Plant Protease Inhibitors Research Articles

Action of plant proteinase inhibitors on enzymes of physiopathological importance

Obtained from leguminous seeds, various plant proteins inhibit animal proteinases, including human, and can be considered for the development of compounds with biological activity. Inhibitors from the Bowman-Birk and plant Kunitz-type family have been characterized by proteinase specificity, primary structure and reactive site. Our group mostly studies the genus Bauhinia, mainly the species bauhinioides, rufa, ungulata and variegata. In some species, more than one inhibitor was characterized, exhibiting different properties. Although proteins from this group share high structural similarity, they present differences in proteinase inhibition, explored in studies using diverse biological models.

Regulation of seed germination and seedling growth by an Arabidopsis phytocystatin isoform, AtCYS6

Phytocystatins are cysteine proteinase inhibitors in plants that are implicated in the endogenous regulation of protein turnover and defense mechanisms against insects and pathogens. A cDNA encoding a phytocystatin called AtCYS6 (Arabidopsis thaliana phytocystatin6) has been isolated. We show that AtCYS6 is highly expressed in dry seeds and seedlings and that it also accumulates in flowers. The persistence of AtCYS6 protein expression in seedlings was promoted by abscisic acid (ABA), a seed germination and post-germination inhibitory phytohormone. This finding was made in transgenic plants bearing an AtCYS6 promoter–β-glucuronidase (GUS) reporter construct, where we found that expression from the AtCYS6 promoter persisted after ABA treatment but was reduced under control conditions and by gibberellin4+7 (GA4+7) treatment during the germination and post-germinative periods. In addition, constitutive over-expression of AtCYS6 retarded germination and seedling growth, whereas these were enhanced in an AtCYS6 knock-out mutant (cys6-2). Additionally, cysteine proteinase activities stored in seeds were inhibited by AtCYS6 in transgenic Arabidopsis. From these data, we propose that AtCYS6 expression is enhanced by the germination inhibitory phytohormone ABA and that it participates in the control of germination rate and seedling growth by inhibiting the activity of stored cysteine proteinases.

Drosomycin, an Innate Immunity Peptide of Drosophila melanogaster, Interacts with the Fly Voltage-gated Sodium Channel

Several peptide families, including insect antimicrobial peptides, plant protease inhibitors, and ion channel gating modifiers, as well as blockers from scorpions, bear a common CSalphabeta scaffold. The high structural similarity between two peptides containing this scaffold, drosomycin and a truncated scorpion beta-toxin, has prompted us to examine and compare their biological effects. Drosomycin is the most expressed antimicrobial peptide in Drosophila melanogaster immune response. A truncated scorpion beta-toxin is capable of binding and inducing conformational alteration of voltage-gated sodium channels. Here, we show that both peptides (i) exhibit anti-fungal activity at micromolar concentrations; (ii) enhance allosterically at nanomolar concentration the activity of LqhalphaIT, a scorpion alpha toxin that modulates the inactivation of the D. melanogaster voltage-gated sodium channel (DmNa(v)1); and (iii) inhibit the facilitating effect of the polyether brevetoxin-2 on DmNa(v)1 activation. Thus, the short CSalphabeta scaffold of drosomycin and the truncated scorpion toxin can maintain more than one bioactivity, and, in light of this new observation, we suggest that the biological role of peptides bearing this scaffold should be carefully examined. As for drosomycin, we discuss the intriguing possibility that it has additional functions in the fly, as implied by its tight interaction with DmNa(v)1.

