Volume 6 Special Issue 1 2012
Protease Inhibitors

ISBN 978-4-903313-94-8

Guest Editor

Raj Deepika Chauhan

Donald Danforth Plant Science Center, ILTAB, Missouri, USA

CONTENTS AND ABSTRACTS

Tzi Bun Ng, Randy Chi Fai Cheung, Jack Ho Wong, Xiu Juan Ye (China) Protease Inhibitors of Botanical Origin (pp 1-10)

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ABSTRACT

Invited Review: The intent of this article is to review recent literature on plant protease inhibitors which can be divided into different types comprising Kunitz, Bowman-Birk, squash and potato inhibitor types. Some of them specifically inhibit trypsin. Others also inhibit chymotrypsin and other proteases. The mode of inhibition can be competitive or noncompetitive. Some of them are characterized by remarkable thermostability and pH stability. In addition to protease inhibitory activity, antifungal, anti-insect, anticancer and /or HIV-1 reverse transcriptase inhibitory activity are present in some plant protease inhibitors.

Tzi Bun Ng, Randy Chi Fai Cheung, Jack Ho Wong, Xiu Juan Ye (China) Isolation and Characterization of Protease Inhibitors from Animal Tissues (pp 11-16)

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ABSTRACT

Invited Mini-Review: The intent of this article is to review protease inhibitors produced from a diversity of animals including cnidarians, annelids, insects, crustaceans, mollusks, fish, amphibians, reptiles and mammals. They can be isolated from various tissues including pancreas, ova, liver, seminal plasma, serum, venom, skin secretion, hemolymph and salivary glands. Animal protease inhibitors display a variety of molecular masses ranging from several to over 50 kilodaltons. Some of them possess different protease specificity and inhibitory constants toward proteases. They play a role in various physiological processes such as reproduction, protection from viral infection, regulation of endogenous and exogenous (pathogen) proteases and anticoagulation activity.

Vishnu Menon, Mala Rao (India) Protease Inhibitors: Emphasizing Functional Aspects of Aspartic Protease Inhibitors (pp 17-30)

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Invited Review: Aspartic proteases are relatively a small group of proteolytic enzymes. Over the last decade, they have received tremendous research interest as potential targets for pharmaceutical intervention as many have been shown to play significant roles in physiological and pathological processes. Despite numerous efforts, however, the only inhibitors for aspartic proteases currently in the market are directed against the HIV protease of viral origin. Nevertheless, several inhibitors including those targeting renin-angiotensin system and β-secretase are in clinical or preclinical developments and few other aspartic proteases are discussed as potential drug targets. Currently the research strategies are focusing on the need for improved comprehension of protease-regulated cascades, along with precise selection of targets and improved inhibitor specificity. There is plethora of synthetic inhibitory compounds targeting aspartic proteases; however there are few reports documented in literature on biologic inhibitors from microorganisms. Protease inhibitors (PIs) are widely distributed in the plant kingdom. One of the important defense strategies that are found in plants to combat predators involves PIs which are particularly effective against phytophagous insects and microorganisms. In plants, these PIs act as anti-metabolic proteins, which interfere with the digestive process of insects. The defensive capabilities of PIs rely on inhibition of proteases present in insect guts or secreted by microorganisms, causing a reduction in the availability of amino acids necessary for their growth and development. The present chapter is a comprehensive state-of-the-art review describing the aspartic protease inhibitors from microbial and plant origin. In addition, the chapter highlights the therapeutic perspectives of aspartic protease inhibitors and biocontrol aspects of protease inhibitors with special emphasis on aspartic protease inhibitors.

Anussorn Wisessing, Kiattawee Choowongkomon (Thailand) Amylase Inhibitors in Plants: Structures, Functions and Applications (pp 31-41)

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ABSTRACT

Invited Review: The a-amylase inhibitors have been purified and characterized from several plant species. Six classes of a-amylase inhibitors have been classified by their structures. They play important roles in the control of endogenous amylases as well as the protection against pathogens and pests. The purification methods, classification, function, inhibition mechanism together with their applications in agricultural, clinical and industrial processes have been discussed in this review.

Vaijayanti A. Tamhane, Manasi Mishra, Neha S. Mahajan, Vidya S. Gupta, Ashok P. Giri (India) Plant Pin-II Family Proteinase Inhibitors: Structural and Functional Diversity (pp 42-58)

