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The BMP signaling pathway plays a crucial role in regulating early embryonic development and tissue homeostasis. SMAD6 encodes a negative regulator of BMP, and rare variants of SMAD6 are recurrently found in individuals with birth defects. However, we observed that a subset of rare pathogenic variants of SMAD6 consistently exhibited positive regulatory effects instead of the initial negative effects on the BMP signaling pathway. We sought to determine whether these SMAD6 variants have common pathogenic mechanisms. Here, we showed that pathogenic SMAD6 variants accompanying this functional reversal exhibit similar increases in deamidation. Mechanistically, increased deamidation of SMAD6 variants promotes the accumulation of the BMP receptor BMPR1A and the formation of new complexes, both of which lead to BMP signaling pathway activation. Specifically, two residues, N262 and N404, in SMAD6 were identified as the crucial sites of deamidation, which was catalyzed primarily by glutamine-fructose-6-phosphate transaminase 2 (GFPT2). Additionally, treatment of cells harboring SMAD6 variants with a deamidase inhibitor restored the inhibitory effect of SMAD6 on the BMP signaling pathway. Conversely, when wild-type SMAD6 was manually simulated to mimic the deamidated state, the reversed function of activating BMP signaling was reproduced. Taken together, these findings show that deamidation of SMAD6 plays a crucial role in the functional reversal of BMP signaling activity, which can be induced by a subset of various SMAD6 variants. Our study reveals a common pathogenic mechanism shared by these variants and provides a potential strategy for preventing birth defects through deamidation regulation, which might prevent the off-target effects of gene editing. [ABSTRACT FROM AUTHOR]

Abstract Background Oncogenic KRAS mutation, the most frequent mutation in non-small cell lung cancer (NSCLC), is an aggressiveness risk factor and leads to the metabolic reprogramming of cancer cells by promoting glucose, glutamine, and fatty acid absorption and glycolysis. Lately, sotorasib was approved by the FDA as a first-in-class KRAS-G12C inhibitor. However, sotorasib still has a derivative barrier, which is not effective for other KRAS mutation types, except for G12C. Additionally, resistance to sotorasib is likely to develop, demanding the need for alternative therapeutic strategies. Methods KRAS mutant, and wildtype NSCLC cells were used in vitro cell analyses. Cell viability, proliferation, and death were measured by MTT, cell counting, colony analyses, and annexin V staining for FACS. Cell tracker dyes were used to investigate cell morphology, which was examined by holotomograpy, and confocal microscopes. RNA sequencing was performed to identify key target molecule or pathway, which was confirmed by qRT-PCR, western blotting, and metabolite analyses by UHPLC-MS/MS. Zebrafish and mouse xenograft model were used for in vivo analysis. Results In this study, we found that nutlin-3a, an MDM2 antagonist, inhibited the KRAS-PI3K/Akt-mTOR pathway and disrupted the fusion of both autophagosomes and macropinosomes with lysosomes. This further elucidated non-apoptotic and catastrophic macropinocytosis associated methuosis-like cell death, which was found to be dependent on GFPT2 of the hexosamine biosynthetic pathway, specifically in KRAS mutant /p53 wild type NSCLC cells. Conclusion These results indicate the potential of nutlin-3a as an alternative agent for treating KRAS mutant/p53 wild type NSCLC cells.

Abstract Glutamine fructose‐6‐phosphate aminotransferase 2 (GFPT2) is a rate‐limiting enzyme in hexosamine biosynthesis involved in the occurrence and progress of many cancers. What role it plays in gastric cancer (GC) is still unclear. In this study, transcriptome sequencing data from the Harbin Medical University (HMU)‐GC cohort and The Cancer Genome Atlas (TCGA) dataset were combined with the HMU‐TCGA training cohort to analyze the biological function and clinical significance of GFPT2. The correlation of GFPT2 with immune cells and stromal cells was analyzed in the GC immune microenvironment through transcriptome sequencing data and a public single‐cell sequencing database. In cell lines, GC tissues, and the tissue microarray, GFPT2 protein expression was confirmed by western blotting and immunohistochemistry. The mRNA of GFPT2 was highly expressed in the tumor (p

