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Frontiers in Microbiology 2022Macroalgae host diverse epiphytic bacterial communities with potential symbiotic roles including important roles influencing morphogenesis and growth of the host,...
Macroalgae host diverse epiphytic bacterial communities with potential symbiotic roles including important roles influencing morphogenesis and growth of the host, nutrient exchange, and protection of the host from pathogens. Macroalgal cell wall structures, exudates, and intra-cellular environments possess numerous complex and valuable carbohydrates such as cellulose, hemi-cellulose, mannans, alginates, fucoidans, and laminarin. Bacterial colonizers of macroalgae are important carbon cyclers, acquiring nutrition from living macroalgae and also from decaying macroalgae. Seaweed epiphytic communities are a rich source of diverse carbohydrate-active enzymes which may have useful applications in industrial bioprocessing. With this in mind, we constructed a large insert fosmid clone library from the metagenome of (Ochrophyta) in which decay was induced. Subsequent sequencing of a fosmid clone insert revealed the presence of a gene encoding a bifunctional phosphomannomutase/phosphoglucomutase (PMM/PGM) enzyme 10L6AlgC, closely related to a protein from the halophilic marine bacterium, sp. 10L6AlgC was subsequently heterologously expressed in and biochemically characterized. The enzyme was found to possess both PMM and PGM activity, which had temperature and pH optima of 45°C and 8.0, respectively; for both activities. The PMM activity had a of 2.229 mM and of 29.35 mM min mg, while the PGM activity had a of 0.5314 mM and a of 644.7 mM min mg. Overall characterization of the enzyme including the above parameters as well as the influence of various divalent cations on these activities revealed that 10L6AlgC has a unique biochemical profile when compared to previously characterized PMM/PGM bifunctional enzymes. Thus 10L6AlgC may find utility in enzyme-based production of biochemicals with different potential industrial applications, in which other bacterial PMM/PGMs have previously been used such as in the production of low-calorie sweeteners in the food industry.
PubMed: 36212884
DOI: 10.3389/fmicb.2022.1000634 -
The Journal of Biological Chemistry Jun 2022Aspergillus fumigatus is the causative agent of invasive aspergillosis, an infection with mortality rates of up to 50%. The glucan-rich cell wall of A. fumigatus is a...
Aspergillus fumigatus is the causative agent of invasive aspergillosis, an infection with mortality rates of up to 50%. The glucan-rich cell wall of A. fumigatus is a protective structure that is absent from human cells and is a potential target for antifungal treatments. Glucan is synthesized from the donor uridine diphosphate glucose, with the conversion of glucose-6-phosphate to glucose-1-phosphate by the enzyme phosphoglucomutase (PGM) representing a key step in its biosynthesis. Here, we explore the possibility of selectively targeting A. fumigatus PGM (AfPGM) as an antifungal treatment strategy. Using a promoter replacement strategy, we constructed a conditional pgm mutant and revealed that pgm is required for A. fumigatus growth and cell wall integrity. In addition, using a fragment screen, we identified the thiol-reactive compound isothiazolone fragment of PGM as targeting a cysteine residue not conserved in the human ortholog. Furthermore, through scaffold exploration, we synthesized a para-aryl derivative (ISFP10) and demonstrated that it inhibits AfPGM with an IC of 2 μM and exhibits 50-fold selectivity over the human enzyme. Taken together, our data provide genetic validation of PGM as a therapeutic target and suggest new avenues for inhibiting AfPGM using covalent inhibitors that could serve as tools for chemical validation.
Topics: Antifungal Agents; Aspergillosis; Aspergillus fumigatus; Glucans; Humans; Phosphoglucomutase
PubMed: 35504355
DOI: 10.1016/j.jbc.2022.102003 -
Experimental and Therapeutic Medicine Oct 2020Non-small cell lung cancer (NSCLC) is a leading cause of mortality worldwide. However, the pathogenesis of NSCLC remains to be fully elucidated. Therefore, the present...
