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Background Protease inhibitors are defense proteins widely distributed in the plant kingdom. By reducing the activity of digestive enzymes in insect guts, they reduce the availability of nutrients and thus impair the growth and development of the attacking herbivore. One well-characterized class of protease inhibitors are Kunitz-type trypsin inhibitors (KTIs), which have been described in various plant species, including Populus spp. Long-lived woody perennials like poplar trees encounter a huge diversity of herbivores, but the specificity of tree defenses towards different herbivore species is hardly studied. We therefore aimed to investigate the induction of KTIs in black poplar (P. nigra) leaves upon herbivory by three different chewing herbivores, Lymantria dispar and Amata mogadorensis caterpillars, and Phratora vulgatissima beetles. Results We identified and generated full-length cDNA sequences of 17 KTIs that are upregulated upon herbivory in black poplar leaves, and analyzed the expression patterns of the eight most up-regulated KTIs via qRT-PCR. We found that beetles elicited higher transcriptional induction of KTIs than caterpillars, and that both caterpillar species induced similar KTI expression levels. Furthermore, KTI expression strongly correlated with the trypsin-inhibiting activity in the herbivore-damaged leaves, but was not dependent on damage severity, i.e. leaf area loss, for most of the genes. Conclusions We conclude that the induction of KTIs in black poplar is controlled at the transcriptional level in a threshold-based manner and is strongly influenced by the species identity of the herbivore. However, the underlying molecular mechanisms and ecological consequences of these patterns remain to be investigated.

Significant efforts are being made to minimize aflatoxin contamination in peanut seeds and one possible strategy is to understand and exploit the mechanisms of plant defense against fungal infection. In this study we have identified and characterized, at biochemical and molecular levels, plant protease inhibitors (PPIs) produced in peanut seeds of the resistant PI 337394 and the susceptible Forman cultivar during Aspergillus parasiticus colonization. With chromatographic methods and 2D-electrophoresis-mass spectrometry we have isolated and identified four variants of Bowman-Birk trypsin inhibitor (BBTI) and a novel Kunitz-type protease inhibitor (KPI) produced in response to A. parasiticus colonization. KPI was detected only in the resistant cultivar, while BBTI was produced in the resistant cultivar in a higher concentration than susceptible cultivar and with different isoforms. The kinetic expression of KPI and BBTI genes along with trypsin inhibitory activity was analyzed in both cultivars during infection. In the susceptible cultivar an early PPI activity response was associated with BBTI occurrence. Meanwhile, in the resistant cultivar a later response with a larger increase in PPI activity was associated with BBTI and KPI occurrence. The biological significance of PPI in seed defense against fungal infection was analyzed and linked to inhibitory properties on enzymes released by the fungus during infection, and to the antifungal effect of KPI. Instituto de Biotecnología Fil: Muller, Virginia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina Fil: Bonacci, Gustavo Roberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina Fil: Batthyany, Carlos. Institut Pasteur de Montevideo. Unidad de Bioquímica y Proteómica Analítica; Uruguay Fil: Amé, María Valeria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina Fil: Carrari, Fernando Oscar. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina Fil: Gieco, Jorge Omar. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Manfredi; Argentina Fil: Asis, Ramón. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina

Plants respond to attacks by herbivores with various defences, which are mounted through the activation of different biochemical pathways that are known to interact. Thus, the attack of a plant by one herbivore species may result in changes in the performances of other species on the same plant. It has been suggested that species with comparable feeding modes induce similar plant defences and such herbivores are therefore expected to have a negative effect on each other's performance. We studied two closely related phytophagous mite species with identical feeding modes. Yet, one of the species (Tetranychus urticae) induces tomato plant defences, whereas the other (T. evansi) reduces them. We found that the "inducing" species benefits from the downregulation of defences by the "reducing" species, which, in turn, suffers from the induction of defences by the inducing species. Moreover, the performances of the two mite species on leaves that were previously attacked by both species simultaneously were intermediate between that on leaves previously attacked by each of the mites separately. The activity of proteinase inhibitor, a defensive compound, was not found to be intermediate in leaves attacked by both species simultaneously--it was almost as high as the activity seen in leaves with defences induced by T. urticae. Oviposition rates of T. urticae showed a nonlinear correlation with inhibitor activity, suggesting that it is potentially problematic to use this activity as an indicator of the level of plant defence. Our results show that herbivores with similar feeding modes have opposite effects on plant defence and differentially affect each other's performance on co-infested plants.

