Search

Infectious spleen and kidney necrosis virus (ISKNV) has brought huge economic loss to the aquaculture industry. Through interfering with the viral replication and proliferation process that depends on host cells, its pathogenicity can be effectively reduced. In this study, we investigated the role of asparagine metabolites in ISKNV proliferation. The results showed that ISKNV infection up-regulated the expression of some key enzymes of the asparagine metabolic pathway in Chinese perch brain (CPB) cells. These key enzymes, including glutamic oxaloacetic transaminase 1/2 (GOT1/2) and malate dehydrogenase1/2 (MDH1/2) associated with the malate-aspartate shuttle (MAS) pathway and asparagine synthetase (ASNS) involved in the asparagine biosynthesis pathway, were up-regulated during ISKNV replication and release stages. In addition, results showed that the production of ISKNV was significantly reduced by inhibiting the MAS pathway or reducing the expression of ASNS by 1.3-fold and 0.6-fold, respectively, indicating that asparagine was a critical limiting metabolite for ISKNV protein synthesis. Furthermore, when asparagine was added to the medium without glutamine, ISKNV copy number was restored to 92% of that in the complete medium, indicating that ISKNV could be fully rescued from the absence of glutamine by supplementing asparagine. The above results indicated that asparagine was a critical factor in limiting the effective replication of ISKNV, which provided a new idea for the treatment of aquatic viral diseases. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Decidualization of the uterine endometrium is a critical process for embryo implantation in mammals, primarily occurring on gestational day 8 in pregnant mice. However, the interplay between the maternal gut microbiome, metabolism, and the uterus at this specific time point remains poorly understood. This study employed a multi-omics approach to investigate the metabolic, gut microbiome, and transcriptomic changes associated with early pregnancy (gestational day 8 (E8)) in mice. Serum metabolomics revealed a distinct metabolic profile at E8 compared to controls, with the differential metabolites primarily enriched in amino acid metabolism pathways. The gut microbial composition showed that E8 mice exhibited higher alpha-diversity and a significant shift in beta-diversity. Specifically, the E8 group displayed a decrease in pathogenic Proteobacteria and an increase in beneficial Bacteroidetes and S24-7 taxa. Transcriptomics identified myriads of distinct genes between the E8 and control mice. The differentially expressed genes were enriched in pathways involved in alanine, aspartate, and glutamate metabolism, PI3K-Akt signaling, and the PPAR signaling pathway. Integrative analysis of the multi-omics data uncovered potential mechanistic relationships among the differential metabolites, gut microbiota, and uterine gene expression changes. Notably, the gene Asns showed strong correlations with specific gut S24-7 and metabolite L-Aspartatic acid, suggesting its potential role in mediating the crosstalk between the maternal environment and embryo development during early pregnancy. These findings provide valuable insights into the complex interplay between the maternal metabolome, the gut microbiome, and the uterine transcriptome in the context of early pregnancy, which may contribute to our understanding of the underlying mechanisms of embryo implantation and development. [ABSTRACT FROM AUTHOR]

Abstract Background The present study aimed to investigate the expression level, biological function, and underlying mechanism of transmembrane protein 176B (TMEM176B) in gastric cancer (GC). Methods TMEM176B expression was detected by quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting (WB). The function of TMEM176B was determined by various in vitro assays including colony formation, 5-ethynyl-2ʹ-deoxyuridine (EdU), Transwell, and flow cytometry. Bioinformatics techniques were then used to elucidate the signaling pathways associated with TMEM176B activity. Tumor formation experiments were conducted on nude mice for in vivo validation of the preceding findings. TMEM176B expression was cross-referenced to clinicopathological parameters and survival outcomes. Results It was observed that TMEM176B was overexpressed in GC cells and tissues. Targeted TMEM176B abrogation inhibited colony formation, proliferation, migration, and invasion but promoted apoptosis in GC cell lines while TMEM176B overexpression had the opposite effects. Subsequent experimental validation disclosed an association between TMEM176B and the phosphatidylinositol 3-carboxykinase (PI3K)-protein kinase B (Akt)-mammalian target of rapamycin (mTOR) signaling axis. Moreover, TMEM176B affects GC cancer progression by regulating asparagine synthetase (ASNS). The in vivo assays confirmed that TMEM176B is oncogenic and the clinical data revealed a connection between TMEM176B expression and the clinicopathological determinants of GC. Conclusion The foregoing results suggest that TMEM176B significantly promotes the development of gastric cancer and is an independent prognostic factor of it.

Colorectal cancer (CRC) is a multi-stage process initiated through the formation of a benign adenoma, progressing to an invasive carcinoma and finally metastatic spread. Tumour cells must adapt their metabolism to support the energetic and biosynthetic demands associated with disease progression. As such, targeting cancer cell metabolism is a promising therapeutic avenue in CRC. However, to identify tractable nodes of metabolic vulnerability specific to CRC stage, we must understand how metabolism changes during CRC development. Here, we use a unique model system – comprising human early adenoma to late adenocarcinoma. We show that adenoma cells transition to elevated glycolysis at the early stages of tumour progression but maintain oxidative metabolism. Progressed adenocarcinoma cells rely more on glutamine-derived carbon to fuel the TCA cycle, whereas glycolysis and TCA cycle activity remain tightly coupled in early adenoma cells. Adenocarcinoma cells are more flexible with respect to fuel source, enabling them to proliferate in nutrient-poor environments. Despite this plasticity, we identify asparagine (ASN) synthesis as a node of metabolic vulnerability in late-stage adenocarcinoma cells. We show that loss of asparagine synthetase (ASNS) blocks their proliferation, whereas early adenoma cells are largely resistant to ASN deprivation. Mechanistically, we show that late-stage adenocarcinoma cells are dependent on ASNS to support mTORC1 signalling and maximal glycolytic and oxidative capacity. Resistance to ASNS loss in early adenoma cells is likely due to a feedback loop, absent in late-stage cells, allowing them to sense and regulate ASN levels and supplement ASN by autophagy. Together, our study defines metabolic changes during CRC development and highlights ASN synthesis as a targetable metabolic vulnerability in later stage disease.

