The metabolic reprogramming of γ-aminobutyrate in oral squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC) is the most common head and neck malignancy. The oncometabolites have been studied in OSCC, but the mechanism of metabolic reprogramming remains unclear. To identify the potential metabolic markers to distinguish malignant oral squamous cell carcinoma (OSCC) tissue from adjacent healthy tissue and study the mechanism of metabolic reprogramming in OSCC. We compared the metabolites between cancerous and paracancerous tissues of OSCC patients by 1HNMR analysis. We established OSCC derived cell lines and analyzed their difference of RNA expression by RNA sequencing. We investigated the metabolism of γ-aminobutyrate in OSCC derived cells by real time PCR and western blotting. Our data revealed that much more γ-aminobutyrate was produced in cancerous tissues of OSCC patients. The investigation based on OSCC derived cells showed that the increase of γ-aminobutyrate was promoted by the synthesis of glutamate beyond the mitochondria. In OSCC cancerous tissue derived cells, the glutamate was catalyzed to glutamine by glutamine synthetase (GLUL), and then the generated glutamine was metabolized to glutamate by glutaminase (GLS). Finally, the glutamate produced by glutamate-glutamine-glutamate cycle was converted to γ-aminobutyrate by glutamate decarboxylase 2 (GAD2). Our study is not only benefit for understanding the pathological mechanisms of OSCC, but also has application prospects for the diagnosis of OSCC.

Clustered de novo start-loss variants in GLUL result in a developmental and epileptic encephalopathy via stabilization of glutamine synthetase.

Glutamine synthetase (GS), encoded by GLUL, catalyzes the conversion of glutamate to glutamine. GS is pivotal for the generation of the neurotransmitters glutamate and gamma-aminobutyric acid and is the primary mechanism of ammonia detoxification in the brain. GS levels are regulated post-translationally by an N-terminal degron that enables the ubiquitin-mediated degradation of GS in a glutamine-induced manner. GS deficiency in humans is known to lead to neurological defects and death in infancy, yet how dysregulation of the degron-mediated control of GS levels might affect neurodevelopment is unknown. We ascertained nine individuals with severe developmental delay, seizures, and white matter abnormalities but normal plasma and cerebrospinal fluid biochemistry with de novo variants in GLUL. Seven out of nine were start-loss variants and two out of nine disrupted 5' UTR splicing resulting in splice exclusion of the initiation codon. Using transfection-based expression systems and mass spectrometry, these variants were shown to lead to translation initiation of GS from methionine 18, downstream of the N-terminal degron motif, resulting in a protein that is stable and enzymatically competent but insensitive to negative feedback by glutamine. Analysis of human single-cell transcriptomes demonstrated that GLUL is widely expressed in neuro- and glial-progenitor cells and mature astrocytes but not in post-mitotic neurons. One individual with a start-loss GLUL variant demonstrated periventricular nodular heterotopia, a neuronal migration disorder, yet overexpression of stabilized GS in mice using in utero electroporation demonstrated no migratory deficits. These findings underline the importance of tight regulation of glutamine metabolism during neurodevelopment in humans.

Abundant urinary amino acids activate glutamine synthetase-encoding glnA by two different mechanisms in Escherichia coli.

