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β-Aspartyl compounds, such as β-aspartyl hydroxamate (serine racemase inhibitor), β-aspartyl-l-lysine (moisture retention), and β-aspartyl-l-tryptophan (immunomodulator) are physiologically active compounds. There is limited literature on the development of effective methods of production of β-aspartyl compounds. In this study, we describe the biochemical characterization of asparagine synthetase (AS) from Streptococcus thermophilus NBRC 13957 (StAS) and the enzymatic synthesis of β-aspartyl compounds using StAS. Recombinant StAS was expressed in Escherichia coli BL21(DE3) and it displayed activity towards hydroxylamine, methylamine, ethylamine, and ammonia, as acceptors of the β-aspartyl moiety. StAS exhibited higher activity toward hydroxylamine and ethylamine as acceptor substrates compared with the enzymes from Lactobacillus delbrueckii NBRC 13953, Lactobacillus reuteri NBRC 15892, and E. coli. The coupling of the synthesis of β-aspartyl compounds by StAS with an ATP-regeneration system using polyphosphate kinase from Deinococcus proteoliticus NBRC 101906 displayed an approximately 2.5-fold increase in the production of β-aspartylhydroxamate from 1.06 mM to 2.53 mM after a 76-h reaction., (Copyright © 2024 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Cancer is a leading concern and important cause of death worldwide. Cancer is a non-communicable illness defined as uncontrolled division of cells. It can develop into metastatic cancer when tumor cells migrate to other organs. In recent years evidence has emerged that the bioavailability of Asn play a crucial role in cancer metastasis. Asn is a non-essential amino acid formed from an ATP dependent catalyzed reaction by the enzyme asparagine synthetase (ASNS), where Asp and Gln are converted to Asn and Glu, respectively. The human ASNS enzyme consist of 561 amino acids, with a molecular weight of 64 KDa. ASNS governs the activation of transcriptional factors that regulate the process of metastasis. In this work the 3D model of ASNS in E. coli (AS-B) and the human ASNS docked with its different ligands have been used to study the 3D mechanism of the conversion of Asp and Gln to Asn and Glu, in human ASNS. The stability evaluation of the docked complexes was checked by molecular dynamic simulation through the bioinformatic tool Desmond. The binding residues and their interactions can be exploited for the development of inhibitors, as well as for finding new drug molecules against ASNS and prevention of metastatic cancer., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Riaz et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Asparaginase-based therapy is a cornerstone in acute lymphoblastic leukemia (ALL) treatment, capitalizing on the methylation status of the asparagine synthetase (ASNS) gene, which renders ALL cells reliant on extracellular asparagine. Contrastingly, ASNS expression in acute myeloid leukemia (AML) has not been thoroughly investigated, despite studies suggesting that AML with chromosome 7/7q deletions might have reduced ASNS levels. Here, we leverage reverse phase protein arrays to measure ASNS expression in 810 AML patients and assess its impact on outcomes. We find that AML with inv(16) has the lowest overall ASNS expression. While AML with deletion 7/7q had ASNS levels slightly lower than those of AML without deletion 7/7q, this observation was not significant. Low ASNS expression correlated with improved overall survival (46 versus 54 weeks, respectively, p = 0.011), whereas higher ASNS levels were associated with better response to venetoclax-based therapy. Protein correlation analysis demonstrated association between ASNS and proteins involved in methylation and DNA repair. In conclusion, while ASNS expression was not lower in patients with deletion 7/7q as initially predicted, ASNS levels were highly variable across AML patients. Further studies are needed to assess whether patients with low ASNS expression are susceptible to asparaginase-based therapy due to their inability to augment compensatory ASNS expression upon asparagine depletion.

Background: Sex-related differences in colorectal (CRC) incidence and mortality are well-documented. However, the impact of sex on metabolic pathways that drive cancer growth is not well understood. High expression of asparagine synthetase (ASNS) is associated with inferior survival for female CRC patients only. Here, we used a CRISPR/Cas9 technology to generate HCT116 ASNS -/- and HCT 116 ASNS +/+ cancer cell lines. We examine the effects of ASNS deletion on tumor growth and the subsequent rewiring of metabolic pathways in male and female Rag2/IL2RG mice., Results: ASNS loss reduces cancer burden in male and female tumor-bearing mice (40% reduction, q < 0.05), triggers metabolic reprogramming including gluconeogenesis, but confers a survival improvement (30 days median survival, q < 0.05) in female tumor-bearing mice alone. Transcriptomic analyses revealed upregulation of G-protein coupled estrogen receptor (GPER1) in tumors from male and female mice with HCT116 ASNS -/- xenograft. Estradiol activates GPER1 in vitro in the presence of ASNS and suppresses tumor growth., Conclusions: Our study indicates that inferior survival for female CRC patients with high ASNS may be due to metabolic reprogramming that sustains tumor growth. These findings have translational relevance as ASNS/GPER1 signaling could be a future therapeutic target to improve the survival of female CRC patients., (© 2024. The Author(s).)

Asparagine is a non-essential amino acid crucial for protein biosynthesis and function, and therefore cell maintenance and growth. Furthermore, this amino acid has an important role in regulating several metabolic pathways, such as tricarboxylic acid cycle and the urea cycle. When compared to normal cells, tumor cells typically present a higher demand for asparagine, making it a compelling target for therapy. In this review article, we investigate different facets of asparagine bioavailability intricate role in malignant tumors raised from solid organs. We take a comprehensive look at asparagine synthetase expression and regulation in cancer, including the impact on tumor growth and metastasis. Moreover, we explore asparagine depletion through L-asparaginase as a potential therapeutic method for aggressive solid tumors, approaching different formulations of the enzyme and combinatory therapies. In summary, here we delve into studies about endogenous and exogenous asparagine availability in solid cancers, analyzing therapeutic implications and future challenges., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: Adult mammalian cardiomyocytes have limited proliferative capacity, but in specifically induced contexts they traverse through cell-cycle reentry, offering the potential for heart regeneration. Endogenous cardiomyocyte proliferation is preceded by cardiomyocyte dedifferentiation (CMDD), wherein adult cardiomyocytes revert to a less matured state that is distinct from the classical myocardial fetal stress gene response associated with heart failure. However, very little is known about CMDD as a defined cardiomyocyte cell state in transition., Methods: Here, we leveraged 2 models of in vitro cultured adult mouse cardiomyocytes and in vivo adeno-associated virus serotype 9 cardiomyocyte-targeted delivery of reprogramming factors ( Oct4 , Sox2 , Klf4 , and Myc ) in adult mice to study CMDD. We profiled their transcriptomes using RNA sequencing, in combination with multiple published data sets, with the aim of identifying a common denominator for tracking CMDD., Results: RNA sequencing and integrated analysis identified Asparagine Synthetase ( Asns ) as a unique molecular marker gene well correlated with CMDD, required for increased asparagine and also for distinct fluxes in other amino acids. Although Asns overexpression in Oct4 , Sox2 , Klf4 , and Myc cardiomyocytes augmented hallmarks of CMDD, Asns deficiency led to defective regeneration in the neonatal mouse myocardial infarction model, increased cell death of cultured adult cardiomyocytes, and reduced cell cycle in Oct4 , Sox2 , Klf4 , and Myc cardiomyocytes, at least in part through disrupting the mammalian target of rapamycin complex 1 pathway., Conclusions: We discovered a novel gene Asns as both a molecular marker and an essential mediator, marking a distinct threshold that appears in common for at least 4 models of CMDD, and revealing an Asns /mammalian target of rapamycin complex 1 axis dependency for dedifferentiating cardiomyocytes. Further study will be needed to extrapolate and assess its relevance to other cell state transitions as well as in heart regeneration., Competing Interests: Disclosures None.

