The aspartic proteinase from the rodent parasite Plasmodium berghei as a potential model for plasmepsins from the human malaria parasite, Plasmodium falciparum

The gene encoding an aspartic proteinase precursor (proplasmepsin) from the rodent malaria parasite Plasmodium berghei has been cloned. Recombinant P. berghei plasmepsin hydrolysed a synthetic peptide substrate and this cleavage was prevented by the general aspartic proteinase inhibitor, isovaleryl pepstatin and by Ro40-4388, a lead compound for the inhibition of plasmepsins from the human malaria parasite Plasmodium falciparum. Southern blotting detected only one proplasmepsin gene in P. berghei. Two plasmepsins have previously been reported in P. falciparum. Here, we describe two further proplasmepsin genes from this species. The suitability of P. berghei as a model for the in vivo evaluation of plasmepsin inhibitors is discussed.     

Novel ways to prevent proteolysis - prophytepsin and proplasmepsin II

The recently determined crystal structures of two aspartic proteinase zymogens, prophytepsin from barley and proplasmepsin II from the malarial parasite Plasmodium falciparum, have provided new insights into zymogen inactivation. Prophytepsin shows a variation of the mechanism of inhibition used by the well-known gastric aspartic proteinase zymogens, whereas proplasmepsin II uses a completely new mode of inactivation.     

Predicting functional residues in Plasmodium falciparum plasmepsins by combining sequence and structural analysis with molecular dynamics simulations

Plasmepsins are aspartic proteases involved in the initial steps of the hemoglobin degradation pathway, a critical stage in the Plasmodium falciparum life cycle during human infection. Thus, they are attractive targets for novel therapeutic compounds to treat malaria, which remains one of the world's biggest health problems. The three-dimensional structures available for P. falciparum plasmepsins II and IV make structure-based drug design of antimalarial compounds that focus on inhibiting plasmepsins possible. However, the structural flexibility of the plasmepsin active site cavity combined with insufficient knowledge of the functional residues and of those determining the specificity of parasitic enzymes is a drawback when designing specific inhibitors. In this study, we have combined a sequence and structural analysis with molecular dynamics simulations to predict the functional residues in P. falciparum plasmepsins. The careful analysis of X-ray structures and 3D models carried out here suggests that residues Y17, V105, T108, L191, L242, Q275, and T298 are important for plasmepsin function. These seven amino acids are conserved across the malarial strains but not in human aspartic proteases. Residues V105 and T108 are localized in a flap of an interior pocket and they only establish contacts with a specific non-peptide achiral inhibitor. We also observed a rapid conformational change in the L3 region of plasmepsins that closes the active site of the enzyme, which explains earlier experimental findings. These results shed light on the role of V105 and T108 residues in plasmepsin specificities, and they should be useful in structure-based design of novel, selective inhibitors that may serve as antimalarial drugs.     

Novel ways to prevent proteolysis — prophytepsin and proplasmepsin II

The recently determined crystal structures of two aspartic proteinase zymogens, prophytepsin from barley and proplasmepsin II from the malarial parasite Plasmodium falciparum, have provided new insights into zymogen inactivation. Prophytepsin shows a variation of the mechanism of inhibition used by the well-known gastric aspartic proteinase zymogens, whereas proplasmepsin II uses a completely new mode of inactivation.     

Genetic disruption of the Plasmodium falciparum digestive vacuole plasmepsins demonstrates their functional redundancy