Growth of uropathogenic Escherichia coli in the bladder induces transcription of glnA which codes for the ammonia-assimilating glutamine synthetase (GS) despite the normally suppressive high ammonia concentration. We previously showed that the major urinary component, urea, induces transcription from the Crp-dependent glnAp1 promoter, but the urea-induced transcript is not translated. Our purpose here was to determine whether the most abundant urinary amino acids, which are known to inhibit GS activity in vitro, also affect glnA transcription in vivo. We found that the abundant amino acids impaired growth, which glutamine and glutamate reversed; this implies inhibition of GS activity. In strains with deletions of crp and glnG that force transcription from the glnAp2 and glnAp1 promoters, respectively, we examined growth and glnA transcription with a glnA-gfp transcriptional fusion and quantitative reverse transcription PCR with primers that can distinguish transcription from the two promoters. The abundant urinary amino acids stimulated transcription from the glnAp2 promoter in the absence of urea but from the glnAp1 promoter in the presence of urea. However, transcription from glnAp1 did not produce a translatable mRNA or GS as assessed by a glnA-gfp translational fusion, enzymatic assay of GS, and Western blot to detect GS antigen in urea-containing media. We discuss these results within the context of the extremely rapid growth of uropathogenic E. coli in urine, the different factors that control the two glnA promoters and possible mechanisms that either overcome or bypass the urea-imposed block of glutamine synthesis during bacterial growth in urine.IMPORTANCEKnowledge of the regulatory mechanisms for genes expressed at the site of infection provides insight into the virulence of pathogenic bacteria. During urinary tract infections-most often caused by Escherichia coli-growth in urine induces the glnA gene which codes for glutamine synthetase. The most abundant urinary amino acids amplified the effect of urea which resulted in hypertranscription from the glnAp1 promoter and, unexpectedly, an untranslated transcript. E. coli must overcome this block in glutamine synthesis during growth in urine, and the mechanism of glutamine acquisition or synthesis may suggest a possible therapy.

Characterizing lysine acetylation of glucokinase.

Glucokinase (GK) catalyzes the phosphorylation of glucose to form glucose-6-phosphate as the substrate of glycolysis for energy production. Acetylation of lysine residues in Escherichia coli GK has been identified at multiple sites by a series of proteomic studies, but the impact of acetylation on GK functions remains largely unknown. In this study, we applied the genetic code expansion strategy to produce site-specifically acetylated GK variants which naturally exist in cells. Enzyme assays and kinetic analyses showed that lysine acetylation decreases the GK activity, mostly resulting from acetylation of K214 and K216 at the entrance of the active site, which impairs the binding of substrates. We also compared results obtained from the glutamine substitution method and the genetic acetyllysine incorporation approach, showing that glutamine substitution is not always effective for mimicking acetylated lysine. Further genetic studies as well as in vitro acetylation and deacetylation assays were performed to determine acetylation and deacetylation mechanisms, which showed that E. coli GK could be acetylated by acetyl-phosphate without enzymes and deacetylated by CobB deacetylase.

Acetylation of xenogeneic silencer H-NS regulates biofilm development through the nitrogen homeostasis regulator in Shewanella.

Adjusting intracellular metabolic pathways and adopting suitable live state such as biofilms, are crucial for bacteria to survive environmental changes. Although substantial progress has been made in understanding how the histone-like nucleoid-structuring (H-NS) protein modulates the expression of the genes involved in biofilm formation, the precise modification that the H-NS protein undergoes to alter its DNA binding activity is still largely uncharacterized. This study revealed that acetylation of H-NS at Lys19 inhibits biofilm development in Shewanella oneidensis MR-1 by downregulating the expression of glutamine synthetase, a critical enzyme in glutamine synthesis. We further found that nitrogen starvation, a likely condition in biofilm development, induces deacetylation of H-NS and the trimerization of nitrogen assimilation regulator GlnB. The acetylated H-NS strain exhibits significantly lower cellular glutamine concentration, emphasizing the requirement of H-NS deacetylation in Shewanella biofilm development. Moreover, we discovered in vivo that the activation of glutamine biosynthesis pathway and the concurrent suppression of the arginine synthesis pathway during both pellicle and attached biofilms development, further suggesting the importance of fine tune nitrogen assimilation by H-NS acetylation in Shewanella. In summary, posttranslational modification of H-NS endows Shewanella with the ability to respond to environmental needs by adjusting the intracellular metabolism pathways.

LncRNA SLC1A5-AS/MZF1/ASCT2 Axis Contributes to Malignant Progression of Hepatocellular Carcinoma.