Background: Oncogenic KRAS mutation, the most frequent mutation in non-small cell lung cancer (NSCLC), is an aggressiveness risk factor and leads to the metabolic reprogramming of cancer cells by promoting glucose, glutamine, and fatty acid absorption and glycolysis. Lately, sotorasib was approved by the FDA as a first-in-class KRAS-G12C inhibitor. However, sotorasib still has a derivative barrier, which is not effective for other KRAS mutation types, except for G12C. Additionally, resistance to sotorasib is likely to develop, demanding the need for alternative therapeutic strategies. Methods: KRAS mutant, and wildtype NSCLC cells were used in vitro cell analyses. Cell viability, proliferation, and death were measured by MTT, cell counting, colony analyses, and annexin V staining for FACS. Cell tracker dyes were used to investigate cell morphology, which was examined by holotomograpy, and confocal microscopes. RNA sequencing was performed to identify key target molecule or pathway, which was confirmed by qRT-PCR, western blotting, and metabolite analyses by UHPLC-MS/MS. Zebrafish and mouse xenograft model were used for in vivo analysis. Results: In this study, we found that nutlin-3a, an MDM2 antagonist, inhibited the KRAS-PI3K/Akt-mTOR pathway and disrupted the fusion of both autophagosomes and macropinosomes with lysosomes. This further elucidated non-apoptotic and catastrophic macropinocytosis associated methuosis-like cell death, which was found to be dependent on GFPT2 of the hexosamine biosynthetic pathway, specifically in KRAS mutant /p53 wild type NSCLC cells. Conclusion: These results indicate the potential of nutlin-3a as an alternative agent for treating KRAS mutant/p53 wild type NSCLC cells. [ABSTRACT FROM AUTHOR]

Glutamine fructose‐6‐phosphate aminotransferase 2 (GFPT2) is a rate‐limiting enzyme in hexosamine biosynthesis involved in the occurrence and progress of many cancers. What role it plays in gastric cancer (GC) is still unclear. In this study, transcriptome sequencing data from the Harbin Medical University (HMU)‐GC cohort and The Cancer Genome Atlas (TCGA) dataset were combined with the HMU‐TCGA training cohort to analyze the biological function and clinical significance of GFPT2. The correlation of GFPT2 with immune cells and stromal cells was analyzed in the GC immune microenvironment through transcriptome sequencing data and a public single‐cell sequencing database. In cell lines, GC tissues, and the tissue microarray, GFPT2 protein expression was confirmed by western blotting and immunohistochemistry. The mRNA of GFPT2 was highly expressed in the tumor (p < 0.001), and GC cells and tumors expressed high levels of GFPT2 protein. Compared to low expression, high GFPT2 mRNA expression was associated with higher levels of tumor invasion, higher pathological stages, and poor prognosis (p = 0.02) in GC patients. In a drug susceptibility analysis, GFPT2 mRNA expression was associated with multiple chemotherapeutic drug sensitivity, including docetaxel, paclitaxel, and cisplatin. Gene enrichment analysis found that GFPT2 was mainly primarily involved in the extracellular matrix receptor interaction pathway. The ESTIMATE, CIBERSORT, and ssGSEA algorithms showed that GFPT2 was associated with immune cell infiltration. In addition, GFPT2 was more likely to be expressed within cancer‐associated fibroblasts (CAFs), and high levels of GFPT2 expression were highly correlated with four CAFs scores (all p < 0.05). Finally, a prognostic model to assess the risk of death in GC patients was constructed based on GFPT2 protein expression and lymph node metastasis rate. In conclusion, GFPT2 plays an essential role in the function of CAFs in GC. It can be used as a biomarker to assess GC prognosis and immune infiltration. [ABSTRACT FROM AUTHOR]

