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     

A genomic perspective of protein kinases in Plasmodium falciparum

Protein kinases are central to regulation of cellular signaling in the eukaryotes. Well-conserved and lineage-specific protein kinases have previously been identified from various completely sequenced genomes of eukaryotes. The current work describes a genome-wide analysis for protein kinases encoded in the Plasmodium falciparum genome. Using a few different profile matching methods, we have identified 99 protein kinases or related proteins in the parasite genome. We have classified these kinases into subfamilies and analyzed them in the context of noncatalytic domains that occur in these catalytic kinase domain-containing proteins. Compared to most eukaryotic protein kinases, these sequences vary significantly in terms of their lengths, inserts in catalytic domains, and co-occurring domains. Catalytic and noncatalytic domains contain long stretches of repeats of positively charged and other polar amino acids. Various components of the cell cycle, including 4 cyclin-dependent kinase (CDK) homologues, 2 cyclins, 1 CDK regulatory subunit, and 1 kinase-associated phosphatase, are identified. Identification of putative mitogen-activated protein (MAP) Kinase and MAP Kinase Kinase of P. falciparum suggests a new paradigm in the highly conserved signaling pathway of eukaryotes. The calcium-dependent kinase family, well represented in P. falciparum, shows varying domain combinations with EF-hands and pleckstrin homology domains. The analysis reveals a new subfamily of protein kinases having limited sequence similarity with previously known subfamilies. A new transmembrane kinase with 6 membrane-spanning regions is identified. Putative apicoplast targeting sequences have been detected in some of these protein kinases, suggesting their export to the apicoplast.     

Deciphering the key residues in Plasmodium falciparum beta-ketoacyl acyl carrier protein reductase responsible for interactions with Plasmodium falciparum acyl carrier protein

The type II fatty acid synthase (FAS) pathway of Plasmodium falciparum is a validated unique target for developing novel antimalarials, due to its intrinsic differences from the typeI pathway operating in humans. beta-Ketoacyl acyl carrier protein (ACP) reductase (FabG) performs the NADPH-dependent reduction of beta-ketoacyl-ACP to beta-hydroxyacyl-ACP, the first reductive step in the elongation cycle of fatty acid biosynthesis. In this article, we report intensive studies on the direct interactions of Plasmodium FabG and Plasmodium ACP in solution, in the presence and absence of its cofactor, NADPH, by monitoring the change in intrinsic fluorescence of P.falciparum FabG (PfFabG) and by surface plasmon resonance. To address the issue of the importance of the residues involved in strong, specific and stoichiometric binding of PfFabG to P.falciparum ACP (PfACP), we mutated Arg187, Arg190 and Arg230 of PfFabG. The activities of the mutants were assessed using both an ACP-dependent and an ACP-independent assay. The affinities of all the PfFabG mutants for acetoacetyl-ACP (the physiological substrate) were reduced to different extents as compared to wild-type PfFabG, but were equally active in biochemical assays with the substrate analog acetoacetyl-CoA. Kinetic analysis and studies of direct binding between PfFabG and PfACP confirmed the identification of Arg187 and Arg230 as critical residues for the PfFabG-PfACP interactions. Our studies thus reveal the significance of the positively charged/hydrophobic patch located adjacent to the active site cavities of PfFabG for interactions with PfACP.     

A Plasmodium falciparum copper-binding membrane protein with copper transport motifs

Formerly known as a hypoendemic malaria country, the Republic of Djibouti declared the goal of pre-eliminating malaria in 2006. The aim of the present study was to evaluate the prevalence of Plasmodium falciparum, Plasmodium vivax and mixed infections in the Djiboutian population by using serological tools and to identify potential determinants of the disease and hotspots of malaria transmission within the country. The prevalence of P. falciparum and P. vivax within the districts of the capital city and the rest of the Republic of Djibouti were assessed using 13 and 2 serological markers, respectively. The relationship between the immune humeral response to P. falciparum and P. vivax and variables such as age, gender, wealth status, urbanism, educational level, distance to rivers/lakes, living area, having fever in the last month, and staying in a malaria-endemic country more than one year was estimated and analysed by questionnaires administered to 1910 Djiboutians. Multivariate ordinal logistic regression models of the immune humeral response were obtained for P. falciparum and P. vivax. The P. falciparum and P. vivax seroprevalence rates were 31.5%, CI95% [29.4-33.7] and 17.5%, CI95% [15.8-19.3], respectively. Protective effects against P. falciparum and P. vivax were female gender, educational level, and never having visited a malaria-endemic area for more than one year. For P. falciparum only, a protective effect was observed for not having a fever in the last month, living more than 1.5 km away from lakes and rivers, and younger ages. This is the first study that assessed the seroprevalence of P. vivax in the Republic of Djibouti. It is necessary to improve knowledge of this pathogen in order to create an effective elimination programme. As supported by recent observations on the subject, the Republic of Djibouti has probably demonstrated a real decrease in the transmission of P. falciparum in the past seven years, which should encourage authorities to improve efforts toward elimination.     

