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Frontiers in Oncology 2023Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive cancer with a poor patient prognosis. Remarkably, PDAC is one of the most aggressive and deadly tumor...
BACKGROUND
Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive cancer with a poor patient prognosis. Remarkably, PDAC is one of the most aggressive and deadly tumor types and is notorious for its resistance to all types of treatment. PDAC resistance is frequently associated with a wide metabolic rewiring and in particular of the glycolytic branch named Hexosamine Biosynthetic Pathway (HBP).
METHODS
Transcriptional and bioinformatics analysis were performed to obtain information about the effect of the HBP inhibition in two cell models of PDAC. Cell count, western blot, HPLC and metabolomics analyses were used to determine the impact of the combined treatment between an HBP's Phosphoglucomutase 3 (PGM3) enzyme inhibitor, named FR054, and erastin (ERA), a recognized ferroptosis inducer, on PDAC cell growth and survival.
RESULTS
Here we show that the combined treatment applied to different PDAC cell lines induces a significant decrease in cell proliferation and a concurrent enhancement of cell death. Furthermore, we show that this combined treatment induces Unfolded Protein Response (UPR), NFE2 Like BZIP Transcription Factor 2 (NRF2) activation, a change in cellular redox state, a greater sensitivity to oxidative stress, a major dependence on glutamine metabolism, and finally ferroptosis cell death.
CONCLUSION
Our study discloses that HBP inhibition enhances, via UPR activation, the ERA effect and therefore might be a novel anticancer mechanism to be exploited as PDAC therapy.
PubMed: 37260977
DOI: 10.3389/fonc.2023.1125855 -
BMC Plant Biology Dec 2022Ginseng polysaccharides, have been used to treat various diseases as an important active ingredient. Nevertheless, the biosynthesis of ginseng polysaccharides is poorly...
BACKGROUND
Ginseng polysaccharides, have been used to treat various diseases as an important active ingredient. Nevertheless, the biosynthesis of ginseng polysaccharides is poorly understood. To elucidate the biosynthesis mechanism of ginseng polysaccharides, combined the transcriptome analysis and polysaccharides content determination were performed on the roots, stems, and leaves collected from four cultivars of ginseng.
RESULTS
The results indicated that the total contents of nine monosaccharides were highest in the roots. Moreover, the total content of nine monosaccharides in the roots of the four cultivars were different but similar in stems and leaves. Glucose (Glc) was the most component of all monosaccharides. In total, 19 potential enzymes synthesizing of ginseng polysaccharides were identified, and 17 enzymes were significantly associated with polysaccharides content. Among these genes, the expression of phosphoglucomutase (PGM), glucose-6-phosphate isomerase (GPI), UTP-glucose-1-phosphate uridylyltransferase (UGP2), fructokinase (scrK), mannose-1-phosphate guanylyltransferase (GMPP), phosphomannomutase (PMM), UDP-glucose 4-epimerase (GALE), beta-fructofuranosidase (sacA), and sucrose synthase (SUS) were correlated with that of MYB, AP2/ERF, bZIP, and NAC transcription factors (TFs). These TFs may regulate the expression of genes involved in ginseng polysaccharides synthesis.
CONCLUSION
Our findings could provide insight into a better understanding of the regulatory mechanism of polysaccharides biosynthesis, and would drive progress in genetic improvement and plantation development of ginseng.
Topics: Transcriptome; Panax; Gene Expression Profiling; Polysaccharides; Monosaccharides
PubMed: 36529733
DOI: 10.1186/s12870-022-03995-x -
Allergy Jun 2022The specificities of IgE and IgG for allergen molecules in patients with inborn errors of immunity (IEI) have not been investigated in detail.
BACKGROUND
The specificities of IgE and IgG for allergen molecules in patients with inborn errors of immunity (IEI) have not been investigated in detail.
OBJECTIVE
To study IgE and IgG antibody specificities in patients with defined hyper-IgE syndromes (HIES) using a comprehensive panel of allergen molecules.
METHODS
We used chips containing micro-arrayed allergen molecules to analyze allergen-specific IgE and IgG levels in sera from two groups of HIES patients: Autosomal recessive mutations in phosphoglucomutase-3 (PGM3); Autosomal dominant negative mutations of STAT3 (STAT3); and age-matched subjects with allergic sensitizations. Assays with rat basophil leukemia cells transfected with human FcεRI were performed to study the biological relevance of IgE sensitizations.
