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Malaria parasite plasmepsins: More than just plain old degradative pepsins

Open AccessPublished:May 04, 2020DOI:https://doi.org/10.1074/jbc.REV120.009309
      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, whereas others, such as the transmission-stage plasmepsins, are minimally understood. Some (e.g. plasmepsin V) have exquisite cleavage sequence specificity; others are fairly promiscuous. Some have canonical pepsin-like aspartic protease features, whereas others have unusual attributes, including the nepenthesin loop of plasmepsin V and a histidine in place of a catalytic aspartate in plasmepsin III. We have learned much about the functioning of these enzymes, but more remains to be discovered about their cellular roles and even their mechanisms of action. Their importance in many key aspects of parasite biology makes them intriguing targets for antimalarial chemotherapy. Further consideration of their characteristics suggests that some are more viable drug targets than others. Indeed, inhibitors of invasion and egress offer hope for a desperately needed new drug to combat this nefarious organism.

      Introduction

      Malaria is caused by protozoan organisms of the genus Plasmodium. The disease is transmitted by infected female Anopheles mosquitos, which inject salivary gland sporozoites into the skin during bloodfeeding. These sporozoites make their way to the liver, replicate, and differentiate into infective merozoites. The 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 (Fig. 1).
      Figure thumbnail gr1
      Figure 1Life 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.
      Aspartic proteases called plasmepsins (Plasmodium pepsins, abbreviated PM) play important roles in each stage of Plasmodium development. Interest in the plasmepsins began when the digestive vacuole plasmepsins (I, II, III, and IV) were found to be important for intraerythrocytic hemoglobin degradation (
      • Goldberg D.E.
      • Slater A.F.G.
      • Beavis R.
      • Chait B.
      • Cerami A.
      • Henderson G.B.
      Hemoglobin degradation in the human malaria pathogen Plasmodium falciparum: a catabolic pathway initiated by a specific aspartic protease.
      ,
      • Francis S.E.
      • Gluzman I.Y.
      • Oksman A.
      • Knickerbocker A.
      • Mueller R.
      • Bryant M.L.
      • Sherman D.R.
      • Russell D.G.
      • Goldberg D.E.
      Molecular characterization and inhibition of a Plasmodium falciparum aspartic hemoglobinase.
      ,
      • Dame J.B.
      • Reddy G.R.
      • Yowell C.A.
      • Dunn B.M.
      • Kay J.
      • Berry C.
      Sequence, expression and modeled structure of an aspartic proteinase from the human malaria parasite Plasmodium falciparum.
      ,
      • Dame J.B.
      • Yowell C.A.
      • Omara-Opyene L.
      • Carlton J.M.
      • Cooper R.A.
      • Li T.
      Plasmepsin 4, the food vacuole aspartic proteinase found in all Plasmodium spp. infecting man.
      ,
      • Banerjee R.
      • Liu J.
      • Beatty W.
      • Pelosof L.
      • Klemba M.
      • Goldberg D.E.
      Four plasmepsins are active in the Plasmodium falciparum food vacuole, including a protease with an active-site histidine.
      ). There followed a major effort to make small-molecule inhibitors to these enzymes, especially PM II, the easiest to express and the first to have a crystal structure (
      • Hill J.
      • Tyas L.
      • Phylip L.H.
      • Kay J.
      • Dunn B.M.
      • Berry C.
      High level expression and characterisation of Plasmepsin II, an aspartic proteinase from Plasmodium falciparum.
      ,
      • Silva A.M.
      • Lee A.Y.
      • Gulnik S.V.
      • Maier P.
      • Collins J.
      • Bhat T.N.
      • Collins P.J.
      • Cachau R.E.
      • Luker K.E.
      • Gluzman I.Y.
      • Francis S.E.
      • Oksman A.
      • Goldberg D.E.
      • Erickson J.W.
      Structure and inhibition of plasmepsin II, a hemoglobin-degrading enzyme from Plasmodium falciparum.
      ). A poor correlation between ability of a compound to kill parasites and potency against isolated enzyme (
      • Meyers M.J.
      • Goldberg D.E.
      Recent advances in plasmepsin medicinal chemistry and implications for future antimalarial drug discovery efforts.
