Tyrosyl-DNA phosphodiesterase 1 (TDP1) and SPRTN protease repair histone 3 and topoisomerase 1 DNA-protein crosslinks in vivo.

DNA-protein crosslinks (DPCs) are frequent and damaging DNA lesions that affect all DNA transactions, which in turn can lead to the formation of double-strand breaks, genomic instability and cell death. At the organismal level, impaired DPC repair (DPCR) is associated with cancer, ageing and neurodegeneration. Despite the severe consequences of DPCs, little is known about the processes underlying repair pathways at the organism level. SPRTN is a protease that removes most cellular DPCs during replication, whereas tyrosyl-DNA phosphodiesterase 1 repairs one of the most abundant enzymatic DPCs, topoisomerase 1-DPC (TOP1-DPC). How these two enzymes repair DPCs at the organism level is currently unknown. We perform phylogenetic, syntenic, structural and expression analysis to compare tyrosyl-DNA phosphodiesterase 1 (TDP1) orthologues between human, mouse and zebrafish. Using the zebrafish animal model and human cells, we demonstrate that TDP1 and SPRTN repair endogenous, camptothecin- and formaldehyde-induced DPCs, including histone H3- and TOP1-DPCs. We show that resolution of H3-DNA crosslinks depends on upstream proteolysis by SPRTN and subsequent peptide removal by TDP1 in RPE1 cells and zebrafish embryos, whereas SPRTN and TDP1 function in different pathways in the repair of endogenous TOP1-DPCs and total DPCs. Furthermore, we have found increased TDP2 expression in TDP1-deficient cells and embryos. Understanding the role of TDP1 in DPCR at the cellular and organismal levels could provide an impetus for the development of new drugs and combination therapies with TOP1-DPC inducing drugs.

Strain Wars: Competitive interactions between SARS-CoV-2 strains are explained by Gibbs energy of antigen-receptor binding.

Since the beginning of the COVID-19 pandemic, SARS-CoV-2 has mutated several times into new strains, with an increased infectivity. Infectivity of SARS-CoV-2 strains depends on binding affinity of the virus to its host cell receptor. In this paper, we quantified the binding affinity using Gibbs energy of binding and analyzed the competition between SARS-CoV-2 strains as an interference phenomenon. Gibbs energies of binding were calculated for several SARS-SoV-2 strains, including Hu-1 (wild type), B.1.1.7 (alpha), B.1.351 (beta), P.1 (Gamma), B.1.36 and B.1.617 (Delta). The least negative Gibbs energy of binding is that of Hu-1 strain, -37.97 kJ/mol. On the other hand, the most negative Gibbs energy of binding is that of the Delta strain, -49.50 kJ/mol. We used the more negative Gibbs energy of binding to explain the increased infectivity of newer SARS-CoV-2 strains compared to the wild type. Gibbs energies of binding was found to decrease chronologically, with appearance of new strains. The ratio of Gibbs energies of binding of mutated strains and wild type was used to define a susceptibility coefficient, which is an indicator of viral interference, where a virus can prevent or partially inhibit infection with another virus.

TEX264 at the intersection of autophagy and DNA repair.

TEX264 (testes expressed gene 264) is a single-pass transmembrane protein, consisting of an N-terminal hydrophobic region, a gyrase inhibitory (GyrI)-like domain, and a loosely structured C terminus. TEX264 was first identified as an endoplasmic reticulum (ER)-resident Atg8-family-binding protein that mediates the degradation of portions of the ER during starvation (i.e., reticulophagy). More recently, TEX264 was identified as a cofactor of VCP/p97 ATPase that promotes the repair of covalently trapped TOP1 (DNA topoisomerase 1)-DNA crosslinks. This review summarizes the current knowledge of TEX264 as a protein with roles in both autophagy and DNA repair and provides an evolutionary and structural analysis of GyrI proteins. Based on our phylogenetic analysis, we provide evidence that TEX264 is a member of a large superfamily of GyrI-like proteins that evolved in bacteria and are present in metazoans, including invertebrates and chordates.Abbreviations: Atg8: autophagy related 8; Atg39: autophagy related 39; Cdc48: cell division cycle 48; CGAS: cyclic GMP-AMP synthase; DPC: DNA-protein crosslinks; DSB: DNA double-strand break; ER: endoplasmic reticulum; GyrI: gyrase inhibitory domain; LRR: leucine-rich repeat; MAFFT: multiple alignment using fast Fourier transform; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MTOR: mechanistic target of rapamycin kinase; STUBL: SUMO targeted ubiquitin ligase; SUMO: small ubiquitin-like modifier; TEX264: testis expressed gene 264; TOP1cc: topoisomerase 1-cleavage complex; UBZ: ubiquitin binding Zn finger domain; VCP: valosin containing protein.