Polycystic kidney disease (PKD) is a genetic disorder characterized by bilateral cyst formation. We showed that PKD cells and kidneys display metabolic alterations, including the Warburg effect and glutaminolysis, sustained in vitro by the enzyme asparagine synthetase (ASNS). Here, we used antisense oligonucleotides (ASO) against Asns in orthologous and slowly progressive PKD murine models and show that treatment leads to a drastic reduction of total kidney volume (measured by MRI) and a prominent rescue of renal function in the mouse. Mechanistically, the upregulation of an ATF4-ASNS axis in PKD is driven by the amino acid response (AAR) branch of the integrated stress response (ISR). Metabolic profiling of PKD or control kidneys treated with Asns-ASO or Scr-ASO revealed major changes in the mutants, several of which are rescued by Asns silencing in vivo. Indeed, ASNS drives glutamine-dependent de novo pyrimidine synthesis and proliferation in cystic epithelia. Notably, while several metabolic pathways were completely corrected by Asns-ASO, glycolysis was only partially restored. Accordingly, combining the glycolytic inhibitor 2DG with Asns-ASO further improved efficacy. Our studies identify a new therapeutic target and novel metabolic vulnerabilities in PKD., (© 2024. The Author(s).)

Background and Aim: The condition of hepatic fibrosis is hazardous. Therefore, it is vital that we investigate the mechanism of hepatic fibrosis to provide new targets for treatment., Methods: Preliminary screening and research was carried out based on our prior results and our speculated role of the particle with quaternary structure arrangement (PAQosome) in hepatic fibrosis. The experiments were conducted using LX-2 or HepG2 cell lines by western blotting, quantitative real-time polymerase chain reaction, luciferase assays, and co-immunoprecipitation and were further validated in the Gene Expression Omnibus (GEO) database., Results: We screened and proved that several subunits of the PAQosome regulate the development of liver fibrosis, including the asparagine synthetase domain-containing 1 upstream open reading frame (ASDURF), prefoldin subunit 4 (PFDN4), prefoldin subunit 5 (PFDN5), unconventional prefoldin RNA polymerase II subunit 5 interactor (URI1), and ubiquitously expressed prefoldin-like chaperone (UXT). ASDURF promotes hepatic fibrosis through the transforming growth factor-β1 (TGFβ1)/Sekelsky mothers against decapentaplegic homologue 3 (Smad3) and NF-κB signaling pathways. ASDURF regulates the expression of asparagine synthetase domain-containing 1 (ASNSD1). PFDN4, PFDN5, URI1, and UXT regulate cell proliferation through the PI3K/AKT pathway, and thus regulate liver fibrosis. A hepatic fibrosis score ≥ F2 was selected as the diagnostic criteria for hepatic fibrosis in the GSE96971 database. The area under the receiver operating characteristic curve of PFDN4, PFDN5, UXT, and ASNSD1 were 0.862 (confidence interval [CI]: 0.6588-1.000), 0.538 (CI: 0.224-0.853), 0.708 (CI: 0.449-0.966), and 0.831 (CI: 0.638-1.000), respectively., Conclusions: These findings demonstrate that the PAQosome is a brand new target for hepatic fibrosis therapy., (© 2023 Journal of Gastroenterology and Hepatology Foundation and John Wiley & Sons Australia, Ltd.)

Asparagine synthetase (ASNS) is a crucial enzyme for the de novo biosynthesis of endogenous asparagine (Asn), and ASNS shows the positive relationship with the growth of several solid tumors. Most of ASNS inhibitors are analogs of transition-state in ASNS reaction, but their low cell permeability hinders their anticancer activity. Therefore, novel ASNS inhibitors with a new pharmacophore urgently need to be developed. In this study, we established and applied a system for in vitro screening of ASNS inhibitors, and found a promising unique bisabolane-type meroterpenoid molecule, bisabosqual A (Bis A), able to covalently modify K556 site of ASNS protein. Bis A targeted ASNS to suppress cell proliferation of human non-small cell lung cancer A549 cells and exhibited a synergistic effect with L-asparaginase (L-ASNase). Mechanistically, Bis A promoted oxidative stress and apoptosis, while inhibiting autophagy, cell migration and epithelial-mesenchymal transition (EMT), impeding cancer cell development. Moreover, Bis A induced negative feedback pathways containing the GCN2-eIF2α-ATF4, PI3K-AKT-mTORC1 and RAF-MEK-ERK axes, but combination treatment of Bis A and rapamycin/torin-1 overcame the potential drug resistance triggered by mTOR pathways. Our study demonstrates that ASNS inhibition is promising for cancer chemotherapy, and Bis A is a potential lead ASNS inhibitor for anticancer development., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Asparaginase is an important agent for the treatment of acute lymphoblastic leukaemia (ALL), but it is occasionally associated with severe adverse events. Thus, for safer and more efficacious therapy, a clinical biomarker predicting asparaginase sensitivity is highly anticipated. Asparaginase depletes serum asparagine by deaminating asparagine into aspartic acid, and ALL cells are thought to be sensitive to asparaginase due to reduced asparagine synthetase (ASNS) activity. We have recently shown that allele-specific methylation of the ASNS gene is highly involved in asparaginase sensitivity in B-precursor ALL (BCP-ALL) by using next-generation sequence (NGS) analysis of bisulphite PCR products of the genomic DNA. Here, we sought to confirm the utility of methylation status of the ASNS gene evaluated with high-performance liquid chromatography (HPLC) analysis of bisulphite PCR products for future clinical applications. In the global methylation status of 23 CpG sites at the boundary region of promoter and exon 1 of the ASNS gene, a strong positive correlation was confirmed between the mean percent methylation evaluated with the HPLC method and that with the NGS method in 79 BCP-ALL cell lines (R 2  = 0.85, p  = 1.3 × 10 -33 ) and in 63 BCP-ALL clinical samples (R 2  = 0.84, p  = 5.0 × 10 -26 ). Moreover, methylation status of the ASNS gene evaluated with the HPLC method was significantly associated with in vitro asparaginase sensitivities as well as gene and protein expression levels of ASNS. These observations indicated that the ASNS gene methylation status evaluated with the HPLC method is a reliable biomarker for predicting the asparaginase sensitivity of BCP-ALL.

Cortical neurogenesis depends on the balance between self-renewal and differentiation of apical progenitors (APs). Here, we study the epigenetic control of AP's division mode by focusing on the enzymatic activity of the histone methyltransferase DOT1L. Combining lineage tracing with single-cell RNA sequencing of clonally related cells, we show at the cellular level that DOT1L inhibition increases neurogenesis driven by a shift of APs from asymmetric self-renewing to symmetric neurogenic consumptive divisions. At the molecular level, DOT1L activity prevents AP differentiation by promoting transcription of metabolic genes. Mechanistically, DOT1L inhibition reduces activity of an EZH2/PRC2 pathway, converging on increased expression of asparagine synthetase (ASNS), a microcephaly associated gene. Overexpression of ASNS in APs phenocopies DOT1L inhibition, and also increases neuronal differentiation of APs. Our data suggest that DOT1L activity/PRC2 crosstalk controls AP lineage progression by regulating asparagine metabolism., (© 2023 The Authors. Published under the terms of the CC BY NC ND 4.0 license.)

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.

Depriving cells of nutrients triggers an energetic crisis, which is resolved by metabolic rewiring and organelle reorganization. Primary cilia are microtubule-based organelles at the cell surface, capable of integrating multiple metabolic and signalling cues, but their precise sensory function is not fully understood. Here we show that primary cilia respond to nutrient availability and adjust their length via glutamine-mediated anaplerosis facilitated by asparagine synthetase (ASNS). Nutrient deprivation causes cilia elongation, mediated by reduced mitochondrial function, ATP availability and AMPK activation independently of mTORC1. Of note, glutamine removal and replenishment is necessary and sufficient to induce ciliary elongation or retraction, respectively, under nutrient stress conditions both in vivo and in vitro by restoring mitochondrial anaplerosis via ASNS-dependent glutamate generation. Ift88-mutant cells lacking cilia show reduced glutamine-dependent mitochondrial anaplerosis during metabolic stress, due to reduced expression and activity of ASNS at the base of cilia. Our data indicate a role for cilia in responding to, and possibly sensing, cellular glutamine levels via ASNS during metabolic stress., (© 2023. The Author(s).)