Background: The present study aimed to investigate the expression level, biological function, and underlying mechanism of transmembrane protein 176B (TMEM176B) in gastric cancer (GC). Methods: TMEM176B expression was detected by quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting (WB). The function of TMEM176B was determined by various in vitro assays including colony formation, 5-ethynyl-2ʹ-deoxyuridine (EdU), Transwell, and flow cytometry. Bioinformatics techniques were then used to elucidate the signaling pathways associated with TMEM176B activity. Tumor formation experiments were conducted on nude mice for in vivo validation of the preceding findings. TMEM176B expression was cross-referenced to clinicopathological parameters and survival outcomes. Results: It was observed that TMEM176B was overexpressed in GC cells and tissues. Targeted TMEM176B abrogation inhibited colony formation, proliferation, migration, and invasion but promoted apoptosis in GC cell lines while TMEM176B overexpression had the opposite effects. Subsequent experimental validation disclosed an association between TMEM176B and the phosphatidylinositol 3-carboxykinase (PI3K)-protein kinase B (Akt)-mammalian target of rapamycin (mTOR) signaling axis. Moreover, TMEM176B affects GC cancer progression by regulating asparagine synthetase (ASNS). The in vivo assays confirmed that TMEM176B is oncogenic and the clinical data revealed a connection between TMEM176B expression and the clinicopathological determinants of GC. Conclusion: The foregoing results suggest that TMEM176B significantly promotes the development of gastric cancer and is an independent prognostic factor of it. [ABSTRACT FROM AUTHOR]

Bifunctional enzymes, characterized by their dual active sites, enable efficient chemical conversion and substrate channeling using elegant coupling mechanisms to coordinate the two active sites. In humans, several bifunctional enzymes synthesize de novo carbon-nitrogen bonds by hydrolyzing glutamine and ATP in distinct active sites. Notable examples include guanosine monophosphate synthetase, cytidine triphosphate synthetase, phosphoribosylformyl-glycinamidine synthase, asparagine synthetase, and nicotinamide adenine dinucleotide synthetase. A more complex example of multifunctional glutamine-hydrolyzing synthetases in humans is carbamoyl phosphate synthetase. These enzymes are crucial for the biosynthesis of amino acids, nucleic acids, and co-factors, thereby playing pivotal roles in human health. This review delineates recent progress in understanding the structural characteristics, regulatory mechanisms, and disease relevance of glutamine-hydrolyzing synthetases in humans. Insights into their catalysis and activity regulation offer potential pathways for developing novel therapeutics.

Infectious spleen and kidney necrosis virus (ISKNV) has brought huge economic loss to the aquaculture industry. Through interfering with the viral replication and proliferation process that depends on host cells, its pathogenicity can be effectively reduced. In this study, we investigated the role of asparagine metabolites in ISKNV proliferation. The results showed that ISKNV infection up-regulated the expression of some key enzymes of the asparagine metabolic pathway in Chinese perch brain (CPB) cells. These key enzymes, including glutamic oxaloacetic transaminase 1/2 (GOT1/2) and malate dehydrogenase1/2 (MDH1/2) associated with the malate-aspartate shuttle (MAS) pathway and asparagine synthetase (ASNS) involved in the asparagine biosynthesis pathway, were up-regulated during ISKNV replication and release stages. In addition, results showed that the production of ISKNV was significantly reduced by inhibiting the MAS pathway or reducing the expression of ASNS by 1.3-fold and 0.6-fold, respectively, indicating that asparagine was a critical limiting metabolite for ISKNV protein synthesis. Furthermore, when asparagine was added to the medium without glutamine, ISKNV copy number was restored to 92% of that in the complete medium, indicating that ISKNV could be fully rescued from the absence of glutamine by supplementing asparagine. The above results indicated that asparagine was a critical factor in limiting the effective replication of ISKNV, which provided a new idea for the treatment of aquatic viral diseases.

Decidualization of the uterine endometrium is a critical process for embryo implantation in mammals, primarily occurring on gestational day 8 in pregnant mice. However, the interplay between the maternal gut microbiome, metabolism, and the uterus at this specific time point remains poorly understood. This study employed a multi-omics approach to investigate the metabolic, gut microbiome, and transcriptomic changes associated with early pregnancy (gestational day 8 (E8)) in mice. Serum metabolomics revealed a distinct metabolic profile at E8 compared to controls, with the differential metabolites primarily enriched in amino acid metabolism pathways. The gut microbial composition showed that E8 mice exhibited higher alpha-diversity and a significant shift in beta-diversity. Specifically, the E8 group displayed a decrease in pathogenic Proteobacteria and an increase in beneficial Bacteroidetes and S24-7 taxa. Transcriptomics identified myriads of distinct genes between the E8 and control mice. The differentially expressed genes were enriched in pathways involved in alanine, aspartate, and glutamate metabolism, PI3K-Akt signaling, and the PPAR signaling pathway. Integrative analysis of the multi-omics data uncovered potential mechanistic relationships among the differential metabolites, gut microbiota, and uterine gene expression changes. Notably, the gene Asns showed strong correlations with specific gut S24-7 and metabolite L-Aspartatic acid, suggesting its potential role in mediating the crosstalk between the maternal environment and embryo development during early pregnancy. These findings provide valuable insights into the complex interplay between the maternal metabolome, the gut microbiome, and the uterine transcriptome in the context of early pregnancy, which may contribute to our understanding of the underlying mechanisms of embryo implantation and development.

AIM: To investigate the expression of asparagine synthetase (ASNS) in cholangiocarcinoma (CCA), and to explore the potential role and molecular mechanisms of ASNS in CCA metastasis. METHODS: A public database was used to analyze ASNS mRNA expression in various tumor tissues. Immunohistochemistry, Western blot and immunofluorescence were used to detect ASNS protein expression in human CCA specimens and murine spontaneous CCA models. The CCK8, wound-healing and Transwell assays were performed to examine the effects of ASNS on CCA cell proliferation, migration and invasion. The effects of ASNS on CCA intrahepatic growth and lung metastasis were investigated by orthotopic implantation and tail vein injection of stable ASNS knockdown CCA cell lines. The ASNS-related pathways in CCA were examined using gene set enrichment analysis, and the underlying molecular mechanisms were verified by immunofluorescence and Western blot. RESULTS: The expression of ASNS in both human and murine CCA tissues was higher than that in adjacent noncancerous tissues. ASNS promoted the proliferation, migration and invasion of human CCA HuCCTl and HCCC-9810 cells in an enzyme activity-independent manner. Bioinformatics analysis showed that p-catenin was enriched in CCA tissues with high expression of ASNS, and ASNS initiated epithelial-mesenchymal transition(EMT) of CCA by promoting the nuclear translocation of p-catenin. Blocking the nuclear translocation of p-catenin with XAV-939 significantly inhibited the invasion and migration of CCA cells. CONCLUSION: ASNS is highly expressed in CCA and promotes EMT by meditating the nuclear translocation of p-catenin. [ABSTRACT FROM AUTHOR]

