Phytocalpains: orthologous calcium-dependent cysteine proteinases

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Vol.8 No.2 February 2003

Phytocalpains: orthologous calciumdependent cysteine proteinases Roge´rio Margis and Ma´rcia Margis-Pinheiro Laborato´rio de Gene´tica Molecular Vegetal, Departamento de Gene´tica, UFRJ, 21944-970 Rio de Janeiro, Brazil

A single calcium-dependent cysteine protease (calpain) gene, essential for aleurone cell development, has been identified recently in maize, although this activity had been described previously in Arabidopsis and maize roots associated with anoxia-induced root-tip death. Calpain genes are ubiquitous in animals and there are up to 12 paralogous genes in humans that exhibit molecular diversity outside of their catalytic domain. Calpain orthologous genes have been identified in 11 plant species. Like their animal counterparts, phytocalpains have significant homology within the catalytic domain, but lack the conserved calcium-binding domain IV, and some members have an N-terminal transmembrane receptor-like domain. Cysteine or thiol proteinases (EC 3.4.22) correspond to all endopeptidyl hydrolases that contain a cysteine residue in their active sites. These proteases have been identified in phylogenetically diverse organisms, such as bacteria, eukaryotic microorganisms, plants and animals [1]. They are involved with multiple processes, ranging from general protein catabolism to regulation of specific protein turnover during germination [2], stress and hypersensitive response [3], programmed cell death and senescence [4]. In many cases, the expression of multiple cysteine proteinases within a single organism is regulated in an independent way [5,6]. The class of cysteine proteases comprises more than 30 families of peptidases, which have been grouped into at least six superfamilies or clans. Members from the six major clans are defined according to the nature and linear organization of the catalytic residues along the primary sequence. The papain superfamily, also known as clan CA, includes the best-known cysteine peptidases: they form a catalytic triad of Cys, His and Asn or Asp catalytic residues in an ordered sequence [7]. These cysteine peptidases are synthesized as preproenzymes and are located in lysosomes or analogous organelles. The most studied cysteine proteinase is papain, from Carica papaya, a typical member of this superfamily. Papain, together with other higher-plant cysteine proteinases and mammalian lysosomal cathepsins B, H, L and S, form the C1A family. In addition, bleomycin hydrolase (family C1B), calpains (family C2), streptopain (family C10) and viral proteases (C6, C21 to C36) are typical members of other families in the papain superfamily [1]. Among these families, calpains Corresponding author: Roge´rio Margis ([email protected]).

are of special interest because they correspond to an evolutionarily distinct branch of the cysteine proteinase family [8]. The calpains are cytoplasmic, calcium-dependent cysteine proteases that differ in requiring micro or milimolar concentrations of Ca2þ for activity and have a highly conserved molecular structure in the catalytic domain [9]. However, the overall molecular diversity of calpain outside of the catalytic domain has attracted interest because of the structural and functional variations that occurred during evolution [10,11]. Several cellular and physiological processes such as cell cycle, apoptosis and long-term potentiation of memory are calpain dependent, but a definite function for these enigmatic proteases has yet to be determined [12]. Calpains are widely distributed among animals, from mammals to invertebrates, and have also been described in fungi and protozoa. However, until recently, no member has been described in either plants or bacteria [7,12]. Searching for calpains in plants As part of an ongoing program to characterize sugarcane ESTs (SUCEST, http://sucest.lad.dcc.unicamp.br/en/) we have identified sugarcane members of the cysteine protease group, sub-sub-class 3.4.22, and correlated their tissue-expression pattern with putative functions [13]. One of these clusters, SCEQRT1030G12.g, has a significant similarity to human calpain-2 (CAPN2), with an e-value of 3.0 e233 and 26% identity over 275 amino acid residues. Subsequently, three other independent clusters (SCCCST2002A03.g, SCEPCL6023G08.g and SCSBSB1051E06.g), covering the full C-terminal region, were identified on the sugarcane EST database. These four clusters were assembled to create a sugarcane calpain clone of 4060 nucleotides (Soff phytoCAPN, GenBank accession AF537609), which was used as a bait sequence on an extensive search for plant calpain orthologous sequences. The general results of a T-Blast-n search [14] performed against 14 different plant cDNA databanks present in the TIGR gene indices [15] and on GenBank (http://www.ncbi.nlm.nih.gov/GenBank/) are presented in Fig. 1a. Calpain homologous sequences were found in 11 different mono- and dicot plant species: maize (Zmay, BE049971), barley (Hvul, TC23295 and TC20083), rice (Osat, TC70280), wheat (Taes, TC24668), soybean (Gmax, BE329863, TC105936 and TC112425), cotton (Ghir, AW187746), tomato (Lesc, TC112058), Medicago (Mtru, BG646186 and BE325588), potato (Stub, BG888762 and