BACKGROUND: Hypoxia is a pivotal factor influencing cellular gene expression and contributing to the malignant progression of tumors. Metabolic anomalies under hypoxic conditions are predominantly mediated by mitochondria. Nonetheless, the exploration of hypoxia-induced long noncoding RNAs (lncRNAs) associated with mitochondria remains largely uncharted. METHODS: We established hypoxia cell models using primary human hepatocytes (PHH) and hepatocellular carcinoma (HCC) cell lines. We isolated mitochondria for high-throughput sequencing to investigate the roles of candidate lncRNAs in HCC progression. We employed in vitro and in vivo assays to evaluate the functions of solute carrier family 1 member 5 antisense lncRNA (SLC1A5-AS). RNA-seq was utilized to scrutinize the comprehensive genome profile regulated by SLC1A5-AS in HCC. Subsequently, quantitative real-time polymerase chain reaction (qRT-PCR) and western blot analysis were utilized to validate the expression of alanine-serine-cysteine transporter 2 (ASCT2, encoded by the SLC1A5 gene), and a glutamine uptake assay was employed to estimate the glutamine uptake capacity of Huh-7 cells after SLC1A5-AS overexpression. To delve into the mechanisms governing the regulation of SLC1A5 expression by SLC1A5-AS, we employed a biotin-labeled SLC1A5-AS probe in conjunction with a western blot assay to confirm the interactions between SLC1A5-AS and candidate transcription factors. Luciferase reporter assays and chromatin immunoprecipitation (ChIP) were utilized to authenticate the effects of the predicted transcription factors on SLC1A5 promoter activity. RESULTS: Following the screening, we identified CTB-147N14.6, derived from the antisense strand of the SLC1A5 gene, which we have named SLC1A5-AS. SLC1A5-AS exhibited significantly elevated expression levels in HCC tissue and was associated with poor prognosis in HCC patients. In vitro and in vivo assays revealed that the overexpression of SLC1A5-AS significantly heightened cell invasion and metastasis. RNA-seq data unveiled SLC1A5-AS involvement in glutamine metabolism, left-handed amino (L-amino) acid transmembrane transporter activity, and the nuclear factor kappa-B (NF-κB) signaling pathway. Overexpression of SLC1A5-AS markedly increased ASCT2 mRNA/protein levels, thereby enhancing glutamine uptake and promoting the growth and metastasis of HCC cells. Mechanistically, higher RNA levels of SLC1A5-AS directly bound with myeloid zinc finger 1 (MZF1), acting as a transcriptional repressor, thus diminishing its binding to the SLC1A5 promoter region. CONCLUSIONS: Our findings unveil a novel role for the lncRNA SLC1A5-AS in glutamine metabolism, suggesting that targeting SLC1A5-AS/MZF1, in conjunction with ASCT2 inhibitor treatment, could be a potential therapeutic strategy for this disease.

Assessment of intra- and inter-genetic diversity in tetraploid and hexaploid wheat genotypes based on omega, gamma and alpha-gliadin profiles.

Durum and bread wheat are well adapted to the Mediterranean Basin. Twenty-three genotypes of each species were grown to evaluate the intra- and inter-genetic diversity based on omega (ω), gamma (γ) and alpha (α)-gliadin profiles. To achieve this purpose, the endosperm storage proteins (both gliadins and glutenins) were extracted from wheat grains and electrophoresed on sodium dodecyl sulfate (SDS)-polyacrylamide gels. The results of SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) revealed nine polymorphic loci out of 16 loci with durum wheat genotypes and nine polymorphic loci out of 18 loci with bead wheat genotypes. The polymorphisms revealed by the SDS-PAGE were 56% and 50% in durum and bread wheat genotypes, respectively. Using the cluster analysis, the durum wheat genotypes were clustered into five groups, while the bread wheat genotypes were grouped into six clusters using un-weighed pair group mean analyses based on ω, γ, and α-gliadins profiles. The 46 durum and bread wheat genotypes were grouped into seven clusters based on the combined ω, γ, and α-gliadins profiles revealed by the SDS-PAGE. The in silico analysis determined the intra-genetic diversity between bread and durum wheat based on the sequences of ω, γ, and α-gliadins. The alignment of ω-gliadin revealed the highest polymorphism (52.1%) between bread and durum wheat, meanwhile, the alignment of γ and α-gliadins revealed very low polymorphism 6.6% and 15.4%, respectively. According to computational studies, all gliadins contain a lot of glutamine and proline residues. The analysis revealed that the bread wheat possessed ω and γ -gliadins with a lower content of proline and a higher content of glutamine than durum wheat. In contrast, durum wheat possessed α-gliadin with a lower content of proline and a higher content of glutamine than bread wheat. In conclusion, the SDS-PAGE, in silico and computational analyses are effective tools to determine the intra- and inter-genetic diversity in tetraploid and hexaploid wheat genotypes based on ω, γ, and α-gliadins profiles.