Effects of Simulated Herbivory on Defensive Compounds in Forage Plants of Norwegian Alpine Rangelands

A field study on the effects of current grazing practices on plants in central Norway found no increase in either phenolic compounds or proteinase inhibitors in plants subjected to grazing by sheep. This could either reflect insufficient damage to the plants due to low grazing intensity or a lack of a long-term response of the plants to grazing. In this study, we tested the hypothesis that damage to forage plants used by sheep and rodents in Norwegian alpine rangelands can stimulate a long-term (at least 2-week) increase in levels of defensive compounds. We used clipping experiments to manipulate the severity and timing of damage to eight species of common plants used by herbivores in Norway. Under greenhouse conditions (i.e., climate-controlled), we subjected mature plants to one of four clipping treatments: control (0% leaf tissue removed), low (10-15% leaf tissue removed), high (70-75% leaf tissue removed), or sustained (15% of leaf tissue removed every other day up to a total removal of 75%, i.e., five clippings over 9 days). Samples were collected 2 weeks after final clipping and analyzed for concentrations of total phenolics, proteinase inhibitors, ratio of total phenolics to soluble proteins, and ratio of proteinase inhibitors to soluble plant proteins. As expected, the different species of plants responded differently to simulated herbivory, but most plants either showed no response to mechanical wounding and tissue loss or had reduced defensive compounds. Thus, our results do not support the hypothesis that herbivory induces a long-term increase in defensive compounds in alpine rangelands of Norway, a result consistent with those from field studies.

Phytocystatins: A comparative analysis of cysteine protease inhibitors in plants

Phytocystatins: A comparative analysis of cysteine protease inhibitors in plants

A dual fluorescent/MALDI chip platform for analyzing enzymatic activity and for protein profiling

Being able to rapidly and sensitively detect specific enzymatic products is important when screening biological samples for enzymatic activity. We present a simple method for assaying protease activity in the presence of protease inhibitors (PIs) by measuring tryptic peptide accumulation on copolymer pMALDI target chips using a dual fluorescence/MALDI-TOF-MS read-out. The small platform of the chip accommodates microliter amounts of sample and allows for rapid protein digestion. Fluorescamine labeling of tryptic peptides is used to indicate the proteolytic activity and is shown to be an affordable, simple process, yielding a strong fluorescence signal with a low background. Subsequent MALDI-TOF-MS analysis, performed in the same sample well, or in a parallel well without adding fluorescamine, detects the specific tryptic peptides and provides confidence in the assay. The dual read-out method was applied to screen the inhibition activity of plant PIs, components of plant defense against herbivores and pathogens. Extracts of PIs from Solanum nigrum and trypsin were applied together to a pMALDI chip on which a suitable substrate was adsorbed. The fluorescence and MALDI-TOF-MS signal decrease were associated with the inhibitory effect of the PIs on trypsin. The developed platform can be modified to screen novel protease inhibitors, namely, those potentially useful for treating or preventing infection by viruses, including HIV and hepatitis C.

Identification and characterization of protease inhibitors in Diplotaxis species

PCR analysis of the genomes of two wild Brassicaceae plants, Diplotaxis muralis and Diplotaxis tenuifolia, demonstrated the presence of several genes coding for potential protease inhibitors, classifiable within the mustard inhibitor family (MSI). This is a small family of plant protease inhibitors named after the mustard trypsin inhibitor MTI-2, the first protease inhibitor characterized in Brassicaceae. From identified sequences two recombinant inhibitors were expressed in Pichia pastoris. In comparison with MTI-2, they show a reduced activity against bovine trypsin. However, when tested against trypsin-like proteases present in the guts of Helicoverpa zea larvae, the Diplotaxis inhibitors and MTI-2 show similar activities, indicating that the usually adopted procedure of reporting activity of plant protease inhibitors against bovine trypsin may lead to wrong estimation of their effect on insect proteases. This issue is of particular relevance when planning the use of PI genes for developing insect resistant plants.

Proteomic Analysis Reveals Suppression of Bark Chitinases and Proteinase Inhibitors in Citrus Plants Affected by the Citrus Sudden Death Disease