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Invited Review: Potato inhibitor II (Pin-II) proteins are plant serine proteinase inhibitors (PIs) that occur in various plant species. Pin-II PIs are characterized by inhibitory repeat domains (IRDs) which form their functional units and show extensive sequence variation. Various studies have been conducted to gauge the occurrence (spatial and temporal) of Pin-II PIs and their appearance in response to biotic stresses mainly herbivore attack. Many hypotheses have been proposed to justify the mechanism which has led to the evolution of Pin-II PIs as well as the selection pressure in force. In spite of ample diversity, these molecules are highlighted by their conserved features with respect to their gene and protein sequence and structure. The structural studies highlight the crucial role of conserved residues in stabilizing the reactive loops and overall the three-dimensional conformation of Pin-II PIs. The remarkable flexibility of reactive loops allows their binding to a wide range of proteinases (either endogenous? or from the pest). Apart from defense, Pin-II PIs have also been speculated to have a significant role in endogenous functions, namely regulation of proteolysis, macromolecular trafficking, programmed cell death and consequently aid the plant growth and development in respective tissue. There have been attempts to test these candidates for their potential in insect control. In vitro experiments and insect bioassays at laboratory scale have given encouraging results and led to the field experiments in order to develop transgenic plants fortified with Pin-II PIs. This has also helped judging the fitness costs that usually transgenic palnts have to pay in return of incorporating the foreign trait.

Tantravahi Srinivasan, P. B. Kirti (India) Protease Inhibitors and Stress Tolerance (pp 59-66)

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Invited Review: Plant protease inhibitors (PIs) are extensively studied for their role in defense against pests and pathogens due to their ability to inhibit specific proteases of the intruder. All classes of PIs were found to play important roles in innate host defense mechanisms and are used to develop transgenic plants resistant to pests and pathogens. Recent reports suggest that they have a role in modulating abiotic stress tolerance also. The induction of PIs was observed in response to not only biotic but also abiotic cues suggesting their possible role in abiotic stress responses and in modulating tolerance to various stresses. During biotic stress, pathogen invasion or damage due to herbivory, induces SA/JA- mediated signalling pathways to combat the attack. Abiotic stress induces injury and different conditions like osmotic stress, oxidative stress etc. as long as the causative factor exists in the milieu. Unlike in the biotic stresses, the causative agent in abiotic stress cannot be countered by the plant. Hence, the abiotic stress has to be tolerated by the plant nullifying all its related damages mostly by adjustment through ABA-dependent, and sometimes through an independent pathway. The overexpression of PIs has been shown to enhance abiotic stress tolerance in the transgenic plants suggesting that their role is multidimensional. In this review, we focus on the possible role of PIs in plant growth, and biotic and abiotic stress tolerance.

Yaxi Liu (China), Jirui Wang (China/USA) Molecular Evolution of Exogenous Alpha-amylase Inhibitors in Triticeae - An Update (pp 67-74)

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ABSTRACT

Invited Review: Exogenous α-amylase inhibitors in seeds and vegetative organs are attractive candidates for the control of seed weevils as these insects are highly dependent on starch as an energy source. In cereal seeds, α-amylase inhibitors proteins were known as one great family on the basis of the homology of their sequences. The overall information on the monomeric, dimeric and tetrameric α-amylase inhibitors strongly support the view that these inhibitors have evolved from a common ancestor gene through duplication and mutation. All α-amylase inhibitors from Triticeae (21 different genomes) have 10 conserved cysteine (5 disulfide bonds) sharing common cysteine skeleton. In the past years the α-amylase inhibitors have been well studied, including large amount of attention that directed towards their evolutionary relationship. Adaptive evolution of monomeric and dimeric α-amylase inhibitor genes and its ecological association has been investigated in wild emmer wheat. They were contributed by both natural selection and co-evolution, which ensures the conserved function as well as the inhibition of a variety of insect amylases. The known aspects of α-amylase inhibitors and their molecular evolution in Triticeae have been discussed in this review.

Amarjit K. Nath, Kanika Chadha, Pratima Sharma (India) Purification and Characterization of a Novel Inhibitor from Poinciana pulcherrima Seeds with Activity towards Pest Digestive Enzymes (pp 75-81)

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Original Research Paper: Six tree legumes were analysed for trypsin inhibitor (TI) activity and inhibition of trypsin enzyme extracted from the Pieris brassicae larvae midgut. TI extracted from Poinciana pulcherrima seeds contained maximum TI activity (trypsin units inhibited (TUI) = 17.24) and it also inhibited gut TI of P. brassicae larvaeto the maximum extent (TUI = 16.61); minimum inhibition of gut TI was observed in seeds of Poinciana regia (TUI = 2.46). The inhibitor was purified to 50.98-fold with 66.46% recovery by ammonium sulphate precipitation and gel filtration chromatography through a Sephadex G-100 column: a single band was observed in native-PAGE. The molecular weight of the purified protein was 17,782 Da as determined by SDS-PAGE and it was a monomer. The purified TI was heat stable over a wide range of temperatures (20-60°C) and had a pH optimum of 7.5. The purified TI hence appears to be of the Bowman-Birk type that lost its activity when treated with different concentrations of 2-mercaptoethanol, indicating the role of disulphide linkages in maintaining its three-dimensional structure. The inhibition was non-competitive and had a K_i value of 0.6 μM, indicating the high affinity of the inhibitor towards its substrate, BApNA. The purified inhibitor suppressed the growth of P. brassicae larvae, judged solely on the basis of the reduction in size and on the weight of excreta. The inhibitor also inhibited trypsin enzyme extracted from the mid-gut of Spodoptera littoralis larvae (36.3 TUI/30 μg of purified inhibitor). The plant genes expressing such TIs can be isolated, cloned and introduced in vegetable crops, thereby conferring resistance and minimizing the devastating crop yield losses caused by various insect pests and pathogens.