RESULTS
Median total IgE levels were significantly lower in the sensitized control group (212.9 kU/L) as compared to PGM3 (5042 kU/L) and STAT3 patients (2561 kU/L). However, PGM3 patients had significantly higher allergen-specific IgE levels and were sensitized to a larger number of allergen molecules as compared to STAT3 patients. Biological relevance of IgE sensitization was confirmed for PGM3 patients by basophil activation testing. PGM3 patients showed significantly lower cumulative allergen-specific IgG responses in particular to milk and egg allergens as compared to STAT3 patients and sensitized controls whereas total IgG levels were comparable to STAT3 patients and significantly higher than in controls.
CONCLUSION
The analysis with multiple micro-arrayed allergen molecules reveals profound differences of allergen-specific IgE and IgG recognition in PGM3 and STAT3 patients which may be useful for classification of IEI and clinical characterization of patients.
Topics: Allergens; Humans; Immunoglobulin E; Immunoglobulin G; Job Syndrome; Mutation
PubMed: 34653276
DOI: 10.1111/all.15143 -
Frontiers in Physiology 2021Thrombosis remains one of the leading causes of morbidity and mortality across the world. Many pathological milieus in the body resulting from multiple risk factors... (Review)
Review
Thrombosis remains one of the leading causes of morbidity and mortality across the world. Many pathological milieus in the body resulting from multiple risk factors escort thrombosis. Hypoxic condition is one such risk factor that disturbs the integrity of endothelial cells to cause an imbalance between anticoagulant and procoagulant proteins. Hypoxia generates reactive oxygen species (ROS) and triggers inflammatory pathways to augment the coagulation cascade. Hypoxia in cells also activates unfolded protein response (UPR) signaling pathways in the endoplasmic reticulum (ER), which tries to restore ER homeostasis and function. But the sustained UPR linked with inflammation, generation of ROS and apoptosis stimulates the severity of thrombosis in the body. Sirtuins, a group of seven proteins, play a vast role in bringing down inflammation, oxidative and ER stress and apoptosis. As a result, sirtuins might provide a therapeutic approach towards the treatment or prevention of hypoxia-induced thrombosis. Sirtuins modulate hypoxia-inducible factors (HIFs) and counteract ER stress-induced apoptosis by attenuating protein kinase RNA-like endoplasmic reticulum kinase (PERK)/Eukaryotic translation initiation factor 2α (eIF2α) pathway activation. It prevents ER-stress mediated inflammation by targeting X-Box Binding Protein 1 (XBP1) and inhibiting nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κβ) signaling through deacetylation. Sirtuins also obstruct nucleotide-binding domain, leucine-rich-containing family, pyrin domain containing 3 (NLRP3) inflammasome activation to reduce the expression of several pro-inflammatory molecules. It protects cells against oxidative stress by targeting nuclear factor erythroid 2-related factor 2 (Nrf2), glutathione (GSH), forkhead box O3 (FOXO3), superoxide dismutase (SOD), catalase (CAT), peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC-1α), glucose-6-phosphate dehydrogenase (G6PD), phosphoglucomutase-2 (PGAM2), and NF-κB, to name few. This review, thus, discusses the potential role of sirtuins as a new treatment for hypoxia-induced thrombosis that involves an intersection of UPR and inflammatory pathways in its pathological manifestation.
PubMed: 34803727
DOI: 10.3389/fphys.2021.733453 -
Biomolecules Dec 2020Phosphoglucomutase 5 (PGM5) in humans is known as a structural muscle protein without enzymatic activity, but detailed understanding of its function is lacking. PGM5...
Phosphoglucomutase 5 (PGM5) in humans is known as a structural muscle protein without enzymatic activity, but detailed understanding of its function is lacking. PGM5 belongs to the alpha-D-phosphohexomutase family and is closely related to the enzymatically active metabolic enzyme PGM1. In the Atlantic herring, , is one of the genes strongly associated with ecological adaptation to the brackish Baltic Sea. We here present the first crystal structures of PGM5, from the Atlantic and Baltic herring, differing by a single substitution Ala330Val. The structure of PGM5 is overall highly similar to structures of PGM1. The structure of the Baltic herring PGM5 in complex with the substrate glucose-1-phosphate shows conserved substrate binding and active site compared to human PGM1, but both PGM5 variants lack phosphoglucomutase activity under the tested conditions. Structure comparison and sequence analysis of PGM5 and PGM1 from fish and mammals suggest that the lacking enzymatic activity of PGM5 is related to differences in active-site loops that are important for flipping of the reaction intermediate. The Ala330Val substitution does not alter structure or biophysical properties of PGM5 but, due to its surface-exposed location, could affect interactions with protein-binding partners.