Mechanisms of metabolic adaptation in the duckweed Lemna gibba: an integrated metabolic, transcriptomic and flux analysis.

BACKGROUND: Duckweeds are small, rapidly growing aquatic flowering plants. Due to their ability for biomass production at high rates they represent promising candidates for biofuel feedstocks. Duckweeds are also excellent model organisms because they can be maintained in well-defined liquid media, usually reproduce asexually, and because genomic resources are becoming increasingly available. To demonstrate the utility of duckweed for integrated metabolic studies, we examined the metabolic adaptation of growing Lemna gibba cultures to different nutritional conditions. RESULTS: To establish a framework for quantitative metabolic research in duckweeds we derived a central carbon metabolism network model of Lemna gibba based on its draft genome. Lemna gibba fronds were grown with nitrate or glutamine as nitrogen source. The two conditions were compared by quantification of growth kinetics, metabolite levels, transcript abundance, as well as by 13C-metabolic flux analysis. While growing with glutamine, the fronds grew 1.4 times faster and accumulated more protein and less cell wall components compared to plants grown on nitrate. Characterization of photomixotrophic growth by 13C-metabolic flux analysis showed that, under both metabolic growth conditions, the Calvin-Benson-Bassham cycle and the oxidative pentose-phosphate pathway are highly active, creating a futile cycle with net ATP consumption. Depending on the nitrogen source, substantial reorganization of fluxes around the tricarboxylic acid cycle took place, leading to differential formation of the biosynthetic precursors of the Asp and Gln families of proteinogenic amino acids. Despite the substantial reorganization of fluxes around the tricarboxylic acid cycle, flux changes could largely not be associated with changes in transcripts. CONCLUSIONS: Through integrated analysis of growth rate, biomass composition, metabolite levels, and metabolic flux, we show that Lemna gibba is an excellent system for quantitative metabolic studies in plants. Our study showed that Lemna gibba adjusts to different nitrogen sources by reorganizing central metabolism. The observed disconnect between gene expression regulation and metabolism underscores the importance of metabolic flux analysis as a tool in such studies.

Yueomyces silvicola sp. nov., a novel ascomycetous yeast species unable to utilize ammonium, glutamate, and glutamine as sole nitrogen sources.

Five yeast strains isolated from tree bark and rotten wood collected in central and southwestern China, together with four Brazilian strains (three from soil and rotting wood collected in an Amazonian rainforest biome and one from Bromeliad collected in Alagoas state) and one Costa Rican strain isolated from a flower beetle, represent a new species closely related with Yueomyces sinensis in Saccharomycetaceae, as revealed by the 26S ribosomal RNA gene D1/D2 domain and the internal transcribed spacer region sequence analysis. The name Yueomyces silvicola sp. nov. is proposed for this new species with the holotype China General Microbiological Culture Collection Center 2.6469 (= Japan Collection of Microorganisms 34885). The new species exhibits a whole-genome average nucleotide identity value of 77.8% with Y. sinensis. The two Yueomyces species shared unique physiological characteristics of being unable to utilize ammonium and the majority of the amino acids, including glutamate and glutamine, as sole nitrogen sources. Among the 20 amino acids tested, only leucine and tyrosine can be utilized by the Yueomyces species. Genome sequence comparison showed that GAT1, which encodes a GATA family protein participating in transcriptional activation of nitrogen-catabolic genes in Saccharomyces cerevisiae, is absent in the Yueomyces species. However, the failure of the Yueomyces species to utilize ammonium, glutamate, and glutamine, which are generally preferred nitrogen sources for microorganisms, implies that more complicated alterations in the central nitrogen metabolism pathway might occur in the genus Yueomyces.

