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RESULTS AND DISCUSSION
Ninety-Two Putative Proteases Are Predicted by Comparative Genomic AnalysisTo gain further insight into the proteolytic machinery of the malaria parasite, the protein sequences in the annotated P. falciparum genome were subjected to an exhaustive search against the Merops protease database, which has a catalog and a structure-based classification of proteases. We adopted a relatively stringent threshold of E 1e-04 for BLASTP to ensure the high coverage with low false-positives. Redundant hits and partial sequences were excluded, resulting in a total of 92 protease homologs (Table 1). As highlighted in the Protease nomenclature column in Table 1, all twelve previously characterized proteases with proteolytic activity are included. In addition, as highlighted in the Gene ID column, 23 out of 25 proteases predicted by first-pass annotation published in PlasmoDB are included, among which subtilases 1 and 2 have been demonstrated to possess proteolytic activity; PFI0660c is not included because the E-score (0.39) of its closest homolog (Bacillus anthracis CAAX amino terminal protease, accession number NP_655263) is far below the cut-off 1e-04; PF11_0314 is not included because it is more likely to possess ATP hydrolytic and regulatory function than protoelytic function based on sequence homology.
The domain/motif organization of predicted proteases was revealed by the InterPro Search. For each putative malaria protease, the known protease sequence or protease domain of the highest similarity was used as a reference for annotation; the catalytic type and protease family were predicted in accordance with the classification in the protease database Merops (http://www.merops.co.uk/merops/merops.htm), and the enzyme was named in accordance with the SWISS-PROT enzyme nomenclature (http://www.expasy.ch/cgi-bin/lists?peptidas.txt) and literatures.
New Catalytic Types and FamiliesProteases are classified into five major clans (Aspartic, Cysteine, Metallo, Serine, and Threonine) based on their catalytic mechanisms. They can be further grouped into distinct families and subfamilies by intrinsic evolutionary relationships (Rawlings and Barrett 1993). Using the comparative database search, we detected a total of 59 new protease homologs, in addition to 12 characterized proteases with proteolytic activity and 21 predicted by official annotation (Table 1). Moreover, a spectrum of conserved core characteristic domains/motifs for specific catalytic classes has been detected in most of the predicted proteases, indicating their potential activity.
The 92 putative proteases belong to 26 families of five clans, compared to the previously reported 12 proteases that belong to six families of four clans (Rosenthal 2002). The distribution (11% aspartic, 36% cysteine, 22% metallo, 17% serine, and 14% threonine) resembles those in other model organisms, supporting the fundamental premise that a prototype protease system is conserved throughout evolution (Rawlings and Barrett 1993; Southan 2001). Our speculation that a large number of potential proteases remain unexplored in the P. falciparum genome appears justified. Undoubtedly, some of the uncharacterized proteases will perform crucial functions in the parasite life cycle as discussed below.
Examples of Potentially Important ProteasesCalpainCalpain is a group of intracellular cysteine proteases that mediate a wide variety of physiological and pathophysiological processes, including signal transduction, cell motility, apoptosis, and cell cycle regulation (Sorimachi et al. 1997; Glading et al. 2002). In P. falciparum, a calpain, yet unidentified, was believed to be essential in merozoite invasion, based on the observation that Calpain inhibitors I and II strongly blocked invasion (Olaya and Wasserman 1991).
We have identified a putative calpain (MAL13P1.310) in the P. falciparum genome, which exhibits high sequence similarity to C. elegans calpain-7 (E=2e-35). Moreover, its ortholog (accession no. EAA19663) has been identified in the newly released genome of the model rodent malaria parasite Plasmodium yoelii yoelii. It possesses a catalytic domain (985–1453) detected by the Hidden Markov Model in the pfam search, with E = 8.0e-13 (Fig. 1). The most intriguing aspect of this domain is the presence of three active sites (Cys1035, His1371, and Asn1391) that constitute a cleft crucial for catalytic activity (Arthur et al. 1995). A multiple alignment of the catalytic regions was produced for the putative plasmodial calpain and the representative human calpains. In addition to the invariable Cys-His-Asn triad, a high degree of identity is also observed in its vicinity, reflecting stringent functional and mechanistic conservation (Fig. 1). Indeed, the experimental demonstration that a single catalytic subunit in rat and chicken calpains possesses a full bona fide proteolytic activity (Yoshizawa et al. 1995) reinforces the potential processing capacity of the putative plasmodial calpain.
Our further phylogenetic analysis of the putative P. falciparum calpain revealed its striking origin, which might have attributed to an alternative Ca2+-independent regulatory mechanism. Figure 2 shows the evolutionary tree inferred by the neighbor-joining (NJ) method using Poisson corrected distance (Saitou and Nei 1987). Evolutionary trees based on Parsimony (PAUP4.0) and Maximum Likelihood (PHYLIP) also yielded topologies and clade structures congruent with NJ (data not shown). Apparently, two putative plasmodial calpains belong to a novel monophyletic group of animal calpain-7 proteases, with 61% bootstrap support. They share the common domain architecture in the calpain-7 clade: lacking any significant similarity to the C-terminal EF-hand Ca2+-binding domain present in most of the essential Ca2+-dependent mammalian calpain subtypes (calpains -1, -2, -3, -9, -11, and Mu/M-type) (Franz et al. 1999). Provided that fungi cysteine protease PalB, the nearest neighbor of calpain-7, contains a PBH domain resembling the Ca2+-binding domain (Denison et al. 1995), one could speculate that the loss of Ca2+ dependency in calpain-7 subtype had been derived from evolutionary events such as domain shuffling, which might be associated with the divergence of mRNA splicing sites (Craik et al. 1983). Such events appear to have occurred close to or prior to the origin of the animal kingdom (Fig. 2).
