An unusual active site has been identified in a family of zinc metalloendopeptidases that includes bacterial protease M and the human and Drosophila insidn-degrading enzymes. All of these enzymes have been characterized as metalloendopeptidases and purified protease HI has been shown to contain stoichiometric levels of zinc. However, all three proteases lack the consensus sequence (EIEXXH) described in the active site of other zinc metalloendopeptidases. Instead, these proteases contain an inversion of this motif, HXXEH. To determine whether this region could represent the active site in these proteins, the two histidines in protease m were individually mutated to arginine and the glutamate was mutated to glutamine. All three mutants were devoid of proteolytic activity toward an exogenous substrate, insulin, as compared to the wild-type protease. Three lines of evidence indicate that this loss of activity in the mutants is not due to distortion of the three-dimensional structure of the protein: (i) the mutants are secreted into the periplasmic space and chromatograph normally; (ia) all three mutants are expressed at levels nearly identical to wild-type protein and do not appear to have an increased susceptibility to proteolysis in the bacteria; and (iii) the mutants compete equally with wild-type protein in a radioimmunoassay. The purified wild-type and glutamate mutants were found to contain stoichiometric amounts of zinc by atomic absorption spectrophotometry, whereas both histidine mutants had negligible zinc signals. These findings are consistent with this region being the active site in this protein, with the histidine residues coordinating the essential zinc atom and the glutamate involved in catalysis.Proteases play a variety of roles in many physiological processes, including fibrinolysis, blood homeostasis, complement activation, digestion, and hormone processing (1). Based on common active sites and mechanisms of action as determined by primary amino acid sequence, inhibitor and substrate profiles, and x-ray crystallographic analyses (2), these proteins are generally classified into one of four families. Representative members of each protease class (i.e., cysteine, serine, aspartyl, and metallo-) have been crystallized and their active sites elucidated (2). For example, the bacterial enzyme thermolysin is the prototypic zinc metalloendopeptidase. X-ray crystallographic studies have shown that this enzyme contains two histidines (His-142 and His-146) and a glutamic acid (Glu-143) at its active site, with the histidines coordinating the binding of a zinc atom and the glutamate acting as a general base in catalysis (3). Based on studies in thermolysin, it has been proposed that the active site sequence in metalloendopeptidases is HEXXH (where X is any amino acid, H is histidine, and E is glutamate). This sequence has been identified in well over 15 zinc-dependent metalloproteases, including aminopeptidases and metalloendopeptidases, many of which show little overall sequence identity to thermolys...

No abstract

Background: In the US, Meningococcal B (MenB) vaccines were first licensed in 2014. In 2015, the Advisory Committee on Immunization Practices recommended that parents of teens talk to their provider about receiving MenB vaccine, rather than issuing a routine recommendation. We assessed parental awareness of MenB vaccines and willingness to vaccinate their teens with MenB vaccines compared to MenACWY vaccines, which have been routinely recommended for many years. Methods: We surveyed parents of teens attending high school in 2017–18 during the Minnesota State Fair. Parents reported via iPad their knowledge of and concern about meningococcal disease and their awareness of and willingness to vaccinate with MenB and MenACWY vaccines. We assessed the relationship between meningococcal disease knowledge and concern, MenB and MenACWY vaccine awareness, and willingness to vaccinate with MenB and MenACWY using adjusted logistic regression. Results: Among 445 parents, the majority had not heard of the newly introduced MenB vaccines Bexsero ® (80.0%; 95% CI: 76.0–83.6) or Trumenba ® (82.0%; 95% CI: 78.1–85.5) or the MenACWY vaccines Menactra ® or Menveo ® (68.8%; 95% CI: 64.2–73.0). The majority were at least somewhat willing to vaccinate their teen with MenB vaccine (89.6%; 95% CI: 86.5, 92.3) and MenACWY vaccine (91.2%; 95% CI: 88.2, 93.7). Awareness of MenB vaccines (OR: 3.8; 95% CI: 1.2–12.2) and concern about meningococcal disease (OR: 3.1; 95% CI: 1.5–6.3) were significantly associated with willingness to vaccinate with MenB vaccine. Conclusions: Awareness of MenB vaccine is lacking among parents of teens but is an important predictor of willingness to vaccinate with the newly licensed MenB vaccines.

A novel active site has been identified in a family of zinc-dependent metalloendopeptidases that includes bacterial proteinase III, the human and Drosophila insulin-degrading enzymes, and the processing-enhancing protein subunit of the mitochondrial processing proteinase. None of these enzymes contains the conserved active site described in most other metalloendopeptidases, HEXXH; instead, all four contain an inversion of this motif, HXXEH. Prior mutagenesis studies of proteinase III indicate that the two histidines are essential for co-ordinating the zinc atom, while all three residues are required for enzyme activity. To identify the third zinc-binding residue in this protein family, three glutamates downstream from the active site were mutated to glutamine in proteinase III. The mutant proteins were expressed and their ability to degrade insulin was compared with the wild-type enzyme. The glutamate-204 mutant was as active as the wild-type protein, the glutamate-162 mutant retained 20% of the activity of the wild-type enzyme and the glutamate-169 mutant was completely devoid of insulin-degrading activity. The purified wild-type and glutamate-204 mutant enzymes were found to contain nearly stoichiometric levels of zinc by atomic absorption spectrophotometry, whereas the glutamate-162 mutant had a slight reduction in the level of zinc, and the glutamate-169 mutant retained less than 0.3 mol of zinc/mol of enzyme. These findings are consistent with glutamate-169 being the third zinc-binding residue in proteinase III.

The insulin receptor is a large cell surface glycoprotein that concentrates insulin at the site of action and also initiates responses to insulin. The receptor is a disulfide-linked oligomer comprised of two alpha and two beta subunits. Signal transduction through the insulin receptor appears to require the activation of an intrinsic tyrosine-specific protein kinase activity. A variety of disorders, both acquired and genetic, are associated with the development of insulin resistance and are frequently the result of cellular defects in insulin receptor structure, function, and action. The recent cloning of several mutant receptors from patients with genetic forms of extreme insulin resistance has increased our understanding of insulin resistance on the molecular level.