Polycyclic aromatic hydrocarbons modulate the activity of Atlantic cod (Gadus morhua) vitamin D receptor paralogs in vitro

© The Author(s), 2021. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Goksoyr, S. O., Goldstone, J., Lille-Langoy, R., Lock, E.-J., Olsvik, P. A., Goksoyr, A., & Karlsen, O. A. Polycyclic aromatic hydrocarbons modulate the activity of Atlantic cod (Gadus morhua) vitamin D receptor paralogs in vitro. Aquatic Toxicology, 238, (2021): 105914, https://doi.org/10.1016/j.aquatox.2021.105914.Vitamin D receptor (VDR) mediates the biological function of the steroid hormone calcitriol, which is the metabolically active version of vitamin D. Calcitriol is important for a wide array of physiological functions, including calcium and phosphate homeostasis. In contrast to mammals, which harbor one VDR encoding gene, teleosts possess two orthologous vdr genes encoding Vdr alpha (Vdra) and Vdr beta (Vdrb). Genome mining identified the vdra and vdrb paralogs in the Atlantic cod (Gadus morhua) genome, which were further characterized regarding their phylogeny, tissue-specific expression, and transactivational properties induced by calcitriol. In addition, a selected set of polycyclic aromatic hydrocarbons (PAHs), including naphthalene, phenanthrene, fluorene, pyrene, chrysene, benzo[a]pyrene (BaP), and 7-methylbenzo[a]pyrene, were assessed for their ability to modulate the transcriptional activity of gmVdra and gmVdrb in vitro. Both gmVdra and gmVdrb were activated by calcitriol with similar potencies, but gmVdra produced significantly higher maximal fold activation. Notably, none of the tested PAHs showed agonistic properties towards the Atlantic cod Vdrs. However, binary exposures of calcitriol together with phenanthrene, fluorene, or pyrene, antagonized the activation of gmVdra, while chrysene and BaP significantly potentiated the calcitriol-mediated activity of both receptors. Homology modeling, solvent mapping, and docking analyses complemented the experimental data, and revealed a putative secondary binding site in addition to the canonical ligand-binding pocket (LBP). Calcitriol was predicted to interact with both binding sites, whereas PAHs docked primarily to the LBP. Importantly, our in vitro data suggest that PAHs can interact with the paralogous gmVdrs and interfere with their transcriptional activities, and thus potentially modulate the vitamin D signaling pathway and contribute to adverse effects of crude oil and PAH exposures on cardiac development and bone deformities in fish.This study was funded by the Research Council of Norway through the ”iCod 2.0: Integrative environmental genomics of Atlantic cod” project (project no. 244564) and the ”dCod 1.0: decoding systems toxicology of Atlantic cod” project (Center for Digital Life Norway project no. 248840)

Conservation and divergence of chemical defense system in the tunicate Oikopleura dioica revealed by genome wide response to two xenobiotics

<p>Abstract</p> <p>Background</p> <p>Animals have developed extensive mechanisms of response to xenobiotic chemical attacks. Although recent genome surveys have suggested a broad conservation of the chemical defensome across metazoans, global gene expression responses to xenobiotics have not been well investigated in most invertebrates. Here, we performed genome survey for key defensome genes in <it>Oikopleura dioica </it>genome, and explored genome-wide gene expression using high density tiling arrays with over 2 million probes, in response to two model xenobiotic chemicals - the carcinogenic polycyclic aromatic hydrocarbon benzo[a]pyrene (BaP) the pharmaceutical compound Clofibrate (Clo).</p> <p>Results</p> <p><it>Oikopleura </it>genome surveys for key genes of the chemical defensome suggested a reduced repertoire. Not more than 23 cytochrome P450 (CYP) genes could be identified, and neither CYP1 family genes nor their transcriptional activator AhR was detected. These two genes were present in deuterostome ancestors. As in vertebrates, the genotoxic compound BaP induced xenobiotic biotransformation and oxidative stress responsive genes. Notable exceptions were genes of the aryl hydrocarbon receptor (AhR) signaling pathway. Clo also affected the expression of many biotransformation genes and markedly repressed genes involved in energy metabolism and muscle contraction pathways.</p> <p>Conclusions</p> <p><it>Oikopleura </it>has the smallest number of CYP genes among sequenced animal genomes and lacks the AhR signaling pathway. However it appears to have basic xenobiotic inducible biotransformation genes such as a conserved genotoxic stress response gene set. Our genome survey and expression study does not support a role of AhR signaling pathway in the chemical defense of metazoans prior to the emergence of vertebrates.</p