The identification of plasmodial calpain has also implicated the existence of calpain-mediated pathways. Its potential cognate targets include host cytoskeletal proteins such as spectrin, integrin, and ezrin. Moreover, the recent discovery of a typical endogenous substrate of calpain, Protein Kinase C (PFL1110c; PFI1685w) in P. falciparum, has provided the support of a parasite-controlled signaling cascade (Doerig et al. 2002).
It is conceivable that the putative protease-active and Ca2+-independent plasmodial calpain may serve as a good antimalarial target for two reasons. First, it may be the central component of crucial signal transduction pathways that affect parasite biology and host-parasite interactions. Second, because it is evolutionarily divergent from the essential subtypes of host calpains, its specific inhibitor may have minimal effects on the host.
MetacaspaseMetacaspase (PF13_0289) is another interesting hypothetical protease. In vertebrates, a cascade of caspases (cysteine aspartyl proteases) is the major modulator of apoptosis (programmed cell death) (Thornberry and Lazbnik 1998; Aravind et al. 1999). Two families of ancient caspase-like proteins (paracaspases and metacaspases) have been found in metazoans, fungi, and protozoa. As shown in the phylogenetic tree (Fig. 3), the putative plasmodial metacaspase occupies a distinct clade constituting paracaspases and metacaspases, which are likely to be the primordial form of 14 subfamilies of vertebrate caspases (bootstrap value = 100%). Interestingly, human paracaspase is capable of interacting with the oncogene Bcl10 and triggering NF-kB activation, indicative of the prone-to-apoptosis property of the ancestral caspase (Uren et al. 2000). Moreover, yeast metacaspase has been demonstrated as an effective executor for apoptosis, suggesting the root of apoptosis dates back to unicellular organisms (Madeo et al. 2002). The multiple alignment clearly reveals that the putative plasmodial metacaspase retains the typical caspase fold, which is centered with the His (404)-Cys (460) catalytic dyad conserved in all representative proteolytically active caspases (Fig. 4). Conversely, considerable sequence diversity is observed in the vicinity of this active site cleft. In particular, yeast metacaspase and the plasmodial homolog exhibit distinct sequence profile to other vertebrate caspases and human paracaspase. Previously, Uren et al. (2000) have postulated that ancient (paracaspases and metacaspases) and vertebrate subtypes differ in substrate-specificity. We have demonstrated that the experimentally confirmed differential substrate-specificity in major vertebrate subtypes is largely determined by the chemical property and configuration of residues situated in the caspase fold (Wang and Gu 2001). Thus, the observed distinct configuration of residues in the active site proximity could account for parasite-specific substrate-preference.
In Plasmodium, the physiological process of apoptosis has never been reported, nor the critical components identified. Nevertheless, the detection of the metacaspase homolog will allow us to investigate the role, if any, of apoptosis and/or analogous signal transduction pathway in the parasite. In addition, since metacaspases have only been found in protozoans, yeasts, and possibly in plants, and are phylogenetically distinct from other caspase subtypes (Fig. 3), the putative plasmodial metacaspase may serve as a potential chemotherapeutic target.
Signal Peptidase 1 (SP1)Signal peptidases (SP) play indispensable roles in protein trafficking and sorting by removing signal peptides from precursors of secretary proteins. This serine protease family consists of two subtypes, SP1 and signalase, based on their distinct structural, functional, and evolutionary features. To date, SPs have been found in bacteria, archaea, fungi, plants, and animals; however, SP has never been reported previously in protists, despite the fact that the dynamic parasite life cycle reflects a need of specific peptidase(s) to process proteins that are translocated across host and parasite membranes. Using the comparative genomic search, we first identified two homologs of signal peptidase, PF13_0118 (SP1) and MAL13P1.167 (signalase) in P. falciparum.
Between two subtypes, SP1 has generated extensive research interest because it represents a novel antibiotic target for its distinct prokaryotic origin and essential functions (Paetzel et al. 2000). We have also identified an ortholog of P. falciparum SP1 in the rodent parasite P. yoelii yoelii genome. Our evolutionary analysis revealed that the two putative plasmodial SP1 have three clusters of homologs: (1) Bacteria SP1; (2) an Arabidopsis chloroplast thylakoidal processing peptidase; and (3) mitochondrial inner membrane peptidases (Imp) found in eukaryotes, which appear to be the nearest neighbor to plasmodial SP1 (Fig. 5). Given the proposed prokaryotic origin of the chloroplast and mitochondrion, malarial SP1 is likely to have evolved via the prokaryotic-specific lineage. Moreover, the potential of its catalytic activity can be inferred from the comparative sequence analysis. The putative SP1 contains the catalytic dyad (Ser175, Lys274) that is invariable across representative SP1 proteins with confirmed signal peptidase activity (Fig. 6). Most notably, this Ser/Lys catalytic dyad mechanism is unique in SP1, compared with the typical Ser/His/Asp triad system in other serine proteases. It seems plausible that the putative plasmodial SP1 has a fundamental role yet to be determined, and represents a promising target given its distant relatedness to the host.
Important Protease-Mediated Pathways Implicated in P. falciparumOur findings suggested at least five new protease-mediated activities: (1) an ATP-dependent ubiquitin-proteasome-mediated cell-cycle control and stress-response system (Verma et al. 2002). Although the mechanism by which proteasomes function in P. falciparum is poorly understood, their importance was suggested by the observed irreversible inhibition on the growth and development of the hepatic and erythrocytic stages of three different Plasmodium species by Lactacystin, a specific threonine protease inhibitor (Gantt et al. 1998). The identification of the clade of threonine proteases and