[EN] Background: For as long as 350 million years, plants and insects have coexisted and developed a set of relationships which affect both organisms at different levels. Plants have evolved various morphological and biochemical adaptations to cope with herbivores attacks. However, Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae) has become the major pest threatening tomato crops worldwide and without the appropriated management it can cause production losses between 80 to 100%. Results: The aim of this study was to investigate the in vivo effect of a serine proteinase inhibitor (BTI-CMe) and a cysteine proteinase inhibitor (Hv-CPI2) from barley on this insect and to examine the effect their expression has on tomato defensive response. We found that larvae fed on the double transgenic plants showed a notable reduction in weight. Moreover, only 56% of the larvae reached the adult stage. The emerged adults showed wings deformities and reduced fertility. We also investigated the effect of proteinase inhibitors ingestion on the insect digestive enzymes. Our results showed a decrease in larval trypsin activity. Transgenes expression had no harmful effect on Nesidiocoris tenuis (Reuter) (Heteroptera: Miridae), a predator of Tuta absoluta, despite transgenic tomato plants attracted the mirid. We also found that barley cystatin expression promoted plant defense by inducing the expression of the tomato endogenous wound inducible Proteinase inhibitor 2 (Pin2) gene, increasing the production of glandular trichomes and altering the emission of volatile organic compounds. Conclusion: Our results demonstrate the usefulness of the co-expression of different proteinase inhibitors for the enhancement of plant resistance to Tuta absoluta., This work was partly supported by grants BIO2013-40747-R and AGL2014-55616-C3 from the Spanish Ministry of Economy and Competitiveness (MINECO)

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.

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.

I'm your Venus: The crystal structure of the human methylamine-induced form of α 2-macroglobulin (α 2M) shows its large central cavity can accommodate two medium-sized proteinases (see structure, front part clipped off to better show central cavity). Twelve major entrances provide access for small substrates to the cavity and the still-active trapped >prey>. The structure unveils the molecular basis of the unique >venus flytrap> mechanism of α 2M. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Phytopathogens and herbivores induce plant defences. Whereas there is evidence that some pathogens suppress these defences by interfering with signalling pathways involved in the defence, such evidence is scarce for herbivores. We found that the invasive spider mite Tetranychus evansi suppresses the induction of the salicylic acid and jasmonic acid signalling routes involved in induced plant defences in tomato. This was reflected in the levels of inducible defence compounds, such as proteinase inhibitors, which in mite-infested plants were reduced to even lower levels than the constitutive levels in herbivore-free plants. Additionally, the spider mite suppressed the release of inducible volatiles, which are implicated in plant defence. Consequently, the mites performed much better on previously attacked plants than on non-attacked plants. These findings provide a new perspective on plant–herbivore interactions, plant protection and plant resistance to invasive species.

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.

