Modulation of fatty acid signalling by cytochrome P ... - Semantic Scholar

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This work was supported by the Wellcome Trust and the Medical Research Council. F.D.B. is an MRC collaborative student with GlaxoWellcome plc, who we thank for assistance with the green fluorescent protein studies. 1 Hammond, S. M., Altshuller, Y. M., Sung, T. C.,

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Rudge, S. A., Rose, K., Engebrecht, J., Morris, A. J. and Frohman, M. A. (1995) J. Biol. Chem. 270, 29640-29643 Ponting, C. P. and Kerr, I. D. (1996) Protein Sci. 5,914-922 Cook, S. J. and Wakelam, M. J. 0. (1991) Cell. Signal. 3, 273-282 Cook, S. J. and Wakelam, M. J. 0. (1992) Biochem. 1.285.247-253 Singer, W. D., Brown, H. A., Jiang, X. and Sternweis, P. C. (1996) J. Biol. Chem. 271, 4504-45 10 Brown, A. H., Gutowski, S., Moomaw, C. R., Slaughter, C. and Sternweis, P. (1993) Cell 75, 1137-1 144 Cockcroft, S., Thomas, G. M. H., Fensome, A., Geny, B., Cunningham, E., Gout, I., Hiles, I., Totty, N. F., Truong, 0. and Hsuan, J. J. (1994) Science 263,523-526 Martin, A., Brown, F. D., Hodgkin, M. N., Bradwell, A. J., Cook, S. J., Hart, M. and Wakelam, M. J. 0. (1996) J. Biol. Chem. 271, 17397-17403 Liscovitch, M., Chalifa, V., Pertiles, P., Chen, C. S. and Cantley, L. C. (1994) J. Biol. Chem. 269, 21 403-21 406 Bowman, E. P., Uhlinger, D. J. and Lambeth, J. D. (1993) J. Biol. Chem. 265,21509-21512 Hammond, S. M., Jenco, J. J., Nakashima, S., Cadwallader, K., Gu, Q., Cook, S., Nozawa, Y.,

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Prestwich, G. D., Frohman, M. A. and Morris, A. J. (1997) J. Biol. Chem. 272,3860-3868 Schmidt, M., Rumenapp, U., Nehls, C., Ott, S., Keller, J., Voneichelstreiber, C. and Jakobs, K. H. (1996) Eur. J. Biochem. 240, 707-712 Schmidt, M., Rumenapp, U., Keller, J., Lohmann, B. and Jakobs, K. H. (1997) Life Sci. 60, 1093-1100 Malcolm, K. C., Elliott, C. M. and Exton, J. H. (1996) J. Biol. Chem. 271, 13135-13139 Ktistakis, N. T., Brown, H. A., Sternweis, P. C. and Roth, M. G. (1995) Proc. Natl. Acad. Sci. U.S.A. 92,4952-4956 Colley, W. C., Sung, T.-C., Roll, R., Jenco, J., Hammond, S. M., Altshuller, Y., Bar-Sagi, D., Morris, A. J. and Frohman, M. A. (1997) Curr. Biol. 7, 191-201 Massenburg, D., Han, J.-S., Liyanage, M., Patton, W. A., Rhee, S. G., Moss, J. and Vaughan, M. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 11718-11722 Banno, Y., Tamiya-Koizumi, K., Oshima, H., Morikawa, A., Yoshida, S. and Nozawa, Y. (1997) J. Biol. Chem. 272,5208-5213 Cross, M. J., Roberts, S., Ridley, A. J., Hodgkin, M.N., Stewart, A., Claesson-Welsh, L. and Wakelam, M. J. 0. (1996) Curr. Biol. 6, 588-597 Pettitt, T. R., Martin, A., Horton, T., Liossis, C., Lord, J. M. and Wakelam, M. J. OP. (1997) J. Biol. Chem. 272, 17354-17359 Stuchfield, J. and Cockcroft, S. (1993) Biochem. J. 293,649-655

Received 31 July 1997

Modulation of fatty acid signalling by cytochrome P-450-mediated hydroxylation C. N. A. Palmer, M. Causevic and C. R. Wolf I.C.R.F. Molecular Pharmacology Unit and Biomedical Research Centre, Ninewells Hospital and Medical School, Dundee DDI 9SY, Scotland, U.K.