The protease SPRTN and SUMOylation coordinate DNA-protein crosslink repair to prevent genome instability.

DNA-protein crosslinks (DPCs) are a specific type of DNA lesion in which proteins are covalently attached to DNA. Unrepaired DPCs lead to genomic instability, cancer, neurodegeneration, and accelerated aging. DPC proteolysis was recently identified as a specialized pathway for DPC repair. The DNA-dependent protease SPRTN and the 26S proteasome emerged as two independent proteolytic systems. DPCs are also repaired by homologous recombination (HR), a canonical DNA repair pathway. While studying the cellular response to DPC formation, we identify ubiquitylation and SUMOylation as two major signaling events in DNA replication-coupled DPC repair. DPC ubiquitylation recruits SPRTN to repair sites, promoting DPC removal. DPC SUMOylation prevents DNA double-strand break formation, HR activation, and potentially deleterious genomic rearrangements. In this way, SUMOylation channels DPC repair toward SPRTN proteolysis, which is a safer pathway choice for DPC repair and prevention of genomic instability.

Zebrafish (Danio rerio) Oatp2b1 as a functional ortholog of the human OATP2B1 transporter.

OATP2B1 belongs to a highly conserved organic anion transporting polypeptide (OATP) family of transporters, involved in the cellular uptake of both endogenous and exogenous compounds. The reported substrates of human OATP2B1 include steroid conjugates, bile salts, and thyroid hormones, as well as pharmaceuticals. Human OATP2B1 has orthologous genes in other vertebrate species, including zebrafish (Danio rerio), a widely used model organism in biomedical and environmental research. Our previous studies showed that zebrafish Oatp2b1 was phylogenetically closest to mammalian OATP2B1/Oatp2b1 and that it shares a similar tissue expression pattern. In this study, we aimed at discovering whether zebrafish Oatp2b1 could be a functional ortholog of human and rodent OATP2B1. To test this hypothesis, our primary goal was to obtain the first in vitro and in silico insights related to the structure and potential substrate preferences of zebrafish Oatp2b1. We generated cells transiently and stably transfected with zebrafish Oatp2b1 cloned from zebrafish liver, constructed an Oatp2b1 homology model, developed transport activity assays with model fluorescent substrate Lucifer yellow, and finally utilized this assay to analyze the interaction of zebrafish Oatp2b1 with both physiological and xenobiotic substances. Apart from structure similarities, our data revealed the strongest interaction of zebrafish Oatp2b1 with bile acids, steroid sulfates, thyroid hormones, and bilirubin, as well as xenobiotics bromosulfophthalein and sulfasalazine, which indicates its functional orthology with human OATP2B1.

Environmental contaminants modulate transport activity of zebrafish organic anion transporters Oat1 and Oat3.

Organic anion transporters (OATs) are transmembrane proteins which belong to SLC22 subfamily. They are responsible for the uptake of various endo- and xenobiotics into the cells of different organs and tissues. Following our previous work on characterization of zebrafish Oat1 and Oat3, in this study we analyzed interaction of various classes of environmental contaminants with these membrane transporters using the transport activity assay with HEK293 Flp-In cell line stably overexpressing zebrafish Oat1 and Oat3, respectively. Based on the initial screening of a series of 36 environmental contaminants on their ability to interact with zebrafish Oat1 and Oat3, the most potent interactors were selected, their IC50 values calculated and type of interaction determined. Finally, to further confirm the type of interaction and initially evaluate their toxic potential, the cytotoxicity assays were performed. Broad ligand selectivity and similarity of zebrafish Oat1 and Oat3 with mammalian orthologs was confirmed and potent interactors among environmental contaminants identified.