Reprogramming of energy metabolism is regarded as one of the hallmarks of cancer; in particular, oncogenic RAS has been shown to be a critical regulator of cancer metabolism. Recently, asparagine metabolism has been heavily investigated as a novel target for cancer treatment. For example, Knott et al. showed that asparagine bioavailability governs metastasis in a breast cancer model. Gwinn et al. reported the therapeutic vulnerability of asparagine biosynthesis in KRAS-driven non-small cell lung cancer. We previously reported that KRAS -mutated CRC cells can adapt to glutamine depletion through upregulation of asparagine synthetase (ASNS), an enzyme that synthesizes asparagine from aspartate. In our previous study, we assessed the efficacy of asparagine depletion using human cancer cell lines. In the present study, we evaluated the clinical relevance of asparagine depletion using a novel patient-derived spheroid xenograft (PDSX) mouse model. First, we examined ASNS expression in 38 spheroid lines and found that 12 lines (12/37, 32.4%) displayed high ASNS expression, whereas 26 lines (25/37, 67.6%) showed no ASNS expression. Next, to determine the role of asparagine metabolism in tumor growth, we established ASNS-knockdown spheroid lines using lentiviral short hairpin RNA constructs targeting ASNS. An in vitro cell proliferation assay demonstrated a significant decrease in cell proliferation upon asparagine depletion in the ASNS-knockdown spheroid lines, and this was not observed in the control spheroids lines. In addition, we examined asparagine inhibition with the anti-leukemia drug L-asparaginase (L-Asp) and observed a considerable reduction in cell proliferation at a low concentration (0.1 U/mL) in the ASNS-knockdown spheroid lines, whereas it exhibited limited inhibition of control spheroid lines at the same concentration. Finally, we used the PDSX model to assess the effects of asparagine depletion on tumor growth in vivo. The nude mice injected with ASNS-knockdown or control spheroid lines were administered with L-Asp once a day for 28 days. Surprisingly, in mice injected with ASNS-knockdown spheroids, the administration of L-Asp dramatically inhibited tumor engraftment. On the other hands, in mice injected with control spheroids, the administration of L-Asp had no effect on tumor growth inhibition at all. These results suggest that ASNS inhibition could be critical in targeting asparagine metabolism in cancers.

Depletion of circulating asparagine with l-asparaginase (ASNase) is a mainstay of leukemia treatment and is under investigation in many cancers. Expression levels of asparagine synthetase (ASNS), which catalyzes asparagine synthesis, were considered predictive of cancer cell sensitivity to ASNase treatment, a notion recently challenged. Using [U- 13 C 5 ]-l-glutamine in vitro and in vivo in a mouse model of B cell lymphomas (BCLs), we demonstrated that supraphysiological or physiological concentrations of asparagine prevent de novo asparagine biosynthesis, regardless of ASNS expression levels. Overexpressing ASNS in ASNase-sensitive BCL was insufficient to confer resistance to ASNase treatment in vivo. Moreover, we showed that ASNase's glutaminase activity enables its maximal anticancer effect. Together, our results indicate that baseline ASNS expression (low or high) cannot dictate BCL dependence on de novo asparagine biosynthesis and predict BCL sensitivity to dual ASNase activity. Thus, except for ASNS-deficient cancer cells, ASNase's glutaminase activity should be considered in the clinic.

Objectives: This study was conducted to investigate the potential anticancer properties of N, N-dibenzyl asparagine (NNDAsp), as an Asparagine (Asp) analog, using colon cancer Caco-2 cell and the normal NCM-460 cell line., Methods: Cell viability rate and levels of produced lactate dehydrogenase (LDH) were achieved upon treatment with NNDAsp compared to Asp treatment using MTT assay and LDH production kit. The protein expression profile of asparagine synthetase (ASNS) was achieved by using ELISA and flow cytometry assay. The levels of released inflammatory cytokines, including interleukin-1 alpha (IL-1α) and IL-1 beta (IL-β), were monitored using an ELISA assay., Results: Our findings showed significant inhibition of colon cancer cell proliferation accompanied by a high level of produced LDH in a dose-dependent of an NNDAsp treatment without detectable toxic effect in normal cells. Interestingly, NNDAsp showed competitive inhibition of ASNS protein expression, in almost 3% of stained cancer cells, compared to 18% and 35% of untreated cells and cells pre-treated with Asp, respectively. Likewise, the concentration of ASNS protein was dramatically depleted in a dose and time-dependent of NNDAsp treatment in comparison with Asp treatment indicated by ELISA assay. Furthermore, as an apoptotic indicator, the expression of P53 and Caspase 3 (Caps3) was significantly increased in Caco-2 cells treated with NNDAsp at both RNA and protein levels. In contrast, their expression was markedly depleted in Asp-treated cells. In addition, the expression of both IL-1α and IL-1 β was markedly increased in Caco-2 cells in a dose and time-dependent of NNDAsp exogenous treatment. Moreover, targeting of ASNS by the Asp analog, NNDAsp, was further confirmed by the docking analysis of inhibitors ligands and crystal structure of ASNS protein., Conclusion: These data provide evidence for the effectiveness of NNDAsp in cancer treatment via selective degradation of ASNS protein expression in colon cancer cells.

The availability of asparagine is the limitation of cell growth and metastasis. Asparagine synthetase (ASNS) was an essential enzyme for endogenous asparagine products. In our study, ASNS-induced asparagine products were essential to maintain tumor growth and colony formations in vitro. But mutated ASNS which defected endogenous asparagine products still upregulated cell invasiveness, which indicated that ASNS promoted invasiveness by alternative pathways. Mechanically, ASNS modulated Wnt signal transduction by promoting GSK3β phosphorylation on ser9 and stabilizing the β-catenin complex, as result, ASNS could promote more β-catenin translocation into nucleus independent of endogenous asparagine. At the same time, ASNS modulated mitochondrial response to Wnt stimuli with increased mitochondrial potential and membrane fusion. In summary, ASNS promoted metastasis depending on Wnt pathway and mitochondrial functions even without endogenous asparagine products., (© 2022. The Author(s).)

