The peroxisome proliferator-activated receptor alpha (PPARalpha) is a ligand-inducible transcription factor, which belongs to the nuclear receptor superfamily. PPARalpha mediates the carcinogenic effects of peroxisome proliferators in rodents. In humans, PPARalpha plays a fundamental role in regulating energy homeostasis via control of lipid metabolism. To study the possible role of chaperone proteins in the regulation of PPARalpha activity, a monoclonal antibody (mAb) was made against PPARalpha and designated as 3B6/PPAR. The specificity of mAb 3B6/PPAR in recognizing PPARalpha was tested in immunoprecipitations using in vitro translated PPAR subtypes. The mAb 3B6/PPAR recognized PPARalpha, failed to bind to PPARbeta or PPARgamma, and is efficient in both immunoprecipitating and visualizing the receptor on protein blots. The immunoprecipitation of PPARalpha in mouse liver cytosol using mAb 3B6/PPAR has resulted in the detection of two co-immunoprecipitated proteins, which are heat shock protein 90 (hsp90) and the hepatitis B virus X-associated protein 2 (XAP2). The concomitant depletion of PPARalpha in hsp90-depleted mouse liver cytosol was also detected. Complex formation between XAP2 and PPARalpha/FLAG was also demonstrated in an in vitro translation binding assay. hsp90 interacts with PPARalpha in a mammalian two-hybrid assay and binds to the E/F domain. Transient expression of XAP2 co-expressed with PPARalpha resulted in down-regulation of a peroxisome proliferator response element-driven reporter gene activity. Taken together, these results indicate that PPARalpha is in a complex with hsp90 and XAP2, and XAP2 appears to function as a repressor. This is the first demonstration that PPARalpha is stably associated with other proteins in tissue extracts and the first nuclear receptor shown to functionally interact with XAP2.
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