Biochemical and structural characterization of Plasmodium falciparum glutamate dehydrogenase 2

Glutamate dehydrogenases (GDHs) play key roles in cellular redox, amino acid, and energy metabolism, thus representing potential targets for pharmacological interventions. Here we studied the functional network provided by the three known glutamate dehydrogenases of the malaria parasite Plasmodium falciparum. The recombinant production of the previously described PfGDH1 as hexahistidyl-tagged proteins was optimized. Additionally, PfGDH2 was cloned, recombinantly produced, and characterized. Like PfGDH1, PfGDH2 is an NADP(H)-dependent enzyme with a specific activity comparable to PfGDH1 but with slightly higher K(m) values for its substrates. The three-dimensional structure of hexameric PfGDH2 was solved to 3.1 ? resolution. The overall structure shows high similarity with PfGDH1 but with significant differences occurring at the subunit interface. As in mammalian GDH1, in PfGDH2 the subunit-subunit interactions are mainly assisted by hydrogen bonds and hydrophobic interactions, whereas in PfGDH1 these contacts are mediated by networks of salt bridges and hydrogen bonds. In accordance with this, the known bovine GDH inhibitors hexachlorophene, GW5074, and bithionol were more effective on PfGDH2 than on PfGDH1. Subcellular localization was determined for all three plasmodial GDHs by fusion with the green fluorescent protein. Based on our data, PfGDH1 and PfGDH3 are cytosolic proteins whereas PfGDH2 clearly localizes to the apicoplast, a plastid-like organelle specific for apicomplexan parasites. This study provides new insights into the structure and function of GDH isoenzymes of P. falciparum, which represent potential targets for the development of novel antimalarial drugs.     

Independent translocation of two micronemal proteins in developing Plasmodium falciparum merozoites

Apical membrane antigen 1 of Plasmodium falciparum (PfAMA1) contains an N-terminal propeptide that is removed prior to the translocation of the mature protein onto the merozoite surface. We localized unprocessed PfAMA1 to the microneme organelles of the intraerythrocytic schizont. The results have suggested that the processed form of PfAMA1 translocates from the microneme compartment independently of another microneme protein, EBA175, which is also involved in the invasion of human erythrocytes.     

Functional characterization of human nucleosome assembly protein 1-like proteins as histone chaperones

Nucleosome Assembly Protein 1 (NAP1) is a highly conserved histone chaperone protein suspected to be involved in the dynamical regulation of the histone H2A-H2B hetero-dimer. However, the exact mechanism by which NAP1-like proteins act is currently unknown. In this work, we characterized the biochemical properties of two human NAP1-like proteins, hNAP1L1 and hNAP1L4, including a previously uncharacterized subtype, with the aim of determining their exact mechanistic role. Both hNAP1L1 and hNAP1L4 were found to be localized mainly to the cytoplasm and a minor population of them was suggested to be in the nucleus. Biochemical analyses demonstrated that both hNAP1L1 and hNAP1L4 mediated nucleosome formation. In addition, hNAP1L1 was shown to possess a significantly greater nucleosome disassembly activity than hNAP1L4, suggesting that hNAP1L1 and hNAP1L4 may play distinct roles in the regulation of histone dynamics. Building upon this initial discovery we also found that histone H2A-H2B and various histone H2A variants-H2B dimers were found to associate with both hNAP1L1 and hNAP1L4 in cell extracts. These results suggest that human NAP1-like proteins play overlapping roles in transport and deposition of histone H2A-H2B or H2A variants-H2B dimers on chromatin and nonoverlapping roles in nucleosome disassembly.     

