Abstract
Repair of a certain type of oxidative DNA damage leads to the release of phosphoglycolate, which is an inhibitor of triose phosphate isomerase and is predicted to indirectly inhibit phosphoglycerate mutase activity. Thus, we hypothesized that phosphoglycolate might play a role in a metabolic DNA damage response. Here, we determined how phosphoglycolate is formed in cells, elucidated its effects on cellular metabolism and tested whether DNA damage repair might release sufficient phosphoglycolate to provoke metabolic effects. Phosphoglycolate concentrations were below 5 µM in wild-type U2OS and HCT116 cells and remained unchanged when we inactivated phosphoglycolate phosphatase (PGP), the enzyme that is believed to dephosphorylate phosphoglycolate. Treatment of PGP knockout cell lines with glycolate caused an up to 500-fold increase in phosphoglycolate concentrations, which resulted largely from a side activity of pyruvate kinase. This increase was much higher than in glycolate-treated wild-type cells and was accompanied by metabolite changes consistent with an inhibition of phosphoglycerate mutase, most likely due to the removal of the priming phosphorylation of this enzyme. Surprisingly, we found that phosphoglycolate also inhibits succinate dehydrogenase with a Ki value of <10 µM. Thus, phosphoglycolate can lead to profound metabolic disturbances. In contrast, phosphoglycolate concentrations were not significantly changed when we treated PGP knockout cells with Bleomycin or ionizing radiation, which are known to lead to the release of phosphoglycolate by causing DNA damage. Thus, phosphoglycolate concentrations due to DNA damage are too low to cause major metabolic changes in HCT116 and U2OS cells.
Highlights
Phosphoglycolate can be formed in all organisms during the repair of DNA damage
Three lines of evidence suggest that the activity on phosphoglycolate might not be the most important function of phosphoglycolate phosphatase (PGP): first, we have previously shown that PGP plays a key role in cellular metabolism by eliminating two glycolytic side products, that otherwise interfere with glycolysis and the pentose phosphate pathway [18] (Supplementary Figure S1B)
We measured phosphoglycolate concentrations in PGP knockout cell lines derived from the HCT116 colorectal cancer and U2OS osteosarcoma cell lines, which were cultured either in the presence or absence of glycolate, a potential biosynthetic precursor of glycolate
Summary
Phosphoglycolate can be formed in all organisms during the repair of DNA damage. Oxidative stress or treatment with certain chemotherapeutic drugs (e.g. Bleomycin) and ionizing radiation leads to the oxidative cleavage of deoxyribose moieties, resulting in DNA 30-ends that carry a phosphoglycolate group [1]. Treatment of PGP knockout cell lines with glycolate caused an up to 500-fold increase in phosphoglycolate concentrations, which resulted largely from a side activity of pyruvate kinase.
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