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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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(A) Electrophoretic mobility shift assays of DNA bindingby eachindividual protein (hPPARα, hLXRα, and hRXRα), amixture of hPPARα and hLXRα proteins, and a mixture ofhPPARα and hLXRα proteins in the presence of anti-PPARαor anti-LXRα. The left side of the gel shows binding to thehACOX PPRE; the middle lane is a no DNA control, and the right sideof the gel shows binding to the hSREBP-1c LXRE. (B) Representativeelectrophoretic mobility shift assays showing DNA binding. The topgel shows PPRE binding for the hPPARα–hLXRα heterodimerin the presence of LCFA or clofibrate (agonist). The bottom gel showsLXRE binding for the hPPARα–hLXRα heterodimer inthe presence of LCFA or T0901317 (agonist). (C) Relative DNA bindingto a PPRE sequence by hPPARα–hLXRα heterodimersin the absence (none) or presence of LCFA. Values are presented relativeto binding to the same response element by hPPARα–hRXRαheterodimers in the presence of clofibrate, a known PPARα agonist.(D) Relative DNA binding to an LXRE sequence by hPPARα–hLXRαheterodimers in the absence (none) or presence of LCFA. Values arepresented relative to binding to the same response element by hLXRα–hRXRαheterodimers in the presence of T0901317, a known LXRα agonist.DNA binding was determined by electrophoretic mobility shift assays,and resulting bands were quantified by densitometry. Values representmeans ± the standard error (n = 4 or 5). Asterisksdenote significant differences due to the addition of ligand: *p < 0.05, and **p < 0.01.
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fig7: (A) Electrophoretic mobility shift assays of DNA bindingby eachindividual protein (hPPARα, hLXRα, and hRXRα), amixture of hPPARα and hLXRα proteins, and a mixture ofhPPARα and hLXRα proteins in the presence of anti-PPARαor anti-LXRα. The left side of the gel shows binding to thehACOX PPRE; the middle lane is a no DNA control, and the right sideof the gel shows binding to the hSREBP-1c LXRE. (B) Representativeelectrophoretic mobility shift assays showing DNA binding. The topgel shows PPRE binding for the hPPARα–hLXRα heterodimerin the presence of LCFA or clofibrate (agonist). The bottom gel showsLXRE binding for the hPPARα–hLXRα heterodimer inthe presence of LCFA or T0901317 (agonist). (C) Relative DNA bindingto a PPRE sequence by hPPARα–hLXRα heterodimersin the absence (none) or presence of LCFA. Values are presented relativeto binding to the same response element by hPPARα–hRXRαheterodimers in the presence of clofibrate, a known PPARα agonist.(D) Relative DNA binding to an LXRE sequence by hPPARα–hLXRαheterodimers in the absence (none) or presence of LCFA. Values arepresented relative to binding to the same response element by hLXRα–hRXRαheterodimers in the presence of T0901317, a known LXRα agonist.DNA binding was determined by electrophoretic mobility shift assays,and resulting bands were quantified by densitometry. Values representmeans ± the standard error (n = 4 or 5). Asterisksdenote significant differences due to the addition of ligand: *p < 0.05, and **p < 0.01.

Mentions: Electrophoretic mobilityshift assays were used to determine whether the hPPARα–hLXRαheterodimer could bind to either PPRE or LXRE sequences. As the RXRαhomodimer binds to both response elements,20,21 hRXRα binding to each response element was used as a positivecontrol. This binding resulted in the strongest band observed foreither response element (Figure 7A). PPARα(in the absence of RXRα or LXRα) showed no binding toeither response element. However, a very weak band was noted for LXRα(in the absence of RXRα or PPARα) binding to the PPRE,and a stronger band was noted for LXRE binding, suggesting that LXRαhomodimers may be able to bind to the LXRE. Although only weak bindingby the PPARα–LXRα heterodimer was noted for PPREbinding, LXRE binding was stronger (Figure 7A). Supershift assays were conducted to ensure that this observedbinding was due to the PPARα–LXRα heterodimer (andnot just LXRα binding). The addition of either a PPARαor LXRα antibody resulted in supershifted bands (Figure 7A). While the LXRα antibody resulted in asingle supershifted band, two bands were noted with the addition ofthe PPARα antibody: one shifted band and one supershifted band(Figure 7A). It is possible that the two bandsrepresent DNA bound by the PPARα–LXRα heterodimer(top band) and DNA bound by LXRα homodimers (lower band). AsPPARα is unable to bind either response element alone, thesedata further indicate DNA binding by the PPARα–LXRαheterodimer.


