Phosphoglycolate has profound metabolic effects but most likely no role in a metabolic DNA response in cancer cell lines

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.

Metabolite Repair Enzymes Control Metabolic Damage in Glycolysis

“…We purified 4P-erythronate phosphatase, identified its gene (PGP), and knocked it out in the human colorectal cancer cell line HCT116, the human osteosarcoma cell line U2OS, and in immortalized human fibroblasts. The PGP-deficient cell lines not only showed a marked increase (5-20-fold) in concentrations of 4P-erythronate but also even stronger increases in 6P-gluconate [10,30], the substrate of the enzyme that is inhibited by 4P-erythronate (Figure 2D) [10,50].…”

“…In some cases, these activities might appear to catalyze novel physiological reactions, albeit at a rate that is far too low to influence the metabolism of the specific metabolites [28,29]. In other cases, activities may be observed with metabolites that are absent from cells [30]. To complicate matters even further, some metabolite repair enzymes have been shown to be multispecific, in other words they act on several different noncanonical metabolites, but they remarkably avoid acting on structurally similar physiological metabolites (see comments below on phosphoglycolate phosphatase, PGP) [10].…”

“…Knocking out PGP in HCT116 cells caused a marked increase in the concentration of 2P-lactate, which in turn perturbed the regulation of glycolysis by inhibiting the synthesis of the glycolytic regulator fructose-2,6BP [10]. The name PGP relates to the fact that this enzyme hydrolyses a third noncanonical metabolite, 2-phosphoglycolate (2P-glycolate), which can be formed when pyruvate kinase erroneously acts on glycolate [30,52] (Figure 1, pathway 4; Figure 2D). Recent work indicates that HCT116 cells produce very little 2P-glycolate unless they are artificially provided with its biochemical precursor glycolate [30].…”

“…The name PGP relates to the fact that this enzyme hydrolyses a third noncanonical metabolite, 2-phosphoglycolate (2P-glycolate), which can be formed when pyruvate kinase erroneously acts on glycolate [30,52] (Figure 1, pathway 4; Figure 2D). Recent work indicates that HCT116 cells produce very little 2P-glycolate unless they are artificially provided with its biochemical precursor glycolate [30]. Lack of destruction of 2P-glycolate under these conditions has several metabolic consequences, including functional inactivation of phosphoglycerate mutase (PGAM) [30], as well as inhibition of succinate dehydrogenase (SDH) [30] and potentially triose phosphate isomerase (TPI) [53].…”

“…Recent work indicates that HCT116 cells produce very little 2P-glycolate unless they are artificially provided with its biochemical precursor glycolate [30]. Lack of destruction of 2P-glycolate under these conditions has several metabolic consequences, including functional inactivation of phosphoglycerate mutase (PGAM) [30], as well as inhibition of succinate dehydrogenase (SDH) [30] and potentially triose phosphate isomerase (TPI) [53]. To what extent accumulation of 2P-glycolate might contribute to the phenotype of PGP loss in normal mammalian cells is still subject to investigation [26,30].…”