SPRTN protease and checkpoint kinase 1 cross-activation loop safeguards DNA replication.

The SPRTN metalloprotease is essential for DNA-protein crosslink (DPC) repair and DNA replication in vertebrate cells. Cells deficient in SPRTN protease exhibit DPC-induced replication stress and genome instability, manifesting as premature ageing and liver cancer. Here, we provide a body of evidence suggesting that SPRTN activates the ATR-CHK1 phosphorylation signalling cascade during physiological DNA replication by proteolysis-dependent eviction of CHK1 from replicative chromatin. During this process, SPRTN proteolyses the C-terminal/inhibitory part of CHK1, liberating N-terminal CHK1 kinase active fragments. Simultaneously, CHK1 full length and its N-terminal fragments phosphorylate SPRTN at the C-terminal regulatory domain, which stimulates SPRTN recruitment to chromatin to promote unperturbed DNA replication fork progression and DPC repair. Our data suggest that a SPRTN-CHK1 cross-activation loop plays a part in DNA replication and protection from DNA replication stress. Finally, our results with purified components of this pathway further support the proposed model of a SPRTN-CHK1 cross-activation loop.

Zebrafish (Danio rerio) Oat1 and Oat3 transporters and their interaction with physiological compounds.

Organic anion transporters (OATs) are membrane proteins within the Solute carrier family 22 (SLC22). They play important roles in cellular uptake of various organic compounds, and due to their expression in barrier tissues of major excretory and non-excretory organs are considered as crucial elements in absorption and distribution of a wide range of endobiotic and xenobiotic compounds. Based on our previous work and initial insights on SLC22 members in zebrafish (Danio rerio), in this study we aimed at in vitro characterization of Oat1 and Oat3 transporters and understanding of their interaction with potential physiological substrates. We first performed synteny analysis to describe in more detail orthological relationship of zebrafish oat1 and oat3 genes. We then developed stable cell lines overexpressing Oat1 and Oat3, and identified Lucifer yellow as Oat1 model fluorescent substrate (Km = 11.4 µM) and 6-carboxyfluorescein as Oat3 model substrate (Km = 5.8 µM). Initial identification performed using the developed assays revealed Kreb's cycle intermediates, bilirubin, bile salts and steroid hormones as the most potent of Oat1 and Oat3 interactors, with IC50 values in micromolar range. Finally, we showed that bilirubin, deoxycholic acid, α-ketoglutarate, pregnenolone, estrone-3-sulfate and corticosterone are in vitro substrates of zebrafish Oat1, and bilirubin and deoxycholic acid are Oat3 substrates. In conclusion, using the approach described, structural and functional similarities of both transporters to human and mammalian orthologs are revealed, their broad ligand selectivity confirmed, potent interactors among endobiotic compounds identified, and first indications of their potential physiological role(s) in zebrafish obtained.

DNA protein crosslink proteolysis repair: From yeast to premature ageing and cancer in humans.

DNA-protein crosslinks (DPCs) are a specific type of DNA lesion consisting of a protein covalently and irreversibly bound to DNA, which arise after exposure to physical and chemical crosslinking agents. DPCs can be bulky and thereby pose a barrier to DNA replication and transcription. The persistence of DPCs during S phase causes DNA replication stress and genome instability. The toxicity of DPCs is exploited in cancer therapy: many common chemotherapeutics kill cancer cells by inducing DPC formation. Recent work from several laboratories discovered a specialized repair pathway for DPCs, namely DPC proteolysis (DPCP) repair. DPCP repair is carried out by replication-coupled DNA-dependent metalloproteases: Wss1 in yeast and SPRTN in metazoans. Mutations in SPRTN cause premature ageing and liver cancer in humans and mice; thus, defective DPC repair has great clinical ramifications. In the present review, we will revise the current knowledge on the mechanisms of DPCP repair and on the regulation of DPC protease activity, while highlighting the most significant unresolved questions in the field. Finally, we will discuss the impact of faulty DPC repair on disease and cancer therapy.

In vitro characterization of zebrafish (Danio rerio) organic anion transporters Oat2a-e.

