Aminopeptidases from Plasmodium falciparum, Plasmodium chabaudi chabaudi and Plasmodium berghei

ABSTRACT. Using fluorogenic substrates and polyacrylamide gels we detected in cell-free extracts of Plasmodium falciparum, Plasmodium chabaudi chabaudi and Plasmodium berghei only a single aminopeptidase. A comparative study of the aminopeptidase activity in each extract revealed that the enzymes have similar specificities and kinetics, a near-neutral pH optima of 7.2 and are moderately thermophilic. Each has an apparent molecular weight of 80,000 ± 10,000, determined by high performance liquid chromatography on a calibrated SW500 column. Whilst the P. c. chabaudi and P. berghei activity co-migrate in native polyacrylamide gels, that of P. falciparum migrates more slowly. The three enzymes can be selectively inhibited by ortho -phenanthroline and are thus metallo-aminopeptidases; however, in contrast to other aminopeptidases the metal co-factor does not appear to be Zn²⁺.     

Nuclease activity of Plasmodium falciparum Alba family protein PfAlba3

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Mitochondrial NADH dehydrogenase from Plasmodium falciparum and Plasmodium berghei

The mitochondrial electron transport system is necessary for growth and survival of malarial parasites in mammalian host cells. NADH dehydrogenase of respiratory complex I was demonstrated in isolated mitochondrial organelles of the human parasite Plasmodium falciparum and the mouse parasite Plasmodium berghei by using the specific inhibitor rotenone on oxygen consumption and enzyme activity. It was partially purified by two sequential steps of fast protein liquid chromatographic techniques from n-octyl glucoside solubilization of the isolated mitochondria of both parasites. In addition, physical and kinetic properties of the malarial enzymes were compared to the host mouse liver mitochondrial respiratory complex I either as intact or as partially purified forms. The malarial enzyme required both NADH and ubiquinone for maximal catalysis. Furthermore, rotenone and plumbagin (ubiquinone analog) showed strong inhibitory effect against the purified malarial enzymes and had antimalarial activity against in vitro growth of P. falciparum. Some unique properties suggest that the enzyme could be exploited as chemotherapeutic target for drug development, and it may have physiological significance in the mitochondrial metabolism of the parasite.     

The ATP-binding cassette (ABC) gene family of Plasmodium falciparum

Multidrug resistance (MDR) in mammalian tumour cells is mediated by P-glycoproteins. The apparent similarities between MDR and the chloroquine-resistance phenotype (CQR) in Plasmodium falciparum suggests that homologous proteins may be involved. In mammals, P-glycoproteins are encoded by mdr genes that are a subset of a super-family characterized by ATP-binding cassettes (ABC). Three genes, pfmdr1, pfmdr2 and pfef3-rl, have been identified in P. falciparum that have homology to the ABC transporter gene family. Each protein encoded by these genes has a distinct structure, suggesting functional differences between the three. Justin Rubio and Alan Cowman here discuss the structure and possible function of the ABC proteins from P. falciparum and evidence that the protein encoded by the pfmdr1 gene can influence quinoline-containing antimalarial drug-resistance phenotypes.     

Drug susceptibility of Plasmodium falciparum from polyclonal isolates

Msp-1 and Msp-2 genes, each present as a unique copy in the genome of Plasmodium, contain polymorphic repeats in bloc 2. We studied allelic polymorphism of Msp-1 and Msp-2 by amplifying bloc 2 with a fluorescent primer, and analysing the fragment generated. We validated this method by mixing two cloned strains: chloroquine-susceptible HB3-Honduras and chloroquine-resistant FCM29-Cameroon. This method was then used to quantify the clones in natural isolates of 19 infected persons during quinine treatment. The fragment analysis method detects efficiently clone numbers and the proportions of each in isolates.     

The heat shock protein 40 family of the malaria parasite Plasmodium falciparum

Few diseases have had such a profound influence on human evolution and history as malaria. Despite intense efforts malaria infection continues to be a major killer. The causative agent of malaria, the unicellular eukaryote Plasmodium, displays a fascinating biology in which ubiquitous cellular concepts are modified to serve the particular needs of the malaria parasite. In this review, we explore how Plasmodium utilizes the heat shock protein 40 system, a chaperone system that ensures correct protein folding under normal and stress conditions. We highlight the peculiarities of the Plasmodium system and discuss whether any components of the system might be exploited for intervention strategies against this debilitating disease.     

