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Summary The proteolytic activity in washings off the Triaenophorus nodulosus cestode tegument and the ability of the worms to inactivate proteolytic enzymes were studied. It was found that the major proteolytic activity in the washing samples is represented by the easily desorbed fraction most probably characterizing the activity of the host’s enzymes. Serine proteinases are an essential part of these enzymes. It was shown that the worms’ incubation medium and their homogenates can inhibit host proteinases and commercial trypsin samples. Suppressive activity of the incubation medium suggests that the inhibitors are rather spontaneously produced by the worms than induced by the presence of proteinases in the surrounding medium. The inhibitor produced by the cestode is hypothesized to be trypsin-specific.

Proteolytic activities in digestive system extracts from the larval midgut of the lesser mulberry pyralid, Glyphodes pyloalis Walker (Lepidoptera: Pyralidae), were analyzed using different specific peptide substrates and proteinase inhibitors. High proteolytic activities were found at pH 10.0 and a temperature of 50° C using azocasein as substrate. The trypsin was active in the pH range of 9.5- 12.0, with its maximum activity at pH 11.5. Ethylene diamine tetraacetic acid had the most inhibitory effect, and 44% inhibition was detected in the presence of this inhibitor. Phenyl methane sulfonyl floride and N-tosyl-L-phe chloromethyl ketone also showed considerable inhibition of larval azocaseinolytic activity, with 40.2 and 35.1% inhibition respectively. These data suggest that the midgut of larvae contains mainly metalloproteases and serine proteases, mainly chymotrypsin. The effect of several metal ions on the activity of proteases showed that NaCl, CaCl2, CoCl2 (5 and 10 mM), and MnCl2 (5mM) reduced the protease activity. The kinetic parameters of trypsin-like proteases using N-benzoyl-L-arg-p-nitroanilide as substrate indicated that the Km and Vmax values of trypsin in the alimentary canal were 50.5 ± 2.0 µM and 116.06 ± 1.96 nmol min(-1) mg(-1) protein, respectively. Inhibition assays showed only small amounts of cysteine proteases were present in the G. pyloalis digestive system. The midgut digestive protease system of G. pyloalis is as diverse as that of any of the other polyphagous lepidopteran insect species, and the midgut of larvae contains mainly metalloproteases. Moreover, serine proteases and chymotrypsin also play main roles in protein digestion. Characterization of the proteolytic properties of the digestive enzymes of G. pyloalis offers an opportunity for developing appropriate and effective pest management strategies via metalloproteases and chymotrypsin inhibitors.

Different tissues of potato, tobacco, and bean plants were screened for anti-fungal protease inhibitor (PI) activity, also following fungal pathogen inoculation or mechanical wounding. A potato (Solanum tuberosum var. Desireé) sprout protein extract showed a strong inhibitory activity against chymotrypsin and Botrytis cinerea fungal proteases, but also on spore germination, hyphal elongation, and development of necrotic lesions. An active mixture of different proteins was affinity column purified and sequenced. Two new anti-fungal genes, PKI1 and PPI3B2, coding, respectively, for a Kunitz-type inhibitor and a Proteinase Inhibitor 1 capable of reducing fungal lesion development, were cloned and partially characterized. Direct effect on leaf necrosis formation was found to be dependent on the anti-chymotrypsin activity of both selected inhibitors. The PKI1 transcript was found to accumulate in untreated sprout tissues, although homologues of this gene seemed to accumulate following Bemisia tabaci attack. In the case of PPI3B2, we provide preliminary evidence that a member of the Proteinase Inhibitor 1 family is active against not only herbivorous insects but also phytopathogenic fungi and foliar lesions caused by them.

Proteinase activity in the midgut of the pentatomid stinkbug Podisus maculiventris was investigated. The optimal pH for adult and nymph proteolysis was pH 6.0 and pH 6.5, respectively. Proteinase activity was characterised using a range of diagnostic inhibitors. Activity of both adult and nymphal gut extracts, detected by the hydrolysis of Z-Phe-Arg-pNA, was inhibited to

The Bowman-Birk inhibitors (BBI) are found widespread in legume species. The primary structure of 23 BBI sequences, isolated from genomic DNA of eight Italian landraces of common bean ( Phaseolus vulgaris L.), is presented in this study. The similarity among the analysed sequences ranged from 92 to 99%. The sequences, though highly similar to those previously described for common bean, showed some distinct features. Differences were detected in number of cysteine residues and in both trypsin and chymotrypsin binding loops. Two variants of trypsin binding loop, differing for the residue at P 2 ′ position (Ile or Arg) and two variants of chymotrypsin binding loop showing at the P 1 position Leu or Phe were found. Although P 2 ′-Ile is the most frequent residue observed in natural BBI sequences as well as in this study, the detection of the variant P 2 ′-Arg in four landraces make them very interesting for further investigations. In fact, sources of different BBI are very interesting due to the different inhibitory activity associated to them.

