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-Life cycle of the malaria parasite. Sporozoites from the salivary glands of an infected mosquito (bottom) make their way to the liver, infect hepatocytes, replicate to thousands of infective merozoites and bud off as merosomes that rupture into the bloodstream. The merozoites invade RBCs, replicate and multiply in the intraerythrocytic cycle. Some differentiate into male and female gametocytes that are taken up into the mosquito, where they develop. In the mosquito midgut, parasites egress from the RBCs as gametes, mate to form zygotes, and differentiate into ookinetes that traverse the midgut and become oocysts. They replicate and differentiate into sporozoites that migrate to the salivary glands, where they are ready for transmission to the next human upon mosquito bite. Points in the life cycle at which plasmepsins are thought to function are labeled.
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Plasmepsins are a group of diverse aspartic proteases in the malaria parasite Plasmodium. Their functions are strikingly multifaceted, ranging from hemoglobin degradation to secretory organelle protein processing for egress, invasion and effector export. Some, particularly the digestive vacuole plasmepsins, have been extensively characterized where...
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Context 1
... merozoites egress into the bloodstream, where they invade red blood cells (RBCs) and set up a continuous intraerythrocytic cycle that amplifies their population, often to overwhelming numbers. Some differentiate into sexual stage parasites, to be taken up by the next mosquito and develop in the mosquito midgut, ultimately migrating to the salivary glands for spread to a new victim ( Figure 1). ...
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... PM V flap is several amino acids longer than the analogous region of the digestive vacuole plasmepsins, a difference that could perhaps underlie PM V's unique substrate specificity. Along the flap (but not predicted to contact the substrate) is an unpaired Cys, which has been implicated in PM V's sensitivity to Hg2+ (118, 120, 121). Connected directly to the flap is an insert made up of two cysteine pairs folded into a cloverleaflike structure called a "nepenthesin insert", which it shares with Nep1 from the pitcher plant Nepenthesia. ...
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... this is often a nucleic acidinteracting motif, it is not clear what purpose such a motif might have in the ER lumen, where all but the non-essential C-terminal tail of PM V is found (109,144). Projecting away from the active site of the enzyme is a poorly structured insert whose length and sequence varies among different Plasmodium species and isolates from different regions (145, 146); mutagenesis studies on recombinant enzyme have not yet revealed a function for this structure (146). Further work has the potential to both uncover novel parasite biology, but also to unearth secrets of biochemistry broadly applicable to other enzymes outside of Plasmodium. ...
Similar publications
During the intravascular stage of infection, the malaria parasite Plasmodium invades a host erythrocyte, multiplies within a parasitophorous vacuole (PV) and exits upon rupture of the PV and erythrocyte membranes in a process known as egress. Both egress and invasion are controlled by effector proteins discharged from specialized secretory organell...
Citations
... Some amount of RBC cytoplasm is ingested by trophozoites and transported to the food vacuole. In this vacuole, hemoglobin is converted into heme and globin (small peptides) by cysteine proteases fal-cipain1-3 and aspartic proteases plasmepsin I-IV (Nasamu et al. 2020;Rosenthal 2004). ...
Malaria remains a global health challenge with significant mortality and morbidity annually, with resistant parasite strains
complicating treatment efforts. There is an acute need for novel antimalarial drugs that can put a stop to the future public
health crisis caused by the multi-drug resistance strains of the Plasmodium parasite. However, the discovery of these new
components is very challenging in the context of the generation of multi-drug resistance properties of malaria. The novel
drugs also need to have several properties involving enhanced therapeutic prospects, successful treatment capabilities, and
novel mechanisms of action that will forestall the resistance. To successfully achieve this aim researchers are trying to
focus on exploring promising malaria targets. Various approaches have been made for the development of drugs for malaria
including the remodelling of existing drugs and the development of novel inhibitors which acts on new targets. Advancement
in the study provides more information on the biology of parasites and the new targets which help in the development
of novel drugs. The present review focuses on the study of novel targets of malaria parasites and subsequent inhibitors of
those particular targets. Some of these targets include malarial protease, various transporter proteins, enzymes involved
in the synthesis of DNA, and nucleic acids like dihydroorotate dehydrogenase, dihydrofolate reductase, apicoplast and
dihydropteroate synthase. Other potential targets are also included in this review such as isoprenoid biosynthesis, farnesyl
transferase of parasite, P. falciparum translational elongation factor 2, and phosphatidyl inositol 4 kinase. These promising
targets have also been summed up along with their corresponding inhibitors for combating multi-drug resistance malaria.
