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Ligand-regulated heterodimerization of peroxisome proliferator-activated receptor α with liver X receptor α.

Balanarasimha M, Davis AM, Soman FL, Rider SD, Hostetler HA - Biochemistry (2014)

Bottom Line: We demonstrated for the first time that the affinity of this interaction and the resulting conformational changes could be altered by endogenous PPARα ligands, namely long chain fatty acids (LCFA) or their coenzyme A thioesters.LCFA had little effect on binding to the PPRE but suppressed binding to the LXRE.Overexpression of both receptors also resulted in transactivation from an LXRE, with decreased levels compared to that of LXRα overexpression alone, and LCFA suppressed transactivation from the LXRE.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Boonshoft School of Medicine, Wright State University , Dayton, Ohio 45435, United States.

ABSTRACT
Peroxisome proliferator-activated receptor α (PPARα) and liver X receptor α (LXRα) are members of the nuclear receptor superfamily that function to regulate lipid metabolism. Complex interactions between the LXRα and PPARα pathways exist, including competition for the same heterodimeric partner, retinoid X receptor α (RXRα). Although data have suggested that PPARα and LXRα may interact directly, the role of endogenous ligands in such interactions has not been investigated. Using in vitro protein-protein binding assays, circular dichroism, and co-immunoprecipitation of endogenous proteins, we established that full-length human PPARα and LXRα interact with high affinity, resulting in altered protein conformations. We demonstrated for the first time that the affinity of this interaction and the resulting conformational changes could be altered by endogenous PPARα ligands, namely long chain fatty acids (LCFA) or their coenzyme A thioesters. This heterodimer pair was capable of binding to PPARα and LXRα response elements (PPRE and LXRE, respectively), albeit with an affinity lower than that of the respective heterodimers formed with RXRα. LCFA had little effect on binding to the PPRE but suppressed binding to the LXRE. Ectopic expression of PPARα and LXRα in mammalian cells yielded an increased level of PPRE transactivation compared to overexpression of PPARα alone and was largely unaffected by LCFA. Overexpression of both receptors also resulted in transactivation from an LXRE, with decreased levels compared to that of LXRα overexpression alone, and LCFA suppressed transactivation from the LXRE. These data are consistent with the hypothesis that ligand binding regulates heterodimer choice and downstream gene regulation by these nuclear receptors.

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Fluorescent protein–protein binding assays withlabeledprotein titrated against increasing concentrations of unlabeled protein.(A) Change in the fluorescence intensity of 25 nM Cy5-labeled hPPARαtitrated with increasing hLXRα concentrations of 0–250nM. (B) Change in the fluorescence intensity of 25 nM Cy3-labeledhLXRα titrated with increasing concentrations of hPPARα.(C) Change in the fluorescence intensity of 25 nM Cy5-labeled hPPARαtitrated with increasing hGR concentrations of 0–250 nM asa control. Insets represent double-reciprocal linear plots of eachbinding curve. Values represent means ± the standard error (n = 3–6).
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fig3: Fluorescent protein–protein binding assays withlabeledprotein titrated against increasing concentrations of unlabeled protein.(A) Change in the fluorescence intensity of 25 nM Cy5-labeled hPPARαtitrated with increasing hLXRα concentrations of 0–250nM. (B) Change in the fluorescence intensity of 25 nM Cy3-labeledhLXRα titrated with increasing concentrations of hPPARα.(C) Change in the fluorescence intensity of 25 nM Cy5-labeled hPPARαtitrated with increasing hGR concentrations of 0–250 nM asa control. Insets represent double-reciprocal linear plots of eachbinding curve. Values represent means ± the standard error (n = 3–6).

Mentions: As the CD spectrum showsonly a change in conformation, protein–protein binding experimentswere conducted to determine the affinity of hPPARα for hLXRα.Each protein was fluorescently labeled with either Cy3 or Cy5 dyeat essentially one dye per protein molecule. Upon titration of Cy5-labeledhPPARα protein with nonfluorescent hLXRα, the fluorescenceintensity decreased, suggesting either a conformational change inCy5-hPPARα or quenching of the Cy5 fluorophore upon hLXRαbinding. This change in fluorescence intensity plotted as a functionof hLXRα concentration resulted in a strongly saturable bindingcurve [Kd = 6 ± 2 nM (Figure 3A)]. Transformation of these values into a double-reciprocalplot resulted in a single straight line, suggesting a single bindingsite (Figure 3A, inset). To ensure that thefluorophore did not alter protein–protein binding, the reverseexperiment was conducted. Upon titration of Cy3-labeled hLXRαwith nonfluorescent hPPARα, the fluorescence intensity increased.This change in fluorescence intensity plotted as a function of hPPARαconcentration also resulted in a saturable binding curve (Figure 3B) at a single binding site (inset), but with slightlyweaker affinity (Kd = 42 ± 16 nM).To determine whether this binding was specific for LXRα andPPARα, Cy5-labeled hLXRα was titrated with nonfluorescenthGR (Figure 3C); however, the shape of thecurve was nonsaturable and almost linear, suggesting only weak ornonspecific binding.