Hydrophobic signalling molecules such as steroid hormones, oxysterols and retinoids are both produced and metabolized by members of the cytochrome P-450 super-family [l]. Many of these reactions modulate highly specialized signals required for the development of multicellular organisms [Z,31 ; however, hydroxylation of fatty acids by cytochrome P-450 occurs in mammals, plants and unicellular microbes, such as Bacillus Abbreviations used: NSAID, non-steroidal anti-inflammatory drug; PPAR, peroxisome-proliferator-activated receptor; RAR, retanoic acid receptor; RXR, retinoid X receptor; T R , thyroid hormone receptor.

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meguterium. T h i s conserved activity is mediated by cytochrome P-450s of the 2 and 4 families in mammals [4,5]and by the bacterial fatty acid hydroxylase, CYP102, found in B. meguterium [6]. A crystal structure has yet to be determined for any mammalian P-450;however, the structure of the haem domain of CYP102 has been solved, and this currently serves as a prototype for the computer modelling of mammalian P-450 structure and function [7,8]. T h e regulation of fatty acid hydroxylases by chemical stimuli has also been conserved throughout evolution. Fatty acid hydroxylases are highly inducible in mammals [4],plants [9] and

The Cell Biology of Lipid Signalling

B. megaterium [lo] by exposure to or treatment with a group of compounds known as peroxisome proliferators (Table 1). These compounds are named for their ability to cause the size and number of hepatic peroxisomes to increase in rodents. Peroxisome proliferators are thought to mimic fatty acids and perturb lipid metabolism, and it appears that induction of fatty acid hydroxylases may represent an adaptive response to these lipid imbalances. CYP4A enzymes are induced during a wide range of pathophysiological states that are known to involve imbalances in lipid homeostasis such as diabetes [ 113, hypertension and starvation. Peroxisome proliferators have been shown to alleviate salt-induced hypertension in the rat, and altered expression of the CYP4A2 gene has been found to co-segregate with hypertension in spontaneously hypertensive rats [12,13]. This has led to the suggestion that CYP4A activation may play a preventative role in the pathology of hypertension. T h e mechanism of this is unknown, and a product of CYP4A2 metabolism, 20-HETE, is itself a potent vasoconstrictor [14]. In mammals and B. megaterium the induction of fatty acid hydroxylase activities by peroxisome proliferators has been shown to be at the Table I Compounds that induce fatty acid hydroxylases Shown is a list of compounds that induce CYP4A activity in the rodent liver. These compounds are structurally diverse and the human population is exposed t o these chemicals both as pharmaceutical agents and as environmental contaminants.

Hypolipidaemicdrugs Clofibrate Fenofibrate Ciprofibrate Wy 14,643 Nafenopin Methylclofenapate Inhibitors of fatty acid metabolism Ibuprofen

ETYA LY I7 I883 Perfluorinated fatty acids Environmental contaminants F'thalate Plasticizers (DEHP) found in 'cling film' etc. Herbicides: lactofen and chlorophenoxy acids (2,4-D and 2,4,5-T)

transcriptional level [ 15,161. In the rabbit there are four members of the CYP4A subfamily, CY4A4, CYP4A5, CYP4A6 and CYP4A7. These genes are differentially regulated, and the encoded proteins have different substrate specificities [4]. CYP4A4 is not present in the liver of normal animals but is dramatically activated in the lung of the pregnant rabbit and is switched off immediately before parturition. This enzyme is specific for the hydroxylation of prostaglandin FZa, and it is thought that this activity is important for the prevention of parturition [4,17,18]. T h e CYP4A6 gene is the most highly activated by treatment with peroxisome proliferators [4]. The 5'-flanking region of this gene contains three responsive elements that bind heterodimers of the peroxisome-proliferator-activated receptor (PPAR) and the retinoid X receptor (RXR), and it would appear that the PPAR is the molecular switch that is responsible for the activation of CYP4A6 transcription in response to peroxisome proliferators and fatty acids [ 191. The requirement of PPARa for the response to peroxisome proliferators in the mouse liver has been confirmed by the generation of mice null for the PPARcr gene [ZO]. In the PPARa-null mice, normal constitutive levels of CYP4A expression are observed, but the treatment of these mice with peroxisome proliferators does not result in an increase in CYP4A activity or in the activity of other hepatic markers of peroxisome proliferation. PPARs are members of the steroid hormone receptor superfamily, and as such they are formed by a well-defined domain structure [2] (Figure 1A). The most highly conserved region of these proteins is a DNA-binding domain that contains two well-defined, zincfinger motifs. The C-terminus comprises a ligand-binding domain and a ligand-dependent transactivation domain (AF2) that is responsible for the ligand-dependent association with transcriptional mediator proteins such as NCOR, TIF, RIP140 and SRCl [21-231. In the study of the responsive elements in the CYP4A6 gene it was observed that sequences immediately upstream of the zinc-finger-binding sites were required for high-efficiency binding of PPARI RXR (Figure 1B) [24]. These sequences were similar to those observed for the monomeric nuclear receptors RevErbA and ROR. Chimaeric studies of the PPAR molecule revealed that a putative helical region adjacent to the zinc fingers (CTE; Figure 1A) could confer monomeric binding to this region [25]. It appears that this

