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Multifunctional Role of Plant Cysteine Proteinases

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Multifunctional Role of Plant Cysteine Proteinases Vol. 51 No. 3/2004 609–624 QUARTERLY Review Multifunctional role of plant cysteine proteinases Małgorzata Grudkowska1 and Barbara Zagdańska1,2½ 1Plant Physiology and Biochemistry Department, Plant Breeding and Acclimatization Institute, Radzików, Warszawa; 2Biochemistry Department, Warsaw Agricultural University, Warszawa, Poland Received: 22 March, 2004; revised: 09 July, 2004; accepted: 11 July, 2004 Key words: cysteine proteinases, localisation, inhibitors, gene expression, cellular functions Cysteine proteinases also referred to as thiol proteases play an essential role in plant growth and development but also in senescence and programmed cell death, in accumulation of storage proteins such as in seeds, but also in storage protein mobili- zation. Thus, they participate in both anabolic and catabolic processes. In addition, they are involved in signalling pathways and in the response to biotic and abiotic stresses. In this review an attempt was undertaken to illustrate these multiple roles of cysteine proteinases and the mechanisms underlying their action. Proteolysis in plants is a complex process in- they rise to 90% of the total proteolytic activ- volving many enzymes and multifarious ity (Wiśniewski & Zagdańska, 2001). They are proteolytic pathways in various cellular com- involved in protein maturation, degradation, partments, with cysteine proteinases playing and protein rebuilt in response to different an essential role. Their share in total proteol- external stimuli and they also play a ysis depends on the kind of plant and its or- house-keeping function to remove abnormal, gan. It amounts up to 30% of total proteolytic misfolded proteins. In each case, the proteoly- activity in mature non-senescing organs. sis by cysteine proteinases is a highly regu- However, the activities of cysteine protein- lated process. The aim of this review is to il- ases respond dramatically to different inter- lustrate the progress in the determination of nal and external stimuli and in some cases the enzyme structure, localisation and gene ½ Corresponding author: Biochemistry Department, Warsaw Agricultural University, Nowoursynowska 159, 02-776 Warszawa, Poland; [email protected] Abbreviations: ER, endoplasmic reticulum; PCD, programmed cell death; SH-EP, sulfhydryl-endo- peptidase; VPE, vacuolar processing enzymes. 610 M. Grudkowska and B. Zagdańska 2004 expression and to highlight the great diver- caspases (family C14) and calpains, the cal- sity of cellular processes in which the cysteine cium-dependent proteinases (family C2), have enzymes are involved. recently been found in plants. Two additional families of cysteine proteinases: ubiquitin C-terminal hydrolases (family C12) and CLASSIFICATION OF CYSTEINE ubiquitin-specific proteinases (C19), the com- PROTEINASES ponents of the ubiquitin-proteasome-depend- ent pathway that catalyze deubiquitination of Peptide hydrolases (peptidase or protease) proteins, have also been detected in plants comprise two groups of enzymes: the (Vierstra, 2003). endopeptidases which act on the interior of peptide chains and the exopeptidases, which cleave peptide bonds on termini of peptide CHARACTERIZATION OF CYSTEINE chains (Barrett, 1994). Exopeptidases have PROTEINASES been differentiated according to their sub- strate specificity as aminopeptidases, acting The family of papain-type proteinases is the at a free N terminus, and carboxypeptidases, most thoroughly investigated among all the which degrade peptides at the C-terminus. cysteine proteinases. Papain is characterized Classification of endopeptidases (protein- by a two-domain structure with the active site ases) is based on the kind of active site resi- between the domains. These enzymes are syn- due (cysteine-, serine-, aspartic-, and metallo- thesized as inactive or less inactive precur- types), and not on the type of their substrate sors which comprise 38–250 amino-acid (Barrett, 1986). The reason for such a classifi- prosequence, an N-terminal signal sequence cation criterion is the often unknown nature and the 220–260 amino-acid mature enzyme. of the substrate. Cysteine proteinases (EC Activation takes place by limited intra- or 3.4.22), endopeptidyl hydrolases with a intermolecular proteolysis (Wiederanders, cysteine residue in their active center are usu- 2003). To date, the amino-acid sequences of ally identified basing on the effect of their ac- more than 50 papain-like and 15 legumain- tive site inhibitors (iodoacetate, iodoaceta- like plant proteinases have been established mide and E64) and activation of the enzymes (Fischer et al., 2000). by thiol compounds. Most cysteine protein- The Arabidopsis thaliana genome encodes ases show acidic pH optima. 