... Thus, proteolysis can only occur on a few less structured sites as exemplified by the main proteolysis site 33-34 at the C-terminal extremity of a helix. Substrate specificity profiles for the digestive plasmepsins of Plasmodium are much better characterized than for schistosome digestive enzymes [10]. Thus, we selected the short peptide Phe-Val-Phe extracted from the substrate specificity study of Plasmodium plasmepsin II [11] because the synthesis did not require further protection/deprotection steps. ...
... Indeed, in a study comparing the genomic structures of the aspartic proteinase family genes, including the cathepsin D gene of schistosome, it was shown that there were exons that were common to both plasmepsins I, II, and IV from Plasmodium falciparum and cathepsin D from schistosome. Both plasmepsins I, II, and IV and cathepsin D have been evidenced to be major actors in hemoglobin degradation in Plasmodium [10] and schistosome [29], respectively. In vitro, the parasite's digestive tract probably contains only a few globules, which could lead to low protease secretion in the digestive tract and hence slow activation of the alkoxyamine prodrugs. ...
The expansion of drug resistant parasites sheds a serious concern on several neglected parasitic diseases. Our recent results on cancer led us to envision the use of peptide-alkoxyamines as a highly selective and efficient new drug against schistosome adult worms, the etiological agents of schistosomiasis. Indeed, the peptide tag of the hybrid compounds can be hydrolyzed by worm’s digestive enzymes to afford a highly labile alkoxyamine which homolyzes spontaneously and instantaneously into radicals—which are then used as a drug against Schistosome adult parasites. This approach is nicely summarized as digging their graves with their forks. Several hybrid peptide-alkoxyamines were prepared and clearly showed an activity: two of the tested compounds kill 50% of the parasites in two hours at a concentration of 100 µg/mL. Importantly, the peptide and alkoxyamine fragments that are unable to generate alkyl radicals display no activity. This strong evidence validates the proposed mechanism: a specific activation of the prodrugs by the parasite proteases leading to parasite death through in situ alkyl radical generation.
... In Plasmodium, plasmepsins (PLMs) are a family of specific aspartic acid proteases involved in bulk protein degradation, and especially hemoglobin digestion. PLM I and II revealed an initial cleavage of hemoglobin (α2β2 tetramer) on the α chain between Phe33 and Leu34 [12]. More generally, PLMs have a rather low substrate selectivity but a strong preference for cleavage between two hydrophobic residues [12]. ...
... PLM I and II revealed an initial cleavage of hemoglobin (α2β2 tetramer) on the α chain between Phe33 and Leu34 [12]. More generally, PLMs have a rather low substrate selectivity but a strong preference for cleavage between two hydrophobic residues [12]. The design of antimalarial drugs based on the inhibition of the digestive vacuole PLM I to IV has been unsuccessful [13][14][15]. ...
... All vacuole PLMs were able to process hemoglobin around pH 5. The initial cleavage of native hemoglobin occurred between Phe33 and Leu34 of the α chain, helping the denaturation of the highly structured hemoglobin molecule so that further proteolysis could proceed and heme could be released [12]. Thus, initially, the authors designed substrates according to the surrounding hemoglobin sequence [16]. ...
The emergence and spread of drug-resistant Plasmodium falciparum parasites shed a serious concern on the worldwide control of malaria, the most important tropical disease in terms of mortality and morbidity. This situation has led us to consider the use of peptide-alkoxyamine derivatives as new antiplasmodial prodrugs that could potentially be efficient in the fight against resistant malaria parasites. Indeed, the peptide tag of the prodrug has been designed to be hydrolysed by parasite digestive proteases to afford highly labile alkoxyamines drugs, which spontaneously and instantaneously homolyse into two free radicals, one of which is expected to be active against P. falciparum. Since the parasite enzymes should trigger the production of the active drug in the parasite’s food vacuoles, our approach is summarized as “to dig its grave with its fork”. However, despite promising sub-micromolar IC50 values in the classical chemosensitivity assay, more in-depth tests evidenced that the anti-parasite activity of these compounds could be due to their cytostatic activity rather than a truly anti-parasitic profile, demonstrating that the antiplasmodial activity cannot be based only on measuring antiproliferative activity. It is therefore imperative to distinguish, with appropriate tests, a genuinely parasiticidal activity from a cytostatic activity.