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e-value

(a) C IIa

Sugarcane

Query

Barley

1.0 e–142

97

98

Cotton

9.0 e–62

82

89

Immature fiber

Medicago

7.0 e–78

78

88

Stem and roots

Potato

3.0 e–56

84

90

Tuber and leaf

Sorghum

6.0 e–68

97

97

Embryos

Soybean

1.0 e–158

65

72

Hypocotyl, root and flower

Tomato

2.0 e–24

61

71

Stem and seedling roots

Wheat

1.0 e–83

97

98

Seedling roots

66

74

Root and above ground

Query

Root, seed and flower Spikes, anthers and leaf

IIb

Arabidopsis

0.0

IIa

IIb

Maize

0.0

85

85

Glume

IIa

IIb

Rice

0.0

81

84

Leaf and endosperm

3.0 e–33

26

41

Ubiquitous

3.0 e–14

28

48

Ubiquitous

e–25

30

46

Ubiquitous

IIa

IIb

III

IIa

IIb

PalB

Hsap Calp13

IIa

IIb

I

cDNA tissue expression pattern

IIa

Hsap Calp7

Hsap Calp2

%I %S

HN IIb

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IV

Sol

7.0

(b)

III IIb – –– Ca+ + ––

–– – + Ca + – –

IV IIa I Sugarcane phytoCAPN

Human CAPN2 TRENDS in Plant Science

Fig. 1. Comparison of phytocalpain domains with those of traditional calpain members. (a) Schematic interlace of identified plant calpain sequences with the structures of three divergent human calpain family members. Relative positions of C, H and N residues from the catalytic triad present in domain II (blue) are indicated. The phytocalpain domains with unknown function, from partial cDNAs (light green) or full cDNAs (dark green), and the N-terminal transmembrane domain (purple) are presented in relation to domains I, II, IV, PalB and Sol of human calpains. The e-value, degree of amino acid identity (% I) and similarity (% S) among sugarcane (used as query sequence) and other plant and human calpains, as the source of ESTs cDNA libraries, are described in separate columns; (b) three-dimensional structures of human calpain-2 [18] and sugarcane phytocalpain predicted three-dimensional model produced with the Swiss-Model ProMod package [36]. The cysteine (yellow), histidine (red) and asparagine (orange) residues that compose the enzyme catalytic triad were drawn in a space-fill format. Relative position of conserved domain II negatively charged residues (2 ) that potentially interact with calcium are shown. The structural domains I, III and IV (human) and subdomains IIa and IIb (in sugarcane and human calpain) were also indicated.

BI435869), sorghum (Sbic, BG464268) and Arabidopsis (Atha, TC144902 and TC144904). A single genomic match, for a calpain homologue, was found in Arabidopsis thaliana chromosome-I (locus At1 g55350). Several calpain features can be identified on the putative protein deduced from this gene, but only at its carboxyl-end. The probable existence of a single calpain gene in plant genomes can be inferred not only by the presence of a single genomic accession in Arabidopsis, but also from the sequence analysis of the distinct but redundant clones obtained from the species-specific cDNA libraries. Recently, calpain full-length cDNA sequences were obtained for Arabidopsis (AY061803), rice (AY062272) and maize (AY061805) [16]. http://plants.trends.com

Expression pattern and function of calpains In humans, the majority of calpain family members have a ubiquitous pattern of expression, occurring in different tissues and cell types such as neurons, myoblasts, corneal or renal cells [17]. Abnormal expression or activity levels of calpains have been correlated with several neuronal and muscular diseases [10,12,17]. Participation of calpain in the eukaryotic cell cycle and apoptosis is correlated with its substrates for degradation, such as the tumor suppressor p53 [12]. The nature of the different cDNA libraries, from which clones were isolated in the 11 plant species, suggests that phytocalpains exhibit a ubiquitous tissue-expression pattern (Fig. 1a). The four sugarcane phytocalpain clusters