Glutaminase (GLS1) gene expression in primary breast cancer.

BACKGROUND: Tumor growth is mediated in part by glutamine, and glutaminase is an enzyme necessary for glutamine catabolism. We studied glutaminase (GLS1) gene expression in primary breast cancer to determine correlations with clinical and tumor characteristics, and gene associations in publicly available databases. A better understanding of glutaminase gene expression may help guide further exploration of glutaminase inhibitors in breast cancer. METHODS: GLS1 mRNA levels were evaluated in The Cancer Genome Atlas (n = 817) and METABRIC (n = 1992) datasets. Associations between GLS1 and tumor subtype (ANOVA followed by post-hoc Tukey test for pairwise comparisons) and selected genes involved in the pathogenesis of breast cancer (Pearson's correlations) were determined in both datasets. In METABRIC, associations with overall survival (Cox proportional hazard model) were determined. For all analyses, p < 0.05 was the threshold for statistical significance. RESULTS: GLS1 expression was significantly higher in triple negative breast cancer (TNBC) than hormone receptor (HR) +/HER2- and HER2+ breast cancer (p < 0.001) and basal versus luminal A, luminal B, and HER2 enriched breast cancer (p < 0.001) in both datasets. In METABRIC, higher GLS1 expression was associated with improved overall survival (HR 0.91, 95% CI: 0.85-0.97, p = 0.005) and this association remained significant in the TNBC subset (HR 0.83, 95% CI: 0.71-0.98, p = 0.032). GLS1 had significant positive gene correlations with immune, proliferative, and basal genes, and inverse correlations with luminal genes and genes involved in metabolism. CONCLUSION: GLS1 expression is highest in TNBC and basal breast cancer, supporting ongoing clinical investigation of GLS1 inhibition in TNBC. GLS1 may have prognostic implications but further research is needed to validate this finding. GLS1 had significant positive gene correlations with immune genes, which may have implications for potential combinations of glutaminase inhibition and immunotherapy.

Bioinformatics and machine learning were used to validate glutamine metabolism-related genes and immunotherapy in osteoporosis patients.

BACKGROUND: Osteoporosis (OP), often referred to as the "silent disease of the twenty-first century," poses a significant public health concern due to its severity, chronic nature, and progressive course, predominantly affecting postmenopausal women and elderly individuals. The pathogenesis and progression of this disease have been associated with dysregulation in tumor metabolic pathways. Notably, the metabolic utilization of glutamine has emerged as a critical player in cancer biology. While metabolic reprogramming has been extensively studied in various malignancies and linked to clinical outcomes, its comprehensive investigation within the context of OP remains lacking. METHODS: This study aimed to identify and validate potential glutamine metabolism genes (GlnMgs) associated with OP through comprehensive bioinformatics analysis. The identification of GlnMgs was achieved by integrating the weighted gene co-expression network analysis and a set of 28 candidate GlnMgs. Subsequently, the putative biological functions and pathways associated with GlnMgs were elucidated using gene set variation analysis. The LASSO method was employed to identify key hub genes, and the diagnostic efficacy of five selected GlnMgs in OP detection was assessed. Additionally, the relationship between hub GlnMgs and clinical characteristics was investigated. Finally, the expression levels of the five GlnMgs were validated using independent datasets (GSE2208, GSE7158, GSE56815, and GSE35956). RESULTS: Five GlnMgs, namely IGKC, TMEM187, RPS11, IGLL3P, and GOLGA8N, were identified in this study. To gain insights into their biological functions, particular emphasis was placed on synaptic transmission GABAergic, inward rectifier potassium channel activity, and the cytoplasmic side of the lysosomal membrane. Furthermore, the diagnostic potential of these five GlnMgs in distinguishing individuals with OP yielded promising results, indicating their efficacy as discriminative markers for OP. CONCLUSIONS: This study discovered five GlnMgs that are linked to OP. They shed light on potential new biomarkers for OP and tracking its progression.

Targeting Glutamine Metabolism through Glutaminase Inhibition Suppresses Cell Proliferation and Progression in Nasopharyngeal Carcinoma.