... Among the four falcipains, falcipain-2 (FP2) and falcipain-3 (FP3) are structurally conserved and crucial for hemoglobinase activity. Apart from cysteine proteases, the plasmodium genome encodes at least ten aspartic proteases, called plasmepsins, of which plasmepsins I-IV are involved in hemoglobin degradation, while plasmepsins V, IX, and X are considered important drug targets [4]. Due to their crucial roles in the erythrocytic stage, targeting protease activity of key proteases is remerging as attractive targets for drug intervention. ...
Proteolytic activity constitutes a fundamental process essential for the survival of the malaria parasite and is thus highly regulated. Falstatin, a protease inhibitor of Plasmodium falciparum, tightly regulates the activity of cysteine hemoglobinases, falcipain-2 and 3 (FP2, FP3), by inhibiting FP2 through a single surface exposed loop. However, the multimeric nature of falstatin and its interaction with FP2 remained unexplored. Here we report that the N-terminal falstatin region is highly disordered, and needs chaperone activity (heat-shock protein 70, HSP70) for its folding. Protein-protein interaction assays showed a significant interaction between falstatin and HSP70. Further, characterization of the falstatin multimer through a series of biophysical techniques identified the formation of a falstatin decamer, which was extremely thermostable. Computational analysis of the falstatin decamer showed the presence of five falstatin dimers, with each dimer aligned in a head-to-tail orientation. Further, the falstatin C-terminal region was revealed to be primarily involved in the oligomerization process. Stoichiometric analysis of the FP2-falstatin multimer showed the formation of a heterooligomeric complex in a 1:1 ratio, with the participation of ten subunits of each protein. Taken together, our results report a novel protease-inhibitor complex and strengthens our understanding of the regulatory mechanisms of major plasmodium hemoglobinases.
... Together, our results uncouple processing by PM V from protein export and indicate that the role of PM V in Plasmodium parasites is broader and likely more ancient than processing exported proteins, as has been postulated previously (74). Its role, therefore, may be more similar to that of its Toxoplasma gondii ortholog, the aspartic protease ASP5, than previously thought. ...
Malaria parasites alter multiple properties of the host erythrocyte by exporting proteins into the host cell. Many exported proteins contain a five-amino acid motif called the Plasmodium export element (PEXEL) that is cleaved by the parasite protease Plasmepsin V (PM V). The presence of a PEXEL is considered a signature of protein export and has been used to identify a large number of exported proteins. The export of proteins becomes essential midway through the intraerythrocytic cycle—preventing protein export blocks parasite development 18–24 h after invasion. However, a genetic investigation revealed that the absence of the PEXEL protein PFA0210c (PF3D7_0104200) causes parasite development to arrest immediately after invasion. We now show that this protein is cleaved by PM V but not exported into the host erythrocyte and instead functions in the parasitophorous vacuole; hence, the protein was renamed PV6. We additionally show that the lysine residue that becomes the N-terminus of PV6 after processing by PM V prevents export. This is the first example of a native Plasmodium falciparum PM V substrate that remains in the parasitophorous vacuole. We also provide evidence suggesting that the parasite may produce at least one additional essential, non-exported PM V substrate. Therefore, the presence of a PEXEL and, hence, processing of a protein by PM V do not always target a protein for export, and PM V likely has a broader function in parasite growth beyond processing exported proteins. Furthermore, we utilized this finding to investigate possible requirements for protein export further.
IMPORTANCE
In the manuscript, the authors investigate the role of the protease Plasmepsin V in the parasite–host interaction. Whereas processing by Plasmepsin V was previously thought to target a protein for export into the host cell, the authors now show that there are proteins cleaved by this protease that are not exported but instead function at the host–parasite interface. This changes the view of this protease, which turns out to have a much broader role than anticipated. The result shows that the protease may have a function much more similar to that of related organisms. The authors also investigate the requirements for protein export by analyzing exported and non-exported proteins and find commonalities between the proteins of each set that further our understanding of the requirements for protein export.