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were obtained from 25 different clones isolated from cDNA libraries: nine flowers from 5 to 20-cm-long stems, five stems, three lateral buds, three roots, two apical meristems from immature plants, seeds and calli and also from plants infected with Acetobacter diazotroficans. The possible phytocalpain substrates in these different plant tissues must be identified to determine what function this enzyme has in plant physiology. In the case of maize, the phytocalpain cDNA was identified as corresponding to the defective kernel 1 (dek1) gene, essential for aleurone cell development [16]. Because present data suggest that phytocalpain is a product from a single gene, silencing phytocalpain should reveal what role this proteinase has in plants others than maize and in tissues others than the aleurone layer. Structural features of calpain members The best-characterized member of the calpain family is the human m-calpain or CAPN-2. Its three-dimensional structure was solved, revealing four major and distinct domains (Fig. 1b) [18]. Domain-I corresponds to an N-terminal a-helix that suffers an autolytic cleavage, before or in parallel to the external substrate proteolysis, as observed with proenzyme activations. The N-terminal domain of other calpain members, such as CAPN7 and 13 (Fig. 1a), must have a completely different sequence and associated function. Domain-II, the catalytic domain, is divided in two subdomains, IIa and IIb [19] (Fig. 1b). In spite of the absence of an EF-hand or other Ca2þ-binding

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motifs in calpains domains IIa and IIb, a recombinant human calpain, composed of domain II only, will show catalytic activity if incubated in the presence of calcium [19]. It has been proposed that calcium neutralizes the negatively charged residues on both sides of domain II and assists in forming the ‘closed’ active site conformation (Fig. 1b). Because of the distance between these subdomains, the catalytic triad formed by a cysteine residue (in domain-IIa) and the histidine and asparagine residues (in domain-IIb) is not assembled, and depends on the presence of calcium to induce the ‘closed’ and active conformation [19]. Domain-III has been considered as a structural linker between the catalytic domain-II and the calcium-binding domain-IV [20]. CAPN2 domain IV (Fig. 1a and 1b) carries the characteristic EF-hand structure, present in calmodulin, troponin-C and other calcium-binding proteins, which is found in six other members of human calpains (CAPN1, 3, 8, 9, 11 and 12) [17]. However, it is absent from several other calpain members, such as human calpain-7 and the homologue to Drosophila, small-optique lobule cysteine proteinase (Fig. 1a). Therefore, the presence of the calcium-binding domain by its self is not enough to characterize a calpain gene; identification must be combined with the nature and context of the cysteine proteinase catalytic domain-II. The level of sequence identity among the phytocalpains ranges from 66 to 97% on sequence fragments 72 to 2159 residues in length (Fig. 1a). The phytocalpains identified

Domain IIa

Domain IIb

* * Mmus palB ..---QTIVSDCSFVASLAISAA. .LVP--THAYAVLDIREFKG. .FIQLKNPWSH-LRW.. Hsap palB/CAPN7 . . - - - Q T I V S D C S F V A S L A I S A A . . L V P - - T H A Y A V L D I R E F K G . . F I Q L K N P W S H - L R W . . Aory palB ..DLVQDMLTDCSVVASLCATTS. .LVS--EHDYAILDMKESKG. .QLLVKNPWAG-ADT..

Hsap Dmel Cele Hsap Hsap

Sol/CAPN13 P2 tra3 CAPN6X CAPN5

..DILQGLLGNCWFLSALAVLAE. ..DICQGVLGNCWLLSALAVLAE. ..DVTQGILGNCWFVSACSALTH. ..QLTQGRLGHKPMVSAFSCLAV. ..DLHQGQVGNCWFVAACSSLAS.

.------HAYSILDVRDVQG. .------HAYSVLDVKDIQG. .LVKG--HAYAVSAVCTIDV. .LLKG--HTYTMTDIRKIRL. .LVKG--HAYAVTDVRKVRL.

.LLRLRNPWGRFS-W.. .LLKLRNPWGHYS-W.. .LIRLQNPWGE-KEW.. .MVRLRNPLGR-QEW.. .MIRLRNPWGE-REW..