OATS/Oats are transmembrane proteins that transport a variety of drugs, environmental toxins and endogenous metabolites into the cell. Zebrafish (Danio rerio) has seven OAT orthologs: Oat1, Oat2a-e and Oat3. In this study we specifically address Oat2 (Slc22a7) family. Conserved synteny analysis showed localization of zebrafish oat2 genes on two chromosomes, 11 and 17. All five zebrafish Oats were localized by live cell imaging in membranes of transiently transfected HEK293-T cells, and Oat2a, b, d, and e were confirmed using western blot analysis. Functional studies using the HEK293T cells overexpressing zebrafish Oats revealed two model fluorescent substrates of three Oats: Lucifer yellow for Oat2a and Oat2d (Km 122, and 49.7µM), and 6-carboxyfluorescein for Oat2b and Oat2d (Km 199.7, and 266.9µM). The initial screening of a series of diverse endo- and xenobiotics showed interaction with a number of compounds, including cGMP and diclofenac (IC50 27.74, and 19.14µM) with Oat2a; estrone-3-sulfate and diclofenac (IC50 30.96, and 12.6µM) with Oat2b; and fumarate and indomethacin (IC50 68.24, and 20.41µM) with Oat2d. This study provides the first comprehensive data set on Oat2 in zebrafish and offers an important basis for more detailed molecular and (eco)toxicological characterizations of these transporters.

Interaction between the zebrafish (Danio rerio) organic cation transporter 1 (Oct1) and endo- and xenobiotics.

Organic cation transporters (OCTs) serve as uptake transporters of numerous endo- and xenobiotics. They have been in the focus of medical toxicological research for more than a decade due to their key role in absorption, distribution, metabolism and excretion due to their expression on basolateral membranes of various barrier tissues. OCTs belong to the SLC22A family within the SLC (Solute carrier) protein superfamily, with three co-orthologs identified in humans (OCT1, 2 and 3), and two Oct orthologs in zebrafish (Oct1 and Oct2). The structural and functional properties of zebrafish Octs, along with their toxicological relevance, have still not been explored. In this study, we performed a functional characterization of zebrafish Oct1 using transient and stable heterologous expression systems and model fluorescent substrates as the basis for interaction studies with a wide range of endo- and xenobiotics. We also conducted a basic topology analysis and homology modeling to determine the structure and membrane localization of Oct1. Finally, we performed an MTT assay to evaluate the toxic effects of the seven interactors identified - oxaliplatin, cisplatin, berberine, MPP+, prazosin, paraquat and mitoxantrone - in human embryonic kidney cells (HEK293T) stably expressing zebrafish Oct1 (HEK293T-drOct1 cells). Our results show that the zebrafish Oct1 structure consists of 12 transmembrane alpha helices, which form the active region with more than one active site. Five new fluorescent substrates of Oct1 were identified: ASP+ (Km=26µM), rhodamine 123 (Km=103.7nM), berberine (Km=3.96µM), DAPI (Km=780nM), and ethidium bromide (Km=97nM). Interaction studies revealed numerous interactors that inhibited the Oct1-dependent uptake of fluorescent substrates. The identified interactors ranged from physiological compounds (mainly steroid hormones) to different classes of xenobiotics, with IC50 values in nanomolar (e.g., pyrimethamine and prazosin) to millimolar range (e.g., cimetidine). Cytotoxicity experiments with HEK293T-drOct1 cells enabled us to identify berberine, oxaliplatin and MPP+ as substrates of Oct1. The data presented in this study provide the first insights into the functional properties of zebrafish Oct1 and offer an important basis for more detailed molecular and ecotoxicological characterizations of this transporter.

DNA-Protein Crosslink Proteolysis Repair.

Proteins that are covalently bound to DNA constitute a specific type of DNA lesion known as DNA-protein crosslinks (DPCs). DPCs represent physical obstacles to the progression of DNA replication. If not repaired, DPCs cause stalling of DNA replication forks that consequently leads to DNA double-strand breaks, the most cytotoxic DNA lesion. Although DPCs are common DNA lesions, the mechanism of DPC repair was unclear until now. Recent work unveiled that DPC repair is orchestrated by proteolysis performed by two distinct metalloproteases, SPARTAN in metazoans and Wss1 in yeast. This review summarizes recent discoveries on two proteases in DNA replication-coupled DPC repair and establishes DPC proteolysis repair as a separate DNA repair pathway for genome stability and protection from accelerated aging and cancer.