      ) suggested that digestive vacuole plasmepsin inhibition was not the mode of parasite killing for these molecules. This ultimately led to the realization that there must be other targets, likely other aspartic proteases, whose inhibition is responsible for the antiplasmodial properties. The search for these targets has uncovered myriad functions for these enzymes. Plasmepsins are involved in bulk protein degradation, secretory protein maturation, egress, invasion, endothelial adherence, and perhaps other processes. A number have been the subject of serious efforts as targets for drug development.
      Plasmepsins (Fig. 2) belong to an ancient family of aspartic proteases—the A1 or pepsin-like family—that is widespread throughout eukaryotes. Among the 10 plasmepsins, the most closely related are the digestive vacuolar plasmepsins, PM I–IV. These proteases are spread across just 16 kilobases of chromosome 14 and share 50–70% amino acid identity. Outside of P. falciparum and related primate-infecting species, these proteases are represented by a single plasmepsin, called PM IV in Plasmodium and ASP1 in the related apicomplexan Toxoplasma gondii (
      • Shea M.
      • Jäkle U.
      • Liu Q.
      • Berry C.
      • Joiner K.A.
      • Soldati-Favre D.
      A family of aspartic proteases and a novel, dynamic and cell-cycle-dependent protease localization in the secretory pathway of Toxoplasma gondii.
      ). PM V is the most diverged plasmepsin, sharing 19–23% amino acid identity with the other plasmepsins. Its structure is bolstered by seven disulfide bonds (compared with two in PM I–IV), bringing it into a separate aspartic protease subfamily from the other plasmepsins—subfamily A1B, with type member Nep1 of the pitcher plant Nepenthesia (
      • Rawlings N.D.
      • Waller M.
      • Barrett A.J.
      • Bateman A.
      MEROPS: the database of proteolytic enzymes, their substrates and inhibitors.
      ). Other apicomplexans also have a single PM V ortholog (ASP5 in Toxoplasma gondii), except for cryptosporidia, which have three (
      • Shea M.
      • Jäkle U.
      • Liu Q.
      • Berry C.
      • Joiner K.A.
      • Soldati-Favre D.
      A family of aspartic proteases and a novel, dynamic and cell-cycle-dependent protease localization in the secretory pathway of Toxoplasma gondii.
      ). Regarding transmission-stage plasmepsins, PM VI and VIII form a clade, sharing 36% amino acid identity with each other. Each has a little-studied ortholog in T. gondii (ASP2 and ASP4 respectively). PM VII has distant homology to PM VI and VIII (31% identity); its uncharacterized Toxoplasma ortholog is ASP6. PM IX and PM X share 37% amino acid identity. Although the two are distinct across Plasmodium and exist on different chromosomes, they are represented by a single T. gondii aspartic protease, ASP3.
      Figure thumbnail gr2
      Figure 2Plasmepsin phylogeny. Sequences for PMs I–X were obtained from PlasmoDB (release 46), aligned using MUSCLE (“Multiple Sequence Comparison by Log-Expectation”, EMBL) (
      • Edgar R.C.
      MUSCLE: multiple sequence alignment with high accuracy and high throughput.
      ), and visualized using iTOL (Interactive Tree of Life) (
      • Letunic I.
      • Bork P.
      Interactive Tree of Life (iTOL) v4: recent updates and new developments.
      ).
      A note on nomenclature: In the literature, plasmepsins are denoted with Roman numerals or Arabic numerals, with or without a space before the number, and plasmepsin III is known as histo-aspartic protease or HAP or PM III (or PMIII or PM3 or PM 3). We suggest going back to a convention initiated in early publications of having Roman numerals after a space. We further suggest that HAP be referred to as PM III for consistency with the other plasmepsins and because its His32 has not been shown to be catalytic. Also, HAP is the name for a gamete fusion protein. Using PM III allows the digestive vacuole plasmepsins in aggregate to be called PM I–IV without ambiguity. An argument for the space before the Roman numeral is that PM V is often referred to in discussions of sending proteins out to the parasitophorous vacuolar membrane or PVM, and PMV gets confusing in this context. These issues are ultimately for those concerned with nomenclature to weigh in on, but in this review, we will use our preferred convention.
      There are also nonplasmepsin aspartic proteases in the Plasmodium genome (see “Other aspartic proteases”). The microgametocyte surface protein (MiGS) has homology to PM V (21% amino acid identity and shared transmembrane domain; 17–20% to other plasmepsins). Additional aspartic proteases in the Plasmodium genome—signal peptide peptidase (SPP) and DNA damage-inducible 1 (Ddi1)—are quite distinct from the plasmepsins. SPP is a presenilin-type aspartic protease (family A22). Ddi1 (family A28) is the most diverged from the other aspartic proteases. It possesses a single catalytic aspartate and likely homodimerizes to complete the active site, an unusual architecture among eukaryotic aspartic proteases (although common to retroviral aspartic proteases).