Citrus sudden death (CSD) is a disease of unknown etiology that greatly affects sweet oranges grafted on Rangpur lime rootstock, the most important rootstock in Brazilian citriculture. We performed a proteomic analysis to generate information related to this plant pathogen interaction. Protein profiles from healthy, CSD-affected and CSD-tolerant stem barks, were generated using two-dimensional gel electrophoresis. The protein spots were well distributed over a pI range of 3.26 to 9.97 and a molecular weight (MW) range from 7.1 to 120 kDa. The patterns of expressed proteins on 2-DE gels made it possible to distinguish healthy barks from CSD-affected barks. Protein spots with MW around 30 kDa and pI values ranging from 4.5 to 5.2 were down-regulated in the CSD-affected root-stock bark. This set of protein spots was identified as chitinases. Another set of proteins, ranging in pI from 6.1 to 9.6 with an MW of about 20 kDa, were also suppressed in CSD-affected rootstock bark; these were identified as miraculin-like proteins, potential trypsin inhibitors. Down-regulation of chitinases and proteinase inhibitors in CSD-affected plants is relevant since chitinases are well-known pathogenesis-related protein, and their activity against plant pathogens is largely accepted.

Recent Findings on the Multifaceted Functionality of Enzyme Inhibition by Natural Compounds: A Review

Enzyme inhibition is a fundamental process to preserve the orderly sequence of events required for life, from seed germination to apoptosis. This review will verse on recent findings showing the various ways in which enzyme inhibition has been incorporated into the arsenal of many organisms, either as an effective defensive weapon or as a factor needed for the establishment of infection, parasitism and/or symbiotic associations, and how this diverse functionality can be exploited for therapeutic uses. It will describe several non-proteinaceous enzyme inhibitors isolated from natural sources or synthesized on the basis of structural or functional similarity to biosynthetic enzymes that have been employed for effective treatment against infections and/or aggresive diseases, such as cancer and AIDS, given their ability to disrupt critical functions of the target organisms or malignant cells. A relation of novel plant proteinase inhibitors (PPIs) with multiple and/or novel functions or engineered to have a highly selectivity on their target enzymes in order to avoid deleterious effects in a multi-trophic level that negatively affect insect pollinators or predators, is included too. This development could eliminate or reduce one of the most pressing concerns about the use of PIs as transgenes in many important crops. Finally, an account of the highly specific inhibition of enzymatic activity by biotechnological (e.g. gene silencing) or biochemical means, which have proven to be powerful tools to reveal novel functions for a variety of enzymes will be made in the context of defense, development, senescence and programmed cell death in plants.

Structural Refinement of Insecticidal Plant Proteinase Inhibitors from Nicotiana alata

Ornamental tobacco (Nicotiana alata) produces a series of 6 kDa proteinase inhibitors belonging to the potato type II inhibitor family. These proteins inhibit trypsin and chymotrypsin, the main digestive enzymes of predatory insects, thus leading to starvation, impaired larval development or death. In this context, the three-dimensional structures of these inhibitors are important for developing novel strategies for pest control. The solution structures of C1 and T1, the two main prototypes of the N. alata inhibitors, were originally determined more than a decade ago (J. Mol. Biol. 242, 231-243 (1994) and Biochemistry 34, 14304-14311 (1995)). Since then methods for NMR structure calculations have evolved considerably. Here we report the refinement of the structures of C1 and T1 with state-of-the-art protocols for NMR structure calculations. This refinement leads to an improved quality of the structures, making them a more reliable basis for the development of novel pesticides and modeling applications.

Chloroplast-like organelles were found in enucleate sieve elements of transgenic plants overexpressing a proteinase inhibitor.

SaPIN2a, a plant proteinase inhibitor from nightshade (Solanum americanum), was located to the enucleate sieve elements (SEs) of phloem. The expressed SaPIN2a in transgenic lettuce showed inhibition of plant endogenous trypsin- and chymotrypsin-like activities, suggesting that SaPIN2a can regulate proteolysis in plant cells. To further investigate the physiological role of SaPIN2a, we produced transgenic nightshade and lettuce plants overexpressing SaPIN2a from the cauliflower mosaic virus (CaMV) 35S promoter using Agrobacterium-mediated transformation. Overexpression of SaPIN2a in transgenic plants was demonstrated by northern blot and western blot analysis. SaPIN2a-overexpressing transgenic nightshade plants showed significantly lower height than wild-type plants. Transmission electron microscopy analysis showed that chloroplast-like organelles with thylakoids, which are not present in enucleate SEs of wild-type plants, were present in the enucleate SEs of SaPIN2a-overexpressing transgenic plants. This finding is discussed in terms of the possible role played by SaPIN2a in the regulation of proteolysis in SEs.

