X-ray Crystal Structure and Specificity of the Plasmodium falciparum Malaria Aminopeptidase PfM18AAP

The malarial aminopeptidases have emerged as promising new drug targets for the development of novel antimalarial drugs. The M18AAP of Plasmodium falciparum malaria is a metallo-aminopeptidase that we show demonstrates a highly restricted specificity for peptides with an N-terminal Glu or Asp residue. Thus, the enzyme may function alongside other aminopeptidases in effecting the complete degradation or turnover of proteins, such as host hemoglobin, which provides a free amino acid pool for the growing parasite. Inhibition of PfM18AAP's function using antisense RNA is detrimental to the intra-erythrocytic malaria parasite and, hence, it has been proposed as a potential novel drug target. We report the X-ray crystal structure of the PfM18AAP aminopeptidase and reveal its complex dodecameric assembly arranged via dimer and trimer units that interact to form a large tetrahedron shape that completely encloses the 12 active sites within a central cavity. The four entry points to the catalytic lumen are each guarded by 12 large flexible loops that could control substrate entry into the catalytic sites. PfM18AAP thus resembles a proteasomal-like machine with multiple active sites able to degrade peptide substrates that enter the central lumen. The Plasmodium enzyme shows significant structural differences around the active site when compared to recently determined structures of its mammalian and human homologs, which provides a platform from which a rational approach to inhibitor design of new malaria-specific drugs can begin.     

Characterization of the Plasmodium falciparum M17 leucyl aminopeptidase. A protease involved in amino acid regulation with potential for antimalarial drug development

Amino acids generated from the catabolism of hemoglobin by intra-erythrocytic malaria parasites are not only essential for protein synthesis but also function in maintaining an osmotically stable environment, and creating a gradient by which amino acids that are rare or not present in hemoglobin are drawn into the parasite from host serum. We have proposed that a Plasmodium falciparum M17 leucyl aminopeptidase (PfLAP) generates and regulates the internal pool of free amino acids and therefore represents a target for novel antimalarial drugs. This enzyme has been expressed in insect cells as a functional 320-kDa homo-hexamer that is optimally active at neutral or alkaline pH, is dependent on metal ions for activity, and exhibits a substrate preference for N-terminally exposed hydrophobic amino acids, particularly leucine. PfLAP is produced by all stages in the intra-erythrocytic developmental cycle of malaria but was most highly expressed by trophozoites, a stage at which hemoglobin degradation and parasite protein synthesis are elevated. The enzyme was located by immunohistochemical methods and by transfecting malaria cells with a PfLAP-green fluorescent protein construct, to the cytosolic compartment of the cell at all developmental stages, including segregated merozoites. Amino acid dipeptide analogs, such as bestatin and its derivatives, are potent inhibitors of the protease and also block the growth of P. falciparum malaria parasites in culture. This study provides a biochemical basis for the antimalarial activity of aminopeptidase inhibitors. Availability of functionally active recombinant PfLAP, coupled with a simple enzymatic readout, will aid medicinal chemistry and/or high throughput approaches for the future design/discovery of new antimalarial drugs.     

Overexpression of leucyl aminopeptidase in Plasmodium falciparum parasites. Target for the antimalarial activity of bestatin

Malaria aminopeptidases are important in the generation and regulation of free amino acids that are used in protein anabolism and for maintaining osmotic stability within the infected erythrocyte. The intraerythrocytic development of malaria parasites is blocked when the activity of aminopeptidases is specifically inhibited by reagents such as bestatin. One of the major aminopeptidases of malaria parasites is a leucyl aminopeptidase of the M17 family. We reasoned that, when this enzyme was the target of bestatin inhibition, its overexpression in malaria cells would lead to a reduced sensitivity to the inhibitor. To address this supposition, transgenic Plasmodium falciparum parasites overexpressing the leucyl aminopeptidase were generated by transfection with a plasmid that housed the full-length gene. Transgenic parasites expressed a 65-kDa protein close to the predicted molecule size of 67.831 kDa for the introduced leucyl aminopeptidase, and immunofluorescence studies localized the protein to the cytosol, the location of the native enzyme. The product of the transgene was shown to be functionally active with cytosolic extracts of transgenic parasites exhibiting twice the leucyl aminopeptidase activity compared with wild-type parasites. In vitro inhibitor sensitivity assays demonstrated that the transgenic parasites were more resistant to bestatin (EC50 64 microM) compared with the parent parasites (EC50 25 microM). Overexpression of genes in malaria parasites would have general application in the identification and validation of targets for antimalarial drugs.     

