Metabolic reprogramming contributes to radioprotection by protein kinase Cδ.

The Journal of Biological Chemistry Pub Date : 2023-10-01 Epub Date: 2023-08-21 DOI:10.1016/j.jbc.2023.105186
Angela M Ohm, Trisiani Affandi, Julie A Reisz, M Cecilia Caino, Angelo D'Alessandro, Mary E Reyland
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Abstract

Loss of protein kinase Cδ (PKCδ) activity renders cells resistant to DNA damaging agents, including irradiation; however, the mechanism(s) underlying resistance is poorly understood. Here, we have asked if metabolic reprogramming by PKCδ contributes to radioprotection. Analysis of global metabolomics showed that depletion of PKCδ affects metabolic pathways that control energy production and antioxidant, nucleotide, and amino acid biosynthesis. Increased NADPH and nucleotide production in PKCδ-depleted cells is associated with upregulation of the pentose phosphate pathway (PPP) as evidenced by increased activation of G6PD and an increase in the nucleotide precursor, 5-phosphoribosyl-1-pyrophosphate. Stable isotope tracing with U-[13C6] glucose showed reduced utilization of glucose for glycolysis in PKCδ-depleted cells and no increase in U-[13C6] glucose incorporation into purines or pyrimidines. In contrast, isotope tracing with [13C5, 15N2] glutamine showed increased utilization of glutamine for synthesis of nucleotides, glutathione, and tricarboxylic acid intermediates and increased incorporation of labeled glutamine into pyruvate and lactate. Using a glycolytic rate assay, we confirmed that anaerobic glycolysis is increased in PKCδ-depleted cells; this was accompanied by a reduction in oxidative phosphorylation, as assayed using a mitochondrial stress assay. Importantly, pretreatment of cells with specific inhibitors of the PPP or glutaminase prior to irradiation reversed radioprotection in PKCδ-depleted cells, indicating that these cells have acquired codependency on the PPP and glutamine for survival. Our studies demonstrate that metabolic reprogramming to increase utilization of glutamine and nucleotide synthesis contributes to radioprotection in the context of PKCδ inhibition.

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代谢重编程有助于蛋白激酶Cδ的辐射保护。
蛋白激酶Cδ(PKCδ)活性的丧失使细胞对DNA损伤剂具有抵抗力,包括辐射;然而,人们对阻力背后的机制知之甚少。在这里,我们询问PKCδ的代谢重编程是否有助于辐射保护。全球代谢组学分析表明,PKCδ的缺失影响控制能量产生和抗氧化剂、核苷酸和氨基酸生物合成的代谢途径。PKCδ缺失细胞中NADPH和核苷酸产生的增加与戊糖磷酸途径(PPP)的上调有关,G6PD的激活增加和核苷酸前体5-磷酸核糖基-1-焦磷酸的增加证明了这一点。用U-[13C6]葡萄糖进行稳定同位素示踪显示,在PKCδ缺失的细胞中,葡萄糖用于糖酵解的利用率降低,而U-[13C6]葡萄糖掺入嘌呤或嘧啶的情况没有增加。相反,[13C5,15N2]谷氨酰胺的同位素示踪显示,谷氨酰胺在核苷酸、谷胱甘肽和三羧酸中间体合成中的利用率增加,标记的谷氨酰胺在丙酮酸和乳酸盐中的结合率增加。使用糖酵解速率测定,我们证实在PKCδ缺失的细胞中厌氧糖酵解增加;这伴随着氧化磷酸化的减少,如使用线粒体应激测定所测定的。重要的是,在照射前用PPP或谷氨酰胺酶的特异性抑制剂预处理细胞,逆转了PKCδ缺失细胞的放射保护作用,表明这些细胞已经获得了对PPP和谷氨酰胺的相互依赖性。我们的研究表明,在PKCδ抑制的情况下,代谢重编程以增加谷氨酰胺和核苷酸合成的利用有助于辐射保护。
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