Selective effect of decreased expression of NCoR and SMRT on PPARα target genes in HuH-7 cells. HuH-7 cells were transfected with control, NCoR, or SMRT siRNA and treated with GW7647 (600 nm) or vehicle (MeSO₂). RNA was extracted, and the expression of NCoR (A), SMRT (B), l-CPT1 (C), PDK4 (D), and mitochondrial HMGCOAS2 (E) genes was measured by real-time quantitative PCR. Each bar is the mean value ± S.D. of triplicate determinations. Statistical differences are indicated (t test; Scramble versus siRNA Me₂SO: **, p < 0.01; ***, p < 0.001; ns, nonsignificant).

PIASy regulates SUMOylation of human PPARα and inhibits its transcriptional activity. A, COS-7 cells were co-transfected with pSG5 control vector or pSG5-hPPARα WT expression vector and with pSG5-SUMO-1-His₆, pSG5-Ubc9, and pcDNA3-FLAG-PIASy expression vectors as described. Empty vector pSG5 was used as negative control. After 48 h, cell extracts were incubated with Ni-NTA beads to isolate histidine-tagged SUMOylated proteins. The SUMOylated hPPARα proteins and hPPARα input proteins were analyzed by Western blotting using anti-PPARα antibodies. The SUMO-1-His₆ input proteins were analyzed by Western blotting using anti-His₆ antibodies. B, HuH-7 cells were co-transfected with pSG5 control vector, pSG5-hPPARα WT expression vectors and with increasing amounts of pcDNA3-FLAG-PIASy expression vectors or pcDNA3-FLAG as controls. After 24 h of transfection, the luciferase and β-galactosidase activities were measured in transfected cell lysates and the ratio luciferase activity/β-galactosidase activity was defined as RLU. Each bar is the mean value ± S.D. of triplicate determinations.

SUMOylation of hPPARα occurs on lysine 185. A, in vitro translated [³⁵S]methionine hPPARα WT, hPPARα K138R, hPPARα K185R, hPPARα K216R, hPPARα K310R, hPPARα K358R, hPPARα K449R proteins, and [³⁵S]methionine reticulocyte lysate were incubated with SUMO E1-activating enzyme, Ubc9, and SUMO-1 WT protein or unconjugatable SUMO-1 mutant protein provided with the in vitro SUMOlink® kit. Proteins were then separated by SDS-PAGE and analyzed by autoradiography. B, COS-7 cells were cotransfected with pSG5 control vector or pSG5-hPPARα WT or pSG5- hPPARα K185R expression vectors and with pSG5-SUMO-1-His₆, pSG5-Ubc9, and pcDNA3-FLAG-PIASy expression vectors or pSG5 vector and pcDNA3-FLAG as controls. After 48 h, COS-7 cells were scraped in ice-cold phosphate-buffered saline. One-tenth of cells were lysed in radioimmune precipitation assay buffer and used as input control. The remaining cells were lysed in denaturant binding buffer, and resulting cell extracts were incubated with Ni-NTA beads to isolate histidine-tagged SUMOylated proteins. The SUMOylated hPPARα proteins and hPPARα input proteins were analyzed by Western blotting using anti-PPARα antibodies. The SUMO-1-His₆ input proteins were analyzed by Western blotting using anti-His₆ antibodies.

The hPPARα K185R mutant displays a lower physical and functional interaction with NCoR compared with hPPARα WT. A, HuH-7 cells were transfected with the J6-TK-Luc, with pSV-β-galactosidase, with the pSG5 control vector, or the pSG5-hPPARα WT or pSG5-hPPARα K185R expression vectors, and with increasing amounts of VP16-AD or VP16-NCoR vectors. The luciferase and β-galactosidase activities were measured in transfected cell lysates, and the ratio luciferase activity/β-galactosidase activity was determined. Results are expressed in fold induction compared with VP16-AD control curves. Each bar is the mean value ± S.D. of triplicate determinations. Statistical differences are indicated (t test; without VP16-NCoR versus with VP16-NCoR: **, p < 0.01; ***, p < 0.001; hPPARα WT versus hPPARα K185R: §§, p < 0.01; §§§, p < 0.001). VP16-AD curves are not represented. B, HuH-7 cells were transfected with the J6-TK-Luc, with pSV-β-galactosidase, with the pSG5 control vector, or the pSG5-hPPARα WT or pSG5-hPPARα K185R expression vectors, and with increasing amounts of pKCR2 control vector or pKR2-NCoR full-length expression vector. The luciferase and β-galactosidase activities were measured in transfected cell lysates, and the ratio luciferase activity/β-galactosidase activity was determined. Then, pKCR2-NCoR curves were compared with their respective pKCR2 control curves, respectively. Results are expressed as relative inhibition. Each bar is the mean value ± S.D. of triplicate determinations. Statistical differences are indicated (t test; without pKR2-NCoR versus with pKR2-NCoR: *, p < 0.05; ns, nonsignificant). pKCR2 curves are not represented. C, HuH-7 cells were cotransfected with pEF-FLAG- hPPARα WT or K185R expression vectors and pKR2-NCoR. Flagged proteins were immunoprecipitated, and associated NCoR proteins were analyzed by Western blotting.