NMR assignments of oxidised thioredoxin from Plasmodium falciparum

During its life cycle, the malaria parasite Plasmodium falciparum is found intracellular to human erythrocytes, where its survival and ability to multiply critically depends on the control of the environment redox state. Thioredoxin is a small protein containing 104 amino acids that is part of the parasite specific redox system. During the catalytic cycle it alternates between a reduced and oxidised form. Here we report the complete resonance assignment of Plasmodium falciparum thioredoxin in its oxidized form by heteronuclear multidimensional spectroscopy. The obtained chemical shifts differ significantly from those reported earlier for this protein in its reduced state.     

Molecular characterisation of drug-resistant Plasmodium falciparum from Thailand

Background  

The increasing levels of Plasmodium falciparum resistance to chloroquine (CQ) in Thailand have led to the use of alternative antimalarials, which are at present also becoming ineffective. In this context, any strategies that help improve the surveillance of drug resistance, become crucial in overcoming the problem.     

A database for Plasmodium falciparum protein models

There is an urgent need for developing alternate strategies to combat Malaria caused by Plasmodium falciparum (P. falciparum) because of growing drug resistance and increased incidents of infection in humans. 3D models of P. falciparum annotated proteins using molecular modeling techniques will enhance our understanding about the mechanism of host parasite interactions for the identification of drug targets and malarial vaccine design. Potential structural templates for P. falciparum annotated proteins were selected from PDB (protein databank) using BLASTP (basic local alignment search tool for proteins). This exercise identified 476 Plasmodium proteins with one or more known structural templates (>or= 40 % identity) for further modeling. The pair-wise sequence alignments generated for protein modeling were manually checked for error. The models were then constructed using MODELLER (a comparative protein modelling program for modelling protein structures) followed by energy minimization in AMBER force field and checked for error using PROCHECK. AVAILABILITY: http://bioinfo.icgeb.res.in/codes/model.html.     

Glycobiology of Plasmodium falciparum

The human malaria parasite, Plasmodium falciparum, has as its only glycoconjugate GPI anchors. These structures, present in essentially all parasite surface proteins, are associated with disease pathology. In contrast, the parasite depends for essential recognition events on saccharides associated with host cell glycoproteins and proteoglycans.     

Double-drug development against antioxidant enzymes from Plasmodium falciparum

Abstract

New drugs against malaria are urgently and continuously needed. Plasmodium parasites are exposed to higher fluxes of reactive oxygen species and need high activities of intracellular antioxidant systems. A most important antioxidative system consists of (di)thiols which are recycled by disulfide reductases (DR), namely both glutathione reductases (GR) of the malarial parasite Plasmodium falciparum and man, and the thioredoxin reductase (TrxR) of P. falciparum. The aim of our interdisciplinary research is to substantiate DR inhibitors as antimalarial agents. Such compounds are active per se but, in addition, they can reverse thiol-based resistance against other drugs in parasites. Reversal of drug resistance by DR inhibitors is currently investigated for the commonly used antimalarial drug chloroquine (CQ). Our recent strategy is based on the synthesis of inhibitors of the glutathione reductases from parasite and host erythrocyte. With the expectation of a synergistic or additive effect, double-headed prodrugs were designed to be directed against two different and essential functions of the malarial parasite P. falciparum, namely glutathione regeneration and heme detoxification. The prodrugs were prepared by linking bioreversibly a GR inhibitor to a 4-aminoquinoline moiety which is known to concentrate in the acidic food vacuole of parasites. Drug-enzyme interaction was correlated with antiparasitic action in vitro on strains resistant towards CQ and in vivo in Plasmodium berghei-infected mice as well as absence of cytotoxicity towards human cells. Because TrxR of P. falciparum was recently shown to be responsible for the residual glutathione disulfide-reducing capacity observed after GR inhibition in P. falciparum, future development of antimalarial drug-candidates that act by perturbing the redox equilibrium of parasites is based on the design of new double-drugs based on TrxR inhibitors as potential antimalarial drug candidates.     