General up regulation of Spodoptera frugiperda trypsins and chymotrypsins allows its adaptation to soybean proteinase inhibitor

The existence of a diverse serine proteinase gene family in lepidopteran insects suggests they play a significant role in the insect adaptation to plant proteinase inhibitors. These proteinases have been shown to be involved in the process of proteolytic digestion in insect larvae. We carried out a selective transcriptome study of midguts from Spodoptera frugiperda larvae fed on a diet supplemented with soybean proteinase inhibitor (SPI). Using subtracted cDNA libraries made of gut-expressed transcripts, a total of 2100 partial sequences were obtained, of those 38% were related to digestive process. Two large and diverse groups of chymotrypsins and trypsins were obtained, and some of these proteinase-encoding genes were further characterized by quantitative RT-PCR. The transcription analyses revealed two groups: one group of genes constitutively expressed in the control larvae that is up regulated by introducing SPI to the diet, and a second group that is absent in the control but is induced by the SPI-rich diet. This observation suggests that adaptation of S. frugiperda to SPI involves de novo synthesis and also up regulation of existing enzymes. Proteases from intestines of larvae reared on a diet with SPI showed insensitivity to the inhibitor. The proteases were also insensitive to a broad-spectrum potato proteinase inhibitor preparation. We propose that adaptation of S. frugiperda to SPI follows a “shotgun” approach, based on a general up regulation of a large set of endoproteinases.

A potato carboxypeptidase inhibitor gene provides pathogen resistance in transgenic rice

A defensive role against insect attack has been traditionally attributed to plant protease inhibitors. Here, evidence is described of the potential of a plant protease inhibitor, the potato carboxypeptidase inhibitor (PCI), to provide resistance to fungal pathogens when expressed in rice as a heterologous protein. It is shown that rice plants constitutively expressing the pci gene exhibit resistance against the economically important pathogens Magnaporthe oryzae and Fusarium verticillioides. A M. oryzae carboxypeptidase was purified by affinity chromatography and further characterized by mass spectrometry. This fungal carboxypeptidase was found to be a novel carboxypeptidase B which was fully inhibited by PCI. Overall, the results indicate that PCI exerts its antifungal activity through the inhibition of this particular fungal carboxypeptidase B. Although pci confers protection against fungal pathogens in transgenic rice, a significant cost in insect resistance is observed. Thus, the weight gain of larvae of the specialist insect Chilo suppressalis (striped stem borer) and the polyphagous insect Spodoptera littoralis (Egyptian cotton worm) fed on pci rice is significantly larger than that of insects fed on wild-type plants. Homology-based modelling revealed structural similarities between the predicted structure of the M. oryzae carboxypeptidase B and the crystal structure of insect carboxypeptidases, indicating that PCI may function not only as an inhibitor of fungal carboxypeptidases, but also as an inhibitor of insect carboxypeptidases. The potential impact of the pci gene in terms of protection against fungal and insect diseases is discussed.

Resistance management in a native plant: nicotine prevents herbivores from compensating for plant protease inhibitors

Plants deploy chemical defenses in complex mixtures, which are thought to be adaptive, but experimental tests have used artificial diets rather than plants. Herbivore attack on Nicotiana attenuata rapidly increases the production and accumulation of trypsin proteinase inhibitors (TPI) and the toxic alkaloid nicotine. By transgenically silencing their respective biosynthetic genes, we were able to abolish TPI activity and reduce inducible nicotine by 85%. Nicotine production was not affected by silencing pi or vice versa, and transformation did not alter levels of other metabolites examined. Spodoptera exigua, a native generalist herbivore that can compensate for heterologous TPI expression, performed better on TPI- or nicotine-deficient plants compared with the wild-type. Because of a compensatory feeding response to TPI when nicotine is absent, larvae performed better on nicotine-deficient plants than they did on plants silenced in both defenses. The antifeedant toxin, nicotine, prevents this compensatory response. We conclude that N. attenuata counters an insect adaptation with a defensive synergism.