Understanding the genomic and epigenetic mechanisms of drug resistance in pediatric acute lymphoblastic leukemia (ALL) is critical for further improvements in treatment outcomes. The role of transcriptomic response in conferring resistance to l-asparaginase (LASP) is poorly understood beyond asparagine synthetase (ASNS). We defined reproducible LASP response genes in LASP-resistant and LASP-sensitive ALL cell lines as well as primary leukemia samples from newly diagnosed patients. Defining target genes of the amino acid stress response-related transcription factor activating transcription factor 4 (ATF4) in ALL cell lines using chromatin immunoprecipitation sequencing (ChIP-seq) revealed 45% of genes that changed expression after LASP treatment were direct targets of the ATF4 transcription factor, and 34% of these genes harbored LASP-responsive ATF4 promoter binding events. SLC7A11 was found to be a response gene in cell lines and patient samples as well as a direct target of ATF4. SLC7A11 was also one of only 2.4% of LASP response genes with basal level gene expression that also correlated with LASP ex vivo resistance in primary leukemia cells. Experiments using chemical inhibition of SLC7A11 with sulfasalazine, gene overexpression, and partial gene knockout recapitulated LASP resistance or sensitivity in ALL cell lines. These findings show the importance of assessing changes in gene expression following treatment with an antileukemic agent for its association with drug resistance and highlight that many response genes may not differ in their basal expression in drug-resistant leukemia cells., (© 2022 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Background: Pancreatic ductal adenocarcinomas (PDACs) is a malignant disorder and is the most common pancreatic cancer type. The malignant cells depend on the uptake of asparagine (Asn) for growth. The synthesis of Asn occurs through the enzyme asparagine synthetase (ASNS). Interestingly, ASNS is known as is direct target of nonsense-mediated RNA decay (NMD). We have previously reported that NMD major factor UPF1 mutations in the pancreatic tumors. However, the relationship between NMD and the level of ASNS is unknown., Method: We constructed point mutations by site-specific mutagenesis. To evaluate NMD magnitude, we assessed the expression ratio of an exogenously expressed wild-type and mutated β-globin mRNA with N39 allele, and five known NMD targets. Then, reverse transcription-polymerase chain reaction (RT-PCR), RT-qPCR and western bolt to determine RNA or protein levels, after knockdown of endogenous UPF1 by small RNA interference in the cells., Results: An RNA editing event (c.3101 A > G) at UPF1 transcripts resulting in an Asparagine (p.1034) changed to a Serine is found in one primary PDAC patient. The edited UPF1 increases the ability of degrading of NMD provoking transcripts, such as β-globin mRNA with N39 allele and 5 out of 5 known endogenous NMD substrate mRNAs, including ASNS. In addition, ASNS mRNA is subjected to NMD degradation by virtue of its possessing uORFs at the 5'UTR. A reduction of endogenous ASNS RNA and the increased protein expression level is found either in the PDAC patient or in the cells with edited UPF1 at c.3101 A > G relative to the controls., Conclusions: This edited UPF1 found in the PDAC results in hyperactivated NMD, which is tightly correlation to elevated expression level of ASNS. The targeting of knockdown of ASNS may improve the antitumor potency in PDACs., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

Asparagine synthetase (ASN) is one of the key enzymes of nitrogen (N) metabolism in plants. The product of ASN is asparagine, which is one of the key compounds involved in N transport and storage in plants. Complete genome-wide analysis and classifications of the ASN gene family have recently been reported in different plants. However, little is known about the systematic analysis and expression profiling of ASN proteins in cotton development and N metabolism. Here, various bioinformatics analysis was performed to identify ASN gene family in cotton. In the cotton genome, forty-three proteins were found that determined ASN genes, comprising of 20 genes in Gossypium hirsutum (Gh), 13 genes in Gossypium arboreum, and 10 genes in Gossypium raimondii. The ASN encoded genes unequally distributed on various chromosomes with conserved glutamine amidotransferases and ASN domains. Expression analysis indicated that the majority of GhASNs were upregulated in vegetative and reproductive organs, fiber development, and N metabolism. Overall, the results provide proof of the possible role of the ASN genes in improving cotton growth, fiber development, and especially N metabolism in cotton. The identified hub genes will help to functionally elucidate the ASN genes in cotton development and N metabolism., (© 2022. The Author(s).)

Asparaginase therapy is a key component of chemotherapy for patients with T-cell acute lymphoblastic leukemia (T-ALL). Asparaginase depletes serum asparagine by deamination into aspartic acid. Normal hematopoietic cells can survive due to asparagine synthetase (ASNS) activity, whereas leukemia cells are supposed to undergo apoptosis due to silencing of the ASNS gene. Because the ASNS gene has a typical CpG island in its promoter, its methylation status in T-ALL cells may be associated with asparaginase sensitivity. Thus, we investigated the significance of ASNS methylation status in asparaginase sensitivity of T-ALL cell lines and prognosis of childhood T-ALL. Sequencing of bisulfite polymerase chain reaction products using next-generation sequencing technology in 22 T-ALL cell lines revealed a stepwise allele-specific methylation of the ASNS gene, in association with an aberrant methylation of a 7q21 imprinted gene cluster. T-ALL cell lines with ASNS hypermethylation status showed significantly higher in vitro l-asparaginase sensitivity in association with insufficient asparaginase-induced upregulation of ASNS gene expression and lower basal ASNS protein expression. A comprehensive analysis of diagnostic samples from pediatric patients with T-ALL in Japanese cohorts (N = 77) revealed that methylation of the ASNS gene was associated with an aberrant methylation of the 7q21 imprinted gene cluster. In pediatric T-ALL patients in Japanese cohorts (n = 75), ASNS hypomethylation status was significantly associated with poor therapeutic outcome, and all cases with poor prognostic SPI1 fusion exclusively exhibited ASNS hypomethylation status. These observations show that ASNS hypomethylation status is associated with asparaginase resistance and is a poor prognostic biomarker in childhood T-ALL., (© 2022 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

There is currently no evidence of the carcinogenic effect of the β-adrenergic agonist ractopamine added in finishing swine and cattle feed for promoting leanness. Nonetheless, it has the capability of stimulating expression of asparagine synthetase (ASNS) through activating transcription factor 5, and many other genes involved in the stress reaction in the skeletal muscle of pigs according to published scientific articles. Because overexpression of ASNS has been detected as a key player in amino acid response and unfolded protein response during the development of not a few malignant diseases, especially those with KRAS mutations, and found to be closely related to tumor proliferation, invasion and metastasis, it seems reasonable to hypothesize that intake of ractopamine residue in meat might bring negative effects to cancer patients., (Copyright © 2021 The Author(s). Published by Wolters Kluwer Health, Inc.)

Asparagine synthetase (ASNS) is a gene on the long arm of chromosome 7 that is copy-number amplified in the majority of glioblastomas. ASNS copy-number amplification is associated with a significantly decreased survival. Using patient-derived glioma stem cells (GSC), we showed that significant metabolic alterations occur in gliomas when perturbing the expression of ASNS, which is not merely restricted to amino acid homeostasis. ASNS-high GSCs maintained a slower basal metabolic profile yet readily shifted to a greatly increased capacity for glycolysis and oxidative phosphorylation when needed. This led ASNS-high cells to a greater ability to proliferate and spread into brain tissue. Finally, we demonstrate that these changes confer resistance to cellular stress, notably oxidative stress, through adaptive redox homeostasis that led to radiotherapy resistance. Furthermore, ASNS overexpression led to modifications of the one-carbon metabolism to promote a more antioxidant tumor environment revealing a metabolic vulnerability that may be therapeutically exploited. IMPLICATIONS: This study reveals a new role for ASNS in metabolic control and redox homeostasis in glioma stem cells and proposes a new treatment strategy that attempts to exploit one vulnerable metabolic node within the larger multilayered tumor network., (©2021 American Association for Cancer Research.)

Nuclear factor erythroid 2-related factor 2 (NRF2) is aberrantly activated in about 93% of pancreatic cancers. Activated NRF2 regulates multiple downstream molecules involved in cancer cell metabolic reprogramming, translational control, and treatment resistance; however, targeting NRF2 for pancreatic cancer therapy remains largely unexplored. In this study, we used the online computational tool CellMiner TM to explore the NCI-60 drug databases for compounds with anticancer activities correlating most closely with the mRNA expression of NQO1, a marker for NRF2 pathway activity. Among the >100,000 compounds analyzed, NSC84167, termed herein as NRF2 synthetic lethality compound-01 (NSLC01), was one of the top hits (r = 0.71, P < 0.001) and selected for functional characterization. NSLC01 selectively inhibited the viabilities of four out of seven conventional pancreatic cancer cell lines and induced dramatic apoptosis in the cells with high NRF2 activation. The selective anticancer activity of NSLC01 was further validated with a panel of nine low-passage pancreatic patient-derived cell lines, and a significant reverse correlation between log(IC 50 ) of NSLC01 and NQO1 expression was confirmed (r = -0.5563, P = 0.024). Notably, screening of a panel of nine patient-derived xenografts (PDXs) revealed six PDXs with high NQO1/NRF2 activation, and NSLC01 dramatically inhibited the viabilities and induced apoptosis in ex vivo cultures of PDX tumors. Consistent with the ex vivo results, NSLC01 inhibited the tumor growth of two NRF2-activated PDX models in vivo (P < 0.01, n = 7-8) but had no effects on the NRF2-low counterpart. To characterize the mechanism of action, we employed a metabolomic isotope tracer assay that demonstrated that NSLC01-mediated inhibition of de novo synthesis of multiple amino acids, including asparagine and methionine. Importantly, we further found that NSLC01 suppresses the eEF2K/eEF2 translation elongation cascade and protein translation of asparagine synthetase. In summary, this study identified a novel compound that selectively targets protein translation and induces synthetic lethal effects in NRF2-activated pancreatic cancers., (© 2021. The Author(s).)

