Current Protein and Peptide Science - Volume 12, Issue 5, 2011

Plant protease inhibitors (PIs) continue to exert interest among plant biochemists and biotechnologists in spite of the (almost complete) failure in the production of transgenic crops with enhanced resistance toward insects. Indeed, plant PIs still offer an ample space for both applicative and basic researches. The huge reservoir provided by the Plant Kingdom, the development of investigative methodologies and biotechnologies and the possibility to apply plant PIs in different fields than crop protection, are at the basis of this ongoing interest. This special issue is aimed at providing readers with an update on the characterization of the numerous families of plant PIs, together with an overview of their possible applications. In addition it gives an interesting example of a co-evolutive adaptation, which allows plants and insects to co-exist for millions of years with mutual benefit. The first four articles introduce plant PIs. Katherine Bateman and Michael James [1] provide a detailed discussion about structural analysis of plant PIs and, when known, of their complexes with proteases. Authors give examples of the threedimensional structures available for almost all the families of PIs, allowing readers to better understand the basic mechanisms of inhibition. The review article by Maria Luiza Oliva et al. [2] reports about the structural and functional properties of one of the largest and most largely known family of plant PIs, the Kunitz family, in Leguminosae plants. Examples of studies on the pharmacological action of Kunitz inhibitors are also given. Alfonso Clemente et al. [3] besides reporting about the structure, inhibitory mechanism, diffusion and nutritional properties of the Bowman-Birk inhibitors in legumes, give a wide and critical review of the potential use of members of this PI family as anti-carginogenic and anti-inflammatory agents. David Turra and Mateo Lorito [4] deal with two PIs families, firstly detected in potato: potato type I (Pin1) and II (Pin2). Together with the structure, activity and protective role of enzymes belonging to these families, authors also outline their physiological role in plants and potential application in medicine. The other main families of plant PIs are described in the review by Volpicella et al. [5] allowing readers to appreciate the variety of inhibitors of which plants provided themselves. In addition the article illustrates examples of inhibitors of the cystatins and mustard inhibitor families, which have been largely modified by directed evolution techniques. A review is dedicated to illustrate the discovery, characteristics and functional role in the induction of plant PIs of systemin, the first plant peptide hormone. The pioneering work on this topic reported by Greg Pearce [6] revolutionized the field of peptide signaling in plants. In the subsequent years, additional systemin-like peptides and other signaling peptides were identified in plants, thus establishing the essential role of signaling peptides in many plant processes. The article by John Gatehouse [7] reports a critical evaluation of the reasons why engineering PIs genes in plants have reached a limited application for increasing resistance of crops toward insects. In addition it analyses the possibility that PIs, used in combination with other insecticidal proteins or thanks to their possible action on different physiological processes than digestion, could have chances to be further employed for plant protection. Two articles follow that focus on natural targets of plant PIs. In the article by Marco Gomes et al. [8] a detailed report about the four main families of plant proteases, together with their biochemical features, physiological role and the potential pharmacological activity is given. Marteen Jongsma and Jules Beeckwilder [9] highlight in their review the adaptive capacity of insects to plant PIs, achieved by the expression of proteases insensitive to inhibition. Authors focus on the structural effects that relatively few modifications in the primary structure of proteases can produce, resulting in lower affinities toward PIs. This successful (by the insect point of view) mechanism is regarded as an aspect of the co-evolution of insect proteases and plant PIs. The last article by Arianna Consiglio et al. [10] is dedicated to the description of a web resource for the retrieval of information available on more than 1,000 plant PIs. The PlantPIs database offers the possibility to obtain information on single PIs in an organized way, useful to either expert scientists or students. Together, the articles in this special issue provide an exciting overview of current research on plant PIs that hopefully will stimulate readers to devise the next generation of experiments in this area.....

This review outlines known examples of the three-dimensional structures of protein proteinase inhibitors from plants. Three families of enzymes, serine proteinases, carboxypeptidases and cysteine proteinases, are targeted by at least a dozen inhibitor families, with the majority of them adopting the standard mechanism of inhibition towards the serine proteinases. All of the inhibitors discussed maintain compact and stable inhibitory domains that bind to the active site of their target proteinases and prevent access to the substrate molecules. One interesting highlight is the knottin group. Three separate inhibitor families utilize the overall knottin fold in a different way. This fold can accommodate extensive sequence variation and for each of the squash, Mirabilis and Potato carboxypeptidase families, the proteinase-binding residues are found at a different location. Plants have also evolved additional strategies to regulate proteinase activity, such as linking inhibitory domains and targeting multiple enzymes at once. The structural aspects of these strategies are discussed in the review.