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activity is repressed by the N-terminal domain of PPAR and could only be utilized in N-terminal truncations and in heterodimers with RXR. We believe that these additional DNA-sequence interactions allow specific signalling in a group of related nuclear receptors (e.g. hepatocyte nuclear factor-4, chicken ovalbumin upstream promotertranscription factor and apolipoprotein regulatory protein-1) that bind to similar DNA motifs [ 19,241. PPARa significantly activates the CYP4A6 gene in gene-transfer experiments without the presence of added inducer. It has been hypothesized that this intrinsic activation may be due to the presence of an endogenous ligand in the tissue-culture system [26]. A point mutation in the putative ligand-binding domain was isolated that reduced the intrinsic activation but was maximally activated by peroxisome proliferators at concentrations about 5-fold higher than those required for the wild-type receptor [26]. These results suggest that this mutant PPAR has a reduced affinity for the activating ligands and is not activated by the low levels of endogenous Figure I PPAR and peroxisome-proliferator-response element (PPRE) structures (A) A schematic illustration of the domain structure of mouse PPAR CI. Shown are the N-terminal A/B domain (AB), the DNAbinding 'zinc finger' domain (ZF), the 'monomeric' DNA-binding domain (CTE), the ligand-binding domain (LIGAND), and the ligand-dependent activation domain (AF2). (B) Peroxisome proliferator response elements (PPREs) are distinct from other nuclear-receptor-binding sites. All PPREs contain an imperfect direct repeat of the zinc-finger binding motif (ZBS) separated by one nucleotide. This is identical to the site utilized by homodimeric nuclear receptors such as HNF-4 and ARP- I . The PPRE also contains an extended binding site (EBS) that is similar to that seen for the monomeric nuclear receptor RevErbA. The binding to the EBS sequence is mediated by the CTE domain of PPAR.

A

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(EBS)lZBSl AQQTCA A AQQTCA

HNF4,ARP-1

AMCT AaOTCA A AQQTCA

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ligand. Recent studies of purified recombinant PPAR protein have confirmed this hypothesis. We have expressed the wild-type and mutant histidine-tagged ligand-binding domains of mouse PPARa and human PPARy in Escherichia coli. These proteins were nickel-affinity purified and assayed for their ability to bind the fluorescent fatty acid cis-parinaric acid. The same mutation in both the a and y isoforms of PPAR leads to a reduced efficiency of binding (Figures 2A and 2B). The wild-type and mutant PPARy proteins were also assayed for binding to a radiolabelled thiazolidinedione compound by rapid filtration. This analysis revealed a I(d of 23 nM for the wild-type protein and 100 nM for the mutant protein. This study confirms that this mutant affects the general ligand-binding properties of PPARs and does not seem to discriminate between ligands. This is not surprising, because this residue is conserved in all PPAR sequences found to date (Figure 2C). The mutated residue lies in a region of the protein that corresponds to helix 3 of RXR [27]. The corresponding Glu-275 residue of RXR projects well into the proposed ligand-binding pocket. The specific interactions of this residue in RXR and the ligand are not known, because of the lack of a ligand bound or holo-RXR structure. The ligand-bound structure has been determined for both the retinoic acid receptor (RAR) [28] and the thyroid hormone receptor (TR) [29]. Examination of these structures has revealed that helix 3 contains many of the most important interactions with both ligands, including the co-ordination of the carboxylate group (Figure 2C). Glu-282 is not conserved in either the RARy or TRa proteins; however, the corresponding position, Ala-225, of TRa is an important contact residue for the binding of thyroid hormone. Lys-336 of RARy, which corresponds to the residue immediately adjacent to Glu-282 of PPARa, is also a contact residue in the binding of all trans retinoic acid. It is of interest to note that the PPAR subfamily is highly divergent over helix 3 when compared with other nuclear receptor subfamilies (Figure 2C). The areas of greatest divergence appear to correspond to the ligand-contact residues of RAR and TR, thus suggesting that this diversity may play a role in the generation of pharmacological selectivity within this subfamily [30]. It is clear that the functional dissection of this region will be of great importance in the rational design of drugs diabetes, obesity and athero" against " sclerosis. The crystallization of mutant and wild-