International audience; Background: Malaria remains a major global health concern. The development of novel therapeutic strategies is critical to overcome the selection of multiresistant parasites. The subtilisin-like protease (SUB1) involved in the egress of daughter Plasmodium parasites from infected erythrocytes and in their subsequent invasion into fresh erythrocytes has emerged as an interesting new drug target.Findings: Using a computational approach based on homology modeling, protein–protein docking and mutation scoring, we designed protein–based inhibitors of Plasmodium vivax SUB1 (PvSUB1) and experimentally evaluated their inhibitory activity. The small peptidic trypsin inhibitor EETI-II was used as scaffold. We mutated residues at specific positions (P4 and P1) and calculated the change in free-energy of binding with PvSUB1. In agreement with our predictions, we identified a mutant of EETI-II (EETI-II-P4LP1W) with a Ki in the medium micromolar range.Conclusions: Despite the challenges related to the lack of an experimental structure of PvSUB1, the computational protocol we developed in this study led to the design of protein-based inhibitors of PvSUB1. The approach we describe in this paper, together with other examples, demonstrates the capabilities of computational procedures to accelerate and guide the design of novel proteins with interesting therapeutic applications.

Targeting allosteric sites is gaining increasing recognition as a strategy for modulating the activity of enzymes, especially in drug design. Here we investigate the mechanisms of allosteric regulation of cathepsin K as a representative of cysteine cathepsins and a promising drug target for the treatment of osteoporosis. Eight novel modifiers are identified by computational targeting of predicted allosteric sites on the surface of the enzyme. All act via hyperbolic kinetic mechanisms in presence of low molecular mass substrates, as expected for allosteric effectors. Two compounds have sizable effects on enzyme activity using interstitial collagen as a natural substrate of cathepsin K and four compounds show a significantly stabilizing effect on cathepsin K. The concept of activity modification space is introduced to obtain a global perspective of the effects elicited by the modifiers. Analysis of the activity modification space reveals that the activity of cathepsin K is regulated via multiple, different allosteric mechanisms.

Although SW-AT-1, a serpin-type trypsin inhibitor from silkworm (Bombyx mori), was identified in previous study, its structure-function relationship has not been studied. In this study, SW-AT-1 was cloned from the body wall of silkworm and expressed in E. coli. rSW-AT-1 inhibited both trypsin and chymotrypsin in a concentration-dependent manner. The association rate constant for rSW-AT-1 and trypsin is 1.31×10−5 M−1s−1, for rSW-AT-1 and chymotrpsin is 2.85×10−6 M−1s−1. Circular dichroism (CD) assay showed 33% α-helices, 16% β-sheets, 17% turns, and 31% random coils in the secondary structure of the protein. Enzymatic and CD analysis indicated that rSW-AT-1 was stable at wide pH range between 4–10, and exhibited the highest activity at weakly acidic or alkaline condition. The predicted three-dimensional structure of SW-AT-1 by PyMOL (v1.4) revealed a deductive reactive centre loop (RCL) near the C-terminus, which was extended from the body of the molecule. In addition to trypsin cleavage site in RCL, matrix-assisted laser desorption ionization time of flight mass spectrometry indicated that the chymotrypsin cleavage site of SW-AT-1 was between F336 and T337 in RCL. Directed mutagenesis indicated that both the N- and C-terminal sides of RCL have effects on the activity, and G327 and E329 played an important role in the proper folding of RCL. The physiological role of SW-AT-1 in the defense responses of silkworm were also discussed.

The molecular interactions between pancreatic lipase (PL) and four tea polyphenols (EGCG analogs), like (−)-epigallocatechin gallate (EGCG), (−)-gallocatechin gallate (GCG), (−)-epicatechin gallate (ECG), and (−)-epigallocatechin (EC), were studied from PL activity, conformation, kinetics and thermodynamics. It was observed that EGCG analogs inhibited PL activity, and their inhibitory rates decreased by the order of EGCG>GCG>ECG>EC. PL activity at first decreased rapidly and then slowly with the increase of EGCG analogs concentrations. α-Helix content of PL secondary structure decreased dependent on EGCG analogs concentration by the order of EGCG>GCG>ECG>EC. EGCG, ECG, and EC could quench PL fluorescence both dynamically and statically, while GCG only quenched statically. EGCG analogs would induce PL self-assembly into complexes and the hydrodynamic radii of the complexes possessed a close relationship with the inhibitory rates. Kinetics analysis showed that EGCG analogs non-competitively inhibited PL activity and did not bind to PL catalytic site. DSC measurement revealed that EGCG analogs decreased the transition midpoint temperature of PL enzyme, suggesting that these compounds reduced PL enzyme thermostability. In vitro renaturation through urea solution indicated that interactions between PL and EGCG analogs were weak and non-covalent.

