Enzymatic depletion of mitochondrial inorganic polyphosphate increases the generation of reactive oxygen species and the activity of the pentose phosphate pathway in mammalian cells.

Abstract

Dysregulated bioenergetics and redox imbalance are well-known underlying mechanisms in the etiopathology of all the major neurodegenerative diseases. One of the metabolic pathways affected in these conditions is the pentose phosphate pathway (PPP). The PPP occurs parallel to glycolysis in the cytosol and contributes to maintaining appropriate cellular redox homeostasis and anabolic biosynthesis. Inorganic polyphosphate (polyP) is a ubiquitous biopolymer shown to be involved in multiple physiological processes. Found highly colocalized with mitochondria, its role in mitochondrial bioenergetics has been previously demonstrated, but the full extent and the involved mechanisms, in mammals, are not fully understood. Here, we investigate the regulatory effects of polyP on the PPP in a mammalian cell model. We used biochemical assays, protein analysis, 13C-NMR, and metabolomics techniques in HEK293 cells, under control conditions and after enzymatical depletion of mitochondrial polyP (MitoPPX cells). Our results indicate that MitoPPX cells have elevated reactive oxygen species levels, characterized by an increased presence of mitochondrial O2⋅− and intracellular H2O2, potentially a consequence of decreased oxidative phosphorylation, previously demonstrated. MitoPPX cells also show increased levels of antioxidant proteins, like superoxide dismutase-2, peroxiredoxin-1, and thioredoxin along with higher glutathione levels. Moreover, MitoPPX cells have higher glucose flux to the PPP as compared to glycolysis, and increased protein levels of PPP enzyme transaldolase, clearly indicating the upregulation of the PPP. These results suggest that mitochondrial polyP plays a key role in the regulation of PPP and maintenance of cellular redox and bioenergetic balance. Our research enhances the current understanding of the extensive effects of polyP on crucial mitochondrial processes thereby affecting cellular physiology. This could contribute towards designing novel pharmacological and therapeutic strategies targeting diseases overlying mitochondrial dysfunction.