The digestive vacuole plasmepsins PfPM1, PfPM2, PfPM4, and PfHAP (a histoaspartic proteinase) are 4 aspartic proteinases among 10 encoded in the Plasmodium falciparum malarial genome. These have been hypothesized to initiate and contribute significantly to hemoglobin degradation, a catabolic function essential to the survival of this intraerythrocytic parasite. Because of their perceived significance, these plasmepsins have been proposed as potential targets for antimalarial drug development. To test their essentiality, knockout constructs were prepared for each corresponding gene such that homologous recombination would result in two partial, nonfunctional gene copies. Disruption of each gene was achieved, as confirmed by PCR, Southern, and Northern blot analyses. Western and two-dimensional gel analyses revealed the absence of mature or even truncated plasmepsins corresponding to the disrupted gene. Reduced growth rates were observed with PfPM1 and PfPM4 knockouts, indicating that although these plasmepsins are not essential, they are important for parasite development. Abnormal mitochondrial morphology also appeared to accompany loss of PfPM2, and an abundant accumulation of electron-dense vesicles in the digestive vacuole was observed upon disruption of PfPM4; however, those phenotypes only manifested in about a third of the disrupted cells. The ability to compensate for loss of individual plasmepsin function may be explained by close similarity in the structure and active site of these four vacuolar enzymes. Our data imply that drug discovery efforts focused on vacuolar plasmepsins must incorporate measures to develop compounds that can inhibit two or more of this enzyme family.     

Structural rationale for the recognition of arginine at P₃ in PEXEL motif containing proteins of Plasmodium falciparum by plasmepsin V

The virulent form of malaria is caused by Plasmodium falciparum that infects red blood cells. In order to survive inside the host, the parasite remodels the infected erythrocytes by exporting more than 300 effector proteins outside the parasitophorous vacuole membrane into the cytosol. The main feature of all the export proteins is the presence of a pentapeptide sequence motif; RxLxE/Q/D. This sequence motif is hydrolysed between L-x and the proteins with the acetylated new N-terminus xE/Q/D are exported. The enzyme responsible for this hydrolysis is plasmepsin V which is one of the ten aspartic proteases in P. falciparum. In order to understand the structural rationale for the specificity of this protease towards cleavage of the above motif, we generated three-dimensional models of seven plasmepsins (I, V to X) for which experimental structures are not available and compared these along with the crystal structures of three P. falciparum plasmepsins (II to IV). The structure comparisons revealed the importance of Tyr13, Glu77 and Ala117 specific to plasmepsin V that facilitates the accommodation of arginine at P? in the RxLxE/Q/D motif. Our analysis correlates the structure-function relationship of plasmepsin V.     

Pepsinogen-like activation intermediate of plasmepsin II revealed by molecular dynamics analysis

Plasmepsins are pharmaceutically relevant aspartic proteases involved in haemoglobin degradation by the malaria causing parasites Plasmodium spp. They are translated as inactive proenzymes, with an elongated prosegment. On prosegment cleavage, plasmepsins undergo a series of hitherto unresolved conformational changes before becoming active. Here, the flexibility of plasmepsin and proplasmepsin and the activation process are investigated by multiple explicit water molecular dynamics simulations. The large N-terminal displacement and the interdomain shift from the proenzyme structure to active plasmepsin are promoted by essential dynamics sampling. An intermediate, stabilized by electrostatic interactions between the catalytic dyad and the N-terminus of mature plasmepsin, is observed along all activation trajectories. Notably, the stabilizing interactions in the activation intermediate of plasmepsin are similar to those in the X-ray structure of pepsinogen. In particular, the catalytic aspartates act as hydrogen bond acceptors for the N-terminal amino group and the Ser2 hydroxyl in plasmepsin, and the side chains of Lys36pro and Tyr9 in pepsinogen. The simulation results are used to suggest in vitro experiments to test the conformational transitions involved in the maturation of plasmepsin, and design small-molecule inhibitors.     

The role of Plasmodium falciparum food vacuole plasmepsins

Plasmepsins (PMs) are thought to have an important function in hemoglobin degradation in the malarial parasite Plasmodium falciparum and have generated interest as antimalarial drug targets. Four paralogous plasmepsins reside in the food vacuole of P. falciparum. Targeted gene disruption by double crossover homologous recombination has been employed to study food vacuole plasmepsin function in cultured parasites. Parasite clones with deletions in each of the individual PM I, PM II, and HAP genes as well as clones with a double PM IV/PM I disruption have been generated. All of these clones lack the corresponding PMs, are viable, and appear morphologically normal. PM II and PM IV/I disruptions have longer doubling times than the 3D7 parental line in rich RPMI medium. This appears to be because of a decreased level of productive progeny rather than an increased cell cycle time. In amino acid-limited medium, all four knockouts exhibit slower growth than the parental strain. Compared with 3D7, knock-out clone sensitivity to aspartic and cysteine protease inhibitors is changed minimally. These results suggest substantial functional redundancy and have important implications for the design of antimalarial drugs. The slow growth phenotype may explain why P. falciparum has maintained four plasmepsin genes with overlapping functions.     

