Glutamine synthetase (GS), the only enzyme responsible for de novo glutamine synthesis, plays a significant role in cancer progression. As an example of the consequences of GS mutations, the R324C variant causes congenital glutamine deficiency, which results in brain abnormalities and neonatal death. However, the influence of GS-deficient mutations on cancer cells remains relatively unexplored. In this study, we investigated the effects of GS and GS-deficient mutations, including R324C and previously unreported K241R, which serve as models for GS inactivation. This study provided intriguing insights into the intricate relationship between GS mutations and cancer cell metabolism. Our findings strongly support recent studies that suggest GS deletion leads to the suppression of diverse signaling cascades associated with glutamine metabolism under glutamine-stripping conditions. The affected processes include DNA synthesis, the citric acid cycle, and reactive oxygen species (ROS) detoxification. This suppression originates from the inherent inability of cells to autonomously synthesize glutamine under glutamine-depleted conditions. As a key source of reduced nitrogen, glutamine is crucial for the formation of purine and pyrimidine bases, which are essential building blocks for DNA synthesis. Furthermore, the citric acid cycle is inhibited by the absence of negatively charged glutamate within the mitochondrial matrix, particularly when glutamine is scarce. This deficiency decreases the flux of α-ketoglutarate (α-KG), a principal driver of the citric acid cycle. Intermediate metabolites of the citric acid cycle directly or indirectly contribute to the generation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, a core component of redox homeostasis. Using the GS_R324C and GS_K241R mutants, we conducted an integrative transcriptomics and metabolomics analysis. The GS mutants with reduced activity activated multiple amino acid biosynthesis pathways, including arginine-proline, glycine-serine-threonine, and alanine-aspartate-glutamate metabolism. This intriguing behavior led us to hypothesize that despite hindrance of the citric acid cycle, abundant intracellular glutamate is redirected through alternative processes, including transamination. Simultaneously, key metabolic enzymes in the amino acid synthesis pathways, such as glutamic-oxaloacetic transaminase 1 (GOT1), glutamic-pyruvic transaminase 2 (GPT2), pyrroline-5-carboxylate reductase 1 (PYCR1), and phosphoserine aminotransferase 1 (PSAT1), exhibited increased mRNA levels. Additionally, GS deficiency appeared to upregulate the expression of glutamine transporters SLC38A2 and SLC1A5. Thus, restricting extracellular amino acids, such as glutamine, induces a stress response while promoting transcription or translation by a select group of genes, thereby facilitating cellular adaptation. However, similar to GS_WT, both GS_R324C and GS_K241R were modulated by glutamine treatment. Among GS-activity-dependent behaviors, the increased expression of numerous aminoacyl-tRNA synthetases (ARSs), which are critical for aminoacyl-tRNA biosynthesis, remains poorly understood. Most ARS-encoding genes are transcriptionally induced by activating transcription factor 4 (ATF4), the expression of which increases under oxidative stress, endoplasmic reticulum stress, hypoxia, and amino acid limitation. In GS-deficient cells, the increased expression of ATF4 was accompanied by pronounced stress caused by glutamine starvation. Thus, ARS upregulation may predominantly arise from increased ATF4 expression in GS-deficient cells. Additionally, transcriptomic analysis revealed the differential expression of specific genes, regardless of GS activity, suggesting that GS is involved in various processes other than glutamine synthesis, including angiogenesis. Although our omics study was limited to H1299 cells, in subsequent experiments, we validated our findings using additional cell lines, including Hepa1-6 and LN-229. To attain a more comprehensive understanding of the impact of the newly identified GS_K241R mutant, our investigation should be extended to various cell types and mouse models. In summary, we identified and investigated GS-deficient mutations in cancer cells and conducted an integrative transcriptomics-metabolomics analysis with comparisons to wild-type GS. This comprehensive approach provided crucial insights into the intricate pathways modulated by GS activity. Our findings advance the understanding of how GS functions in the context of reprogrammed cellular metabolism, particularly during glutamine deprivation. The altered metabolism triggered by elevated glutamate levels arising from GS mutations highlights the remarkable plasticity of cancer cell metabolism. Notably, considering the increasing research focus on GS as a potential therapeutic target in various cancer types, the findings of this study could provide innovative perspectives for drug development and the formulation of clinical treatment strategies. 