BACKGROUND: Glutaminase (GLS), the key enzyme involved in glutamine metabolism, has been identified as a critical player in tumor growth and progression. The GLS inhibitor CB-839 has entered several clinical trials against a variety of tumors. OBJECTIVE: Our study aimed to investigate the role and underlying mechanism of GLS and its inhibitor CB-839 in nasopharyngeal carcinoma (NPC). METHODS: The expression, downstream genes, and signaling pathways of GLS in NPC were determined by real-time polymerase chain reaction (RT-PCR), PCR array, western blotting (WB), and immunohistochemical staining (IHC), and the phenotype of GLS was confirmed by in vivo experiments of subcutaneous tumor formation in mice and in vitro experiments of functional biology, including Cell Counting Kit-8 (CCK-8), colony formation, flow cytometry, transwell migration, and Boyden invasion assay. Finally, it was also verified whether the treatment of NPC cells by GLS inhibitor CB-839 can change various biological functions and protein expression to achieve the purpose of blocking tumor progression. RESULTS: GLS was remarkably overexpressed in NPC cells and tissues, predicting a poor overall survival of NPC patients. GLS promoted cell cycle, proliferation, colony formation, migratory, and invasive capacities by regulating Cyclin D2 (CCND2) via PI3K/AKT/mTOR pathway in NPC in vitro and in vivo. Notably, CB-839 showed an effective anti-NPC tumor effect by blocking the biological functions of the tumor. CONCLUSION: The first innovative proof is that GLS promotes cell proliferation by regulating CCND2 via PI3K/AKT/mTOR pathway in NPC, and GLS inhibitor CB-839 may serve as a new potential therapeutic target for NPC treatment.

Transcriptional metabolic reprogramming implements meiotic fate decision in mouse testicular germ cells.

Nutrient starvation drives yeast meiosis, whereas retinoic acid (RA) is required for mammalian meiosis through its germline target Stra8. Here, by using single-cell transcriptomic analysis of wild-type and Stra8-deficient juvenile mouse germ cells, our data show that the expression of nutrient transporter genes, including Slc7a5, Slc38a2, and Slc2a1, is downregulated in germ cells during meiotic initiation, and this process requires Stra8, which binds to these genes and induces their H3K27 deacetylation. Consequently, Stra8-deficient germ cells sustain glutamine and glucose uptake in response to RA and exhibit hyperactive mTORC1/protein kinase A (PKA) activities. Importantly, expression of Slc38a2, a glutamine importer, is negatively correlated with meiotic genes in the GTEx dataset, and Slc38a2 knockdown downregulates mTORC1/PKA activities and induces meiotic gene expression. Thus, our study indicates that RA via Stra8, a chordate morphogen pathway, induces meiosis partially by generating a conserved nutrient restriction signal in mammalian germ cells by downregulating their nutrient transporter expression.

STAG2 inactivation reprograms glutamine metabolism of BRAF-mutant thyroid cancer cells.

STAG2, an important subunit in cohesion complex, is involved in the segregation of chromosomes during the late mitosis and the formation of sister chromatids. Mutational inactivation of STAG2 is a major cause of the resistance of BRAF-mutant melanomas to BRAF/MEK inhibitors. In the present study, we found that STAG2 was frequently down-regulated in thyroid cancers compared with control subjects. By a series of in vitro and in vivo studies, we demonstrated that STAG2 knockdown virtually had no effect on malignant phenotypes of BRAF-mutant thyroid cancer cells such as cell proliferation, colony formation and tumorigenic ability in nude mice compared with the control. In addition, unlike melanoma, STAG2 knockdown also did not affect the sensitivity of these cells to MEK inhibitor. However, we surprisingly found that STAG2-knockdown cells exhibited more sensitive to glutamine deprivation or glutaminase inhibitor BPTES compared with control cells. Mechanistically, knocking down STAG2 in BRAF-mutant thyroid cancer cells decreases the protein stability of c-Myc via the ERK/AKT/GSK3ß feedback pathway, thereby impairing glutamine metabolism of thyroid cancer cells by down-regulating its downstream targets such as SCL1A5, GLS and GLS2. Our data, taken together, demonstrate that STAG2 inactivation reprograms glutamine metabolism of BRAF-mutant thyroid cancer cells, thereby improving their cellular response to glutaminase inhibitor. This study will provide a potential therapeutic strategy for BRAF-mutant thyroid cancers.

