Quantitative flux analysis reveals folate-dependent NADPH production.
Quantitative flux analysis reveals folate-dependent NADPH production.
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三磷酸腺苷是动物机械和电子工作(如肌肉收缩、神经元放电)的主要能量来源。对于化学工作，NADPH也有同样重要的作用，它为氧化还原防御和还原生物合成提供动力。从葡萄糖合成NADPH的最直接途径是磷酸戊糖氧化途径(OxPPP)，苹果酸酶有时也很重要。虽然糖酵解和氧化磷酸化对ATP产生的相对贡献已经得到了广泛的分析，但对NADPH代谢的类似分析还很少。在这里，我们展示了通过液-质联用直接跟踪从标记底物进入NADPH的氘的能力，并将该方法与碳标记和数学模型相结合来测量细胞质中的NADPH通量。在增殖细胞中，对胞浆NADPH的最大贡献是oxPPP。令人惊讶的是，几乎相似的贡献来自丝氨酸驱动的一碳代谢，其中亚甲基四氢叶酸氧化为10-甲酰基-四氢叶酸与NADP+还原为NADPH相耦合。此外，线粒体一碳代谢的示踪显示，10-甲酰-四氢叶酸完全氧化生成NADPH。由于叶酸代谢以前不被认为是NADPH的产生，因此通过敲除亚甲基四氢叶酸脱氢酶(MTHFD)基因来确认其功能意义。胞浆或线粒体MTHFD同工酶的耗尽导致细胞NADPH/NADP+和GSH/GSSG比值降低，细胞对氧化应激的敏感性增加。因此，虽然叶酸代谢对增殖细胞的重要性早已被认识到，并归因于它为核酸合成产生一个碳单位的功能，但这一途径的另一个关键功能是产生还原能力。
ATP is the dominant energy source in animals for mechanical and electrical work (e.g., muscle contraction, neuronal firing). For chemical work, there is an equally important role for NADPH, which powers redox defense and reductive biosynthesis. The most direct route to produce NADPH from glucose is the oxidative pentose phosphate pathway (oxPPP), with malic enzyme sometimes also important. While the relative contribution of glycolysis and oxidative phosphorylation to ATP production has been extensively analyzed, similar analysis of NADPH metabolism has been lacking. Here we demonstrate the ability to directly track, by liquid chromatography-mass spectrometry, the passage of deuterium from labeled substrates into NADPH, and combine this approach with carbon labeling and mathematical modeling to measure cytosolic NADPH fluxes. In proliferating cells, the largest contributor to cytosolic NADPH is the oxPPP. Surprisingly a nearly comparable contribution comes from serine-driven one-carbon metabolism, where oxidation of methylene tetrahydrofolate to 10-formyl-tetrahydrofolate is coupled to reduction of NADP+ to NADPH. Moreover, tracing of mitochondrial one-carbon metabolism revealed complete oxidation of 10-formyl-tetrahydrofolate to make NADPH. Since folate metabolism has not previously been considered an NADPH producer, confirmation of its functional significance was undertaken through knockdown of methylenetetrahydrofolate dehydrogenase (MTHFD) genes. Depletion of either the cytosolic or mitochondrial MTHFD isozyme resulted in decreased cellular NADPH/NADP+ and GSH/GSSG ratios and increased cell sensitivity to oxidative stress. Thus, while the importance of folate metabolism for proliferating cells has been long recognized and attributed to its function of producing one carbon units for nucleic acid synthesis, another crucial function of this pathway is generating reducing power.
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DOI：
10.1126/science.1211485
发表时间：
2011-12-02
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Anastasiou D;Poulogiannis G;Asara JM;Boxer MB;Jiang JK;Shen M;Bellinger G;Sasaki AT;Locasale JW;Auld DS;Thomas CJ;Vander Heiden MG;Cantley LC
通讯作者：
Cantley LC
影响因子：
7.4
作者：
Melamud E;Vastag L;Rabinowitz JD
通讯作者：
Rabinowitz JD
影响因子：
3.9
作者：
Price, NE;Cook, PF
通讯作者：
Cook, PF
影响因子：
6.3
作者：
Brekke, Eva M. F.;Walls, Anne B.;Sonnewald, Ursula
通讯作者：
Sonnewald, Ursula
影响因子：
2.9
作者：
Pawelek, PD;MacKenzie, RE
通讯作者：
MacKenzie, RE