Non-small cell lung cancer (NSCLC) is a leading cause of mortality worldwide. However, the pathogenesis of NSCLC remains to be fully elucidated. Therefore, the present study aimed to explore the differential expression of mRNAs and microRNAs (miRNAs/miRs) in NSCLC and to determine how these RNA molecules interact with one another to affect disease progression. Differentially expressed genes (DEGs) and differentially expressed miRNAs (DEMs) were identified from the GSE18842, GSE32863 and GSE29250 datasets downloaded from the Gene Expression Omnibus (GEO database). Functional and pathway enrichment analysis were performed based on Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. STRING, Cytoscape and MCODE were applied to construct a protein-protein interaction (PPI) network and to screen hub genes. The interactions between miRNAs and mRNAs were predicted using miRWalk 3.0 and a miRNA-mRNA regulatory network was constructed. The prognostic value of the identified hub genes was then evaluated via Kaplan-Meier survival analyses using datasets from The Cancer Genome Atlas. A total of 782 DEGs and 46 DEMs were identified from the 3 GEO datasets. The enriched pathways and functions of the DEGs and target genes of the DEMs included osteoclast differentiation, cell adhesion, response to a drug, plasma membrane, extracellular exosome and protein binding. A subnetwork composed of 11 genes was extracted from the PPI network and the genes in this subnetwork were mainly involved in the cell cycle, cell division and DNA replication. A miRNA-gene regulatory network was constructed with 247 miRNA-gene pairs based on 6 DEMs and 210 DEGs. Kaplan-Meier survival analysis indicated that the expression of ubiquitin E2 ligase C, cell division cycle protein 20, DNA topoisomerase IIα, aurora kinase A and B, cyclin B2, maternal embryonic leucine zipper kinase, slit guidance ligand 3, phosphoglucomutase 5, endomucin, cysteine dioxygenase type 1, dihydropyrimidinase-like 2, miR-130b, miR-1181 and miR-127 was significantly associated with overall survival of patients with lung adenocarcinoma. In the present study, a miRNA-mRNA regulatory network in NSCLC was established, which may provide future avenues for scientific exploration and therapeutic targeting of NSCLC.
PubMed: 32855723
DOI: 10.3892/etm.2020.9105 -
Journal of Oncology 2021Metabolic reprogramming of aerobic glycolysis is a hallmark of cancer cells. Regulators of aerobic glycolysis have become targets for cancer diagnosis and therapy....
Metabolic reprogramming of aerobic glycolysis is a hallmark of cancer cells. Regulators of aerobic glycolysis have become targets for cancer diagnosis and therapy. However, the regulators of aerobic glycolysis in breast cancer development have not been well elucidated. Here, we show that the phosphoglucomutase (PGM) family member PGM5 promotes conversion of glucose-1-phosphate (G1P) into glucose-6-phosphate (G6P) and inhibits breast cancer cell proliferation and migration through regulating aerobic glycolysis. In breast cancer patients, PGM5 is significantly downregulated, and its low expression is a predictor of poor prognosis. MicroRNA-1224-3p (miR-1224-3p) inhibits the PGM5 level through directly targeting its 3'-untranslated region and suppresses PGM5-mediated breast cancer cell proliferation, migration, and glycolytic function. Moreover, the miR-1224-3p/PGM5 axis regulates the expression of cell cycle- and apoptosis-related genes and the markers of epithelial-mesenchymal transition (EMT), a process involved in migration and metastasis of cancer cells. Taken together, our results indicate that miR-1224-3p/PGM5 axis plays important roles in breast cancer cell proliferation, migration, and aerobic glycolysis and may be a potential target for breast cancer therapy.
PubMed: 33986801
DOI: 10.1155/2021/5529770 -
Applied Microbiology and Biotechnology Aug 2019Biotechnological industry strives to develop anaerobic bioprocesses fueled by abundant and cheap carbon sources, like sucrose. However, oxygen-limiting conditions often...
Biotechnological industry strives to develop anaerobic bioprocesses fueled by abundant and cheap carbon sources, like sucrose. However, oxygen-limiting conditions often lead to by-product formation and reduced ATP yields. While by-product formation is typically decreased by gene deletion, the breakdown of oligosaccharides with inorganic phosphate instead of water could increment the ATP yield. To observe the effect of oxygen limitation during sucrose consumption, a non-fermentative Escherichia coli K-12 strain was transformed with genes enabling sucrose assimilation. It was observed that the combined deletion of the genes adhE, adhP, mhpF, ldhA, and pta abolished the anaerobic growth using sucrose. Therefore, the biomass-specific conversion rates were obtained using oxygen-limited continuous cultures. Strains performing the breakdown of the sucrose by hydrolysis (SUC-HYD) or phosphorolysis (SUC-PHOSP) were studied in such conditions. An experimentally validated in silico model, modified to account for plasmid and protein burdens, was employed to calculate carbon and electron consistent conversion rates. In both strains, the biomass yields were lower than expected and, strikingly, SUC-PHOSP showed a yield lower than SUC-HYD. Flux balance analyses indicated a significant increase in the non-growth-associated ATP expenses by comparison with the growth on glucose. The observed fructose-1,6-biphosphatase and phosphoglucomutase activities, as well as the concentrations of glycogen, suggest the operation of ATP futile cycles triggered by a combination of the oxygen limitation and the metabolites released during the sucrose breakdown.