Double-drug development against antioxidant enzymes from Plasmodium falciparum

New drugs against malaria are urgently and continuously needed. Plasmodium parasites are exposed to higher fluxes of reactive oxygen species and need high activities of intracellular antioxidant systems. A most important antioxidative system consists of (di)thiols which are recycled by disulfide reductases (DR), namely both glutathione reductases (GR) of the malarial parasite Plasmodium falciparum and man, and the thioredoxin reductase (TrxR) of P. falciparum. The aim of our interdisciplinary research is to substantiate DR inhibitors as antimalarial agents. Such compounds are active per se but, in addition, they can reverse thiol-based resistance against other drugs in parasites. Reversal of drug resistance by DR inhibitors is currently investigated for the commonly used antimalarial drug chloroquine (CQ). Our recent strategy is based on the synthesis of inhibitors of the glutathione reductases from parasite and host erythrocyte. With the expectation of a synergistic or additive effect, double-headed prodrugs were designed to be directed against two different and essential functions of the malarial parasite P. falciparum, namely glutathione regeneration and heme detoxification. The prodrugs were prepared by linking bioreversibly a GR inhibitor to a 4-aminoquinoline moiety which is known to concentrate in the acidic food vacuole of parasites. Drug-enzyme interaction was correlated with antiparasitic action in vitro on strains resistant towards CQ and in vivo in Plasmodium berghei-infected mice as well as absence of cytotoxicity towards human cells. Because TrxR of P. falciparum was recently shown to be responsible for the residual glutathione disulfide-reducing capacity observed after GR inhibition in P. falciparum, future development of antimalarial drug-candidates that act by perturbing the redox equilibrium of parasites is based on the design of new double-drugs based on TrxR inhibitors as potential antimalarial drug candidates.     

Molecular models of protein kinase 6 from Plasmodium falciparum

Cyclin-dependent kinases (CDKs) have been identified as potential targets for development of drugs, mainly against cancer. These studies generated a vast library of chemical inhibitors of CDKs, and some of these molecules can also inhibit kinases identified in the Plasmodium falciparum genome. Here we describe structural models for Protein Kinase 6 from P. falciparum (PfPK6) complexed with Roscovitine and Olomoucine. These models show clear structural evidence for differences observed in the inhibition, and may help designing inhibitors for PfPK6 generating new potential drugs against malaria. Figure Ribbon diagram of PfPK6 complexed with a roscovitine and b olomoucine     

The crystal structure of superoxide dismutase from Plasmodium falciparum

Background  

Superoxide dismutases (SODs) are important enzymes in defence against oxidative stress. In Plasmodium falciparum, they may be expected to have special significance since part of the parasite life cycle is spent in red blood cells where the formation of reactive oxygen species is likely to be promoted by the products of haemoglobin breakdown. Thus, inhibitors of P. falciparum SODs have potential as anti-malarial compounds. As a step towards their development we have determined the crystal structure of the parasite's cytosolic iron superoxide dismutase.     

Immunogenicity of synthetic peptides derived from Plasmodium falciparum proteins

To obtain antibodies suitable to be used in an antigen-capture assay, we have identified, synthesized, and evaluated a series of peptides from different Plasmodium falciparum excretory-secretory proteins: glutamate-rich protein (GLURP); histidine-rich protein 2; histidine-rich protein 3; Falciparum interspersed repeat antigen and, serine-rich antigen homologous. Conformational as well as antigenic predictions were performed using the ANTHEPROT package. Chemical synthesis was carried out by the multiple manual synthesis using the t-boc strategy. The peptides were used as antigens for the preparation of polyclonal antibodies in rabbits. Out of the 14 peptide constructs, eight by ELISA and, six by MABA elicited antibodies that showed correspondence between the predictive study and the immunogenicity obtained in rabbits. All antipeptide (GLURP, HRP2, and FIRA) antisera were found to bind to the corresponding synthetic sequence in an ELISA assay. The binding activity and specificity of antibodies were determined by Western blot with supernatant culture from P. falciparum. Anti-GLURP (IMT-94 and IMT-200) antisera bound to five molecules present in supernatant with molecular weight of 73, 82, 116, 124, and 128 kDa. Anti-HRP2 (IMT-192) antisera recognized a band of 58 kDa. In both cases, the specific molecules were inhibited by preincubation with the homologous peptide. Anti-HRP3, anti-FIRA neither anti-SERPH antisera showed reactivity. Anti-peptides HRP2 antibodies recognized the recombinant protein present in Parasight-F test. The same way, synthetic peptides from HRPII molecule were recognized by monoclonal antibody present in the Parasight-F assay. Our results confirm the potential value of synthetic peptides when inducing monospecific polyclonal antibodies for the development of diagnostic tests based on the capture of antigens.