The NAD(+)-dependent Sir2p histone deacetylase is a negative regulator of chromosomal DNA replication

The establishment of DNA synthesis during the S phase is a multistep process that occurs in several stages beginning in late mitosis. The first step is the formation of a large prereplicative complex (pre-RC) at individual replication origins and occurs during exit from mitosis and entry into G1 phase. To better understand the genetic requirements for pre-RC formation, we selected chromosomal suppressors of a temperature-sensitive cdc6-4 mutant defective for pre-RC assembly. Loss-of-function mutations in the chromatin-modifying genes SIR2, and to a lesser extent in SIR3 and SIR4, suppressed the cdc6-4 temperature-sensitive lethality. This suppression was independent of the well-known silencing roles for the SIR proteins at the HM loci, at telomeres, or at the rDNA locus. A deletion of SIR2 uniquely rescued both the DNA synthesis defect of the cdc6-4 mutant and its severe plasmid instability phenotype for many origins. A SIR2 deletion suppressed additional initiation mutants affecting pre-RC assembly but not mutants that act subsequently. These findings suggest that Sir2p negatively regulates the initiation of DNA replication through a novel mechanism and reveal another connection between proteins that initiate DNA synthesis and those that establish silent heterochromatin in budding yeast.     

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.     

Biochemical characterization of the two nucleosome assembly proteins from Plasmodium falciparum

The human malaria parasite Plasmodium falciparum contains two nucleosome assembly proteins, which we have termed PfNAPS and PfNAPL. We have over-expressed, purified and characterized these proteins using biochemical and biophysical techniques. PfNAPS and PfNAPL exist as dimers in solution and circular dichroism studies suggest that they may have different three-dimensional protein structures. ELISA-based binding data also suggest that PfNAPS and PfNAPL preferentially interact with the H3-H4 tetramer histones over H2A and H2B histones. We show that the parasite lysate phosphorylates only PfNAPL and this phosphorylation can be inhibited by heparin suggesting a potential role of casein kinase II in this process. Immuno-fluorescence experiments revealed that both PfNAPS and PfNAPL were expressed in all erythrocytic stages of the parasite. PfNAPL was predominantly localised in the cytoplasm in asexual and sexual stages of the parasite. PfNAPS did not co-localise with PfNAPL and was more intimately associated with the parasite nucleus, most strikingly in P. falciparum gametocytes. Taken together, our data show that although PfNAPS and PfNAPL share histone chaperone acitivities, they are regulated differently by phosphorylation and are spatially segregated within the parasite. These proteins are therefore likely to play non-redundant roles as nucleosome assembly motors in the parasite.     

SirT3 is a nuclear NAD+-dependent histone deacetylase that translocates to the mitochondria upon cellular stress

In humans, there are at least seven Sir2-like proteins (SirT1-7) with diverse functions, including the regulation of chromatin structure, and metabolism. SirT3 levels have been shown to correlate with extended life span, to localize to the mitochondria, and to be highly expressed in brown adipose tissue. In humans, SirT3 exists in two forms, a full-length protein of approximately 44 kDa and a processed polypeptide lacking 142 amino acids at its N terminus. We found that SirT3 not only localizes to the mitochondria, but also to the nucleus under normal cell growth conditions. Both the full-length and processed forms of SirT3 target H4-K16 for deacetylation in vitro and can deacetylate H4-K16 in vivo when recruited to a gene. Using a highly specific antibody against the N terminus of SirT3, we found that SirT3 is transported from the nucleus to the mitochondria upon cellular stress. This includes DNA damage induced by Etoposide and UV-irradiation, as well as overexpression of SirT3 itself.     

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.     

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.     

Characterization of mimivirus NAD+-dependent DNA ligase

Mimivirus, a parasite of Acanthamoeba polyphaga, is the largest DNA virus known; it encodes a cornucopia of proteins with imputed functions in DNA replication, modification, and repair. Here we produced, purified, and characterized mimivirus DNA ligase (MimiLIG), an NAD+-dependent nick joining enzyme homologous to bacterial LigA and entomopoxvirus DNA ligase. MimiLIG is a 636-aa polypeptide composed of an N-terminal NAD+ specificity module (domain Ia), linked to nucleotidyltransferase, OB-fold, helix-hairpin-helix, and BRCT domains, but it lacks the tetracysteine Zn-binding module found in all bacterial LigA enzymes. MimiLIG requires conserved domain Ia residues Tyr36, Asp46, Tyr49, and Asp50 for its initial reaction with NAD+ to form the ligase-AMP intermediate, but not for the third step of phosphodiester formation at a preadenylylated nick. MimiLIG differs from bacterial LigA enzymes in that its activity is strongly dependent on the C-terminal BRCT domain, deletion of which reduced its specific activity in nick joining by 75-fold without affecting the ligase adenylylation step. The DeltaBRCT mutant of MimiLIG was impaired in sealing at a preadenylylated nick. We propose that eukaryal DNA viruses acquired the NAD+-dependent ligases by horizontal transfer from a bacterium and that MimiLIG predates entomopoxvirus ligase, which lacks both the tetracysteine and BRCT domains. We speculate that the dissemination of NAD+-dependent ligase from bacterium to eukaryotic virus might have occurred within an amoebal host.     