Seed proteins that inhibit proteinases are classified in families based on amino acid sequence similarity, nature of reactive site and mechanism of action, and are used as tools for investigating proteinases in physiological and pathological events. More recently, the plant Kunitz family of inhibitors with two disulphide bridges was enlarged with members containing variable number of cysteine residues, ranging from no cysteine at all to more than four residues. The characteristic of these proteins, as well the interactions with their target proteinases, are briefly discussed.

Bowman-Birk inhibitors and their variants (BBI) from legumes, such as soybean, pea, lentil and chickpea, are a class of naturally-occurring protease inhibitors which have potential health-promoting properties within the gastrointestinal tract. BBI can resist both acidic conditions and the action of proteolytic enzymes, and transit through the stomach and small intestine without major degradation, permitting significant amounts to reach the large intestine in active form to exert their reported anti-carcinogenic and anti-inflammatory properties. These potential pharmacological benefits have been linked recently to the intrinsic ability of BBI to inhibit serine proteases, and the data suggest that both trypsin- and chymotrypsin- like proteases involved in carcinogenesis should be considered as potential targets of BBI. However, the therapeutic targets and the action mechanisms of BBI remain unknown. Their elucidation will provide insights into the properties of these plant protease inhibitors as colorectal chemopreventive agents, providing a strong base for the development of legume crops and their products as pro-nutritional, health-promoting food. The deployment of modern genomic tools and genome sequence information are underpinning studies of natural and induced polymorphism in BBI. Genetic markers for BBI variants with improved properties can be exploited ultimately in legume breeding programmes to assist the introgression of such variant genes and the development of superior genotypes for human nutrition.

Serine protease inhibitors (PIs) are a large and complex group of plant proteins. Members of the Potato type I (Pin1) and II (Pin2) proteinase inhibitor families are among the first and most extensively characterized plant PIs. Many insects and phytopathogenic microorganisms use intracellular and extracellular serine proteases playing important roles in pathogenesis. Plants, however, are able to fight these pathogens through the activation of an intricate defence system that leads to the accumulation of various PIs, including Pin1 and Pin2. Several transgenic plants over-expressing members of the Pin1 and Pin2 families have been obtained in the last twenty years and their enhanced defensive capabilities demonstrated against insects, fungi and bacteria. Furthermore, plants genetically engineered with Pin1 and Pin2 showed altered regulation of different physiological processes (e.g., dehydratation response, programmed cell death, growth, trichome density and branching), supporting an endogenous role in various plant species in addition to the well established defensive one. This review summarizes the current knowledge about Pin1 and Pin2 structure, the role of these proteins in plant defence and physiology, and their potential exploitation in biotechnology.

Plant protease inhibitors (PIs) are generally small proteins present in high concentrations in storage tissues (tubers and seeds), and to a lower level in leaves. Even if most of them are active against serine and cysteine proteases, PIs active against aspartic proteases and carboxypeptidases have also been identified. Inhibitors of serine proteases are further classifiable in several families on the basis of their structural features. They comprise the families known as Bowman- Birk, Kunitz, Potato I and Potato II, which are the subject of review articles included in this special issue. In the present article we aim to give an overview of other families of plant PIs, active either against serine proteases or other class of proteases, describing their distribution, activity and main structural characteristics.

Systemin, an 18-amino acid signaling peptide isolated from tomato leaves, has been found to be an integral component of the jasmonic acid signaling pathway, leading to the synthesis of protease inhibitors (PIs). The discovery of systemin has led to a search for other peptide signals involved in defense in the Solanaceae and in other plant families. A new class of peptides having similar signaling properties but little sequence homology to systemin have been found and termed hydroxyproline-rich glycopeptide systemins (HypSys). These small (18-20 amino acids) glycopeptides, like systemin, are derived from larger precursor proteins (proHypSys) and until recently were thought to function only in protection from herbivore attack. However, HypSys glycopeptides isolated from petunia induced the defensin gene, known for its involvement in pathogen defense. More recently, a HypSys glycopeptide was isolated from sweet potato, a member of the Convolvulaceae family and found to induce the sporamin gene which codes for the major storage protein in tubers with trypsin inhibitor activity. These recent discoveries expand the function and range of the HypSys family of glycopeptides and establish these unique inducible signaling molecules as potential components of defense pathways throughout the Eudicots. Herein we review the signaling and structural properties of systemin and the HypSys glycopeptides and their roles in the induction of PIs.

