Retinoic acid (RA) modulates transcription of numerous target genes, thereby regulating a myriad of biological processes. It is well established that RA functions by activating retinoic acid receptors (RARs), which, in turn, control cell differentiation, proliferation, and apoptosis. However, perplexing reports of diverse and sometime opposing actions of RA have been published. Hence, while RA induces apoptosis and inhibits cell growth in some settings, it potentiates proliferation and acts as an anti-apoptotic agent in others. These observations raise the possibility that signaling pathways other than RAR may be involved in mediating RA activities. Here we show that RA is a high affinity ligand for another nuclear receptor, namely the orphan receptor peroxisome proliferator-activated receptor (PPAR) ␤/␦. We demonstrate that while RA does not activate PPAR␣ and PPAR␥, it binds to PPAR␤/␦ with nanomolar affinity, modulates the conformation of the receptor, promotes interaction with the coactivator SRC-1, and efficiently activates PPAR␤/␦-mediated transcription. Transcriptional signaling by RA is thus exerted by a dual pathway, providing a rationale for understanding divergent cellular responses to this hormone.

Human thymidine kinase 2 (hTK2) phosphorylates pyrimidine deoxyribonucleosides to the corresponding nucleoside monophosphates, using a nucleotide triphosphate as a phosphate donor. In this study, hTK2 was cloned and expressed at high levels in Escherichia coli as a fusion protein with maltose-binding protein. Induction of a heat-shock response by ethanol and coexpression of plasmid-encoded GroEL/ES chaperonins at 28 degrees C minimized the nonspecific aggregation of the hybrid protein and improved the recovery of three homooligomeric forms of the properly folded enzyme, i.e., dimer > tetramer > hexamer. The dimer and the tetramer were isolated in stable and highly purified forms after proteolytic removal of the fusion partner. Both oligomers contained a substoichiometric amount of deoxyribonucleotide triphosphates (dTTP > dCTP > dATP), known to be strong feedback inhibitors of the enzyme. Steady-state kinetic studies were consistent with the presence of endogenous inhibitors, and both oligomeric forms revealed a lag phase of at least approximately 5 min, which was abolished on preincubation with substrate (dThd or dCyd). The rather similar kinetic properties of the two oligomeric forms indicate that the basic functional unit is a dimer. Molecular docking experiments with a modeled hTK2 three-dimensional structure accurately predicted the binding positions at the active site of the natural substrates (dThd, dCyd, and ATP) and inhibitors (dTTP and dCTP), with highly conserved orientations obtained for all ligands. The calculated relative nonbonded interaction energies are in agreement with the biochemical data and show that the inhibitor complexes have lower stabilization energies (higher affinity) than the substrates.

The peroxisome proliferator-activated receptor ␣ (PPAR␣) is a ligand-activated transcription factor and a key regulator of lipid homeostasis. Numerous fatty acids and eicosanoids serve as ligands and activators for PPAR␣. Here we demonstrate that S-hexadecyl-CoA, a nonhydrolyzable palmitoyl-CoA analog, antagonizes the effects of agonists on PPAR␣ conformation and function in vitro. In electrophoretic mobility shift assays, S-hexadecyl-CoA prevented agonist-induced binding of the PPAR␣-retinoid X receptor ␣ heterodimer to the acylCoA oxidase peroxisome proliferator response element. PPAR␣ bound specifically to immobilized palmitoylCoA and Wy14643, but not BRL49653, abolished binding. S-Hexadecyl-CoA increased in a dose-dependent and reversible manner the sensitivity of PPAR␣ to chymotrypsin digestion, and the S-hexadecyl-CoA-induced sensitivity required a functional PPAR␣ ligand-binding pocket. S-Hexadecyl-CoA prevented ligand-induced interaction between the co-activator SRC-1 and PPAR␣ but increased recruitment of the nuclear receptor corepressor NCoR. In cells, the concentration of free acylCoA esters is kept in the low nanomolar range due to the buffering effect of high affinity acyl-CoA-binding proteins, especially the acyl-CoA-binding protein. By using PPAR␣ expressed in Sf21 cells for electrophoretic mobility shift assays, we demonstrate that S-hexadecylCoA was able to increase the mobility of the PPAR␣-containing heterodimer even in the presence of a molar excess of acyl-CoA-binding protein, mimicking the conditions found in vivo.Members of the nuclear receptor superfamily mediate liganddependent transactivation of genes controlling development, differentiation, and homeostasis in response to nutritional, metabolic, and hormonal signals (1). The peroxisome proliferator-activated receptor ␣ (PPAR␣, 1 NR1C1 (2)) belongs to the nuclear hormone receptor superfamily (3). Through heterodimerization with the retinoid X receptors (4) (NR2B1-3) and binding to DR-1 response elements, PPAR␣ regulates transcription of several genes encoding enzymes involved in lipid metabolism (5, 6). Accordingly, PPAR␣ is predominantly expressed in tissues with a high turnover of fatty acids (7).Activation of nuclear receptor-mediated transcription involves an agonist-dependent release of co-repressors and recruitment of co-activators. Accumulating evidence obtained by x-ray crystallography has revealed a significant ligand-dependent conformational change involving repositioning of the conserved AF-2 helix in the ligand-binding domains of nuclear receptors (8 -11). This ligand-induced conformational change has been demonstrated to be a determining event governing interactions with co-activators and co-repressors (see reviewed in Ref. 15). The crystal structures of the 17) have revealed an overall folding pattern similar to that observed for other nuclear receptor ligand-binding domains (8 -11). However, the PPAR ligand-binding pocket is substantially larger than those of other nuclear receptors, and this may in part explain the obse...

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