Plasmodium falciparum expresses a multidrug resistance-associated protein

Plasmodium falciparum proteins that efflux toxic metabolic products such as oxidised glutathione (GSSG) are possible targets for anti-malarial drug development. Proteins capable of transporting GSSG and glutathione conjugates include the multidrug resistance-associated transporters (MRPs). A gene, PFA0590w, encoding a MRP homologue, has been identified in P. falciparum. Here we show the presence of full-length mRNA (5.5 kb) of this PfMRP in trophozoites by RT-PCR and Northern blotting. A polyclonal anti-PfMRP antibody generated against two unique, hydrophilic peptides in the predicted sequence produced a strong immunoreactive protein band of 210-215 kDa on Western blots of schizonts of chloroquine-sensitive and chloroquine-resistant strains, confirming expression of PfMRP protein. Using confocal microscopy the protein was seen to be localised at the edge of the schizonts with no obvious staining of the food vacuole. We suggest that PfMRP may act as the GSSG transporter in the parasite plasma membrane.     

Plasmodium falciparum merozoite surface protein 1.

In addition to the major carbohydrate moieties of the glycosylphosphatidylinositol (GPI) anchor, we report that Plasmodium falciparum merozoite surface protein 1 (MSP-1) bears O-GlcNAc modifications predominantly in beta-anomeric configuration, in both the C- and N-terminal portions of the protein. Subcellular fractionation of parasitized erythrocytes in the late trophozoite/schizont stage reveals that GPI-anchored C-terminal fragments of MSP-1 are recovered in Triton X-100 resistant, low-density membrane fractions. Our results suggest that O-GlcNAc-modified MSP-1 N-terminal fragments tend to localize within the parasitophorous vacuolar membrane while GPI-anchored MSP-1 C-terminal fragments associate with low-density, Triton X-100 resistant membrane domains (rafts), redistribute in the parasitized erythrocyte and are eventually shed as membrane vesicles that also contain the endogenous, GPI-linked CD59.     

Plasmodium falciparum: glycosylation status of Plasmodium falciparum circumsporozoite protein expressed in the baculovirus system

We expressed the main surface antigen of Plasmodium falciparum sporozoites, the circumsporozoite protein (CSP), in High Five (Trichoplusia ni) insect cells using the baculovirus system. Significant amounts of the recombinant protein could be obtained, as judged by SDS-PAGE, Western blot, and immunofluorescence analysis. The cellular localization for recombinant CSP was determined by immunofluorescence. The high fluorescence signal of the permeabilized cells, relative to that of fixed nonpermeabilized cells, revealed a clear intracellular localization of this surface antigen. Analysis of possible posttranslational modifications of CSP showed that this recombinant protein is only N-glycosylated in the baculovirus system. Although DNA-sequence analysis revealed a GPI-cleavage/attachment site, no GPI anchor could be demonstrated. These analyses show that the glycosylation status of this recombinant protein may not reflect its native form in P. falciparum. The impact of these findings on vaccine development will be discussed.     