Biochemical characterization of Plasmodium falciparum Sir2, a NAD+-dependent deacetylase

In Plasmodium falciparum, the causative agent of cerebral malaria, silent information regulator 2 (Sir2) has been implicated in pathogenesis through its role in var gene silencing. P. falciparum Sir2 (PfSir2) in addition to the catalytic core, has a 13 residue N-terminal and 4 residue C-terminal extension over the shorter Archaeoglobus fulgidus Sir2. In this paper, we highlight our studies aimed at understanding the kinetic mechanism of PfSir2 and the role of N- and C-terminal extensions in protein function and oligomerization. Bisubstrate kinetic analysis showed that PfSir2 exhibits a rapid equilibrium ordered sequential mechanism, with peptide binding preceding NAD(+). This study also reports on surfactin as a novel Sir2 inhibitor exhibiting competitive inhibition with respect to NAD(+) and uncompetitive inhibition with acetylated peptide. This inhibition pattern with surfactin provides further support for ordered binding of substrates. Surfactin was also found to be a potent inhibitor of intra-erythrocytic growth of P. falciparum with 50% inhibitory concentration in the low micromolar range. PfSir2, like the yeast homologs (yHst2 and Sir2p), is a trimer in solution. However, dissociation of trimer to monomers in the presence of NAD(+) is characteristic of the parasite enzyme. Oligomerization studies on N- and/or C-terminal deletion constructs of PfSir2 highlight the role of C-terminus of the protein in mediating homotrimerization. N-terminal deletion resulted in reduced catalytic efficiency although substrate affinity was not altered in the constructs. Interestingly, deletion of both the ends relaxed NAD(+) specificity.     

Purification and characterization of an aminopeptidase from Plasmodium falciparum

A soluble aminopeptidase from Plasmodium falciparum was purified by high performance liquid chromatography. The enzyme has a molecular weight of 100 000 and pI 6.8. Activity can be monitored conveniently with L-alanine-p-nitroanilide or L-leucine-p-nitroanilide at 405 nm or with L-leucine-7-amido-4-methylcoumarin in a fluorescence assay. The enzyme is inhibited by bestatin and phosphoramidone but not by leupeptin, chymostatin, antipain or pepstatin. pH-rate studies indicated the presence of a group on the free enzyme, pKa = 6.6, which must be in the conjugate base form for activity. The aminopeptidase has an essential sulfhydryl group at the active site which is rapidly modified by Hg2+ or Zn2+, is slowly modified by p-hydroxymercuribenzoate, but is not accessible to iodoacetamide or N-ethylmaleimide. The aminopeptidase is inhibited noncompetitively by chloroquine, mefloquine and quinacrine (Ki = 410, 280 and 20 microM, respectively) but is not inhibited by quinine or primaquine. Hemin does not inhibit. Complexation of hemin with quinacrine prevents inhibition by quinacrine.     

Identification of proteins from Plasmodium falciparum that are homologous to reticulocyte binding proteins in Plasmodium vivax

Plasmodium falciparum infections can be fatal, while P. vivax infections usually are not. A possible factor involved in the greater virulence of P. falciparum is that this parasite grows in red blood cells (RBCs) of all maturities whereas P. vivax is restricted to growth in reticulocytes, which represent only approximately 1% of total RBCs in the periphery. Two proteins, expressed at the apical end of the invasive merozoite stage from P. vivax, have been implicated in the targeting of reticulocytes for invasion by this parasite. A search of the P. falciparum genome databases has identified genes that are homologous to the P. vivax rbp-1 and -2 genes. Two of these genes are virtually identical over a large region of the 5' end but are highly divergent at the 3' end. They encode high-molecular-mass proteins of >300 kDa that are expressed in late schizonts and localized to the apical end of the merozoite. To test a potential role in merozoite invasion of RBCs, we analyzed the ability of these proteins to bind to mature RBCs and reticulocytes. No binding to mature RBCs or cell preparations enriched for reticulocytes was detected. We identified a parasite clone that lacks the gene for one of these proteins, showing that the gene is not required for normal in vitro growth. Antibodies to these proteins can inhibit merozoite invasion of RBCs.     

