@article{kollitz_zhang_hawkins_whitfield_reif_kullman_2016, title={Evolutionary and Functional Diversification of the Vitamin D Receptor-Lithocholic Acid Partnership}, volume={11}, ISSN={["1932-6203"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-85005987339&partnerID=MN8TOARS}, DOI={10.1371/journal.pone.0168278}, abstractNote={The evolution, molecular behavior, and physiological function of nuclear receptors are of particular interest given their diverse roles in regulating essential biological processes. The vitamin D receptor (VDR) is well known for its canonical roles in calcium homeostasis and skeletal maintenance. Additionally, VDR has received an increased amount of attention due to the discovery of numerous non-calcemic functions, including the detoxification of lithocholic acid. Lithocholic acid is a toxic metabolite of chenodeoxycholic acid, a primary bile acid. The partnership between the VDR and lithocholic acid has been hypothesized to be a recent adaptation that evolved to mediate the detoxification and elimination of lithocholic acid from the gut. This partnership is speculated to be limited to higher vertebrates (birds and mammals), as lower vertebrates do not synthesize the parent compound of lithocholic acid. However, the molecular functions associated with the observed insensitivity of basal VDRs to lithocholic acid have not been explored. Here we characterize canonical nuclear receptor functions of VDRs from select species representing key nodes in vertebrate evolution and span a range of bile salt phenotypes. Competitive ligand binding assays revealed that the receptor’s affinity for lithocholic acid is highly conserved across species, suggesting that lithocholic acid affinity is an ancient and non-adaptive trait. However, transient transactivation assays revealed that lithocholic acid-mediated VDR activation might have evolved more recently, as the non-mammalian receptors did not respond to lithocholic acid unless exogenous coactivator proteins were co-expressed. Subsequent functional assays indicated that differential lithocholic acid-mediated receptor activation is potentially driven by differential protein-protein interactions between VDR and nuclear receptor coregulator proteins. We hypothesize that the vitamin D receptor-lithocholic acid partnership evolved as a by-product of natural selection on the ligand-receptor partnership between the vitamin D receptor and the native VDR ligand: 1α,25-dihydroxyvitamin D3, the biologically active metabolite of vitamin D3.}, number={12}, journal={PLOS ONE}, publisher={Public Library of Science (PLoS)}, author={Kollitz, Erin M. and Zhang, Guozhu and Hawkins, Mary Beth and Whitfield, G. Kerr and Reif, David M. and Kullman, Seth W.}, editor={Zhang, ChiEditor}, year={2016}, month={Dec} }
 @article{kollitz_zhang_hawkins_whitfield_reif_kullman_2015, title={Molecular Cloning, Functional Characterization, and Evolutionary Analysis of Vitamin D Receptors Isolated from Basal Vertebrates}, volume={10}, ISSN={["1932-6203"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-84929469049&partnerID=MN8TOARS}, DOI={10.1371/journal.pone.0122853}, abstractNote={The vertebrate genome is a result of two rapid and successive rounds of whole genome duplication, referred to as 1R and 2R. Furthermore, teleost fish have undergone a third whole genome duplication (3R) specific to their lineage, resulting in the retention of multiple gene paralogs. The more recent 3R event in teleosts provides a unique opportunity to gain insight into how genes evolve through specific evolutionary processes. In this study we compare molecular activities of vitamin D receptors (VDR) from basal species that diverged at key points in vertebrate evolution in order to infer derived and ancestral VDR functions of teleost paralogs. Species include the sea lamprey (Petromyzon marinus), a 1R jawless fish; the little skate (Leucoraja erinacea), a cartilaginous fish that diverged after the 2R event; and the Senegal bichir (Polypterus senegalus), a primitive 2R ray-finned fish. Saturation binding assays and gel mobility shift assays demonstrate high affinity ligand binding and classic DNA binding characteristics of VDR has been conserved