32 papain-type (C1 family) cysteine protein- Rawlings & Barrett (1993), who based classi- ases which can be classified into eight main fication of proteinases on the structural and groups (senescence- and stress-induced, evolutionary relationship, have grouped the aleurain, cathepsin-b like, bromelain-like, enzymes into families and clans. A clan com- KDEL, telo sequences, actinidain-like) based prises a group of families of an evolutionary on the sequence similarity to other cysteine relationship. The classification proposed by proteinases (Simpson, 2001). Plant papain- those authors is continuously brought up to type enzymes (C1) are synthesized as small date (MEROPS peptidases database, preproproteins of 40–50 kDa prepropeptides http://merops.sanger.ac.uk). Cysteine pro- that undergo proteolytic processing of the pre teinases being labeled with the prefix C and and pro peptides to yield mature, fully active comprise more than 40 families of peptidases enzymes (22–35 kDa). The structural rela- grouped into at least six superfamilies or tionships between eukaryotic papain clans. Most plant cysteine proteinases belong proteinase proparts and their role in regula- to the papain (C1) and legumain (C13) fami- tion of enzyme activity, correct intracellular lies. The members of cysteine proteinases: targeting and folding of the mature enzyme Vol. 51 Multifunctional role of plant cysteine proteinases 611 has been described recently by Wiederanders with the classification of plant vacuoles into (2003). The prerequisite for catalysis by protein-storage and lytic vacuoles (Kinoshita papain are not only the Cys 25 and His 159 et al., 1995a; 1995b; 1999). An immuno- residues but also the Asn 175 residue impor- cytochemical analysis confirmed the specific tant for the proper orientation of the His side localization of bVPE in the protein storage chain (Beers et al., 2004). Besides, it has been vacuoles and gVPE in the lytic vacuoles. How- found that members of the papain group of ever, there are a few exceptions, e.g. barley proteinases preferentially cleave peptide grain legumain (nucellain), although found in bonds with Arg in P1 position (Fischer et al., the cell wall of the nucellus, is related to the 2000) or Phe at the P2 position (Menard & seed-specific enzymes (Linnestad et al., Storer, 1998). 1998). Calpains (C2) are cytoplasmic, calcium-de- Caspases, belonging to the C14 class of spe- pendent cysteine proteinases requiring mi- cific cysteine proteinases show a high speci- cro- or millimolar concentrations of Ca2+ for ficity with an absolute requirement for an activity, with a highly conserved molecular Asp residue adjacent to the cleavage site and structure in the catalytic site. To date, only a recognition sequence of at least four amino three reports on the identification of cal- acids N-terminal to the cleavage site. The cium-dependent enzymes in plants have been available data suggest that a true caspase-like published: i.e. in Arabidopsis roots (Safadi et proteolytic activity is present in plants al., 1997), and in root tips (Subbaiah et al., (Woltering et al., 2002). Caspase-like protein- 2000) and the grain aleurone layer of Zea ases in plants are inhibited by specific mays (Wang et al., 2003). caspase inhibitors and are resistant to typical Legumains are a newly discovered group of cysteine proteinase inhibitors (del Pozo & cysteine proteinases (C13) isolated from ma- Lam, 1998; Lam & del Pozo, 2000). turing Ricinus communis seeds (Hara-Nishi- mura et al., 1991), Glycine max cotyledons (Scott et al., 1992), germinating Vicia sativa SYNTHESIS AND INTRACELLULAR seeds (Becker et al., 1995) and in different or- TRANSFER OF CYSTEINE gans of A. thaliana (Kinoshita et al., 1995a; PROTEINASES 1995b). These enzymes belong to the aspara- ginyl-specific subclass of the cysteine Cysteine proteinases are synthesized at endopeptidase family which cleave peptide membrane bound polysomes in the cytoplasm bonds with Asn or Asp (less efficiently) in the as large precursors with short N-terminal and P1¢ positions at the C-terminal flank (Becker much longer C-terminal propeptides. The in- et al., 1995). They are active only at acidic pH active proenzymes enter the lumen of the (Müntz & Shutov, 2002). Plant legumains are endoplasmic reticulum (ER) and are trans- usually called vacuolar processing enzymes ported to the vacuole or cell wall. Most soluble (VPE) but they are also present in the cell plant proteins have a C-terminal KDEL or wall and their function is not restricted to pre- HDEL tetrapeptide sequence recognized by cursor protein processing but also includes the ERD2-KDEL receptor on the Golgi appa- protein breakdown in the vacuole or cell wall ratus (Okamoto et al., 2003) and are trans- (Müntz et al., 2002). Comparison of se- ported to the trans-Golgi network. However, quences
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