Biotechnology and Molecular Biology Review Vol. 2 (3), pp. 068-085, August 2007 Available online at http://www.academicjournals.org/BMBR ISSN 1538-2273 © 2007 Academic Journals Standard Review Plant protease inhibitors: a defense strategy in plants Huma Habib and Khalid Majid Fazili* Department of Biotechnology, The University of Kashmir, P/O Naseembagh, Hazratbal, Srinagar -190006, Jammu and Kashmir, India. Accepted 7 July, 2007 Proteases, though essentially indispensable to the maintenance and survival of their host organisms, can be potentially damaging when overexpressed or present in higher concentrations, and their activities need to be correctly regulated. An important means of regulation involves modulation of their activities through interaction with substances, mostly proteins, called protease inhibitors. Some insects and many of the phytopathogenic microorganisms secrete extracellular enzymes and, in particular, enzymes causing proteolytic digestion of proteins, which play important roles in pathogenesis. Plants, however, have also developed mechanisms to fight these pathogenic organisms. One important line of defense that plants have to fight these pathogens is through various inhibitors that act against these proteolytic enzymes. These inhibitors are thus active in endogenous as well as exogenous defense systems. Protease inhibitors active against different mechanistic classes of proteases have been classified into different families on the basis of significant sequence similarities and structural relationships. Specific protease inhibitors are currently being overexpressed in certain transgenic plants to protect them against invaders. The current knowledge about plant protease inhibitors, their structure and their role in plant defense is briefly reviewed. Key words: Proteases, enzymes, protease inhibitors, serpins, cystatins, pathogens, defense. Table of content 1. Introduction 2. Families of PPIs 2.1 Serpin (Serine PI) family 2.2 Bowman Birk inhibitors (BBIs) family 2.3 Kunitz family 2.4 Squash inhibitors 2.5 Cereal trypsin/-amylase inhibitors 2.6 Mustard (Sinapis) trypsin inhibitor (MSI) 2.7 Potato type I PIs (PI 1) 2.8 Potato type II PIs (PI 2) 2.9 Cysteine PIs (CYS), the cystatin superfamily 2.10 Aspartyl and metallocarboxypeptidase inhibitors 3. Plant protection 4. Transgenic plants 5. Resistance to inhibitors 6. Concluding remarks 7. Acknowledgement 8. References INTRODUCTION Proteolytic enzymes, also called proteases, are the enzy- mes that catalyse the hydrolytic cleavage of specific peptide bonds in their target proteins. These enzymes *Corresponding author. E-mail: [email protected] or are widely distributed in nearly all plants, animals and [email protected]. Phone: 009419003881; +91-194- microorganisms (Joanitti et al., 2006; Neurath, 1989; 2102154; Fax: +91-194-2428723. Valueva and Mosolov, 2004; Christeller, 2005; Haq et al., Abreviations: BBI, Bowman-Birk inhibitor; BTI, Barley trypsin 2004; Supuran et al., 2002; Mosolov and Valueva, 2005; inhibitor; CPI, Cysteine protease inhibitor; CpTi, Cowpea trypsin Mosolov et al., 2001; Lawrence and Koundal, 2002; inhibitor; Cmps, Cucurbita maxima phloem serpin; MSI, Mustard Ryan, 1990). In higher organisms, nearly 2% of the trypsin inhibitor; PI, Protease inhibitor; PPI, Plant PI; PI1, Potato genes code for these enzymes (Barrett et al., 2001). type1PI; PI2, Potato type2 PI; PVy, Potato virus Y; SFTI, Being essentially indispensable to the maintenance and Sunflower trypsin inhibitor; TEV, Tobaco etch virus. Habeeb and Khalid 069 Figure 1A. A diagrammatic model for control of enzyme activity at the level of synthesis. Enzymes may be synthesized as inactive preproteins and are processed to be activated. E represents enzyme, S represents substrate and P represents the products. Figure 1B. Model showing enzymes activity regulation by virtue of substrate specificity. E,S and P have the same meaning as in Figure 1A. survival of their host organism, proteases play key roles their cognate enzymes, and are called protease inhibitors in many biological processes. The proteolytic events (PIs). Trypsin can be considered as a prototype of the catalysed by these enzymes serve as mediators of signal class of enzymes synthesized as inactive precursors. initiation, transmission and termination in many of the Synthesised as trypsinogen, it requires proteolytic pro- cellular events such as inflammation, apoptosis, blood cessing to be activated. Once activated trysin acts clotting and hormone processing pathways (Ivanov et al., specifically only on peptide bonds whose carboxyl func- 2006). Despite the fact that these enzymes are indispen- tions are contributed by lysine or arginine residues. sable to the cells and organisms that host them, they may Further