色谱2025，Vol.43Issue(3)：207-219,13.DOI:10.3724/SP.J.1123.2024.04003
细胞转录组学和代谢组学整合策略表征突变影响谷氨酰胺合成酶酶活的关键代谢通路
Integrative transcriptomics-metabolomics approach to identify metabolic pathways regulated by glutamine synthetase activity
摘要
Abstract
Glutamine synthetase(GS),the only enzyme responsible for de novo glutamine syn-thesis,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-defi-cient 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 ba-ses,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,par-ticularly 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 phos-phate(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-aspar-tate-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 ap-peared to upregulate the expression of glutamine transporters SLC38A2 and SLC1A5.Thus,re-stricting extracellular amino acids,such as glutamine,induces a stress response while promo-ting transcription or translation by a select group of genes,thereby facilitating cellular adapta-tion.However,similar to GS＿WT,both GS＿R324C and GS＿K241R were modulated by gluta-mine 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 reticu-lum 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 up-regulation 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 gluta-mine 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 identi-fied 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 con-ducted 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 re-programmed cellular metabolism,particularly during glutamine deprivation.The altered metab-olism 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.关键词
谷氨酰胺合成酶突变/转录组学/代谢组学/肺癌/谷氨酰胺Key words
glutamine synthetase mutation/transcriptomics/metabolomics/lung cancer/glutamine分类
化学引用本文复制引用
凌婷,石京,冯婷泽,裴劭君,李思怿,朴海龙..细胞转录组学和代谢组学整合策略表征突变影响谷氨酰胺合成酶酶活的关键代谢通路[J].色谱,2025,43(3):207-219,13.基金项目
国家重点研究与发展计划(2022YFA0806503) （2022YFA0806503）
国家自然科学基金(81972625) （81972625）
大连市科技创新基金(2019J12SN52) （2019J12SN52）
辽宁振兴人才计划(XLYC2002035).National Key Research and Development Program of China(No.2022YFA0806503) （XLYC2002035）
National Natural Sci-ence Foundation of China(No.81972625) （No.81972625）
Dalian Science and Technology Innovation Funding(No.2019J12SN52) （No.2019J12SN52）
Liaoning Revitalization Talents Program(No.XLYC2002035). （No.XLYC2002035）
