RESUMO
The peroxisome proliferator-activated receptor alpha (PPARalpha) is a ligand-activated transcription factor and a key regulator of lipid homeostasis. Numerous fatty acids and eicosanoids serve as ligands and activators for PPARalpha. Here we demonstrate that S-hexadecyl-CoA, a nonhydrolyzable palmitoyl-CoA analog, antagonizes the effects of agonists on PPARalpha conformation and function in vitro. In electrophoretic mobility shift assays, S-hexadecyl-CoA prevented agonist-induced binding of the PPARalpha-retinoid X receptor alpha heterodimer to the acyl-CoA oxidase peroxisome proliferator response element. PPARalpha bound specifically to immobilized palmitoyl-CoA and Wy14643, but not BRL49653, abolished binding. S-Hexadecyl-CoA increased in a dose-dependent and reversible manner the sensitivity of PPARalpha to chymotrypsin digestion, and the S-hexadecyl-CoA-induced sensitivity required a functional PPARalpha ligand-binding pocket. S-Hexadecyl-CoA prevented ligand-induced interaction between the co-activator SRC-1 and PPARalpha but increased recruitment of the nuclear receptor co-repressor NCoR. In cells, the concentration of free acyl-CoA 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 PPARalpha expressed in Sf21 cells for electrophoretic mobility shift assays, we demonstrate that S-hexadecyl-CoA was able to increase the mobility of the PPARalpha-containing heterodimer even in the presence of a molar excess of acyl-CoA-binding protein, mimicking the conditions found in vivo.
Assuntos
Acil Coenzima A/farmacologia , Coenzima A/farmacologia , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Receptores Citoplasmáticos e Nucleares/química , Receptores Citoplasmáticos e Nucleares/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Acil-CoA Oxidase , Animais , Linhagem Celular , Cromatografia de Afinidade , Proteínas de Ligação a DNA/efeitos dos fármacos , Dimerização , Genes Reporter , Glutationa Transferase/genética , Histona Acetiltransferases , Ligantes , Camundongos , Modelos Moleculares , Coativador 1 de Receptor Nuclear , Oxirredutases/química , Oxirredutases/metabolismo , Biossíntese de Proteínas , Conformação Proteica , Ratos , Receptores Citoplasmáticos e Nucleares/efeitos dos fármacos , Receptores do Ácido Retinoico/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Receptores X de Retinoides , Spodoptera , Transativadores/metabolismo , Fatores de Transcrição/efeitos dos fármacos , Transcrição Gênica , TransfecçãoRESUMO
We report a protocol for the rapid identification of DNA-binding proteins. Immobilized DNA probes harboring a specific sequence motif are incubated with cell or nuclear extract. Proteins are analyzed directly off the solid support by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The determined molecular masses are often sufficient for identification. If not, the proteins are subjected to mass spectrometric peptide mapping followed by database searches. Apart from protein identification, the protocol also yields information on posttranslational modifications. The protocol was validated by the identification of known prokaryotic and eukaryotic DNA-binding proteins, and its use provided evidence that poly(ADP-ribose) polymerase exhibits DNA sequence-specific binding to DNA.