RESUMO
The uncoupling protein from brown adipose tissue (UCP1) is a transporter that catalyzes a regulated discharged of the mitochondrial proton gradient. The proton conductance in UCP1 is inhibited by nucleotides and activated by fatty acids. We have recently shown that all-trans-retinoic acid (ATRA) is a high-affinity activator of UCP1. In the present report, we have set to analyze the structural requirements for the ligands that activate UCP1 and particularly the specificity for different retinoids. For this purpose, we have developed a new protocol to determine the activity of UCP1 in respiring yeast mitochondria that can be adapted for high-throughput screenings. Our results evidence differences between the structural requirements for the activation by fatty acids and retinoids. Thus, although all active retinoids must possess a carboxylate, the introduction of additional polar groups renders them inactive. The linear and rigid structure of these molecules suggests the existence of a long hydrophobic binding pocket. We postulate that the access to the retinoid binding site must occur from the lipid bilayer and this could be at the interface between two transmembrane alpha-helices.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Membrana/metabolismo , Mitocôndrias/efeitos dos fármacos , Retinoides/farmacologia , Proteínas de Transporte/agonistas , Proteínas de Transporte/genética , Ácidos Graxos/química , Ácidos Graxos/farmacologia , Canais Iônicos , Proteínas de Membrana/agonistas , Proteínas de Membrana/genética , Mitocôndrias/metabolismo , Proteínas Mitocondriais , Estrutura Molecular , NAD/metabolismo , Consumo de Oxigênio , Proteínas Recombinantes/metabolismo , Retinoides/química , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteína Desacopladora 1RESUMO
Retinoic acid is a potent activator of the uncoupling protein-1 (UCP1) both at the gene and mitochondrial level. Irradiation with ultraviolet light can be used to directly photolabel proteins with retinoic acid. The procedure has been applied to investigate its interaction with UCP1 isolated from brown adipose tissue mitochondria. All-trans-retinoic acid binds to UCP1 with high affinity and the labeling is only partially protected by guanosine diphosphate. Ubiquinone (UQ) has been described to be an obligatory cofactor for uncoupling protein function and we demonstrate that it greatly increases the affinity of UCP1 for retinoic acid. Data support the notion of a direct interaction between UQ and retinoic acid.