Objective: To investigate the relationship between m RNA expression of G protein signal regulated protein 2 (GPSM2), glutamine fructose-6-phosphate transaminase 2 (GFPT2), nucleolar small RNA 51 (SNORA51) and clinicopathological features of hepatocellular carcinoma (HCC) tissues and their effects on prognosis. Methods: 60 patients with HCC who were diagnosed and treated with surgical resection in Affiliated Hospital of Inner Mongolia Medical University from January 2017 to December 2018 were selected. Real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) was used to detect the m RNA expressions of GPSM2, GFPT2and SNORA51 in HCC tissues and paracancer tissues, and the relationship between the m RNA expressions of GPSM2, GFPT2 and SNORA51 and the clinicopathological characteristics of patients with HCC were analyzed. After 3 years of follow-up, Kaplan-Meier survival curve was used to analyze the prognosis of patients with HCC in different groups, and univariate and multivariate Logistic regression was used to analyze the influencing factors of prognosis of patients with HCC. Results: The m RNA expression levels of GPSM2, GFPT2 and SNORA51 in HCC tissues were significantly higher than those in paracancer tissues (P<0.05). The proportion of vascular invasion and TNM stage Ⅲ in the GPSM2 m RNA high expression group, GFPT2 m RNA high expression group and SNORA51mRNA high expression group were significantly higher than those in the GPSM2 m RNA low expression group and GFPT2m RNA low expression group and SNORA51mRNA low expression group (P<0.05) . Kaplan-Meier analysis showed that the 3-year survival rate of GPSM2 m RNA low expression group, GFPT2 m RNA low expression group, SNORA51mRNA low expression group were significantly higher than those in the GPSM2 m RNA high expression group, GFPT2 m RNA high expression group and SNORA51mRNA high expression group (P<0.05). The results of multivariate Logistic regression analysis model showed that vascular invasion, TNM stage Ⅲ, GPSM2 m RNA high expression, GFPT2 m RNA high expression and SNORA51 m RNA high expression were the risk factors for death of patients with HCC (P<0.05). Conclusion: The abnormally high expression of GPSM2, GFPT2 and SNORA51in HCC tissues is associated with clinicopathological features such as vascular invasion and TNM stage, and the high expression of GPSM2, GFPT2 and SNORA51 is a risk factor for death of patients with HCC. [ABSTRACT FROM AUTHOR]

Purpose: The purpose of this study is to investigate the underlying relationship of diagnosis and therapy between glutamine-fructose-6-phosphate transaminase 2 (GFPT2) and various cancers., Methods: The Cancer Genome Atlas (TCGA) database was utilized to get gene expression RNAseq and clinical data for 33 tumors. The immunotherapeutic cohorts, including GSE35640, GSE78220, GSE67501, GSE181815, and IMvigor210, were derived from the Gene Expression Omnibus database (GEO) and a previously released article. Differential expression analysis of GFPT2 was performed using several clinical factors, and prognostic analysis was performed using Cox proportional hazard regression. In addition, the Cell type Identification By Estimating Relative Subsets Of RNA transcripts (CIBERSORT) and the Estimation of STromal and Immune cells in MAlignant Tumor tissues utilizing Expression data (ESTIMATE) algorithms were used to investigate the connection between GFPT2 and the tumor microenvironment. This approach additionally incorporated dynamic immunological indicators, such as tumor mutational burden (TMB) and microsatellite instability (MSI). In addition, a correlation between GFPT2 expression and the effectiveness of anticancer drugs was plotted for discussion., Results: GFPT2 expression significantly differed in 11 out of 33 cancers. Although the distinct correlation between GFPT2 expression and clinical parameters had no wide distribution in pan-cancer, it demonstrated the potential prognostic validity of gene expression. GFPT2 demonstrated a strong correlation with immune infiltration, immune modulators, and immunerelated biomarkers. Furthermore, a variance analysis demonstrated a significant relationship between GFPT2 and the efficacy of immunotherapy. In addition, GFPT2 was associated with increased sensitivity of drugs such as Olaparib and Lenvatinib and decreased sensitivity of drugs such as Nilotinib., Conclusion: Collectively, GFPT2 is potentially useful as a biomarker for prognostic prediction and immune infiltration in a variety of malignancies ,and could lead to exciting new approaches to personalized oncotherapy., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Respiratory syncytial virus (RSV), or human orthopneumovirus, is a negative-sense RNA virus that is the causative agent of severe lower respiratory tract infections in children and is associated with exacerbations of adult lung disease. The mechanisms how severe and/or repetitive virus infections cause declines in pulmonary capacity are not fully understood. We have recently discovered that viral replication triggers epithelial plasticity and metabolic reprogramming involving the hexosamine biosynthetic pathway (HBP). In this study, we examine the relationship between viral induced innate inflammation and the activation of hexosamine biosynthesis in small airway epithelial cells. We observe that RSV induces ~2-fold accumulation of intracellular UDP-GlcNAc, the end-product of the HBP and the obligate substrate of N glycosylation. Using two different silencing approaches, we observe that RSV replication activates the HBP pathway in a manner dependent on the RELA proto-oncogene (65 kDa subunit). To better understand the effect of RSV on the cellular N glycoproteome, and its RELA dependence, we conduct affinity enriched LC-MS profiling in wild-type and RELA-silenced cells. We find that RSV induces the accumulation of 171 N glycosylated peptides in a RELA-dependent manner; these proteins are functionally enriched in integrins and basal lamina formation. To elaborate this mechanism of HBP expression, we demonstrate that RSV infection coordinately induces the HBP pathway enzymes in a manner requiring RELA; these genes include Glutamine-Fructose-6-Phosphate Transaminase 1 (GFPT)-1/2, Glucosamine-Phosphate N-Acetyltransferase (GNPNAT)-1, phosphoglucomutase (PGM)-3 and UDP-N-Acetylglucosamine Pyrophosphorylase (UAP)-1. Using small-molecule inhibitor(s) of 8-oxoguanine DNA glycosylase1 (OGG1), we observe that OGG1 is also required for the expression of HBP pathway. In proximity ligation assays, RSV induces the formation of a nuclear and mitochondrial RELA∙OGG1 complex. In co-immunoprecipitaton (IP) experiments, we discover that RSV induces Ser 536-phosphorylated RELA to complex with OGG1. Chromatin IP experiments demonstrate a major role of OGG1 in supporting the recruitment of RELA and phosphorylated RNA Pol II to the HBP pathway genes. We conclude that the RELA∙OGG1 complex is an epigenetic regulator mediating metabolic reprogramming and N glycoprotein modifications of integrins in response to RSV. These findings have implications for viral-induced adaptive epithelial responses. [ABSTRACT FROM AUTHOR]