To examine the physiological change in the growth suppression and abortion of parthenocarpic cucumber fruit, the expression of candidate marker genes of sugar starvation in relation to growth activity was examined. Fruits that failed to start exponential growth seemed to eventually abort. Hexose concentration of fruits was low in growth-suppressed fruit and increased in normally growing fruit consistent with the vacuolization. The correlation matrix indicated that the transcript levels of the genes, except CsaV3_6G046050 and CsaV3_5G032930, had a highly significant negative correlation with the relative growth rate in fruit length and had highly significant mutual positive correlations, suggesting that the asparagine synthetase gene, Cucumis sativus putative CCCH-type zinc finger protein CsSEF1, C. sativus BTB/POZ domain-containing protein At1g63850-like, CsaV3_3G000800, CsaV3_3G041280, and CsaV3_7G032930 are good markers of sugar starvation in cucumber fruit. The expression of candidate marker genes together with the hexose analysis strongly suggests that severe sugar starvation is occurring in growth-suppressed fruit. [ABSTRACT FROM AUTHOR]

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. Synopsis: The epigenetic modifier DOT1L represses expression of asparagine synthetase to preserve the apical progenitor state and prevents microcephaly by premature neuronal differentiation. DOT1L influences the balance between symmetric and asymmetric cell divisions.DOT1L prevents expression of ASNS in apical progenitors in concert with PRC2.DOT1L regulates apical progenitor metabolism. [ABSTRACT FROM AUTHOR]

L-asparaginase, which hydrolyzes asparagine into aspartic acid and ammonia, is frequently used to treat acute lymphoblastic leukaemia in children. When combined with other chemotherapy drugs, the event-free survival rate is 90%. Due to immunogenicity and drug resistance, however, not all patients benefit from it, restricting the use of L-asparaginase therapy in other haematological cancers. To solve the problem of immunogenicity, several L-ASNase variants have emerged, such as Erwinia-ASNase and PEG-ASNase. However, even when Erwinia-ASNase is used as a substitute for E. coli-ASNase or PEG-ASNase, allergic reactions occur in 3%-33% of patients. All of these factors contributed to the development of novel L-ASNases. Additionally, L-ASNase resistance mechanisms, such as the methylation status of ASNS promoters and activation of autophagy, have further emphasized the importance of personalized treatment for paediatric haematological neoplasms. In this review, we discussed the metabolic effects of L-ASNase, mechanisms of drug resistance, applications in non-ALL leukaemia, and the development of novel L-ASNase. [ABSTRACT FROM AUTHOR]

Storing potato tubers at cold temperatures, either for transport or continuity of supply, is associated with the conversion of sucrose to reducing sugars. When cold-stored cut tubers are processed at high temperatures, with endogenous asparagine, acrylamide is formed. Acrylamide is classified as a carcinogen. Potato processors prefer cultivars which accumulate fewer reducing sugars and thus less acrylamide on processing, and suitable processing cultivars may not be available. We used CRISPR-Cas9 to disrupt the genes encoding vacuolar invertase (VInv) and asparagine synthetase 1 (AS1) of cultivars Atlantic and Desiree to reduce the accumulation of reducing sugars and the production of asparagine after cold storage. Three of the four guide RNAs employed induced mutation frequencies of 17–98%, which resulted in deletions, insertions and substitutions at the targeted gene sites. Eight of ten edited events had mutations in at least one allele of both genes; for two, only the VInv was edited. No wild-type allele was detected in both genes of events DSpco7, DSpFN4 and DSpco12, suggesting full allelic mutations. Tubers of two Atlantic and two Desiree events had reduced fructose and glucose concentrations after cold storage. Crisps from these and four other Desiree events were lighter in colour and included those with 85% less acrylamide. These results demonstrate that multiplex CRISPR-Cas9 technology can generate improved potato cultivars for healthier processed potato products. [ABSTRACT FROM AUTHOR]

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 (R2 = 0.85, p = 1.3 × 10-33) and in 63 BCP-ALL clinical samples (R2 = 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. [ABSTRACT FROM AUTHOR]

Abstract 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.

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. [ABSTRACT FROM AUTHOR]

Colorectal cancer (CRC) is a multi-stage process initiated through the formation of a benign adenoma, progressing to an invasive carcinoma and finally metastatic spread. Tumour cells must adapt their metabolism to support the energetic and biosynthetic demands associated with disease progression. As such, targeting cancer cell metabolism is a promising therapeutic avenue in CRC. However, to identify tractable nodes of metabolic vulnerability specific to CRC stage, we must understand how metabolism changes during CRC development. Here, we use a unique model system - comprising human early adenoma to late adenocarcinoma. We show that adenoma cells transition to elevated glycolysis at the early stages of tumour progression but maintain oxidative metabolism. Progressed adenocarcinoma cells rely more on glutamine-derived carbon to fuel the TCA cycle, whereas glycolysis and TCA cycle activity remain tightly coupled in early adenoma cells. Adenocarcinoma cells are more flexible with respect to fuel source, enabling them to proliferate in nutrient-poor environments. Despite this plasticity, we identify asparagine (ASN) synthesis as a node of metabolic vulnerability in late-stage adenocarcinoma cells. We show that loss of asparagine synthetase (ASNS) blocks their proliferation, whereas early adenoma cells are largely resistant to ASN deprivation. Mechanistically, we show that late-stage adenocarcinoma cells are dependent on ASNS to support mTORC1 signalling and maximal glycolytic and oxidative capacity. Resistance to ASNS loss in early adenoma cells is likely due to a feedback loop, absent in late-stage cells, allowing them to sense and regulate ASN levels and supplement ASN by autophagy. Together, our study defines metabolic changes during CRC development and highlights ASN synthesis as a targetable metabolic vulnerability in later stage disease., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Abstract 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.

