Aspartic proteases of Plasmodium vivax are highly conserved in wild isolates

The plasmepsins are the aspartic proteases of malaria parasites. Treatment of aspartic protease inhibitor inhibits hemoglobin hydrolysis and blocks the parasite development in vitro suggesting that these proteases might be exploited their potentials as antimalarial drug targets. In this study, we determined the genetic variations of the aspartic proteases of Plasmodium vivax (PvPMs) of wild isolates. Two plasmepsins (PvPM4 and PvPM5) were cloned and sequenced from 20 P. vivax Korean isolates and two imported isolates. The sequences of the enzymes were highly conserved except a small number of amino acid substitutions did not modify key residues for the function or the structure of the enzymes. The high sequence conservations between the plasmepsins from the isolates support the notion that the enzymes could be reliable targets for new antimalarial chemotherapeutics.     

A programmed cell death pathway in the malaria parasite Plasmodium falciparum has general features of mammalian apoptosis but is mediated by clan CA cysteine proteases

Several recent discoveries of the hallmark features of programmed cell death (PCD) in Plasmodium falciparum have presented the possibility of revealing novel targets for antimalarial therapy. Using a combination of cell-based assays, flow cytometry and fluorescence microscopy, we detected features including mitochondrial dysregulation, activation of cysteine proteases and in situ DNA fragmentation in parasites induced with chloroquine (CQ) and staurosporine (ST). The use of the pan-caspase inhibitor, z-Val-Ala-Asp-fmk (zVAD), and the mitochondria outer membrane permeabilization (MOMP) inhibitor, 4-hydroxy-tamoxifen, enabled the characterization of a novel CQ-induced pathway linking cysteine protease activation to downstream mitochondrial dysregulation, amplified protease activity and DNA fragmentation. The PCD features were observed only at high (μM) concentrations of CQ. The use of a new synthetic coumarin-labeled chloroquine (CM-CQ) showed that these features may be associated with concentration-dependent differences in drug localization. By further using cysteine protease inhibitors z-Asp-Glu-Val-Asp-fmk (zDEVD), z-Phe-Ala-fmk (zFA), z-Phe-Phe-fmk (zFF), z-Leu-Leu-Leu-fmk (zLLL), E64d and CA-074, we were able to implicate clan CA cysteine proteases in CQ-mediated PCD. Finally, CQ induction of two CQ-resistant parasite strains, 7G8 and K1, reveals the existence of PCD features in these parasites, the extent of which was less than 3D7. The use of the chemoreversal agent verapamil implicates the parasite digestive vacuole in mediating CQ-induced PCD.     

Proteases as regulators of pathogenesis: Examples from the Apicomplexa

The diverse functional roles that proteases play in basic biological processes make them essential for virtually all organisms. Not surprisingly, proteolysis is also a critical process required for many aspects of pathogenesis. In particular, obligate intracellular parasites must precisely coordinate proteolytic events during their highly regulated life cycle inside multiple host cell environments. Advances in chemical, proteomic and genetic tools that can be applied to parasite biology have led to an increased understanding of the complex events centrally regulated by proteases. In this review, we outline recent advances in our knowledge of specific proteolytic enzymes in two medically relevant apicomplexan parasites: Plasmodium falciparum and Toxoplasma gondii. Efforts over the last decade have begun to provide a map of key proteotolyic events that are essential for both parasite survival and propagation inside host cells. These advances in our molecular understanding of proteolytic events involved in parasite pathogenesis provide a foundation for the validation of new networks and enzyme targets that could be exploited for therapeutic purposes. This article is part of a Special Issue entitled: Proteolysis 50 years after the discovery of lysosome.     

Structural mapping of the ClpB ATPases of Plasmodium falciparum: Targeting protein folding and secretion for antimalarial drug design