Topics: Adenosine Triphosphate; Anaerobiosis; Computer Simulation; Escherichia coli K12; Gene Deletion; Metabolic Engineering; Oxygen; Sucrose
PubMed: 31147757
DOI: 10.1007/s00253-019-09909-6 -
Bioengineered Apr 2022Long non-coding RNAs (lncRNAs) have been widely studied and play crucial roles in cervical cancer (CC) progression. Here, we investigated the function and mechanism of...
Long non-coding RNAs (lncRNAs) have been widely studied and play crucial roles in cervical cancer (CC) progression. Here, we investigated the function and mechanism of lncRNA PGM5-AS1 action in CC cells. Using real-time quantitative polymerase chain reaction or western blotting, PGM5-AS1 and decorin (DCN) were downregulated in CC tissues and cells, whereas miR-4284 was upregulated. Luciferase assay, RNA pull-down assay, and western blotting showed that PGM5-AS1 could sponge miR-4284 to upregulate DCN expression in CC cells. Additionally, cell functional experiments showed that PGM5-AS1 overexpression led to decreased proliferation, migration, and invasion of CC cells. However, the inhibitory effect of PGM5-AS1 overexpression on CC cells was partly relieved by DCN knockdown because of the targeting interaction between PGM5-AS1, miR-4284, and DCN. In summary, this study identified that PGM5-AS1 negatively regulates CC cell malignancy by targeting miR-4284/DCN.
Topics: Cell Line, Tumor; Cell Proliferation; Cytoskeletal Proteins; Decorin; Female; Gene Expression Regulation, Neoplastic; Humans; MicroRNAs; Phosphoglucomutase; RNA, Long Noncoding; Uterine Cervical Neoplasms
PubMed: 35420507
DOI: 10.1080/21655979.2022.2062088 -
Veterinary Research Jul 2020Lipooligosaccharides (LOSs) are virulence determinants of Glaesserella parasuis, a pathogen of the respiratory tract of pigs. We previously reported that disruption of...
Lipooligosaccharides (LOSs) are virulence determinants of Glaesserella parasuis, a pathogen of the respiratory tract of pigs. We previously reported that disruption of the galU or galE gene in G. parasuis results in increased sensitivity to porcine serum, indicating that the galactose catabolism pathway is required for polysaccharide formation in G. parasuis. Here, we evaluated the role of the HAPS_0849 gene in LOS synthesis. The G. parasuis SC096 HAPS_0849 mutant produced a highly truncated LOS molecule, although a small fraction of intact LOS was still observed, and this mutant was found to be more sensitive to serum than the parental strain. HAPS_0849 was overexpressed and purified for biochemical assays, and this protein exhibited phosphoglucomutase (PGM) activity. Heterologous expression of a pgm gene from Escherichia coli in the HAPS_0849 mutant led to restoration of the wild-type LOS glycoform, further demonstrating the PGM function of HAPS_0849 in G. parasuis. The autoagglutination and biofilm formation ability of this strain were also investigated. Disruption of HAPS_0849 led to an increased tendency to autoagglutinate and form more biofilms, and these enhanced phenotypes were observed in the absence of glucose. In addition, LOSs from HAPS_0849, galU and lgtB mutants had similar truncated glycoforms, while LOSs from the galE and lex-1 mutants exhibited another type of defective LOS pattern. These findings imply that HAPS_0849 may function upstream of GalU in the generation of glucose 1-phosphate. In conclusion, our results preliminarily described the functions of HAPS_0849 in G. parasuis, and this gene was partially required for LOS synthesis.
