Critical Role of Residues Defining the Ligand Binding Pocket in Hepatocyte Nuclear Factor-4α

Aggelidou, Eleni; Ιορδανίδου, Παναγιώτα; Tsantili, Panayota; Παπαδόπουλος, Γεώργιος; Hadzopoulou-Cladaras, Margarita

doi:10.1074/jbc.m401120200

Cited by 11 publications

(37 citation statements)

References 32 publications

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“…A comparison of the HNF4α sequence from S. aurata to that reported for other vertebrates revealed a high degree of conservation among fish, amphibians, 24 birds, and mammals. All residues reported as being important for the structure and function of AF-1, AF-2 and LBD domains were fully conserved in S. aurata HNF4α (Green et al, 1998;Kistanova et al, 2001;Eeckhoute et al, 2003b;Aggelidou et al, 2004;Iordanidou et al, 2005). The results of the phylogenetic tree analysis are consistent with the taxonomic hierarchy.…”

Section: Discussionsupporting

confidence: 74%

Hepatocyte Nuclear Factor 4α Transactivates the Mitochondrial Alanine Aminotransferase Gene in the Kidney of Sparus aurata

et al. 2011

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Alanine aminotransferase (ALT) plays an important role in amino acid metabolism and gluconeogenesis. The preference of carnivorous fish for protein amino acids instead of carbohydrates as a source of energy lead us to study the transcriptional regulation of the mitochondrial ALT (mALT) gene and to characterize the enzyme kinetics and modulation of mALT expression in kidney of gilthead sea bream (Sparus aurata) under different nutritional and hormonal conditions. 5'-deletion analysis of mALT promoter in transiently transfected HEK293 cells, site-directed mutagenesis and electrophoretic mobility shift assays allowed us to identify HNF4α as a new factor involved in the transcriptional regulation of mALT expression. Quantitative RT-PCR assays showed that starvation and the administration of streptozotocin (STZ) decreased HNF4α levels in the kidney of S. aurata, leading to down-regulation of mALT transcription. Analysis of the tissue distribution showed that kidney, liver and intestine were the tissues with higher mALT and HNF4α expression. Kinetic analysis indicates that mALT enzyme is more efficient in catalyzing the conversion of L-alanine to pyruvate than the reverse reaction. From these results we conclude that HNF4α transactivates the mALT promoter and that the low levels of mALT expression found in the kidney of starved and STZ-treated fish result from a decreased expression of HNF4α. Our findings suggest that the mALT isoenzyme plays a major role in oxidazing dietary amino acids, and points to ALT as a target for a biotechnological action to spare protein and optimize the use of dietary nutrients for fish culture.3

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Section: Discussionsupporting

confidence: 74%

Hepatocyte Nuclear Factor 4α Transactivates the Mitochondrial Alanine Aminotransferase Gene in the Kidney of Sparus aurata

et al. 2011

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“…To investigate further the involvement of these amino acid residues in coactivator function, we used transient transfection analysis and interaction assays. We demonstrate that distinct residues in the LBD pocket, previously shown to control transcriptional activity (21), may be involved in coactivator and ligand interactions, although some residues may be critical for both functions.…”

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confidence: 76%

Section: Methodsmentioning

confidence: 99%

“…The pCMXGAL-LBD point mutations in residues Ser-181 and Met-182 in H3, Arg-212 in H4, Leu-219, Leu-220, and Arg-226 in H5, Ile-338 in H10, and Ile-346 in H11 were generated by PCR-mediated site-directed mutagenesis, as described previously (21). The pcDNA3.1-LBD mutants have also been described (21). The fulllength point mutants pcDNA3.1-LBD R168G/K170N/K171N and E363K were generated by the PCR-mediated site-directed mutagenesis method (21) by using forward primers 5Ј-GAC ATT GGG GCC AAT AAT ATT GCC 3Ј (R168G/K170N/K171N) and 5Ј-C CTG CTG CAG AAG ATG CTG CTT GG-3Ј (E363K) and reverse primers 5Ј-GGC AAT ATT ATT GGC CCC AAT GTC-3Ј (R168G/K170N/K171N) and 5Ј-CC AAG CAG CAT CTT CTG CAG CAG G-3Ј (E363K), respectively.…”