Activated protein C modulates T-cell metabolism and epigenetic FOXP3 induction via α-ketoglutarate.

A direct regulation of adaptive immunity by the coagulation protease activated protein C (aPC) has recently been established. Preincubation of T cells with aPC for 1 hour before transplantation increases FOXP3+ regulatory T cells (Tregs) and reduces acute graft-versus-host disease (aGVHD) in mice, but the underlying mechanism remains unknown. Because cellular metabolism modulates epigenetic gene regulation and plasticity in T cells, we hypothesized that aPC promotes FOXP3+ expression by altering T-cell metabolism. To this end, T-cell differentiation was assessed in vitro using mixed lymphocyte reaction or plate-bound α-CD3/CD28 stimulation, and ex vivo using T cells isolated from mice with aGVHD without and with aPC preincubation, or analyses of mice with high plasma aPC levels. In stimulated CD4+CD25- cells, aPC induces FOXP3 expression while reducing expression of T helper type 1 cell markers. Increased FOXP3 expression is associated with altered epigenetic markers (reduced 5-methylcytosine and H3K27me3) and reduced Foxp3 promoter methylation and activity. These changes are linked to metabolic quiescence, decreased glucose and glutamine uptake, decreased mitochondrial metabolism (reduced tricarboxylic acid metabolites and mitochondrial membrane potential), and decreased intracellular glutamine and α-ketoglutarate levels. In mice with high aPC plasma levels, T-cell subpopulations in the thymus are not altered, reflecting normal T-cell development, whereas FOXP3 expression in splenic T cells is reduced. Glutamine and α-ketoglutarate substitution reverse aPC-mediated FOXP3+ induction and abolish aPC-mediated suppression of allogeneic T-cell stimulation. These findings show that aPC modulates cellular metabolism in T cells, reducing glutamine and α-ketoglutarate levels, which results in altered epigenetic markers, Foxp3 promoter demethylation and induction of FOXP3 expression, thus favoring a Treg-like phenotype.

Glutamine and amino acid metabolism as a prognostic signature and therapeutic target in endometrial cancer.

INTRODUCTION: Endometrial cancer (EC) is the most common female reproductive system cancer in developed countries with growing incidence and associated mortality, which may be due to the growing prevalence of obesity. Metabolism reprogramming including glucose, amino acid, and lipid remodeling is a hallmark of tumors. Glutamine metabolism has been reported to participate in tumor proliferation and development. This study aimed to develop a glutamine metabolism-related prognostic model for EC and explore potential targets for cancer treatment. METHOD: Transcriptomic data and survival outcome of EC were retrieved from The Cancer Genome Atlas (TCGA). Differentially expressed genes related to glutamine metabolism were recognized and utilized to build a prognostic model by univariate and multivariate Cox regressions. The model was confirmed in the training, testing, and the entire cohort. A nomogram combing prognostic model and clinicopathologic features was established and tested. Moreover, we explored the effect of a key metabolic enzyme, PHGDH, on the biological behavior of EC cell lines and xenograft model. RESULTS: Five glutamine metabolism-related genes, including PHGDH, OTC, ASRGL1, ASNS, and NR1H4, were involved in prognostic model construction. Kaplan-Meier curve suggested that patients recognized as high risk underwent inferior outcomes. The receiver operating characteristic (ROC) curve showed the model was sufficient to predict survival. Enrichment analysis recognized DNA replication and repair dysfunction in high-risk patients whereas immune relevance analysis revealed low immune scores in the high-risk group. Finally, a nomogram integrating the prognostic model and clinical factors was created and verified. Further, knockdown of PHGDH showed cell growth inhibition, increasing apoptosis, and reduced migration. Promisingly, NCT-503, a PHGDH inhibitor, significantly repressed tumor growth in vivo (p = 0.0002). CONCLUSION: Our work established and validated a glutamine metabolism-related prognostic model that favorably evaluates the prognosis of EC patients. DNA replication and repair may be the crucial point that linked glutamine metabolism, amino acid metabolism, and EC progression. High-risk patients stratified by the model may not be sufficient for immune therapy. PHGDH might be a crucial target that links serine metabolism, glutamine metabolism as well as EC progression.

