Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.24.27 (thermolysin)
 1,894 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The protective antigen (PA) component of anthrax toxin contains two sites that are uniquely sensitive to proteolytic cleavage. Cleavage at the sequence RKKR167 by the cellular protease furin is absolutely required for toxicity, whereas cleavage by chymotrypsin or thermolysin at the sequence FFD315 inactivates the protein, apparently by blocking the ability of PA to translocate the catalytic moieties of the toxins, lethal factor (LF) and edema factor (EF), to the cytosol of eukaryotic cells. To specify the role of the chymotrypsin-sensitive site of PA in the translocation of LF, we altered residues 313-315. None of the mutations in this region interfered with the ability of PA to bind to its cellular receptor, be cleaved by cell surface furin, and bind LF. Substitution of Ala for Asp315 or for both Phe313 and Phe314 reduced the ability of PA to intoxicate cells in the presence of LF by 3- and 7-fold, respectively. Substitution of Phe313 by Cys greatly reduced the rate of LF translocation and delayed toxicity. The rate at which the Cys-substituted PA killed cells was increased significantly by blocking the sulfhydryl group with iodoacetamide, suggesting that this added Cys interacts with cellular proteins and slows translocation of LF. Deletion of the 2 Phe rendered PA completely non-toxic. This deleted PA protein lacked the ability shown by native PA to form oligomers on cells and in solution and to induce release of 86Rb from Chinese hamster ovary cells. These results suggest that the chymotrypsin-sensitive site in PA is required for membrane channel formation and translocation of LF into the cytosol. PA double mutants were constructed that cannot be cleaved at either the furin or chymotrypsin sites. These PA proteins were more stable in Bacillus anthracis culture supernatants and may therefore be useful as a replacement for PA in anthrax vaccines.
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PMID:The chymotrypsin-sensitive site, FFD315, in anthrax toxin protective antigen is required for translocation of lethal factor. 796 69

The insect metalloproteinase inhibitor (IMPI) from the greater wax moth, Galleria mellonella, represents the first and to date only specific inhibitor of microbial metalloproteinases reported from animals. Here, we report on the characterization including carbohydrate analysis of two recombinant constructs encoded by impi cDNA either upstream or downstream of the furin cleavage site identified. rIMPI-1, corresponding to native IMPI purified from hemolymph, is encoded by the N-terminal part of the impi sequence, whereas rIMPI-2 is encoded by its C-terminal part. rIMPI-1 is glycosylated at N48 with GlcNAc2Man3, showing fucosylation to different extents. Similarly, rIMPI-2 is glycosylated at N149 with GlcNAc2Man3, but is fully fucosylated. rIMPI-1 represents a promising template for the design of second-generation antibiotics owing to its specific activity against thermolysin-like metalloproteinases produced by human pathogenic bacteria such as Vibrio vulnificus. In contrast, rIMPI-2 does not inhibit bacterial metalloproteinases, but is moderately active against recombinant human matrix metalloproteinases (MMPs). Both microbial metalloproteinases and MMPs induce expression of the impi gene when injected into G. mellonella larvae. These findings provide evidence that the impi gene encodes two distinct inhibitors, one inhibiting microbial metalloproteinases and contributing to innate immunity, the other putatively mediating regulation of endogenous MMPs during metamorphosis.
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PMID:The insect metalloproteinase inhibitor gene of the lepidopteran Galleria mellonella encodes two distinct inhibitors. 1721 56