Undecaprenyl Phosphate Phosphatase Activity of Undecaprenol Kinase Regulates the Lipid Pool in Gram-Positive Bacteria

Bacteria cell walls contain many repeating glycan structures, such as peptidoglycans, lipopolysaccharides, teichoic acids, and capsular polysaccharides. Their synthesis starts in the cytosol, and they are constructed from a glycan lipid carrier, undecaprenyl phosphate (C₅₅P), which is essential for cell growth and survival. The lipid derivative undecaprenol (C₅₅OH) is predominant in many Gram-positive bacteria but has not been detected in Gram-negative bacteria; its origin and role have thus remained unknown. Recently, a homologue of diacylglycerol kinase (DgkA) in <i>Escherichia coli</i> (<i>E. coli</i>) was demonstrated to be an undecaprenol kinase (UK) in the Gram-positive bacterium <i>Streptococcus mutans</i> (<i>S. mutans</i>). In this study, we found that <i>S. mutans</i> UK was not only an undecaprenol kinase but also a Mg-ADP-dependent undecaprenyl phosphate phosphatase (UpP), catalyzing the hydrolysis of C₅₅P to C₅₅OH and a free inorganic phosphate. Furthermore, the naturally undetectable C₅₅OH was observed in <i>E. coli</i> cells expressing <i>S. mutans dgkA</i>, supporting the phosphatase activity of UK/UpP <i>in vivo</i>. These two activities were indispensable to each other and utilized identical essential residues binding to their substrates, suggesting that both activities share the same active site and might involve a direct phosphoryl transfer mechanism. This study revealed a unique membrane enzyme displaying bifunctional activities determined by substrate binding and C₅₅OH production. The reciprocal conversion of C₅₅P and the undecaprenol pool efficiently regulate cell wall synthesis, especially in Gram-positive bacteria

Single Site <i>N</i>‑Glycosylation of B Cell Maturation Antigen (BCMA) Inhibits γ‑Secretase-Mediated Shedding and Improves Surface Retention and Cell Survival

B cell maturation antigen (BCMA), a member of the tumor necrosis factor receptor (TNFR) family, on the cell surface plays a key role in maintaining the survival of plasma cells and malignant as well as inflammatory accessory cells. Therefore, targeting BCMA or disrupting its interaction with ligands has been a potential approach to cancer therapy. BCMA contains a single N-glycosylation site, but the function of N-glycan on BCMA is not understood. Here, we found that the N-glycosylation of BCMA promoted its cell-surface retention while removing the N-glycan increased BCMA secretion through γ-secretase-mediated shedding. Addition of γ-secretase inhibitor prevented nonglycosylated BCMA from shedding and protected cells from dexamethasone and TRAIL-induced apoptosis

DH-PS induced IL-1ra production <i>in vivo</i>.

<p>BALB/c mice (n = 3 for each group) were injected intraperitoneally with DH-PS (100 or 300 μg/mouse) or PBS only. Sera collected at 0 (before injection), 2 and 18 hours were used for the measurements of IL-1ra by ELISA assay. Results were presented as mean concentrations with error bars showing the standard deviation of three mice. Statistically significant difference: * compared with PBS-treated group, p<0.05.</p

DH-PS induced more IL-1ra production than F3 in human CD14⁺ cells and THP-1 cells.

<p>(A) Human CD14⁺ cells isolated from one healthy donor were cultured (2×10⁶ cells/ml) with increasing concentrations of DH-PS or F3 for 18 hours and supernatants were collected for the measurements of IL-1ra. (B) Human CD14⁺ cells were cultured with DH-PS (100 μg/ml) or F3 (100 μg/ml) and supernatants were collected at the indicated time points for IL-1ra measurements. (C) THP-1 cells were cultured (2×10⁶ cells/ml) with increasing concentrations of DH-PS or F3 for 18 hours and supernatants were collected for the measurements of IL-1ra. In A and C, X-axis represented the concentration of DH-PS (μg/ml). Concentration 0 represented the use of PBS only as vehicle control. Results were presented as mean concentrations of IL-1ra with error bars showing the standard deviation of triplicate. Statistically significant difference: * compared with F3-treated group, p<0.05. # compared with F3-treated group, p<0.005. (D) THP-1 cells were cultured with DH-PS (100 μg/ml) or F3 (100 μg/ml) and cells were collected at the indicated time points for the assessment of mRNA expression of IL-1ra.</p

DH-PS induced IL-1ra production through MAPK, PI3K and NF-κB.

