Elastase

As a strong heritability component has recently been identified in a whole exome sequencing study in SARS-CoV-2 infected COVID-19 patients, the ongoing identification of host susceptibility factors appears to be a worthwhile endeavour. In this article, α1-antitrypsin deficiency (A1AT deficiency) is proposed to represent a host susceptibility background in some COVID-19 patients. A1AT possesses general anti-inflammatory and immune-modulating properties in addition to maintaining the protease/antiprotease balance in the lung. A1AT deficiency is a common, underdiagnosed, inherited disorder. Associated immunological and clinical responses show overlap with post infectious residual problems experienced by some COVID patients. Research hypotheses to investigate the possibility of a A1AT deficiency-COVID 19 link include 1) investigating whether there is a causal connection between pathogenic A1AT deficiency alleles and COVID morbidity and mortality based on shared immunopathological features 2) whether altered glycosylation in AIAT deficiency has an effect on viral envelope glycosylation properties/gender glycosylation differences/hyposmia? 3) Is the COVID associated Kawasaki-like multisystem inflammatory syndrome in children (MIS-C) similar to AIAT - associated Kawasaki syndrome? 4) Do post- COVID infection disease states underlie the triggering of ongoing AIAT associated pathogenicity in various ways in specific A1AT pathogenic allele variant carriers? 5) Do international infection hotspots overlap with the geographical distribution of specific A1AT pathogenic allele variants? A1AT deficiency has previously been shown to alter host responses to atypical mycobacteria and HIV infectivity. It appears urgent to clarify whether superimposed COVID-19 infection in A1AT deficiency carriers results in an altered, more severe or prolonged illness. Demonstration of a pathophysiological relationsip between the two conditions will allow consideration of an additional therapeutic option in COVID 19 infection, the FDA-approved medication (Prolastin) (purified A1AT obtained from pooled human serum) is available for the neutrophil - driven immunopathology associated with genetic AIAT deficient disorders.

Since the vegetarian diet lacks the substrate for pancreatic elastase-1 as an enzyme, a decreased secretion of this enzyme could be expected. We aimed therefore to assess the changes of exocrine pancreatic secretion in a prospective way in a group of healthy omnivores who modified their diet by abstaining from meat for 1 month. Twenty healthy omnivores (fourteen females and six males) were used in the study. The nutrient intake was assessed for 7 d before commencing the study (omnivore diet) and after 1 month of dietary modification (modified diet; meat excluded). Similarly, the faecal output of pancreatic enzymes (elastase-1, chymotrypsin and lipase) was assessed before and 1 month after the period of dietary modification. Statistical differences between two points of the assessment (paired data) were calculated with the use of the Wilcoxon rank test. The relationship between the changes of faecal enzyme output and the changes in nutrient intake was assessed using multiple regressi...

Micromethods are described to determine in 10 min the activity of the five most common pancreatic zymogens: amylase, lipase, trypsin, chymotrypsin, and elastase. Progress of the reactions is monitored at 405 nm, allowing the kinetic determination of the five enzymes on a single 96-well microtiter plate. Amylase activity is measured by the release of p-nitrophenol from a chemically defined substrate. Linearity of the assay is from 10 to 360 UIL of amylase, and activities as low as 0.4 U/L can be easily measured by extending the period of incubation up to 24 h. Chymotrypsin, trypsin, and elastase activities are monitored by the release of p-nitroanilide from specific substrates, and activities are from 25 to 6,500, 15 to 260, and 20 to 600 U/L, respectively. Finally, lipase is determined by the clearing of a commercially available stabilized emulsion of triolein. The lipase determination can be performed from 90 to 3,600 U/L. When microplate methods were compared with conventional procedures, a perfect correspondence was found between the two types of procedure. Factors necessary to convert microplate results to those of conventional assays are provided. These microassays make possible the rapid and simultaneous determination of the three main types of pancreatic hydrolases (a glycohydrolase, three proteases, and a lipase) with <5 microliter of pancreatic juice by kinetic analysis. They could be easily adopted as routine assays in most research laboratories.