Topics: Animals; Catalytic Domain; Fishes; Glucosephosphates; Phosphoglucomutase; Protein Binding; Substrate Specificity
PubMed: 33287293
DOI: 10.3390/biom10121631 -
The Journal of Biological Chemistry Feb 2022The malaria-causing parasite Plasmodium falciparum is responsible for over 200 million infections and 400,000 deaths per year. At multiple stages during its complex life...
The malaria-causing parasite Plasmodium falciparum is responsible for over 200 million infections and 400,000 deaths per year. At multiple stages during its complex life cycle, P. falciparum expresses several essential proteins tethered to its surface by glycosylphosphatidylinositol (GPI) anchors, which are critical for biological processes such as parasite egress and reinvasion of host red blood cells. Targeting this pathway therapeutically has the potential to broadly impact parasite development across several life stages. Here, we characterize an upstream component of parasite GPI anchor biosynthesis, the putative phosphomannomutase (PMM) (EC 5.4.2.8), HAD5 (PF3D7_1017400). We confirmed the PMM and phosphoglucomutase activities of purified recombinant HAD5 by developing novel linked enzyme biochemical assays. By regulating the expression of HAD5 in transgenic parasites with a TetR-DOZI-inducible knockdown system, we demonstrated that HAD5 is required for malaria parasite egress and erythrocyte reinvasion, and we assessed the role of HAD5 in GPI anchor synthesis by autoradiography of radiolabeled glucosamine and thin layer chromatography. Finally, we determined the three-dimensional X-ray crystal structure of HAD5 and identified a substrate analog that specifically inhibits HAD5 compared to orthologous human PMMs in a time-dependent manner. These findings demonstrate that the GPI anchor biosynthesis pathway is exceptionally sensitive to inhibition in parasites and that HAD5 has potential as a specific, multistage antimalarial target.
Topics: Animals; Erythrocytes; Glycosylphosphatidylinositols; Humans; Malaria, Falciparum; Phosphotransferases (Phosphomutases); Plasmodium falciparum; Protozoan Proteins
PubMed: 34973333
DOI: 10.1016/j.jbc.2021.101550 -
Biodesign Research 2022Maltose is a natural -(1,4)-linked disaccharide with wide applications in food industries and microbial fermentation. However, maltose has scarcely been used for...
Maltose is a natural -(1,4)-linked disaccharide with wide applications in food industries and microbial fermentation. However, maltose has scarcely been used for biosynthesis, possibly because its phosphorylation by maltose phosphorylase (MP) yields -glucose 1-phosphate (-G1P) that cannot be utilized by -phosphoglucomutase (-PGM) commonly found in synthetic enzymatic biosystems previously constructed by our group. Herein, we designed an synthetic enzymatic reaction module comprised of MP, -phosphoglucomutase (-PGM), and polyphosphate glucokinase (PPGK) for the stoichiometric conversion of each maltose molecule to two glucose 6-phosphate (G6P) molecules. Based on this synthetic module, we further constructed two synthetic biosystems to produce bioelectricity and fructose 1,6-diphosphate (FDP), respectively. The 14-enzyme biobattery achieved a Faraday efficiency of 96.4% and a maximal power density of 0.6 mW/cm, whereas the 5-enzyme FDP-producing biosystem yielded 187.0 mM FDP from 50 g/L (139 mM) maltose by adopting a fed-batch substrate feeding strategy. Our study not only suggests new application scenarios for maltose but also provides novel strategies for the high-efficient production of bioelectricity and value-added biochemicals.
PubMed: 37850132
DOI: 10.34133/2022/9806749 -
Science (New York, N.Y.) Jan 2021Metabolic pathways differ across species but are expected to be similar within a species. We discovered two functional, incompatible versions of the galactose pathway in...
Metabolic pathways differ across species but are expected to be similar within a species. We discovered two functional, incompatible versions of the galactose pathway in We identified a three-locus genetic interaction for growth in galactose, and used precisely engineered alleles to show that it arises from variation in the galactose utilization genes , , and phosphoglucomutase (), and that the reference allele of is incompatible with the alternative alleles of the other genes. Multiloci balancing selection has maintained the two incompatible versions of the pathway for millions of years. Strains with alternative alleles are found primarily in galactose-rich dairy environments, and they grow faster in galactose but slower in glucose, revealing a trade-off on which balancing selection may have acted.