Caseinolytic chaperones and proteases (Clp) belong to the AAA+ protein superfamily and are part of the protein quality control machinery in cells. The eukaryotic parasite Plasmodium falciparum, the causative agent of malaria, has evolved an elaborate network of Clp proteins including two distinct ClpB ATPases. ClpB1 and ClpB2 are involved in different aspects of parasitic proteostasis. ClpB1 is present in the apicoplast, a parasite-specific and plastid-like organelle hosting various metabolic pathways necessary for parasite growth. ClpB2 localizes to the parasitophorous vacuole membrane where it drives protein export as core subunit of a parasite-derived protein secretion complex, the Plasmodium Translocon of Exported proteins (PTEX); this process is central to parasite virulence and survival in the human host. The functional associations of these two chaperones with parasite-specific metabolism and protein secretion make them prime drug targets. ClpB proteins function as unfoldases and disaggregases and share a common architecture consisting of four domains—a variable N-terminal domain that binds different protein substrates, followed by two highly conserved catalytic ATPase domains, and a C-terminal domain. Here, we report and compare the first crystal structures of the N terminal domains of ClpB1 and ClpB2 from Plasmodium and analyze their molecular surfaces. Solution scattering analysis of the N domain of ClpB2 shows that the average solution conformation is similar to the crystalline structure. These structures represent the first step towards the characterization of these two malarial chaperones and the reconstitution of the entire PTEX to aid structure-based design of novel anti-malarial drugs.     

Disruption of a mitochondrial protease machinery in Plasmodium falciparum is an intrinsic signal for parasite cell death

The ATP-dependent ClpQY protease system in Plasmodium falciparum is a prokaryotic machinery in the parasite. In the present study, we have identified the complete ClpQY system in P. falciparum and elucidated its functional importance in survival and growth of asexual stage parasites. We characterized the interaction of P. falciparum ClpQ protease (PfClpQ) and PfClpY ATPase components, and showed that a short stretch of residues at the C terminus of PfClpY has an important role in this interaction; a synthetic peptide corresponding to this region antagonizes this interaction and interferes with the functioning of this machinery in the parasite. Disruption of ClpQY function by this peptide caused hindrance in the parasite growth and maturation of asexual stages of parasites. Detailed analyses of cellular effects in these parasites showed features of apoptosis-like cell death. The peptide-treated parasites showed mitochondrial dysfunction and loss of mitochondrial membrane potential. Dysfunctioning of mitochondria initiated a cascade of reactions in parasites, including activation of VAD–FMK-binding proteases and nucleases, which resulted in apoptosis-like cell death. These results show functional importance of mitochondrial proteases in the parasite and involvement of mitochondria in programmed cell death in the malaria parasites.     

Computational identification of putative miRNAs and their target genes in pathogenic amoeba Naegleria fowleri

Naegleria fowleri is a parasitic unicellular free living eukaryotic amoeba. The parasite spreads through contaminated water and causes primary amoebic meningoencephalitis (PAM). Therefore, it is of interest to understand its molecular pathogenesis. Hence, we analyzed the parasite genome for miRNAs (microRNAs) that are non-coding, single stranded RNA molecules. We identified 245 miRNAs using computational methods in N. fowleri, of which five miRNAs are conserved. The predicted miRNA targets were analyzed by using miRanda (software) and further studied the functions by subsequently annotating using AmiGo (a gene ontology web tool).     

Partial purification and characterization of a cysteine protease inhibitor from the plerocercoid of Spirometra erinacei

Helminthic cysteine proteases are well known to play critical roles in tissue invasion, nutrient uptake, and immune evasion of the parasites. In the same manner, the sparganum, the plerocercoid of Spirometra mansoni, is also known to secrete a large amount of cysteine proteases. However, cysteine protease inhibitors regulating the proteolytic activities of the cysteine protease are poorly illustrated. In this regard, we partially purified an endogenous cysteine protease inhibitor from spargana and characterized its biochemical properties. The cysteine protease inhibitor was purified by sequential chromatographies using Resource Q anion exchanger and Superdex 200 HR gel filtration from crude extracts of spargana. The molecular weight of the purified protein was estimated to be about 11 kD on SDS-PAGE. It was able to inhibit papain and 27 kDa cysteine protease of spargana with the ratio of 25.7% and 49.1%, respectively, while did not inhibit chymotrypsin. This finding suggests that the cysteine protease inhibitor of spargana may be involved in regulation of endogenous cysteine proteases of the parasite, rather than interact with cysteine proteases from their hosts.     