Proteinase inhibitors which act on the digestive enzymes of insect herbivores are a basic mechanism of plant defence. Attempts to exploit this defence mechanism in plant genetic engineering have used over-expression of both endogenous and exogenous inhibitors. While significant protection against insect pests has been routinely achieved, the engineered plants do not show levels of resistance considered commercially viable. As a result of selective pressures, insect herbivores have developed multiple mechanisms of adaptation to overcome the defensive effects of plant proteinase inhibitors. Common polyphagous crop pests are well adapted to deal with a range of different inhibitors, which have only limited effects on fitness as a result. A range of strategies have been attempted to improve effectiveness of proteinase inhibitors as antimetabolites towards insects, including selection for inhibitory activity against insect digestive enzymes, mutagenesis for novel inhibitory activity, and engineering inhibitors with multiple functions. However, proteinase inhibitor genes have only been used in transgenic crops in combination with other insecticidal genes. In Chinese genetically engineered cotton varieties which express Bt toxins as an insecticidal protein against lepidopteran larvae, the CpTI (cowpea trypsin inhibitor) gene has been employed as a second transgene to improve protection. This gene combination represents the only commercial deployment of a proteinase inhibitor transgene to date, with Bt/CpTI cotton grown on over 0.5 million hectares in 2005. Future prospects for using proteinase inhibitor genes to enhance insect resistance in transgenic crops will require reassessment of their mechanisms of action, particularly in affecting processes other than digestion, as exemplified by effects on sap-feeding hemipteran pests.

Proteinases play a fundamental metabolic role during the life cycle in the plant kingdom. By interacting with endogenous or exogenous inhibitors, the proteolytic activity is modulated to meet metabolic requirements. By probing proteolytic enzymes with their inhibitors, it is possible to identify novel functions unrelated to their proteolytic activity. A group of plant proteolytic enzymes stands as a line of defence against environmental changes as their activation is triggered following various types of stress. On the other hand, plants also contain proteinase inhibitors as countermeasures for their protection against insects and pests. Both proteinases and inhibitors emerge as useful tools to combat human diseases. This review focuses on the biochemical characterization of plant proteinases, their inhibitors, the pharmacological potential of proteinases and inhibitors, and new putative emerging functions of proteolytically inhibited proteinases.

Plants are at the basis of the food chain, but there is no such thing as a “free lunch” for herbivores. To promote reproductive success, plants evolved multi-layered defensive tactics to avoid or discourage herbivory. To the detriment of plants, herbivores, in turn, evolved intricate strategies to find, eat, and successfully digest essential plant parts to raise their own offspring. In this battle the digestive tract is the arena determining final victory or defeat as measured by growth or starvation of the herbivore. Earlier, specific molecular opponents were identified as proteases and inhibitors: digestive proteases of herbivores evolved structural motifs to occlude plant protease inhibitors, or alternatively, the insects evolved proteases capable of specifically degrading the host plant inhibitors. In response plant inhibitors evolved hyper-variable and novel protein folds to remain active against potential herbivores. At the level of protease regulation in herbivorous insects, it was shown that inhibition-insensitive digestive proteases are up-regulated when sensitive proteases are inhibited. The way this regulation operates in mammals is known as negative feedback by gut-luminal factors, so-called ‘monitor peptides’ that are sensitive to the concentration of active enzymes. We propose that regulation of gut enzymes by endogenous luminal factors has been an open invitation to plants to “hijack” this regulation by evolving receptor antagonists, although yet these plant factors have not been identified. In future research the question of the co-evolution of insect proteases and plant inhibitors should, therefore, be better approached from a systems level keeping in mind that evolution is fundamentally opportunistic and that the plant's fitness is primarily improved by lowering the availability of essential amino acids to an herbivore by any available mechanism.

PlantPIs is a web querying system for a database collection of plant protease inhibitors data. Protease inhibitors in plants are naturally occurring proteins that inhibit the function of endogenous and exogenous proteases. In this paper the design and development of a web framework providing a clear and very flexible way of querying plant protease inhibitors data is reported. The web resource is based on a relational database, containing data of plants protease inhibitors publicly accessible, and a graphical user interface providing all the necessary browsing tools, including a data exporting function. PlantPIs contains information extracted principally from MEROPS database, filtered, annotated and compared with data stored in other protein and gene public databases, using both automated techniques and domain expert evaluations. The data are organized to allow a flexible and easy way to access stored information. The database is accessible at http://www.plantpis.ba.itb.cnr.it/.