... molecular Docking was performed using Autodock 4.2 software. the three dimensional structure of protein molecules, namely, falcipain 2(pDb ID:3bpF), plasmepsin II (pDb ID: ILF3), decaprenylphosphoryl-beta-D-ribose oxidase (pDb ID: 4p8Y), histone deacetylase HDAC 3 (pDb ID: 4A64), COVID-19 main protease (pDb ID:6LU7), 11beta-hydroxysteroid dehydrogenase-1(pDb ID: 3OQ1), 5-lipoxygenase activating protein (pDb ID: 6NCF), cathepsin (pDb ID: 1CS6), thioredoxin reductase from Entamoeba histolytica (4CCR), and human alpha thrombin (pDb ID :1D3t), were retrieved from protein plasmepsin (1LF3) Asp protease of P. falciparum is involved in the degradation of hemoglobin, secretory organelle protein processing for egress, invasion, and effector export 21,22 . ...
Nature has always been a source of drug candidates. Since ancient times, people have been using plants and their metabolites for various medicinal purposes. Glaucarubinone is a quassinoid present in the family Simaroubaceae. Simarouba glauca, also known as Laxmitaru or paradise tree is grouped under the family Simaroubaceae, Glaucarubinone present in S. glauca is known for its medicinal property. Molecular docking methods are widely used to investigate the interactions between a drug candidate and its target, and to discern the therapeutic action to design new drug candidate with enhanced activities. The information generated from docking studies helps to obtain an insight into interactions of drug candidate with amino acid in the active site of the target proteins, and to predict the binding energy of ligands to the target. By molecular Dynamic Simulation, the flexibility and the conformational stability of target proteins-glaucarubinone complex is confirmed.
... During the growth phase of the Plasmodium parasite, HAP is responsible for catalyzing the degradation of erythrocyte hemoglobin at specific peptide bonds, which serve as cleavage sites in the degradation pathway. This degradation process provides the parasite with essential amino acids for protein nutrient enrichment [5] , whereas the other plasmepsins play different roles [6] . ...
Aspartic proteases can hydrolyze peptide bonds, making them potential targets for drug development against malaria parasites. In particular, inhibiting the histoaspartic protease (HAP) can disrupt the growth phase of Plasmodium falciparum and its ability to degrade hemoglobin for protein synthesis. Compound 5, specifically designed as 2-(2-benzoyl-4-methylphenoxy)quinoline-3-carbaldehyde, served as the basis for designing 50 hypothetical compounds (A1-A50). These compounds were subjected to in silico screening to assess their toxicity profiles, pharmacokinetics, bioactivity scores, and theoretical binding affinities, as a part of the drug design protocol. Out of the 50 compounds, nine lead candidates showed no toxicity to human cells. In addition, ten standard reference antimalarial drugs were included in this study for comparison. The highest binding energies were observed for compound A5 (−11.2 kcal/mol) and A31 (−11.3 kcal/mol), surpassing the performance of mefloquine, the best reference drug, which ranked ninth with a binding energy of (−9.6 kcal/mol). Compound A31 did not exhibit the evidence of interaction with either Asp215 or His32, whereas compound A5 displayed π-π stacking interactions with His32. Mefloquine also did not show any interaction with Asp215 or His32. Moreover, compound A5 demonstrated greater hydrophobic interactions at the active site with most binding residues, except for Lys7 in the hydrophobic region. This characteristic suggests that compound A5 may have the ability to adopt a smaller surface area, exhibit increased biological activity, and have reduced interactions with water, which could facilitate slower clearance. Based on the assessment of various drug-likeness parameters, compound A5 (2-(2-benzoyl-4-methylphenoxy)-7-methylquinoline-3-carbaldehyde) is a potential lead candidate for the development of a new antimalarial drug.
... It was not immediately obvious how knockdown of PTEX reduces Hb peptides and so we postulated that PTEX could either be involved in the uptake of Hb or involved in the trafficking of the early acting falcipain and plasmepsin Hb proteases to the cytostome en route to the food vacuole / digestive vacuole [34][35][36]. These Hb proteases show a certain level of redundancy where knocking out one protease can sometimes be rescued by another protease, but not all of them can be knocked out simultaneously (extensively reviewed in [37]). The cytostome is an invagination of both the PPM and the PVM from which Hb containing vesicles bud off for transport to the food vacuole where the majority of Hb digestion occurs [38,39]. ...