Suppression of the pathogen-inducible Medicago truncatula putative protease-inhibitor MtTi2 does not influence root infection by Aphanomyces euteiches but results in transcriptional changes from wildtype roots

During the parasitic interaction between the model legume Medicago truncatula and the oomycete Aphanomyces euteiches, a plant protease inhibitor (PI)-encoding gene, MtTi2, was found to be induced. Sequence and database analyses showed that MtTi2 belongs to a group of at least four PIs, which are all specifically expressed upon pathogen infection or elicitor treatment. Reporter analysis showed that the MtTi2 promoter is activated in infected root tissues and revealed a distinct promoter area involved in this transcriptional induction. In order to elucidate the function of MtTi2, RNA i-mediated silencing experiments were carried out. After introduction of double-stranded MtTi2 RNA into M. truncatula roots, no endogenous transcripts were observed, indicating efficient gene suppression. Since some plant protease inhibitors were shown to suppress pathogen growth, the MtTi2 i-phenotype was analyzed with respect to pathogen spreading after root infection. No altered pathogen development was observed in MtTi2i-roots as compared to control roots, indicating that MtTi2 does not directly influence the pathogen. In order to investigate whether suppression of MtTi2 results in transcriptional changes, transcriptome profiles of MtTi2 i roots and control roots were analyzed using an 8000 gene M. truncatula microarray. A cluster analysis of genes regulated in roots upon infection in two MtTi2 i-lines or two vector lines revealed groups of genes showing different regulation in MtTi2 i- and wild type roots. Hence, it is likely that MtTi2 has endogenous functions other than directly suppressing the pathogen. Furthermore, the transcriptome approach revealed insight into the transcriptional changes in response to A. euteiches infection.

Resisting degradation by human elastase: Commonality of design features shared by ‘canonical’ plant and bacterial macrocyclic protease inhibitor scaffolds

A previously unexplained difference in the resistance to enzymatic hydrolysis of 11-mer Bowman–Birk-type inhibitors of human leukocyte elastase that differ in P1 is found to correlate with the strength of a particular intramolecular hydrogen bond within the inhibitor. This transannular hydrogen bond stabilizes the side chain of the conserved P2 Thr in a ‘canonical’ +60°-rotamer χ 1 conformation and thereby directs it for a close interaction with the enzyme’s catalytic His. As the implications of this NMR analysis are neither limited to this macrocyclic scaffold derived from plant proteins nor to a particular serine protease, we present a unified analysis with inhibitory bacterial depsipeptides of 7–12 residues in length that share key design features for which we propose communal functional explanations.

Inhibition of Proteinase Inhibitor Transcripts by Leptinotarsa decemlineata Regurgitant in Solanum lycopersicum

One mechanism by which plants defend themselves against insect herbivores is the production of plant proteinase inhibitors, which can inhibit digestion in the midgut, thus affecting growth and survival. In this work, the effect of Colorado potato beetle (CPB) [Leptinotarsa decemlineata (Say)] regurgitant on Solanum lycopersicum defenses was investigated. When regurgitant from fourth-instar CPB was applied to wounded S. lycopersicum leaves, the wound-induced transcripts for the proteinase inhibitors pin1 and pin2 were reduced. Boiling the regurgitant abolished its ability to reduce the pin transcripts. Ultrafiltration of the regurgitant demonstrated that it contained a component between 10 and 30 kDa molecular weight that inhibited wound-induced pin1 and pin2 expression, suggesting that it may be a protein. This may represent a mechanism that the CPB has evolved to elude the plant's induced response to infestation.