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. [ABSTRACT FROM AUTHOR]

Mutations of KRAS gene are found in various types of cancer, including colorectal cancer (CRC). Despite intense efforts, no pharmacological approaches are expected to be effective against KRAS-mutant cancers. Macropinocytosis is an evolutionarily conserved actin-dependent endocytic process that internalizes extracellular fluids into large vesicles called macropinosomes. Recent studies have revealed macropinocytosis's important role in metabolic adaptation to nutrient stress in cancer cells harboring KRAS mutations. Here we showed that KRAS-mutant CRC cells enhanced macropinocytosis for tumor growth under nutrient-depleted conditions. We also demonstrated that activation of Rac1 and phosphoinositide 3-kinase were involved in macropinocytosis of KRAS-mutant CRC cells. Furthermore, we found that macropinocytosis was closely correlated with asparagine metabolism. In KRAS-mutant CRC cells engineered with knockdown of asparagine synthetase, macropinocytosis was accelerated under glutamine-depleted condition, and albumin addition could restore the glutamine depletion-induced growth suppression by recovering the intracellular asparagine level. Finally, we discovered that the combination of macropinocytosis inhibition and asparagine depletion dramatically suppressed the tumor growth of KRAS-mutant CRC cells in vivo. These results indicate that dual blockade of macropinocytosis and asparagine bioavailability could be a novel therapeutic strategy for KRAS-mutant cancers. [ABSTRACT FROM AUTHOR]

SUMMARY: Phosphorus (P) deficiency adversely affects nodule development as reflected by reduced nodule fresh weight in legume plants. Though mechanisms underlying nodule adaptation to P deficiency have been studied extensively, it remains largely unknown which regulator mediates nodule adaptation to P deficiency. In this study, GUS staining and quantitative reverse transcription‐PCR analysis reveal that the SPX member GmSPX5 is preferentially expressed in soybean (Glycine max) nodules. Overexpression of GmSPX5 enhanced soybean nodule development particularly under phosphate (Pi) sufficient conditions. However, the Pi concentration was not affected in soybean tissues (i.e., leaves, roots, and nodules) of GmSPX5 overexpression or suppression lines, which distinguished it from other well‐known SPX members functioning in control of Pi homeostasis in plants. Furthermore, GmSPX5 was observed to interact with the transcription factor GmNF‐YC4 in vivo and in vitro. Overexpression of either GmSPX5 or GmNF‐YC4 significantly upregulated the expression levels of five asparagine synthetase‐related genes (i.e., GmASL2–6) in soybean nodules. Meanwhile, yeast one‐hybrid and luciferase activity assays strongly suggested that interactions of GmSPX5 and GmNF‐YC4 activate GmASL6 expression through enhancing GmNF‐YC4 binding of the GmASL6 promoter. These results not only demonstrate the GmSPX5–GmNF‐YC4–GmASL6 regulatory pathway mediating soybean nodule development, but also considerably improve our understanding of SPX functions in legume crops. Significance Statement: The soybean (Glycine max) SPX member GmSPX5 is preferentially expressed in nodules. GmSPX5 overexpression or suppression did not affect the phosphate (Pi) concentration in soybean tissues, which distinguished it from other SPX members functioning in control of Pi homeostasis in plants. Furthermore, GmSPX5 interacted with GmNF‐YC4, and their overexpression led to upregulation of the expression of five asparagine synthetase‐related genes in soybean nodules. These results suggest that the GmSPX5–GmNF‐YC4 regulatory pathway mediates soybean nodule growth. [ABSTRACT FROM AUTHOR]

Asparagine is an efficient nitrogen transport and storage carrier. Asparagine synthesis occurs by the amination of aspartate which is catalyzed by asparagine synthetase (ASN) in plants. Complete genome-wide analysis and classifications of the ASN gene family have recently been reported in different plants. However, systematic analysis and expression profiles of these genes have not been performed in apple (Malus domestica). Here, a comprehensive bioinformatics approach was applied to identify MdASNs in apple. Then, plant phylogenetic tree, chromosome location, conserved protein motif, gene structure, and expression pattern of MdASNs were analyzed. Five members were identified and distributed on 4 chromosomes with conserved GATase-7 and ASN domains. Expression analysis indicated that all MdASNs mRNA accumulated at the highest level in reproductive organs, namely flowers or fruits, which may be associated with the redistribution of free amino acids in plant metabolic organs and reservoirs. Additionally, most of MdASNs were dramatically up-regulated under various nitrogen supplies, especially in the aboveground part. Taken together, MdASNs may be assigned to be responsible for the nitrogen metabolism and asparagine synthesis in apple.

Asparagine and glutamine depletion operated by the drug Asparaginase (ASNase) has revolutionized therapy in pediatric patients affected by Acute Lymphoblastic Leukemia (ALL), bringing remissions to a remarkable 90 % of cases. However, the knowledge of the proproliferative role of asparagine in adult and solid tumors is still limited. We have here analyzed the effect of ASNase on three adenocarcinoma cell lines (A549, lung adenocarcinoma, MCF-7, breast cancer, and 786-O, kidney cancer). In contrast to MCF-7 cells, 786-O and A549 cells proved to be a relevant target for cell cycle perturbation by asparagine and glutamine shortage. Indeed, when the cell-cycle was analyzed by flow cytometry, A549 showed a canonical response to asparaginase, 786-O cells, instead, showed a reduction of the percentage of cells in the G1 phase and an increase of those in the S-phase. Despite an increased number of PCNA and RPA70 positive nuclear foci, BrdU and EdU incorporation was absent or strongly delayed in treated 786-O cells, thus indicating a readiness of replication forks unmatched by DNA synthesis. In 786-O asparagine synthetase was reduced following treatment and glutamine synthetase was totally absent. Interestingly, DNA synthesis could be recovered by adding Gln to the medium. MCF-7 cells showed no significant changes in the cell cycle phases, in DNA-bound PCNA and in total PCNA, but a significant increase in ASNS and GS mRNA and protein expression. The collected data suggest that the effect observed on 786-O cells following ASNase treatment could rely on mechanisms which differ from those well-known and described for leukemic blasts, consisting of a complete block in the G1/S transition in proliferating cells and on an increase on non-proliferative (G0) blasts., Competing Interests: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:Maristella Maggi has patent A HIGHLY STABLE, PROTEASE-RESISTANT E. COLI ASPARAGINASE issued to University of Pavia. Claudia Scotti has patent A HIGHLY STABLE, PROTEASE-RESISTANT E. COLI ASPARAGINASE issued to University of Pavia. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.)

Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer. HCC cells consume large amounts of glutamine to survive, but can adapt to glutamine depletion in the presence of an exogenous asparagine. L-asparaginase (ASNase) converts glutamine and asparagine to glutamate and aspartate, respectively, and has been used to treat leukemia. Here we examined the effects of ASNase treatment on HCC cells and explored the potential impact of combining ASNase with the tyrosine kinase inhibitor lenvatinib (Len) for HCC treatment. Cell viability and death of HCC cell lines treated with either Len or ASNase alone or with Len and ASNase combined were determined. We assessed mRNA and protein expression levels of glutamine synthetase (GS) and asparagine synthetase (ASNS) by real-time quantitative PCR and immunoblotting. The antitumor effect of the combination therapy relative to Len or ASNase monotherapy was also evaluated in a xenograft tumor mouse model. ASNase treatment inhibited growth of SNU387 and SNU398 HCC cells, which have low GS and high ASNS expression levels, respectively, but did not clearly inhibit growth of the other cell lines. Len plus ASNase combination therapy synergistically inhibited proliferation and induced oxidative stress leading to cell death of some HCC cells lines. However, cell death of Huh7 cells, which express ASCT2, an important glutamine transporter for cancer cells, was not affected by the combination treatment. In a xenograft model, Len combined with ASNase significantly attenuated tumor development relative to mice treated with Len or ASNase alone. ASNase-mediated targeting of two amino acids, glutamine and asparagine, which are indispensable for HCC survival, induces oxidative stress and can be a novel cancer treatment option that exerts a synergistic effect when used in combination with Len. [ABSTRACT FROM AUTHOR]

Introduction: Canagliflozin (CANA) is a sodium-glucose cotransporter 2 inhibitor that was recently approved for treating diabetes. However, its effects on liver function are not well understood. The function of asparagine synthetase (ASNS) has been studied in several cancers but not in liver injury. Therefore, we investigated the connection between CANA and ASNS in alleviating damage (i.e., their hepatoprotective effect) in a rat liver injury model. Methods: The rat model of liver injury was established using carbon tetrachloride treatment. Rats with liver injury were administered CANA orally for 8 weeks daily. After week 8, peripheral blood was collected to measure serum alanine aminotransferase, aspartate aminotransferase, and lactate dehydrogenase levels. Liver histopathology was examined using hematoxylin and eosin staining to determine the degree of liver injury. Protein expression in the rat livers was examined using Western blotting. Results: CANA treatment decreased serum alanine aminotransferase, aspartate aminotransferase, and lactate dehydrogenase levels compared with those of the untreated group, demonstrating diminished liver injury. Mechanistically, CANA treatment activated AMP-activated protein kinase (AMPK), leading to increased nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2) and activating transcription factor 4 (ATF4), which upregulated ASNS expression in liver-injured rats. Conclusion: CANA significantly alleviated liver injury by activating the AMPK/Nrf2/ATF4 axis and upregulating ASNS expression, indicating its potential for treating patients with type 2 diabetes mellitus with impaired liver function. [ABSTRACT FROM AUTHOR]

Purpose: Glutamine plays an important role in cell viability and growth of various tumors. For the fetal subtype of hepatoblastoma, growth inhibition through glutamine depletion was shown. We studied glutamine depletion in embryonal cell lines of hepatoblastoma carrying different mutations. Since asparagine synthetase was identified as a prognostic factor and potential therapeutic target in adult hepatocellular carcinoma, we investigated the expression of its gene ASNS and of the gene GLUL, encoding for glutamine synthetase, in hepatoblastoma specimens and cell lines and investigated the correlation with overall survival. Methods: We correlated GLUL and ASNS expression with overall survival using publicly available microarray and clinical data. We examined GLUL and ASNS expression by RT-qPCR and by Western blot analysis in the embryonal cell lines Huh-6 and HepT1, and in five hepatoblastoma specimens. In the same cell lines, we investigated the effects of glutamine depletion. Hepatoblastoma biopsies were examined for histology and CTNNB1 mutations. Results: High GLUL expression was associated with a higher median survival time. Independent of mutations and histology, hepatoblastoma samples showed strong GLUL expression and glutamine synthesis. Glutamine depletion resulted in the inhibition of proliferation and of cell viability in both embryonal hepatoblastoma cell lines. ASNS expression did not correlate with overall survival. Conclusion: Growth inhibition resulting from glutamine depletion, as described for the hepatoblastoma fetal subtype, is also detected in established embryonal hepatoblastoma cell lines carrying different mutations. At variance with adult hepatocellular carcinoma, in hepatoblastoma asparagine synthetase has no prognostic significance. [ABSTRACT FROM AUTHOR]

The loss‐of‐function variants of the human asparagine synthetase (ASNS) gene cause asparagine synthetase deficiency (ASNSD). Diagnosis of ASNSD requires genetic tests because a specific biochemical diagnostic for ASNSD is not available. There are a few reports describing the functional evaluation of ASNS variants. Therefore, in vitro methods are needed to evaluate the detected variants in patients. In this report, five types of human ASNS proteins (wild‐type and our reported four variants: p.Leu145Ser, p.Leu247Trp, p.Val489Asp, and p.Trp541Cysfs*5) were expressed in silkworm using a baculoviral expression system. An enzymatic activity assay of ASNS was performed, and the concentration of asparagine by ninhydrin and High Performance Liquid Chromatography methods using the purified recombinant proteins was measured. We established ASNS deficient HEK293 cells using the CRISPR/Cas9 method and evaluated the growth of cells without asparagine after transduction of ASNS variants with a lentiviral expression system. The four ASNS variants displayed significantly low enzymatic activity. The ASNS deficient HEK293 cells transduced with wild‐type ASNS grew without asparagine, whereas cells transduced with the variants did not grow or showed significantly slower growth than cells transduced with wild‐type ASNS. Herein, we established a method for evaluating the enzymatic activity of the recombinant human ASNS variants. The results of the cell‐based assay corroborated the results of the enzymatic activity. These methods should enable the evaluation of the pathogenicity of ASNS variants. [ABSTRACT FROM AUTHOR]