Xlae Mmus Hsap Hsap Lmaj Hsap Hsap Hsap Hsap Mmus

CAPN mCAPN2 mCAPN2 µCAPN1 CAPN CAPN10 CAPN11 CAPN9 CAPN3 CAPN3

..DIRQGALGDCWLLAAIASLTL. ..DICQGALGDCWLLAAIASLTL. ..DICQGALGDCWLLAAIASLTL. ..DICQGALGDCWLLAAIASLTL. ..EVEQGELGDCWLMCAVATLAE. ..QVKQGLLGDCWFLCACAALQK. ..DICQGILGDCWLLAAIGSLTT. ..DICQGELGDCWLLAAIASLTL. ..DICQGDLGDCWFLAAIACLTL. ..DICQGDLGDCWLLAAIACLTL.

.LVKG--HAYSVTGAEEVLY. .LVKG--HAYSVTGAEEVES. .LVKG--HAYSVTGAEEVES. .LVKG--HAYSVTGAKQVNY. .FLPG--HAYSVLDVKEFQ-. .---GEFHAFIVSDLRELQ-. .LVRG--HAYSVTGLQDVHY. .LIKG--HAYSVTGIDQVSF. .LVKG--HAYSVTGLEEALF. .LVKG--HAYSVTGLEEALF.

.LIRVRNPWGEV-EW.. .LIRIRNPWGQV-EW.. .LIRIRNPWGEV-EW.. .LIRMRNPWGEV-EW.. .LICLRNPWVHGSGW.. .LLRIQNPWGR-RCW.. .LIRVRNPWGRI-EW.. .LIRIRNPWGQV-EW.. .LVRLRNPWGQV-EW.. .LVRLRNPWGQV-EW..

Soff Hvul Mtru Gmax Taes Atha Zmay Osat

phytoCAPN phytoCAPN phytoCAPN phytoCAPN phytoCAPN phytoCAPN phytoCAPN phytoCAPN

..DVCQGRLGDCWFLSAVAVLTE. ..---------------------. ..---------------------. ..DVCQGRLGDCWFLSAVAVLAE. ..---------------------. ..DVCQGRLGDCWFLSAVAVLTE. ..DVCQGRLGDCWFLSAVAVLTE. ..DVCQGRLGDCWFLSAVAVLTE.

.IVQG--HAYSILQVREVDG. .IVQG--HAYSILQIREVDG. .-------------------. .-------------------. .IVQG--HAYSILQIREVDG. .IVQG--HAYSVLQVREVDG. .IVQG--HAYSILQVREVDG. .IVQG--HAYSILQVREVDG.

.LIQIRNPWANEVEW.. .LVQIRNPWANEVEW.. .LVQIRNPWANEVEW.. .--------------.. .LVQIRNPWANEVEW.. .LVQIRNPWANEVEW.. .LIQIRNPWANEVEW.. .LVQIRNPWANEVEW..

Consensus

..DI QG LGDCW LAAIA LT . .LV G

HAYSVT VREV

. .LIRIRNPWG

EW..

TRENDS in Plant Science

Fig. 2. Alignment of calpain sequences. Analysis was performed based on a pattern-induced multi-alignment algorithm with sequential clustering, covering the calpain catalytic domains IIa and IIb that contain residues from the catalytic triad (arrow and black background). Semi-conservative residues, present in . 50% of the aligned sequences, are highlighted in blue. Characteristic aspartic and proline residues specifically found in members of the calpain family are indicated by an asterisk and underlined in the consensus sequence. Regions corresponding to domains IIa and IIb are indicated by black bars on the top of the alignment. http://plants.trends.com