Metalloprotease SPRTN/DVC1 Orchestrates Replication-Coupled DNA-Protein Crosslink Repair.

The cytotoxicity of DNA-protein crosslinks (DPCs) is largely ascribed to their ability to block the progression of DNA replication. DPCs frequently occur in cells, either as a consequence of metabolism or exogenous agents, but the mechanism of DPC repair is not completely understood. Here, we characterize SPRTN as a specialized DNA-dependent and DNA replication-coupled metalloprotease for DPC repair. SPRTN cleaves various DNA binding substrates during S-phase progression and thus protects proliferative cells from DPC toxicity. Ruijs-Aalfs syndrome (RJALS) patient cells with monogenic and biallelic mutations in SPRTN are hypersensitive to DPC-inducing agents due to a defect in DNA replication fork progression and the inability to eliminate DPCs. We propose that SPRTN protease represents a specialized DNA replication-coupled DPC repair pathway essential for DNA replication progression and genome stability. Defective SPRTN-dependent clearance of DPCs is the molecular mechanism underlying RJALS, and DPCs are contributing to accelerated aging and cancer.

Functional characterization of rainbow trout (Oncorhynchus mykiss) Abcg2a (Bcrp) transporter.

ABCG2 (BCRP - breast cancer resistance protein) belongs to the ATP-binding cassette (ABC) superfamily. It plays an important role in the disposition and elimination of xeno- and endobiotics and/or their metabolites in mammals. Likewise, the protective role of ABC transporters, including Abcg2, has been reported for aquatic organisms. In our previous study we have cloned the full gene sequence of rainbow trout (Oncorhynchus mykiss) Abcg2a and showed its high expression in liver and primary hepatocytes. Based on those insights, the main goal of this study was to perform a detailed functional characterization of trout Abcg2a using insect ovary cells (Spodoptera frugiperda, Sf9) as a heterologous expression system. Membrane vesicles preparations from Sf9 cells were used for the ATPase assay determinations and basic biochemical properties of fish Abcg2a versus human ABCG2 have been compared. A series of 39 physiologically and/or environmentally relevant substances was then tested on interaction with trout Abcg2a and human ABCG2. Correlation analysis reveals highly similar pattern of activation and inhibition. Significant activation of trout Abcg2a ATPase was observed for prazosin, doxorubicine, sildenafil, furosemid, propranolol, fenofibrate and pheophorbide. Pesticides showed either a weak activation (malathione) or strong (endosulfan) to weak (chlorpyrifos, fenoxycarb, DDE) inhibition of trout Abcg2a ATPase while the highest activation was obtained for benzo(a)pyrene, curcumine and testosterone. In conclusion, data from this study offer the first characterization of fish Abcg2a, reveal potent interactors among physiologically or environmentally relevant substances and point to similarities regarding strengths and interactor preferences between human ABCG2 and fish Abcg2a.

Phylogenetic, syntenic, and tissue expression analysis of slc22 genes in zebrafish (Danio rerio).

BACKGROUND: SLC22 protein family is a member of the SLC (Solute carriers) superfamily of polyspecific membrane transporters responsible for uptake of a wide range of organic anions and cations, including numerous endo- and xenobiotics. Due to the lack of knowledge on zebrafish Slc22 family, we performed initial characterization of these transporters using a detailed phylogenetic and conserved synteny analysis followed by the tissue specific expression profiling of slc22 transcripts. RESULTS: We identified 20 zebrafish slc22 genes which are organized in the same functional subgroups as human SLC22 members. Orthologies and syntenic relations between zebrafish and other vertebrates revealed consequences of the teleost-specific whole genome duplication as shown through one-to-many orthologies for certain zebrafish slc22 genes. Tissue expression profiles of slc22 transcripts were analyzed using qRT-PCR determinations in nine zebrafish tissues: liver, kidney, intestine, gills, brain, skeletal muscle, eye, heart, and gonads. Our analysis revealed high expression of oct1 in kidney, especially in females, followed by oat3 and oat2c in females, oat2e in males and orctl4 in females. oct1 was also dominant in male liver. oat2d showed the highest expression in intestine with less noticeable gender differences. All slc22 genes showed low expression in gills, and moderate expression in heart and skeletal muscle. Dominant genes in brain were oat1 in females and oct1 in males, while the highest gender differences were determined in gonads, with dominant expression of almost all slc22 genes in testes and the highest expression of oat2a. CONCLUSIONS: Our study offers the first insight into the orthology relationships, gene expression and potential role of Slc22 membrane transporters in zebrafish. Clear orthological relationships of zebrafish slc22 and other vertebrate slc22 genes were established. slc22 members are mostly highly conserved, suggesting their physiological and toxicological importance. One-to-many orthologies and differences in tissue expression patterns of zebrafish slc22 genes in comparison to human orthologs were observed. Our expression data point to partial similarity of zebrafish versus human Slc22 members, with possible compensatory roles of certain zebrafish transporters, whereas higher number of some orthologs implies potentially more diverse and specific roles of these proteins in zebrafish.