Computational analysis of plasmepsin IV bound to an allophenylnorstatine inhibitor

The plasmepsin proteases from the malaria parasite Plasmodium falciparum are attracting attention as putative drug targets. A recently published crystal structure of Plasmodium malariae plasmepsin IV bound to an allophenylnorstatine inhibitor [Clemente, J.C. et al. (2006) Acta Crystallogr. D 62, 246-252] provides the first structural insights regarding interactions of this family of inhibitors with plasmepsins. The compounds in this class are potent inhibitors of HIV-1 protease, but also show nM binding affinities towards plasmepsin IV. Here, we utilize automated docking, molecular dynamics and binding free energy calculations with the linear interaction energy LIE method to investigate the binding of allophenylnorstatine inhibitors to plasmepsin IV from two different species. The calculations yield excellent agreement with experimental binding data and provide new information regarding protonation states of active site residues as well as conformational properties of the inhibitor complexes.     

Ectopic Expression of a Neospora caninum Kazal Type Inhibitor Triggers Developmental Defects in Toxoplasma and Plasmodium

Regulated proteolysis is known to control a variety of vital processes in apicomplexan parasites including invasion and egress of host cells. Serine proteases have been proposed as targets for drug development based upon inhibitor studies that show parasite attenuation and transmission blockage. Genetic studies suggest that serine proteases, such as subtilisin and rhomboid proteases, are essential but functional studies have proved challenging as active proteases are difficult to express. Proteinaceous Protease Inhibitors (PPIs) provide an alternative way to address the role of serine proteases in apicomplexan biology. To validate such an approach, a Neospora caninum Kazal inhibitor (NcPI-S) was expressed ectopically in two apicomplexan species, Toxoplasma gondii tachyzoites and Plasmodium berghei ookinetes, with the aim to disrupt proteolytic processes taking place within the secretory pathway. NcPI-S negatively affected proliferation of Toxoplasma tachyzoites, while it had no effect on invasion and egress. Expression of the inhibitor in P. berghei zygotes blocked their development into mature and invasive ookinetes. Moreover, ultra-structural studies indicated that expression of NcPI-S interfered with normal formation of micronemes, which was also confirmed by the lack of expression of the micronemal protein SOAP in these parasites. Our results suggest that NcPI-S could be a useful tool to investigate the function of proteases in processes fundamental for parasite survival, contributing to the effort to identify targets for parasite attenuation and transmission blockage.     

Trypanosoma cruzi: sequence polymorphism of the gene encoding the Tc52 immunoregulatory-released factor in relation to the phylogenetic diversity of the species

We have previously identified a Trypanosoma cruzi gene encoding a protein named Tc52 sharing structural and functional properties with the thioredoxin and glutaredoxin family involved in thiol-disulfide redox reactions. Gene targeting strategy and immunological studies allowed showing that Tc52 is among T. cruzi virulence factors. Taking into account that T. cruzi has a genetic variability that might be important determinant that governs the different behaviour of T. cruzi clones in vitro and in vivo, we thought it was of interest to analyse the sequence polymorphism of Tc52 gene in several reference clones. The DNA sequences of 12 clones which represent the whole genetic diversity of T. cruzi allowed showing that 40 amino-acid positions over 400 analysed are targets for mutations. A number of residues corresponding to putative amino-acids playing a role in GSH binding and/or enzymatic function and others located nearby are subject to mutations. Although the immunological analysis showed that Tc52 is present in parasite extracts from different clones, it is possible that the amino-acid differences could affect the enzymatic and/or the immunomodulatory function of Tc52 variants and therefore the parasite phenotype.     

Serine proteases in schistosomes and other trematodes

Trematodes, also known as flukes, are phylogenetically ancient parasitic organisms. Due to their importance as human and veterinary parasites, their proteins have been investigated extensively as drug and vaccine targets. Among those, proteases, as crucial enzymes for parasite survival, are considered candidate molecules for anti-parasitic interventions. Surprisingly however, trematode serine proteases, in comparison with other groups of proteases, are largely neglected. Genes encoding serine proteases have been identified in trematode genomes in significant abundance, but the biological roles and biochemical functions of these proteases are poorly understood. However, increasing volumes of genomic and proteomic studies, and accumulated experimental evidence, indicate that this class of proteases plays a substantial role in host–parasite interactions and parasite survival. Here, we discuss in detail serine proteases at genomic and protein levels, and their known or hypothetical functions.     

