93G diet with 0, 1 or 2 % n-3 PUFA (EPA+DHA) relative to the total energy intake for 12 weeks. Rats were surgically ovariectomised at week 8, and, after 1-week recovery, rats were injected with 17ß-oestradiol-3-benzoate (E2) or maize oil for the last 3 weeks. Supplementation with n-3 PUFA and E2 injection significantly increased the ratio of the hepatic expression of phosphorylated AMP activated protein kinase (p-AMPK):AMP activated protein kinase (AMPK) and decreased sterol regulatory element-binding protein-2, 3-hydroxy-3-methylglutaryl coenzyme A reductase and proprotein convertase subtilisin/kexin type 9. Supplementation with n-3 PUFA increased hepatic expression of cholesterol 7α-hydroxylase (CYP7A1), sterol 12α-hydroxylase (CYP8B1) and sterol 27-hydroxylase (CYP27A1); however, E2 injection decreased CYP7A1 and CYP8B1 but not CYP27A1. Additionally, E2 injection increased hepatic expression of oestrogen receptor-α and ß. In conclusion, n-3 PUFA supplementation and E2 injection had synergic hypocholesterolaemic effects by down-regulating hepatic cholesterol synthesis (n-3 PUFA and oestrogen) and up-regulating bile acid synthesis (n-3 PUFA) in ovariectomised rats.

Atorvastatin induces bile acid-synthetic enzyme Cyp7a1 by suppressing FXR signaling in both liver and intestine in mice.

Statins are effective cholesterol-lowering drugs to treat CVDs. Bile acids (BAs), the end products of cholesterol metabolism in the liver, are important nutrient and energy regulators. The present study aims to investigate how statins affect BA homeostasis in the enterohepatic circulation. Male C57BL/6 mice were treated with atorvastatin (100 mg/kg/day po) for 1 week, followed by BA profiling by ultra-performance LC-MS/MS. Atorvastatin decreased BA pool size, mainly due to less BA in the intestine. Surprisingly, atorvastatin did not alter total BAs in the serum or liver. Atorvastatin increased the ratio of 12α-OH/non12α-OH BAs. Atorvastatin increased the mRNAs of the BA-synthetic enzymes cholesterol 7α-hydroxylase (Cyp7a1) (over 10-fold) and cytochrome P450 27a1, the BA uptake transporters Na⁺/taurocholate cotransporting polypeptide and organic anion transporting polypeptide 1b2, and the efflux transporter multidrug resistance-associated protein 2 in the liver. Noticeably, atorvastatin suppressed the expression of BA nuclear receptor farnesoid X receptor (FXR) target genes, namely small heterodimer partner (liver) and fibroblast growth factor 15 (ileum). Furthermore, atorvastatin increased the mRNAs of the organic cation uptake transporter 1 and cholesterol efflux transporters Abcg5 and Abcg8 in the liver. The increased expression of BA-synthetic enzymes and BA transporters appear to be a compensatory response to maintain BA homeostasis after atorvastatin treatment. The Cyp7a1 induction by atorvastatin appears to be due to suppressed FXR signaling in both the liver and intestine.

Structural characterization of human cholesterol 7α-hydroxylase.

Hepatic conversion to bile acids is a major elimination route for cholesterol in mammals. CYP7A1 catalyzes the first and rate-limiting step in classic bile acid biosynthesis, converting cholesterol to 7α-hydroxycholesterol. To identify the structural determinants that govern the stereospecific hydroxylation of cholesterol, we solved the crystal structure of CYP7A1 in the ligand-free state. The structure-based mutation T104L in the B' helix, corresponding to the nonpolar residue of CYP7B1, was used to obtain crystals of complexes with cholest-4-en-3-one and with cholesterol oxidation product 7-ketocholesterol (7KCh). The structures reveal a motif of residues that promote cholest-4-en-3-one binding parallel to the heme, thus positioning the C7 atom for hydroxylation. Additional regions of the binding cavity (most distant from the access channel) are involved to accommodate the elongated conformation of the aliphatic side chain. Structural complex with 7KCh shows an active site rigidity and provides an explanation for its inhibitory effect. Based on our previously published data, we proposed a model of cholesterol abstraction from the membrane by CYP7A1 for metabolism. CYP7A1 structural data provide a molecular basis for understanding of the diversity of 7α-hydroxylases, on the one hand, and cholesterol-metabolizing enzymes adapted for their specific activity, on the other hand.