Low-complexity regions in Plasmodium falciparum proteins

Full-sequence data available for Plasmodium falciparum chromosomes 2 and 3 are exploited to perform a statistical analysis of the long tracts of biased amino acid composition that characterize the vast majority of P. falciparum proteins and to make a comparison with similarly defined tracts from other simple eukaryotes. When the relatively minor subset of prevalently hydrophobic segments is discarded from the set of low-complexity segments identified by current segmentation methods in P. falciparum proteins, a good correspondence is found between prevalently hydrophilic low-complexity segments and the species-specific, rapidly diverging insertions detected by multiple-alignment procedures when sequences of bona fide homologs are available. Amino acid preferences are fairly uniform in the set of hydrophilic low-complexity segments identified in the two P. falciparum chromosomes sequenced, as well as in sequenced genes from Plasmodium berghei, but differ from those observed in Saccharomyces cerevisiae and Dictyostelium discoideum. In the two plasmodial species, amino acid frequencies do not correlate with properties such as hydrophilicity, small volume, or flexibility, which might be expected to characterize residues involved in nonglobular domains but do correlate with A-richness in codons. An effect of phenotypic selection versus neutral drift, however, is suggested by the predominance of asparagine over lysine.     

Characterisation of two novel proteins from the asexual stage of Plasmodium falciparum, H101 and H103

The merozoite surface of the pathogenic malaria parasite Plasmodium falciparum is comprised of proteins that are important for the identification and invasion of human red cells. Merozoite surface protein (MSP)3 is a polymorphic protein associated with the surface of merozoites and is also a vaccine candidate. A distinct feature of the MSP3 sequence is three blocks of alanine-rich heptad repeats that are predicted to form an intramolecular coiled-coil. Three orthologues of MSP3 that also contain alanine-rich heptad repeats have been described in P. vivax and we therefore searched the P. falciparum genome database for MSP3 paralogues. We have identified two genes, H101 and H103 related to MSP3, however like another MSP3 paralogue, MSP6, H101 and H103 do not contain heptad repeats. H101 and H103 are expressed during the asexual cycle and immunofluorescence indicates H103 localises to the merozoite surface as a peripheral membrane protein. Transfected parasite lines that express truncated forms of H101 or H103 were viable and grew at the same rate as the parental parasite line. This result may reflect redundancy in function among members of the MSP3/MSP6 gene family as has been described for other families of paralogue genes in P. falciparum.     

Molecular characterization of bifunctional hydroxymethyldihydropterin pyrophosphokinase-dihydropteroate synthase from Plasmodium falciparum

A 2118-base pair gene encoding the bifunctional hydroxymethyldihydropterin pyrophosphokinase-dihydropteroate syntheses of Plasmodium falciparum (pfPPPK-DHPS) was expressed under the control of the T5 promoter in a DHPS-deficient Escherichia coli strain. The enzyme was purified to near homogeneity using nickel affinity chromatography followed by gel filtration and migrates as an intense band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with apparent mass of approximately 83 kDa. Gel filtration suggested that the native pfPPPK-DHPS might exist as a tetramer of identical subunits. The enzyme was found to be Mg2+ - and ATP-dependent and had optimal temperature ranging from 37 to 45 degrees C with peak activity at pH 10. Sodium chloride and potassium chloride at 0.2 and 0.4 M, respectively, activated the activity of the enzyme but higher salt concentrations were inhibitory. Guanidine-HCl and urea inhibited the enzyme activity by 50% at 0.25 and 0.9 M, respectively. Kinetic properties of the recombinant pfPPPK-DHPS were investigated. Sulfathiazole and dapsone were potent inhibitors of pfPPPK-DHPS, whilst sulfadoxine, sulfanilamide, sulfacetamide and p-aminosalicylic acid were less inhibitory. Our construct provides an abundant source of recombinant pfPPPK-DHPS for crystallization and drug screening.     

Biochemical characterization of Plasmodium falciparum hypoxanthine-guanine-xanthine phosphorybosyltransferase: role of histidine residue in substrate selectivity

