RESUMEN
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.
Asunto(s)
Nucleótidos de Desoxiadenina/química , Nucleótidos de Desoxicitosina/química , Escherichia coli/genética , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/aislamiento & purificación , Timidina Quinasa/química , Timidina Quinasa/aislamiento & purificación , Nucleótidos de Timina/química , Sitios de Unión , Línea Celular Tumoral , Chaperonina 10/biosíntesis , Chaperonina 10/genética , Chaperonina 60/biosíntesis , Chaperonina 60/genética , Clonación Molecular , Medios de Cultivo Condicionados , Nucleótidos de Desoxiadenina/análisis , Nucleótidos de Desoxicitosina/análisis , Dimerización , Inhibidores Enzimáticos/química , Escherichia coli/enzimología , Escherichia coli/crecimiento & desarrollo , Etanol/química , Humanos , Isoenzimas/antagonistas & inhibidores , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/aislamiento & purificación , Cinética , Modelos Moleculares , Proteínas Recombinantes de Fusión/antagonistas & inhibidores , Proteínas Recombinantes de Fusión/genética , Solubilidad , Espectrometría de Fluorescencia , Especificidad por Sustrato , Timidina Quinasa/antagonistas & inhibidores , Timidina Quinasa/genética , Nucleótidos de Timina/análisis , Triptófano/químicaRESUMEN
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) beta/delta. We demonstrate that while RA does not activate PPARalpha and PPARgamma, it binds to PPARbeta/delta with nanomolar affinity, modulates the conformation of the receptor, promotes interaction with the coactivator SRC-1, and efficiently activates PPARbeta/delta-mediated transcription. Transcriptional signaling by RA is thus exerted by a dual pathway, providing a rationale for understanding divergent cellular responses to this hormone.