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.     

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 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.     

Molecular characterization of a 2-Cys peroxiredoxin from the human malaria parasite Plasmodium falciparum.

We have identified the 2-Cys peroxiredoxin (PfPrx-1) from the human malaria parasite Plasmodium falciparum. The PfPrx-1 showed the highest identity at amino acid level to the type II Prx among the currently known six subfamilies of mammalian Prx. The sequence identity between the PfPrx-1 and the previously reported 1-Cys Prx of P. falciparum (PfPrx-2), which corresponded to mammalian type VI Prx, was 25%. This suggests that the parasite possesses two Prx subfamilies. The PfPrx-1 showed significant sequence similarities with those of 2-Cys peroxiredoxins of plants in the BLASTX search. This may reflect the consequences of a genetic transfer from an algal endosymbiont to the parasite nucleus during evolution. The recombinant PfPrx-1 protein (rPfPrx-1) was expressed as a histidine fusion protein in Escherichia coli and purified with Ni chromatography. The rPfPrx-1 existed as dimers under non-reducing conditions and dissociated into monomers in the presence of dithiothreitol. The PfPrx-1 protein also exists as a dimer in the parasites themselves. The reduction of the oxidized enzyme by the donation of electrons from E. coli thioredoxin (Trx)/Trx reductase system was demonstrated in its reaction with H(2)O(2), using the rPfPrx-1 protein. These results suggested that the PfPrx-1 can act as a terminal peroxidase of the parasite Trx system. An elevated expression of the PfPrx-1 protein seen in the trophozoite, the stage with active metabolism, suggests an association of the parasite Trx system with its intracellular redox control.     

Molecular cloning, expression, characterization and mutation of Plasmodium falciparum guanylate kinase

The present work describes cloning, expression, purification, characterization, and mutation of Plasmodium falciparum guanylate kinase (PlasmoDB ID PFI1420w). Amino-acid sequence alignment revealed important differences especially in K42-V51, Y73-A77, and F100-L110, which include residues important for kinase activity, and at helix 3, which is important for domain movements. The catalytic efficiency for dGMP was 22-fold lower than that for GMP, whose value is the lowest among known guanylate kinases. dGMP was found to a competitive inhibitor for GMP with K_i = 0.148 mM and a mixed-type inhibitor with regard to ATP with measured K_i = 0.4 mM. The specificity constant (K_cat/K_m) of the four examined mutants varied for natural substrate GMP/dGMP, indicating the involvement of different mechanisms in substrate recognition and subsequent loop-domain movement. These results show that P. falciparum guanylate kinase is structurally and biochemically distinct from other guanylate kinases and could be a possible target in drug development.     

Expression, purification, biochemical characterization and inhibition of recombinant Plasmodium falciparum aldolase

The energy metabolism of the blood stage form of the human malaria parasite Plasmodium falciparum is adapted to the host cell. Like erythrocytes, P. falciparum merozoites lack a functional citric acid cycle. Generation of ATP depends therefore fully on the glycolytic pathway. Aldolase is a key enzyme of this pathway and a high degree of sequence diversity between parasite and host makes it a potential drug target. We have expressed the enzyme in its tetrameric form in Escherichia coli and the catalytic constants Vmax and Km of the recombinant enzyme correspond to the constants of parasite-derived aldolase. Rabbit antibodies against the recombinant P. falciparum aldolase inhibit the natural enzyme and no cross-reaction with human aldolase is detectable. Both the recombinant and the natural protein bind to the cytosolic domain of the band 3 membrane protein in vitro. A 19-residue synthetic peptide corresponding to the sequence of the binding domain of band 3 is an inhibitor when included in the binding assay. In addition, this peptide inhibits the catalytic activity of recombinant P. falciparum aldolase when assayed in a buffer system devoid of anions such as chloride or phosphate. The band 3-derived peptides compete with the aldolase substrate fructose-1,6-diphosphate for binding, suggesting that both reagents have a high affinity for the substrate pocket. A similar sequence motif exists in P. falciparum actin II. A 19-residue peptide corresponding to this sequence is also an inhibitor which could suggest that the P. falciparum aldolase can associate with the cytoskeleton of the parasite or of the host.     

Isolation and characterization of rhoptries of Plasmodium falciparum.