Background: Understanding the determinants of free asparagine concentration in wheat grain is necessary to reduce levels of the processing contaminant acrylamide in baked and toasted wheat products. Although crop management strategies can help reduce asparagine concentrations, breeders have limited options to select for genetic variation underlying this trait. Asparagine synthetase enzymes catalyse a critical step in asparagine biosynthesis in plants and, in wheat, are encoded by five homeologous gene triads that exhibit distinct expression profiles. Within this family, TaASN2 genes are highly expressed during grain development but TaASN-B2 is absent in some varieties., Results: Natural genetic diversity in the asparagine synthetase gene family was assessed in different wheat varieties revealing instances of presence/absence variation and other polymorphisms, including some predicted to affect the function of the encoded protein. The presence and absence of TaASN-B2 was determined across a range of UK and global common wheat varieties and related species, showing that the deletion encompassing this gene was already present in some wild emmer wheat genotypes. Expression profiling confirmed that TaASN2 transcripts were only detectable in the grain, while TaASN3.1 genes were highly expressed during the early stages of grain development. TaASN-A2 was the most highly expressed TaASN2 homeologue in most assayed wheat varieties. TaASN-B2 and TaASN-D2 were expressed at similar, lower levels in varieties possessing TaASN-B2. Expression of TaASN-A2 and TaASN-D2 did not increase to compensate for the absence of TaASN-B2, so total TaASN2 expression was lower in varieties lacking TaASN-B2. Consequently, free asparagine concentrations in field-produced grain were, on average, lower in varieties lacking TaASN-B2, although the effect was lost when free asparagine accumulated to very high concentrations as a result of sulphur deficiency., Conclusions: Selecting wheat genotypes lacking the TaASN-B2 gene may be a simple and rapid way for breeders to reduce free asparagine concentrations in commercial wheat grain.

In peripheral nerves, Schwann cells form myelin and provide trophic support to axons. We previously showed that the mitochondrial protein prohibitin 2 can localize to the axon-Schwann-cell interface and is required for developmental myelination. Whether the homologous protein prohibitin 1 has a similar role, and whether prohibitins also play important roles in Schwann cell mitochondria is unknown. Here, we show that deletion of prohibitin 1 in Schwann cells minimally perturbs development, but later triggers a severe demyelinating peripheral neuropathy. Moreover, mitochondria are heavily affected by ablation of prohibitin 1 and demyelination occurs preferentially in cells with apparent mitochondrial loss. Furthermore, in response to mitochondrial damage, Schwann cells trigger the integrated stress response, but, contrary to what was previously suggested, this response is not detrimental in this context. These results identify a role for prohibitin 1 in myelin integrity and advance our understanding about the Schwann cell response to mitochondrial damage.

T cell receptor (TCR) triggering by antigen results in metabolic reprogramming that, in turn, facilitates the exit of T cells from quiescence. The increased nutrient requirements of activated lymphocytes are met, in part, by upregulation of cell surface transporters and enhanced uptake of amino acids, fatty acids, and glucose from the environment. However, the role of intracellular pathways of amino acid biosynthesis in T cell activation is relatively unexplored. Asparagine is a nonessential amino acid that can be synthesized intracellularly through the glutamine-hydrolyzing enzyme asparagine synthetase (ASNS). We set out to define the requirements for uptake of extracellular asparagine and ASNS activity in CD8+ T cell activation. At early time points of activation in vitro, CD8+ T cells expressed little or no ASNS, and, as a consequence, viability and TCR-stimulated growth, activation, and metabolic reprogramming were substantially impaired under conditions of asparagine deprivation. At later time points (more than 24 hours of activation), TCR-induced mTOR-dependent signals resulted in ASNS upregulation that endowed CD8+ T cells with the capacity to function independently of extracellular asparagine. Thus, our data suggest that the coordinated upregulation of ASNS expression and uptake of extracellular asparagine is involved in optimal T cell effector responses.

Very low-density lipoprotein receptor (VLDLR) is a member of the LDL receptor family that is involved in the uptake of VLDL into cells. Increased hepatic VLDLR under endoplasmic reticulum (ER) stress has been shown to cause fatty liver. In this study, the effect of dietary protein restriction on hepatic VLDLR and the role of VLDLR in fatty liver were investigated using Vldlr knockout (KO) mice. Growing wild-type (WT) and KO mice were fed a control diet containing 20% ​​protein or a low protein diet containing 3% protein for 11 days. In WT mice, the amount of hepatic Vldlr mRNA and VLDLR protein increased by approximately 8- and 7-fold, respectively, due to protein restriction. Vldlr mRNA and protein levels increased in both type 1 and type 2 VLDLR. However, neither Vldlr mRNA nor protein levels were significantly increased in heart, muscle, and adipose tissue, demonstrating that VLDLR increase due to protein restriction occurred in a liver-specific manner. Increased liver triglyceride levels during protein restriction occurred in KO mice to the same extent as in WT mice, indicating that increased VLDLR during protein restriction was not the main cause of fatty liver, which was different from the case of ER stress.

Recently, human asparagine synthetase has been found to be associated with the mitotic spindle. However, this event cannot be seen in yeast because yeast takes a different cell division process via closed mitosis (there is no nuclear envelope breakdown to allow the association between any cytosolic enzyme and mitotic spindle). To find out if yeast asparagine synthetase can also (but hiddenly) have this feature, the coding sequences of green fluorescent protein (GFP) and nuclear localization signal (NLS) were introduced downstream of ASN1 and ASN2, encoding asparagine synthetases Asn1p and Asn2p, respectively, in the yeast genome having mCherrry coding sequence downstream of TUB1 encoding alpha-tubulin, a building block of the mitotic spindle. The genomically engineered yeast strains showed co-localization of Asn1p-GFP-NLS (or Asn2p-GFP-NLS) and Tub1p-mCherry in dividing nuclei. In addition, an activity-disrupted mutation was introduced to ASN1 (or ASN2). The yeast mutants still exhibited co-localization between defective asparagine synthetase and mitotic spindle, indicating that the biochemical activity of asparagine synthetase is not required for its association with the mitotic spindle. Furthermore, nocodazole treatment was used to depolymerize the mitotic spindle, resulting in lack of association between the enzyme and the mitotic spindle. Although yeast cell division undergoes closed mitosis, preventing the association of its asparagine synthetase with the mitotic spindle, however, by using yeast constructs with re-localized Asn1/2p have suggested the moonlighting role of asparagine synthetase in cell division of higher eukaryotes., Competing Interests: The authors declare no competing interests.

