TY - JOUR

T1 - Acyl-CoA Esters Antagonize the Effects of Ligands on Peroxisome Proliferator-activated Receptor a Conformation, DNA Binding, and Interaction with Co-factors

AU - Elholm, M

AU - Dam, I

AU - Jorgensen, C

AU - Krogsdam, A M

AU - Holst, D

AU - Kratchmarova, I

AU - Gottlicher, M

AU - Gustafsson, J A

AU - Berge, R

AU - Flatmark, T

AU - Knudsen, J

AU - Mandrup, S

AU - Kristiansen, K

N1 - Keywords: Acyl Coenzyme A; Acyl-CoA Oxidase; Animals; Cell Line; Chromatography, Affinity; Coenzyme A; DNA-Binding Proteins; Dimerization; Genes, Reporter; Glutathione Transferase; Histone Acetyltransferases; Ligands; Mice; Models, Molecular; Oxidoreductases; Protein Biosynthesis; Protein Conformation; Rats; Receptors, Cytoplasmic and Nuclear; Receptors, Retinoic Acid; Recombinant Proteins; Retinoid X Receptors; Spodoptera; Trans-Activators; Transcription Factors; Transcription, Genetic; Transfection

PY - 2001

Y1 - 2001

N2 - 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.

AB - 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.

U2 - 10.1074/jbc.M101073200

DO - 10.1074/jbc.M101073200

M3 - Journal article

C2 - 11279171

VL - 276

SP - 21410

EP - 21416

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 24

ER -