Roles for two aminopeptidases in vacuolar hemoglobin catabolism in Plasmodium falciparum

During the erythrocytic stage of its life cycle, the human malaria parasite Plasmodium falciparum catabolizes large quantities of host-cell hemoglobin in an acidic organelle, the food vacuole. A current model for the catabolism of globin-derived oligopeptides invokes peptide transport out of the food vacuole followed by hydrolysis to amino acids by cytosolic aminopeptidases. To test this model, we have examined the roles of four parasite aminopeptidases during the erythrocytic cycle. Localization of tagged aminopeptidases, coupled with biochemical analysis of enriched food vacuoles, revealed the presence of amino acid-generating pathways in the food vacuole as well as the cytosol. Based on the localization data and in vitro assays, we propose a specific role for one of the plasmodial enzymes, aminopeptidase P, in the catabolism of proline-containing peptides in both the vacuole and the cytosol. We establish an apparent requirement for three of the four aminopeptidases (including the two food vacuole enzymes) for efficient parasite proliferation. To gain insight into the impact of aminopeptidase inhibition on parasite development, we examined the effect of the presence of amino acids in the culture medium of the parasite on the toxicity of the aminopeptidase inhibitor bestatin. The ability of bestatin to block parasite replication was only slightly affected when 19 of 20 amino acids were withdrawn from the medium, indicating that exogenous amino acids cannot compensate for the loss of aminopeptidase activity. Together, these results support the development of aminopeptidase inhibitors as novel chemotherapeutics directed against malaria.     

Fingerprinting the substrate specificity of M1 and M17 aminopeptidases of human malaria, Plasmodium falciparum

Background

Plasmodium falciparum, the causative agent of human malaria, expresses two aminopeptidases, PfM1AAP and PfM17LAP, critical to generating a free amino acid pool used by the intraerythrocytic stage of the parasite for proteins synthesis, growth and development. These exopeptidases are potential targets for the development of a new class of anti-malaria drugs.

Methodology/Principal Findings

To define the substrate specificity of recombinant forms of these two malaria aminopeptidases we used a new library consisting of 61 fluorogenic substrates derived both from natural and unnatural amino acids. We obtained a detailed substrate fingerprint for recombinant forms of the enzymes revealing that PfM1AAP exhibits a very broad substrate tolerance, capable of efficiently hydrolyzing neutral and basic amino acids, while PfM17LAP has narrower substrate specificity and preferentially cleaves bulky, hydrophobic amino acids. The substrate library was also exploited to profile the activity of the native aminopeptidases in soluble cell lysates of P. falciparum malaria.

Conclusions/Significance

This data showed that PfM1AAP and PfM17LAP are responsible for majority of the aminopeptidase activity in these extracts. These studies provide specific substrate and mechanistic information important for understanding the function of these aminopeptidases and could be exploited in the design of new inhibitors to specifically target these for anti-malaria treatment.     

