Abstract
Recent studies have revealed that the oxidative pentose phosphate pathway (PPP), malic enzyme (ME), and folate metabolism are the three major routes for generating cellular NADPH, a key cofactor involved in redox control and reductive biosynthesis. Many tumor cells exhibit altered NADPH metabolism to fuel their rapid proliferation. However, little is known about how NADPH metabolism is coordinated in tumor cells. Here we report that ME1 increases the PPP flux by forming physiological complexes with 6-phosphogluconate dehydrogenase (6PGD). We found that ME1 and 6PGD form a hetero-oligomer that increases the capability of 6PGD to bind its substrate 6-phosphogluconate. Through activating 6PGD, ME1 enhances NADPH generation, PPP flux, and tumor cell growth. Interestingly, although ME1 could bind either the dimer-defect mutant 6PGD (K294R) or the NADP+-binding defect 6PGD mutants, only 6PGD (K294R) activity was induced by ME1. Thus, ME1/6PGD hetero-complexes may mimic the active oligomer form of 6PGD. Together, these findings uncover a direct cross-talk mechanism between ME1 and PPP, may reveal an alternative model for signaling transduction via protein conformational simulation, and pave the way for better understanding how metabolic pathways are coordinated in cancer.
Highlights
Recent studies have revealed that the oxidative pentose phosphate pathway (PPP), malic enzyme (ME), and folate metabolism are the three major routes for generating cellular NADPH, a key cofactor involved in redox control and reductive biosynthesis
To explore the intrinsic relationship among NADPH metabolic pathways, we examined whether ME1 coordinates PPP to
U2OS cells stably expressing ME1 or control vector were cultured in medium containing [1,2-13C2] glucose, and the oxidative PPP flux was measured on the basis of the rate of glucose consumption and the generation of 13C-labeling lactate (M1) determined by liquid chromatography-mass spectrometry (LC-MS)
Summary
To explore the intrinsic relationship among NADPH metabolic pathways, we examined whether ME1 coordinates PPP to. Both mutants were able to markedly increase 6PGD enzymatic activity and 6PGD-mediated NADPH generation in vitro (Fig. 3F, supplemental Fig. 2B) These effects were observed in 293T cells. Like wild-type ME1, these mutant forms of ME1 increased the number of colonies (Fig. 3H) Together, these findings suggest that ME1 promotes cell proliferation independent of its enzymatic activity, correlating with its effect on 6PGD (Fig. 3G). Immunoprecipitation assays showed that this mutant 6PGD lost the ability to bind to ME1 (Fig. 4F, fourth lane), and consistently, no enhanced 6PGD enzymatic activity was observed when ME1 was present (Fig. 4E, supplemental Fig. 4C) These data suggest that direct protein-protein association is required for ME1-mediated 6PGD activation
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