across vertebrate evolution. Concentration response curves in transient transfection assays reveal EC50 values in the low nanomolar range, however maximum transactivational efficacy varies significantly between receptor orthologs. Protein-protein interactions were investigated using co-transfection, mammalian 2-hybrid assays, and mutations of coregulator activation domains. We then combined these results with our previous study of VDR paralogs from 3R teleosts into a bioinformatics analysis. Our results suggest that 1, 25D3 acts as a partial agonist in basal species. Furthermore, our bioinformatics analysis suggests that functional differences between VDR orthologs and paralogs are influenced by differential protein interactions with essential coregulator proteins. We speculate that we may be observing a change in the pharmacodynamics relationship between VDR and 1, 25D3 throughout vertebrate evolution that may have been driven by changes in protein-protein interactions between VDR and essential coregulators.}, number={4}, journal={PLOS ONE}, author={Kollitz, Erin M. and Zhang, Guozhu and Hawkins, Mary Beth and Whitfield, G. Kerr and Reif, David M. and Kullman, Seth W.}, year={2015}, month={Apr} }
 @article{kollitz_hawkins_whitfield_kullmam_2014, title={Functional diversification of vitamin D receptor paralogs in teleost fish after a whole genome duplication event}, volume={155}, DOI={10.1210/en.2014-1505}, abstractNote={The diversity and success of teleost fishes (Actinopterygii) has been attributed to three successive rounds of whole-genome duplication (WGD). WGDs provide a source of raw genetic material for evolutionary forces to act upon, resulting in the divergence of genes with altered or novel functions. The retention of multiple gene pairs (paralogs) in teleosts provides a unique opportunity to study how genes diversify and evolve after a WGD. This study examines the hypothesis that vitamin D receptor (VDR) paralogs (VDRα and VDRβ) from two distantly related teleost orders have undergone functional divergence subsequent to the teleost-specific WGD. VDRα and VDRβ paralogs were cloned from the Japanese medaka (Beloniformes) and the zebrafish (Cypriniformes). Initial transactivation studies using 1α, 25-dihydroxyvitamin D3 revealed that although VDRα and VDRβ maintain similar ligand potency, the maximum efficacy of VDRβ was significantly attenuated compared with VDRα in both species. Subsequent analyses revealed that VDRα and VDRβ maintain highly similar ligand affinities; however, VDRα demonstrated preferential DNA binding compared with VDRβ. Protein-protein interactions between the VDR paralogs and essential nuclear receptor coactivators were investigated using transactivation and mammalian two-hybrid assays. Our results imply that functional differences between VDRα and VDRβ occurred early in teleost evolution because they are conserved between distantly related species. Our results further suggest that the observed differences may be associated with differential protein-protein interactions between the VDR paralogs and coactivators. We speculate that the observed functional differences are due to subtle ligand-induced conformational differences between the two paralogs, leading to divergent downstream functions.}, number={12}, journal={Endocrinology}, author={Kollitz, E. M. and Hawkins, M. B. and Whitfield, G. K. and Kullmam, Seth}, year={2014}, pages={4641–4654} }
 @article{krasowski_ai_hagey_kollitz_kullman_reschly_ekins_2011, title={The evolution of farnesoid X, vitamin D, and pregnane X receptors: insights from the green-spotted pufferfish (Tetraodon nigriviridis) and other non-mammalian species}, volume={12}, ISSN={["1471-2091"]}, DOI={10.1186/1471-2091-12-5}, abstractNote={The farnesoid X receptor (FXR), pregnane X receptor (PXR), and vitamin D receptor (VDR) are three closely related nuclear hormone receptors in the NR1H and 1I subfamilies that share the property of being activated by bile salts. Bile salts vary significantly in structure across vertebrate species, suggesting that receptors binding these molecules may show adaptive evolutionary changes in response. We have previously shown that FXRs from the sea lamprey (Petromyzon