type PPAR ligand-binding domains will facilitate such endeavours. As mentioned before, the response to peroxisome proliferators precedes the evolution of nuclear receptors. This response to lipid perturbation is found in bacilli and in plants, organisms that do not contain any members of the nuclear-receptor superfamily. The CYP102 gene of B. megaterium is regulated by a protein known as Bm3R1 [16,31]. Bm3R1 is a helix-loop-helix DNA-binding protein that is related to the tetracycline repressor, TetR [32]. T h e binding of Bm3R1 to its regulatory sequence is disrupted by fatty acids, peroxisome proliferators and nonsteroidal anti-inflammatory drugs (NSAIDs) [ 10,161. This leads to the transcriptional activation of the CYP102 operon. Studies using fluorescent and radiolabelled fatty acids have shown that Bm3R1 interacts directly with the inducing agents and functions as a sensor of fatty acids (C. N. A. Palmer, E. h e n , V, Hughes and C. R. Wolf, unpublished work). The most potent of

inducing compounds are the polyunsaturated fatty acids, including linoleic and arachidonic acid (Figure 3A). These polyunsaturated fatty M, whereas satuacids induce effectively at rated fatty acids, peroxisome proliferators and NSAIDs induce CYP102 at 10-4-10-3 M. The polyunsaturated fatty acids are highly toxic to B. megaterium (Figure 3B) and only induce CYP102 activity over a very narrow range of concentrations. This response is transient, which indicates that the fatty acids are metabolically inactivated. It has been known for a long time that long-chain fatty acids are substrates for the CYP102 enzyme [6]. In fact, the oxidation of arachidonic acid by CYP102 is the most efficient reaction known to be performed by a cytochrome P-450, with a turnover rate of 3000 mol of arachidonic acid/mol of CYP102 per min [33]. This is 100-1000-fold greater than the rate of metabolism by mammalian P-450s and may be attributed to the efficient coupling of NADPH hydrolysis by the fused reductase domain of

Figure 2 Mutation of the PPAR ligand-binding domain results in reduced binding to the fluorescent fatty acid cis-parinaric acid (A) Purified, recombinant mouse PPARs (3 pM) were assayed for binding to 3 pM cis-parinaric acid (CPA) in I ml of 25 mM TrisiHCI, pH 8.0, at room temperature. The fluorescence was measured by excitation at 3 I 8 nm and emission at 4 I0 nm. The values obtained with protein alone and CPA alone were combined and deducted from the result obtained with the protein and CPA. Purified trypsinogen (5 pM; Sigma) was assayed as a negative control. (6) Purified, recombinant human PPARy and the corresponding glycine mutant (hPPARgamma-G) (800 nM) were assayed for binding to 300 nM CPA under the above conditions. (C) Alignment of helix 3 amino acid sequences. Important ligant-contact residues are underlined. Conserved regions within the PPAR subfamily are boxed.

