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New Delhi metallo-β-lactamase-1 (NDM-1) has attracted extensive attention for its high catalytic activities of hydrolyzing almost all β-lactam antibiotics. NDM-1 shows relatively higher similarity to subclass B1 metallo-β-lactamases (MβLs), but its residue at position 229 is identical to that of B2/B3 MβLs, which is a Tyr instead of a B1-MβL-conserved Trp. To elucidate the possible role of Y229 in the bioactivity of NDM-1, we performed mutagenesis study and molecular dynamics (MD) simulations. Although residue Y229 is spatially distant from the active site and not contacting directly with the substrate or zinc ions, the Y229W mutant was found to have higher kcat and Km values than those of wild-type NDM-1, resulting in 1 ∼ 7 fold increases in k(cat) /K(m) values against tested antibiotics. In addition, our MD simulations illustrated the enhanced flexibility of Loop 2 upon Y229W mutation, which could increase the kinetics of both substrate entrance (kon) and product egress (koff). The enhanced flexibility of Loop 2 might allow the enzyme to adjust the geometry of its active site to accommodate substrates with different structures, broadening its substrate spectrum. This study indicated the possible role of the residue at position 229 in the evolution of NDM-1.

Various natural polysaccharides are capable of activating the immune system and therefore can be employed as biological response modifiers in anti-tumor therapy. We previously found a homogenous polysaccharide from the mycelium of marine fungus Phoma herbarum YS4108, named YCP, exhibiting strong in vivo antitumor ability via enhancement of the host immune responses. To further elucidate the role of YCP as a biological response modifier, the immunomodulating activities of YCP in B cells was investigated in the current study. We demonstrated that stimulation of YCP with murine splenic B cells resulted in cell proliferation and generation of IgM antibody response. Binding of YCP to B cells was a direct, saturable and reversible event and required TLR2 and TLR4 involvement. TLR2 and TLR4 defunctionalization by either antibody blocking or allele-specific mutation significantly impaired the B-cell proliferative and IgM responses to YCP. YCP interaction with TLR2 and TLR4 led to the activation of intracellular p38, ERK and JNK, as well as the translocation of transcriptional factor NF-κB into nucleus. Furthermore, specific inhibitors of p38, ERK, JNK and NF-κB could attenuate the ability of YCP to induce B cell proliferation and IgM production. Taken together, this study has indicated for the first time the immunostimulating properties of YCP on B cells through a receptor-mediated mechanism, which involves TLR2 and TLR4 and resultant activation of MAPK and NF-κB signaling pathways, thereby highlighting the role of YCP as an efficacious biological response modifier in oncologic immunotherapy.

Th17 cells have been reported to produce proinflammatory cytokines like Interleukin-17, IL-22, and regarded as important players in various inflammatory diseases. One of the IL-12 cytokine family cytokines, IL-23, composed of p19 and p40 subunit, is known for its potential to promote Th17 development and IL-17 producing, and the IL-23/IL-17 pathway is considered to be potential therapeutic target for autoimmune inflammation responses. Knockout mice deficient in either IL-23 or IL-17 related genes can suppress the allergic responses. Several IL-23 or IL-17 neutralizing agents are being evaluated in vitro or in vivo to disrupt the IL-23/IL-17 axis. Herein, we report that prokaryotically expressed soluble IL-23 receptor cytokine-binding homology region as an endogenous extracellular receptor analogue could be a natural antagonist against IL-23/IL-17 axis. We provide evidence that IL23R-CHR can bind to IL-23 in a dose-dependent manner in vitro, and block IL-23 signal by IL23R-CHR reducing the RORγt expression, which in turn lowers the expression of IL-17/IL-22, thus protecting naive CD4+ T cells against Th17 development. Together, this study indicates the importance of IL-23 pathway in Th17 development and the negative regulation of Th17 development by IL23R-CHR, and highlights the important roles of the soluble receptor extracellular region in the therapeutic strategy of neutralizing IL-23.