The enzyme hypoxanthine-guanine phosphorybosyltransferase (HGPRT) in the malarial parasite Plasmodium falciparum (Pf) is central to the salvage pathway for purine nucleotide biosynthesis and is a potential antimalarial chemotherapeutic target. The pH profile of the enzyme activity using xanthine as a substrate shows the possible involvement of a histidine residue in the activity of the enzyme. Chemical modification studies using diethylpyrocarbonate (DEPC) also corroborate this hypothesis. A comparative sequence alignment of Pf HGPRT with the human, Tricomonus foetus and Toxoplasma gondii HGPRT, coupled with the 3D structural alignment between these enzymes indicated that a histidine residue at position 196 of the Pf HGPRT sequence was located in the close proximity to the active site. Site directed mutagenesis of this histidine residue to lysine (the corresponding residue in the human enzyme) specifically abrogated xanthine and guanine utilization of the enzyme without affecting the conversion of hypoxanthine to its corresponding nucleotide. The mechanism of action for this enzyme was evaluated by steady state kinetics for the substrates xanthine, guanine and PRPP and product inhibition studies. The results indicate the possibility of ping-pong mechanism for the enzyme in contrast to the ternary complex mechanism followed by the human enzyme. These results show that the difference in human and malarial HGPRT can be gainfully exploited to design specific inhibitor for this enzyme.     

Heterologous expression of proteins from Plasmodium falciparum: results from 1000 genes

As part of a structural genomics initiative, 1000 open reading frames from Plasmodium falciparum, the causative agent of the most deadly form of malaria, were tested in an E. coli protein expression system. Three hundred and thirty-seven of these targets were observed to express, although typically the protein was insoluble. Sixty-three of the targets provided soluble protein in yields ranging from 0.9 to 406.6 mg from one liter of rich media. Higher molecular weight, greater protein disorder (segmental analysis, SEG), more basic isoelectric point (pI), and a lack of homology to E. coli proteins were all highly and independently correlated with difficulties in expression. Surprisingly, codon usage and the percentage of adenosines and thymidines (%AT) did not appear to play a significant role. Of those proteins which expressed, high pI and a hypothetical annotation were both strongly and independently correlated with insolubility. The overwhelmingly important role of pI in both expression and solubility appears to be a surprising and fundamental issue in the heterologous expression of P. falciparum proteins in E. coli. Twelve targets which did not express in E. coli from the native gene sequence were codon-optimized through whole gene synthesis, resulting in the (insoluble) expression of three of these proteins. Seventeen targets which were expressed insolubly in E. coli were moved into a baculovirus/Sf-21 system, resulting in the soluble expression of one protein at a high level and six others at a low level. A variety of factors conspire to make the heterologous expression of P. falciparum proteins challenging, and these observations lay the groundwork for a rational approach to prioritizing and, ultimately, eliminating these impediments.     

Biochemical and electron paramagnetic resonance study of the iron superoxide dismutase from Plasmodium falciparum

Recombinant iron-containing superoxide dismutase (Fe-SOD) from Plasmodium falciparum was produced in a SOD-deficient strain of Escherichia coli, purified and characterised. The enzyme is a dimer, which contains 1.7 Fe equivalents and is sensitive to hydrogen peroxide (H(2)O(2)). Electron paramagnetic resonance (EPR) analysis showed two different signals, reflecting the presence of two different types of high-spin Fe sites with different symmetries. The role of the W71 residue during inactivation by H(2)O(2) of the P. falciparum Fe-SOD was studied by site-directed mutagenesis. First, the W71V mutation led to a change in the relative proportion of the two Fe-based EPR signals. Second, the mutant protein was almost as active as the wild-type (WT) protein but more sensitive to heat inactivation. Third, resistance to H(2)O(2) was only slightly increased indicating that W71 was marginally responsible for the sensitivity of Fe-SOD to H(2)O(2). A molecular model of the subunit was designed to assist in interpretation of the results. The fact that the parasite SOD does not belong to classes of SOD present in humans may provide a novel approach for the design of antimalarial drugs.     

Extremely diverged actin proteins in Plasmodium falciparum

In a previous paper the nucleotide sequence of a complementary DNA coding for a Plasmodium falciparum actin protein (pf-actin I) has been described. Here we present evidence that the genome of this human malaria parasite encodes for still another actin protein (pf-actin II). Via nucleotide sequence analysis of its coding DNA we established the amino acid sequence of this protein. This sequence was compared with the pf-actin I sequence and those of a number of other actins. The comparative studies revealed that the amino acid sequence of pf-actin II is very diverged from the actins known thus far. The mutual amino acid sequence similarity between both Plasmodium actins is also very poor and in fact the observed value is the lowest ever seen between actins within one species. Furthermore, the studies suggest that the actin genes from sporozoa and ciliated protozoa, but not those from amoebae, have evolved from a common primitive ancestor. It is likely, however, that during evolution the actin sequences in these protozoa are not as well conserved as in other eukaryotic lineages.     