Low and high affinity amino acid H+-cotransporters for cellular import of neutral and charged amino acids

Amides and acidic amino acids represent the major long distance transport forms of organic nitrogen. Six amino acid permeases (AAPs) from Arabidopsis mediating transport of a wide spectrum of amino acids were isolated. AAPs are distantly related to plasma membrane amino acid transport systems N and A and to vesicular transporters such as VGAT from mammals. A detailed comparison of the properties by electrophysiology after heterologous expression in Xenopus oocytes shows that, although capable of recognizing and transporting a wide spectrum of amino acids, individual AAPs differ with respect to specificity. Apparent substrate affinities are influenced by structure and net charge and vary by three orders of magnitude. AAPs mediate cotransport of neutral amino acids with one proton. Uncharged forms of acidic and basic amino acids are cotransported with one proton. Since all AAPs are differentially expressed, different tissues may be supplied with a different spectrum of amino acids. AAP3 and AAP5 are the only transporters mediating efficient transport of the basic amino acids. In vivo competition shows that the capability to transport basic amino acids in planta might be overruled by excess amides and acidic amino acids in the apoplasm. With the exception of AAP6, AAPs do not recognize aspartate; only AAP6 has an affinity for aspartate in the physiologically relevant range. This property is due to an overall higher affinity of AAP6 for neutral and acidic amino acids. Thus AAP6 may serve a different role either in cooperating with the lower affinity systems to acquire amino acids in the low concentration range, as a system responsible for aspartate transport or as an uptake system from the xylem. In agreement, a yeast mutant deficient in acidic amino acid uptake at low aspartate concentrations was complemented only by AAP6. Taken together, the AAPs transport neutral, acidic and cationic amino acids, including the major transport forms, i.e. glutamine, asparagine and glutamate. Increasing proton concentrations strongly activate transport of amino acids. Thus the actual apoplasmic concentration of amino acids and the pH will determine what is transported in vivo, i.e. major amino acids such as glutamine, asparagine, and glutamate will be mobilized preferentially.     

A family of removable cassettes designed to obtain antibiotic-resistance-free genomic modifications of Escherichia coli and other bacteria

The gene coding for the cytosolic glyceraldehyde-3-phosphate dehydrogenase (GAPDH; EC 1.2.1.12) was isolated from Plasmodium falciparum. The gene contains 1 intron and the A+T content is characteristic for the codon usage of P. falciparum. The predicted open reading frame codes for 337 amino acids (36651Da) and is 63.5% identical to the human erythrocytic GAPDH. GAPDH sequences from several field isolates of P. falciparum displayed 100% conservation. Phylogenetic analysis supports the hypothesis that dinoflagellates and Plasmodium are closely related. The protein encoded by the pfGAPDH was expressed recombinantly in Escherichia coli and exhibited enzymatic activity with NAD(+) but not with NADP(+) as cofactor. Antiserum raised against the recombinantly expressed enzyme detected specifically all developmental stages of cultured P. falciparum blood-stage parasites.     

AAP1 transports uncharged amino acids into roots of Arabidopsis

Amino acids are available to plants in some soils in significant amounts, and plants frequently make use of these nitrogen sources. The goal of this study was to identify transporters involved in the uptake of amino acids into root cells. Based on the fact that high concentrations of amino acids inhibit plant growth, we hypothesized that mutants tolerating toxic levels of amino acids might be deficient in the uptake of amino acids from the environment. To test this hypothesis, we employed a forward genetic screen for Arabidopsis thaliana mutants tolerating toxic concentrations of amino acids in the media. We identified an Arabidopsis mutant that is deficient in the amino acid permease 1 (AAP1, At1g58360) and resistant to 10 mm phenylalanine and a range of other amino acids. The transporter was localized to the plasma membrane of root epidermal cells, root hairs, and throughout the root tip of Arabidopsis. Feeding experiments with [(14)C]-labeled neutral, acidic and basic amino acids showed significantly reduced uptake of amino acids in the mutant, underscoring that increased tolerance of aap1 to high levels of amino acids is coupled with reduced uptake by the root. The growth and uptake studies identified glutamate, histidine and neutral amino acids, including phenylalanine, as physiological substrates for AAP1, whereas aspartate, lysine and arginine are not. We also demonstrate that AAP1 imports amino acids into root cells when these are supplied at ecologically relevant concentrations. Together, our data indicate an important role of AAP1 for efficient use of nitrogen sources present in the rhizosphere.     