The major digestive proteinase activities of a new sugar beet pest, Aubeonymus mariaefranciscae Roudier (Coleoptera: Curculionidae), were characterized. Both larvae and adults of A. mariaefranciscae were found to use a complex proteolytic system for protein digestion based on at least trypsin-, chymotrypsin-, elastase-, cathepsin D, leucine aminopeptidase-, carboxypeptidase A- and carboxypeptidase B-like activities. An azocaseinolytic activity at pH 5.0-7.0 was identified, that was not affected by specific inhibitors and activators, making its classification in any of the mechanistic classes established not possible. According to this proteolytic profile, several serine proteinase inhibitors were tested in vitro and in vivo to establish their potential as resistance factors against A. mariaefranciscae. Larvae fed from neonate to pupation on diets containing 0.2% (w/w) soybean Bowman-Birk trypsin-chymotrypsin inhibitor, soybean Kunitz trypsin inhibitor, turkey egg white trypsin inhibitor, or lima bean trypsin inhibitor endure lower survival rates and display significant delays in the developmental time to pupation and to adult emergence. Interestingly, the most significant levels of mortality (about 90%) occurred with larvae fed on diets containing a combination of two or three inhibitors, suggesting a synergistic toxicity.

Cathepsin L is a lysosomal cysteine proteinase primarily devoted to the metabolic turnover of intracellular proteins. However, accumulating evidence suggests that this endopeptidase might also be implicated in the regulation of other important biological functions, including bone resorption in normal and pathological conditions. These findings support the concept that cathepsin L, in concert with other proteolytic enzymes involved in bone remodeling processes, could contribute to facilitate bone metastasis formation. In support of this hypothesis, recent studies indicate that cathepsin L can foster this process by triggering multiple mechanisms which, in part, differ from those of the major cysteine proteinase of osteoclasts, namely cathepsin K. Therefore, cathepsin L can be regarded as an additional target in the treatment of patients with metastatic bone disease. This review discusses the clinical and therapeutic implications related to these findings.

We extracted proteinase inhibitors from the nacre of the oyster Pinctada margaritifera with water. Mixing the nacre powder with water for 20 h led to a water-soluble fraction [0.24% (wt/wt) of nacre]. After dialysis of the water-soluble matrix through 6- to 8-kDa and 0.5-kDa membranes, the proteinase inhibitors were divided into low and high molecular weight fractions that contained inhibitors of papain, bovine cathepsin B, and human cathepsin L. We studied the heterogeneity of the inhibitors after separating the low molecular weight fraction according to charge and hydrophobicity. After multistep purification, mass spectrometry analysis revealed that a potent inhibitory fraction contained several molecules. This observation demonstrates the difficulties encountered in attempting to isolate individual metabolites from the complex mixture of molecules present in nacre matrix. Interestingly, the low molecular weight fraction contained specific inhibitors that could discern between cathepsin B and cathepsin L. The nacre organic inhibitors were active against several cysteine proteinases, yet they were more specific in relation to serine proteinases, because only proteinase K was inhibited. These results demonstrate, for the first time, the presence of active proteinase inhibitors in the mollusc shell, and it is possible that these inhibitors may play a role in either protection of proteins involved in shell formation or in defense against parasites, or both.

Rationale: The chemokine interleukin-8 is implicated in the development of bronchopulmonary dysplasia in preterm infants. The 77-amino acid isoform of interleukin-8 (interleukin-877) is a less potent chemoattractant than other shorter isoforms. Although interleukin-877 is abundant in the preterm circulation, its regulation in the preterm lung is unknown. Objectives: To study expression and processing of pulmonary interleukin-877 in preterm infants who did and did not develop bronchopulmonary dysplasia. Methods: Total interleukin-8 and interleukin-877 were measured in bronchoalveolar lavage fluid from preterm infants by immunoassay. Neutrophil serine proteases were used to assess processing. Neutrophil chemotaxis assays and degranulation of neutrophil matrix metalloproteinase-9 were used to assess interleukin-8 function. Main Results: Peak total interleukin-8 and interleukin-877 concentrations were increased in infants who developed bronchopulmonary dysplasia compared to those who did not. Shorter forms of interleukin-8 predominated in the preterm lung (96.3% No-bronchopulmonary dysplasia vs 97.1% bronchopulmonary dysplasia, p>0.05). Preterm bronchoalveolar lavage fluid significantly converted exogenously added interleukin-877 to shorter isoforms (p

