Department of Genetics, University Medical Center Utrecht, Utrecht University, Utrecht, 3584, CX, the Netherlands; Center for Molecular Medicine, University Medical Center Utrecht, Utrecht University, Utrecht, 3584, CX, the Netherlands; Department of Pediatrics, University Medical Center Utrecht, Utrecht University, Utrecht, 3584, CX, the Netherlands.
Department of Genetics, University Medical Center Utrecht, Utrecht University, Utrecht, 3584, CX, the Netherlands; Center for Molecular Medicine, University Medical Center Utrecht, Utrecht University, Utrecht, 3584, CX, the Netherlands.
Biochim Biophys Acta Gen Subj. 2020 Mar;1864(3):129484. doi: 10.1016/j.bbagen.2019.129484. Epub 2019 Nov 14.
High glutaminase (GLS;EC3.5.1.2) activity is an important pathophysiological phenomenon in tumorigenesis and metabolic disease. Insight into the metabolic consequences of high GLS activity contributes to the understanding of the pathophysiology of both oncogenic pathways and inborn errors of glutamate metabolism. Glutaminase catalyzes the conversion of glutamine into glutamate, thereby interconnecting many metabolic pathways.
We developed a HEK293-based cell-model that enables tuning of GLS activity by combining the expression of a hypermorphic GLS variant with incremental GLS inhibition. The metabolic consequences of increasing GLS activity were studied by metabolic profiling using Direct-Infusion High-Resolution Mass-Spectrometry (DI-HRMS).
Of 12,437 detected features [m/z], 109 features corresponding to endogenously relevant metabolites were significantly affected by high GLS activity. As expected, these included strongly decreased glutamine and increased glutamate levels. Additionally, increased levels of tricarboxylic acid (TCA) intermediates with a truncation of the TCA cycle at the level of citrate were detected as well as increased metabolites of transamination reactions, proline and ornithine synthesis and GABA metabolism. Levels of asparagine and nucleotide metabolites showed the same dependence on GLS activity as glutamine. Of the nucleotides, especially metabolites of the pyrimidine thymine metabolism were negatively impacted by high GLS activity, which is remarkable since their synthesis depend both on aspartate (product of glutamate) and glutamine levels. Metabolites of the glutathione synthesizing γ-glutamyl-cycle were either decreased or unaffected.
By providing a metabolic fingerprint of increasing GLS activity, this study shows the large impact of high glutaminase activity on the cellular metabolome.
高谷氨酰胺酶（GLS；EC3.5.1.2）活性是肿瘤发生和代谢疾病中的一个重要病理生理现象。深入了解高 GLS 活性的代谢后果有助于理解致癌途径和谷氨酸代谢先天错误的病理生理学。谷氨酰胺酶催化谷氨酰胺转化为谷氨酸，从而将许多代谢途径相互连接。
我们开发了一种基于 HEK293 的细胞模型，通过组合表达超活力 GLS 变体和递增 GLS 抑制来调节 GLS 活性。通过使用直接进样高分辨率质谱（DI-HRMS）进行代谢谱分析研究了增加 GLS 活性的代谢后果。
在检测到的 12437 个特征[m/z]中，有 109 个与内源性相关代谢物显著相关的特征受到高 GLS 活性的显著影响。正如预期的那样，这包括谷氨酰胺水平的大幅降低和谷氨酸水平的增加。此外，还检测到三羧酸（TCA）循环中柠檬酸水平截断的 TCA 中间产物以及转氨基反应、脯氨酸和鸟氨酸合成和 GABA 代谢的增加代谢物。天冬酰胺和核苷酸代谢物的水平与谷氨酰胺对 GLS 活性的依赖性相同。在核苷酸中，特别是嘧啶胸腺嘧啶代谢物的代谢物受到高 GLS 活性的负面影响，这很明显，因为它们的合成既依赖于谷氨酸（谷氨酸的产物）又依赖于谷氨酰胺水平。谷胱甘肽合成的γ-谷氨酰环的代谢物要么减少，要么不受影响。
本研究通过提供增加 GLS 活性的代谢指纹，展示了高谷氨酰胺酶活性对细胞代谢组的巨大影响。