      Here, we review the state of knowledge on the plasmepsins, noting outstanding questions in the field. This review will focus on P. falciparum, the deadliest human malaria parasite, for which extensive biological studies have been performed; reference to other species will be made as relevant. Study of the diverse plasmepsins has taught us much about the biology of the malaria parasite and a little about the protein chemistry of aspartic proteases and is pointing the way to exciting new inhibitors that are being developed as antimalarial chemotherapeutic agents.

      Digestive vacuole plasmepsins: Plasmepsins I–IV

      Intraerythrocytic malaria parasites digest hemoglobin at a prodigious rate (
      • Francis S.E.
      • Sullivan Jr., D.J.
      • Goldberg D.E.
      Hemoglobin metabolism in the malaria parasite Plasmodium falciparum.
      ). This catabolic process provides nutrients for the parasite and has been proposed to maintain osmotic balance in the host cell (
      • Francis S.E.
      • Sullivan Jr., D.J.
      • Goldberg D.E.
      Hemoglobin metabolism in the malaria parasite Plasmodium falciparum.
      ,
      • Lew V.L.
      • Tiffert T.
      • Ginsburg H.
      Excess hemoglobin digestion and the osmotic stability of Plasmodium falciparum-infected red blood cells.
      ,
      • Sherman I.W.
      • Tanigoshi L.
      Incorporation of 14C-amino-acids by malaria (Plasmodium lophurae) IV. In vivo utilization of host cell haemoglobin.
      ). Hemoglobin is ingested by Plasmodium through an endocytic structure called the cytostome, which spans the plasma membrane and the vacuolar membrane surrounding the parasite (Fig. 3A) (
      • Abu Bakar N.
      • Klonis N.
      • Hanssen E.
      • Chan C.
      • Tilley L.
      Digestive-vacuole genesis and endocytic processes in the early intraerythrocytic stages of Plasmodium falciparum.
      ,
      • Aikawa M.
      • Hepler P.K.
      • Huff C.G.
      • Sprinz H.
      The feeding mechanism of avian malarial parasites.
      ). Hemoglobin-containing vesicles pinch off and fuse with the digestive vacuole, an acidic organelle containing 10 proteases that function in a semi-ordered pathway to degrade the hemoglobin to small peptides and amino acids (
      • Goldberg D.E.
      • Slater A.F.G.
      • Cerami A.
      • Henderson G.B.
      Hemoglobin degradation in the malaria parasite Plasmodium falciparum: an ordered process in a unique organelle.
      ,
      • Kolakovich K.A.
      • Gluzman I.Y.
      • Duffin K.L.
      • Goldberg D.E.
      Generation of hemoglobin peptides in the acidic digestive vacuole of Plasmodium falciparum implicates peptide transport in amino acid production.
      ,
      • Rudzinska M.A.
      • Trager W.
      • Bray R.S.
      Pinocytotic uptake and the digestion of hemoglobin in malaria parasites.
      ) (Fig. 3B). Some of these peptides are thought to be exported out of the digestive vacuole for terminal degradation in the cytoplasm (
      • Kolakovich K.A.
      • Gluzman I.Y.
      • Duffin K.L.
      • Goldberg D.E.
      Generation of hemoglobin peptides in the acidic digestive vacuole of Plasmodium falciparum implicates peptide transport in amino acid production.
      ,
      • Lewis I.A.
      • Wacker M.
      • Olszewski K.L.
      • Cobbold S.A.
      • Baska K.S.
      • Tan A.
      • Ferdig M.T.
      • Llinás M.
      Metabolic QTL analysis links chloroquine resistance in Plasmodium falciparum to impaired hemoglobin catabolism.
      ,
      • Lee A.H.
      • Dhingra S.K.
      • Lewis I.A.
      • Singh M.K.
      • Siriwardana A.
      • Dalal S.
      • Rubiano K.
      • Klein M.S.
      • Baska K.S.
      • Krishna S.
      • Klemba M.
      • Roepe P.D.
      • Llinás M.
      • Garcia C.R.S.
      • Fidock D.A.
