A Multiplex Molecular Cell-Based Sensor to Detect Ligands of PPARs: An Optimized Tool for Drug Discovery in Cyanobacteria.

Cyanobacteria produce a wealth of secondary metabolites. Since these organisms attach fatty acids into molecules in unprecedented ways, cyanobacteria can serve as a novel source for bioactive compounds acting as ligands for Peroxisome Proliferator-Activated Receptors (PPAR). PPARs (PPARα, PPARß/δ and PPARγ) are ligand-activated nuclear receptors, involved in the regulation of various metabolic and cellular processes, thus serving as potential drug targets for a variety of pathologies. Yet, given that PPARs' agonists can have pan-, dual- or isoform-specific action, some controversy has been raised over currently approved drugs and their side effects, highlighting the need for novel molecules. Here, we expand and validate a cell-based PPAR transactivation activity biosensor, and test it in a screening campaign to guide drug discovery. Biosensor upgrades included the use of different reporter genes to increase signal intensity and stability, a different promoter to modulate reporter gene expression, and multiplexing to improve efficiency. Sensor's limit of detection (LOD) ranged from 0.36-0.89 nM in uniplex and 0.89-1.35 nM in multiplex mode. In triplex mode, the sensor's feature screening, a total of 848 fractions of 96 cyanobacteria extracts were screened. Hits were confirmed in multiplex mode and in uniplex mode, yielding one strain detected to have action on PPARα and three strains to have dual action on PPARα and -ß.

Antifouling Marine Coatings with a Potentially Safer and Sustainable Synthetic Polyphenolic Derivative.

The development of harmless substances to replace biocide-based coatings used to prevent or manage marine biofouling and its unwanted consequences is urgent. The formation of biofilms on submerged marine surfaces is one of the first steps in the marine biofouling process, which facilitates the further settlement of macrofoulers. Anti-biofilm properties of a synthetic polyphenolic compound, with previously described anti-settlement activity against macrofoulers, were explored in this work. In solution this new compound was able to prevent biofilm formation and reduce a pre-formed biofilm produced by the marine bacterium, Pseudoalteromonas tunicata. Then, this compound was applied to a marine coating and the formation of P. tunicata biofilms was assessed under hydrodynamic conditions to mimic the marine environment. For this purpose, polyurethane (PU)-based coating formulations containing 1 and 2 wt.% of the compound were prepared based on a prior developed methodology. The most effective formulation in reducing the biofilm cell number, biovolume, and thickness was the PU-based coating containing an aziridine-based crosslinker and 2 wt.% of the compound. To assess the marine ecotoxicity impact of this compound, its potential to disrupt endocrine processes was evaluated through the modulation of two nuclear receptors (NRs), peroxisome proliferator-activated receptor γ (PPARγ), and pregnane X receptor (PXR). Transcriptional activation of the selected NRs upon exposure to the polyphenolic compound (10 µM) was not observed, thus highlighting the eco-friendliness towards the addressed NRs of this new dual-acting anti-macro- and anti-microfouling agent towards the addressed NRs.

The Preservation of PPARγ Genome Duplicates in Some Teleost Lineages: Insights into Lipid Metabolism and Xenobiotic Exploitation.

Three peroxisome proliferator-activated receptor paralogues (PPARα, -ß and -γ) are currently recognized in vertebrate genomes. PPARγ is known to modulate nutrition, adipogenesis and immunity in vertebrates. Natural ligands of PPARγ have been proposed; however, the receptor also binds synthetic ligands such as endocrine disruptors. Two paralogues of PPARα and PPARß have been documented in teleost species, a consequence of the 3R WGD. Recently, two PPARγ paralogue genes were also identified in Astyanax mexicanus. We aimed to determine whether the presence of two PPARγ paralogues is prevalent in other teleost genomes, through genomic and phylogenetic analysis. Our results showed that besides Characiformes, two PPARγ paralogous genes were also identified in other teleost taxa, coinciding with the teleost-specific, whole-genome duplication and with the retention of both genes prior to the separation of the Clupeocephala. To functionally characterize these genes, we used the European sardine (Sardina pilchardus) as a model. PPARγA and PPARγB display a different tissue distribution, despite the similarity of their functional profiles: they are unresponsive to tested fatty acids and other human PPARγ ligands yet yield a transcriptional response in the presence of tributyltin (TBT). This observation puts forward the relevance of comparative analysis to decipher alternative binding architectures and broadens the disruptive potential of man-made chemicals for aquatic species.

An ancestral nuclear receptor couple, PPAR-RXR, is exploited by organotins.

Environmental chemicals have been reported to greatly disturb the endocrine and metabolic systems of multiple animal species. A recent example involves the exploitation of the nuclear receptor (NR) heterodimeric pair composed by PPAR/RXR (peroxisome proliferator-activated receptor/retinoid X receptor), which shows lipid perturbation in mammalian species. While gene orthologues of both of these receptors have been described outside vertebrates, no functional characterization of PPAR has been carried in protostome lineages. We provide the first functional analysis of PPAR in Patella sp. (Mollusca), using model obesogens such as tributyltin (TBT), triphenyltin (TPT), and proposed natural ligands (fatty acid molecules). To gain further insights, we used site-directed mutagenesis to PPAR and replaced the tyrosine 277 by a cysteine (the human homologous amino acid and TBT anchor residue) and an alanine. Additionally, we explored the alterations in the fatty acid profiles after an exposure to the model obesogen TBT, in vivo. Our results show that TBT and TPT behave as an antagonist of Patella sp. PPAR/RXR and that the tyrosine 277 is important, but not essential in the response to TBT. Overall, these results suggest a relation between the response of the mollusc PPAR-RXR to TBT and the lipid profile alterations reported at environmentally relevant concentrations. Our findings highlight the importance of comparative analysis between protostome and deuterostome lineages to decipher the differential impact of environmental chemicals.