Background: Recently, increasing evidence has suggested that Glutamine-fructose-6-phosphate transaminase 2 (GFPT2) is related to carcinogenesis. However, the potential roles of GFPT2 in colon cancer still need to be fully investigated. Methods: We examined the protein levels of GFPT2 by immunohistochemistry (IHC) in tissues collected from 83 patients with colon cancer. We further detected GFBPT2 protein levels by Western Blot assay. We checked the relationship between GFPT2 expression levels and overall survival (OS), stromal and immune scores and immune components from The Cancer Gene Atlas (TCGA) database. GFBP2-related pathways were validated in the Cancer Cell Line Encyclopedia (CCLE) database. Expression of GFPT2 in single cell subpopulations was calculated from The Tumor Immune Single Cell Center (TISCH). The levels of GFPT2 and drug sensitivity data were performed from CellMiner dataset. Results: GFPT2 was highly expressed and correlated with poor pathological features in 83 colon cancer patients. Moreover, increased GFPT2 expression was significantly associated with poorer OS in 329 colon adenocarcinoma (COAD) patients. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed the differentially expressed genes of GFPT2 were mostly enriched in focal adhesion, ECM receptor interaction, JAK/STAT signaling pathway and immune related pathways. In addition, GFPT2 expression was correlated with the tumor microenvironment (TME). GFPT2 expression was linked to cancer-associated fibroblasts (CAFs)-associated factors and epithelial-mesenchymal transition (EMT)-related factors. GFPT2 was positively correlated with immunosuppressive cells and regulated immunosuppressive factors and T-cell exhaustion. Finally, our data suggested that the expression of GFPT2 may be a judgment of the sensitivity of a certain class of drugs. Conclusions: Our work reveals the roles of GFPT2 in tumorigenesis, particularly in immune response, TME and drug resistance, which are crucial for the development of customized cancer therapies. [ABSTRACT FROM AUTHOR]