Background: The prognostic factors of skull base chordoma associated with outcomes of patients after surgery remain inadequately identified. This study was designed to identify a novel prognostic factor for patients with skull base chordoma. Method: Using a proteomic technique, the tumor biomarkers that were upregulated in the rapid-recurrence group of chordoma were screened and then narrowed down by bioinformatic analysis. Finally one potential biomarker was chosen for validation by immunohistochemistry using tissue microarray (TMA). A total of 187 patients included in TMA were randomly divided into two cohorts, the training cohort included 93 patients and the validation cohort included 94 patients. Kaplan-Meier survival analysis was used to assess the patients' survival. Univariable and multivariable Cox regression analysis were used to identify prognostic factors predicting recurrence-free survival (RFS). CCK-8 assay, clonal formation assay and transwell assay were used to test the effect of asparagine synthetase (ASNS) on the proliferation, migration and invasion in chordoma cell lines. Results: Among 146 upregulated proteins, ASNS was chosen as a potential prognostic biomarker after bioinformatics analysis. The H-scores of ASNS ranged from 106.27 to 239.58 in TMA. High expression of ASNS was correlated with shorter RFS in both the training cohort (p = 0.0093) and validation cohort (p < 0.001). Knockdown of ASNS by small interfering RNA (siRNA) inhibited the growth, colony formation, migration and invasion of chordoma cells in vitro. Conclusion: This study indicates that high expression of ASNS is correlated with poor prognosis of patients with skull base chordoma. ASNS may be a useful prognostic factor for patients with skull base chordoma. [ABSTRACT FROM AUTHOR]

BackgroundThe prognostic factors of skull base chordoma associated with outcomes of patients after surgery remain inadequately identified. This study was designed to identify a novel prognostic factor for patients with skull base chordoma.MethodUsing a proteomic technique, the tumor biomarkers that were upregulated in the rapid-recurrence group of chordoma were screened and then narrowed down by bioinformatic analysis. Finally one potential biomarker was chosen for validation by immunohistochemistry using tissue microarray (TMA). A total of 187 patients included in TMA were randomly divided into two cohorts, the training cohort included 93 patients and the validation cohort included 94 patients. Kaplan-Meier survival analysis was used to assess the patients’ survival. Univariable and multivariable Cox regression analysis were used to identify prognostic factors predicting recurrence-free survival (RFS). CCK-8 assay, clonal formation assay and transwell assay were used to test the effect of asparagine synthetase (ASNS) on the proliferation, migration and invasion in chordoma cell lines.ResultsAmong 146 upregulated proteins, ASNS was chosen as a potential prognostic biomarker after bioinformatics analysis. The H-scores of ASNS ranged from 106.27 to 239.58 in TMA. High expression of ASNS was correlated with shorter RFS in both the training cohort (p = 0.0093) and validation cohort (p < 0.001). Knockdown of ASNS by small interfering RNA (siRNA) inhibited the growth, colony formation, migration and invasion of chordoma cells in vitro.ConclusionThis study indicates that high expression of ASNS is correlated with poor prognosis of patients with skull base chordoma. ASNS may be a useful prognostic factor for patients with skull base chordoma.

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.

Summary: Free asparagine is the precursor for acrylamide, which forms during the baking, toasting and high‐temperature processing of foods made from wheat. In this study, CRISPR/Cas9 was used to knock out the asparagine synthetase gene, TaASN2, of wheat (Triticum aestivum) cv. Cadenza. A 4‐gRNA polycistronic gene was introduced into wheat embryos by particle bombardment and plants were regenerated. T1 plants derived from 11 of 14 T0 plants were shown to carry edits. Most edits were deletions (up to 173 base pairs), but there were also some single base pair insertions and substitutions. Editing continued beyond the T1 generation. Free asparagine concentrations in the grain of plants carrying edits in all six TaASN2 alleles (both alleles in each genome) were substantially reduced compared with wildtype, with one plant showing a more than 90 % reduction in the T2 seeds. A plant containing edits only in the A genome alleles showed a smaller reduction in free asparagine concentration in the grain, but the concentration was still lower than in wildtype. Free asparagine concentration in the edited plants was also reduced as a proportion of the free amino acid pool. Free asparagine concentration in the T3 seeds remained substantially lower in the edited lines than wildtype, although it was higher than in the T2 seeds, possibly due to stress. In contrast, the concentrations of free glutamine, glutamate and aspartate were all higher in the edited lines than wildtype. Low asparagine seeds showed poor germination but this could be overcome by exogenous application of asparagine. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract Background Free asparagine is the precursor for acrylamide formation during cooking and processing of grains, tubers, beans and other crop products. In wheat grain, free asparagine, free glutamine and total free amino acids accumulate to high levels in response to sulphur deficiency. In this study, RNA-seq data were acquired for the embryo and endosperm of two genotypes of bread wheat, Spark and SR3, growing under conditions of sulphur sufficiency and deficiency, and sampled at 14 and 21 days post anthesis (dpa). The aim was to provide new knowledge and understanding of the genetic control of asparagine accumulation and breakdown in wheat grain. Results There were clear differences in gene expression patterns between the genotypes. Sulphur responses were greater at 21 dpa than 14 dpa, and more evident in SR3 than Spark. TaASN2 was the most highly expressed asparagine synthetase gene in the grain, with expression in the embryo much higher than in the endosperm, and higher in Spark than SR3 during early development. There was a trend for genes encoding enzymes of nitrogen assimilation to be more highly expressed in Spark than SR3 when sulphur was supplied. TaASN2 expression in the embryo of SR3 increased in response to sulphur deficiency at 21 dpa, although this was not observed in Spark. This increase in TaASN2 expression was accompanied by an increase in glutamine synthetase gene expression and a decrease in asparaginase gene expression. Asparagine synthetase and asparaginase gene expression in the endosperm responded in the opposite way. Genes encoding regulatory protein kinases, SnRK1 and GCN2, both implicated in regulating asparagine synthetase gene expression, also responded to sulphur deficiency. Genes encoding bZIP transcription factors, including Opaque2/bZIP9, SPA/bZIP25 and BLZ1/OHP1/bZIP63, all of which contain SnRK1 target sites, were also expressed. Homeologues of many genes showed differential expression patterns and responses, including TaASN2. Conclusions Data on the genetic control of free asparagine accumulation in wheat grain and its response to sulphur supply showed grain asparagine levels to be determined in the embryo, and identified genes encoding signalling and metabolic proteins involved in asparagine metabolism that respond to sulphur availability.