The pancreas avidly takes up and synthesizes the amino acid asparagine (Asn), in part, to maintain an active translational machinery that requires incorporation of the amino acid. The de novo synthesis of Asn in the pancreas occurs through the enzyme asparagine synthetase (ASNS). The pancreas has the highest expression of ASNS of any organ, and it can further upregulate ASNS expression in the setting of amino acid depletion. ASNS expression is driven by an intricate feedback network within the integrated stress response (ISR), which includes the amino acid response (AAR) and the unfolded protein response (UPR). Asparaginase is a cancer chemotherapeutic drug that depletes plasma Asn. However, asparaginase-associated pancreatitis (AAP) is a major medical problem and could be related to pancreatic Asn depletion. In this review, we will provide an overview of ASNS and then describe its role in pancreatic health and in the exocrine disorders of pancreatitis and pancreatic cancer. We will offer the overarching perspective that a high abundance of ASNS expression is hardwired in the exocrine pancreas to buffer the high demands of Asn for pancreatic digestive enzyme protein synthesis, that perturbations in the ability to express or upregulate ASNS could tip the balance towards pancreatitis, and that pancreatic cancers exploit ASNS to gain a metabolic survival advantage., Competing Interests: Declaration of competing interest SZH has equity in Prevcon, serves on the scientific advisory board for Atias, and has research funding from Servier. CT and MSK declare no conflict of interest., (Copyright © 2020 IAP and EPC. Published by Elsevier B.V. All rights reserved.)

Nitrogen (N) is a major limiting factor affecting crop yield in unfertilized soil. Thus, cultivars with a high N use efficiency (NUE) and good grain protein content (GPC) are needed to fulfill the growing food demand and to reduce environmental burden. This is especially true for rice (Oryza sativa L.) that is cultivated with a high input of N fertilizer and is a primary staple food crop for more than half of the global population. Here, we report that rice asparagine synthetase 1 (OsASN1) is required for grain yield and grain protein contents under both N-sufficient (conventional paddy fields) and N-limiting conditions from analyses of knockout mutant plants. In addition, we show that overexpression (OX) of OsASN1 results in better nitrogen uptake and assimilation, and increased tolerance to N limitation at the seedling stage. Under field conditions, the OsASN1 OX rice plants produced grains with increased N and protein contents without yield reduction compared to wild-type (WT) rice. Under N-limited conditions, the OX plants displayed increased grain yield and protein content with enhanced photosynthetic activity compared to WT rice. Thus, OsASN1 can be an effective target gene for the development of rice cultivars with higher grain protein content, NUE, and grain yield under N-limiting conditions., (© The Author(s) 2020. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.)

Clostridium difficile 630 possesses a cryptic but functional gene cluster vanG Cd homologous to the vanG operon of Enterococcus faecalis . Expression of vanG Cd in the presence of subinhibitory concentrations of vancomycin is accompanied by peptidoglycan amidation on the meso -DAP residue. In this paper, we report the presence of two potential asparagine synthetase genes named asnB and asnB2 in the C. difficile genome whose products were potentially involved in this peptidoglycan structure modification. We found that asnB expression was only induced when C. difficile was grown in the presence of vancomycin, yet independently from the vanG Cd resistance and regulation operons. In addition, peptidoglycan precursors were not amidated when asnB was inactivated. No change in vancomycin MIC was observed in the asnB mutant strain. In contrast, overexpression of asnB resulted in the amidation of most of the C. difficile peptidoglycan precursors and in a weak increase of vancomycin susceptibility. AsnB activity was confirmed in E. coli . In contrast, the expression of the second asparagine synthetase, AsnB2, was not induced in the presence of vancomycin. In summary, our results demonstrate that AsnB is responsible for peptidoglycan amidation of C. difficile in the presence of vancomycin.

Asparagine synthetase (ASNS) catalyses the ATP-dependent conversion of aspartate to asparagine. However, both the regulation and biological functions of asparagine in tumour cells remain largely unknown. Here, we report that p53 suppresses asparagine synthesis through the transcriptional downregulation of ASNS expression and disrupts asparagine-aspartate homeostasis, leading to lymphoma and colon tumour growth inhibition in vivo and in vitro. Moreover, the removal of asparagine from culture medium or the inhibition of ASNS impairs cell proliferation and induces p53/p21-dependent senescence and cell cycle arrest. Mechanistically, asparagine and aspartate regulate AMPK-mediated p53 activation by physically binding to LKB1 and oppositely modulating LKB1 activity. Thus, we found that p53 regulates asparagine metabolism and dictates cell survival by generating an auto-amplification loop via asparagine-aspartate-mediated LKB1-AMPK signalling. Our findings highlight a role for LKB1 in sensing asparagine and aspartate and connect asparagine metabolism to the cellular signalling transduction network that modulates cell survival.

Activating transcription factor 4 (ATF4) is a master transcriptional regulator of the integrated stress response (ISR) that enables cell survival under nutrient stress. The mechanisms by which ATF4 couples metabolic stresses to specific transcriptional outputs remain unknown. Using functional genomics, we identified transcription factors that regulate the responses to distinct amino acid deprivation conditions. While ATF4 is universally required under amino acid starvation, our screens yielded a transcription factor, Zinc Finger and BTB domain-containing protein 1 (ZBTB1), as uniquely essential under asparagine deprivation. ZBTB1 knockout cells are unable to synthesize asparagine due to reduced expression of asparagine synthetase (ASNS), the enzyme responsible for asparagine synthesis. Mechanistically, ZBTB1 binds to the ASNS promoter and promotes ASNS transcription. Finally, loss of ZBTB1 sensitizes therapy-resistant T cell leukemia cells to L-asparaginase, a chemotherapeutic that depletes serum asparagine. Our work reveals a critical regulator of the nutrient stress response that may be of therapeutic value., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background : Cancer cells survive and develop under nutrient deficient microenvironment caused by low blood supply. Although anaerobic metabolism could function through the enhanced uptake of glucose, other mechanisms of tolerance to glucose deficient conditions might be required. Materials and Methods : Expression of asparagine synthetase (ASNS) under normal glucose and glucose-deprived conditions was examined. Cancer cell proliferation and migration were evaluated by in vitro and in vivo assays. In addition, the relationship between ASNS expression and cancer stages was also analyzed. Results : Expression of ASNS was enhanced under glucose deficient conditions. In vitro assays indicated that ASNS could promote the proliferation and migration abilities of esophageal squamous cell carcinoma (ESCC) cells under glucose deficient condition. In mechanism, 2 critical effectors during nutrient deprivation, NRF2 and ATF4, were upregulated and demonstrated to promote ASNS expression. Clinically, high level of ASNS was significantly associated with ESCC with advanced stages and metastasis. In vivo , ASNS could promote tumor growth and metastasis in mouse xenograft models. Conclusion : This study uncovered that glucose deprivation induces the overexpression of ASNS in ESCC cells, which in turn causes cancer cell tolerance to nutrient stress and promotes cancer development. The illustration of the mechanism sheds deep insight on how cell biology was regulated in response to the conditions of limited nutrient availability., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Tumor cells adapt to nutrient-limited environments by inducing gene expression that ensures adequate nutrients to sustain metabolic demands. For example, during amino acid limitations, ATF4 in the amino acid response induces expression of asparagine synthetase (ASNS), which provides for asparagine biosynthesis. Acute lymphoblastic leukemia (ALL) cells are sensitive to asparagine depletion, and administration of the asparagine depletion enzyme l-asparaginase is an important therapy option. ASNS expression can counterbalance l-asparaginase treatment by mitigating nutrient stress. Therefore, understanding the mechanisms regulating ASNS expression is important to define the adaptive processes underlying tumor progression and treatment. Here we show that DNA hypermethylation at the ASNS promoter prevents its transcriptional expression following asparagine depletion. Insufficient expression of ASNS leads to asparagine deficiency, which facilitates ATF4-independent induction of CCAAT-enhancer-binding protein homologous protein (CHOP), which triggers apoptosis. We conclude that chromatin accessibility is critical for ATF4 activity at the ASNS promoter, which can switch ALL cells from an ATF4-dependent adaptive response to ATF4-independent apoptosis during asparagine depletion. This work may also help explain why ALL cells are most sensitive to l-asparaginase treatment compared with other cancers., (© 2019 Jiang et al.)