The Surface-Associated and Secreted MopE Protein of Methylococcus capsulatus (Bath) Responds to Changes in the Concentration of Copper in the Growth Medium

Expression of surface-associated and secreted protein MopE of the methanotrophic bacterium Methylococcus capsulatus (Bath) in response to the concentration of copper ions in the growth medium was investigated. The level of protein associated with the cells and secreted to the medium changed when the copper concentration in the medium varied and was highest in cells exposed to copper stress

The Methylococcus capsulatus (Bath) Secreted Protein, MopE*, Binds Both Reduced and Oxidized Copper

Under copper limiting growth conditions the methanotrophic bacterium Methylococcus capsulatus (Bath) secrets essentially only one protein, MopE*, to the medium. MopE* is a copper-binding protein whose structure has been determined by X-ray crystallography. The structure of MopE* revealed a unique high affinity copper binding site consisting of two histidine imidazoles and one kynurenine, the latter an oxidation product of Trp130. In this study, we demonstrate that the copper ion coordinated by this strong binding site is in the Cu(I) state when MopE* is isolated from the growth medium of M. capsulatus. The conclusion is based on X-ray Near Edge Absorption spectroscopy (XANES), and Electron Paramagnetic Resonance (EPR) studies. EPR analyses demonstrated that MopE*, in addition to the strong copper-binding site, also binds Cu(II) at two weaker binding sites. Both Cu(II) binding sites have properties typical of non-blue type II Cu (II) centres, and the strongest of the two Cu(II) sites is characterised by a relative high hyperfine coupling of copper (A(parallel to) = 20 mT). Immobilized metal affinity chromatography binding studies suggests that residues in the N-terminal part of MopE* are involved in forming binding site(s) for Cu(II) ions. Our results support the hypothesis that MopE plays an important role in copper uptake, possibly making use of both its high (Cu(I) and low Cu(II) affinity properties

X-ray Absorption Near Edge Structure analysis of endogenously expressed CorA.

<p>XANES spectrum of crystalline CorA, recorded in steps of 0.7-edge feature at 8984 eV, typical for Cu(I) compounds (marked with an arrowhead).</p

MALDI-TOF MS and MS/MS analyses of the PA-gel excised 26 kDa polypeptide.

<p>a) MALDI-TOF MS spectrum of trypsin-produced peptides derived from the 26 kDa polypeptide excised from the outer membrane fraction of low-copper grown <i>M</i>. <i>album</i> BG8. Monoistopic peaks are labeled with their respective <i>m/z</i> ratio. b) MALDI-TOF MS/MS spectrum of the <i>m/z</i> 2625.37 ion, indicating the observed fragmentation pattern and the sequence ion assignment.</p

Overall view and copper-binding site of CorA.

<p>a) Stereo plot of CorA, illustrating the 15 β-strands, Cu(I)(yellow sphere), Ca²⁺ (green sphere), and residues coordinating the metal ions (ball-and-stick models). b) Superimposition of CorA (red) and MopE* (blue). The copper and calcium in CorA are illustrated as yellow and green spheres, respectively. The metals in MopE* are coloured blue. Because of the superposition calcium appears as blue and copper as yellow. The 15 β-strands of CorA superimpose well on 15 of the 21 strands found in MopE*. Also the residues involved in copper and calcium binding superimpose well. c) Stereo plot illustrating the 2foDfc electron density, contoured at 1 σ in the copper binding site, demonstrating the oxidation of tryptophan to kynurenine at residue 62. The copper and the coordinating water are illustrated as yellow and red spheres, respectively. The polyethylene glycol molecule shown is found near the kynurenine residue in all CorA molecules in the crystal structure.</p

SDS-PAGE analyses of protein fractions obtained from NaCl extraction of a) the outer membrane and b) of whole fermentor-grown cells.

<p>12.5% PA-gels were used and stained with Coomassie Brilliant Blue R-250. a) Lane 1, <i>M. album</i> BG8 outer membrane fraction; lane 2, Tris-HCl wash of low ionic strength; lane 3, 1 M NaCl extract of the outer membrane fraction; lane 4, outer membrane fraction after treatment with 1 M NaCl. b) Lane 1, <i>M. album</i> BG8 cells resuspended in buffer of low ionic strength; lane 2, Tris-HCl wash of low ionic strength; lane 3, 1 M NaCl extract of whole cells; lane 4, whole cells after treatment with 1 M NaCl. CorA is indicated in both a) and b). Molecular mass markers are indicated.</p