Asparagine synthetase catalyses the transfer of an amino group from glutamine to aspartate to form glutamate and asparagine. The accumulation of free (nonprotein) asparagine in crops has implications for food safety because free asparagine is the precursor for acrylamide, a carcinogenic contaminant that forms during high‐temperature cooking and processing. Here we review publicly available genome data for asparagine synthetase genes from species of the Pooideae subfamily, including bread wheat and related wheat species (Triticum and Aegilops spp.), barley (Hordeum vulgare) and rye (Secale cereale) of the Triticeae tribe. Also from the Pooideae subfamily: brachypodium (Brachypodium dIstachyon) of the Brachypodiae tribe. More diverse species are also included, comprising sorghum (Sorghum bicolor) and maize (Zea mays) of the Panicoideae subfamily and rice (Oryza sativa) of the Ehrhartoideae subfamily. The asparagine synthetase gene families of the Triticeae species each comprise five genes per genome, with the genes assigned to four groups: 1, 2, 3 (subdivided into 3.1 and 3.2) and 4. Each species has a single gene per genome in each group, except that some bread wheat varieties (genomes AABBDD) and emmer wheat (Triticum dicoccoides; genomes AABB) lack a group 2 gene in the B genome. This raises questions about the ancestry of cultivated pasta wheat and the B genome donor of bread wheat, suggesting that the hybridisation event that gave rise to hexaploid bread wheat occurred more than once. In phylogenetic analyses, genes from the other species cluster with the Triticeae genes, but brachypodium, sorghum and maize lack a group 2 gene, while rice has only two genes, one group 3 and one group 4. This means that TaASN2, the most highly expressed asparagine synthetase gene in wheat grain, has no equivalent in maize, rice, sorghum or brachypodium. An evolutionary pathway is proposed in which a series of gene duplications gave rise to the five genes found in modern Triticeae species. [ABSTRACT FROM AUTHOR]

Asparagine synthetase (ASNS) and CTP synthase (CTPS) are two metabolic enzymes that catalyze the biosynthesis of asparagine and CTP, respectively. Both CTPS and ASNS have been identified to form cytoophidia in Saccharomyces cerevisiae. Glutamine is a common substrate for both these enzymes, and they play an important role in glutamine homeostasis. Here, we find that the ASNS cytoophidia are shorter than the CTPS cytoophidia, and that disruption of ASNS shortens the length of CTPS cytoophidia. However, the deletion of CTPS has no effect on the formation and length of ASNS cytoophidia, or on the ASNS protein level. We also find that Asn1 overexpression induces the formation of a multi-dot structure in diauxic phase which suggests that the increased protein level may trigger cytoophidia formation. Collectively, our results reveal a connection between ASNS cytoophidia and CTPS cytoophidia. [ABSTRACT FROM AUTHOR]

IpaH enzymes are bacterial E3 ligases targeting host proteins for ubiquitylation. Two autoinhibition modes of IpaH enzymes have been proposed based on the relative positioning of the Leucine-rich repeat domain (LRR) with respect to the NEL domain. In mode 1, substrate-binding competitively displaces the interactions between theLRR and NEL to relieve autoinhibition. However, the molecular basis for mode 2 is unclear. Here, we present the crystal structures of Shigella IpaH9.8 and the LRR of IpaH9.8 in complex with the substrate of human guanylate-binding protein 1 (hGBP1). A hydrophobic cluster in the C-terminus of IpaH9.8LRR forms a hydrophobic pocket involved in binding the NEL domain, and the binding is important for IpaH9.8 autoinhibition. Substrate-binding destabilizes the hydrophobic cluster by inducing conformational changes of IpaH9.8LRR. Arg166 and Phe187 in IpaH9.8LRR function as sensors for substrate-binding. Collectively, our findings provide insights into the molecular mechanisms for the actication of IpaH9.8 in autoinhibition mode 2. Ye, Xiong et al. present crystal structures of bacterial E3 ubiquitin ligase IpaH9.8 and IpaH9.8LRR–hGBP1. They find that substrate-binding destabilizes the hydrophobic cluster to relieve the autoinhibition of IpaH9.8. This study provides insights into the mechanisms underlying substrate-induced activation of IpaH9.8. [ABSTRACT FROM AUTHOR]

GABA released from heterogeneous types of interneurons acts in a complex spatio-temporal manner on postsynaptic targets in the networks. In addition to GABA, a large fraction of GABAergic cells also express neuromodulator peptides. Somatostatin (SOM) containing interneurons, in particular, have been recognized as key players in several brain circuits, however, the action of SOM and its downstream network effects remain largely unknown. Here, we used optogenetics, electrophysiologic, anatomical and behavioral experiments to reveal that the dendrite-targeting, SOM+ GABAergic interneurons demonstrate a unique layer-specific action in the medial entorhinal cortex (MEC) both in terms of GABAergic and SOM-related properties. We show that GABAergic and somatostatinergic neurotransmission originating from SOM+ local interneurons preferentially inhibit layerIII-V pyramidal cells, known to be involved in memory formation. We propose that this dendritic GABA–SOM dual inhibitory network motif within the MEC serves to selectively modulate working-memory formation without affecting the retrieval of already learned spatial navigation tasks. Miklós Kecskés et al. show that somatostatin-expressing interneurons in the medial entorhinal cortex regulate deep-layer pyramidal neurons and impact short-term memory in mice. [ABSTRACT FROM AUTHOR]

Asparagine Synthetase (ASNS) catalyzes the synthesis of the non-essential amino acid asparagine (Asn) from aspartate (Asp) and glutamine (Gln). ASNS expression is highly regulated at the transcriptional level, being induced by both the Amino Acid Response (AAR) and the Unfolded Protein Response (UPR) pathways. Lack of ASNS protein expression is a hallmark of Acute Lymphoblastic Leukemia (ALL) blasts, which, therefore, are auxotrophic for Asn. This peculiarity is the rationale for the use of bacterial L-Asparaginase (ASNase) for ALL therapy, the first example of anti-cancer treatment targeting a tumor-specific metabolic feature. Other hematological and solid cancers express low levels of ASNS and, therefore, should also be Asn auxotrophs and ASNase sensitive. Conversely, in the last few years, several reports indicate that in some cancer types ASNS is overexpressed, promoting cell proliferation, chemoresistance, and a metastatic behavior. However, enhanced ASNS activity may constitute a metabolic vulnerability in selected cancer models, suggesting a variable and tumor-specific role of the enzyme in cancer. Recent evidence indicates that, beyond its canonical role in protein synthesis, Asn may have additional regulatory functions. These observations prompt a re-appreciation of ASNS activity in the biology of normal and cancer tissues, with particular attention to the fueling of Asn exchange between cancer cells and the tumor microenvironment.

