Functional characterization of SLC39 family members ZIP5 and ZIP10 in overexpressing HEK293 cells reveals selective copper transport activity

Polesel, Marcello; Inglés-Prieto, Álvaro; Christodoulaki, Eirini; Ferrada, Evandro; Doucerain, C.; Altermatt, Patrick; Knecht, Michelle; Kühn, Michael; Steck, Anna-Lena; Wilhelm, Maria; Manolova, Vania

doi:10.1007/s10534-022-00474-6

Cited by 10 publications

(3 citation statements)

References 40 publications

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“…In terms of iron regulation, studies have shown that Slc39a14 can transport non-transferrin bound iron (NTBI), and increased iron accumulation has been observed in HEK293 human kidney cells and Sf9 insect cells overexpressing mouse Slc39a14 [ 49 ]. In contrast, iron accumulation was reduced in AML12 liver cells when Slc39a14 expression was inhibited.…”

Section: Discussionmentioning

confidence: 99%

Inhibition of Slc39a14/Slc39a8 reduce vascular calcification via alleviating iron overload induced ferroptosis in vascular smooth muscle cells

Aierken,

He,

et al. 2024

Cardiovasc Diabetol

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Background Vascular calcification (VC) is an independent risk factor for cardiovascular diseases. Recently, ferroptosis has been recognised as a novel therapeutic target for cardiovascular diseases. Although an association between ferroptosis and vascular calcification has been reported, the role and mechanism of iron overload in vascular calcification are still poorly understood. Specifically, further in-depth research is required on whether metalloproteins SLC39a14 and SLC39a8 are involved in ferroptosis induced by iron overload. Methods R language was employed for the differential analysis of the dataset, revealing the correlation between ferroptosis and calcification. The experimental approaches encompassed both in vitro and in vivo studies, incorporating the use of iron chelators and models of iron overload. Additionally, gain- and loss-of-function experiments were conducted to investigate iron’s effects on vascular calcification comprehensively. Electron microscopy, immunofluorescence, western blotting, and real-time polymerase chain reaction were used to elucidate how Slc39a14 and Slc39a8 mediate iron overload and promote calcification. Results Ferroptosis was observed in conjunction with vascular calcification (VC); the association was consistently confirmed by in vitro and in vivo studies. Our results showed a positive correlation between iron overload in VSMCs and calcification. Iron chelators are effective in reversing VC and iron overload exacerbates this process. The expression levels of the metal transport proteins Slc39a14 and Slc39a8 were significantly upregulated during calcification; the inhibition of their expression alleviated VC. Conversely, Slc39a14 overexpression exacerbates calcification and promotes intracellular iron accumulation in VSMCs. Conclusions Our research demonstrates that iron overload occurs during VC, and that inhibition of Slc39a14 and Slc39a8 significantly relieves VC by intercepting iron overload-induced ferroptosis in VSMCs, providing new insights into the VC treatment. Graphical Abstract

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Section: Discussionmentioning

confidence: 99%

Inhibition of Slc39a14/Slc39a8 reduce vascular calcification via alleviating iron overload induced ferroptosis in vascular smooth muscle cells

Aierken,

He,

et al. 2024

Cardiovasc Diabetol

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“…The Jump In T-REx human embryonic kidney 293 cell line (HEK293) expressing doxycycline-inducible human wild-type SLCs was generated by RESOLUTE, as described previously ( Polesel et al, 2023 ). After thawing, HEK293 cells stably expressing all constructs were selected in DMEM medium supplemented with 10% FBS, 5% penicillin/streptomycin, 0.0005% blasticidin, and 4% geneticin for 1 week.…”

Section: Methodsmentioning

confidence: 99%

Advancing drug discovery through assay development: a survey of tool compounds within the human solute carrier superfamily

Digles,

Ingles-Prieto,

Dvorak

et al. 2024

Front. Pharmacol.

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With over 450 genes, solute carriers (SLCs) constitute the largest transporter superfamily responsible for the uptake and efflux of nutrients, metabolites, and xenobiotics in human cells. SLCs are associated with a wide variety of human diseases, including cancer, diabetes, and metabolic and neurological disorders. They represent an important therapeutic target class that remains only partly exploited as therapeutics that target SLCs are scarce. Additionally, many small molecules reported in the literature to target SLCs are poorly characterized. Both features may be due to the difficulty of developing SLC transport assays that fulfill the quality criteria for high-throughput screening. Here, we report one of the main limitations hampering assay development within the RESOLUTE consortium: the lack of a resource providing high-quality information on SLC tool compounds. To address this, we provide a systematic annotation of tool compounds targeting SLCs. We first provide an overview on RESOLUTE assays. Next, we present a list of SLC-targeting compounds collected from the literature and public databases; we found that most data sources lacked specificity data. Finally, we report on experimental tests of 19 selected compounds against a panel of 13 SLCs from seven different families. Except for a few inhibitors, which were active on unrelated SLCs, the tested inhibitors demonstrated high selectivity for their reported targets. To make this knowledge easily accessible to the scientific community, we created an interactive dashboard displaying the collected data in the RESOLUTE web portal (https://re-solute.eu). We anticipate that our open-access resources on assays and compounds will support the development of future drug discovery campaigns for SLCs.

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“…The Jump In T-REx human embryonic kidney 293 cell line (HEK293-JI) expressing doxycycline-inducible human SPNS2 were generated by RESOLUTE as described previously 46 . Briefly, codon-optimized sequences of wild type and mutant SPNS2 in pDONR221 (Addgene #132307) were subcloned into a modified pJTI R4 DEST CMV TO pA plasmid (Thermo Fisher Scientific) containing Twin-Strep-Tag (IBA Lifesciences) and HA epitopes (SH tag) at the N or the C terminus of SPNS2 as indicated.…”

Section: Cell Lines and Culture Conditionsmentioning

confidence: 99%

Transport and inhibition of the sphingosine-1-phosphate exporter SPNS2

Sauer,

Li,

Pike

et al. 2023

Preprint

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Sphingosine-1-phosphate (S1P) is a signaling lysolipid critical to heart development, immunity, and hearing. Accordingly, mutations in the S1P transporter SPNS2 are associated with reduced white cell count and hearing defects. SPNS2 also exports the S1P-mimicking FTY720-P (Fingolimod) and thereby is central to the pharmacokinetics of this drug when treating multiple sclerosis. Here, we use a combination of cryo-electron microscopy, immunofluorescence, in vitro binding and in vivo S1P export assays, and molecular dynamic simulations to probe SPNS2’s substrate binding and transport. These results reveal the transporter’s binding mode to its native substrate S1P, the therapeutic FTY720-P, and the SPNS2-targeting inhibitor 33p. Further capturing an inward-facing apo state, our structures illuminate the protein’s mechanism for exchange between inward-facing and outward-facing conformations. Finally, using these structural, localization, and S1P transport results, we identify how pathogenic mutations ablate the protein’s export activity and thereby lead to hearing loss.

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Functional characterization of SLC39 family members ZIP5 and ZIP10 in overexpressing HEK293 cells reveals selective copper transport activity

Cited by 10 publications

References 40 publications

Inhibition of Slc39a14/Slc39a8 reduce vascular calcification via alleviating iron overload induced ferroptosis in vascular smooth muscle cells

Inhibition of Slc39a14/Slc39a8 reduce vascular calcification via alleviating iron overload induced ferroptosis in vascular smooth muscle cells

Advancing drug discovery through assay development: a survey of tool compounds within the human solute carrier superfamily

Transport and inhibition of the sphingosine-1-phosphate exporter SPNS2

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