As obligate intracellular pathogens, viruses rely on the host cell machinery to replicate efficiently, with the host metabolism extensively manipulated for this purpose. High-throughput small interfering RNA (siRNA) screens provide a systematic approach for the identification of novel host-virus interactions. Here, we report a large-scale screen for host factors important for human cytomegalovirus (HCMV), consisting of 6,881 siRNAs. We identified 47 proviral factors and 68 antiviral factors involved in a wide range of cellular processes, including the mediator complex, proteasome function, and mRNA splicing. Focused characterization of one of the hits, asparagine synthetase (ASNS), demonstrated a strict requirement for asparagine for HCMV replication which leads to an early block in virus replication before the onset of DNA amplification. This effect is specific to HCMV, as knockdown of ASNS had little effect on herpes simplex virus 1 or influenza A virus replication, suggesting that the restriction is not simply due to a failure in protein production. Remarkably, virus replication could be completely rescued 7 days postinfection with the addition of exogenous asparagine, indicating that while virus replication is restricted at an early stage, it maintains the capacity for full replication days after initial infection. This study represents the most comprehensive siRNA screen for the identification of host factors involved in HCMV replication and identifies the nonessential amino acid asparagine as a critical factor in regulating HCMV virus replication. These results have implications for control of viral latency and the clinical treatment of HCMV in patients. IMPORTANCE HCMV accounts for more than 60% of complications associated with solid organ transplant patients. Prophylactic or preventative treatment with antivirals, such as ganciclovir, reduces the occurrence of early onset HCMV disease. However, late onset disease remains a significant problem, and prolonged treatment, especially in patients with suppressed immune systems, greatly increases the risk of antiviral resistance. Very few antivirals have been developed for use against HCMV since the licensing of ganciclovir, and of these, the same viral genes are often targeted, reducing the usefulness of these drugs against resistant strains. An alternative approach is to target host genes essential for virus replication. Here we demonstrate that HCMV replication is highly dependent on levels of the amino acid asparagine and that knockdown of a critical enzyme involved in asparagine synthesis results in severe attenuation of virus replication. These results suggest that reducing asparagine levels through dietary restriction or chemotherapeutic treatment could limit HCMV replication in patients., (Copyright © 2019 Lee et al.)

We and others have reported that the anticancer activity of L-asparaginase (ASNase) against asparagine synthetase (ASNS)-positive cell types requires ASNase glutaminase activity, whereas anticancer activity against ASNS-negative cell types does not. Here, we attempted to disentangle the relationship between asparagine metabolism, glutamine metabolism, and downstream pathways that modulate cell viability by testing the hypothesis that ASNase anticancer activity is based on asparagine depletion rather than glutamine depletion per se. We tested ASNase wild-type (ASNase WT ) and its glutaminase-deficient Q59L mutant (ASNase Q59L ) and found that ASNase glutaminase activity contributed to durable anticancer activity against xenografts of the ASNS-negative Sup-B15 leukemia cell line in NOD/SCID gamma mice, whereas asparaginase activity alone yielded a mere growth delay. Our findings suggest that ASNase glutaminase activity is necessary for durable, single-agent anticancer activity in vivo , even against ASNS-negative cancer types., (©2019 American Association for Cancer Research.)

New anticancer platinum(II) compounds with distinctive modes of action are appealing alternatives to combat the drug resistance and improve the efficacy of clinically used platinum chemotherapy. Herein, we describe a rare example of an antitumor Pt II complex targeting a tumor-associated protein, rather than DNA, under cellular conditions. Complex [(bis-NHC)Pt(bt)]PF 6 (1 a; Hbt=1-(3-hydroxybenzo[b]thiophen-2-yl)ethanone) overcomes cisplatin resistance in cancer cells and displays significant tumor growth inhibition in mice with higher tolerable doses compared to cisplatin. The cellular Pt species shows little association with DNA, and localizes in the cytoplasm as revealed by nanoscale secondary ion mass spectrometry. An unbiased thermal proteome profiling experiment identified asparagine synthetase (ASNS) as a molecular target of 1 a. Accordingly, 1 a treatment reduced the cellular asparagine levels and inhibited cancer cell proliferation, which could be reversed by asparagine supplementation. A bis-NHC-ligated Pt species generated from the hydrolysis of 1 a forms adducts with thiols and appears to target an active-site cysteine of ASNS., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

l-Asparagine synthetase (AS) acts in asparagine formation and can be classified into two families: AS-A or AS-B. AS-A is mainly found in prokaryotes and can synthetize asparagine from ammonia. Distinct from other eukaryotes, Trypanosoma cruzi produces an AS-A. AS-A from Trypanosoma cruzi (Tc-AS-A) differs from prokaryotic AS-A due to its ability to catalyze asparagine synthesis using both glutamine and ammonia as nitrogen sources. Regarding these peculiarities, this work uses several biophysical techniques to provide data concerning the Tc-AS-A in-solution behavior. Tc-AS-A was produced as a recombinant and purified by three chromatography steps. Circular dichroism, dynamic light scattering, and analytical size exclusion chromatography showed that Tc-AS-A has the same fold and quaternary arrangement of prokaryotic AS-A. Despite the tendency of protein to aggregate, stable dimers were obtained when solubilization occurred at pH ≤ 7.0. We also demonstrate the protective efficacy against T. cruzi infection in mice immunized with Tc-AS-A. Our results indicate that immunization with Tc-AS-A might confer partial protection to infective forms of T. cruzi in this particular model., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The sex-determining region Y (SRY)-box (SOX) family has a crucial role in carcinogenesis and cancer progression. However, the role of SOX12 and the mechanism by which it is dysregulated in colorectal cancer (CRC) remain unclear. Here we analyzed SOX12 expression patterns in two independent CRC cohorts (cohort I, n = 390; cohort II, n = 363) and found that SOX12 was significantly upregulated in CRC, indicating a poor prognosis in CRC patients. Overexpression of SOX12 promoted CRC cell proliferation and metastasis, whereas downregulation of SOX12 hampered CRC aggressiveness. Mechanistically, SOX12 facilitated asparagine synthesis by transactivating glutaminase (GLS), glutamic oxaloacetic transaminase 2 (GOT2), and asparagine synthetase (ASNS). Downregulation of GLS, GOT2, and ASNS blocked SOX12-mediated CRC cell proliferation and metastasis, whereas ectopic expression of GLS, GOT2, and ASNS attenuated the SOX12 knockdown-induced suppression of CRC progression. In addition, serial deletion, site-directed mutagenesis, luciferase reporter, and chromatin immunoprecipitation (ChIP) assays indicated that hypoxia-inducible factor 1α (HIF-1α) directly binds to the SOX12 promoter and induces SOX12 expression. Administration of L-asparaginase decreased SOX12-mediated tumor growth and metastasis. In human CRC samples, SOX12 expression positively correlated with GLS, GOT2, ASNS, and HIF-1α expression. Based on these results, SOX12 may serve as a prognostic biomarker and L-asparaginase represents a potential novel therapeutic agent for CRC.

Recent work has found that many metabolic enzymes have the ability to polymerize in response to metabolic changes or environmental stress. This ability to polymerize is well conserved for the few metabolic enzyme paralogs that have been studied in yeast. Here we describe the first set of paralogs, Asn1p and Asn2p, that have differential assembly behavior. Asn1p and Asn2p both co-assemble into filaments in response to nutrient limitation. However, the ability of Asn2p to form filaments is strictly dependent on the presence of Asn1p. Using mutations that block enzyme activity but have differential effects on Asn1p polymerization, we have found that Asn1p polymers are unlikely to have acquired a moonlighting function. Together these results provide a novel system for understanding the regulation and evolution of metabolic enzyme polymerization.

