PFAS介导的DRP1脱酰胺化将线粒体动力学与嘌呤生物合成相偶联

Cancer cells dynamically remodel mitochondrial networks to balance energy production and biosynthetic precursor generation, but how mitochondrial dynamics are coordinated with specific biosynthetic pathways during cell cycle progression remains unclear. Phosphoribosylformylglycinamidine synthase (PFAS) is a key enzyme in de novo purine synthesis. It forms purinosome with other purine synthetic enzymes to support rapid nucleotide synthesis. As the scaffold of this mitochondria-adjacent multienzyme complex, how PFAS links mitochondrial dynamics to purine synthesis remains unknown. We found that PFAS deletion markedly impaired cancer cell proliferation and depleted the nucleotide pool. Moreover, PFAS deletion led to reduced cellular energy production and disrupted redox balance. Interestingly, hypoxanthine supplementation in PFAS-deleted cells successfully restored nucleotide levels to control levels, but it did not rescue redox balance or cell proliferation, indicating a purine-independent function of PFAS. Moreover, [U- 13 C]glucose isotope tracing experiments demonstrated that PFAS deletion reduced glucose-derived carbon entry into the TCA cycle, consistent with suppressed oxidative metabolism. In parallel, [U- 13 C]glutamine tracing revealed that glutamine contribution to TCA intermediates and aspartate was increased upon PFAS depletion, indicating a shift toward glutamine-supported anaplerosis. Furthermore, mitochondrial proteomics showed that PFAS deletion caused a compensatory upregulation of enzymes in tricarboxylic acid (TCA) cycle and electron transport chain. PFAS-deletion-mediated metabolic defects suggested an impaired mitochondrial function. We therefore examined mitochondrial morphology in control and PFAS-deleted cells. Indeed, transmission electron microscopy revealed strikingly increased fragmentated mitochondria in PFAS-deficient cells. We then focus on the molecular mechanisms by which PFAS regulates mitochondrial morphology. LC-MS/MS and biochemical assays demonstrated that PFAS is a bona fide deamidase of DRP1. Deamidated DRP1 (N267D/N268D) completely lost GTPase activity and showed impaired GTP binding activity, further failing to drive mitochondrial fission. Interestingly, DRP1 deamidation increased during G1 phase and peaked in S phase, coinciding with mitochondrial elongation and the heightened nucleotide synthesis. Thus, PFAS-mediated DRP1 deamidation couples mitochondrial networks to the metabolic demand of proliferative cells. Collectively, the de novo purine synthetic enzyme PFAS deamidates DRP1 to regulate mitochondrial morphology, further promoting mitochondrial oxidative phosphorylation and nucleotide synthesis. These findings uncover a previously unrecognized link between nucleotide biosynthesis and mitochondrial dynamics, providing a framework for targeting the PFAS-DRP1 axis in cancer.