BackgroundRecently, increasing evidence has suggested that Glutamine-fructose-6-phosphate transaminase 2 (GFPT2) is related to carcinogenesis. However, the potential roles of GFPT2 in colon cancer still need to be fully investigated.MethodsWe examined the protein levels of GFPT2 by immunohistochemistry (IHC) in tissues collected from 83 patients with colon cancer. We further detected GFBPT2 protein levels by Western Blot assay. We checked the relationship between GFPT2 expression levels and overall survival (OS), stromal and immune scores and immune components from The Cancer Gene Atlas (TCGA) database. GFBP2-related pathways were validated in the Cancer Cell Line Encyclopedia (CCLE) database. Expression of GFPT2 in single cell subpopulations was calculated from The Tumor Immune Single Cell Center (TISCH). The levels of GFPT2 and drug sensitivity data were performed from CellMiner dataset.ResultsGFPT2 was highly expressed and correlated with poor pathological features in 83 colon cancer patients. Moreover, increased GFPT2 expression was significantly associated with poorer OS in 329 colon adenocarcinoma (COAD) patients. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed the differentially expressed genes of GFPT2 were mostly enriched in focal adhesion, ECM receptor interaction, JAK/STAT signaling pathway and immune related pathways. In addition, GFPT2 expression was correlated with the tumor microenvironment (TME). GFPT2 expression was linked to cancer-associated fibroblasts (CAFs)-associated factors and epithelial-mesenchymal transition (EMT)-related factors. GFPT2 was positively correlated with immunosuppressive cells and regulated immunosuppressive factors and T-cell exhaustion. Finally, our data suggested that the expression of GFPT2 may be a judgment of the sensitivity of a certain class of drugs.ConclusionsOur work reveals the roles of GFPT2 in tumorigenesis, particularly in immune response, TME and drug resistance, which are crucial for the development of customized cancer therapies.

In response to cardiac injury, increased activity of the hexosamine biosynthesis pathway (HBP) is linked with cytoprotective as well as adverse effects depending on the type and duration of injury. Glutamine-fructose amidotransferase (GFAT; gene name gfpt) is the rate-limiting enzyme that controls flux through HBP. Two protein isoforms exist in the heart called GFAT1 and GFAT2. There are conflicting data on the relative importance of GFAT1 and GFAT2 during stress-induced HBP responses in the heart. Using neonatal rat cardiac cell preparations, targeted knockdown of GFPT1 and GFPT2 were performed and HBP activity measured. Immunostaining with specific GFAT1 and GFAT2 antibodies was undertaken in neonatal rat cardiac preparations and murine cardiac tissues to characterise cell-specific expression. Publicly available human heart single cell sequencing data was interrogated to determine cell-type expression. Western blots for GFAT isoform protein expression were performed in human cardiomyocytes derived from induced pluripotent stem cells (iPSCs). GFPT1 but not GFPT2 knockdown resulted in a loss of stress-induced protein O-GlcNAcylation in neonatal cardiac cell preparations indicating reduced HBP activity. In rodent cells and tissue, immunostaining for GFAT1 identified expression in both cardiac myocytes and fibroblasts whereas immunostaining for GFAT2 was only identified in fibroblasts. Further corroboration of findings in human heart cells identified an enrichment of GFPT2 gene expression in cardiac fibroblasts but not ventricular myocytes whereas GFPT1 was expressed in both myocytes and fibroblasts. In human iPSC-derived cardiomyocytes, only GFAT1 protein was expressed with an absence of GFAT2. In conclusion, these results indicate that GFAT1 is the primary cardiomyocyte isoform and GFAT2 is only present in cardiac fibroblasts. Cell-specific isoform expression may have differing effects on cell function and should be considered when studying HBP and GFAT functions in the heart. • GFAT2 is not expressed in cardiac myocytes. • GFAT2 is abundantly expressed in cardiac fibroblasts. • GFAT1 is expressed in both cardiac myocytes and fibroblasts. • GFAT1 regulates the stress-induced increase in cardiac O-GlcNAcylation. [ABSTRACT FROM AUTHOR]

