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 low pH-induced fusion of influenza virus with intact erythrocyte plasma membranes is preceded by a delay time following pH reduction, that is itself pH- and temperature dependent. At 37 degrees C/pH 4.8, lipid mixing between virus and target membranes begins < 2 s after pH reduction, whereas at 4 degrees C/pH 4.8, fusion does not commence until > 10 min after pH reduction. We have found that within this time period at 4 degrees C, a population of virus acquires the capacity to subsequently undergo fusion with high efficiency at elevated temperatures and pH 7.4. Both the kinetics and the extent of this pH 7.4 fusion depend upon the time of pre-incubation at pH 4.8/4 degrees C. Incubation at pH 7.4/4 degrees C, following this pre-incubation does not result in fusion, but the capacity to fuse at pH 7.4/37 degrees C is retained for a time period exceeding 1 h. The longevity of this fusion committed state makes it amenable to biochemical and immunological analysis. We have shown that it is insensitive to dithiothreitol, neuraminidase and trypsin, but is incapacitated by thermolysin or protease K. We conclude that only the HA2 sub-unit of influenza haemagglutinin is a necessary protein component of later stages of the fusion pathway.
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PMID:A long-lived state for influenza virus-erythrocyte complexes committed to fusion at neutral pH. 139 18

New classes of mutants of influenza virus A/seal/Mass/1/80 are described in which the haemagglutinins (HA) have lost their protease cleavability by trypsin, but can be activated by elastase, chymotrypsin or thermolysin in different cell types. The same proteases that were required for activation of infectivity of the mutants also activated haemolysis and cell-fusing properties. The protease activation (pa)-mutants were non-pathogenic for chickens, but induced a protective immune response against a highly pathogenic challenge virus. The failure of the mutants to be activated by trypsin, but instead to be activated by the other proteases employed, was related to amino acid exchanges around the HA cleavage site. The cleavability of the chymotrypsin and elastase pa-mutants is most likely determined by replacement of Arg-1 by neutral amino acids such as Ile, Thr, Met or Leu, depending on the substrate specificity of the activating proteases. Cleavage activation of the thermolysin pa-mutants, on the other hand, became possible by insertion of a single Leu residue at position 4 of the HA2 polypeptide, which compensates for the loss of the Gly residue at the N terminus of the fusion peptide due to thermolysin cleavage. The correction of the mutations in revertants confirmed the conclusions drawn from sequence analyses of the pa-mutants.
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PMID:Trypsin-resistant protease activation mutants of an influenza virus. 789 52

Influenza virus A/seal/Mass/1/80 (H7N7) mutants were obtained; the hemagglutinins (HAs) of the mutants were not activated by trypsin, as in the wild-type virus, but by thermolysin. The mutants grew efficiently under multiple replication cycle conditions and formed plaques in chicken embryo cells only when thermolysin was added to the culture medium. They exhibited hemolytic activity and induced protective immunity in chickens after an asymptomatic course of infection. Nucleotide sequencing of the HA gene and direct amino acid sequencing showed that insertion of a single leucine into the fusion peptide of the HA2 chain close to the cleavage site and a shift of the cleavage site toward the C terminus by one amino acid were responsible for the changes in the biological properties of the thermolysin activation mutants. Revertants could be obtained when trypsin or trypsin-like endoproteases were present in the virus-producing system.
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PMID:Thermolysin activation mutants with changes in the fusogenic region of an influenza virus hemagglutinin. 793 38

As a step toward studying membrane fusion with a simplified molecule, the ectodomain, residues 1-185, of the membrane-anchored subunit HA2 of the influenza virus haemagglutinin (HA) was solubilized by adding the very polar FLAG octapeptide (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys) to the N-terminal HA2 fusion peptide. The resulting chimeric protein, F185, when expressed in bacteria, folded spontaneously into a soluble trimer, with a high alpha-helical content and a high melting temperature, structural characteristics of the low-pH-induced conformation of HA2. Removal of the FLAG octapeptide by proteolysis with enterokinase converted the soluble molecule to one that aggregated, bound nonionic detergent, and bound to lipid vesicles, properties of the low-pH-induced conformation of HA. Thermolysin treatment of the aggregated protein removed the nonpolar fusion peptide, regenerating soluble trimers of HA2 (residues 24-185), which is analogous to thermolysin treatment of HA in the low-pH-induced conformation. Thermolysin treatment also dissociates F185 from the detergent-protein complex by removing the fusion peptide. These results suggest that highly polar peptides can be fused to the membrane-binding regions of membrane proteins to increase their solubility. They also indicate that ectodomains of HA2 made in bacteria have membrane-binding properties similar to those of the same ectodomain generated by low-pH treatment of HA isolated from virus.
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PMID:A polar octapeptide fused to the N-terminal fusion peptide solubilizes the influenza virus HA2 subunit ectodomain. 975 51

The proteolysis of flu virions of the strain A/Puerto Rico/8/34 (subtype H1N1) by enzymes of various classes was studied to develop an approach to the study of the structural organization and interaction of the basic protein components of the virion environment: hemagglutinin (HA), transmembrane homotrimeric glycoprotein, and matrix protein M1 forming a layer under the lipid membrane. Among the tested proteolytic enzymes and enzymic preparations (thermolysin, trypsin, chymotrypsin, subtilisin Carlsberg, pronase, papain, and bromelain), the cysteine proteases bromelain and papain and the enzymic preparation pronase efficiently deleted HA ectodomains, while chymotrypsin, trypsin, and subtilisin Carlsberg deleted only a part of them. An analysis by MALDI TOF mass spectrometry allowed us to locate the sites of HA hydrolysis by various enzymic preparations. Bromelain, papain, trypsin, and pronase split the polypeptide chain after the K177 residue located before the transmembrane domain (HA2 185-211). Subtilisin Carlsberg hydrolyzed the peptide bond at other neighboring points: after L178 (a basic site) or V176. The hydrolytic activity of bromelain measured by a highly specific chromogenic substrate of cysteine proteases Glp-Phe-Ala-pNA was almost three times higher in the presence of 5 mM beta-mercaptoethanol than in the presence of 50 mM. However, the complete removal of exodomains of HA, HA, and low-activity enzyme by the HA high- and low-activity enzyme required identical time intervals. In the absence of the reducing reagent, the removal of HA by bromelain proceeded a little more slowly and was accompanied by significant fragmentation of protein Ml1. The action of trans-epoxysuccinyl-L-leucylamido)butane (E-64), a specific inhibitor of cysteine proteases, and HgCl2 on the hydrolysis of proteins HA and M1 by bromelain was investigated.
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PMID:[Flu virion as a substrate for proteolytic enzymes]. 1867 93