Topics: Bacterial Proteins; Escherichia coli; Gene Expression Regulation, Bacterial; Haemophilus parasuis; Lipopolysaccharides; Microorganisms, Genetically-Modified; Phosphoglucomutase
PubMed: 32736655
DOI: 10.1186/s13567-020-00822-9 -
Scientific Reports Mar 2020Human phosphoglucomutase 1 (PGM1) is an evolutionary conserved enzyme that belongs to the ubiquitous and ancient α-D-phosphohexomutases, a large enzyme superfamily with...
Human phosphoglucomutase 1 (PGM1) is an evolutionary conserved enzyme that belongs to the ubiquitous and ancient α-D-phosphohexomutases, a large enzyme superfamily with members in all three domains of life. PGM1 catalyzes the bi-directional interconversion between α-D-glucose 1-phosphate (G1P) and α-D-glucose 6-phosphate (G6P), a reaction that is essential for normal carbohydrate metabolism and also important in the cytoplasmic biosynthesis of nucleotide sugars needed for glycan biosynthesis. Clinical studies have shown that mutations in the PGM1 gene may cause PGM1 deficiency, an inborn error of metabolism previously classified as a glycogen storage disease, and PGM1 deficiency was recently also shown to be a congenital disorder of glycosylation. Here we present three crystal structures of the isoform 2 variant of PGM1, both as a free enzyme and in complex with its substrate and product. The structures show the longer N-terminal of this PGM1 variant, and the ligand complex structures reveal for the first time the detailed structural basis for both G1P substrate and G6P product recognition by human PGM1. We also show that PGM1 and the paralogous gene PGM5 are the results of a gene duplication event in a common ancestor of jawed vertebrates, and, importantly, that both PGM1 isoforms are conserved and of functional significance in all vertebrates. Our finding that PGM1 encodes two equally conserved and functionally important isoforms in the human organism should be taken into account in the evaluation of disease-related missense mutations in patients in the future.
Topics: Animals; Catalytic Domain; Cytoplasm; Glucose-6-Phosphate; Glucosephosphates; Glycogen Storage Disease; Glycosylation; Humans; Ligands; Mutation, Missense; Phosphoglucomutase; Phosphotransferases (Phosphomutases); Protein Isoforms; Vertebrates
PubMed: 32221390
DOI: 10.1038/s41598-020-62548-0 -
AMB Express Mar 2020Lentinan is a Lentinus edodes secondary metabolite that can regulate human immune function, but yields are low. Here, the effects of Ca and Na on L. edodes lentinan...
Lentinan is a Lentinus edodes secondary metabolite that can regulate human immune function, but yields are low. Here, the effects of Ca and Na on L. edodes lentinan content were investigated. Metal ion concentrations and induction times were optimized according to mycelial biomass, and intracellular polysaccharide (IPS), extracellular polysaccharide (EPS), and total polysaccharide (TPS) content. The activities and gene expression of phospho-glucose isomerase (PGI), phosphoglucomutase (PGM), and UDP-glcpyrophosphorylase (UGP) were also measured. Ca and Na concentration and induction time affected biomass, IPS, and EPS concentrations. Na increased EPS, IPS and TPS, while Ca increased biomass, IPS, and TPS. During fermentation, mycelial biomass varied greatly under Ca induction, while IPS, EPS and TPS varied greatly under Na induction. PGM and UGP activities increased in the presence of Na, while PGI increased with Ca. Compared to control samples, pgi and pgm expression under Na was greater at days 45 and 60, respectively, while under Ca, ugp expression was greater at day 45. IPS content correlated significantly with enzyme activity, while EPS correlated with PGM activity. Our data contributes to better understanding how Na and Ca affect mycelial growth and secondary metabolite production, and of polysaccharide biosynthesis mechanisms of L. edodes.
PubMed: 32170413
DOI: 10.1186/s13568-020-00985-w -
Current Biology : CB Aug 2022Starch metabolism is linked to plant growth, yet blocking its biosynthesis has species-specific consequences. In a new study, plastidial phosphoglucomutase is knocked...
Starch metabolism is linked to plant growth, yet blocking its biosynthesis has species-specific consequences. In a new study, plastidial phosphoglucomutase is knocked out in aspen trees using CRISPR-Cas9, limiting starch production and altering photosynthesis, but growth, bud break and wood production proceed unaffected.
Topics: Carbohydrate Metabolism; Photosynthesis; Plant Leaves; Plastids; Starch; Trees
PubMed: 35998602
DOI: 10.1016/j.cub.2022.07.024