Human neutrophil proteinases (elastase, proteinase-3, and cathepsin-G) are released at sites of acute inflammation. We hypothesized that these inflammation-associated proteinases can affect cell signaling by targeting proteinase-activated receptor-2 (PAR 2). The PAR family of G protein-coupled receptors is triggered by a unique mechanism involving the proteolytic unmasking of an N-terminal self-activating tethered ligand (TL). Proteinases can either activate PAR signaling by unmasking the TL sequence or disarm the receptor for subsequent enzyme activation by cleaving downstream from the TL sequence. We found that none of neutrophil elastase, cathepsin-G, and proteinase-3 can activate G q-coupled PAR 2 calcium signaling; but all of these proteinases can disarm PAR 2 , releasing the N-terminal TL sequence, thereby preventing G q-coupled PAR 2 signaling by trypsin. Interestingly, elastase (but neither cathepsin-G nor proteinase-3) causes a TL-independent PAR 2-mediated activation of MAPK that, unlike the canonical trypsin activation, does not involve either receptor internalization or recruitment of ␤-arrestin. Cleavage of synthetic peptides derived from the extracellular N terminus of PAR 2 , downstream of the TL sequence, demonstrated distinct proteolytic sites for all three neutrophil-derived enzymes. We conclude that in inflammation, neutrophil proteinases can modulate PAR 2 signaling by preventing/disarming the G q /calcium signal pathway and, via elastase, can selectively activate the p44/42 MAPK pathway. Our data illustrate a new mode of PAR regulation that involves biased PAR 2 signaling by neutrophil elastase and a disarming/silencing effect of cathepsin-G and proteinase-3. Neutrophil infiltration represents a rapid innate immune defense mechanism that accompanies acute inflammation.

The Kunitz-type protease inhibitor ShPI-1 inhibits human neutrophil elastase (HNE, K i = 2.35Á10 −8 M) but does not interact with the porcine pancreatic elastase (PPE); whereas its P1 site variant, ShPI-1/K13L, inhibits both HNE and PPE (K i = 1.3Á10 −9 M, and K i = 1.2Á10 −8 M, respectively). By employing a combination of molecular modeling tools, e.g., structural alignment, molecular dynamics simulations and Molecular Mechanics Generalized-Born/ Poisson-Boltzmann Surface Area free energy calculations, we showed that D226 of HNE plays a critical role in the interaction of this enzyme with ShPI-1 through the formation of a strong salt bridge and hydrogen bonds with K13 at the inhibitor's P1 site, which compensate the unfavorable polar-desolvation penalty of the latter residue. Conversely, T226 of PPE is unable to establish strong interactions with K13, thereby precluding the insertion of K13 side-chain into the S1 subsite of this enzyme. An alternative conformation of K13 site-chain placed at the entrance of the S1 subsite of PPE, similar to that observed in the crystal structure of ShPI-1 in complex with chymotrypsin (PDB: 3T62), is also unfavorable due to the lack of stabilizing pair-wise interactions. In addition, our results suggest that the higher affinity of ShPI-1/K13L for both elastases mainly arises from the lower polar-desolvation penalty of L13 compared to that of K13, and not from stronger pair-wise interactions of the former residue with those of each enzyme. These results provide insights into the PPE and HNE inhibition and may contribute to the design of more potent and/or specific inhibitors toward one of these proteases.

Buddlejol (1), a new sterol, has been isolated from the ethyl acetate soluble fraction of the antispasmodic plant Buddleja asiatica along with stigmasterol (2), lignoceric acid (3), taraxerol (4) and a-amyrin (5), respectively. The structure of Buddlejol (1) was established as (24S)-stigmast-5,22-diene-7b-ethoxy-3b-ol by spectral analysis and comparison with closely related structures. Buddlejol revealed to be a competitive inhibitor of chymotrypsin with the Ki value of 10.60 lM as indicated by Lineweaver-Burk and Dixon plots and their re-plots against its chymotrypsin inhibition assay, while the other compounds showed less inhibitory potential. The bioassayguided isolation was stimulated by the preliminary cytotoxic screening of various fractions of B. asiatica.