The recognition of carbohydrate moieties by cells of the innate immune system is emerging as an essential element in antifungal immunity, but despite the number and diversity of lectins expressed by innate immune cells, few carbohydrate receptors have been characterized. Mincle, a C-type lectin, is expressed predominantly on macrophages, and is here shown to play a role in macrophage responses to the yeast Candida albicans. After exposure to the yeast in vitro, Mincle localized to the phagocytic cup, but it was not essential for phagocytosis. In the absence of Mincle, production of TNF-α by macrophages was reduced, both in vivo and in vitro. In addition, mice lacking Mincle showed a significantly increased susceptibility to systemic candidiasis. Thus, Mincle plays a novel and nonredundant role in the induction of inflammatory signaling in response to C. albicans infection.
Discovery of glycan-competitive galectin-3-binding compounds that attenuate lung fibrosis in a murine model and that block intracellular galectin-3 accumulation at damaged vesicles, hence revealing galectin-3-glycan interactions involved in fibrosis progression and in intracellular galectin-3 activities, is reported. 3,3'-Bis-(4-aryltriazol-1-yl)thiodigalactosides were synthesized and evaluated as antagonists of galectin-1, -2, -3, and -4 N-terminal, -4 C-terminal, -7 and -8 N-terminal, -9 N-terminal, and -9 C-terminal domains. Compounds displaying low-nanomolar affinities for galectins-1 and -3 were identified in a competitive fluorescence anisotropy assay. X-ray structural analysis of selected compounds in complex with galectin-3, together with galectin-3 mutant binding experiments, revealed that both the aryltriazolyl moieties and fluoro substituents on the compounds are involved in key interactions responsible for exceptional affinities towards galectin-3. The most potent galectin-3 antagonist was demonstrated to act in an assay monitoring galectin-3 accumulation upon amitriptyline-induced vesicle damage, visualizing a biochemically/medically relevant intracellular lectin-carbohydrate binding event and that it can be blocked by a small molecule. The same antagonist administered intratracheally attenuated bleomycin-induced pulmonary fibrosis in a mouse model with a dose/response profile comparing favorably with that of oral administration of the marketed antifibrotic compound pirfenidone.
The crystal structure of a Ca(2+)-binding domain (dVI) of rat m-calpain has been determined at 2.3 A resolution, both with and without bound Ca2+. The structures reveal a unique fold incorporating five EF-hand motifs per monomer, three of which bind calcium at physiological calcium concentrations, with one showing a novel EF-hand coordination pattern. This investigation gives us a first view of the calcium-induced conformational changes, and consequently an insight into the mechanism of calcium induced activation in calpain. The crystal structures reveal a dVI homodimer which provides a preliminary model for the subunit dimerization in calpain.
We used NMR spectroscopy, molecular modeling and infectivity competition assays to investigate the key interactions between the spike protein (VP8(*)) from 'sialidase-insensitive' human Wa and 'sialidase-sensitive' porcine CRW-8 rotaviruses and the glycans of gangliosides G(M1) and G(D1a). Our data provide strong evidence that N-acetylneuraminic acid is a key determinant for binding of these rotaviruses. This is in contrast to the widely accepted paradigm that sialic acids are irrelevant in host cell recognition by sialidase-insensitive rotaviruses.
The structural differences could be correlated with the observed substrate specificities of caspase-1, caspase-3 and caspase-8, as determined from kinetic experiments. This information will help us to understand the role of the various caspases in the propagation of the apoptotic signal. The information gained from this investigation should be useful for the design of specific inhibitors.
The past decade has seen an alarming worldwide increase in resistance to beta-lactam antibiotics among many pathogenic bacteria, which is due mainly to plasmid- or chromosomally encoded beta-lactamases that specifically cleave penicillin and cephalosporins, rendering them inactive. There is therefore a need to develop new strategies in the design of effective inhibitors of beta-lactamase. All the small-molecule inhibitors in clinical use are not very effective and are rapidly degraded. Furthermore, newly characterized mutants of the plasmid-mediated beta-lactamase TEM-1 are highly resistant to these small-molecule inhibitors, including clavulanic acid and tazobactam. It has been shown that Streptomyces clavuligerus produces an exocellular beta-lactamase inhibitory protein (BLIP; M(r) 17.5 K). Here we present data defining BLIP as the most effective known inhibitor of a variety of beta-lactamases, with Ki values in the subnanomolar to picomolar range. To identify those features in BLIP that make it such a potent inhibitor, we have determined its molecular structure at 2.1 A resolution. BLIP is a relatively flat molecule with a unique fold, comprising a tandem repeat of a 76-amino-acid domain. Each domain consists of a helix-loop-helix motif that packs against a four-stranded antiparallel beta-sheet (Fig. 1a). To our knowledge, BLIP is the first example of a protein inhibitor having two similarly folded domains that interact with and inhibit a single target enzyme.
Galectin-3 is a multifunctional carbohydrate-binding protein that has roles in cancer progression. In addition to carbohydrate-dependent extracellular functions, galectin-3 participates in carbohydrate-independent intracellular signalling pathways, including apoptosis, via protein-protein interactions, some of which engage the carbohydrate-binding groove. When ligands bind within this site, conformational rearrangements are induced and information on unliganded galectin-3 is therefore valuable for structure-based drug design. Removal of cocrystallized lactose from the human galectin-3 carbohydrate-recognition domain was achieved via crystal soaking, but took weeks despite low affinity. Pre-soaking to remove lactose enabled the subsequent binding of cryoprotectant glycerol, whereas when the lactose was not removed a priori the glycerol could not displace it in the short cryosoaking time frame. This slow diffusion of lactose out of the crystals contrasts with the entrance of glycerol, which takes place within minutes. The importance of the removal of incumbent ligands prior to attempts to introduce alternative ligands is indicated, even for proteins exhibiting low affinity for ligands, and has significance for ligand exchange in structure-based drug design.
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