Respiratory syncytial virus (RSV), or human orthopneumovirus, is a negative-sense RNA virus that is the causative agent of severe lower respiratory tract infections in children and is associated with exacerbations of adult lung disease. The mechanisms how severe and/or repetitive virus infections cause declines in pulmonary capacity are not fully understood. We have recently discovered that viral replication triggers epithelial plasticity and metabolic reprogramming involving the hexosamine biosynthetic pathway (HBP). In this study, we examine the relationship between viral induced innate inflammation and the activation of hexosamine biosynthesis in small airway epithelial cells. We observe that RSV induces ~2-fold accumulation of intracellular UDP-GlcNAc, the end-product of the HBP and the obligate substrate of N glycosylation. Using two different silencing approaches, we observe that RSV replication activates the HBP pathway in a manner dependent on the RELA proto-oncogene (65 kDa subunit). To better understand the effect of RSV on the cellular N glycoproteome, and its RELA dependence, we conduct affinity enriched LC-MS profiling in wild-type and RELA-silenced cells. We find that RSV induces the accumulation of 171 N glycosylated peptides in a RELA-dependent manner; these proteins are functionally enriched in integrins and basal lamina formation. To elaborate this mechanism of HBP expression, we demonstrate that RSV infection coordinately induces the HBP pathway enzymes in a manner requiring RELA; these genes include Glutamine-Fructose-6-Phosphate Transaminase 1 (GFPT)-1/2, Glucosamine-Phosphate N-Acetyltransferase (GNPNAT)-1, phosphoglucomutase (PGM)-3 and UDP-N-Acetylglucosamine Pyrophosphorylase (UAP)-1. Using small-molecule inhibitor(s) of 8-oxoguanine DNA glycosylase1 (OGG1), we observe that OGG1 is also required for the expression of HBP pathway. In proximity ligation assays, RSV induces the formation of a nuclear and mitochondrial RELA∙OGG1 complex. In co-immunoprecipitaton (IP) experiments, we discover that RSV induces Ser 536-phosphorylated RELA to complex with OGG1. Chromatin IP experiments demonstrate a major role of OGG1 in supporting the recruitment of RELA and phosphorylated RNA Pol II to the HBP pathway genes. We conclude that the RELA∙OGG1 complex is an epigenetic regulator mediating metabolic reprogramming and N glycoprotein modifications of integrins in response to RSV. These findings have implications for viral-induced adaptive epithelial responses.

Abstract Purpose Asthenozoospermia (ASZ) is a condition characterized by reduced sperm motility in semen affecting approximately 19% of infertile men. Major risk factors, particularly gene mutations, still remain unknown. The main aim of the present study was to identify novel genes and mutations that may influence human sperm motility. Methods Whole-exome sequencing (WES) was performed on a large pedigree of infertile men (n = 5) followed by bioinformatics analyses. Candidate pathogenic variants were screened in a control cohort of 400 ancestry-matched Iranian fertile men, 30 unrelated men with idiopathic ASZ, and public databases. Results A rare mutation in GFPT2 gene (c.1097G > A; p.Arg366Gln) located in the SIS 1 domain was segregated with the phenotype and was consistent with autosomal recessive inheritance. The in silico analyses revealed that the mutation might affect the function of SIS 1 domain and abolish its carbohydrate-binding ability. Conclusion Homozygosity of the GFPT2 p.Arg366Gln mutation was associated with increased levels of reactive oxygen species (ROS) in spermatozoa and decreased sperm motility. Highlights • Asthenozoospermia is a complex genetic disease with a relatively unknown etiology. • The WES approach is efficient in identifying potential causal variants in many pedigrees in which traditional methods had failed to detect them. • We identified a rare variant in GFPT2 in six ASZ cases, affecting conserved amino acid, and propose this gene as a novel ASZ-implicated gene. • This study not only sheds light on the underlying pathobiology of AZS but also provides a basis for more efficient diagnosis in a clinical setting [ABSTRACT FROM AUTHOR]