Postprandial response and tissue distribution of the bile acid synthesis-related genes, cyp7a1, cyp8b1 and shp, in rainbow trout Oncorhynchus mykiss.

In mammals, cholesterol 7α-hydroxylase (CYP7A1) and sterol 12α-hydroxylase (CYP8B1) are rate-limiting enzymes in bile acid synthesis. In addition, a small heterodimer partner (SHP) is also known to inhibit bile acid synthesis via the suppression of CYP7A1 and CYP8B1 expression. However, little information is currently available regarding primary structure of the genes involved in bile acid synthesis in fish. We therefore cloned cyp7a1, cyp8b1 and shp genes from rainbow trout and obtained cDNAs encoding two isoforms each of Cyp7a1 (-1 and -2), Cyp8b1 (-1 and -2) and Shp (-1 and -2). Both cyp7a1-1 and -2 encoded proteins of 512 amino acids. Trout cyp7a1-1 was expressed not only primarily in the kidney, pyloric caecum and mid-gut, but also weakly in the liver, eye, gill and ovary. cyp7a1-2 was highly expressed in the liver, pyloric caecum and mid-gut. cyp8b1-1 and -2, which encoded proteins of 512 and 509 amino acids, respectively, were principally expressed in the liver. Both shp-1 and -2, which encoded proteins of 288 and 290 amino acids, respectively, were strongly expressed in the liver, but shp-2 was also highly expressed in the gallbladder and digestive tract. The temporal changes in the expression of cyp7a1-1/-2, cyp8b1-1/-2 and shp-1/-2 in the liver were assessed after consumption of a single meal. Expression of cyp7a1-1/-2 and cyp8b1-1/-2 increased within 3h post feeding (hpf) when the stomach was still approximately 84% full and the gallbladder was almost completely empty. Although the expression of shp-1 did not change after feeding, the expression pattern of shp-2 was inversely related to the expression patterns of cyp7a1-1/-2 and cyp8b1-1/-2. Specifically, shp-2 expression decreased until 3 hpf before returning to initial levels at 24 hpf. These findings suggest that Cyp7a1s/8b1s and Shp-2 function antagonistically in bile acid synthesis in rainbow trout.

Cytochrome P450 7A1 cholesterol 7alpha-hydroxylation: individual reaction steps in the catalytic cycle and rate-limiting ferric iron reduction.

Cytochrome P450 (P450) 7A1 is well known as the cholesterol 7α-hydroxylase, the first enzyme involved in bile acid synthesis from cholesterol. The human enzyme has been reported to have the highest catalytic activity of any mammalian P450. Analyses of individual steps of cholesterol 7α-hydroxylation reaction revealed several characteristics of this reaction: (i) two-step binding of cholesterol to ferric P450, with an apparent K(d) of 0.51 µM, (ii) a rapid reduction rate in the presence of cholesterol (∼10 s(-1) for the fast phase), (iii) rapid formation of a ferrous P450-cholesterol-O(2) complex (29 s(-1)), (iv) the lack of a non-competitive kinetic deuterium isotope effect, (v) the lack of a kinetic burst, and (vi) the lack of a deuterium isotope effect when the reaction was initiated with the ferrous P450-cholesterol complex. A minimum kinetic model was developed and is consistent with all of the observed phenomena and the rates of cholesterol 7α-hydroxylation and H(2)O and H(2)O(2) formation. The results indicate that the first electron transfer step, although rapid, becomes rate-limiting in the overall P450 7A1 reaction. This is a different phenomenon compared with other P450s that have much lower rates of catalysis, attributed to the much more efficient substrate oxidation steps in this reaction.