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)

(A) Electrophoretic mobility shift assays of DNA bindingby eachindividual protein (hPPARα, hLXRα, and hRXRα), amixture of hPPARα and hLXRα proteins, and a mixture ofhPPARα and hLXRα proteins in the presence of anti-PPARαor anti-LXRα. The left side of the gel shows binding to thehACOX PPRE; the middle lane is a no DNA control, and the right sideof the gel shows binding to the hSREBP-1c LXRE. (B) Representativeelectrophoretic mobility shift assays showing DNA binding. The topgel shows PPRE binding for the hPPARα–hLXRα heterodimerin the presence of LCFA or clofibrate (agonist). The bottom gel showsLXRE binding for the hPPARα–hLXRα heterodimer inthe presence of LCFA or T0901317 (agonist). (C) Relative DNA bindingto a PPRE sequence by hPPARα–hLXRα heterodimersin the absence (none) or presence of LCFA. Values are presented relativeto binding to the same response element by hPPARα–hRXRαheterodimers in the presence of clofibrate, a known PPARα agonist.(D) Relative DNA binding to an LXRE sequence by hPPARα–hLXRαheterodimers in the absence (none) or presence of LCFA. Values arepresented relative to binding to the same response element by hLXRα–hRXRαheterodimers in the presence of T0901317, a known LXRα agonist.DNA binding was determined by electrophoretic mobility shift assays,and resulting bands were quantified by densitometry. Values representmeans ± the standard error (n = 4 or 5). Asterisksdenote significant differences due to the addition of ligand: *p < 0.05, and **p < 0.01.
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Related In: Results  -  Collection

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fig7: (A) Electrophoretic mobility shift assays of DNA bindingby eachindividual protein (hPPARα, hLXRα, and hRXRα), amixture of hPPARα and hLXRα proteins, and a mixture ofhPPARα and hLXRα proteins in the presence of anti-PPARαor anti-LXRα. The left side of the gel shows binding to thehACOX PPRE; the middle lane is a no DNA control, and the right sideof the gel shows binding to the hSREBP-1c LXRE. (B) Representativeelectrophoretic mobility shift assays showing DNA binding. The topgel shows PPRE binding for the hPPARα–hLXRα heterodimerin the presence of LCFA or clofibrate (agonist). The bottom gel showsLXRE binding for the hPPARα–hLXRα heterodimer inthe presence of LCFA or T0901317 (agonist). (C) Relative DNA bindingto a PPRE sequence by hPPARα–hLXRα heterodimersin the absence (none) or presence of LCFA. Values are presented relativeto binding to the same response element by hPPARα–hRXRαheterodimers in the presence of clofibrate, a known PPARα agonist.(D) Relative DNA binding to an LXRE sequence by hPPARα–hLXRαheterodimers in the absence (none) or presence of LCFA. Values arepresented relative to binding to the same response element by hLXRα–hRXRαheterodimers in the presence of T0901317, a known LXRα agonist.DNA binding was determined by electrophoretic mobility shift assays,and resulting bands were quantified by densitometry. Values representmeans ± the standard error (n = 4 or 5). Asterisksdenote significant differences due to the addition of ligand: *p < 0.05, and **p < 0.01.
Mentions: Electrophoretic mobilityshift assays were used to determine whether the hPPARα–hLXRαheterodimer could bind to either PPRE or LXRE sequences. As the RXRαhomodimer binds to both response elements,20,21 hRXRα binding to each response element was used as a positivecontrol. This binding resulted in the strongest band observed foreither response element (Figure 7A). PPARα(in the absence of RXRα or LXRα) showed no binding toeither response element. However, a very weak band was noted for LXRα(in the absence of RXRα or PPARα) binding to the PPRE,and a stronger band was noted for LXRE binding, suggesting that LXRαhomodimers may be able to bind to the LXRE. Although only weak bindingby the PPARα–LXRα heterodimer was noted for PPREbinding, LXRE binding was stronger (Figure 7A). Supershift assays were conducted to ensure that this observedbinding was due to the PPARα–LXRα heterodimer (andnot just LXRα binding). The addition of either a PPARαor LXRα antibody resulted in supershifted bands (Figure 7A). While the LXRα antibody resulted in asingle supershifted band, two bands were noted with the addition ofthe PPARα antibody: one shifted band and one supershifted band(Figure 7A). It is possible that the two bandsrepresent DNA bound by the PPARα–LXRα heterodimer(top band) and DNA bound by LXRα homodimers (lower band). AsPPARα is unable to bind either response element alone, thesedata further indicate DNA binding by the PPARα–LXRαheterodimer.

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