Specific inhibition of the aspartate aminotransferase of Plasmodium falciparum

Aspartate aminotransferases (AspATs; EC 2.6.1.1) catalyze the conversion of aspartate and α-ketoglutarate into oxaloacetate and glutamate and are key enzymes in the nitrogen metabolism of all organisms. Recent findings suggest that the plasmodial enzyme [Plasmodium falciparum aspartate aminotransferase (PfAspAT)] may also play a pivotal role in energy metabolism and in the de novo biosynthesis of pyrimidines. However, while PfAspAT is a potential drug target, the high homology between the active sites of currently available AspAT structures hinders the development of specific inhibitors of these enzymes. In this article, we report the X-ray structure of the PfAspAT homodimer at a resolution of 2.8 Å. While the overall fold is similar to the currently available structures of other AspATs, the structure presented shows a significant divergence in the conformation of the N-terminal residues. Deletion of these divergent PfAspAT N-terminal residues results in a loss of activity for the recombinant protein, and addition of a peptide containing these 13 N-terminal residues results in inhibition both in vitro and in a lysate isolated from cultured parasites, while the activity of human cytosolic AspAT is unaffected. The finding that the divergent N-terminal amino acids of PfAspAT play a role in catalytic activity indicates that specific inhibition of the enzyme may provide a lead for the development of novel compounds in the treatment of malaria. We also report on the localization of PfAspAT to the parasite cytosol and discuss the implications of the role of PfAspAT in the supply of malate to the parasite mitochondria.     

Identification of histidine residues important in the catalysis and structure of aspartyl aminopeptidase

Aspartyl aminopeptidase (DAP), a widely distributed and abundant cytosolic enzyme, removes glutamyl or aspartyl residues from N-terminal acidic amino acid-containing peptides. DAP is a member of the M18 family of the MH clan of cocatalytic metallopeptidases. The human and mouse enzymes have been cloned. We have identified 8 highly homologous eukaryotic sequences that are probable aspartyl aminopeptidases. Eight histidine residues of human DAP were sequentially mutated to phenylalanine. Mutation of His94, His170, and His440 abolished enzymatic activity. His94 and His440 are postulated to be involved in binding cocatalytic zinc atoms by homology with other members of the MH clan. Mutation of His352 dramatically reduced enzyme activity. Gel-filtration analysis of the His352 mutant revealed destabilization of the quaternary structure and dissociation of the native 440-kDa enzyme. Mutation of His33 and of histidines residing in a cluster at residues 349, 359, and 363 all decreased k(cat). These studies reveal an important role for histidine residues both in catalysis and in the structural integrity of DAP.     

A Plasmodium falciparum dipeptidyl aminopeptidase I participates in vacuolar hemoglobin degradation

Intraerythrocytic growth of the human malaria parasite Plasmodium falciparum requires the catabolism of large amounts of host cell hemoglobin. Endoproteolytic digestion of hemoglobin to short oligopeptides occurs in an acidic organelle called the food vacuole. How amino acids are generated from these peptides is not well understood. To gain insight into this process, we have studied a plasmodial ortholog of the lysosomal exopeptidase cathepsin C. The plasmodial enzyme dipeptidyl aminopeptidase 1 (DPAP1) was enriched from parasite extract by two different approaches and was shown to possess hydrolytic activity against fluorogenic dipeptide substrates. To localize DPAP1 we created a transgenic parasite line expressing a chromosomally encoded DPAP1-green fluorescent protein fusion. Green fluorescent protein fluorescence was observed in the food vacuole of live transgenic parasites, and anti-DPAP1 antibody labeled the food vacuole in parasite cryosections. Together these data implicate DPAP1 in the generation of dipeptides from hemoglobin-derived oligopeptides. To assess the significance of DPAP1, we attempted to ablate DPAP1 activity from blood stage parasites by truncating the chromosomal DPAP1-coding sequence. The inability to disrupt the coding sequence indicates that DPAP1 is important for asexual proliferation. The proenzyme form of DPAP1 was found to accumulate in the parasitophorous vacuole of mature parasites. This observation suggests a trafficking route for DPAP1 through the parasitophorous vacuole to the food vacuole.     

