Novel Green Fluorescent Polyamines to Analyze ATP13A2 and ATP13A3 Activity in the Mammalian Polyamine Transport System

Abstract

Cells acquire polyamines putrescine (PUT), spermidine (SPD) and spermine (SPM) via the complementary actions of polyamine uptake and synthesis pathways. The endosomal P5B-type ATPases ATP13A2 and ATP13A3 emerge as major determinants of mammalian polyamine uptake. Our biochemical evidence shows that fluorescently labeled polyamines are genuine substrates of ATP13A2. They can be used to measure polyamine uptake in ATP13A2- and ATP13A3-dependent cell models resembling radiolabeled polyamine uptake. We further report that ATP13A3 enables faster and stronger cellular polyamine uptake than does ATP13A2. We also compared the uptake of new green fluorescent PUT, SPD and SPM analogs using different coupling strategies (amide, triazole or isothiocyanate) and fluorophores (symmetrical BODIPY, BODIPY-FL and FITC). ATP13A2 promotes the uptake of various SPD and SPM analogs, whereas ATP13A3 mainly stimulates the uptake of PUT and SPD conjugates. However, the polyamine linker and coupling position on the fluorophore impacts the transport capacity, whereas replacing the fluorophore affects polyamine selectivity. The highest uptake in ATP13A2 or ATP13A3 cells is observed with BODIPY-FL-amide conjugated to SPD, whereas BODIPY-PUT analogs are specifically taken up via ATP13A3. We found that P5B-type ATPase isoforms transport fluorescently labeled polyamine analogs with a distinct structure–activity relationship (SAR), suggesting that isoform-specific polyamine probes can be designed.

ATP13A4 upregulation drives the elevated polyamine transport system in the breast cancer cell line MCF7

“…This suggests that the relative uptake capacity towards BODIPY-labeled polyamine analogs is informative, but not conclusive for deducing the substrate specificity of ATP13A4. Our data in the MCF7 and MCF10A cell models with ATP13A4 expression suggest that ATP13A4 presents a broad polyamine specificity, similar to what has been described for ATP13A3 [3,19]. Indeed, we observed ATP13A4transport dependent BODIPY-putrescine, -spermidine and -spermine uptake that is competitive with MGBG uptake, a toxic polyamine analog.…”

“…Fluorescently labeled polyamines are genuine substrates of P5B-ATPases stimulating their ATPase activity [19]. They behave remarkably similar as radiolabeled polyamines for ATP13A2 or ATP13A3-dependent uptake in cells, although subtle differences have also been observed [19].…”

“…Fluorescently labeled polyamines are genuine substrates of P5B-ATPases stimulating their ATPase activity [19]. They behave remarkably similar as radiolabeled polyamines for ATP13A2 or ATP13A3-dependent uptake in cells, although subtle differences have also been observed [19]. This suggests that the relative uptake capacity towards BODIPY-labeled polyamine analogs is informative, but not conclusive for deducing the substrate specificity of ATP13A4.…”

Kufor-Rakeb Syndrome-Associated Psychosis: A Novel Loss-of-FunctionATP13A2Variant and Response to Treatment

“…This protocol is based on a previous publication [15], with minor modifications. Briefly, 70% confluent SH-SY5Y cells in a 12-well plate were incubated (30 min, 37 °C) either with 5 µM 14 C-labeled spermine (3139-50 µCi, ARC) or with a mixture of 5 µM 14 C-spermine and 100 µM unlabeled spermine in cell culture medium.…”

“…Detection was performed by means of chemiluminescence (Bio-Rad ChemiDoc) and protein levels were quantified with Image Lab software. 14 C-labeled polyamine uptake -This protocol is based on a previous publication [15], with minor modifications. Briefly, 70% confluent SH-SY5Y cells in a 12-well plate were incubated (30 min, 37 °C) either with 5 µM ADP-Glo assay -ATPase activity was assessed using a commercially available luminescence assay (ADP-Glo Max assay, V7002, Promega) according to manufacturer's instructions.…”