Breast cancer cells that have undergone partial epithelial-mesenchymal transition (EMT) are believed to be more invasive than cells that have completed EMT. To study metabolic reprogramming in different mesenchymal states, we analyzed protein expression following EMT in the breast epithelial cell model D492 with single-shot LFQ supported by a SILAC proteomics approach. The D492 EMT cell model contains three cell lines: the epithelial D492 cells, the mesenchymal D492M cells, and a partial mesenchymal, tumorigenic variant of D492 that overexpresses the oncogene HER2. The analysis classified the D492 and D492M cells as basal-like and D492HER2 as claudin-low. Comparative analysis of D492 and D492M to tumorigenic D492HER2 differentiated metabolic markers of migration from those of invasion. Glutamine-fructose-6-phosphate transaminase 2 (GFPT2) was one of the top dysregulated enzymes in D492HER2. Gene expression analysis of the cancer genome atlas showed that GFPT2 expression was a characteristic of claudin-low breast cancer. siRNA-mediated knockdown of GFPT2 influenced the EMT marker vimentin and both cell growth and invasion in vitro and was accompanied by lowered metabolic flux through the hexosamine biosynthesis pathway (HBP). Knockdown of GFPT2 decreased cystathionine and sulfide:quinone oxidoreductase (SQOR) in the transsulfuration pathway that regulates H 2 S production and mitochondrial homeostasis. Moreover, GFPT2 was within the regulation network of insulin and EGF, and its expression was regulated by reduced glutathione (GSH) and suppressed by the oxidative stress regulator GSK3-β. Our results demonstrate that GFPT2 controls growth and invasion in the D492 EMT model, is a marker for oxidative stress, and associated with poor prognosis in claudin-low breast cancer., Competing Interests: Conflict of interest The authors declare no competing interests., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

In response to cardiac injury, increased activity of the hexosamine biosynthesis pathway (HBP) is linked with cytoprotective as well as adverse effects depending on the type and duration of injury. Glutamine-fructose amidotransferase (GFAT; gene name gfpt) is the rate-limiting enzyme that controls flux through HBP. Two protein isoforms exist in the heart called GFAT1 and GFAT2. There are conflicting data on the relative importance of GFAT1 and GFAT2 during stress-induced HBP responses in the heart. Using neonatal rat cardiac cell preparations, targeted knockdown of GFPT1 and GFPT2 were performed and HBP activity measured. Immunostaining with specific GFAT1 and GFAT2 antibodies was undertaken in neonatal rat cardiac preparations and murine cardiac tissues to characterise cell-specific expression. Publicly available human heart single cell sequencing data was interrogated to determine cell-type expression. Western blots for GFAT isoform protein expression were performed in human cardiomyocytes derived from induced pluripotent stem cells (iPSCs). GFPT1 but not GFPT2 knockdown resulted in a loss of stress-induced protein O-GlcNAcylation in neonatal cardiac cell preparations indicating reduced HBP activity. In rodent cells and tissue, immunostaining for GFAT1 identified expression in both cardiac myocytes and fibroblasts whereas immunostaining for GFAT2 was only identified in fibroblasts. Further corroboration of findings in human heart cells identified an enrichment of GFPT2 gene expression in cardiac fibroblasts but not ventricular myocytes whereas GFPT1 was expressed in both myocytes and fibroblasts. In human iPSC-derived cardiomyocytes, only GFAT1 protein was expressed with an absence of GFAT2. In conclusion, these results indicate that GFAT1 is the primary cardiomyocyte isoform and GFAT2 is only present in cardiac fibroblasts. Cell-specific isoform expression may have differing effects on cell function and should be considered when studying HBP and GFAT functions in the heart., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Myocardial infarction (MI) is a major atherosclerotic cardiovascular disease which represents a leading cause of death worldwide. Kruppel-like factor 5 (KLF5) is a member of the kruppel-like transcription factor family which has been reported with pro-apoptotic functions in myocardial cells. This work focuses on the function of KLF5 in the pathogenesis of MI and the molecules involved. A mouse model with MI was established. Hypoxia/reoxygenation (H/R)-treated H9C2 cells were applied for in vitro experiments. A KLF5-specific inhibitor ML264 was administrated in cell and animal models. ML264 significantly reduced apoptosis, expression of fibrosis-related markers, reactive oxygen species in the H/R-treated H9C2 cells, and it reduced myocardial injury, infarct size, apoptosis and fibrosis in the myocardial tissues in model mice through specific downregulation of KLF5. A microRNA (miRNA) microarray analysis was performed, which suggested miR-27a as the most upregulated miRNA in the H/R-treated cells after ML264 treatment. miR-27a mimic reduced apoptosis and fibrosis in H/R-treated cells, while miR-27a inhibition blocked the protective roles of ML264. The integrated bioinformatic analyses and luciferase assays confirmed glutamine fructose-6-phosphate transaminase 2 (GFPT2) mRNA as a target of miR-27a. Overexpression of GFPT2 counteracted the protective functions of miR-27a against MI through the activation of the TGF-β/Smad2/3 signaling pathway. To conclude, this study evidenced that KLF5 possibly induces cell and tissue damage in MI through downregulation of miR-27a and the subsequent activation of GFPT2/TGF-β/Smad2/3 axis. This study may offer novel thoughts into MI treatment.