Microtubule associated motor proteins of Plasmodium falciparum merozoites

We have studied the occurrence, stage specificity and cellular location of key molecules associated with microtubules in Plasmodium falciparum merozoites. Antibodies to gamma tubulin, conventional kinesin and cytoplasmic dynein were used to determine the polarity of merozoite microtubules (mt), the stage specificity of the motor proteins and their location during merozoite development. We conclude that the minus ends of the mts are located at their apical pole. Kinesin was present throughout the lifecycle, appearing as a distinct crescent at the apex of developing merozoites. The vast majority of cytoplasmic dynein reactivity occurred in late merogony, also appearing at the merozoite apex. Destruction of mt with dinitroanilines did not affect the cellular location of kinesin or dynein. In invasion assays, dynein inhibitors reduced the number of ring stage parasites. Our results show that both conventional kinesin and cytoplasmic dynein are abundant, located at the negative pole of the merozoite mt and, intriguingly, appear there only in very late merogony, prior to merozoite release and invasion.     

Identification and characterization of the Plasmodium falciparum RhopH2 ortholog in Plasmodium vivax

Plasmodium vivax is one of the most important human malaria species that is geographically widely endemic and potentially affects a larger number of people than its more notorious cousin, Plasmodium falciparum. During invasion of red blood cells, the parasite requires the intervention of high molecular weight complex rhoptry proteins (RhopH) that are also essential for cytoadherence. PfRhopH2, a member of the RhopH multigene family, has been characterized as being crucial during P. falciparum infection. This study describes identifying and characterizing the pfrhoph2 orthologous gene in P. vivax (hereinafter named pvrhoph2). The PvRhopH2 is a 1,369-amino acid polypeptide encoded by PVX_099930 gene, for which orthologous genes have been identified in other Plasmodium species by bioinformatic approaches. Both P. falciparum and P. vivax genes contain nine introns, and there is a high degree of similarity between the deduced amino acid sequences of the two proteins. Moreover, PvRhopH2 contains a signal peptide at its N-terminus and 12 cysteines predominantly in its C-terminal half. PvRhopH2 is localized in one of the apical organelles of the merozoite, the rhoptry, and the localization pattern is similar to that of PfRhopH2 in P. falciparum. The recombinant PvRhopH2 protein is recognized by serum antibodies of patients naturally exposed to P. vivax, suggesting that PvRhopH2 is immunogenic in humans.     

Purification and characterization of Plasmodium falciparum succinate dehydrogenase

Succinate dehydrogenase (SDH), a Krebs cycle enzyme and complex II of the mitochondrial electron transport system was purified to near homogeneity from the human malarial parasite Plasmodium falciparum cultivated in vitro by FPLC on Mono Q, Mono S and Superose 6 gel filtration columns. The malarial SDH activity was found to be extremely labile. Based on Superose 6 FPLC, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and nondenaturing-PAGE analyses, it was demonstrated that the malarial enzyme had an apparent native molecular mass of 90 +/- 8 kDa and contained two major subunits with molecular masses of 55 +/- 6 and 35 +/- 4 kDa (n = 8). The enzymatic reaction required both succinate and coenzyme Q (CoQ) for its maximal catalysis with Km values of 3 and 0.2 microM, and k(cat) values of 0.11 and 0.06 min(-1), respectively. Catalytic efficiency of the malarial SDH for both substrates were found to be relatively low (approximately 600-5000 M(-1) s(-1)). Fumarate, malonate and oxaloacetate were found to inhibit the malarial enzyme with Ki values of 81, 13 and 12 microM, respectively. The malarial enzyme activity was also inhibited by substrate analog of CoQ, 5-hydroxy-2-methyl-1,4-naphthoquinone, with a 50% inhibitory concentration of 5 microM. The quinone had antimalarial activity against the in vitro growth of P. falciparum with a 50% inhibitory concentration of 0.27 microM and was found to completely inhibit oxygen uptake of the parasite at a concentration of 0.88 microM. A known inhibitor of mammalian mitochondrial SDH, 2-thenoyltrifluoroacetone. had no inhibitory effect on both the malarial SDH activity and the oxygen uptake of the parasite at a concentration of 50 microM. Many properties observed in the malarial SDH were found to be different from the host mammalian enzyme.     