Background Chilo partellus is an important insect pest infesting sorghum and maize. The larvae internalize in the stem, rendering difficulties in pest management. We investigated the effects of Capsicum annuum proteinase inhibitors (CanPIs) on C. partellus larvae by in-vitro and in-vivo experiments. Methods Recombinant CanPI-7 (with four-Inhibitory Repeat Domains, IRDs), -22 (two-IRDs) and insect proteinase activities were estimated by proteinase assays, dot blot assays and in gel activity assays. Feeding bioassays of lab reared C. partellus with CanPI-7 and -22 were performed. C. partellus proteinase gene expression was done by RT-PCR. In-silico structure prediction of proteinases and CanPI IRDs was carried out, their validation and molecular docking was done for estimating the interaction strength. Results Larval proteinases of C. partellus showed higher activity at alkaline pH and expressed few proteinase isoforms. Both CanPIs showed strong inhibition of C. partellus larval proteinases. Feeding bioassays of C. partellus with CanPIs revealed a dose dependent retardation of larval growth, reduction of pupal mass and fecundity, while larval and pupal periods increased significantly. Ingestion of CanPIs resulted in differential up-regulation of C. partellus proteinase isoforms, which were sensitive to CanPI-7 but were insensitive to CanPI-22. In-silico interaction studies indicated the strong interaction of IRD-9 (of CanPI-22) with Chilo proteinases tested. Conclusions Of the two PIs tested, CanPI-7 prevents induction of inhibitor insensitive proteinases in C. partellus so it can be explored for developing C. partellus tolerance in sorghum. General significance Ingestion of CanPIs, effectively retards C. partellus growth; while differentially regulating the proteinases., Graphical abstract fx1, Highlights • CanPI-7 and -22 ingestion led to dose-dependent growth and development retardation in Chilo partellus. • Ingestion of CanPIs showed up-regulation of proteinase activity and differential proteinase isoforms in C. partellus. • CanPI-7/-22 induced differential proteinases of C. partellus were sensitive to CanPI-7 and were insensitive to CanPI-22. • Molecular interaction studies of C. partellus proteinases and CanPIs identified a potent inhibitor.

1. 1. We have investigated the kinetics of inhibition of cathepsin D in heart, liver and skeletal muscle of CD-1 mice following administration of 25, 50, 100 and 200 mg/kg i.p. of pepstatin A, a specific inhibitor of this protease. 2. 2. In the liver, a significant inhibition of cathepsin D occurred up to at least 15 days, whereas, in heart and skeletal muscle, this inhibition lasted for a much shorter period of time. 3. 3. These results show that the recovery of enzyme activity to normal values is dose-dependent and that, at the same dose level, marked differences occur in the recovery of enzyme activity in these organ tissues, the liver being the most sensitive one. © 1988.

The present study describes the main characteristics of the proteolytic activities of the velvetbean caterpillar, Anticarsia gemmatalis Hübner, and their sensitivity to proteinase inhibitors and activators. Midguts of last instar larvae reared on an artificial diet were homogenized in 0.15 M NaCl and centrifuged at 14,000 g for 10 min at 4 degrees C and the supernatants were used in enzymatic assays at 30 degrees C, pH 10.0. Basal total proteolytic activity (azocasein hydrolysis) was 1.14 +/- 0.15 absorbance variation min(-1) mg protein(-1), at 420 nm; basal trypsin-like activity (N-benzoyl-L-arginine-p-nitroanilide, BApNA, hydrolysis) was 0.217 +/- 0.02 mmol p-nitroaniline min(-1) mg protein(-1). The maximum proteolytic activities were observed at pH 10.5 using azocasein and at pH 10.0 using BApNA, this pH being identical to the midgut pH of 10.0. The maximum trypsin-like activity occurred at 50 degrees C, a temperature that reduces enzyme stability to 80 and 60% of the original, when pre-incubated for 5 and 30 min, respectively. Phenylmethylsulfonyl fluoride inhibited the proteolytic activities with an IC50 of 0.39 mM for azocasein hydrolysis and of 1.35 mM for BApNA hydrolysis. Benzamidine inhibited the hydrolysis with an IC50 of 0.69 and 0.076 mM for azocasein and BApNA, respectively. The absence of cysteine-proteinases is indicated by the fact that 2-mercaptoethanol and L-cysteine did not increase the rate of azocasein hydrolysis. These results demonstrate the presence of serine-proteinases and the predominance of trypsin-like activity in the midgut of Lepidoptera insects, now also detected in A. gemmatalis, and suggest this enzyme as a major target for pest control based on disruption of protein metabolism using proteinase inhibitors.