Bioinsecticidal activity of Archidendron ellipticum trypsin inhibitor on growth and serine digestive enzymes during larval development of Spodoptera litura

The roles of serine proteases involved in the digestion mechanism of the cutworm Spodoptera litura (Lepidoptera: Noctuidae) were examined ( in vitro and in vivo) following feeding of plant protease inhibitors. A trypsin inhibitor from Archidendron ellipticum (AeTI) was purified by ammonium sulfate fractionation, ion-exchange chromatography and size-exclusion chromatography (HPLC) and its bioinsecticidal properties against S. litura were compared with Soybean Kunitz trypsin inhibitor (SBTI). AeTI inhibited the trypsin-like activities of the midgut proteases of fifth instar larvae of S. litura by over 70%. Dixon plot analysis revealed competitive inhibition of larval midgut trypsin and chymotrypsin by AeTI, with an inhibition constant ( K i) of 3.5 × 10 − 9 M and 1.5 × 10 − 9 M, respectively. However, inhibitor kinetics using double reciprocal plots for both trypsin and chymotrypsin inhibitions demonstrated a mixed inhibition pattern. Feeding experiments conducted on different (neonate to ultimate) instars suggested a dose-dependent decrease for both the larval body weight as well as % survival of larva fed on diet containing 50, 100 and 150 μM AeTI. Influence of AeTI on the larval gut physiology indicated a 7-fold decrease of trypsin-like protease activity and a 5-fold increase of chymotrypsin-like protease activity, after being fed with a diet supplemented with 150 μM AeTI. This study suggests that although the early (1st to 3rd) larval instars of S. litura are susceptible to the trypsin inhibitory action of AeTI, the later instars may facilitate the development of new serine proteases, insensitive to the inhibitor.

In vitro assay of plant protease inhibitors from four different sources on digestive proteases of rohu, Labeo rohita (Hamilton), fingerlings

The in vitro inhibitory effect of protease inhibitors from four seed extracts (soybean, grasspea, black gram and horse gram) on digestive proteases of rohu was assessed by enzyme inhibition assay and substrate sodium dodecyl sulphate-polyacrylamide gel electrophoresis. High proteolytic activity was detected in the intestinal extract of rohu (Labeo rohita) fingerlings at two different pH ranges (8–8.5 and 10–11). That protein digestion occurs mainly in the alkaline condition in this fish without a stomach is evident from very high trypsin activity (0.95±0.04 benzoyl-dl-arginine-p-nitroanilide U mg protein−1) in the intestine. In case of grass pea seed, more than 50% inhibition of alkaline protease activity was recorded when the ratio of inhibitor to enzyme was 9.41 μg U−1. More than 40% inhibition of protease activity was recorded in case of horse gram seed when the ratio of inhibitor to enzyme was 5.51 μg U−1. Black gram at 11.0 μg U−1 and soybean seed proteins at 62.75 μg U−1 resulted in 50% and more than 30% inhibition of digestive protease activity in rohu fingerlings respectively. A plot of the inhibition values obtained by changing the relative concentrations of enzyme/inhibitor resulted in different dose–response curves for different protein sources. The use of substrate gel electrophoresis allowed the visualization of the aforementioned differences in inhibition. Each seed extract produced a characteristic profile of protease inhibition. It is concluded that protease inhibitors present in plant protein sources adversely affect the digestive proteases in fish and hence there is a need to eliminate/reduce the amount of such inhibitors through proper processing before incorporation into aquafeeds.

Isolation and characterization of a novel mung bean protease inhibitor with antipathogenic and anti-proliferative activities

A novel protease inhibitor, designated mungoin, with both antifungal and antibacterial activities, and exhibiting a molecular mass of 10 kDa in SDS-polyacrylamide gel electrophoresis, was isolated from mung bean ( Phaseolus mungo) seeds. The isolation procedure involved a combination of extraction, ammonium sulfate precipitation, ion exchange chromatography on CM-Sephadex, and high-performance liquid chromatography (HPLC) on SP-Toyopearl. Its isoelectric point was estimated to be 9.8 by isoelectric focusing. Its N-terminal amino acid sequence was determined to be EMPGKPACLDTDDFCYKP, demonstrating some resemblance to the C-terminal sequences of other protease inhibitors and inhibitor precursors from leguminous plants. It exerted a potent inhibitory action toward a variety of fungal species including Physalospora piricola, Mycosphaerella arachidicola, Botrytis cinerea, Pythium aphanidermatum, Sclerotium rolfsii and Fusarium oxysporum, as well as an antibacterial action against Staphylococcus aureus. In addition, this novel plant protease inhibitor displayed anti-proliferative activity toward tumor cells.