PPARy282 IFQGCQFRSVEAVQIETEYAK RXRK 265 VTNICQAADKQLFTL .VEWAK

mb TRa:

PPARG: P

10,

I

227 MDKESEUTSCIIKI.VEFAK 215 FSEEIllKUTPAITBV.VDFAK

lfn]K$IETJ H C C SVET EL EFAK C C TVE EL EFA

RXRa: 265 VTNICQAADKQLFT. LVEWAK RXRP: VTNICQAADKQLFT.LVEWAK W b VTNICEAADKQLFT.LVEWAK

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CYP102. The concept that hydroxylation of fatty acids by CYP102 leads to an attenuation of their inducing ability has been established by the demonstration that the CYP102 0-1 hydroxylation product of phytanic acid is greatly reduced in its ability to induce CYP102 [34]. CYP102 is Figure 3

6. rnegateriurn fatty acid hydroxylase transcriptional activity (A) Polyunsaturated fatty acids activate the transcription of a gene encoding a fatty acid hydroxylase in 6. rnegateriurn. Cultures of a strain of 6. rnegaterium carrying a CYP I OZ//I-galactosidase reporter construct were treated for I h with increasing concentrations of arachidonic acid (o), linoleic acid (0), and oletc acid (0). Cell lysates were prepared from these cultures and these were assayed for /I-galactosidase activity and total protein content. The activities are expressed as a percentage relative to the activity obtained with 100 pM nafenopin. (6) Induction of fatty acid hydroxylase activity protects 6. megateriurn from toxic fatty acids. Cultures of 6. rnegaterium A.T.C.C. 14583 were treated with nafenopin ( I00 pM) or solvent alone (DMSO) for I h. These cultures were then subdivided and either treated with linoleic acid (20 pM) or solvent alone. The growth rate was monitored by absorbance at 600 nrn. Cultures containing and nafenopin alone ( 0 )grew normally. Culsolvent alone (0) tures treated with linoleic acid and pretreated with nafenopin (A) grew at a marginally reduced rate. Cultures treated with linoleic acid alone ( 0 ) displayed marked lysis.

A

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therefore a powerful mediator of fatty acid clearance in B. megaterium, and it is possible that this enzyme may mediate the detoxification of unsaturated fatty acids. Indeed, when exponentially growing cultures of B. megaterium are treated with 20 pM linoleic acid visible lysis occurs (C. N. A. Palmer, E. h e n , V. Hughes and C. R. Wolf, unpublished work). The cultures then recover and re-enter logarithmic growth. In cultures that have been treated with the peroxisome proliferator nafenopin, the bacteria are resistant to the linoleic acid toxicity (Figure 3B). The role of Bm3R1 and CYP102 in this response has been confirmed by the use of a mutant strain of B. megaterium that is resistant to fatty acids and a specific CYP102 inhibitor that abolishes the adaptive response to fatty acids (C. N. A. Palmer, E. h e n , V. Hughes and C. R. Wolf, unpublished work). These data collectively demonstrate that the CYP102 operon appears to mediate an effective adaptive response for the detoxification of toxic fatty acids. B. subtilis also displays a similar sensitivity to unsaturated fatty acids, and its genome contains a region that is similar to the CYP102 operon of B. megaterium (C. N. A. Palmer, E. h e n , V. Hughes and C. R. Wolf, unpublished work). This is of interest because other investigators have reported that unsaturated fatty acids inhibit the two-component signal transduction pathways that control sporulation [35]. This inhibition has been demonstrated in vitro with the histidine-protein kinase, KinA, and occurs at the same concentrations as we have observed for toxicity in B. megaterium and B. subtilis. It is therefore tempting to speculate that inhibition of the sporulation or related signalling pathways such as osmoregulation, may play a role in the toxicity observed with unsaturated fatty acids. The sensing and metabolism of the fatty acids at concentrations directly below the concentrations required for the inhibition of these pathways provides an illustration of the elegance and efficiency of the action of the proteins encoded by the CYP102 operon. In summary, fatty acid hydroxylases have been conserved throughout evolution and are an adaptive response to fatty acid overload. The direct sensing of fatty acids by DNA-bound transcription factors has also been utilized throughout evolution; however, the structures of the lipid-sensing proteins are completely different. In mammals, the fatty acid sensor is a transcription factor whose interaction with the


transcriptional machinery is enhanced by fatty acid binding; in bacilli the response system involves derepression in which the DNA-bound repressor is displaced from t h e regulatory sequences. T h i s appears to be a n intriguing example of convergent evolution that fulfils a similar role in both bacilli a n d mammals. This work was supported by BBSRC ROPA award No. MOLO4650.

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