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. 2022 Mar 31;11(4):685.
doi: 10.3390/antiox11040685.

Enzymatic Depletion of Mitochondrial Inorganic Polyphosphate (polyP) Increases the Generation of Reactive Oxygen Species (ROS) and the Activity of the Pentose Phosphate Pathway (PPP) in Mammalian Cells

Vedangi Hambardikar  1 , Mariona Guitart-Mampel  1 , Ernest R Scoma  1 , Pedro Urquiza  1 , Gowda G A Nagana  2 , Daniel Raftery  2 , John A Collins  3 , Maria E Solesio  1
Affiliations

Enzymatic Depletion of Mitochondrial Inorganic Polyphosphate (polyP) Increases the Generation of Reactive Oxygen Species (ROS) and the Activity of the Pentose Phosphate Pathway (PPP) in Mammalian Cells

Vedangi Hambardikar et al. Antioxidants (Basel). .

Abstract

Inorganic polyphosphate (polyP) is an ancient biopolymer that is well preserved throughout evolution and present in all studied organisms. In mammals, it shows a high co-localization with mitochondria, and it has been demonstrated to be involved in the homeostasis of key processes within the organelle, including mitochondrial bioenergetics. However, the exact extent of the effects of polyP on the regulation of cellular bioenergetics, as well as the mechanisms explaining these effects, still remain poorly understood. Here, using HEK293 mammalian cells under Wild-type (Wt) and MitoPPX (cells enzymatically depleted of mitochondrial polyP) conditions, we show that depletion of polyP within mitochondria increased oxidative stress conditions. This is characterized by enhanced mitochondrial O2- and intracellular H2O2 levels, which may be a consequence of the dysregulation of oxidative phosphorylation (OXPHOS) that we have demonstrated in MitoPPX cells in our previous work. These findings were associated with an increase in basal peroxiredoxin-1 (Prx1), superoxide dismutase-2 (SOD2), and thioredoxin (Trx) antioxidant protein levels. Using 13C-NMR and immunoblotting, we assayed the status of glycolysis and the pentose phosphate pathway (PPP) in Wt and MitoPPX cells. Our results show that MitoPPX cells display a significant increase in the activity of the PPP and an increase in the protein levels of transaldolase (TAL), which is a crucial component of the non-oxidative phase of the PPP and is involved in the regulation of oxidative stress. In addition, we observed a trend towards increased glycolysis in MitoPPX cells, which corroborates our prior work. Here, for the first time, we show the crucial role played by mitochondrial polyP in the regulation of mammalian redox homeostasis. Moreover, we demonstrate a significant effect of mitochondrial polyP on the regulation of global cellular bioenergetics in these cells.