Rodent and nonrodent malaria parasites differ in their phospholipid metabolic pathways

Malaria, a disease affecting humans and other animals, is caused by a protist of the genus Plasmodium. At the intraerythrocytic stage, the parasite synthesizes a high amount of phospholipids through a bewildering number of pathways. In the human Plasmodium falciparum species, a plant-like pathway that relies on serine decarboxylase and phosphoethanolamine N-methyltransferase activities diverts host serine to provide additional phosphatidylcholine and phosphatidylethanolamine to the parasite. This feature of parasitic dependence toward its host was investigated in other Plasmodium species. In silico analyses led to the identification of phosphoethanolamine N-methyltransferase gene orthologs in primate and bird parasite genomes. However, the gene was not detected in the rodent P. berghei, P. yoelii, and P. chabaudi species. Biochemical experiments with labeled choline, ethanolamine, and serine showed marked differences in biosynthetic pathways when comparing rodent P. berghei and P. vinckei, and human P. falciparum species. Notably, in both rodent parasites, ethanolamine and serine were not significantly incorporated into phosphatidylcholine, indicating the absence of phosphoethanolamine N-methyltransferase activity. To our knowledge, this is the first study to highlight a crucial difference in phospholipid metabolism between Plasmodium species. The findings should facilitate efforts to develop more rational approaches to identify and evaluate new targets for antimalarial therapy.     

Immunolocalization and characterization of two novel proteases in Leishmania donovani: Putative roles in host invasion and parasite development

Two novel intracellular proteases having identical molecular mass (58 kDa) were purified from virulent Indian strain of Leishmania donovani by a combination of aprotinin-agarose affinity chromatography, ion exchange chromatography and finally continuous elution electrophoresis. Both of these proteases migrate in SDS-PAGE as a single homogeneous bands suggesting monomeric nature of these proteases. The enzyme activity of one of the proteases was inhibited by serine protease inhibitor aprotinin and another one was inhibited by metalloprotease inhibitor 1, 10 phenanthroline. The purified enzymes were thus of serine protease (SP-Ld) and metalloprotease (MP-Ld) type. The optimal pH for protease activity is 8.0 and 7.5 for SP-Ld and MP-Ld respectively. The temperature optimum for SP-Ld is 28 °C and for MP-Ld is 37 °C showing their thermostability upto 60 °C. Broad substrate (both natural and synthetic) specificity and the effect of Ca²⁺ upon these enzymes suggested novelty of these proteases. Kinetic data indicate that SP-Ld is of trypsin like as BAPNA appears to be the best substrate and MP-Ld seems to be collagenase type as it degrades azocoll with maximum efficiency. Both immunofluorescence and immune-gold electron microscopy studies revealed that the SP-Ld is localized in the flagellar pocket as well as at the surface of the parasite, whereas MP-Ld is located extensively near the flagellar pocket region. This work also suggests that the uses of anti SP-Ld and anti MP-Ld antibodies are quite significant in interfering with the process of parasite invasion and multiplication respectively. Thus the major role of SP-Ld could be predicted in invasion process as it down regulates the phagocytic activity of macrophages, and MP-Ld appears to play important roles in parasitic development.     

Squamous cell carcinoma antigen 1 is an inhibitor of parasite-derived cysteine proteases

The physiological significance of the squamous cell carcinoma antigens 1 (SCCA1) and SCCA2, members of the ovalbumin serpin family, remains unresolved. In this study, we examined whether SCCA1 or SCCA2 inhibits protozoa- or helminth-derived cysteine proteases. SCCA1, but not SCCA2, potently inhibited the cysteine protease activities of CPB2.8 from Leishmania mexicana, cruzain from Trypanosoma cruzi, rhodesain from Trypanosoma brucei rhodesience, and cathepsin L2 from Fasciola hepatica. The inhibitory activities of SCCA1 were due to its resistance to cleavage by the cysteine proteases. The findings indicate that induction of cysteine protease inhibitors might be a novel defense mechanism against parasite development.     

Metacaspases

Metacaspases are cysteine-dependent proteases found in protozoa, fungi and plants and are distantly related to metazoan caspases. Although metacaspases share structural properties with those of caspases, they lack Asp specificity and cleave their targets after Arg or Lys residues. Studies performed over the past 10 years have demonstrated that metacaspases are multifunctional proteases essential for normal physiology of non-metazoan organisms. This article provides a comprehensive overview of the metacaspase function and molecular regulation during programmed cell death, stress and cell proliferation, as well as an analysis of the first metacaspase-mediated proteolytic pathway. To prevent further misapplication of caspase-specific molecular probes for measuring and inhibiting metacaspase activity, we provide a list of probes suitable for metacaspases.     