Mapping selectivity and specificity of active site of plasmepsins from Plasmodium falciparum using molecular interaction field approach

In the search for selectivity, the aspartic proteases are known to be a very difficult case because the enzymes of this family are not only sequentially but structurally also very similar. To gain insight into the selectivity and specificity of the aspartic proteases family we characterized the binding sites of four malarial aspartic protease (plasmepsin I, plasmepsin II, plasmepsin IV, P. vivax plasmepsin) and two human aspartic proteases (cathepsin D and pepsin) with the intention of identifying the regions that could be potential sites for obtaining selectivity using molecular interaction field approach.     

Novel uncomplexed and complexed structures of plasmepsin II,an aspartic protease from Plasmodium falciparum

Malaria remains a human disease of global significance and a major cause of high infant mortality in endemic nations. Parasites of the genus Plasmodium cause the disease by degrading human hemoglobin as a source of amino acids for their growth and maturation. Hemoglobin degradation is initiated by aspartic proteases, termed plasmepsins, with a cleavage at the alpha-chain between residues Phe33 and Leu34. Plasmepsin II is one of the four catalytically active plasmepsins that has been identified in the food vacuole of Plasmodium falciparum. Novel crystal structures of uncomplexed plasmepsin II as well as the complex with a potent inhibitor have been refined with data extending to resolution limits of 1.9A and 2.7A, and to R factors of 17% and 18%, respectively. The inhibitor, N-(3-[(2-benzo[1,3]dioxol-5-yl-ethyl)[3-(1-methyl-3-oxo-1,3-dihydro-isoindol-2-yl)-propionyl]-amino]-1-benzyl-2-(hydroxypropyl)-4-benzyloxy-3,5-dimethoxy-benzamide, belongs to a family of potent non-peptidic inhibitors that have large P1' groups. Such inhibitors could not be modeled into the binding cavity of the structure of plasmepsin II in complex with pepstatin A. Our structures reveal that the binding cavities of the new complex and uncomplexed plasmepsin II are considerably more open than that of the pepstatin A complex, allowing for larger heterocyclic groups in the P1', P2' and P2 positions. Both complexed and uncomplexed plasmepsin II crystallized in space group P2, with one monomer in the asymmetric unit. The structures show extensive interlocking of monomers around the crystallographic axis of symmetry, with areas in excess of 2300A(2) buried at the interface, and a loop of one monomer interacting with the binding cavity of the 2-fold related monomer. Electron density for this loop is only fully ordered in the complexed structure.     

Flap flexibility amongst plasmepsins I,II, III,IV, and V: Sequence,structural, and molecular dynamics analyses

Herein, for the first time, we comparatively report the opening and closing of apo plasmepsin I – V. Plasmepsins belong the aspartic protease family of enzymes, and are expressed during the various stages of the P. falciparum lifecycle, the species responsible for the most lethal and virulent malaria to infect humans. Plasmepsin I, II, IV and HAP degrade hemoglobin from infected red blood cells, whereas plasmepsin V transport proteins crucial to the survival of the malaria parasite across the endoplasmic reticulum. Flap‐structures covering the active site of aspartic proteases (such as HIV protease) are crucial to the conformational flexibility and dynamics of the protein, and ultimately control the binding landscape. The flap‐structure in plasmepsins is made up of a flip tip in the N‐terminal lying perpendicular to the active site, adjacent to the flexible loop region in the C‐terminal. Using molecular dynamics, we propose three parameters to better describe the opening and closing of the flap‐structure in apo plasmepsins. Namely, the distance, d₁, between the flap tip and the flexible region; the dihedral angle, ?, to account for the twisting motion; and the TriCα angle, θ₁. Simulations have shown that as the flap‐structure twists, the flap and flexible region move apart opening the active site, or move toward each other closing the active site. The data from our study indicate that of all the plasmepsins investigated in the present study, Plm IV and V display the highest conformational flexibility and are more dynamic structures versus Plm I, II, and HAP. Proteins 2015; 83:1693–1705. © 2015 Wiley Periodicals, Inc.     

