Environmental contaminants modulate transport activity of zebrafish (Danio rerio) multidrug and toxin extrusion protein 3 (Mate3/Slc47a2.1)

Lana Vujica; Jovica Lončar; Lana Mišić; Bono Lučić; Katarina Radman; Ivan Mihaljević; Branimir Bertoša; Josip Mesarić; Marina Horvat; Tvrtko Smital

doi:10.1016/j.scitotenv.2023.165956

Environmental contaminants modulate transport activity of zebrafish (Danio rerio) multidrug and toxin extrusion protein 3 (Mate3/Slc47a2.1)

Sci Total Environ. 2023 Nov 25:901:165956. doi: 10.1016/j.scitotenv.2023.165956. Epub 2023 Aug 2.

Authors

Affiliations

¹ Laboratory for Molecular Ecotoxicology, Division for Marine and Environmental Research, Ruđer Bošković Institute, Bijenička cesta 54, 10 000 Zagreb, Croatia.
² NMR Center, Ruđer Bošković Institute, Bijenička cesta 54, 10 000 Zagreb, Croatia.
³ Department of Chemistry, Faculty of Science, University of Zagreb, Horvatovac 102a, 10 000 Zagreb, Croatia.
⁴ Centre for Informatics and Computing, Ruđer Bošković Institute, Bijenička cesta 54, 10 000 Zagreb, Croatia.
⁵ Laboratory for Molecular Ecotoxicology, Division for Marine and Environmental Research, Ruđer Bošković Institute, Bijenička cesta 54, 10 000 Zagreb, Croatia. Electronic address: smital@irb.hr.

PMID: 37541507
DOI: 10.1016/j.scitotenv.2023.165956

Abstract

Zebrafish Mate3 is one of six co-orthologs of human multidrug and toxin extrusion proteins. It is highly expressed in the kidneys, intestine, testes, and brain of males. Initial interaction studies showed its interaction with xenobiotic compounds, suggesting a role in the efflux of toxic compounds. In this study, we aimed to test various environmental contaminants for their interaction with zebrafish Mate3. We developed a stable zebrafish Mate3 cell line and optimized a high-throughput screening assay using DAPI and ASP⁺ as fluorescent model substrates. To gain insight into the structure and function of the Mate3 protein and relate these to the results of the DAPI and ASP⁺ transport measurements, we predicted its 3D structure using the AlphaFold2 algorithm. A 3D structure with high per residue confidence scores with 13 transmembrane segments (TMs) was obtained, with topology and mutual positioning characteristic of the Mate protein family in a shape open to the extracellular part. Molecular docking methods were used to identify DAPI and ASP⁺ binding sites on the surface and in the center of the protein cavity. Because our kinetics experiments combined with molecular docking indicated that there may be additional active sites in zebrafish Mate3, additional cytotoxicity experiments were performed and highly potent Mate3 interactors were identified from a set of 55 different environmental contaminants. Our results suggest that some of the identified interactors may be of environmental concern, as their interaction with Mate3 could lead to an impairment of its normal efflux function, making fish more sensitive to harmful substances commonly released into the aquatic environment. Finally, the quality of zebrafish Mate3 structures predicted by the AlphaFold2 algorithm opens up the possibility of successfully using this tool for in silico research on transport preferences of other Mate proteins.

Keywords: (3-6max) zebrafish; AlphaFold2 3D structure; In vitro interaction studies; Mate3; Molecular docking.