Abstract
The mitochondrial Ca2+ uptake in trypanosomatids shares biochemical characteristics with that of animals. However, the composition of the mitochondrial Ca2+ uniporter complex (MCUC) in these parasites is quite peculiar, suggesting lineage-specific adaptations. In this work, we compared the inhibitory activity of ruthenium red (RuRed) and Ru360, the most commonly used MCUC inhibitors, with that of the recently described inhibitor Ru265, on Trypanosoma cruzi, the agent of Chagas disease. Ru265 was more potent than Ru360 and RuRed in inhibiting mitochondrial Ca2+ transport in permeabilized cells. When dose-response effects were investigated, an increase in sensitivity for Ru360 and Ru265 was observed in TcMICU1-KO and TcMICU2-KO cells as compared with control cells. In the presence of RuRed, a significant increase in sensitivity was observed only in TcMICU2-KO cells. However, application of Ru265 to intact cells did not affect growth and respiration of epimastigotes, mitochondrial Ca2+ uptake in Rhod-2-labeled intact cells, or attachment to host cells and infection by trypomastigotes, suggesting a low permeability for this compound in trypanosomes.
Highlights
The mitochondria have a fundamental role in intracellular Ca2+ homeostasis, buffering cytosolic Ca2+ increases arising from both influx from the extracellular space and release from intracellular organelles
The most important findings of this work are: (1) Ru360 and Ru265 are nM inhibitors of the mitochondrial Ca2+ uniporter (MCU) complex of T. cruzi with Ru265 being the most effective, with an IC50 of only 26.3 ± 2.0 nM; (2) ablation of either TcMICU1 or TcMICU2 increased the sensitivity of TcMCU complex to Ru360 and Ru265; and (3) application of Ru265 to intact cells did not significantly affect epimastigotes growth or respiration, mitochondrial Ca2+ uptake in intact cells, and trypomastigote attachment and invasion of host cells, suggesting low permeability in trypanosomes
It is important to note that while ruthenium red (RuRed) is effective in the submicromolar range [34], Ru360 is effective with an IC50 between 0.2 and 2 nM [25] in mammalian mitochondria, suggesting that the TcMCU complex is less sensitive to Ru360
Summary
The mitochondria have a fundamental role in intracellular Ca2+ homeostasis, buffering cytosolic Ca2+ increases arising from both influx from the extracellular space and release from intracellular organelles. The discovery that Trypanosoma cruzi, the agent of Chagas disease, possesses a mitochondrial Ca2+ uptake mechanism with similar characteristics to those of animal mitochondria [9,10], combined with the finding of the absence of the channel in Saccharomyces cerevisiae [11] and the availability of the genomes of these species, was important for the molecular identification, first of a modulator of the channel, mitochondrial Ca2+ uptake 1 (MICU1) [12] and of the pore subunit of the channel or MCU [7,8] After this discovery several accessory proteins were found, like MCU regulator 1 (MCUR1) [13], MICU2 and MICU3 [14], MCUb [15], and essential MCU regulator (EMRE) [16], comprising an MCU complex (uniplex or holocomplex). All these characteristics are suggestive of the parallel evolution of the complex in trypanosomes [24], which belong to the Discoba supergroup of eukaryotes, and animal cells, which belong to the Opisthokonta supergroup
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