Section: Methodsmentioning

confidence: 99%

“…However, mutagenesis in the positively charged amino acid residues R168G/K170N/K171N that lie in the hinge (D) region had a small effect in HNF-4␣-mediated transactivation, which was enhanced in the presence of coactivator. Furthermore, in a recent study, we have shown that point mutations in residues that are located in helices H3, H5, H10, and H11, which come in contact with the ligand, cause a dramatic decrease in HNF-4␣ activity (21). Structural modeling showed that these residues play a significant role in maintaining the structural integrity of the HNF-4␣ LBD.…”

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confidence: 91%

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Distinct Amino Acid Residues May Be Involved in Coactivator and Ligand Interactions in Hepatocyte Nuclear Factor-4α

Ιορδανίδου

Aggelidou

Demetriades

et al. 2005

Journal of Biological Chemistry

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Hepatocyte nuclear factor-4 (HNF-4) is a transcription factor of the nuclear hormone receptor superfamily that is constitutively active without the addition of exogenous ligand. Crystallographic analysis of the HNF-4␣ and HNF-4␥ ligand binding domains (LBDs) demonstrated the presence of endogenous ligands that may act as structural cofactors for HNF-4. It was also proposed by crystallographic studies that a combination of ligand and coactivator might be required to lock the receptor in its active state. We previously showed that mutations in amino acid residues Ser-181 and Met-182 in H3, Leu-219 and Leu-220 and Arg-226 in H5, Ileu-338 in H10, and Ileu-346 in H11, which line the LBD pocket in HNF-4␣ and come in contact with the ligand, impair its transactivation potential. In the present study, physical and functional interaction assays were utilized with two different coactivators, PGC-1 and SRC-3, to address the role of coactivators in HNF-4 function. We show that the integrity of the hinge (D) domain of HNF-4␣ and the activation function (AF)-2 activation domain region are critical for coactivation. Surprisingly, a different mode of coactivation is observed among the LBD point mutants that lack transcriptional activity. In particular, coactivation is maintained in mutants Ser-181, Arg-226, and Ile-346 but is abolished in mutants Met-182, Leu-219, and Ile-338. Physical interactions confirm this pattern of activation, implying that distinct amino acid residues may be involved in coactivator and ligand interactions, although some residues may be critical for both functions. Our results provide evidence and expand predictions based on the crystallographic data as to the role of coactivators in HNF-4␣ constitutive transcriptional activity.

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Drosophila HNF4 Regulates Lipid Mobilization and β-Oxidation

et al. 2009

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Summary Drosophila HNF4 (dHNF4) is the single ancestral ortholog of a highly conserved subfamily of nuclear receptors that includes two mammalian receptors, HNFα and HNFγ, and 269 members in C. elegans. We show here that dHNF4 null mutant larvae are sensitive to starvation. Starved mutant larvae consume glycogen normally, but retain lipids in their midgut and fat body, and have increased levels of long chain fatty acids, suggesting that they are unable to efficiently mobilize stored fat for energy. Microarray studies support this model, indicating reduced expression of genes that control lipid catabolism and β-oxidation. A GAL4-dHNF4;UAS-lacZ ligand sensor can be activated by starvation or exogenous long chain fatty acids, suggesting that dHNF4 is responsive to dietary signals. Taken together, our results support a feed-forward model for dHNF4, in which fatty acids released from triglycerides activate the receptor, inducing enzymes that drive fatty acid oxidation for energy production.

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Critical Role of Residues Defining the Ligand Binding Pocket in Hepatocyte Nuclear Factor-4α

Cited by 11 publications

References 32 publications

Hepatocyte Nuclear Factor 4α Transactivates the Mitochondrial Alanine Aminotransferase Gene in the Kidney of Sparus aurata

Hepatocyte Nuclear Factor 4α Transactivates the Mitochondrial Alanine Aminotransferase Gene in the Kidney of Sparus aurata

Distinct Amino Acid Residues May Be Involved in Coactivator and Ligand Interactions in Hepatocyte Nuclear Factor-4α

Drosophila HNF4 Regulates Lipid Mobilization and β-Oxidation

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