Plasmepsin II, an acidic hemoglobinase from the Plasmodium falciparum food vacuole, is active at neutral pH on the host erythrocyte membrane skeleton.

Plasmepsin II, an aspartic protease from the human intraerythrocytic parasite Plasmodium falciparum, is involved in degradation of the host cell hemoglobin within the acidic food vacuole of the parasite. Previous characterization of enzymatic activities from Plasmodium soluble extracts, responsible for in vitro hydrolysis of erythrocyte spectrin, had shown that the hydrolysis process occurred at pH 5.0 and involved aspartic protease(s) cleaving mainly within the SH3 motif of the spectrin alpha-subunit. Therefore, we used a recombinant construct of the erythroid SH3 motif as substrate to investigate the involvement of plasmepsins in spectrin hydrolysis. Using specific anti-plasmepsin II antibodies in Western blotting experiments, plasmepsin II was detected in chromatographic fractions enriched in the parasite SH3 hydrolase activity. Involvement of plasmepsin II in hydrolysis was demonstrated by mass spectrometry identification of cleavage sites in the SH3 motif, upon hydrolysis by Plasmodium extract enzymatic activity, and by recombinant plasmepsin II. Furthermore, recombinant plasmepsin II digested native spectrin at pH 6.8, either purified or situated in erythrocyte ghosts. Additional degradation of actin and protein 4.1 from ghosts was observed. Specific antibodies were used in confocal imaging of schizont-infected erythrocytes to localize plasmepsin II in mature stages of the parasite development cycle; antibodies clearly labeled the periphery of the parasites. Taken together, these results strongly suggest that, in addition to hemoglobin degradation, plasmepsin II might be involved in cytoskeleton cleavage of infected erythrocytes.     

X-ray structure of plasmepsin II complexed with a potent achiral inhibitor

The malaria parasite Plasmodium falciparum degrades host cell hemoglobin inside an acidic food vacuole during the blood stage of the infectious cycle. A number of aspartic proteinases called plasmepsins (PMs) have been identified to play important roles in this degradation process and therefore generated significant interest as new antimalarial targets. Several x-ray structures of PMII have been described previously, but thus far, structure-guided drug design has been hampered by the fact that only inhibitors comprising a statine moiety or derivatives thereof have been published. Our drug discovery efforts to find innovative, cheap, and easily synthesized inhibitors against aspartic proteinases yielded some highly potent non-peptidic achiral inhibitors. A highly resolved (1.6 A) x-ray structure of PMII is presented, featuring a potent achiral inhibitor in an unprecedented orientation, contacting the catalytic aspartates indirectly via the "catalytic" water. Major side chain rearrangements in the active site occur, which open up a new pocket and allow a new binding mode of the inhibitor. Moreover, a second inhibitor molecule could be located unambiguously in the active site of PMII. These newly obtained structural insights will further guide our attempts to improve compound properties eventually leading to the identification of molecules suitable as antimalarial drugs.     

Effects on growth, hemoglobin metabolism and paralogous gene expression resulting from disruption of genes encoding the digestive vacuole plasmepsins of Plasmodium falciparum