A Prospero-related homeodomain protein is a novel co-regulator of hepatocyte nuclear factor 4alpha that regulates the cholesterol 7alpha-hydroxylase gene.

Prox1, an early specific marker for developing liver and pancreas in foregut endoderm has recently been shown to interact with alpha-fetoprotein transcription factor and repress cholesterol 7alpha-hydroxylase (CYP7A1) gene transcription. Using a yeast two-hybrid assay, we found that Prox1 strongly and specifically interacted with hepatocyte nuclear factor (HNF)4alpha, an important transactivator of the human CYP7A1 gene in bile acid synthesis and phosphoenolpyruvate carboxykinase (PEPCK) gene in gluconeogenesis. A real time PCR assay detected Prox1 mRNA expression in human primary hepatocytes and HepG2 cells. Reporter assay, GST pull-down, co-immunoprecipitation, and yeast two-hybrid assays identified a specific interaction between the N-terminal LXXLL motif of Prox1 and the activation function 2 domain of HNF4alpha. Prox1 strongly inhibited HNF4alpha and peroxisome proliferators-activated receptor gamma coactivator-1alpha co-activation of the CYP7A1 and PEPCK genes. Knock down of the endogenous Prox1 by small interfering RNA resulted in significant increase of CYP7A1 and PEPCK mRNA expression and the rate of bile acid synthesis in HepG2 cells. These results suggest that Prox1 is a novel co-regulator of HNF4alpha that may play a key role in the regulation of bile acid synthesis and gluconeogenesis in the liver.

Cholesterol-metabolizing cytochromes P450.

By catalyzing the first steps in different pathways of cholesterol degradation, cytochromes P450 (P450s) 7A1, 27A1, 11A1, and 46A1 play key roles in cholesterol homeostasis. CYP7A1 is a microsomal liver-specific enzyme that converts cholesterol to 7alpha-hydroxycholesterol. CYP27A1 is a ubiquitously expressed mitochondrial P450 that metabolizes cholesterol to 27-hydroxycholesterol. CYP11A1 also resides in mitochondria but is expressed mainly in steroidogenic tissues, where it catalyzes the conversion of cholesterol to pregnenolone. Finally, CYP46A1 is a brain-selective microsomal monooxygenase producing 24S-hydroxycholesterol from cholesterol. Catalytic efficiencies of cholesterol-metabolizing P450s vary significantly and probably reflect physiological requirements of different organs for the rate of cholesterol turnover. P450s 7A1, 27A1, 11A1, and 46A1 represent a unique system for elucidation of how different enzymes have adapted to fit their specific roles in cholesterol elimination. Studies of cholesterol-metabolizing P450s suggest that their activities could be modulated post-translationally and that they should also be considered as targets for regulation of cholesterol homeostasis.

Cholesterol 7alpha-hydroxylase is phosphorylated at multiple amino acids.

The activity of cholesterol 7alpha-hydroxylase (gpCYP7A1), the rate limiting enzyme in bile acid synthesis, has been postulated to be regulated by phosphorylation/dephosphorylation. This study has found that several kinase activators rapidly reduce the amount of bile acid produced by the human hepatoma cell line, HepG2, and that gpCYP7A1 from HepG2 cell extracts eluted in the phosphoprotein fraction of FeIII columns. After incubating the HepG2 cells with radioactive orthophosphate, the band identified as gpCYP7Al on immunoblots was strongly labeled. Recombinant gpCYP7A was expressed as 6xHIS fusion polypeptides and subjected to kinase assays. The locations of phosphorylation were mapped further by screening synthetic peptides against AMP-activated protein kinase (AMPK), c-Jun N-terminal kinase, protein kinase A, and a panel of nine protein kinase C isoforms. AMPK, also known as 3-hydroxy-3-methylglutaryl coenzyme A reductase kinase, phosphorylated cholesterol 7alpha-hydroxylase, suggesting a potential mechanism of coordination of cholesterol synthesis and degradation.