      Evidence for regulation of hemoglobin metabolism and intracellular ionic flux by the Plasmodium falciparum chloroquine resistance transporter.
      ).
      Figure thumbnail gr3
      Figure 3Hemoglobin ingestion and digestion. A, electron micrograph of a P. falciparum-infected erythrocyte. C, cytostome; V, vesicle; DV, digestive vacuole. Adapted from Ref.
      • Goldberg D.E.
      • Slater A.F.G.
      • Cerami A.
      • Henderson G.B.
      Hemoglobin degradation in the malaria parasite Plasmodium falciparum: an ordered process in a unique organelle.
      . This research was originally published in Proceedings of the National Academy of Sciences of the United States of America. Goldberg, D. E., Slater, A. F. G., Cerami, A., and Henderson, G. B. Hemoglobin degradation in the malaria parasite Plasmodium falciparum: an ordered process in a unique organelle. Proc. Natl. Acad. Sci. U.S.A. 1990; 87:2931–2935. © United States National Academy of Sciences. B, semi-ordered pathway of hemoglobin degradation. Plasmepsins and falcipains are involved in the initial steps of catabolism and are partially redundant. Falcilysin recognizes oligopeptides. DPAP1 cleaves two residues off of the N terminus of hemoglobin fragments. Aminopeptidases finish the digestion, resulting in free amino acids. Heme is liberated during the initial steps of proteolysis; most is sequestered in the digestive vacuole as hemozoin. C, plasmepsin targeting pathway follows the hemoglobin internalization route. Plasmepsins are made in the ER and traverse the secretory system as type II integral membrane protein precursors (ball and stick). They traffic to the cytostome, a hemoglobin ingestion apparatus that spans the two membranes at the parasite surface. They are internalized with their substrate cargo (hemoglobin) and are delivered to the digestive vacuole surface, where they are cleaved by falcipain-2. PPM, parasite plasma membrane. Black bars, hemozoin crystals that accumulate after heme release.
      The degradative process may start in transport vesicles and commences early after erythrocyte invasion (
      • Abu Bakar N.
      • Klonis N.
      • Hanssen E.
      • Chan C.
      • Tilley L.
      Digestive-vacuole genesis and endocytic processes in the early intraerythrocytic stages of Plasmodium falciparum.
      ). Heme is liberated by degradation and most is sequestered as a crystalline lattice of β-hematin dimers called hemozoin (
      • Sigala P.A.
      • Goldberg D.E.
      The peculiarities and paradoxes of plasmodium heme metabolism.
      ). Some antimalarial drugs like chloroquine appear to function by blocking heme sequestration, leading to toxic heme build-up (
      • Sullivan D.J.
      Quinolines block every step of malaria heme crystal growth.
      ). A network of proteins implicated in the hemoglobin endocytosis process has recently been defined and includes the artemisinin-resistance protein Kelch13 (
      • Birnbaum J.
      • Scharf S.
      • Schmidt S.
      • Jonscher E.
      • Hoeijmakers W.A.M.
      • Flemming S.
      • Toenhake C.G.
      • Schmitt M.
      • Sabitzki R.
      • Bergmann B.
      • Fröhlke U.
      • Mesén-Ramírez P.
      • Blancke Soares A.
      • Herrmann H.
      • Bártfai R.
      • Spielmann T.
      A Kelch13-defined endocytosis pathway mediates artemisinin resistance in malaria parasites.
      ). This finding has yielded a cogent mechanistic explanation for artemisinin resistance. Mutations in Kelch13 and other proteins cause decreased hemoglobin ingestion and digestion, yielding less artemisinin-activating heme (
      • Birnbaum J.
      • Scharf S.
      • Schmidt S.
      • Jonscher E.
      • Hoeijmakers W.A.M.
      • Flemming S.
      • Toenhake C.G.
      • Schmitt M.
      • Sabitzki R.
      • Bergmann B.
      • Fröhlke U.
      • Mesén-Ramírez P.
      • Blancke Soares A.
      • Herrmann H.
      • Bártfai R.
      • Spielmann T.
      A Kelch13-defined endocytosis pathway mediates artemisinin resistance in malaria parasites.
      ,
      • Yang T.
      • Yeoh L.M.
      • Tutor M.V.
      • Dixon M.W.
      • McMillan P.J.
      • Xie S.C.
      • Bridgford J.L.
      • Gillett D.L.
      • Duffy M.F.
      • Ralph S.A.
      • McConville M.J.
