Acyl-CoA esters antagonize the effects of ligands on peroxisome proliferator-activated receptor alpha conformation, DNA binding, and interaction with Co-factors.
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.
Elholm
M
M
Department of Biochemistry, Center for Experimental BioInformatics, University of Southern Denmark, DK-5320 Odense M., Denmark.
Dam
I
I
Jorgensen
C
C
Krogsdam
A M
AM
Holst
D
D
Kratchmarova
I
I
Gottlicher
M
M
Gustafsson
J A
JA
Berge
R
R
Flatmark
T
T
Knudsen
J
J
Mandrup
S
S
Kristiansen
K
K
D000214:Acyl Coenzyme A / Q000494:pharmacology*; D042962:Acyl-CoA Oxidase; D000818:Animals; D002460:Cell Line; D002846:Chromatography, Affinity; D003065:Coenzyme A / Q000494:pharmacology*; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism*; D019281:Dimerization; D017930:Genes, Reporter; D005982:Glutathione Transferase / Q000235:genetics; D051548:Histone Acetyltransferases; D008024:Ligands; D051379:Mice; D008958:Models, Molecular; D056920:Nuclear Receptor Coactivator 1; D010088:Oxidoreductases / Q000737:chemistry / Q000378:metabolism; D014176:Protein Biosynthesis; D011487:Protein Conformation; D051381:Rats; D018160:Receptors, Cytoplasmic and Nuclear / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism*; D018168:Receptors, Retinoic Acid / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D047488:Retinoid X Receptors; D018411:Spodoptera; D015534:Trans-Activators / Q000378:metabolism; D014157:Transcription Factors / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism*; D014158:Transcription, Genetic; D014162:Transfection
D000214:Acyl Coenzyme A; D004268:DNA-Binding Proteins; D008024:Ligands; D018160:Receptors, Cytoplasmic and Nuclear; D018168:Receptors, Retinoic Acid; D011994:Recombinant Proteins; D047488:Retinoid X Receptors; C067607:S-(3-oxohexadecyl)-coenzyme A; D015534:Trans-Activators; D014157:Transcription Factors; D010088:Oxidoreductases; D042962:Acyl-CoA Oxidase; D051548:Histone Acetyltransferases; C538861:Ncoa1 protein, mouse; D056920:Nuclear Receptor Coactivator 1; D005982:Glutathione Transferase; D003065:Coenzyme A