A key element of Plasmodium biology and pathogenesis is the trafficking of ~10% of the parasite proteome into the host red blood cell (RBC) it infects. To cross the parasite-encasing parasitophorous vacuole membrane, exported proteins utilise a channel-forming protein complex termed the Plasmodium translocon of exported proteins (PTEX). PTEX is obligatory for parasite survival, both in vitro and in vivo, suggesting that at least some exported proteins have essential metabolic functions. However, to date only one essential PTEX-dependent process, the new permeability pathways, has been described. To identify other essential PTEX-dependant proteins/processes, we conditionally knocked down the expression of one of its core components, PTEX150, and examined which pathways were affected. Surprisingly, the food vacuole mediated process of haemoglobin (Hb) digestion was substantially perturbed by PTEX150 knockdown. Using a range of transgenic parasite lines and approaches, we show that two major Hb proteases; falcipain 2a and plasmepsin II, interact with PTEX core components, implicating the translocon in the trafficking of Hb proteases. We propose a model where these proteases are translocated into the PV via PTEX in order to reach the cytostome, located at the parasite periphery, prior to food vacuole entry. This work offers a second mechanistic explanation for why PTEX function is essential for growth of the parasite within its host RBC.
... We also identified various proteases, for example, SUB2, DPAP1, DPAP2, M1AAP, M16, as well as PMI, PMIII, PMX, the majority of which were so far linked to hemoglobin processing (reviewed in, e.g., Arisue et al., 2020;Goldberg, 2005;Nasamu et al., 2020;Rosenthal, 2002;Siqueira-Neto et al., 2018;Trenholme et al., 2010). ...
Transmission of malaria parasites to the mosquito is mediated by sexual precursor cells, the gametocytes. Upon entering the mosquito midgut, the gametocytes egress from the enveloping erythrocyte while passing through gametogenesis. Egress follows an inside‐out mode during which the membrane of the parasitophorous vacuole (PV) ruptures prior to the erythrocyte membrane. Membrane rupture requires exocytosis of specialized egress vesicles of the parasites; that is, osmiophilic bodies (OBs) involved in rupturing the PV membrane, and vesicles that harbor the perforin‐like protein PPLP2 (here termed P‐EVs) required for erythrocyte lysis. While some OB proteins have been identified, like G377 and MDV1/Peg3, the majority of egress vesicle‐resident proteins is yet unknown. Here, we used high‐resolution imaging and BioID methods to study the two egress vesicle types in Plasmodium falciparum gametocytes. We show that OB exocytosis precedes discharge of the P‐EVs and that exocytosis of the P‐EVs, but not of the OBs, is calcium sensitive. Both vesicle types exhibit distinct proteomes with the majority of proteins located in the OBs. In addition to known egress‐related proteins, we identified novel components of OBs and P‐EVs, including vesicle‐trafficking proteins. Our data provide insight into the immense molecular machinery required for the inside‐out egress of P. falciparum gametocytes.
... Altogether ten A1 members are encoded by P. falciparum, known as plasmepsins (PM) I-X. Molecular phylogenetic analysis groups these isozymes into six clades (A-F), which correspond to specific functions within its life cycle [20][21][22]. ...
... Accordingly, gene knockout (KO) resulted in parasites that produced oocysts but failed to develop into sporozoites. As expected, in vitro growth in RBCs showed no impairment [20]. Thus, ASP2 may be involved in piroplasmid oocyst or kinete maturation and/or penetration and migration of kinetes through tick tissues, providing a suitable target for the development of transmission-blocking interventions. ...
... The piroplasmid ASP5 is expected to localize to the endoplasmic reticulum, based on findings with its plasmodial ortholog, PM V [20]. In B. microti, it has been shown to be expressed throughout the life cycle in the mouse and tick host [23]. ...
Piroplasmids of the genera Babesia, Theileria, and Cytauxzoon are tick-transmitted parasites with a high impact on animals and humans. They have complex life cycles in their definitive arthropod and intermediate vertebrate hosts involving numerous processes, including invasion of, and egress from, host cells, parasite growth, transformation, and migration. Like other parasitic protozoa, piroplasmids are equipped with different types of protease to fulfill many of such essential processes. Blockade of some key proteases, using inhibitors or antibodies, hinders piroplasmid growth, highlighting their potential usefulness in drug therapies and vaccine development. A better understanding of the functional significance of these enzymes will contribute to the development of improved control measures for the devastating animal and human diseases caused by these pathogens.