marinus) and zebrafish (Danio rerio) are activated by planar bile alcohols found in these two species. In this report, we characterize FXR, PXR, and VDR from the green-spotted pufferfish (Tetraodon nigriviridis), an actinopterygian fish that unlike the zebrafish has a bile salt profile similar to humans. We utilize homology modelling, docking, and pharmacophore studies to understand the structural features of the Tetraodon receptors.Tetraodon FXR has a ligand selectivity profile very similar to human FXR, with strong activation by the synthetic ligand GW4064 and by the primary bile acid chenodeoxycholic acid. Homology modelling and docking studies suggest a ligand-binding pocket architecture more similar to human and rat FXRs than to lamprey or zebrafish FXRs. Tetraodon PXR was activated by a variety of bile acids and steroids, although not by the larger synthetic ligands that activate human PXR such as rifampicin. Homology modelling predicts a larger ligand-binding cavity than zebrafish PXR. We also demonstrate that VDRs from the pufferfish and Japanese medaka were activated by small secondary bile acids such as lithocholic acid, whereas the African clawed frog VDR was not.Our studies provide further evidence of the relationship between both FXR, PXR, and VDR ligand selectivity and cross-species variation in bile salt profiles. Zebrafish and green-spotted pufferfish provide a clear contrast in having markedly different primary bile salt profiles (planar bile alcohols for zebrafish and sterically bent bile acids for the pufferfish) and receptor selectivity that matches these differences in endogenous ligands. Our observations to date present an integrated picture of the co-evolution of bile salt structure and changes in the binding pockets of three nuclear hormone receptors across the species studied.}, journal={BMC BIOCHEMISTRY}, author={Krasowski, Matthew D. and Ai, Ni and Hagey, Lee R. and Kollitz, Erin M. and Kullman, Seth W. and Reschly, Erica J. and Ekins, Sean}, year={2011}, month={Feb} }
 @article{howarth_hagey_law_ai_krasowski_ekins_moore_kollitz_hinton_kullmam_2010, title={Two farnesoid X receptor alpha isoforms in Japanese medaka (Oryzias latipes) are differentially activated in vitro}, volume={98}, DOI={10.1016/j.aquatox.2010.02.020}, abstractNote={The nuclear receptor farnesoid X receptor alpha (FXRα, NR1H4) is activated by bile acids in multiple species including mouse, rat, and human and in this study we have identified two isoforms of Fxrα in Japanese medaka (Oryzias latipes), a small freshwater teleost. Both isoforms share a high amino acid sequence identity to mammalian FXRα (∼70% in the ligand-binding domain). Fxrα1 and Fxrα2 differ within the AF1 domain due to alternative splicing at the fourth intron-exon boundary. This process results in Fxrα1 having an extended N-terminus compared to Fxrα2. A Gal4DBD-FxrαLBD fusion construct was activated by chenodeoxycholic, cholic, deoxycholic and lithocholic acids, and the synthetic agonist GW4064 in transient transactivation assays. Activation of the Gal4DBD-FxrαLBD fusion construct was enhanced by addition of PGC-1α, as demonstrated through titration assays. Surprisingly, when the full-length versions of the two Fxrα isoforms were compared in transient transfection assays, Fxrα2 was activated by C24 bile acids and GW4064, while Fxrα1 was not significantly activated by any of the compounds tested. Since the only significant difference between the full-length constructs was sequence in the AF1 domain, these experiments highlight a key functional region in the Fxrα AF1 domain. Furthermore, mammalian two-hybrid studies demonstrated the ability of Fxrα2, but not Fxrα1, to interact with PGC-1α and SRC-1, and supported our results from the transient transfection reporter gene activation assays. These data demonstrate that both mammalian and teleost FXR (Fxrα2 isoform) are activated by primary and secondary bile acids.}, number={3}, journal={Aquatic Toxicology (Amsterdam, Netherlands)}, author={Howarth, D. L. and Hagey, L. R. and Law, S. H. W. and Ai, N. and Krasowski, M. D. and Ekins, S. and Moore, J. T. and Kollitz, E. M. and Hinton, D. E. and Kullmam, Seth}, year={2010}, pages={245–255} }