谷氨酰胺合成酶(GS)是细胞中唯一可以从头合成谷氨酰胺的酶,在癌症代谢中扮演着重要角色。GS酶活缺陷突变往往导致严重的代谢疾病甚至是死亡。理解GS酶活降低对生理功能的影响可能为靶向治疗提供新的方向,同时对其内在机制的探索可以为治疗干预开辟新的途径。已报道的GS酶活缺陷突变包括R324C(第324位精氨酸突变为半胱氨酸)和R341C(第341位精氨酸突变为半胱氨酸)等,本研究新发现了GS的另一个酶活缺陷突变位点K241(第241位赖氨酸)。为了探究GS酶活缺陷突变带来的影响,本研究利用双组学技术对GS的酶活缺陷突变R324C和K241R(第241位赖氨酸突变为精氨酸)在肺癌细胞中的功能进行研究,通过对转录组和代谢组数据的整合揭示了GS酶活缺陷突变对一些重要生物过程的显著影响。GS的缺陷突变阻碍了细胞周期和多种氨基酸代谢途径。除了谷氨酰胺合成受到抑制,精氨酸-脯氨酸代谢、甘氨酸-丝氨酸-酪氨酸代谢以及天冬氨酸-谷氨酸-丙氨酸代谢在GS突变的细胞中表现出更强的活力,同时,氨酰-tRNA的生物合成途径也显著被激活。进一步的研究发现,GS酶活缺陷带来氨基酸代谢的改变源自谷氨酸的重新定向和相关代谢酶的表达变化,同时,氨酰-tRNA生物合成的激活源自谷氨酰胺在GS酶活缺陷细胞中带来的能量应激激活了特定蛋白的表达,如转录因子4(ATF4)。此外,细胞表型实验表明,GS酶活缺陷细胞的迁移能力低于野生型细胞。以上结果揭示了GS酶活缺陷突变体细胞中的代谢重编程现象,突出了癌细胞代谢的复杂性和适应性。

The natural amino acid asparagine (Asn) is required by cells to sustain function and proliferation. Healthy cells can synthesize Asn through asparagine synthetase (ASNS) activity, whereas specific cancer and genetically diseased cells are forced to obtain asparagine from the extracellular environment. ASNS catalyzes the ATP-dependent synthesis of Asn from aspartate by consuming glutamine as a nitrogen source. Asparagine Synthetase Deficiency (ASNSD) is a disease that results from biallelic mutations in the ASNS gene and presents with congenital microcephaly, intractable seizures, and progressive brain atrophy. ASNSD often leads to premature death. Although clinical and cellular studies have reported that Asn deprivation contributes to the disease symptoms, the global metabolic effects of Asn deprivation on ASNSD-derived cells have not been studied. We analyzed two previously characterized cell culture models, lymphoblastoids and fibroblasts, each carrying unique ASNS mutations from families with ASNSD. Metabolomics analysis demonstrated that Asn deprivation in ASNS-deficient cells led to disruptions across a wide range of metabolites. Moreover, we observed significant decrements in TCA cycle intermediates and anaplerotic substrates in ASNS-deficient cells challenged with Asn deprivation. We have identified pantothenate, phenylalanine, and aspartate as possible biomarkers of Asn deprivation in normal and ASNSD-derived cells. This work implies the possibility of a novel ASNSD diagnostic via targeted biomarker analysis of a blood draw.

Asparagine synthetase (ASNS) catalyzes synthesis of asparagine (Asn) and Glu from Asp and Gln in an ATP-dependent reaction. Asparagine synthetase deficiency (ASNSD) results from biallelic mutations in the ASNS gene. Affected children exhibit congenital microcephaly, continued brain atrophy, seizures, and often premature mortality. However, the underlying mechanisms are unclear. This report describes a compound heterozygotic ASNSD child with two novel mutations in the ASNS gene, c.1118G>T (paternal) and c.1556G>A (maternal), that lead to G373V or R519H ASNS variants. Structural mapping suggested that neither variant participates directly in catalysis. Growth of cultured fibroblasts from either parent was unaffected in Asn-free medium, whereas growth of the child's cells was suppressed by about 50%. Analysis of Asn levels unexpectedly revealed that extracellular rather than intracellular Asn correlated with the reduced proliferation during incubation of the child's cells in Asn-free medium. Our attempts to ectopically express the G373V variant in either HEK293T or JRS cells resulted in minimal protein production, suggesting instability. Protein expression and purification from HEK293T cells revealed reduced activity for the R519H variant relative to WT ASNS. Expression of WT ASNS in ASNS-null JRS cells resulted in nearly complete rescue of growth in Asn-free medium, whereas we observed no proliferation for the cells expressing either the G373V or R519H variant. These results support the conclusion that the coexpression of the G373V and R519H ASNS variants leads to significantly reduced Asn synthesis, which negatively impacts cellular growth. These observations are consistent with the ASNSD phenotype.