Peroxisome proliferator-activated receptor ␣ (PPAR␣) is a key regulator of lipid homeostasis in hepatocytes and target for fatty acids and hypolipidemic drugs. How these signaling molecules reach the nuclear receptor is not known; however, similarities in ligand specificity suggest the liver fatty acid binding protein (L-FABP) as a possible candidate. In localization studies using laser-scanning microscopy, we show that L-FABP and PPAR␣ colocalize in the nucleus of mouse primary hepatocytes. Furthermore, we demonstrate by pull-down assay and immunocoprecipitation that L-FABP interacts directly with PPAR␣. In a cell biological approach with the aid of a mammalian two-hybrid system, we provide evidence that L-FABP interacts with PPAR␣ and PPAR␥ but not with PPAR␤ and retinoid X receptor-␣ by protein-protein contacts. In addition, we demonstrate that the observed interaction of both proteins is independent of ligand binding. P eroxisome proliferator-activated receptor ␣ (PPAR␣) is a nuclear target for fatty acids, hypolipidemic drugs, and other peroxisome proliferators (1-4) and initiates gene expression of enzymes involved in lipid metabolism (5, 6). Two further subtypes of this receptor exist, namely PPAR␤ and PPAR␥, of which the latter is implicated to play a role in adipogenesis and adipocyte fatty acid metabolism upon activation by fatty acids and antidiabetic thiazolidindiones (7). The mechanism and pathway by which fatty acids and respective drugs as signaling molecules reach their destination are not known, but assuming targeted transport in hepatocytes where all three subtypes of this receptor are expressed (8), liver fatty acid binding protein (L-FABP) is a candidate to serve as shuttle for these ligands. This hypothesis is based on several observations. First, the 14.4-kDa L-FABP, which is supposed to play a role in intracellular lipid trafficking (9, 10), is abundant in the cytosol and is also found inside the nucleus of liver cells (11), the presumed place of PPAR activation. Second, L-FABP binds fatty acids and hypolipidemic drugs that have been identified as PPAR␣ agonists (2-4) as well as BRL48,482, an antidiabetic thiazolidindione, all with dissociation constants in the micro to nanomolar range (12-14). Third, hypolipidemic drugs are able to induce expression of L-FABP and ␤-oxidative enzymes via PPAR␣ (15). This link between multiple ligand interactions and gene expression was revealed recently by us by identifying branchedchain phytanic acid as a ligand for L-FABP and PPAR␣ and by demonstrating that this fatty acid induced L-FABP expression via activation of PPAR␣ (6). Thus, L-FABP might be part of the PPAR␣ targeted signal transduction pathway, for which two alternative mechanisms can be envisaged (10, 16); L-FABP forms a cytosolic sink for the signaling molecules, thus acting as negative regulator of their concentrations available for PPAR␣ activation in the nucleus (i.e., increased PPAR␣ activation results from decreased intracellular L-FABP concentrations). Alternatively, L-FABP itself transp...

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We describe the crystal structure of human epidermal-type fatty acid binding protein (E-FABP) that was recently found to be highly upregulated in human psoriatic keratinocytes. To characterize E-FABP with respect to ligand-binding properties and tertiary structure, we cloned the respective cDNA, overexpressed the protein in Escherichia coli and purified it to homogeneity by a combination of ion-exchange and size-exclusion chromatographic steps with a yield of 30 mg/L broth. The purified protein revealed a 5-fold higher affinity for stearic acid than for oleic and arachidonic acids. The crystal structure of recombinant human E-FABP was determined to 2.05 A and refined to an R(factor) of 20.7%. The initial residual electron density maps clearly showed the presence of a ligand, which was identified as endogenous bacterial fatty acid. Within a central cavity of 252 A(3), this ligand is bound in a U-shaped conformation, its carboxyl group interacting with tyrosine 131 and arginines 129 and 109, the latter via an ordered water molecule. The E-FABP crystal structure is unique in the FABP family because of the presence of a disulfide bridge between cysteines 120 and 127 that may be physiologically as well as pathophysiologically relevant. Cysteines 67 and 87 are also in close vicinity but in contrast do not form a disulfide bridge. We postulate that this protein belongs to a particular FABP subfamily whose members share common structural as well as functional features.