The Echinodermata PPAR: Functional characterization and exploitation by the model lipid homeostasis regulator tributyltin.

The wide ecological relevance of lipid homeostasis modulators in the environment has been increasingly acknowledged. Tributyltin (TBT), for instance, was shown to cause lipid modulation, not only in mammals, but also in fish, molluscs, arthropods and rotifers. In vertebrates, TBT is known to interact with a nuclear receptor heterodimer module, formed by the retinoid X receptor (RXR) and the peroxisome proliferator-activated receptor (PPAR). These modulate the expression of genes involved in lipid homeostasis. In the present work, we isolated for the first time the complete coding region of the Echinodermata (Paracentrotus lividus) gene orthologues of PPAR and RXR and evaluated the ability of a model lipid homeostasis modulator, TBT, to interfere with the lipid metabolism in this species. Our results demonstrate that TBT alters the gonadal fatty acid composition and gene expression patterns: yielding sex-specific responses in fatty acid levels, including the decrease of eicosapentaenoic acid (C20:5 n-3, EPA) in males, and increase of arachidonic acid (20:4n-6, ARA) in females, and upregulation of long-chain acyl-CoA synthetase (acsl), ppar and rxr. Furthermore, an in vitro test using COS-1 cells as host and chimeric receptors with the ligand binding domain (LBD) of P. lividus PPAR and RXR shows that organotins (TBT and TPT (Triphenyltin)) suppressed activity of the heterodimer PPAR/RXR in a concentration-dependent manner. Together, these results suggest that TBT acts as a lipid homeostasis modulator at environmentally relevant concentrations in Echinodermata and highlight a possible conserved mode of action via the PPAR/RXR heterodimer.

Evolutionary Exploitation of Vertebrate Peroxisome Proliferator-Activated Receptor γ by Organotins.

Globally persistent man-made chemicals display ever-growing ecosystemic consequences, a hallmark of the Anthropocene epoch. In this context, the assessment of how lineage-specific gene repertoires influence organism sensitivity toward endocrine disruptors is a central question in toxicology. A striking example highlights the role of a group of compounds known as obesogens. In mammals, most examples involve the modulation of the nuclear receptor peroxisome proliferator-activated receptor γ (PPARγ). To address the structural and biological determinants of PPARγ exploitation by a model obesogen, tributyltin (TBT), in chordates, we employed comparative genomics, transactivation and ligand binding assays, homology modeling, and site-directed-mutagenesis. We show that the emergence of multiple PPARs (α, ß and γ) in vertebrate ancestry coincides with the acquisition of TBT agonist affinity, as can be deduced from the conserved transactivation and binding affinity of the chondrichthyan and mammalian PPARγ. The amphioxus single-copy PPAR is irresponsive to TBT; as well as the investigated teleosts, this is a probable consequence of a specific mutational remodeling of the ligand binding pocket. Our findings endorse the modulatory ability of man-made chemicals and suggest an evolutionarily diverse setting, with impacts for environmental risk assessment.

Cloning and functional characterization of a retinoid X receptor orthologue in Platynereis dumerilii: An evolutionary and toxicological perspective.

In the present work we provide the first isolation and functional characterization of a RXR orthologue in an annelid species, the Platynereis dumerilii. Using an in vitro luciferase reporter assay we evaluated the annelid receptor ability to respond to ligand 9-cis-retinoic acid, TBT and TPT. Our results show that the annelid RXR responds to 9-cis-retinoic acid and to the organotins by activating reporter gene transcription. The findings suggest a conserved mode of action of the receptor in response to a common signaling molecule and modulation by organotin compounds between vertebrates and Lophotrochozoans.

Genes belonging to the insulin and ecdysone signaling pathways can contribute to developmental time, lifespan and abdominal size variation in Drosophila americana.

Even within a single genus, such as Drosophila, cases of lineage-specific adaptive evolution have been found. Therefore, the molecular basis of phenotypic variation must be addressed in more than one species group, in order to infer general patterns. In this work, we used D. americana, a species distantly-related to D. melanogaster, to perform an F2 association study for developmental time (DT), chill-coma recovery time (CRT), abdominal size (AS) and lifespan (LS) involving the two strains (H5 and W11) whose genomes have been previously sequenced. Significant associations were found between the 43 large indel markers developed here and DT, AS and LS but not with CRT. Significant correlations are also found between DT and LS, and between AS and LS, that might be explained by variation at genes belonging to the insulin and ecdysone signaling pathways. Since, in this F2 association study a single marker, located close to the Ecdysone receptor (EcR) gene, explained as much as 32.6% of the total variation in DT, we performed a second F2 association study, to determine whether large differences in DT are always due to variation in this genome region. No overlapping signal was observed between the two F2 association studies. Overall, these results illustrate that, in D. americana, pleiotropic genes involved in the highly-conserved insulin and ecdysone signaling pathways are likely responsible for variation observed in ecologically relevant phenotypic traits, although other genes are also involved.