Characterization of the amino-terminal activation domain of peroxisome proliferator-activated receptor alpha. Importance of alpha-helical structure in the transactivating function.
The transactivating function of the A/B region of mouse peroxisome proliferator-activated receptor alpha (PPARalpha; NR1C1) was characterized. The truncated version of PPARalpha lacking the A/B region had 60-70% lower transactivating function than full-length PPARalpha in both the presence and absence of the peroxisome proliferator ciprofibrate. When tethered to the yeast Gal4 DNA-binding domain, the A/B region exhibited the significant ligand-independent transactivating function, AF-1 activity. The first 44 amino acid residues were necessary for maximal transactivation, and the minimally essential region was further delimited to amino acids 15-44. This region is highly enriched with acidic residues, but mutational analyses showed that the protein structure, rather than the negative charge itself, was important for the AF-1 activity. An alpha-helical configuration was predicted for this region, and a CD spectrum analysis of the synthetic peptides showed that mutant sequences with higher AF-1 activity have higher helical contents and vice versa. The most active mutant, in which Met(31) was replaced with Leu, was approximately 5-fold more potent than the wild-type A/B region. These findings indicate that the AF-1 region of PPARalpha is an acidic activation domain and that the helix-forming property is implicated in the transactivating function.
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Department of Life Science, Faculty of Science, Himeji Institute of Technology, 3-2-1 Koto, Kamigori, Hyogo 678-1297, Japan.
Osada
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Yumoto
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Osumi
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T
D000595:Amino Acid Sequence; D002942:Circular Dichroism; D006367:HeLa Cells; D006801:Humans; D008156:Luciferases / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D017433:Protein Structure, Secondary; D018160:Receptors, Cytoplasmic and Nuclear / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014157:Transcription Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015533:Transcriptional Activation; D014162:Transfection
D010455:Peptides; D018160:Receptors, Cytoplasmic and Nuclear; D014157:Transcription Factors; D008156:Luciferases; D020928:Mitogen-Activated Protein Kinases