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to date align mainly with the IIa and IIb catalytic domains, some of them also have extensions that cover the more variable domains I and III, but no sequence determined to date corresponds with the calcium-binding domain IV. The accuracy of the ending regions in plant calpain homologues presented in Fig. 1 and the absence of a domain IV can be affirmed because partial cDNAs from sugarcane, Medicago and barley, identified to date have a conserved EAL/V sequence followed by a stop codon and a poly-A tract, as well as the more recently identified full-length cDNAs from rice, maize and Arabidopsis. The N-terminal region of deduced sequences of Arabidopsis, rice and maize phytocalpains share a common transmembrane domain and it has been suggested that it can act as a receptor-like domain [16]. Phytocalpains might require an inhibitor The tuning of proteolytic processes driven by the calpains is not just a consequence of their differential expression levels. It is also dependent on the presence or absence of their specific inhibitor calpastatin [21]. This inhibitor can be expressed in different forms; owing to alternative splicing, two proteins differing in their N-terminal sequences can be expressed in mouse tissues [22], or alternatively undergo a mechanism of reversible phosphorylation that changes the intracellular localization and aggregation state [23]. Calpain crystallographic data [24] and studies with small synthetic oligopeptides from calpastatin region A and C [25] have shown that the protease-inhibitor interaction is calcium dependent and involves the classical EF-hand motifs present in calpain domains IV and VI, from the large and small sub-unit, respectively. No plant calpastatin homologue sequences have been identified yet in spite of an extensive search of general or plant-specific databases. In eight different plant species, domain IV is absent in all identified phytocalpains, suggesting that if an inhibitor exists, it would have evolved differently from that of the calpastatin and consequently with different structural domains. Curiously, recent data has demonstrated that engineered forms of chicken cystatin and stefin-B, members of a different class of inhibitor, can also interact with the catalytic subunit and inhibit CAPN2 [26]. Therefore, it is feasible that phytocystatin, a family of cysteine proteinase inhibitors [27], could play the same role in plants that calpastatin plays in animals. Evolutionary aspects of the calpain family Several authors have speculated about the evolutionary history of protease families [8,28,29]. Their origin can be viewed as the evolution from a single, general-purpose ancestral protease to multiple paralogous enzymes with increased specificity through the process of repeated gene duplication [8,30]. The papain superfamily originated early in eukaryote evolution and might have occurred before the eukaryote – prokaryote divergence [28]. The evolution of the calpain family in animals produced a series of paralogous genes that evolved to form calpain proteinases with singular characteristics [29]. The heterodimer nature of calpains is considered a recent event and is absent in invertebrates, whereas the first major calpain http://plants.trends.com

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92 99

Osat phytoCAPN Hvul phytoCAPN Zmay phytoCAPN 100 97 Soff phytoCAPN Atha phytoCAPN Gmax phytoCAPN 85 96 Mtru phytoCAPN 98 100 100

100 100 100

98

100 99 100 100 95 100 99 100

99

100 98 100

0.4

0.3

0.2

0.1

Lmaj CAPN Ncra CAPN Mmus CAPN10 Rnor CAPN10 Hsap CAPN10 Hsap CAPN13 Mmus CAPN6 Hsap CAPN6X Mmus CAPN5 Hsap CAPN5 Cele tra3 Mmus CAPN12 Dmel P2 Mmus CAPN9 Hsap CAPN9 Mmus CAPN3 Hsap CAPN3 Hsap CAPN11 Mmus CAPN8 Rnor CAPN8 Xlae CAPN8 Mmus CPAN2 Hsap CAPN2 Mmus CAPN1 Hsap CAPN1 Aory PALB Hsap PALB CAPN7 Mmus PALB CAPN7 EC 3.4.22.1 CathepsinB EC 3.4.22.2 Papain 0.0 TRENDS in Plant Science

Fig. 3. Phylogenetic unrooted tree of calpains from different organisms. The tree was built using the neighbor-joining and amino acid p-distance methods with a pair-wise deletion option performed on the pattern-induced multi-alignment of domains IIa and IIb. The bootstrap test with 5000 replications was used to assure the topology of tree branches. Analysis was performed based on the alignment of 35 different calpain sequences and human cathepsin B and papain as the cysteine proteinase out groups. Seven sequences were derived from plants (Osat: rice, Hvul: barley; Zmay: corn, Soff: sugarcane, Atha: Arabidopsis, Gmax: soybean and Mtru: Medicago) and 28 from other eukaryotic organisms (Lmaj: Leishmania; Ncra: Neurospora, Mmus: mouse, Rnor: rat, Hsap: man, Cele: Caenorhabditis elegans, Dmel: Drosophila melanogaster, Xlae: Xenopus laevii, Aory: Aspergilus oryzae). Major branch clusters, corresponding to phytocalpains (green) and PALB/CAPN7 (purple) and all other CAPNs classes (black) were signaled with filled circles. Mono and dicot plants formed two distinct clusters inside the phytocalpain branch (green unfilled circles). The bootstrap replication values .85% are indicated in the main branching points and a scale corresponding to the genetic distance is at the bottom of the tree.

