Functional and structural characterization of Streptococcus pneumoniae pyruvate kinase involved in fosfomycin resistance

Glycolysis is the primary metabolic pathway in the strictly fermentative Streptococcus pneumoniae, which is a major human pathogen associated with antibiotic resistance. Pyruvate kinase (PYK) is the last enzyme in this pathway that catalyzes the production of pyruvate from phosphoenolpyruvate (PEP) and plays a crucial role in controlling carbon flux; however, while S. pneumoniae PYK (SpPYK) is indispensable for growth, surprisingly little is known about its functional properties. Here, we report that compromising mutations in SpPYK confers resistance to the antibiotic fosfomycin, which inhibits the peptidoglycan synthesis enzyme MurA, implying a direct link between PYK and cell wall biogenesis. The crystal structures of SpPYK in the apo and ligand-bound states reveal key interactions that contribute to its conformational change as well as residues responsible for the recognition of PEP and the allosteric activator fructose 1,6-bisphosphate (FBP). Strikingly, FBP binding was observed at a location distinct from previously reported PYK effector binding sites. Furthermore, we show that SpPYK could be engineered to become more responsive to glucose 6-phosphate instead of FBP by sequence and structure-guided mutagenesis of the effector binding site. Together, our work sheds light on the regulatory mechanism of SpPYK and lays the groundwork for antibiotic development that targets this essential enzyme.Taguchi A., Nakashima R., Nishino K.. Functional and structural characterization of Streptococcus pneumoniae pyruvate kinase involved in fosfomycin resistance. Journal of Biological Chemistry 299, 104892 (2023); https://doi.org/10.1016/j.jbc.2023.104892

Structural Basis of Nucleotide Selectivity in Pyruvate Kinase

Taguchi A., Nakashima R., Nishino K. Structural Basis of Nucleotide Selectivity in Pyruvate Kinase. Journal of Molecular Biology 436, 168708 (2024); https://doi.org/10.1016/j.jmb.2024.168708.Nucleoside triphosphates are indispensable in numerous biological processes, with enzymes involved in their biogenesis playing pivotal roles in cell proliferation. Pyruvate kinase (PYK), commonly regarded as the terminal glycolytic enzyme that generates ATP in tandem with pyruvate, is also capable of synthesizing a wide range of nucleoside triphosphates from their diphosphate precursors. Despite their substrate promiscuity, some PYKs show preference towards specific nucleotides, suggesting an underlying mechanism for differentiating nucleotide bases. However, the thorough characterization of this mechanism has been hindered by the paucity of nucleotide-bound PYK structures. Here, we present crystal structures of Streptococcus pneumoniae PYK in complex with four different nucleotides. These structures facilitate direct comparison of the protein-nucleotide interactions and offer structural insights into its pronounced selectivity for GTP synthesis. Notably, this selectivity is dependent on a sequence motif in the nucleotide recognition site that is widely present among prokaryotic PYKs, particularly in Firmicutes species. We show that pneumococcal cell growth is significantly impaired when expressing a PYK variant with compromised GTP and UTP synthesis activity, underscoring the importance of PYK in maintaining nucleotide homeostasis. Our findings collectively advance our understanding of PYK biochemistry and prokaryotic metabolism

Applicability of Licorice Extracts for Treatment of Oral Diseases, Evaluated by Simplified In Vitro Assay Systems with Oral Cells

Licorice extracts contain various useful substances for oral health. Alkaline extract showed potent anti‐HIV activity, whereas flavonoid‐rich water extracts showed potent anti‐HSV activity, closely correlated with polarizability, ionization potential, a number of ring systems, atomic number and mass. Licorice flavonoids showed higher tumor‐specificity against human oral squamous cell carcinoma as compared with human normal oral mesenchymal cells. Glycyrrhiza, at noncytotoxic concentrations, potently inhibited the IL‐1β‐induced inflammation in cultured human gingival and periodontal ligament fibroblasts. Glycyrrhizin, a major component of Glycyrrhiza, showed the highest UV‐protected activity. The results suggest the possible applicability of licorice extracts for several oral diseases and cosmetic products

