Very-Long-Chain and Branched-Chain Fatty Acyl-CoAs are High Affinity Ligands for the Peroxisome Proliferator-Activated Receptor α (PPARα)

Peroxisome proliferator-activated receptors (PPAR) are ligand-activated members of the steroid/thyroid nuclear hormone receptor superfamily. PPAR have been identified in several species, with at least three isotypes recognized (α, β/δ, γ). Each isotype, encoded by a different gene, is differentially expressed in select tissues and expression levels depend on cellular processes (rev. in (1). Although there is some overlap in ligand specificity, unique isotype binding occurs by a variety of xenobiotics depends upon specific amino acid substitutions within the PPAR ligand-binding pocket (1,2). Independent of ligand binding, a basal level of transcription occurs by PPAR forming a heterodimer with the retinoid X receptor (RXR) which binds to a specific DNA response element (PPRE) within the target genes (3,4). Ligand binding to one and, even more so, both receptors greatly potentiates transcription (rev. in (4). PPRE exist within many of the genes involved in fatty acid oxidation and cell differentiation, suggesting that PPAR are responsible for regulating transcription of these genes (5). An especially important role of PPARα is inducing transcription of multiple peroxisomal enzymes required for β-oxidation of very-long-chain fatty acids (VLCFA) and branched-chain fatty acids (BCFA). VLCFA and BCFA accumulation are associated with human neurological disorders including Zellweger syndrome, X-linked adrenoleukodystrophy, Refsum disease, and MFP-2 deficiency (6-8). These genetic disorders arise from defects in enzymes of the peroxisomal β-oxidation pathway which result in accumulation of VLCFA, BCFA, and their thioesters (6-8). Both VLCFA and isoprenoid-derived BCFA preferentially undergo peroxisomal β-oxidation following acylation of the respective fatty acids to CoA thioesters. The classical peroxisomal β-oxidation pathway catalyzed by palmitoyl-CoA oxidase (AOX), L-bifunctional protein (L-PBE), and 3-ketoacyl-CoA thiolase (PTL) is responsible for the oxidation of straight-chain fatty acids (9). All three enzymes of the classical peroxisomal β-oxidation system are transcriptionally activated by PPARα ligands (rev. in (10). In contrast, although the branched-chain phytanic acid is considered a β-oxidation pathway precursor, a different set of peroxisomal enzymes are utilized for oxidation of BCFA. For example, phytanic acid must first undergo α-oxidation to produce pristanoyl-CoA before entering the β-oxidation cycle (11). BCFA-CoAs are then utilized by the branched-chain acyl-CoA oxidase, D-bifunctional protein (D-PBE), and sterol carrier protein-x (SCP-x). Both D-PBE (9) and SCP-x (12,13) are inducible upon administration of branched-chain PPARα ligands. While there is evidence indicating that VLCFA and BCFA are peroxisome proliferators and may be PPARα ligands, it is not completely clear whether the unesterified VLCFA and BCFA directly and/or metabolites thereof activate PPARα. For example, early transactivation assays show: (i) PPARα is activated by polyunsaturated (20:4, 20:5, 22:6), but not monounsaturated (24:1, 22:1) VLCFA (3), (ii) n-3 polyunsaturated VLCFA elicited similar PPARα activation levels as n-6 polyunsaturated VLCFA (3). These findings of VLCFA being potential PPARα ligands are supported by gel-shift assays (14) and co-activator-dependent receptor ligand assays (15). Likewise, transactivation studies with branched-chain fatty acids also show PPARα activation (11). It must be noted that most of these assays utilize very high concentrations of VLCFA (i.e. 30-100 μM) and BCFA (20 μM)--several orders of magnitude higher than concentrations of unesterified fatty acids detected in nuclei of living cells (16,17). These data suggested that affinities of PPARα for these ligands are very weak, i.e. Kds in the μM range, and the free fatty acids may not represent the endogenous ligands. The finding that, since unesterified fatty acids are potent detergents, they are rapidly activated/metabolized to their CoA thioesters in cells and tissues (rev. (18-20), suggests that metabolites such as the CoA thioesters may represent the endogenous PPARα ligands. This possibility is supported by recent findings in mice. For instance, peroxisome proliferators increase the level of CoA thioesters of the peroxisome proliferators in mice (21,22). Inhibitors of fatty acid thioesterification such as 2-bromopalmitic acid inhibit the effects of bezafibrate on peroxisomal proliferation (23). Mice with a disruption of the AOX gene exhibit peroxisomal proliferation, elevated serum levels of VLCFA, as well as elevated VLCFA-CoAs, hyperactivation of PPARα, and up-regulation of down-stream genes which contain PPRE (23). On the other hand, ablation of the adrenoleukodystrophy gene inhibits VLCFA transport into peroxisomes and results in elevated serum VLCFA, unaltered liver VLCFA-CoA, and normal PPARα activity (10). Taken together with other studies (24,25) these data in both normal and gene-targeted mice suggest that intermediates of peroxisomal β-oxidation are PPARα ligands. In summary, while many of the above experiments are suggestive that the CoA thioesters of VLCFA and BCFA represent functional ligands for PPARα, it remains to be shown whether the VLCFA, BCFA, or their CoA thioesters exhibit characteristic hallmarks of endogenous ligands that bind to PPARα: (i) high (Kds in nM range) affinity, (ii) physically alter the structure/conformation of the PPARα protein, and (iii) affect co-factor recruitment PPARα (26). The objective of the present study was to begin to resolve these issues through use of direct ligand binding assays (i.e. non-fluorescent ligand binding assays based on quenching of tyrosine emission), circular dichroism to characterize potential ligand-induced changes in PPARα secondary structure, and co-immunoprecipitation to study co-factor recruitment.