Nuclear receptors are a family of transcription factors with structurally conserved ligand binding domains that regulate their activity. Despite intensive efforts to identify ligands, most nuclear receptors are still "orphans." Here, we demonstrate that the ligand binding pocket of the Drosophila nuclear receptor E75 contains a heme prosthetic group. E75 absorption spectra, resistance to denaturants, and effects of site-directed mutagenesis indicate a single, coordinately bound heme molecule. A correlation between the levels of E75 expression and the levels of available heme suggest a possible role as a heme sensor. The oxidation state of the heme iron also determines whether E75 can interact with its heterodimer partner DHR3, suggesting an additional role as a redox sensor. Further, the E75-DHR3 interaction is also regulated by the binding of NO or CO to the heme center, suggesting that E75 may also function as a diatomic gas sensor. Possible mechanisms and roles for these interactions are discussed.
Nuclear receptors are a large family of transcription factors that play major roles in development, metamorphosis, metabolism and disease. To determine how, where and when nuclear receptors are regulated by small chemical ligands and/or protein partners, we have used a 'ligand sensor' system to visualize spatial activity patterns for each of the 18 Drosophila nuclear receptors in live developing animals. Transgenic lines were established that express the ligand binding domain of each nuclear receptor fused to the DNA-binding domain of yeast GAL4. When combined with a GAL4-responsive reporter gene, the fusion proteins show tissue-and stage-specific patterns of activation. We show that these responses accurately reflect the presence of endogenous and exogenously added hormone, and that they can be modulated by nuclear receptor partner proteins. The amnioserosa, yolk, midgut and fat body, which play major roles in lipid storage, metabolism and developmental timing, were identified as frequent sites of nuclear receptor activity. We also see dynamic changes in activation that are indicative of sweeping changes in ligand and/or co-factor production. The screening of a small compound library using this system identified the angular psoralen angelicin and the insect growth regulator fenoxycarb as activators of the Ultraspiracle (USP) ligand-binding domain. These results demonstrate the utility of this system for the functional dissection of nuclear receptor pathways and for the development of new receptor agonists and antagonists that can be used to modulate metabolism and disease and to develop more effective means of insect control.
Most cases of cystic fibrosis (CF) are attributable to the F508del allele of CFTR, which causes the protein to be retained in the endoplasmic reticulum (ER) and subsequently degraded. One strategy for CF therapy is to identify corrector compounds that help traffic F508del-CFTR to the cell surface. Pharmacological chaperones, or correctors that bind specifically to F508del-CFTR and restore function, would be the most promising drug development candidates, but few pharmacological chaperones exist for F508del-CFTR. Using differential scanning fluorimetry (DSF), we have surveyed corrector compounds and identified one, RDR1, which binds directly to the first nucleotide binding domain (NBD1) of F508del-CFTR. We show that RDR1 treatment partially rescues F508del-CFTR function in both cells and in an F508del-CF mouse model. Thus, RDR1 is a pharmacological chaperone of F508del-CFTR and represents a novel scaffold for drug development.
Background: Defective folding and trafficking of -cell ATP-sensitive potassium (K ATP ) channels causes congenital hyperinsulinism. Results: Carbamazepine improves the processing and surface expression of trafficking-impaired K ATP channels harboring a subset of sulfonylurea receptor 1 mutations. Conclusion: Carbamazepine is a novel corrector of K ATP channels. Significance: Carbamazepine may be used to treat congenital hyperinsulinism caused by defective K ATP channel trafficking.
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