      • Tilley L.
      • Cobbold S.A.
      Decreased K13 abundance reduces hemoglobin catabolism and proteotoxic stress, underpinning artemisinin resistance.
      ).

      Discovery

      Involvement of aspartic proteases in hemoglobin digestion was suspected nearly 50 years ago from the characterization of crude parasite extracts (
      • Levy M.R.
      • Siddiqui W.A.
      • Chou S.C.
      Acid protease activity in Plasmodium falciparumP. knowlesi and ghosts of their respective host red cells.
      ,
      • Levy M.R.
      • Chou S.C.
      Activity and some properties of an acid proteinase from normal and Plasmodium berghei-infected red cells.
      ). The older literature contains numerous reports of hemoglobin-degrading acidic protease activities in various Plasmodium species, and some were shown to be blocked by the canonical aspartic protease inhibitor pepstatin A (
      • Levy M.R.
      • Siddiqui W.A.
      • Chou S.C.
      Acid protease activity in Plasmodium falciparumP. knowlesi and ghosts of their respective host red cells.
      ,
      • Levy M.R.
      • Chou S.C.
      Activity and some properties of an acid proteinase from normal and Plasmodium berghei-infected red cells.
      ,
      • Sherman I.W.
      • Tanigoshi L.
      The proteases of Plasmodium: a cathepsin D-like enzyme from Plasmodium lophurae.
      ,
      • Gyang F.N.
      • Poole B.
      • Trager W.
      Peptidases from Plasmodium falciparum cultured in vitro.
      ,
      • Aissi E.
      • Charet P.
      • Bouquelet S.
      • Biguet J.
      Endoprotease in Plasmodium yoelii nigeriensis.
      ,
      • Sato K.
      • Fukabori Y.
      • Suzuki M.
      Plasmodium berghei: a study of globinolytic enzyme in erythrocytic parasite.
      ,
      • Hempelmann E.
      • Wilson R.J.M.
      Endopeptidases from Plasmodium knowlesi.
      ,
      • Bailly E.
      • Savel J.
      • Mahouy G.
      • Jaureguiberry G.
      Plasmodium falciparum: isolation and characterization of a 55-kDa protease with a cathepsin D-like activity from P. falciparum.
      ,
      • Vander Jagt D.L.
      • Hunsaker L.A.
      • Campos N.M.
      Characterization of a hemoglobin-degrading, low molecular weight protease from Plasmodium falciparum.
      ,
      • vander Jagt D.L.
      • Hunsaker L.A.
      • Campos N.M.
      • Scaletti J.V.
      Localization and characterization of hemoglobin-degrading aspartic proteinases from the malarial parasite Plasmodium falciparum.
      ). Their cellular roles remained unclear until studies on isolated digestive vacuoles showed the ability to degrade hemoglobin (
      • Goldberg D.E.
      • Slater A.F.G.
      • Cerami A.
      • Henderson G.B.
      Hemoglobin degradation in the malaria parasite Plasmodium falciparum: an ordered process in a unique organelle.
      ,
      • Choi I.
      • Mego J.L.
      Intravacuolar proteolysis in Plasmodium falciparum digestive vacuoles is similar to intralysosomal proteolysis in mammalian cells.
      ), and protease inhibitor profiling of hemoglobinase activity from highly purified digestive vacuoles revealed a central role for aspartic proteases (
      • Goldberg D.E.
      • Slater A.F.G.
      • Cerami A.
      • Henderson G.B.
      Hemoglobin degradation in the malaria parasite Plasmodium falciparum: an ordered process in a unique organelle.
      ). Soon thereafter, two aspartic proteases (now called PM I and II) were purified from large-scale digestive vacuole preparations and shown to cleave hemoglobin (
      • Goldberg D.E.
      • Slater A.F.G.
      • Beavis R.
      • Chait B.
      • Cerami A.
      • Henderson G.B.
      Hemoglobin degradation in the human malaria pathogen Plasmodium falciparum: a catabolic pathway initiated by a specific aspartic protease.
      ,
      • Francis S.E.
      • Gluzman I.Y.
      • Oksman A.
      • Knickerbocker A.
      • Mueller R.
      • Bryant M.L.
      • Sherman D.R.
      • Russell D.G.
      • Goldberg D.E.
      Molecular characterization and inhibition of a Plasmodium falciparum aspartic hemoglobinase.
      ,
      • Gluzman I.Y.