Cholesterol binding to cytochrome P450 7A1, a key enzyme in bile acid biosynthesis.

The conversion of cholesterol to 7alpha-hydroxycholesterol catalyzed by cytochrome P450 7A1 (CYP7A1) initiates the major pathway for cholesterol elimination in mammals. In the present work we focused on identification of determinants of the CYP7A1 substrate specificity inside the active site using a homology model with a novel P450-fold, site-directed mutagenesis, and substrate-binding and kinetic studies. Forty-one mutants, encompassing twenty-six amino acid residues, were generated and characterized, and of these, seven residues appear to determine cholesterol binding in the active site. In addition, four cholesterol derivatives were used as active site probes in the wild type and the seven mutant enzymes, and the spectral binding constants and products were analyzed. It was concluded that Asn288 in the I helix plays a key role in the P450-cholesterol contacts by hydrogen bonding to the steroid 3beta-hydroxyl, while Val280 and Ala284 are beside and the Trp283 is above the steroid nucleus orienting the cholesterol molecule. Leu360 and Ala358 between the K helix and the beta1-4 strand and Leu485 in the beta4 sheet-turn appear to define the size of the active site over the heme pyrrole ring A, thus limiting the orientation and size of the substrate at the steroid A ring. Additionally, the A358V mutant was found to form two new products, one being 7beta-hydroxycholesterol. Our data indicate that a tight fit of cholesterol in the enzyme active site is in part responsible for the high efficiency of cholesterol turnover by CYP7A1.

Estrogen receptor alpha regulates expression of the orphan receptor small heterodimer partner.

Hormonal status can influence diverse metabolic pathways. Small heterodimer partner (SHP) is an orphan nuclear receptor that can modulate the activity of several transcription factors. Estrogens are here shown to directly induce expression of the SHP in the mouse and rat liver and in human HepG2 cells. SHP is rapidly induced within 2 h following treatment of mice with ethynylestradiol (EE) or the estrogen receptor alpha (ERalpha)-selective compound propyl pyrazole triol (PPT). SHP induction by these estrogens is completely absent in ERalphaKO mice. Mutation of the human SHP promoter defined HNF-3, HNF-4, GATA, and AP-1 sites as important for basal activity, whereas EE induction required two distinct elements located between -309 and -267. One of these elements contains an estrogen response element half-site that bound purified ERalpha, and ERalpha with a mutated DNA binding domain was unable to stimulate SHP promoter activity. This ERalpha binding site overlaps the known farnesoid X receptor (FXR) binding site in the SHP promoter, and the combination of EE plus FXR agonists did not produce an additive induction of SHP expression in mice. Surprisingly, induction of SHP by EE did not inhibit expression of the known SHP target genes cholesterol 7alpha-hydroxylase (CYP7A1) or sterol 12alpha-hydroxylase (CYP8B1). However, the direct regulation of SHP expression may provide a basis for some of the numerous biological effects of estrogens.

Effects of compatible solutes on mammalian cytochrome P450 stability.

The sensitivity of pig cytochrome P450c17 (CYP17), an endoplasmic reticulum membrane-bound enzyme, towards heat denaturation (48 degrees C) was measured by the P450-to-P420 spectral transition indicating conformational labilization of the protein. Both sucrose and glucose have comparable and increasingly protective effects at concentrations ranging from 100 to 800 mM, while ectoine, a novel zwitterionic compatible solute which regulates bacterial osmoadaptation and stabilizes cytoplasmic enzymes, has a strong labilizing effect on CYP17 and favours its proteolytic inactivation possibly by electrostatic derangements. Sucrose or glucose, but not ectoine, can therefore eventually be proposed as compatible stabilizers of cytochrome P450 structures.