Four of the plasmepsins of Plasmodium falciparum are localised in the digestive vacuole (DV) of the asexual blood stage parasite (PfPM1, PfPM2, PfPM4 and PfHAP), and each of these aspartic proteinases has been successfully targeted by gene disruption. This study describes further characterisation of the single-plasmepsin knockout mutants, and the creation and characterisation of double-plasmepsin knockout mutants lacking complete copies of pfpm2 and pfpm1 or pfhap and pfpm2. Double-plasmepsin knockout mutants were created by transfecting pre-existing knockout mutants with a second plasmid knockout construct. PCR and Southern blot analysis demonstrate the integration of a large concatamer of each plasmid construct into the targeted gene. All mutants have been characterised to assess the involvement of the DV plasmepsins in sustaining growth during the asexual blood stage. Analyses reaffirmed that knockout mutants Deltapfpm1 and Deltapfpm4 had lower replication rates in the asexual erythrocytic stage than the parental line (Dd2), but double-plasmepsin knockout mutants lacking intact copies of either pfpm2 and pfpm1, or pfpm2 and pfhap, had normal growth rates compared with Dd2. The amount of crystalline hemozoin produced per parasite during the asexual cycle was measured in each single-plasmepsin knockout to estimate the effect of each DV plasmepsin on hemoglobin digestion. Only Deltapfpm4 had a statistically significant reduction in hemozoin accumulation, indicating that hemoglobin digestion was impaired in this mutant. In the single-plasmepsin knockouts, no statistically significant differences were found in the steady state levels of mRNA from the remaining intact DV plasmepsin genes. Disruption of a DV plasmepsin gene does not affect the accumulation of mRNA encoding the remaining paralogous plasmepsins, and Western blot analysis confirmed that the accumulation of the paralogous plasmepsins in each knockout mutant was similar among all clones examined.     

Active site specificity of plasmepsin II.

Members of the aspartic proteinase family of enzymes have very similar three-dimensional structures and catalytic mechanisms. Each, however, has unique substrate specificity. These distinctions arise from variations in amino acid residues that line the active site subsites and interact with the side chains of the amino acids of the peptides that bind to the active site. To understand the unique binding preferences of plasmepsin II, an enzyme of the aspartic proteinase class from the malaria parasite, Plasmodium falciparum, chromogenic octapeptides having systematic substitutions at various positions in the sequence were analyzed. This enabled the design of new, improved substrates for this enzyme (Lys-Pro-Ile-Leu-Phe*Nph-Ala/Glu-Leu-Lys, where * indicates the cleavage point). Additionally, the crystal structure of plasmepsin II was analyzed to explain the binding characteristics. Specific amino acids (Met13, Ser77, and Ile287) that were suspected of contributing to active site binding and specificity were chosen for site-directed mutagenesis experiments. The Met13Glu and Ile287Glu single mutants and the Met13Glu/Ile287Glu double mutant gain the ability to cleave substrates containing Lys residues.     

Parasite killing in Plasmodium vivax malaria by nitric oxide: implication of aspartic protease inhibition

Nitric oxide (NO) is known to possess antiparasitic activity towards Plasmodium species. Parasite proteases are currently considered to be promising targets for antimalarial chemotherapy. In the present study, we have studied the inhibitory effect of NO on the activity of plasmepsin in Plasmodium vivax, the pepsin-like aspartic protease which is believed to be involved in the cleavage during hemoglobin degradation in Plasmodium falciparum. NO donors (+/-) (E)-4-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide (NOR-3), S-nitrosoglutathione (GSNO), and sodium nitroprusside (SNP) were found to inhibit this plasmepsin activity in a dose-dependent manner in purified P. vivax aspartic protease enzyme extracts. This inhibitory effect may be attributable to the nitrosylation of the cysteine residue at the catalytic site. However, an inhibitor of aspartic protease activity, namely pepstatin, was also found to inhibit (IC50 3 microM ) the enzyme activity, which we have used as a positive control. Our results therefore provide novel insights into the pathophysiological mechanisms, and will be useful for designing strategies for selectively upregulating NO production in P. vivax infections for antimalarial chemotherapy and also biochemical adaptations of the malaria parasite for survival in the host erythrocytes with a better understanding of the protease substrate interactions.     

Synthesis, biological evaluation, and modeling studies of inhibitors aimed at the malarial proteases plasmepsins I and II

The increasing resistance of the malarial parasite to antimalarial drugs is a major contributor to the reemergence of the disease and increases the need for new drug targets. The two aspartic proteases, plasmepsins I and II, from Plasmodium falciparum have recently emerged as potential targets. In an effort to inhibit these hemoglobinases, a series of inhibitors encompassing a basic hydroxyethylamine transition state isostere as a central fragment were prepared. The synthesized compounds were varied in the P1' position and exhibited biological activities in the range of 31 to >2000 nM. To try to rationalize the results, molecular docking and 3D-QSAR analysis were used.     