Genetic and structural characterization of PvSERA4: potential implication as therapeutic target for Plasmodium vivax malaria

Plasmodium vivax malaria is geographically the most widely distributed and prevalent form of human malaria. The development of drug resistance by the parasite to existing drugs necessitates higher focus to explore and identify new drug targets. Plasmodial proteases have key roles in parasite biology and are involved in nutritional uptake, egress from infected reticulocytes, and invasion of the new target erythrocytes. Serine repeat antigens (SERA) of Plasmodium are parasite proteases that remain attractive drug targets and are important vaccine candidates due to their high expression profiles in the blood stages. SERA proteins have a unique putative papain-like cysteine protease motif that has either serine or cysteine in its active site. In P. vivax, PvSERA4 is the highest transcribed member of this multigene family. In this study, we have investigated the genetic polymorphism of PvSERA4 central protease domain and deduced its 3D model by homology modeling and also performed MD simulations to acquire refined protein structure. Sequence analysis of protease domain of PvSERA4 from Indian field isolates reveals that the central domain is highly conserved. The high sequence conservation of the PvSERA4 enzyme domain coupled with its high expression raises the possibility of it having a critical role in parasite biology and hence, being a reliable target for new selective inhibitor-based antimalarial chemotherapeutics. The 3D model showed the presence of an unusual antiparallel Beta hairpin motif between catalytic residues similar to hemoglobin binding motif of Plasmodial hemoglobinases. Our PvSERA4 model will aid in designing structure-based inhibitors against this enzyme.     

A role of falcipain-2, principal cysteine proteases of Plasmodium falciparum in merozoite egression

The process of merozoite release in Plasmodium falciparum involves rupture of the parasitophorous vacuole membrane and erythrocyte plasma membrane. Through the use of protease inhibitors that halt the merozoite release, a number of parasite proteases, especially serine, aspartic, and cysteine proteases, have been implicated in the schizont rupture. To understand the precise role of cysteine proteases in the merozoite release, in the present study, we treated P. falciparum cultures with siRNAs corresponding to falcipain-1, falcipain-2, and falcipain-3, the three papain-family proteases of the parasite. Treatment of malaria parasites with either of the falcipain siRNAs considerably reduced parasite growth. Morphological examination of the siRNA treated parasite cultures revealed that most of the parasites in falcipain-2 siRNA treated cultures were arrested at schizont stage. Analysis of a transgenic P. falciparum line expressing chimeric-GFP upon treatment with falcipain-2 siRNA revealed block in the rupture of erythrocyte membrane at the time of merozoite egression. These results suggest that falcipain-2 is an important parasitic protease that participates in hemoglobin degradation and in the merozoite release.     

Aspartic proteases of Plasmodium falciparum and other parasitic protozoa as drug targets

All parasitic protozoa contain multiple proteases, some of which are attracting attention as drug targets. Aspartic proteases are already the targets of some clinically useful drugs (e.g. chemotherapy of HIV infection) and a variety of factors make these enzymes appealing to those seeking novel antiparasite therapies. This review provides a critical analysis of the current knowledge on Plasmodium aspartic proteases termed plasmepsins, proposes a definitive nomenclature for this group of enzymes, and compares these enzymes with aspartic proteases of humans and other parasitic protozoa. The present status of attempts to obtain specific inhibitors of the parasite enzymes that will be useful as drugs is outlined and suggestions for future research priorities are proposed.     

Plasmodium berghei: plasmodium perforin-like protein 5 is required for mosquito midgut invasion in Anopheles stephensi

During its life cycle the malarial parasite Plasmodium forms three invasive stages which have to invade different and specific cells for replication to ensue. Invasion is vital to parasite survival and consequently proteins responsible for invasion are considered to be candidate vaccine/drug targets. Plasmodium perforin-like proteins (PPLPs) have been implicated in invasion because they contain a predicted pore-forming domain. Ookinetes express three PPLPs, and one of them (PPLP3) has previously been shown to be essential for mosquito midgut invasion. In this study we show through phenotypic analysis of loss-of-function mutants that PPLP5 is equally essential for mosquito infection. Deltapplp5 ookinetes cannot invade midgut epithelial cells, but subsequent parasite development is rescued if the midgut is bypassed by injection of ookinetes into the hemocoel. The indistinguishable phenotypes of Deltapplp5 and Deltapplp3 ookinetes strongly suggest that these two proteins contribute to a common process.