Localisation of hypoxanthine phosphoribosyl transferase in the malaria parasite Plasmodium falciparum.

The enzyme hypoxanthine phosphoribosyl transferase of the human malaria parasite Plasmodium falciparum has been located in parasites and parasite-infected erythrocytes by antibody probing. The probe was a polyclonal rabbit antiserum made against the parasite enzyme made in Escherichia coli. The enzyme is associated with membrane-bound compartments in merozoites and asexual blood parasites. In particular, indirect immunofluorescence studies reveal the enzyme localized in vesicle-like structures within the cytoplasm of the infected erythrocyte. This is the first time a P. falciparum protein of defined metabolic function has been tracked to a site outside the parasite cytosol. Studies on the targeting of the enzyme using a cell-free system suggests that the protein reaches its destination via a route different from the normal secretory pathway.     

Plasmodium falciparum carbonic anhydrase is a possible target for malaria chemotherapy

Plasmodiumfalciparum is responsible for the majority of life-threatening cases of human malaria. The global emergence of drug-resistant malarial parasites necessitates identification and characterization of novel drug targets. Carbonic anhydrase (CA) is present at high levels in human red cells and in P. falciparum. Existence of at least three isozymes of the alpha < class was demonstrated in P. falciparum and a rodent malarial parasite Plasmodium berghei. The major isozyme CA1 was purified and partially characterized from P. falciparum (PfCA1). A search of the malarial genome database yielded an open reading frame similar to the alpha-CAs from various organisms, including human. The primary amino acid sequence of the PfCA1 has 60% identity with a rodent parasite Plasmodium yoelii enzyme (PyCA). The single open reading frames encoded 235 and 252 amino acid proteins for PfCA1 and PyCA, respectively. The highly conserved active site residues were also found among organisms having alpha-CAs. The PfCA1 gene was cloned, sequenced and expressed in Escherichia coli. The purified recombinant PfCA1 enzyme was catalytically active. It was sensitive to acetazolamide and sulfanilamide inhibition. Kinetic properties of the recombinant PfCA1 revealed the authenticity to the wild type enzyme purified from P. falciparum in vitro culture. Furthermore, the PfCA1 inhibitors acetazolamide and sulfanilamide showed good antimalarial effect on the in vitro growth of P. falciparum. Our molecular tools developed for the recombinant enzyme expression will be useful for developing potential antimalarials directed at P. falciparum carbonic anhydrase.     

Proteolipid of vacuolar H(+)-ATPase of Plasmodium falciparum: cDNA cloning, gene organization and complementation of a yeast null mutant

Vacuolar H(+)-ATPase (V-ATPase), an electrogenic proton pump, is highly expressed in Plasmodium falciparum, the human malaria parasite. Although V-ATPase-driven proton transport is involved in various physiological processes in the parasite, the overall features of the V-ATPase of P. falciparum, including the gene organization and biogenesis, are far less known. Here, we report cDNA cloning of proteolipid subunit c of P. falciparum, the smallest and most highly hydrophobic subunit of V-ATPase. RT-PCR analysis as well as Northern blotting indicated expression of the proteolipid gene in the parasite cells. cDNA, which encodes a complete reading frame comprising 165 amino acids, was obtained, and its deduced amino acid sequence exhibits 52 and 57% similarity to the yeast and human counterparts, respectively. Southern blot analysis suggested the presence of a single copy of the proteolipid gene, with 5 exons and 4 introns. Upon transfection of the cDNA into a yeast null mutant, the cells became able to grow at neutral pH, accompanied by vesicular accumulation of quinacrine. In contrast, a mutated proteolipid with replacement of glutamate residue 138 with glutamine did not lead to recovery of the growth ability or vesicular accumulation of quinacrine. These results indicated that the cDNA actually encodes the proteolipid of P. falciparum and that the proteolipid is functional in yeast.     