Recombinant antibodies specific for the Plasmodium falciparum histidine-rich protein 2

Early diagnosis and appropriate treatment are key elements of malaria control programs in endemic areas. A major step forward in recent years has been the production and use of rapid diagnostic tests (RDTs) in settings where microscopy is impracticable. Many current RDTs target the Plasmodium falciparum histidine-rich protein 2 (PfHRP2) released in the plasma of infected individuals. These RDTs have had an indisputably positive effect on malaria management, but still present several limitations, including the poor characterization of the commercial monoclonal antibodies (mAbs) used for PfHRP2 detection, variable sensitivity and specificity, and high costs. RDT use is further limited by impaired stability caused by temperature fluctuations during transport and uncontrolled storage in field-based facilities. To circumvent such drawbacks, an alternative could be the development of well-characterized, stabilized recombinant antibodies, with high binding affinity and specificity. Here, we report the characterization of the cDNA sequences encoding the Fab fragment of F1110 and F1546, two novels anti-PfHRP2 mAbs. FabF1546 was produced in the Escherichia coli periplasm. Its properties of binding to the parasite and to a recombinant PfHRP-2 antigen were similar to those of the parental mAb. As the affinity and stability of recombinant antibodies can be improved by protein engineering, our results open a novel approach for the development of an improved RDT for malaria diagnosis.     

Recombinant antibodies specific for the Plasmodium falciparum histidine-rich protein 2

Early diagnosis and appropriate treatment are key elements of malaria control programs in endemic areas. A major step forward in recent years has been the production and use of rapid diagnostic tests (RDTs) in settings where microscopy is impracticable. Many current RDTs target the Plasmodium falciparum histidine-rich protein 2 (PfHRP2) released in the plasma of infected individuals. These RDTs have had an indisputably positive effect on malaria management, but still present several limitations, including the poor characterization of the commercial monoclonal antibodies (mAbs) used for PfHRP2 detection, variable sensitivity and specificity and high costs. RDT use is further limited by impaired stability caused by temperature fluctuations during transport and uncontrolled storage in field-based facilities. To circumvent such drawbacks, an alternative could be the development of well-characterized, stabilized recombinant antibodies, with high binding affinity and specificity. Here, we report the characterization of the cDNA sequences encoding the Fab fragment of F1110 and F1546, two novel anti-PfHRP2 mAbs. FabF1546 was produced in the Escherichia coli periplasm. Its properties of binding to the parasite and to a recombinant PfHRP-2 antigen were similar to those of the parental mAb. As the affinity and stability of recombinant antibodies can be improved by protein engineering, our results open a novel approach for the development of an improved RDT for malaria diagnosis.Key words: Plasmodium falciparum, malaria, histidine-rich protein, monoclonal antibodies, recombinant Fab, rapid diagnostic test     

1H-NMR studies of synthetic polypeptide models of Plasmodium falciparum circumsporozoite protein tandemly repeated sequence

The major immunodominant region of the coating protein of Plasmodium falciparum sporozoites contains multiple tandem copies of the sequence Asn-Ala-Asn-Pro (NANP). Current efforts for the development of an antisporozoite vaccine are focused on the synthesis of polypeptides reproducing part of the circumsporozoite protein repeat sequence and, in an attempt to relate conformational properties and biological response, 1H-nmr one- and two-dimensional studies of the synthetic models (NANP)2NA and (NANP)6 were carried out in water and water/methanol mixtures, at 400 and 500 MHz. In water, (NANP)6 undergoes fast conformational averaging. At variance, in water/methanol, the molecule appears to adopt an extensive structure, but detailed analysis is impaired by high spectral degeneracy. Based on the results obtained with (NANP)2NA and from preliminary experiments in water/trifluoroethanol, an interpretation is suggested for the (NANP)6 data in water/methanol in terms of a mixed sequence of beta I-turns and half-turns (or/and gamma I-turns) around the positions Ni-1-Pi-Ni + 1.     

Celastrol inhibits Plasmodium falciparum enoyl-acyl carrier protein reductase

Enoyl-acyl carrier protein reductase (ENR), a critical enzyme in type II fatty acid biosynthesis, is a promising target for drug discovery against hepatocyte-stage Plasmodium falciparum. In order to identify PfENR-specific inhibitors, we docked 70 FDA-approved, bioactive, and/or natural product small molecules known to inhibit the growth of whole-cell blood-stage P. falciparum into several PfENR crystallographic structures. Subsequent in vitro activity assays identified a noncompetitive low-micromolar PfENR inhibitor, celastrol, from this set of compounds.     