Trafficking of plasmepsin II to the food vacuole of the malaria parasite Plasmodium falciparum

A family of aspartic proteases, the plasmepsins (PMs), plays a key role in the degradation of hemoglobin in the Plasmodium falciparum food vacuole. To study the trafficking of proPM II, we have modified the chromosomal PM II gene in P. falciparum to encode a proPM II-GFP chimera. By taking advantage of green fluorescent protein fluorescence in live parasites, the ultrastructural resolution of immunoelectron microscopy, and inhibitors of trafficking and PM maturation, we have investigated the biosynthetic path leading to mature PM II in the food vacuole. Our data support a model whereby proPM II is transported through the secretory system to cytostomal vacuoles and then is carried along with its substrate hemoglobin to the food vacuole where it is proteolytically processed to mature PM II.     

Plasmodium vivax: collaborative roles for plasmepsin 4 and vivapains in hemoglobin hydrolysis

Plasmepsins, a family of aspartic proteases of Plasmodium species, are known to participate in a wide variety of cellular processes essential for parasite survival. Therefore, the plasmepsins of malaria parasites have been recognized as attractive antimalarial drug targets. Although the plasmepsins of P. falciparum have been extensively characterized, the plasmepsins of P. vivax are currently not well known. To expand our understanding of the plasmepsins of P. vivax, we characterized plasmepsin 4 of P. vivax (PvPM4). The bacterially expressed recombinant PvPM4 was insoluble, but it was easily refolded into a soluble protein. The processing of PvPM4 into a mature enzyme occurred through autocatalytic activity under acidic conditions in a pepstatin A-sensitive manner, in which process a portion of prodomain was essential for correct folding. PvPM4 could hydrolyze native human hemoglobin at acidic pHs, but preferred denatured hemoglobin as a substrate. PvPM4 acted synergistically with vivapain-2 and vivapain-3, cysteine proteases of P. vivax, in the hydrolysis of hemoglobin. The vivapains also mediated processing of PvPM4 into a mature enzyme. These results collectively suggest that PvPM4 is an active hemoglobinase of P. vivax that works collaboratively with vivapains to enhance the parasite’s ability to hydrolyze hemoglobin.     

Structural insights into the activation and inhibition of histo-aspartic protease from Plasmodium falciparum

Histo-aspartic protease (HAP) from Plasmodium falciparum is a promising target for the development of novel antimalarial drugs. The sequence of HAP is highly similar to those of pepsin-like aspartic proteases, but one of the two catalytic aspartates, Asp32, is replaced with histidine. Crystal structures of the truncated zymogen of HAP and of the complex of the mature enzyme with inhibitor KNI-10395 have been determined at 2.1 and 2.5 ? resolution, respectively. As in other proplasmepsins, the propeptide of the zymogen interacts with the C-terminal domain of the enzyme, forcing the N- and C-terminal domains apart, thereby separating His32 and Asp215 and preventing formation of the mature active site. In the inhibitor complex, the enzyme forms a tight domain-swapped dimer, not previously seen in any aspartic proteases. The inhibitor is found in an unprecedented conformation resembling the letter U, stabilized by two intramolecular hydrogen bonds. Surprisingly, the location and conformation of the inhibitor are similar to those of the fragment of helix 2 comprising residues 34p-38p in the prosegments of the zymogens of gastric aspartic proteases; a corresponding helix assumes a vastly different orientation in proplasmepsins. Each inhibitor molecule is in contact with two molecules of HAP, interacting with the carboxylate group of the catalytic Asp215 of one HAP protomer through a water molecule, while also making a direct hydrogen bond to Glu278A' of the other protomer. A comparison of the shifts in the positions of the catalytic residues in the inhibitor complex presented here with those published previously gives further hints regarding the enzymatic mechanism of HAP.