RESUMEN
Carnitine/acylcarnitine translocase (CACT) is a mitochondrial-membrane carrier proteins that mediates the transport of acylcarnitines into the mitochondrial matrix for their oxidation by the mitochondrial fatty acid-oxidation pathway. CACT deficiency causes a variety of pathological conditions, such as hypoketotic hypoglycemia, cardiac arrest, hepatomegaly, hepatic dysfunction and muscle weakness, and it can be fatal in newborns and infants. Here we report that expression of the Cact gene is induced in mouse skeletal muscle after 24h of fasting. To gain insight into the control of Cact gene expression, we examine the transcriptional regulation of the mouse Cact gene. We show that the 5'-flanking region of this gene is transcriptionally active and contains a consensus sequence for the estrogen-related receptor (ERR), a member of the nuclear receptor family of transcription factors. This sequence binds ERRαin vivo and in vitro and is required for the activation of Cact expression by the peroxisome proliferator-activated receptor gamma coactivator (PGC)-1/ERR axis. We also demonstrate that XTC790, the inverse agonist of ERRα, specifically blocks Cact activation by PGC-1ß in C2C12 cells.
Asunto(s)
Carnitina Aciltransferasas/genética , Regulación Enzimológica de la Expresión Génica , Receptores de Estrógenos/metabolismo , Transactivadores/metabolismo , Activación Transcripcional , Región de Flanqueo 5'/genética , Animales , Sitios de Unión , Ayuno , Expresión Génica , Células HEK293 , Humanos , Ratones , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/enzimología , Nitrilos/farmacología , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma , Receptores de Estrógenos/agonistas , Tiazoles/farmacología , Transactivadores/genética , Factores de Transcripción , Transcripción Genética/efectos de los fármacos , Receptor Relacionado con Estrógeno ERRalfaRESUMEN
Human carnitine palmitoyltransferase 1B (CPT1B) is a highly malonyl-CoA-sensitive enzyme (IC50=0.097 microm) and has a positive determinant (residues 18-28) of malonyl-CoA inhibition. By contrast, rat carnitine palmitoyltransferase 1A is less sensitive to malonyl-CoA inhibition (IC(50)=1.9 microm), and has both a positive (residues 1-18) and a negative (residues 18-28) determinant of its inhibition. Interestingly, pig CPT1B shows a low degree of malonyl-CoA sensitivity (IC(50)=0.804 microm). Here, we examined whether any additional molecular determinants affect malonyl-CoA inhibition of CPT1B. We show that the malonyl-CoA sensitivity of CPT1B is determined by the length (either 50 or 128 residues) of the N-terminal region constructed by recombining pig and human enzymes. We also show that the N-terminal region of pig CPT1B carries a single positive determinant of malonyl-CoA sensitivity, but that this is located between residues 1 and 18 of the N-terminal segment. Importantly, we found a single amino acid variation (D17E) relevant to malonyl-CoA sensitivity. Thus, Asp17 is specifically involved, under certain assay conditions, in the high malonyl-CoA sensitivity of the human enzyme, whereas the naturally occurring variation, Glu17, is responsible for both the low malonyl-CoA sensitivity and high carnitine affinity characteristics of the pig enzyme. This is the first demonstration that a single naturally occurring amino acid variation can alter CPT1B enzymatic properties.