Ligand-regulated heterodimerization of peroxisome proliferator-activated receptor α with liver X receptor α.

Balanarasimha M, Davis AM, Soman FL, Rider SD, Hostetler HA - Biochemistry (2014)

Fluorescent protein–protein binding assays withlabeledprotein titrated against increasing concentrations of unlabeled protein.(A) Change in the fluorescence intensity of 25 nM Cy5-labeled hPPARαtitrated with increasing hLXRα concentrations of 0–250nM. (B) Change in the fluorescence intensity of 25 nM Cy3-labeledhLXRα titrated with increasing concentrations of hPPARα.(C) Change in the fluorescence intensity of 25 nM Cy5-labeled hPPARαtitrated with increasing hGR concentrations of 0–250 nM asa control. Insets represent double-reciprocal linear plots of eachbinding curve. Values represent means ± the standard error (n = 3–6).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4007980&req=5

fig3: Fluorescent protein–protein binding assays withlabeledprotein titrated against increasing concentrations of unlabeled protein.(A) Change in the fluorescence intensity of 25 nM Cy5-labeled hPPARαtitrated with increasing hLXRα concentrations of 0–250nM. (B) Change in the fluorescence intensity of 25 nM Cy3-labeledhLXRα titrated with increasing concentrations of hPPARα.(C) Change in the fluorescence intensity of 25 nM Cy5-labeled hPPARαtitrated with increasing hGR concentrations of 0–250 nM asa control. Insets represent double-reciprocal linear plots of eachbinding curve. Values represent means ± the standard error (n = 3–6).
Mentions: As the CD spectrum showsonly a change in conformation, protein–protein binding experimentswere conducted to determine the affinity of hPPARα for hLXRα.Each protein was fluorescently labeled with either Cy3 or Cy5 dyeat essentially one dye per protein molecule. Upon titration of Cy5-labeledhPPARα protein with nonfluorescent hLXRα, the fluorescenceintensity decreased, suggesting either a conformational change inCy5-hPPARα or quenching of the Cy5 fluorophore upon hLXRαbinding. This change in fluorescence intensity plotted as a functionof hLXRα concentration resulted in a strongly saturable bindingcurve [Kd = 6 ± 2 nM (Figure 3A)]. Transformation of these values into a double-reciprocalplot resulted in a single straight line, suggesting a single bindingsite (Figure 3A, inset). To ensure that thefluorophore did not alter protein–protein binding, the reverseexperiment was conducted. Upon titration of Cy3-labeled hLXRαwith nonfluorescent hPPARα, the fluorescence intensity increased.This change in fluorescence intensity plotted as a function of hPPARαconcentration also resulted in a saturable binding curve (Figure 3B) at a single binding site (inset), but with slightlyweaker affinity (Kd = 42 ± 16 nM).To determine whether this binding was specific for LXRα andPPARα, Cy5-labeled hLXRα was titrated with nonfluorescenthGR (Figure 3C); however, the shape of thecurve was nonsaturable and almost linear, suggesting only weak ornonspecific binding.

Bottom Line: We demonstrated for the first time that the affinity of this interaction and the resulting conformational changes could be altered by endogenous PPARα ligands, namely long chain fatty acids (LCFA) or their coenzyme A thioesters.LCFA had little effect on binding to the PPRE but suppressed binding to the LXRE.Overexpression of both receptors also resulted in transactivation from an LXRE, with decreased levels compared to that of LXRα overexpression alone, and LCFA suppressed transactivation from the LXRE.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Boonshoft School of Medicine, Wright State University , Dayton, Ohio 45435, United States.

ABSTRACT
Peroxisome proliferator-activated receptor α (PPARα) and liver X receptor α (LXRα) are members of the nuclear receptor superfamily that function to regulate lipid metabolism. Complex interactions between the LXRα and PPARα pathways exist, including competition for the same heterodimeric partner, retinoid X receptor α (RXRα). Although data have suggested that PPARα and LXRα may interact directly, the role of endogenous ligands in such interactions has not been investigated. Using in vitro protein-protein binding assays, circular dichroism, and co-immunoprecipitation of endogenous proteins, we established that full-length human PPARα and LXRα interact with high affinity, resulting in altered protein conformations. We demonstrated for the first time that the affinity of this interaction and the resulting conformational changes could be altered by endogenous PPARα ligands, namely long chain fatty acids (LCFA) or their coenzyme A thioesters. This heterodimer pair was capable of binding to PPARα and LXRα response elements (PPRE and LXRE, respectively), albeit with an affinity lower than that of the respective heterodimers formed with RXRα. LCFA had little effect on binding to the PPRE but suppressed binding to the LXRE. Ectopic expression of PPARα and LXRα in mammalian cells yielded an increased level of PPRE transactivation compared to overexpression of PPARα alone and was largely unaffected by LCFA. Overexpression of both receptors also resulted in transactivation from an LXRE, with decreased levels compared to that of LXRα overexpression alone, and LCFA suppressed transactivation from the LXRE. These data are consistent with the hypothesis that ligand binding regulates heterodimer choice and downstream gene regulation by these nuclear receptors.

Show MeSH
Related in: MedlinePlus