Topics: Alleles; Galactokinase; Galactose; Metabolic Networks and Pathways; Monosaccharide Transport Proteins; Phosphoglucomutase; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Selection, Genetic; Trans-Activators
PubMed: 33479156
DOI: 10.1126/science.aba0542 -
A facile and robust T7-promoter-based high-expression of heterologous proteins in Bacillus subtilis.Bioresources and Bioprocessing May 2022To mimic the Escherichia coli T7 protein expression system, we developed a facile T7 promoter-based protein expression system in an industrial microorganism Bacillus...
To mimic the Escherichia coli T7 protein expression system, we developed a facile T7 promoter-based protein expression system in an industrial microorganism Bacillus subtilis. This system has two parts: a new B. subtilis strain SCK22 and a plasmid pHT7. To construct strain SCK22, the T7 RNA polymerase gene was inserted into the chromosome, and several genes, such as two major protease genes, a spore generation-related gene, and a fermentation foam generation-related gene, were knocked out to facilitate good expression in high-density cell fermentation. The gene of a target protein can be subcloned into plasmid pHT7, where the gene of the target protein was under tight control of the T7 promoter with a ribosome binding site (RBS) sequence of B. subtilis (i.e., AAGGAGG). A few recombinant proteins (i.e., green fluorescent protein, α-glucan phosphorylase, inositol monophosphatase, phosphoglucomutase, and 4-α-glucanotransferase) were expressed with approximately 25-40% expression levels relative to the cellular total proteins estimated by SDS-PAGE by using B. subtilis SCK22/pHT7-derived plasmid. A fed-batch high-cell density fermentation was conducted in a 5-L fermenter, producing up to 4.78 g/L inositol monophosphatase. This expression system has a few advantageous features, such as, wide applicability for recombinant proteins, high protein expression level, easy genetic operation, high transformation efficiency, good genetic stability, and suitability for high-cell density fermentation.
PubMed: 38647747
DOI: 10.1186/s40643-022-00540-4 -
Journal of Bacteriology Jun 2021Capsular polysaccharide (CPS) is a major virulence determinant for many human-pathogenic bacteria. Although the essential functional roles for CPS in bacterial virulence...
Capsular polysaccharide (CPS) is a major virulence determinant for many human-pathogenic bacteria. Although the essential functional roles for CPS in bacterial virulence have been established, knowledge of how CPS production is regulated remains limited. Streptococcus pneumoniae (pneumococcus) CPS expression levels and overall thickness change in response to available oxygen and carbohydrate. These nutrients in addition to transition metal ions can vary significantly between host environmental niches and infection stage. Since the pneumococcus must modulate CPS expression among various host niches during disease progression, we examined the impact of the nutritional transition metal availability of manganese (Mn) and zinc (Zn) on CPS production. We demonstrate that increased Mn/Zn ratios increase CPS production via Mn-dependent activation of the phosphoglucomutase Pgm, an enzyme that functions at the branch point between glycolysis and the CPS biosynthetic pathway in a transcription-independent manner. Furthermore, we find that the downstream CPS protein CpsB, an Mn-dependent phosphatase, does not promote aberrant dephosphorylation of its target capsule-tyrosine kinase CpsD during Mn stress. Together, these data reveal a direct role for cellular Mn/Zn ratios in the regulation of CPS biosynthesis via the direct activation of Pgm. We propose a multilayer mechanism used by the pneumococcus in regulating CPS levels across various host niches. Evolving evidence strongly indicates that maintenance of metal homeostasis is essential for establishing colonization and continued growth of bacterial pathogens in the vertebrate host. In this study, we demonstrate the impact of cellular manganese/zinc (Mn/Zn) ratios on bacterial capsular polysaccharide (CPS) production, an important virulence determinant of many human-pathogenic bacteria, including Streptococcus pneumoniae. We show that higher Mn/Zn ratios increase CPS production via the Mn-dependent activation of the phosphoglucomutase Pgm, an enzyme that functions at the branch point between glycolysis and the CPS biosynthetic pathway. The findings provide a direct role for Mn/Zn homeostasis in the regulation of CPS expression levels and further support the ability of metal cations to act as important cellular signaling mediators in bacteria.
Topics: Bacterial Capsules; Gene Expression Regulation, Bacterial; Glycolysis; Homeostasis; Humans; Ions; Manganese; Mutation; Phosphoglucomutase; Phosphorylation; Pneumococcal Infections; Polysaccharides, Bacterial; Streptococcus pneumoniae; Virulence Factors; Zinc
PubMed: 33875543
DOI: 10.1128/JB.00602-20