As a rate-limiting enzyme of the hexosamine biosynthesis pathway (HBP), which is responsible for glycosylation, Glutamine fructose-6-phosphate amidotransferase 2 (GFPT2) is involved in human breast and lung tumorigenesis. However, whether GFTP2 is associated with tumor metastasis remains unclear. Here, we found that GFPT2 promoted the proliferation, migration, invasion and metastasis of colorectal cancer (CRC) cells. Mechanically, p65 acted as an upstream transcription factor of GFPT2 and regulated its expression and function. In turn, GFPT2 enhanced the glycosylation of p65, which led to the nuclear translocation of p65 and then activated NF-κB pathway. Thus, GFTP2 and p65 formed a positive feedback loop to promote the progression of CRC. In addition, GFPT2 was up-regulated in CRC tissues and closely related with liver metastasis (P<0.0001) and tumor stage (P=0.0184). High expression of GFPT2 predicted poor prognosis for CRC patients. Moreover, GFTP2 expression was positively linked with O-linked N-acetylglucosamine transferase in CRC tissues. Our study reveals a new mechanism of GFPT2 in CRC metastasis and provides a new target therapeutic target to deter metastasis., Competing Interests: None., (AJCR Copyright © 2020.)

The hexosamine biosynthetic pathway (HBP), a branch of glucose metabolism, provides a substrate for glycosylation modification, which has a wide-ranging effect on cellular functions. Glutamine-fructose-6-phosphate transaminase 2 (GFPT2) has been reported to regulate the HBP as the first and rate-limiting enzyme. Given the inverse association between GFPT2 expression and survival of patients with serous ovarian cancer (SOC) observed in The Cancer Genome Atlas (TCGA) database, we attempted to investigate the role of GFPT2 and its related mechanisms in SOC. The results showed that GFPT2 was over-expressed in SOC tissues, and positive correlations with advanced stage (FIGO III/IV), suboptimal removal rate and poor survival were observed in 90 SOC patients. Cell migration and invasion were also inhibited in GFPT2 knockdown SKOV3 and HEY cells. The levels of O-linked β-N-acetylglucosamine (O-GlcNAc) and intranuclear β-catenin were evaluated and the observed increase in O-GlcNAcylation induced by GFPT2 may contribute to epithelial-mesenchymal transition (EMT). These data provide novel insights into the function of GFPT2 and O-GlcNAcylation in the EMT and thus the invasiveness SOC. [ABSTRACT FROM AUTHOR]

PPP2R2A is one of the regulatory subunits of the PP2A phosphatase complexes, and previous studies showed that its upregulation promotes cancer cell survival and growth. In this research, we used the tandem affinity purification and the HPLC-Chip-ESI/MS/MS mass spectrometry to screen the PPP2R2A-binding proteins and the results indicated that the GFPT-1/-2 were the potential partners of PPP2R2A. We further validated the interaction between PPP2R2A and GFPT-1/-2 through GST Pull-down, co-immunoprecipitation and immunofluorescence assays. And we found that knockdown of PPP2R2A by lentivirus-mediated shRNA enhanced the phosphorylation of GFPT2, whereas the phosphorylation of GFPT1 had no significant change. GFPT2 is a rate-limiting enzyme in the hexosamine pathway. Our results showed that the knockdown of PPP2R2A promoted the total cellular O-GlcNAcylation in MDA-MB-231 breast cancer cells. These results suggest that PPP2R2A interacts with GFPT1/2, and leads to the phosphorylation of GFPT2, which can regulate the cellular O-GlcNAcylation.