Traffic jams: protein transport in Plasmodium falciparum

Protein targeting in malaria parasites is a complex process, involving several cellular compartments that distinguish these cells from more familiar systems, such as yeast or mammals. At least a dozen distinct protein destinations are known. The best studied of these is the vestigial chloroplast (the apicoplast), but new tools promise rapid progress in understanding how Plasmodium falciparum and related apicomplexan parasites traffic proteins to their invasion-related organelles, and how they modify the host by trafficking proteins into its cytoplasm and plasma membrane. Here, Giel van Dooren and colleagues discuss recent insights into protein targeting via the secretory pathway in this fascinating and important system. This topic emerged as a major theme at the Molecular Approaches to Malaria conference, Lorne, Australia, 2-5 February 2000.     

Protein farnesyltransferase and protein prenylation in Plasmodium falciparum

Comparison of the malaria parasite and mammalian protein prenyltransferases and their cellular substrates is important for establishing this enzyme as a target for developing antimalarial agents. Nineteen heptapeptides differing only in their carboxyl-terminal amino acid were tested as alternative substrates of partially purified Plasmodium falciparum protein farnesyltransferase. Only NRSCAIM and NRSCAIQ serve as substrates, with NRSCAIM being the best. Peptidomimetics, FTI-276 and GGTI-287, inhibit the transferase with IC(50) values of 1 and 32 nm, respectively. Incubation of P. falciparum-infected erythrocytes with [(3)H]farnesol labels 50- and 22-28-kDa proteins, whereas [(3)H]geranylgeraniol labels only 22-28-kDa proteins. The 50-kDa protein is shown to be farnesylated, whereas the 22-28-kDa proteins are geranylgeranylated, irrespective of the labeling prenol. Protein labeling is inhibited more than 50% by either 5 microm FTI-277 or GGTI-298. The same concentration of inhibitors also inhibits parasite growth from the ring stage by 50%, decreases expression of prenylated proteins as measured with prenyl-specific antibody, and inhibits parasite differentiation beyond the trophozoite stage. Furthermore, differentiation specific prenylation of P. falciparum proteins is demonstrated. Protein labeling is detected predominantly during the trophozoite to schizont and schizont to ring transitions. These results demonstrate unique properties of protein prenylation in P. falciparum: a limited specificity of the farnesyltransferase for peptide substrates compared with mammalian enzymes, the ability to use farnesol to label both farnesyl and geranylgeranyl moieties on proteins, differentiation specific protein prenylation, and the ability of peptidomimetic prenyltransferase inhibitors to block parasite differentiation.     

Some conditions apply: Systems for studying Plasmodium falciparum protein function

Malaria, caused by infection with Plasmodium parasites, remains a significant global health concern. For decades, genetic intractability and limited tools hindered our ability to study essential proteins and pathways in Plasmodium falciparum, the parasite associated with the most severe malaria cases. However, recent years have seen major leaps forward in the ability to genetically manipulate P. falciparum parasites and conditionally control protein expression/function. The conditional knockdown systems used in P. falciparum target all 3 components of the central dogma, allowing researchers to conditionally control gene expression, translation, and protein function. Here, we review some of the common knockdown systems that have been adapted or developed for use in P. falciparum. Much of the work done using conditional knockdown approaches has been performed in asexual, blood-stage parasites, but we also highlight their uses in other parts of the life cycle and discuss new ways of applying these systems outside of the intraerythrocytic stages. With the use of these tools, the field’s understanding of parasite biology is ever increasing, and promising new pathways for antimalarial drug development are being discovered.     

Fitting hidden Markov models of protein domains to a target species: application to Plasmodium falciparum

Background

The identification of gene sets that are significantly impacted in a given condition based on microarray data is a crucial step in current life science research. Most gene set analysis methods treat genes equally, regardless how specific they are to a given gene set.

Results

In this work we propose a new gene set analysis method that computes a gene set score as the mean of absolute values of weighted moderated gene t-scores. The gene weights are designed to emphasize the genes appearing in few gene sets, versus genes that appear in many gene sets. We demonstrate the usefulness of the method when analyzing gene sets that correspond to the KEGG pathways, and hence we called our method P athway A nalysis with D own-weighting of O verlapping G enes (PADOG). Unlike most gene set analysis methods which are validated through the analysis of 2-3 data sets followed by a human interpretation of the results, the validation employed here uses 24 different data sets and a completely objective assessment scheme that makes minimal assumptions and eliminates the need for possibly biased human assessments of the analysis results.