Advances in Transient-Liquid-Phase Bonding of Ultra-high Temperature ZrC Ceramics

AbstractFull exploitation of the many attractive engineering properties of ultra-high temperature ceramics (UHTCs) requires that they can be joined. This paper explores progress in identifying joining strategies based on the use of transient liquid phases (TLPs). Wetting studies are used to explore the suitability of specific liquids for joining, while bonding studies provide the ultimate test. Sintering aids in the UHTC provide a major potential obstacle to successful joining, and dissolved impurities in the TLP can also complicate the joining process. Nonetheless, we show that well-bonding interfaces can be achieved when ZrC ceramics are bonded at 1673 K using a Ni/Nb/Ni multilayer interlayer

Detection of tissue T-cell in patients with chronic active hepatitis using fragmented sheep red blood cell (SRBC) membrane.

Fragmented sheep red blood cell (SRBC) membrane was used for detection of T-cells in liver biopsy specimens from patients with chronic active hepatitis. SRBC was separated with Lymphoprep, sonicated, then filtered through a 3 mu Millipore-membrane as a fragmented SRBC reagent. Tissue T-cells were stained by an indirect immunofluorescent technique using SRBC reagent and fluorescein isothiocyanate (FITC)-labelled rabbit anti-SRBC. Positively staining lymphocytes were present in portal tracts and in areas of piecemeal necrosis. There also seemed to be a positive correlation between the number of positively staining lymphocytes and the activity of chronic hepatitis; numerous lymphocytes being stained in areas of severe piecemeal necrosis. Our findings suggest that the fragmented SRBC technique for detection of T-cells is reliable and reproducible, that it could be used as a clinical routine method, and that it is useful for further elucidating the nature of host immune reactions on tissue levels.

The involvement of Gab1 and PI 3-kinase in β1 integrin signaling in keratinocytes

金沢大学大学院医学系研究科血管分子科学The control of the stem cell compartment in epidermis is closely linked to the regulation of keratinocyte proliferation and differentiation. β1 integrins are expressed 2-fold higher by stem cells than transit-amplifying cells. Signaling from these β1 integrins is critical for the regulation of the epidermal stem cell compartment. To clarify the functional relevance of this differential expression of β1 integrins, we established HaCaT cells with high β1integrin expression by repeated flow cytometric sorting of this population from the parental cell line. In these obtained cells expressing β1 integrins by 5-fold, MAPK activation was markedly increased. Regarding the upstream of MAPK, Gab1 phosphorylation was also higher with high β1 integrin expression, while Shc phosphorylation was not altered. In addition, enhanced phosphatidylinositol 3-kinase activation was also observed. These observations suggest that Gab1 and phosphatidylinositol 3-kinase play pivotal roles in the β1 integrin-mediated regulation of the epidermal stem cell compartment. © 2007 Elsevier Inc. All rights reserved

Tissue immune complexes demonstrated in the liver of patients with chronic aggressive hepatitis using FITC-labelled human Clq.

Immune complexes in liver specimens from 10 patients with chronic liver diseases [2 with chronic persistent hepatitis (CPH), 3 with chronic aggressive hepatitis (CAH) of moderate activity, 3 with CAH of severe activity, and 2 with liver cirrhosis] were examined by a technique of direct immunofluorescence using FITC-labelled human purified Clq (FITC-Clq). FITC-Clq bound to the nuclei of all cells in liver tissue. After DNase treatment, positive nuclei were absent, but positive staining with FITC-Clq remained in amorphous deposits and hepatic cell membranes in the areas of piecemeal necrosis of four CAH patients. Since FITC-Clq could not be demonstrated in the liver tissue of CPH and liver cirrhosis which contained no piecemeal necrosis, positive fluorescence in the liver of CAH patients was thought to indicate immune complexes bound to FITC-Clq. The fact that these positive substances, however, were few in number, may be the result of physiological mechanisms of immune clearance which rapidly eliminate immune complexes from the body.</p