The first characterization of multidrug and toxin extrusion (MATE/SLC47) proteins in zebrafish (Danio rerio).

Multidrug and toxin extrusion (MATE) proteins are involved in the extrusion of endogenous compounds and xenobiotics across the plasma membrane. They are conserved from bacteria to mammals, with different numbers of genes within groups. Here, we present the first data on identification and functional characterization of Mate proteins in zebrafish (Danio rerio). Phylogenetic analysis revealed six Mates in teleost fish, annotated as Mate3-8, which form a distinct cluster separated from the tetrapod MATEs/Mates. Synteny analysis showed that zebrafish mate genes are orthologous to human MATEs. Gene expression analysis revealed that all the mate transcripts were constitutively and differentially expressed during embryonic development, followed by pronounced and tissue-specific expression in adults. Functional analyses were performed using transport activity assays with model substrates after heterologous overexpression of five zebrafish Mates in HEK293T cells. The results showed that zebrafish Mates interact with both physiological and xenobiotic substances but also substantially differ with respect to the interacting compounds and interaction strength in comparison to mammalian MATEs/Mates. Taken together, our data clearly indicate a potentially important role for zebrafish Mate transporters in zebrafish embryos and adults and provide a basis for detailed functional characterizations of single zebrafish Mate transporters.

Characterization of glutathione-S-transferases in zebrafish (Danio rerio).

Glutathione-S-transferases (GSTs) are one of the key enzymes that mediate phase II of cellular detoxification. The aim of our study was a comprehensive characterization of GSTs in zebrafish (Danio rerio) as an important vertebrate model species frequently used in environmental research. A detailed phylogenetic analysis of GST superfamily revealed 27 zebrafish gst genes. Further insights into the orthology relationships between human and zebrafish GSTs/Gsts were obtained by the conserved synteny analysis. Expression of gst genes in six tissues (liver, kidney, gills, intestine, brain and gonads) of adult male and female zebrafish was determined using qRT-PCR. Functional characterization was performed on 9 cytosolic Gst enzymes after overexpression in E. coli and subsequent protein purification. Enzyme kinetics was measured for GSH and a series of model substrates. Our data revealed ubiquitously high expression of gstp, gstm (except in liver), gstr1, mgst3a and mgst3b, high expression of gsto2 in gills and ovaries, gsta in intestine and testes, gstt1a in liver, and gstz1 in liver, kidney and brain. All zebrafish Gsts catalyzed the conjugation of GSH to model GST substrates 1-chloro-2,4-dinitrobenzene (CDNB) and monochlorobimane (MCB), apart from Gsto2 and Gstz1 that catalyzed GSH conjugation to dehydroascorbate (DHA) and dichloroacetic acid (DCA), respectively. Affinity toward CDNB varied from 0.28 mM (Gstp2) to 3.69 mM (Gstm3), while affinity toward MCB was in the range of 5 µM (Gstt1a) to 250 µM (Gstp1). Affinity toward GSH varied from 0.27 mM (Gstz1) to 4.45 mM (Gstt1a). Turnover number for CDNB varied from 5.25s(-1) (Gstt1a) to 112s(-1) (Gstp2). Only Gst Pi enzymes utilized ethacrynic acid (ETA). We suggest that Gstp1, Gstp2, Gstt1a, Gstz1, Gstr1, Mgst3a and Mgst3b have important role in the biotransformation of xenobiotics, while Gst Alpha, Mu, Pi, Zeta and Rho classes are involved in the crucial physiological processes. In summary, this study provides the first comprehensive analysis of GST superfamily in zebrafish, presents new insight into distinct functions of individual Gsts, and offers methodological protocols that can be used for further verification of interaction of environmental contaminants with fish Gsts.