Asparagine is one of the important amino acids for long-distance transport of nitrogen (N) in plants. However, little is known about the effect of asparagine on plant development, especially in crops. Here, a new T-DNA insertion mutant, asparagine synthetase 1 ( asn1 ), was isolated and showed a different plant height, root length, and tiller number compared with wild type (WT). In asn1 , the amount of asparagine decreased sharply while the total nitrogen (N) absorption was not influenced. In later stages, asn1 showed reduced tiller number, which resulted in suppressed tiller bud outgrowth. The relative expression of many genes involved in the asparagine metabolic pathways declined in accordance with the decreased amino acid concentration. The CRISPR/Cas9 mutant lines of OsASN1 showed similar phenotype with asn1 . These results suggest that OsASN1 is involved in the regulation of rice development and is specific for tiller outgrowth.

Synthetic beta-adrenergic agonists (BA) have broad biomedical and agricultural application for increasing lean body mass, yet a poor understanding of the biology underpinning these agents is limiting further drug discovery potential. Growing female pigs (77 ± 7 kg) were administered the BA, Ractopamine (20 ppm in feed), or the recombinant growth hormone (GH), Reporcin (10 mg/48 hrs injected) for 1, 3, 7, 13 (n = 10 per treatment, per time point) or 27 days (n = 15 per treatment). Using RNA-sequencing and inferred pathway analysis, we examined temporal changes to the Longissimus Dorsi skeletal muscle transcriptome (n = 3 per treatment, per time point) relative to a feed-only control cohort. Gene expression changes were affirmed by quantitative-PCR on all samples (n = 164). RNA-sequencing analysis revealed that BA treatment had greater effects than GH, and that asparagine synthetase (Asns) was the 5 th most significantly increased gene by BA at day 3. ASNS protein expression was dramatically increased by BA treatment at day 7 (p < 0.05). The most significantly increased gene at day 3 was activating transcription factor 5 (Atf5), a transcription factor known to regulate ASNS gene expression. Gene and protein expression of Atf4, another known regulator of Asns expression, was not changed by BA treatment. Expression of more than 20 known Atf4 target genes were increased by BA treatment, suggesting that BA treatment induces an integrated stress response (ISR) in skeletal muscle of pigs. In support of this, mRNA expression of sestrin-2 (Sesn2) and cyclin-dependant kinase 1 alpha (Cdkn1a), two key stress-responsive genes and negative regulators of cellular growth, were also strongly increased from day 3 of BA treatment. Finally, tRNA charging was the most significantly enriched pathway induced by BA treatment, suggesting alterations to the translational capacity/efficiency of the muscle. BA-mediated changes to the skeletal muscle transcriptome are highly indicative of an integrated stress response (ISR), particularly genes relating to amino acid biosynthesis and protein translational capacity.

Asparagine synthetase (ASNS) and CTP synthase (CTPS) are two metabolic enzymes crucial for glutamine homeostasis. A genome-wide screening in Saccharomyces cerevisiae reveal that both ASNS and CTPS form filamentous structures termed cytoophidia. Although CTPS cytoophidia were well documented in recent years, the filamentation of ASNS is less studied. Using the budding yeast as a model system, here we confirm that two ASNS proteins, Asn1 and Asn2, are capable of forming cytoophidia in diauxic and stationary phases. We find that glucose deprivation induces ASNS filament formation. Although ASNS and CTPS form distinct cytoophidia with different lengths, both structures locate adjacently to each other in most cells. Moreover, we demonstrate that the Asn1 cytoophidia colocalize with the Asn2 cytoophidia, while Asn2 filament assembly is largely dependent on Asn1. In addition, we are able to alter Asn1 filamentation by mutagenizing key sites on the dimer interface. Finally, we show that ASN1D330V promotes filamentation. The ASN1D330V mutation impedes cell growth in an ASN2 knockout background, while growing normally in an ASN2 wild-type background. Together, this study reveals a connection between ASNS and CTPS cytoophidia and the differential filament-forming capability between two ASNS paralogs., Competing Interests: The authors have declared that no competing interests exist.

General control nonderepressible 2 (GCN2) plays a major role in the cellular response to amino acid limitation. Although maintenance of amino acid homeostasis is critical for tumor growth, the contribution of GCN2 to cancer cell survival and proliferation is poorly understood. In this study, we generated GCN2 inhibitors and demonstrated that inhibition of GCN2 sensitizes cancer cells with low basal-level expression of asparagine synthetase (ASNS) to the antileukemic agent l-asparaginase (ASNase) in vitro and in vivo. We first tested acute lymphoblastic leukemia (ALL) cells and showed that treatment with GCN2 inhibitors rendered ALL cells sensitive to ASNase by preventing the induction of ASNS, resulting in reduced levels of de novo protein synthesis. Comprehensive gene-expression profiling revealed that combined treatment with ASNase and GCN2 inhibitors induced the stress-activated MAPK pathway, thereby triggering apoptosis. By using cell-panel analyses, we also showed that acute myelogenous leukemia and pancreatic cancer cells were highly sensitive to the combined treatment. Notably, basal ASNS expression at protein levels was significantly correlated with sensitivity to combined treatment. These results provide mechanistic insights into the role of GCN2 in the amino acid response and a rationale for further investigation of GCN2 inhibitors for the treatment of cancer., Competing Interests: Conflict of interest statement: The authors are employees of Takeda Pharmaceutical Company Limited.

Early life events are associated with the susceptibility to chronic diseases in adult life. Perturbations of endoplasmic reticulum (ER) homeostasis activate the unfolded protein response (UPR), which contributes to the development of metabolic alterations. Our aim was to evaluate liver UPR in an animal model of intrauterine growth restriction (IUGR). A significantly increased expression of X-box binding protein-1 spliced (XBP1s) mRNA (p<0.01), Endoplasmic Reticulum-localized DnaJ homologue (Erdj4) mRNA (p<0.05) and Bip/GRP78-glucose-regulated protein 78 (Bip) mRNA (p<0.05) was observed in the liver of IUGR rats at birth. Furthermore, the expression of gluconeogenesis genes and lipogenesis genes were significantly upregulated (p<0.05) in IUGR pups. At 105 d, IUGR male rats showed significantly reduced glucose tolerance (p<0.01). A significant decreased expression of XBP1s mRNA (p<0.01) and increased expression of double-stranded RNA-dependent protein kinase-like ER kinase (PERK) and Asparagine synthetase (ASNS) (p<0.05) was observed in the liver of IUGR male adult rats. Liver focal steatosis and periportal fibrosis were observed in IUGR rats. These findings show for the first time that fetal exposure to uteroplacental insufficiency is associated with the activation of hepatic UPR and suggest that UPR signaling may play a role in the metabolic risk., Competing Interests: The authors have declared that no competing interests exist.

Using a functional model of breast cancer heterogeneity, we previously showed that clonal sub-populations proficient at generating circulating tumour cells were not all equally capable of forming metastases at secondary sites. A combination of differential expression and focused in vitro and in vivo RNA interference screens revealed candidate drivers of metastasis that discriminated metastatic clones. Among these, asparagine synthetase expression in a patient's primary tumour was most strongly correlated with later metastatic relapse. Here we show that asparagine bioavailability strongly influences metastatic potential. Limiting asparagine by knockdown of asparagine synthetase, treatment with l-asparaginase, or dietary asparagine restriction reduces metastasis without affecting growth of the primary tumour, whereas increased dietary asparagine or enforced asparagine synthetase expression promotes metastatic progression. Altering asparagine availability in vitro strongly influences invasive potential, which is correlated with an effect on proteins that promote the epithelial-to-mesenchymal transition. This provides at least one potential mechanism for how the bioavailability of a single amino acid could regulate metastatic progression.