Comparison of the SUMOylation consensus site in mouse, rat, and human PPARα. PPARα primary protein sequences in mouse, rat, and human were compared by using BLASTP algorithm. (*) represents identical amino acids and (:) represents different amino acids.

Human PPARα is a substrate for SUMO-1 modification in vitro and in HuH-7 cells. A, protein sequence of hPPARα (NM Q07869) was analyzed with the SUMOylation prediction site algorithm SUMOplot^TM prediction. Numbers correspond to amino acids position. The A/B domain contains AF-1 ligand-independent transcriptional activity; C, DNA binding domain; D, hinge region; E, ligand binding domain containing AF-2 ligand-dependent transcriptional activity. B, [³⁵S]methionine hPPARα protein was incubated with GST or GST-Ubc9 proteins. Complexes were precipitated with glutathione-Sepharose, and proteins were analyzed by autoradiography. C, [³⁵S]methionine hPPARα WT protein or [³⁵S]methionine reticulocyte lysate (as control) were incubated with SUMO E1-activating enzyme, Ubc9, and SUMO-1 WT protein or unconjugatable SUMO-1 mutant protein provided by the SUMOlink® kit. Proteins were separated by SDS-PAGE and analyzed by autoradiography. D, HuH-7 cells were transfected with pSG5-hPPARα WT expression vector and/or pSG5-SUMO-1-His₆ expression vectors. After 24 h, cells were lysed in denaturating conditions with HCl-guanidinium. Lysates were incubated with Ni-NTA beads and subsequently eluted with loading buffer. Proteins were separated by SDS-PAGE and analyzed by Western blotting using anti-PPARα antibodies. E, HuH-7 cells were transfected with pSG5 control vector or pSG5-hPPARα WT expression vector, and with pSG5-SUMO-1-His₆ expression vectors. Cells were treated with Me₂SO as control or GW7647 (600 nm). After 24 h, a SUMOylation test was performed as described above. F, HuH-7 cells were transfected with pSG5-SUMO-1-His₆ expression vector and treated with GW7647 for 24 h. SUMOylated proteins were analyzed by Western blotting using an anti-His₆ antibodies.

Effect of SUMO pathway modulation on PPARα target genes in HuH-7 cells. HuH-7 cells were transfected with siRNA Ubc9 and/or siRNA hPPARα. Then RNA was extracted, and the expression of l-CPT1 (A), PDK4 (B), and mitochondrial HMGCOAS2 (C) genes was measured by real-time quantitative PCR. HuH-7 cells were cotransfected with pSG5-hPPARα WT and/or pcDNA3-FLAG-PIASy expression vectors or pSG5 vector and/or pcDNA3-FLAG as controls. After 24 h of transfection, cells were treated with Me₂SO or GW7647 (600 nm) in DMEM medium 0.2% fetal calf serum, 0.2% BSA, for 24 h. Then RNA was extracted and the expression of l-FABP (D), PDK4 (E), and mitochondrial HMGCOAS2 (F) was measured by real-time quantitative PCR. Each bar is the mean value ± S.D. of triplicate determinations. Statistical differences are indicated (t test; Control versus PIASy: *, p < 0.05; **, p < 0.01; ns, nonsignificant).

The transcriptional activity of hPPARα K185R is increased compared with hPPARα WT. A, HuH-7 cells were transfected with the pSG5 control vector, or the pSG5-hPPARα WT or pSG5-hPPARα K185R expression vectors, with the pSV-β-galactosidase, with the reporter vector J6-TK-Luc. After 24 h of transfection, cells were treated with Me₂SO or GW7647 (600 nm) in DMEM medium 0.2% SVF, 0.2% BSA for 24 h. The luciferase and β-galactosidase activities were measured in transfected cell lysates, and the ratio luciferase activity/β-galactosidase activity was defined as RLU. PPARα protein amounts were evaluated by Western blotting. Each bar is the mean value ± S.D. of triplicate determinations. Statistical differences are indicated (t test; PPARα WT versus PPARα K185R: ***, p < 0.001). B, PPARα protein from transfection assay cell lysates was analyzed by Western blotting using anti-PPARα antibodies.

The hPPARα WT and hPPARα K185R proteins display a similar physical and functional interaction profile with SMRT. A, HuH-7 cells were transfected with the J6-TK-Luc, with pSV-β-galactosidase, with the pSG5 control vector, or the pSG5-hPPARα WT or pSG5-hPPARα K185R expression vectors, and with increasing amounts of VP16-AD or VP16-SMRT vectors. The luciferase and β-galactosidase activities were measured in transfected cell lysates, and the ratio luciferase activity/β-galactosidase activity was determined. Results are expressed in fold induction compared with VP16-AD control curves. Each bar is the mean value ± S.D. of triplicate determinations. VP16-AD curve are not represented. B, HuH-7 cells were transfected with the J6-TK-Luc, with pSV-β-galactosidase, with the pSG5 control vector, or the pSG5-hPPARα WT or pSG5-hPPARα K185R expression vectors, and with increasing amounts of pCI control vector or pCI-SMRT expression vector. The luciferase and β-galactosidase activities were measured in transfected cell lysates, and the ratio luciferase activity/β-galactosidase activity was determined. Then, pCI-SMRT curves were compared with their respective pCI control curves. Results are expressed as relative inhibition. Each bar is the mean value ± S.D. of triplicate determinations. pCI curves are not represented.