<p>(A) THP-1 cells (2×10⁶ cells/ml) were pretreated with the indicated concentrations of inhibitors for various kinases including ERK/ELK, JNK, p38 MAPK, PI3K and NFκB (0.01, 0.1 and 1 μM, except for ERK and PI3K: 0.1, 1 and 10 μM) or DMSO (0.1%) as control for 60 minutes and cultured with DH-PS (100 μg/ml) for another 18 hrs (B) Human CD14⁺ cells were pretreated with the indicated concentrations of inhibitors or DMSO as control like (A) and cultured with DH-PS (100 μg/ml) for another 18 hrs. Supernatants were harvested for IL-1ra measurements. Results were presented as fold of control (Y-axis) derived from the mean values of IL-1ra concentrations of inhibitor-treated groups divided by DMSO control group and error bars showed the standard deviation of triplicate. Statistically significant difference (Mean concentrations of IL-1ra were used for the comparisons): * compared with DMSO-treated group, p<0.05. # compared with DMSO-treated group, p<0.005. (C) THP-1 cells (2×10⁶ cells/ml) were pretreated with inhibitors for ERK/ELK (10 μM), JNK (1 μM), p38 MAPK (1 μM), PI3K (10 μM) and NFκB (1 μM) or DMSO (0.1%) as control and cultured with DH-PS (100 μg/ml) for another 12 hrs. Cells were collected for the assessment of mRNA expression of IL-1ra.</p

Investigation of SSEA‑4 Binding Protein in Breast Cancer Cells

SSEA-4, a sialyl-glycolipid, has been commonly used as a pluripotent human embryonic stem cell marker, and its expression is correlated with the metastasis of some malignant tumors. However, there is no in-depth functional study related to the receptor and the role of this glycolipid. Here, we report the identification of an SSEA-4-binding protein in a breast cancer cell line, MCF-7. By using affinity capture and glycan microarray techniques, the intracellular FK-506 binding protein 4 (FKBP4) was identified to bind directly to SSEA-4. The biological significance of SSEA-4/FKBP4 interaction was investigated

DH-PS elicited the productions of cytokines and chemokines <i>in vivo</i>.

<p>BALB/c mice (n = 3 for DH-PS and PBS group) were injected intraperitoneally with DH-PS (300 μg/mouse) or PBS only. Sera collected at 0 (before injection), 2 and 18 hours were used for the measurements of cytokines and chemokines. Y-axis represented the mean concentrations (Conc.) of cytokines/chemokines with error bars showing the standard deviation of three mice. Statistically significant difference: * compared with PBS-treated group, p<0.01. # compared with PBS-treated group, p<0.001.</p

DH-PS induced IL-1ra production in human PBMC, monocytes, but not neutrophils.

<p>(A) Human PBMCs were cultured (2×10⁶ cells/ml) with increasing concentrations of DH-PS for 18 hrs and the supernatants were harvested for IL-1ra measurements. (B) Human monocytes (CD14⁺ cells) were cultured (1×10⁶ cells/ml) with increasing concentrations of DH-PS for 18 hrs and the supernatants were harvested for IL-1ra measurements. (C) Neutrophils were cultured (1×10⁵ cells/ml) with the increasing concentrations of DH-PS for 3 or 24 hours and supernatants were harvested for IL-1ra measurements. Y-axis represented the mean concentration of IL-1ra (pg/ml). X-axis represented the concentration of DH-PS (μg/ml). Concentration 0 represented the use of PBS only as vehicle control. Results were presented as mean values with error bars showing the standard deviation of triplicate. Statistically significant difference: * compared with PBS-treated group, p<0.05. # compared with PBS-treated group, p<0.005.</p

DH-PS dose-dependently induced IL-1ra production in human monocytic cell line THP-1 cells.

<p>(A) THP-1 cells were cultured with increasing concentrations of DH-PS at different cell densities for 18 hrs and supernatants were harvested for IL-1ra measurements. X-axis represented the concentration of DH-PS (μg/ml). Concentration 0 represented the use of PBS only as vehicle control. (B) THP-1 cells were cultured (2×10⁶ cells/ml) with DH-PS (100 μg/ml) or PBS and supernatants were collected in indicated time points for IL-1ra measurements. Results were presented as mean values with error bars showing the standard deviation of triplicate. Statistically significant difference: * compared with PBS-treated group, p<0.05. # compared with PBS-treated group, p<0.005. (C) THP-1 cells were cultured (2×10⁶ cells/ml) with DH-PS (100 μg/ml) and cells were collected in indicated time points for the assessment of mRNA expression of IL-1ra by RT-PCR.</p

Dengue virus infection is through a cooperative interaction between a mannose receptor and CLEC5A on macrophage as a multivalent hetero-complex.

<p><b>(a)</b> Binding model of dengue virus with MR/DC-SIGN and CLEC5A on GM-MDM cell membrane. <b>(b)</b> The strong avidity receptor captures dengue virus to facilitate the interaction with a weak-binding signal receptor in proximity to induce innate immune response.</p