Interaction of environmental contaminants with zebrafish organic anion transporting polypeptide, Oatp1d1 (Slco1d1).

Polyspecific transporters from the organic anion transporting polypeptide (OATP/Oatp) superfamily mediate the uptake of a wide range of compounds. In zebrafish, Oatp1d1 transports conjugated steroid hormones and cortisol. It is predominantly expressed in the liver, brain and testes. In this study we have characterized the transport of xenobiotics by the zebrafish Oatp1d1 transporter. We developed a novel assay for assessing Oatp1d1 interactors using the fluorescent probe Lucifer yellow and transient transfection in HEK293 cells. Our data showed that numerous environmental contaminants interact with zebrafish Oatp1d1. Oatp1d1 mediated the transport of diclofenac with very high affinity, followed by high affinity towards perfluorooctanesulfonic acid (PFOS), nonylphenol, gemfibrozil and 17α-ethinylestradiol; moderate affinity towards carbaryl, diazinon and caffeine; and low affinity towards metolachlor. Importantly, many environmental chemicals acted as strong inhibitors of Oatp1d1. A strong inhibition of Oatp1d1 transport activity was found by perfluorooctanoic acid (PFOA), chlorpyrifos-methyl, estrone (E1) and 17ß-estradiol (E2), followed by moderate to low inhibition by diethyl phthalate, bisphenol A, 7-acetyl-1,1,3,4,4,6-hexamethyl-1,2,3,4 tetrahydronapthalene and clofibrate. In this study we identified Oatp1d1 as a first Solute Carrier (SLC) transporter involved in the transport of a wide range of xenobiotics in fish. Considering that Oatps in zebrafish have not been characterized before, our work on zebrafish Oatp1d1 offers important new insights on the understanding of uptake processes of environmental contaminants, and contributes to the better characterization of zebrafish as a model species.

Molecular characterization of zebrafish Oatp1d1 (Slco1d1), a novel organic anion-transporting polypeptide.

The organic anion-transporting polypeptide (OATP/Oatp) superfamily includes a group of polyspecific transporters that mediate transport of large amphipathic, mostly anionic molecules across cell membranes of eukaryotes. OATPs/Oatps are involved in the disposition and elimination of numerous physiological and foreign compounds. However, in non-mammalian species, the functional properties of Oatps remain unknown. We aimed to elucidate the role of Oatp1d1 in zebrafish to gain insights into the functional and structural evolution of the OATP1/Oatp1 superfamily. We show that diversification of the OATP1/Oatp1 family occurs after the emergence of jawed fish and that the OATP1A/Oatp1a and OATP1B/Oatp1b subfamilies appeared at the root of tetrapods. The Oatp1d subfamily emerged in teleosts and is absent in tetrapods. The zebrafish Oatp1d1 is similar to mammalian OATP1A/Oatp1a and OATP1B/Oatp1b members, with the main physiological role in transport and balance of steroid hormones. Oatp1d1 activity is dependent upon pH gradient, which could indicate bicarbonate exchange as a mode of transport. Our analysis of evolutionary conservation and structural properties revealed that (i) His-79 in intracellular loop 3 is conserved within OATP1/Oatp1 family and is crucial for the transport activity; (ii) N-glycosylation impacts membrane targeting and is conserved within the OATP1/Oatp1 family with Asn-122, Asn-133, Asn-499, and Asn-512 residues involved; (iii) the evolutionarily conserved cholesterol recognition interaction amino acid consensus motif is important for membrane localization; and (iv) Oatp1d1 is present in dimeric and possibly oligomeric form in the cell membrane. In conclusion, we describe the first detailed characterization of a new Oatp transporter in zebrafish, offering important insights into the functional evolution of the OATP1/Oatp1 family and the physiological role of Oatp1d1.