check on the activity of trypsin is due to its be potentially damaging when overexpressed or present interaction with antitrypsin, the protein inhibitor of the in higher concentrations. For this reason the activities of activated form (Laskowski and Qasim, 1999). these enzymes need to be strictly regulated and PIs are of very common occurrence. They have been controlled (Rawlings et al. 2004a). The synthesis of these isolated and characterized from a large number of enzymes as inactive pre-proteins (Figure 1A), and their organisms, including plants, animals and microorganisms substrate specificity (Figure 1B) keeps a control on their (Valueva and Mosolov, 2004; Christeller, 2005; Haq et activities, but it does not fulfill the desired level of al., 2004; Supuran et al., 2002; Mosolov and Valueva, regulation, and the fact remains, that cells and organisms 2005; Mosolov et al., 2001). Naturally occurring PIs are require additional means of control. One important control essential for regulating the activity of their corresponding mechanism involves interaction of the active enzymes proteases and play key regulatory roles in many biolo- with proteins that inhibit their activities (Figure 2). These gical processes. For a few PIs, functions other than inhibitors form less active or fully inactive complexes with blocking protease action have also been found, such as 070 Biotechnol. Mol. Biol. Rev. Families of PIs PIs have been grouped into families and subfamilies and into different clans on the basis of sequence relationship and the relationship of protein folds of the inhibitory domains or units. An inhibitor domain is defined as the segment of the amino acid sequence containing a single reactive site after removal of any parts that are not directly involved in the inhibitor activity. On the basis of sequence homologies of their inhibitor domains, PIs have been classified into 48 families (Rawlings et al., 2004b). Proteins containing a single inhibitor unit are termed simple inhibitors, and those that contain multiple inhibitor units are termed complex inhibitors. A total of 11 families belong to the latter category and contain between 2 and 15 inhibitory domains. Most of these are homotypic, containing inhibitor units from a single family, some are however heterotypic, and contain inhibitor units from different families (Richardson et al., 2001; Trexler et al., Figure 2. Diagrammatic representation of the different modes 2001, 2002). On the basis of tertiary structure, 31 of the of protease inhibition (A) Direct blockage of the active center 48 families have been assigned to 26 clans, indicating canonical inhibition of trysin like proteases (B) Indirect that a large proportion of families show no relationships in blockage of the active center (C) Adjacent or and exocite their three dimensional structures. binding (D) Allosteric interaction. Peptidases have been classified on the basis of structural and evolutionary relationships into different families and clans (Rawlings and Barrett, 1993). These growth factor activities, receptor clearance signaling or families have been divided into five groups based on involvement in carcinogenesis (Qi et al., 2005). A number chemistry of the catalytic site of enzymes (Barrett, 1986, of inherited diseases such as emphysema, and epilepsy 1994; Grudkowska and Zagdanska, 2004). The families result from abnormalities in PIs (Lomas et al., 2002; of PIs could not, however, be grouped on the basis of the Ritchie, 2003; Lehesjoki, 2003; Bitoun et al., 2002) catalytic type of enzymes inhibited, since a number of PIs are of common occurrence in the plant kingdom. families contain cross-class inhibitors (Rawlings et al., Plant PIs (PPIs) are generally small proteins that have 2004b). The proteins in family 13 Kunitz-type PPIs mainly been described as occurring in storage tissues, generally inhibit serine peptidases, but they also include such as tubers and seeds, but they have also been found inhibitors of cysteine and aspartate proteases (Heibges et in the aerial parts of plants (De Leo et al., 2002). They al., 2003). Family 14, the serpin family, mostly contain are also induced in plants in response to injury or attack inhibitors of serine proteases, but it also contains inhibi- by insects or pathogens (Ryan, 1990). In plants, these tors of some cysteine proteases. In the past, however, PIs act as anti-metabolic proteins, which interfere with the PIs have been classified into serine, cysteine, aspartate digestive process of insects. One of the important and metallocarboxy PIs. (De Leo et al., 2002; Laskowski defense strategies that are found in plants to combat et al., 2003; Koiwa et al., 1997). PIs that are active predators involves PIs which are in particular effective against