Keywords: ROS; antioxidants; mammalian bioenergetics; mitochondria; mitochondrial inorganic polyphosphate; pentose phosphate pathway; polyP.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Mitochondria isolated from MitoPPX cells show decreased levels of polyP. However, the enzymatical depletion of the polymer within the organelle does not have a deleterious effect on cell viability in HEK293 cells. (A) Graph showing the results of the MTT cell viability assays. Note that no significant differences in terms of cell viability were found between Wt and MitoPPX cells in this experiment. (B) Graph showing the concentration of polyP in mitochondria isolated from Wt and MitoPPX cells. To demonstrate the decreased levels of the polymer in mitochondria isolated from MitoPPX cells, polyP was measured using DAPI fluorescence after the isolation of the organelles. PolyP concentrations were determined based on extrapolation from the standard curve, which was prepared with exogenous polyP. Data in the graph are shown as mean ± standard deviation of triplicates obtained from at least three independent experiments (n = 3). Unpaired t-tests were used to detect significant differences between Wt and MitoPPX cells. Abbreviations used in this figure: MitoPPX: cells expressing the exopolyphosphatase enzyme in mitochondria; Wt: Wild-type cells; PolyP: inorganic polyphosphate, MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-dypheniyltretazolium bromide, DAPI: 4′-6-diamidino-2-phenylindole.
Figure 2
Figure 2
MitoPPX cells show increased levels of ROS. (A) Representative confocal images showing increased levels of O2−, measured by MitoSox Red, in MitoPPX cells, when compared with the Wt cells. (B) Quantification of the confocal images obtained after the incubation of samples with MitoSox Red. (C) Quantification of the fluorescence signal in Wt and MitoPPX cells after the incubation of the samples with Amplex Red to measure H2O2 generation. Data in all graphs are shown as mean ± standard deviation of at least 60 fields (MitoSox Red); triplicates were obtained from at least three independent experiments. Unpaired t-tests were used to detect significant differences between Wt and MitoPPX cells. Scale bar: 200 μM. Abbreviations used in this figure: ROS: reactive oxygen species; MitoPPX: cells expressing the exopolyphosphatase enzyme in mitochondria; Wt: Wild-type cells; O2−: superoxide; H2O2: hydrogen peroxide.
Figure 3
Figure 3
Antioxidant levels are increased in MitoPPX cells. (A) Non-reducing immunoblotting was performed to detect the basal levels of SOD2, Prx1, Trx, Nrx, Prx3, and Nrf2 in MitoPPX and Wt cells. Representative membranes are shown. β-actin was used as a loading control. (B) Densitometric analysis of basal SOD2, Prx1, Trx, Nrx, Prx3, and Nrf2 protein levels. Data shown as mean ± standard deviation from three independent experiments are presented. Unpaired t-tests were used to detect significant differences between Wt and MitoPPX cells. Abbreviations used in this figure: SOD: superoxide dismutase 2; Prx1: peroxiredoxine1; Trx: thioredoxin; Nrx: nucleoredoxin; Prx3: peroxiredoxine 3; Nrf2: nuclear factor erythroid 2-related factor 2; MitoPPX: cells expressing the exopolyphosphatase enzyme on mitochondria; Wt: Wild-type cells.
Figure 3
Figure 3
Antioxidant levels are increased in MitoPPX cells. (A) Non-reducing immunoblotting was performed to detect the basal levels of SOD2, Prx1, Trx, Nrx, Prx3, and Nrf2 in MitoPPX and Wt cells. Representative membranes are shown. β-actin was used as a loading control. (B) Densitometric analysis of basal SOD2, Prx1, Trx, Nrx, Prx3, and Nrf2 protein levels. Data shown as mean ± standard deviation from three independent experiments are presented. Unpaired t-tests were used to detect significant differences between Wt and MitoPPX cells. Abbreviations used in this figure: SOD: superoxide dismutase 2; Prx1: peroxiredoxine1; Trx: thioredoxin; Nrx: nucleoredoxin; Prx3: peroxiredoxine 3; Nrf2: nuclear factor erythroid 2-related factor 2; MitoPPX: cells expressing the exopolyphosphatase enzyme on mitochondria; Wt: Wild-type cells.
Figure 4
Figure 4
Mitochondrial depletion of polyP increases the protein levels of TAL. (A) Graph showing the percentage of glucose flux through the PPP, measured using NMR, in Wt and MitoPPX cells. Data in all graphs are shown as average ± standard deviation of four independent NMR experiments, which were conducted with samples obtained on four separate days. Unpaired t-tests were used to detect significant differences between Wt and MitoPPX cells. (B) Non-reducing immunoblotting was performed to detect the basal levels of G6PD and PGD (oxidative phase of the PPP) and TK and TAL (non-oxidative phase of the PPP) in Wt and MitoPPX cells. Representative immunoblots are shown. β-actin was used as a loading control. (C) Densitometric analysis of basal G6PD, PGD, TK, and TAL protein levels. Data shown as mean ± standard deviation from n = 3 independent experiments are presented. Unpaired t-tests were used to detect significant differences between Wt and MitoPPX cells. Abbreviations used in this figure: TAL: transaldolase; PPP: pentose phosphate pathway; NMR: nuclear magnetic resonance; G6PD: glucose-6-phosphate dehydrogenase; PGD: phosphogluconate dehydrogenase; TK: transketolase; MitoPPX: cells expressing the exopolyphosphatase enzyme in mitochondria; Wt: Wild-type cells.
Figure 4
Figure 4
Mitochondrial depletion of polyP increases the protein levels of TAL. (A) Graph showing the percentage of glucose flux through the PPP, measured using NMR, in Wt and MitoPPX cells. Data in all graphs are shown as average ± standard deviation of four independent NMR experiments, which were conducted with samples obtained on four separate days. Unpaired t-tests were used to detect significant differences between Wt and MitoPPX cells. (B) Non-reducing immunoblotting was performed to detect the basal levels of G6PD and PGD (oxidative phase of the PPP) and TK and TAL (non-oxidative phase of the PPP) in Wt and MitoPPX cells. Representative immunoblots are shown. β-actin was used as a loading control. (C) Densitometric analysis of basal G6PD, PGD, TK, and TAL protein levels. Data shown as mean ± standard deviation from n = 3 independent experiments are presented. Unpaired t-tests were used to detect significant differences between Wt and MitoPPX cells. Abbreviations used in this figure: TAL: transaldolase; PPP: pentose phosphate pathway; NMR: nuclear magnetic resonance; G6PD: glucose-6-phosphate dehydrogenase; PGD: phosphogluconate dehydrogenase; TK: transketolase; MitoPPX: cells expressing the exopolyphosphatase enzyme in mitochondria; Wt: Wild-type cells.
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