Marri Swathi, Soundappan S. Mohanraj, Chilka Srinivas, Elaprolu R. Prasad, Vantaku Venkatrao, Nalini Mallikarjuna, Aparna Dutta-Gupta, Kollipara Padmasree (India) Cajanus cajan (cv. C11): An Important Non-Host Resource for Proteinase Inhibitors Active against Larval Midgut Proteinases of Lepidopteran Insect Pest Achaea janata (pp 82-85)

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Original Research Paper: Our previous studies indicated that seeds of various cultivars and wild relatives of pigeonpea possessed proteinase inhibitors (PIs) which were active against midgut trypsin-like proteinases of lepidopteran insects. In the present study a detailed analysis of PIs distribution in mature seeds of five different cultivars of Cajanus cajan and thirteen different accessions of Cajanus platycarpus was carried out. Further the inhibitory potential of these PIs against larval midgut trypsin-like proteinases of Achaea janata was also evaluated. Among the chosen cultivars and C. platycarpus accessions, the cultivar ‘ICP 7118’ also called as ‘C11’ showed highest inhibitory potential against A. janata larval midgut trypsin-like proteinases (AJPs). The inhibitory potential of PIs from ‘C11’ was 25 and 106 fold higher against AJPs, compared to bovine pancreatic trypsin and chymotrypsin, respectively. Further, the activity profile of PIs of ‘C11’ on gelatin-polyacrylamide gel demonstrated the presence of higher number of trypsin inhibitor isoforms as well as stronger inhibitory potential against AJPs, when compared with C. platycarpus accessions. Thus, the results from the present investigation confirmed that the cultivar ‘C11’ of C. cajan is the best non-host resource for PIs active against AJPs and these PIs can be readily exploited further as ecofriendly pesticide in the management of this non-host lepidopteran insect pest A. janata using transgenic technology.

Huma Habib, Mohammad Afzal Zargar, Khalid Majid Fazili (India) Cloning and Expression of Trypsin Inhibitor Gene Ti from Pea (Pisum sativum L.) cv. ‘Arkel’ in Escherichia coli DH5α Cells (pp 86-90)

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Original Research Paper: Protease inhibitors (PIs) play key regulatory roles in many biological processes. The single gene advantage associated with these inhibitors make them ideal candidates for gene transfer to produce pest resistant recombinant plants. The aqueous extracts from pea (Pisum sativum) displayed prominent trypsin inhibitory activity. For cloning of trypsin inhibitor gene (ti) into bacterial hosts, cDNA was first prepared from the RNA isolated from pea seedlings. Amplification of the Ti gene was carried out using two sets of primers, the 5′-primer contained EcoRI restriction sequence, and the 3′- primer contained HindIII restriction sites. The purified amplicons were cloned into pet 27b+ expression vector using EcoRI and HindIII restriction enzymes. The constructed vectors were transformed into Escherichia coli DH5α cells. The recombinant cells were grown in LB medium containing kanamycin and treated with IPTG to induce the expression of the cloned Ti gene. The expression profile of the cells revealed highly intense 12.6 kDa band in the induced samples. The gel print technique and Dot blot assay revealed that the protein showed significant inhibitory activity towards trypsin and enzyme assay with synthetic substrates showed that the protein caused 90 ± 3% inhibition of trypsin.

Taqi Ahmed Khan, Mohd Mazid (India), Jaime A. Teixeira da Silva (Japan), Firoz Mohammad (India), Mohd Nasir Khan (Kingdom of Saudi Arabia) Role of NO-Mediated H₂O₂ Signaling under Abiotic Stress (Heavy Metal)-Induced Oxidative Stress in Plants: An Overview (pp 91-107)

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Review: Environmental stress, like heavy metal, constitutes the most significant factor leading to a substantial and unpredictable decrease in crop yield in agriculture. Hydrogen peroxide (H₂O₂) and nitrogen oxide (NO) are produced as primary signals in a stress signal cascade. H₂O₂ accumulation can lead to either enhanced expression of antioxidant enzymes or increased expression of other defense proteins. Alternatively, it can initiate programmed cell death, particularly when NO is also produced, depending on the intensity of the oxidative signal or oxidative load exerted by heavy metal toxicity in tissues. In this review, we examine the regulatory role of H₂O₂ and NO signaling in oxidative stress induced by heavy metals in plants, exemplified by a number of research studies. Finally, H₂O₂ and NO signaling in response to heavy metals may provide some clues to improving crop productivity.