Phage display is a powerful tool with which to adapt the specificity of protease inhibitors. To this end, a library of variants of the potato protease inhibitor PI2 was introduced in a canonical phagemid vector. Although PI2 is a natural trypsin inhibitor, we were unable to select trypsin-binding variants from the library. Instead, only mutants carrying deletions or amber stop codons were found. Bacteria carrying these mutations had a much faster growth rate than those carrying the wt PI2-encoding gene, even when the promoter was repressed. To overcome these problems, two new phagemid vectors for g3-mediated phage display were constructed. The first vector has a lower plasmid copy number, as compared to the canonical vector. Bacteria harboring this new vector are much less affected by the presence of the PI2-g3 fusion gene, which appears from a markedly reduced growth retardation. A second vector was equipped with the promoter of the Escherichia coli psp operon, instead of the lac promoter, to control the PI2-g3 gene fusion expression. The psp promoter is induced upon helper phage infection. A phagemid vector with this promoter controlling a PI2-g3 gene fusion did not affect the viability of the host. Furthermore, both new vectors were shown to produce phage particles that display the inhibitor protein and were therefore considered suitable for phage display. The inhibitor library was introduced in both new vectors. Trypsin-binding phages with inhibitory sequences were selected, instead of sequences with stop codons or deletions. This demonstrates the usefulness of these new vectors for phage display of proteins that affect the viability of E. coli.

The proteinase inhibitor set in skeletal muscle is poorly characterized at present. This study was aimed to investigate in mouse skeletal muscle 1) the tissue-associated counterpart, if any, of serum protease inhibitors (which may also play antiproteolytic functions in tissues) and 2) calpastatin, a tissue inhibitor of calcium-activated neutral proteases (calpains). Triton-extracts were prepared from muscle homogenates of mice, which had been perfused extensively with phosphate buffered saline (PBS) (under deep anesthesia) to remove blood inhibitors. Among various inhibitors tested, the following muscle-associated inhibitors were identified by western-blotting: alpha-2-macroglobulin (185, 165, 35 kDa), alpha-1-antitrypsin (52 kDa), inter-alpha-trypsin inhibitor (220, 180 kDa) and calpastatin (70 kDa). Combined light microscope and confocal immunohistochemical experiments revealed that, in all muscles examined (soleus, plantaris, extensor digitorum longus) the above specific immunoreactivities were localized outside the muscle fibers (in periendomysium, blood vessel wall) as well as within them. Inter-alpha-trypsin inhibitor, however, completely lacked the intracellular localization. This wide distribution of proteinase inhibitors suggests that numerous muscular structures may be normally protected from unwanted proteolysis, thus providing an essential background for further studies on pathological models with altered proteolysis (m. dystrophy, denervation atrophy, etc.).

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.

Background Proteolytic degradation of the extracellular matrix (ECM) is a key event in tumour metastasis and invasion. Matrix metalloproteinases (MMPs) are a family of endopeptidases that degrade most of the components of the ECM. Several broad-spectrum MMP inhibitors (MMPIs) have been developed, but have had little success due to side effects. Thus, it is important to develop mathematical methods to provide new drug treatment strategies. Matrix metalloproteinase 2 (MMP2) activation occurs via a mechanism involving complex formation that consists of membrane type 1 MMP (MT1-MMP), tissue inhibitor of matrix metalloproteinase 2 (TIMP2) and MMP2. Here, we focus on developing a method for analysing the complex formation process. Results We used control analysis to investigate inhibitor responses in complex formation processes. The essence of the analysis is to define the response coefficient which measures the inhibitory efficiency, a small fractional change of concentration of a targeting molecule in response to a small fractional change of concentration of an inhibitor. First, by using the response coefficient, we investigated models for general classes of complex formation processes: chain reaction systems composed of ordered steps, and chain reaction systems and site-binding reaction systems composed of unordered multi-branched steps. By analysing the ordered step models, we showed that parameter-independent inequalities between the response coefficients held. For the unordered multi-branched step models, we showed that independence of the response coefficients with respect to equilibrium constants held. As an application of our analysis, we discuss a mathematical model for the MMP2 activation process. By putting the experimentally derived parameter values into the model, we were able to conclude that the TIMP2 and MMP2 interaction is the most efficient interaction to consider in selecting inhibitors. Conclusions Our result identifies a new drug target in the process of the MMP2 activation. Thus, our analysis will provide new insight into the design of more efficient drug strategies for cancer treatment.