Purification and properties of recombinant Plasmodium falciparum S-adenosyl-L-homocysteine hydrolase

Recombinant S-adenosyl-L-homocysteine (SAH) hydrolase of the malaria parasite Plasmodium falciparum was expressed in Escherichia coli, purified to homogeneity and characterized. Comparison of the malaria parasite SAH hydrolase with that derived from the human gene indicated marked differences in kcat values. The values of both forward and reverse reactions of P. falciparum SAH hydrolase are more than 21-fold smaller than those of the human enzyme. Km values of the parasite and human SAH enzymes are 1.2 and 7.8 microM, respectively. On the other hand, IC50 values of neplanocin A, a strong inhibitor of SAH hydrolase and a growth inhibitor of P. falciparum, are 101 nM for the parasite enzyme and 47 nM for human enzyme. P. falciparum SAH hydrolase has been thought to be a target for a chemotherapeutic agent against malaria. This study may make it possible to develop a specific inhibitor for the parasite SAH hydrolase.     

Glucose-6-phosphate metabolism in Plasmodium falciparum

Malaria is still one of the most threatening diseases worldwide. The high drug resistance rates of malarial parasites make its eradication difficult and furthermore necessitate the development of new antimalarial drugs. Plasmodium falciparum is responsible for severe malaria and therefore of special interest with regard to drug development. Plasmodium parasites are highly dependent on glucose and very sensitive to oxidative stress; two observations that drew interest to the pentose phosphate pathway (PPP) with its key enzyme glucose-6-phosphate dehydrogenase (G6PD). A central position of the PPP for malaria parasites is supported by the fact that human G6PD deficiency protects to a certain degree from malaria infections. Plasmodium parasites and the human host possess a complete PPP, both of which seem to be important for the parasites. Interestingly, there are major differences between parasite and human G6PD, making the enzyme of Plasmodium a promising target for antimalarial drug design. This review gives an overview of the current state of research on glucose-6-phosphate metabolism in P. falciparum and its impact on malaria infections. Moreover, the unique characteristics of the enzyme G6PD in P. falciparum are discussed, upon which its current status as promising target for drug development is based.     

Plasmodium falciparum: induction, selection, and characterization of pyrimethamine-resistant mutants

We have selected eight pyrimethamine resistant mutants of a cloned, drug sensitive, Plasmodium falciparum malaria parasite, strain FCR3. The mutants exhibited resistance to between 10 and 200 times higher concentrations of drug than the wild type parasite. The mutants were selected from cultured parasites that were either unmutagenized or N-methyl-N'-nitro-N-nitrosoguanidine mutagenized. One mutant was shown to contain a mutant dihydrofolate reductase enzyme in parasite extracts that exhibited (1) a five- to ninefold reduction in its binding of methotrexate, (2) an undetectable enzyme activity based on the spectrophotometric conversion of dihydrofolate to tetrahydrofolate, and (3) essentially normal amounts of the parasite's bifunctional thymidylate synthetase-dihydrofolate reductase enzyme. Other mutants exhibited both normal dihydrofolate reductase specific activity and normal enzyme sensitivity to the inhibitory activity of the drug.     

The H89 cAMP-dependent protein kinase inhibitor blocks Plasmodium falciparum development in infected erythrocytes.

In Plasmodium falciparum, the causative agent of human malaria, the catalytic subunit gene of cAMP-dependent protein kinase (Pfpka-c) exists as a single copy. Interestingly, its expression appears developmentally regulated, being at higher levels in the pathogenic asexual stages than in the sexual forms of parasite that are responsible for transmission to the mosquito vector. Within asexual parasites, PfPKA activity can be readily detected in schizonts. Similar to endogenous PKA activity of noninfected red blood cells, the parasite enzyme can be stimulated by cAMP and inhibited by protein kinase inhibitor.Importantly, ex vivo treatment of infected erythrocytes with the classical PKA-C inhibitor H89 leads to a block in parasite growth. This suggests that the PKA activities of infected red blood cells are essential for parasite multiplication. Finally, structural considerations suggest that drugs targeting the parasite, rather than the erythrocyte enzyme, might be developed that could help in the fight against malaria.