A novel cytochrome P450 expressed primarily in brain.

hct-1 (hippocampal transcript) was detected in a differential screen of a rat hippocampal cDNA library. Expression of hct-1 was enriched in the formation but was also detected in rat liver and kidney, though at much lower levels; expression was barely detectable in testis, ovary, and adrenal. In liver, unlike brain, expression was sexually dimorphic; hepatic expression was greatly reduced in female rats. In mouse, brain expression was widespread, with the highest levels being detected in corpus callosum; only low levels were detected in liver. Sequence analysis of rat and mouse hct-1 cDNAs revealed extensive homologies with cytochrome P450s (CYPs), a diverse family of heme-binding monooxygenases that metabolize a range of substrates including steroids, fatty acids, and xenobiotics. Among the CYPs, hct-1 is most similar (39% at the amino acid sequence) to cholesterol 7 alpha-hydroxylase (CYP7) and contains a postulated steroidogenic domain present in other steroid-metabolizing CYPs but clearly represents a type of CYP not previously reported. Genomic Southern analysis suggests that a single gene corresponding to hct-1 is present in mouse, rat, and human. hct-1 is unusual in that, unlike all other CYPs described, the primary site of expression is in the brain. Similarity to CYP7 and other steroid-metabolizing CYPs may argue that hct-1 (CYP7B) plays a role in steroid metabolism in brain, notable because of the documented ability of brain-derived steroids (neurosteroids) to modulate cognitive function in vivo.

Structure and nucleotide sequences of the human cholesterol 7 alpha-hydroxylase gene (CYP7).

The human cholesterol 7 alpha-hydroxylase gene (CYP7) spans about 11 kb of the genome and contains six exons and five introns. Nucleotide sequences of a 5'-upstream region to exon III (5535 bp), a 5'-upstream EcoRI fragment (2575 bp), and an EcoRI fragment covering intron V to exon VI (2319 bp) have been determined. A comparison of our sequences with those reported previously unveiled numerous sequencing discrepancies that are apparently due to sequencing errors. There are only one confirmed and one possible genetic polymorphism in the promoter of this highly conserved human gene. The proximal promoter contained many consensus recognition sequences for liver-enriched transcription factors and steroid hormone receptors that may play important roles in regulation of the CYP7 gene transcription by bile acids, cholesterol, and hormones.

Transcriptional regulation of the human cholesterol 7 alpha-hydroxylase gene.

As an initial step toward understanding the transcriptional regulation of cholesterol 7 alpha-hydroxylase (CYP7) in man, we isolated and functionally characterized the 5'-flanking region of the human CYP7 gene. The nucleotide sequences of the first exon and 1.6 kb preceding the exon were determined and found to contain a TATA box at position -30, a modified CAAT box at position -92, three potential hepatocyte nuclear factor 3 (HNF-3) recognition sites at nucleotides -316, -288, and -255, respectively, and a modified sterol response element at position -271. DNA sequences containing 1.3 kb of the 5'-flanking region and 29 nucleotides of the first exon were linked to the chloramphenicol acetyltransferase gene and transiently transfected into several cell lines. Promoter activity was very strong in the human hepatoma cell line HepG2 but absent in cells of nonhepatic origin. Mutational analysis of the promoter identified several regions that function in the transcriptional regulation of CYP7. Introduction of a fragment containing the region from -432 to -220 upstream of a heterologous promoter, in either orientation, resulted in a tremendous stimulation of activity in HepG2 cells. DNase I footprint analysis identified three regions within this fragment which were protected from digestion. The overexpression of HNF-3 in HepG2 cells resulted in a 4-fold stimulation of CYP7 transcriptional activity. We suggest that the region between -432 and -220 functions as a cell-specific enhancer whose activity is controlled, in part, by HNF-3.