Molecular cloning and characterization of Plasmodium falciparum transketolase

The pentose phosphate pathway (PPP) is an important metabolic pathway for yielding reducing power in the form of NADPH and production of pentose sugar needed for nucleic acid synthesis. Transketolase, the key enzyme of non-oxidative arm of PPP, plays a vital role in the survival/replication of the malarial parasite. This enzyme in Plasmodium falciparum is a novel drug target as it has least homology with the human host. In the present study, the P. falciparum transketolase (PfTk) was expressed, localized and biochemically characterized. The recombinant PfTk harboring transketolase activity catalyzed the oxidation of donor substrates, fructose-6-phosphate (F6P) and hydroxypyruvate (HP), with K(m)(app) values of 2.25 and 4.78 mM, respectively. p-Hydroxyphenylpyruvate (HPP) was a potent inhibitor of PfTk, when hydroxypyruvate was used as a substrate, exhibiting a K(i) value of 305 microM. At the same time, noncompetitive inhibition was observed with F6P. The native PfTk is a hexamer with subunit molecular weight of 70kDa, which on treatment with low concentrations of guanidine hydrochloride (GdmCl) dissociated into functionally active dimers. This protein was localized in the cytosol and nucleus of the parasite as studied by confocal microscopy. A model structure of PfTk was constructed based on the crystal structure of the transketolases of Saccharomyces cerevisae, Leishmania mexicana and Escherichia coli to assess the structural homology. Consistent with the homology modeling predictions, CD analysis indicated that PfTk is composed of 39% alpha-helices and 26% beta-sheets. The availability of a structural model of PfTk and the observed differences in its kinetic properties compared to the host enzyme may facilitate designing of novel inhibitors of PfTk with potential anti-malarial activity.     

Molecular cloning and characterization of Plasmodium falciparum transportin

A cDNA encoding transportin, a protein involved in the nuclear import of M9 nuclear localization signal-bearing proteins, has been cloned from the malaria parasite Plasmodium falciparum. The complete cDNA consists of 3,667 bp encoding 1,136 amino acid residues. Amino acid sequence analysis revealed that Ran-GTP and M9 binding domains are highly conserved in P. falciparum, suggesting that the transportin-mediated nuclear transport pathway exists in this protozoan parasite. Southern blot analysis revealed that the transportin gene exists as a single copy in the malarial genome.     

Plasmodium falciparum infection-induced changes in erythrocyte membrane proteins

Over the past decade, advances in proteomic and mass spectrometry techniques and the sequencing of the Plasmodium falciparum genome have led to an increasing number of studies regarding the parasite proteome. However, these studies have focused principally on parasite protein expression, neglecting parasite-induced variations in the host proteome. Here, we investigated P. falciparum-induced modifications of the infected red blood cell (iRBC) membrane proteome, taking into account both host and parasite proteome alterations. Furthermore, we also determined if some protein changes were associated with genotypically distinct P. falciparum strains. Comparison of host membrane proteomes between iRBCs and uninfected red blood cells using fluorescence-based proteomic approaches, such as 2D difference gel electrophoresis revealed that more than 100 protein spots were highly up-represented (fold change increase greater than five) following P. falciparum infection for both strains (i.e. RP8 and Institut Pasteur Pregnancy Associated Malaria). The majority of spots identified by mass spectrometry corresponded to Homo sapiens proteins. However, infection-induced changes in host proteins did not appear to affect molecules located at the outer surface of the plasma membrane. The under-representation of parasite proteins could not be attributed to deficient parasite protein expression. Thus, this study describes for the first time that considerable host protein modifications were detected following P. falciparum infection at the erythrocyte membrane level. Further analysis of infection-induced host protein modifications will improve our knowledge of malaria pathogenesis.