Abstract Background Our previous studies concluded that metabolic disorder in the basal portions of rice shoots caused by a lack of cytosolic glutamine synthetase1;2 (GS1;2) resulted in a severe reduction in the outgrowth of tillers. Rice mutants lacking GS1;2 (gs1;2 mutants) showed a remarkable reduction in the contents of both glutamine and asparagine in the basal portions of shoots. In the current study, we attempted to reveal the mechanisms for this decrease in asparagine content using rice mutants lacking either GS1;2 or asparagine synthetase 1 (AS1). The contributions of the availability of glutamine and asparagine to the outgrowth of rice tillers were investigated. Results Rice has two AS genes, and the enzymes catalyse asparagine synthesis from glutamine. In the basal portions of rice shoots, expression of OsAS1, the major species in this tissue, was reduced in gs1;2 mutants, whereas OsAS2 expression was relatively constant. OsAS1 was expressed in phloem companion cells of the nodal vascular anastomoses connected to the axillary bud vasculatures in the basal portions of wild-type shoots, whereas cell-specific expression was markedly reduced in gs1;2 mutants. OsAS1 was up-regulated significantly by NH4 + supply in the wild type but not in gs1;2 mutants. When GS reactions were inhibited by methionine sulfoximine, OsAS1 was up-regulated by glutamine but not by NH4 +. The rice mutants lacking AS1 (as1 mutants) showed a decrease in asparagine content in the basal portions of shoots. However, glutamine content and tiller number were less affected by the lack of AS1. Conclusion These results indicate that in phloem companion cells of the nodal vascular anastomoses, asparagine synthesis is largely dependent on glutamine or its related metabolite-responsive AS1. Thus, the decrease in glutamine content caused by a lack of GS1;2 is suggested to result in low expression of OsAS1, decreasing asparagine content. However, the availability of asparagine generated from AS1 reactions is apparently less effective for the outgrowth of tillers. With respect to the tiller number and the contents of glutamine and asparagine in gs1;2 and as1 mutants, the availability of glutamine rather than asparagine in basal portions of rice shoots may be required for the outgrowth of rice tillers.

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. [ABSTRACT FROM AUTHOR]

Women have a lower incidence of colorectal cancer (CRC) than men, however, they have a higher incidence of right-sided colon cancer (RCC). This is of concern as patients with RCC have the poorest clinical outcomes among all CRC patients. Aberrant metabolism is a known hallmark and therapeutic target for cancer. We propose that metabolic subphenotypes exist between CRCs due to intertumoral molecular and genomic variation, and differences in environmental milieu of the colon which vary between the sexes. Metabolomics analysis of patient colon tumors (n = 197) and normal tissues (n = 39) revealed sex-specific metabolic subphenotypes dependent on anatomic location. Tumors from women with RCC were nutrient-deplete, showing enhanced energy production to fuel asparagine synthesis and amino acid uptake. The clinical importance of our findings were further investigated in an independent data set from The Cancer Genomic Atlas, and demonstrated that high asparagine synthetase (ASNS) expression correlated with poorer survival for women. This is the first study to show a unique, nutrient-deplete metabolic subphenotype in women with RCC, with implications for tumor progression and outcomes in CRC patients. [ABSTRACT FROM AUTHOR]

Asparagine Synthetase (ASNS) catalyzes the synthesis of the non-essential amino acid asparagine (Asn) from aspartate (Asp) and glutamine (Gln). ASNS expression is highly regulated at the transcriptional level, being induced by both the Amino Acid Response (AAR) and the Unfolded Protein Response (UPR) pathways. Lack of ASNS protein expression is a hallmark of Acute Lymphoblastic Leukemia (ALL) blasts, which, therefore, are auxotrophic for Asn. This peculiarity is the rationale for the use of bacterial L-Asparaginase (ASNase) for ALL therapy, the first example of anti-cancer treatment targeting a tumor-specific metabolic feature. Other hematological and solid cancers express low levels of ASNS and, therefore, should also be Asn auxotrophs and ASNase sensitive. Conversely, in the last few years, several reports indicate that in some cancer types ASNS is overexpressed, promoting cell proliferation, chemoresistance, and a metastatic behavior. However, enhanced ASNS activity may constitute a metabolic vulnerability in selected cancer models, suggesting a variable and tumor-specific role of the enzyme in cancer. Recent evidence indicates that, beyond its canonical role in protein synthesis, Asn may have additional regulatory functions. These observations prompt a re-appreciation of ASNS activity in the biology of normal and cancer tissues, with particular attention to the fueling of Asn exchange between cancer cells and the tumor microenvironment. [ABSTRACT FROM AUTHOR]

This Phase IIb (NCT02195180) open-label study evaluated erythrocyte-encapsulated asparaginase (eryaspase) in combination with chemotherapy in second-line advanced pancreatic adenocarcinoma. Eligible patients were randomized 2:1 to either eryaspase in combination with gemcitabine or mFOLFOX6 (eryaspase arm), or to gemcitabine or mFOLFOX6 alone (control arm). Co-primary endpoints were overall survival (OS) and progression-free survival (PFS) in patients with low asparagine synthetase (ASNS) expression. Secondary endpoints included OS and PFS in the entire population. 141 patients were randomized (eryaspase arm, n = 95; control arm, n = 46). Median OS and PFS in patients with low ASNS expression were 6.2 months (95% CI, 5.1–8.8) in the eryaspase arm versus 4.9 months (3.1–7.1) in the control arm (HR, 0.63; 95% CI, 0.39–1.01; P = 0.056) and 2.0 months (95% CI, 1.8–3.4) in the eryaspase arm versus 1.8 months (1.4–3.8) in the control arm (HR, 0.67; 95% CI, 0.40–1.12; P = 0.127), respectively. In the entire population, median OS and PFS for the eryaspase arm versus control were 6.0 months versus 4.4 months (HR, 0.60; P = 0.008) and 2.0 months versus 1.6 months (HR, 0.56; 95% CI, 0.37–0.84; P = 0.005), respectively. The combination of eryaspase and chemotherapy was well tolerated. The most frequent Grade 3/4 adverse events in the eryaspase arm (n = 93) were gamma-glutamyltransferase increase (16 [17.2%]), neutropenia (12 [12.9%]), and physical health deterioration (12 [12.9%]). Eryaspase in combination with chemotherapy is associated with improvements in OS and PFS, irrespective of ASNS expression in second-line advanced pancreatic adenocarcinoma. A Phase III trial is underway. • Eryaspase provides encapsulated asparaginase within erythrocytes to minimize toxicity. • Asparagine synthetase (ASNS) appears to be a prognostic indicator of ASNase activity. • Eryaspase plus chemotherapy improved patient survival, irrespective of ASNS expression. [ABSTRACT FROM AUTHOR]