Conclusions

PADOG significantly improves gene set ranking and boosts sensitivity of analysis using information already available in the gene expression profiles and the collection of gene sets to be analyzed. The advantages of PADOG over other existing approaches are shown to be stable to changes in the database of gene sets to be analyzed. PADOG was implemented as an R package available at: http://bioinformaticsprb.med.wayne.edu/PADOG/or http://www.bioconductor.org.     

A novel 40-kDa membrane-associated EF-hand calcium-binding protein in Plasmodium falciparum.

A 40-kDa sexual stage radiolabeled surface protein of Plasmodium falciparum, Pfs40, was previously identified as a potential target antigen of transmission blocking immunity by an immunogenetic approach. Synthetic oligonucleotide "guessmers," based on microsequenced tryptic peptides of Pfs40 purified by two-dimensional gel electrophoresis, were used to clone the full length cDNA and genomic DNA encoding Pfs40. The deduced amino acid sequence predicted an integral membrane protein containing five EF-hand calcium-binding domains. The biological activity of one or more of these domains was confirmed by binding of 45Ca to both native and recombinant Pfs40. Antisera to recombinant Pfs40 immunoprecipitated the native radiolabeled 40-kDa surface protein. The predicted noncytosolic membrane-associated localization of Pfs40 is unique within the EF-hand calcium-binding protein superfamily.     

A microprocessor-based control unit for Plasmodium falciparum culture apparatus

We describe a microprocessor–based control unit for apparatuswhich effects changes of culture medium on Plasmodium falciparumparasites maintained in vitro. The unit has required minimalattention during the two years it has been in operation andit has markedly facilitated the production of mature sexualstages of Plasmodium falciparum on a routine basis. Received on September 2, 1986; accepted on October 27, 1986     

15-Deoxyspergualin modulates Plasmodium falciparum heat shock protein function

Heat shock proteins are essential for the survival of all cells. The C-terminal EEVD motif of Hsp70 has previously been implicated in binding 15-deoxyspergualin (DSG), an immunosuppressant with antimalarial activity whose mechanism of action is uncertain. We report the cloning, overexpression, and characterization of three members of the heat shock family, PfHsp70-1 (an Hsp70 protein with a C-terminal EEVD motif), PfHsp70-2 (an Hsp70 protein without the EEVD motif), and PfHsp70 interacting protein. The chaperone activity of PfHsp70-1, and PfHsp70-2 was enhanced by ATP and by PfHip. Interestingly, while binding of protein substrates to PfHsp70-1, PfHsp70-2 and PfHip was unaffected in the presence of DSG, the ATP enhanced chaperone activity of PfHsp70-1 but not PfHsp70-2 was stimulated further by DSG. Our finding suggests that the binding partner of DSG in the parasite cellular milieu is PfHsp70-1 and paves the way for the elucidation of the mechanism of antimalarial action of DSG.     

Characterization of a membrane-associated rhoptry protein of Plasmodium falciparum

Invasive forms of apicomplexan parasites contain secretory organelles called rhoptries that are essential for entry into host cells. We present a detailed characterization of an unusual rhoptry protein of the human malaria parasite Plasmodium falciparum, the rhoptry-associated membrane antigen (RAMA) that appears to have roles in both rhoptry biogenesis and host cell invasion. RAMA is synthesized as a 170-kDa protein in early trophozoites, several hours before rhoptry formation and is transiently localized within the endoplasmic reticulum and Golgi within lipid-rich microdomains. Regions of the Golgi membrane containing RAMA bud to form vesicles that later mature into rhoptries in a process that is inhibitable by brefeldin A. Other rhoptry proteins such as RhopH3 and RAP1 are found in close apposition with RAMA suggesting direct protein-protein interactions. We suggest that RAMA is involved in trafficking of these proteins into rhoptries. In rhoptries, RAMA is proteolytically processed to give a 60-kDa form that is anchored in the inner face of the rhoptry membrane by means of the glycosylphosphatidylinositol anchor. The p60 RAMA form is discharged from the rhoptries of free merozoites and binds to the red blood cell membrane by its most C-terminal region. In early ring stages RAMA is found in association with the parasitophorous vacuole.