Rhoptries have been isolated from Plasmodium falciparum schizont-infected erythrocytes by isopycnic density centrifugation. Gradient fractions were analyzed by immunoblotting with antibodies against two polypeptides of 140 and 110 kDa, known to be components of the rhoptry. The proteins were present primarily in fractions with a density of 1.16 g ml-1. Electron microscopy of these fractions indicated they were enriched in rhoptries. For the most part, the isolated organelle retained in situ morphology, although some rhoptries were distorted, indicating the structure of some of the organelles is not rigid. Electrophoretic analysis of the rhoptry fractions indicated the presence of a number of proteins, many of which have not been identified to date. Properties of proteins in the isolated rhoptry were examined using the 140 and 110 kDa proteins as representative markers. Both proteins are present in a complex with a 130-kDa protein, as all three co-immunoprecipitate. At the late schizont stage, the rhoptry proteins are present in two distinct forms; a soluble form with an Mr of 480 000 which would correspond to a single copy of the 140/130/110 kDa complex and a form that can be sedimented at 130 000 x g. Properties of the sedimentable form suggest that the proteins are included in structures that resemble membranes. Ionic detergents were required to solubilize the proteins while high concentrations of NaCl and Na2CO3 resulted in only partial solubilization. Furthermore, treatment of disrupted rhoptries with phospholipase A and C resulted in the release of proteins into the soluble form.     

Isolation and characterization of a cysteine proteinase gene of Plasmodium falciparum.

We have previously identified a 28-kDa cysteine proteinase of Plasmodium falciparum trophozoites that appears to be an essential malarial hemoglobinase and a potential target for antimalarial chemotherapy. The trophozoite cysteine proteinase (TCP) shares a number of biochemical properties with the lysosomal cysteine proteinase cathepsin L. To isolate the gene encoding TCP, we synthesized degenerate oligonucleotides based on two amino acid sequences of cathepsin L that are well conserved among papain-family cysteine proteinases, and used the oligonucleotides to prime the polymerase chain reaction (PCR) with P. falciparum genomic DNA. A 549-bp DNA fragment was amplified by PCR. This fragment was used as a hybridization probe to screen a lambda gt11 library of P. falciparum genomic DNA and isolate a 1.8-kb genomic clone (C1.8) that encoded an intact malarial cysteine proteinase gene. The sequence of C1.8 predicted a 67-kDa protein containing a typical signal sequence, a large pro sequence, and a 26.8-kDa mature proteinase with 37% amino acid identity to cathepsin L. Antisera directed against a peptide encoded by C1.8 recognized a 28-kDa trophozoite protein on immunoblots. In a Northern analysis, C1.8 hybridized predominantly with RNA from rings, the life-cycle stage immediately preceding the trophozoite stage. Taken together, these results strongly suggest that the P. falciparum cysteine proteinase gene we have isolated and characterized encodes TCP.     

Purification and characterization of culture-derived exoantigens of Plasmodium falciparum

An 83 kDa glycoprotein and a 100 kDa glycoprotein have been purified from the supernatant fluid of in vitro cultures of Plasmodium falciparum by conventional cation-exchange liquid chromatography, size exclusion high performance liquid chromatography, and anion-exchange high performance liquid chromatography. Both proteins exist as dimers in the native state and have been identified as parasite antigens by Western immunoblotting and by their specific reactivity in the indirect enzyme-linked immunosorbent assay. The N-terminal amino acid sequence of these two proteins has been determined and they are at least 90% homologous. The use of monospecific rabbit antisera raised against the individual pure proteins confirm their cross-reactivity. We postulate that the 83 kDa protein is a specific processing product of the larger 100 kDa protein. The presence of these proteins in the culture supernatant suggests they could both be derived from the merozoite surface coat and are potential protective antigens.     

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.     

Biochemical and immunological characterization of bacterially expressed and refolded Plasmodium falciparum 42-kilodalton C-terminal merozoite surface protein 1

Protection against Plasmodium falciparum can be induced by vaccination in animal models with merozoite surface protein 1 (MSP1), which makes this protein an attractive vaccine candidate for malaria. In an attempt to produce a product that is easily scaleable and inexpensive, we expressed the C-terminal 42 kDa of MSP1 (MSP1(42)) in Escherichia coli, refolded the protein to its native form from insoluble inclusion bodies, and tested its ability to elicit antibodies with in vitro and in vivo activities. Biochemical, biophysical, and immunological characterization confirmed that refolded E. coli MSP1(42) was homogeneous and highly immunogenic. In a formulation suitable for human use, rabbit antibodies were raised against refolded E. coli MSP1(42) and tested in vitro in a P. falciparum growth invasion assay. The antibodies inhibited the growth of parasites expressing either homologous or heterologous forms of P. falciparum MSP1(42). However, the inhibitory activity was primarily a consequence of antibodies directed against the C- terminal 19 kDa of MSP1 (MSP1(19)). Vaccination of nonhuman primates with E. coli MSP1(42) in Freund's adjuvant protected six of seven Aotus monkeys from virulent infection with P. falciparum. The protection correlated with antibody-dependent mechanisms. Thus, this new construct, E. coli MSP1(42), is a viable candidate for human vaccine trials.