RBM4 dictates ESCC cell fate switch from cellular senescence to glutamine-addiction survival through inhibiting LKB1-AMPK-axis.

Cellular senescence provides a protective barrier against tumorigenesis in precancerous or normal tissues upon distinct stressors. However, the detailed mechanisms by which tumor cells evade premature senescence to malignant progression remain largely elusive. Here we reported that RBM4 adversely impacted cellular senescence to favor glutamine-dependent survival of esophageal squamous cell carcinoma (ESCC) cells by dictating the activity of LKB1, a critical governor of cancer metabolism. The level of RBM4 was specifically elevated in ESCC compared to normal tissues, and RBM4 overexpression promoted the malignant phenotype. RBM4 contributed to overcome H-RAS- or doxorubicin-induced senescence, while its depletion caused P27-dependent senescence and proliferation arrest by activating LKB1-AMPK-mTOR cascade. Mechanistically, RBM4 competitively bound LKB1 to disrupt the LKB1/STRAD/MO25 heterotrimeric complex, subsequently recruiting the E3 ligase TRIM26 to LKB1, promoting LKB1 ubiquitination and degradation in nucleus. Therefore, such molecular process leads to bypassing senescence and sustaining cell proliferation through the activation of glutamine metabolism. Clinically, the ESCC patients with high RBM4 and low LKB1 have significantly worse overall survival than those with low RBM4 and high LKB1. The RBM4 high/LKB1 low expression confers increased sensitivity of ESCC cells to glutaminase inhibitor CB-839, providing a novel insight into mechanisms underlying the glutamine-dependency to improve the efficacy of glutamine inhibitors in ESCC therapeutics.

Genome-wide comparative analysis of the valine glutamine motif containing genes in four Ipomoea species.

BACKGROUND: Genes with valine glutamine (VQ) motifs play an essential role in plant growth, development, and resistance to biotic and abiotic stresses. However, little information on the VQ genes in sweetpotato and other Ipomoea species is available. RESULTS: This study identified 55, 58, 50 and 47 VQ genes from sweetpotato (I. batatas), I.triflida, I. triloba and I. nil, respectively. The phylogenetic analysis revealed that the VQ genes formed eight clades (I-VII), and the members in the same group exhibited similar exon-intron structure and conserved motifs distribution. The distribution of the VQ genes among the chromosomes of Ipomoea species was disproportional, with no VQ genes mapped on a few of each species' chromosomes. Duplication analysis suggested that segmental duplication significantly contributes to their expansion in sweetpotato, I.trifida, and I.triloba, while the segmental and tandem duplication contributions were comparable in I.nil. Cis-regulatory elements involved in stress responses, such as W-box, TGACG-motif, CGTCA-motif, ABRE, ARE, MBS, TCA-elements, LTR, and WUN-motif, were detected in the promoter regions of the VQ genes. A total of 30 orthologous groups were detected by syntenic analysis of the VQ genes. Based on the analysis of RNA-seq datasets, it was found that the VQ genes are expressed distinctly among different tissues and hormone or stress treatments. A total of 40 sweetpotato differentially expressed genes (DEGs) refer to biotic (sweetpotato stem nematodes and Ceratocystis fimbriata pathogen infection) or abiotic (cold, salt and drought) stress treatments were detected. Moreover, IbVQ8, IbVQ25 and IbVQ44 responded to the five stress treatments and were selected for quantitative reverse-transcription polymerase chain reaction (qRT-PCR) analysis, and the results were consistent with the transcriptome analysis. CONCLUSIONS: Our study may provide new insights into the evolution of VQ genes in the four Ipomoea genomes and contribute to the future molecular breeding of sweetpotatoes.