Many inflammatory diseases may be linked to pathologically elevated signaling via the receptor for lipopolysaccharide (LPS), toll-like receptor 4 (TLR4). There has thus been great interest in the ...discovery of TLR4 inhibitors as potential anti-inflammatory agents. Recently, the structure of TLR4 bound to the inhibitor E5564 was solved, raising the possibility that novel TLR4 inhibitors that target the E5564-binding domain could be designed. We utilized a similarity search algorithm in conjunction with a limited screening approach of small molecule libraries to identify compounds that bind to the E5564 site and inhibit TLR4. Our lead compound, C34, is a 2-acetamidopyranoside (MW 389) with the formula C.sub.17 H.sub.27 NO.sub.9, which inhibited TLR4 in enterocytes and macrophages in vitro, and reduced systemic inflammation in mouse models of endotoxemia and necrotizing enterocolitis. Molecular docking of C34 to the hydrophobic internal pocket of the TLR4 co-receptor MD-2 demonstrated a tight fit, embedding the pyran ring deep inside the pocket. Strikingly, C34 inhibited LPS signaling ex-vivo in human ileum that was resected from infants with necrotizing enterocolitis. These findings identify C34 and the beta-anomeric cyclohexyl analog C35 as novel leads for small molecule TLR4 inhibitors that have potential therapeutic benefit for TLR4-mediated inflammatory diseases.
Amino acid racemases catalyze the stereoinversion of the chiral Cα to produce the D-enantiomers that participate in biological processes, such as cell wall construction in prokaryotes. Within this ...large protein family, bacterial proline racemases have been extensively studied as a model of enzymes acting with a pyridoxalphosphate-independent mechanism. Here we report the crystal structure of the proline racemase from the human parasite Trypanosoma cruzi (TcPRACA), a secreted enzyme that triggers host B cell polyclonal activation, which prevents specific humoral immune responses and is crucial for parasite evasion and fate. The enzyme is a homodimer, with each monomer folded in two symmetric α/α subunits separated by a deep crevice. The structure of TcPRACA in complex with a transition-state analog, pyrrole-2carboxylic acid, reveals the presence of one reaction center per monomer, with two Cys residues optimally located to perform acid/base catalysis through a carbanion stabilization mechanism. Mutation of the catalytic Cys residues abolishes the enzymatic activity but preserves the mitogenic properties of the protein. In contrast, inhibitor binding promotes the closure of the interdomain crevice and completely abrogates B cell proliferation, suggesting that the mitogenic properties of TcPRACA depend on the exposure of transient epitopes in the ligand-free enzyme.
Deep-sea microbes exhibit immunosilence Funk, Michael
Science (American Association for the Advancement of Science),
03/2021, Letnik:
371, Številka:
6535
Journal Article
Effects of lentinan on nuclear factor-kappaB (NF-kappaB) activity in liver of burn rats with sepsis were investigated. To mimic the clinical sepsis after burn, rats were subjected to 30% ...full-thickness scald injury, followed by intraperitoneal (i.p.) injection of lipopolysaccharide (LPS). Seventy-two adult rats were randomly divided into six groups: the normal control group (n=12); the burn sepsis group (n=12); the burn sepsis with positive drugs; the burn sepsis with low-dose lentinan treatment group (50.0 mg/kg, n=12); the burn sepsis with middle-dose lentinan treatment group (100.0 mg/kg, n=12) and the burn sepsis with high-dose lentinan treatment group (200.0 mg/kg, n=12). Expression of NF-kappaB in the liver was measured with western blot analysis. The morphology of liver was evaluated with hematoxylin and eosin staining. The expression of NF-kappaB significantly increased in the liver of burn rats with sepsis. Compared with the burn sepsis group, lentinan treatment obviously reduced the damage of hepatic cell morphology, and decreased the activity of NF-kappaB significantly in the medium and high concentrations of lentinan treatment groups (P<0.05). Most importantly, treatment with lentinan was able to reverse the increased concentration of IL-4, IL-6, IL-10 and TNF-alpha in plasma which was induced by LPS. Lentinan treatment can significantly decrease the expression of NF-kappaB in the liver of burn rats with sepsis. Key words: lentinan, sepsis, liver, NF-kappaB, burn, injury
Fibroblast growth factor (FGF) 13, a member of the FGF11 subfamily, is a kind of intracrine protein similar to other family members including FGF11, FGF12, and FGF14. Unlike classical FGF, FGF13 ...exerts its bioactivities independent of fibroblast growth factor receptors (FGFRs). However, the effect of exogenous administration of FGF13 still remains further investigated. In the present study, we established an
Escherichia coli
expression system for the large-scale production of FGF13 and then obtained two isoform proteins including recombinant human FGF13A (rhFGF13A) and rhFGF13B with a purity greater than 90% by column chromatography, respectively. Otherwise, soluble analysis indicated that both rhFGF13A and rhFGF13B expressed in
E. coli
BL21 (DE3) pLysS were soluble. Furthermore, cellular-based experiments demonstrated that rhFGF13A, rather than rhFGF13B, could promote the proliferation of NIH3T3 cells in the presence of heparin. Mechanistically, the mitogenic effect of FGF13 was mediated by activation of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK), but not p38. Moreover, blockage of FGFRs also significantly attenuated the mitogenic effects of rhFGF13A, implying that FGFRs are still related to FGF13. Thus, our research shows that exogenous FGF13 can act as secreted FGF to participate in cell signal transmission and heparin is still required as an ancillary cofactor for the mitogenic effects of FGF13, which may help people to discover more potential functions of FGF13 in cell life activities.
In bovine mammary epithelial cells (BMECs), a cascade of inflammatory reactions induced by lipopolysaccharide (LPS) has been shown to result in cell injury and apoptosis. The present study aims to ...reveal the protective effect of ferulic acid (FA) on LPS-induced BMEC apoptosis and explore its potential molecular mechanisms. First, we showed that FA had low cytotoxicity to BMECs and significantly decreased cell apoptosis and the proinflammatory response induced by LPS. Next, FA blocked LPS-induced oxidative stress by restoring the balance of the redox state and inhibiting mitochondrial dysfunction, the main contributor to LPS-induced apoptosis and ROS generation. Furthermore, the relief of inflammation and redox disturbance in the FA preconditioning group were accompanied by weaker NF-κB activation, enhanced Nrf2 activation and maintained cell viability compared to the LPS group. When BMECs were treated with FA alone, we observed that Nrf2 activation was induced before the inhibition of NF-κB activation and that the Keap1-Nrf2 relationship was disturbed. We concluded that FA prevented LPS-induced BMEC apoptosis by reversing the dominant relationship between NF-κB and Nrf2. Keywords: BMEC, LPS, inflammation, oxidative stress, FA, NF-κB and Nrf2 signals
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