gene duplication and further acquisition of the calmodulinlike domain was predicted to have occurred somewhere between 1000 million and 670 million years ago, after the plant– animal and fungus – animal split occurred [29,31]. Because there is variation in the calpain domains between the family members, sequence alignment (Fig. 2) and a phylogenetic tree (Fig. 3) were built based just on the regions corresponding to catalytic domains IIa and IIb. The phylogenetic analysis of the calpain members of cysteine proteinases from protists, plants, and animals produced four major clusters, which is supported statistically by the internal branch test [32,33] (Fig. 3).

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Nevertheless, animal calpains with the calcium-binding domain IV formed a major central group (depicted in black) that also included members of CAPN5, 6 and 10. The plant-derived sequences were included in a major cluster from which palB/CAPN7 members (depicted in purple) were excluded. This topology, reinforced by the use of cathepsin-B and papain as cysteine proteinase outgroups, identify these plant sequences as phytocalpains, constituting a new group inside the family C2, CA clan [7] of cysteine peptidases. To date, only two reports have identified calciumdependent proteolytic activity in plants [34,35]. Both used gel assays (zimograms), associated with different incubation conditions in the presence or absence of calcium and cysteine proteinase inhibitors, and have detected the calcium-dependent proteinase in Arabidopsis roots [34] and associated with anoxia-induced root-tip death in maize [35]. The data discussed here indicates that extensive work should be done to better characterize and understand the presumably distinct roles that this cysteine proteinase family has in plants. A comprehensive analysis of calpain gene expression pattern in different species should provide new insights on phytocalpain functions. In addition, the functional characterization of the N-terminal domain as a receptor, analysis of intracellular localization, coupled with the identification of endogenous membrane associates or soluble calpain substrates should considerably advance our understanding of the physiological role of phytocalpain in plant development. Acknowledgements We wish to thank FAPESP for support and development of the SUCEST project.

References 1 Rawlings, N.D. and Barrett, A.J. (1994) Families of cysteine peptidases. Methods Enzymol. 244, 461 – 486 2 Schmid, M. et al. (2001) The ricinosomes of senescing plant tissue bud from the endoplasmic reticulum. Proc. Natl. Acad. Sci. U. S. A. 98, 5353 – 5358 3 del Pozo, O. and Lam, E. (1998) Caspases and programmed cell death in the hypersensitive response of plants to pathogens. Curr. Biol. 8, 1129 – 1132 4 Drake, R. et al. (1996) Isolation and analysis of cDNAs encoding tomato cysteine proteases expressed during leaf senescence. Plant Mol. Biol. 30, 755 – 767 5 Linthorst, H.J. et al. (1993) Circadian expression and induction by wounding of tobacco genes for cysteine proteinase. Plant Mol. Biol. 21, 685 – 694 6 Lidgett, A.J. et al. (1995) Isolation and expression pattern of a cDNA encoding a cathepsin B-like protease from Nicotiana rustica. Plant Mol. Biol. 29, 379 – 384 7 Rawlings, N.D. and Barrett, A.J. (2000) MEROPS: the peptidase database. Nucleic Acids Res. 28, 323 – 325 8 Berti, P.J. and Storer, A.C. (1995) Alignment/phylogeny of the papain superfamily of cysteine proteases. J. Mol. Biol. 246, 273 – 283 9 Croall, D.E. and DeMartino, G.N. (1991) Calcium-activated neutral protease (calpain) system: structure, function, and regulation. Physiol. Rev. 71, 813 – 847 10 Ono, Y. et al. (1998) Structure and physiology of calpain, an enigmatic protease. Biochem. Biophys. Res. Commun. 245, 289– 294 11 Ohno, S. et al. (1984) Evolutionary origin of a calcium-dependent protease by fusion of genes for a thiol protease and a calcium-binding protein? Nature 312, 566 – 570

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12 Carafoli, E. and Molinari, M. (1998) Calpain: a protease in search of a function? Biochem. Biophys. Res. Commun. 247, 193 – 203 13 Correa, G.C. et al. (2001) Identification, classification and expression pattern analysis of sugarcane cysteine proteinases. Genet. Mol. Biol. 24, 275 – 283 14 Altschul, S.F. et al. (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25, 3389– 3402 15 Quackenbush, J. et al. (2001) The TIGR Gene Indices: analysis of gene transcript sequences in highly sampled eukaryotic species. Nucleic Acids Res. 29, 159– 164 16 Lid, S.E. et al. (2002) The defective kernel 1 (dek1) gene required for aleurone cell development in the endosperm of maize grains encodes a membrane protein of the calpain gene superfamily. Proc. Natl. Acad. Sci. U. S. A. 99, 5460 – 5465 17 Huang, Y. and Wang, K.K. (2001) The calpain family and human disease. Trends Mol. Med. 7, 355– 362 18 Strobl, S. et al. (2000) The crystal structure of calcium-free human m-calpain suggests an electrostatic switch mechanism for activation by calcium. Proc. Natl. Acad. Sci. U. S. A. 97, 588– 592 19 Hata, S. et al. (2001) Domain II of m-calpain is a Ca2þ-dependent cysteine protease. FEBS Lett. 501, 111 – 114 20 Sorimachi, H. and Suzuki, K. (2001) The structure of calpain. J. Biochem. (Tokyo) 129, 653 – 664 21 Maki, M. et al. (1991) Calpastatins: biochemical and molecular biological studies. Biomed. Biochim. Acta 50, 509 – 516 22 Takano, J. et al. (2000) Four types of calpastatin isoforms with distinct amino-terminal sequences are specified by alternative first exons and differentially expressed in mouse tissues. J. Biochem. (Tokyo) 128, 83 – 92 23 Averna, M. et al. (2001) Changes in intracellular calpastatin localization are mediated by reversible phosphorylation. Biochem. J. 354, 25 – 30 24 Lin, G.D. et al. (1997) Crystal structure of calcium bound domain VI of ˚ resolution and its role in enzyme assembly, regulation, calpain at 1.9 A and inhibitor binding. Nat. Struct. Biol. 4, 539 – 547 25 Takano, E. et al. (1995) Preference of calcium-dependent interactions between calmodulin-like domains of calpain and calpastatin subdomains. FEBS Lett. 362, 93 – 97 26 Diaz, B.G. et al. (2001) Cystatins as calpain inhibitors: engineered chicken cystatin- and stefin B-kininogen domain 2 hybrids support a cystatin-like mode of interaction with the catalytic subunit of mu-calpain. Biol. Chem. 382, 97 – 107 27 Margis, R. et al. (1998) Structural and phylogenetic relationships among plant and animal cystatins. Arch. Biochem. Biophys. 359, 24 – 30 28 Hughes, A.L. (1994) Evolution of cysteine proteinases in eukaryotes. Mol. Phylogenet. Evol. 3, 310 – 321 29 Jekely, G. and Friedrich, P. (1999) The evolution of the calpain family as reflected in paralogous chromosome regions. J. Mol. Evol. 49, 272– 281 30 Creighton, T.E. and Darby, N.J. (1989) Functional evolutionary divergence of proteolytic enzymes and their inhibitors. Trends Biochem. Sci. 14, 319 – 324 31 Dear, N. et al. (1997) A new subfamily of vertebrate calpains lacking a calmodulin-like domain: implications for calpain regulation and evolution. Genomics 45, 175 – 184 32 Kumar, S. et al. (1994) MEGA: molecular evolutionary genetics analysis software for microcomputers. Comput. Appl. Biosci. 10, 189– 191 33 Sitnikova, T. et al. (1995) Interior-branch and bootstrap tests of phylogenetic trees. Mol. Biol. Evol. 12, 319– 333 34 Subbaiah, C.C. et al. (2000) A Ca2þ-dependent cysteine protease is associated with anoxia-induced root tip death in maize. J. Exp. Bot. 51, 721– 730 35 Safadi, F. et al. (1997) Partial purification and characterization of a Ca2þ-dependent proteinase from Arabidopsis roots. Arch. Biochem. Biophys. 348, 143 – 151 36 Schwede, T. et al. (2000) Protein structure computing in the genomic era. Res. Microbiol. 151, 107 – 112