      • Francis S.E.
      • Oksman A.
      • Smith C.E.
      • Duffin K.L.
      • Goldberg D.E.
      Order and specificity of the Plasmodium falciparum hemoglobin degradation pathway.
      ). After release of the P. falciparum genome, two more plasmepsins were shown to be present in the digestive vacuole, PM III and PM IV (
      • Dame J.B.
      • Yowell C.A.
      • Omara-Opyene L.
      • Carlton J.M.
      • Cooper R.A.
      • Li T.
      Plasmepsin 4, the food vacuole aspartic proteinase found in all Plasmodium spp. infecting man.
      ,
      • Banerjee R.
      • Liu J.
      • Beatty W.
      • Pelosof L.
      • Klemba M.
      • Goldberg D.E.
      Four plasmepsins are active in the Plasmodium falciparum food vacuole, including a protease with an active-site histidine.
      ,
      • Coombs G.H.
      • Goldberg D.E.
      • Klemba M.
      • Berry C.
      • Kay J.
      • Mottram J.C.
      Aspartic proteases of Plasmodium falciparum and other parasitic protozoa as drug targets.
      ).

      Function

      There is extensive functional redundancy among the digestive vacuole plasmepsins and between these enzymes and the cysteine proteases called falcipains. Knockouts of individual P. falciparum plasmepsins have minor effects on parasite growth in culture (
      • Liu J.
      • Gluzman I.Y.
      • Drew M.E.
      • Goldberg D.E.
      The role of Plasmodium falciparum food vacuole plasmepsins.
      ,
      • Omara-Opyene A.L.
      • Moura P.A.
      • Sulsona C.R.
      • Bonilla J.A.
      • Yowell C.A.
      • Fujioka H.
      • Fidock D.A.
      • Dame J.B.
      Genetic disruption of the Plasmodium falciparum digestive vacuole plasmepsins demonstrates their functional redundancy.
      ,
      • Bonilla J.A.
      • Moura P.A.
      • Bonilla T.D.
      • Yowell C.A.
      • Fidock D.A.
      • Dame J.B.
      Effects on growth, hemoglobin metabolism and paralogous gene expression resulting from disruption of genes encoding the digestive vacuole plasmepsins of Plasmodium falciparum.
      ,
      • Liu J.
      • Istvan E.S.
      • Gluzman I.Y.
      • Gross J.
      • Goldberg D.E.
      Plasmodium falciparum ensures its amino acid supply with multiple acquisition pathways and redundant proteolytic enzyme systems.
      ), whereas knockout of all four leads to a substantial growth defect, although the knockout line is still viable (
      • Bonilla J.A.
      • Bonilla T.D.
      • Yowell C.A.
      • Fujioka H.
      • Dame J.B.
      Critical roles for the digestive vacuole plasmepsins of Plasmodium falciparum in vacuolar function.
      ). Knockout of the single Plasmodium berghei digestive vacuole plasmepsin yields a similar phenotype in the mouse model (
      • Spaccapelo R.
      • Janse C.J.
      • Caterbi S.
      • Franke-Fayard B.
      • Bonilla J.A.
      • Syphard L.M.
      • Di Cristina M.
      • Dottorini T.
      • Savarino A.
      • Cassone A.
      • Bistoni F.
      • Waters A.P.
      • Dame J.B.
      • Crisanti A.
      Plasmepsin 4-deficient Plasmodium berghei are virulence attenuated and induce protective immunity against experimental malaria.
      ,
      • Lin J.W.
      • Spaccapelo R.
      • Schwarzer E.
      • Sajid M.
      • Annoura T.
      • Deroost K.
      • Ravelli R.B.G.
      • Aime E.
      • Capuccini B.
      • Mommaas-Kienhuis A.M.
      • O'Toole T.
      • Prins F.
      • Franke-Fayard B.M.D.
      • Ramesar J.
      • Chevalley-Maurel S.
      • et al.
      Replication of Plasmodium in reticulocytes can occur without hemozoin formation, resulting in chloroquine resistance.
      ). Cysteine protease inhibitors are more potent in the P. falciparum knockouts (
      • Liu J.
      • Gluzman I.Y.
      • Drew M.E.
      • Goldberg D.E.
      The role of Plasmodium falciparum food vacuole plasmepsins.
      ,
      • Bonilla J.A.
      • Bonilla T.D.
      • Yowell C.A.
      • Fujioka H.
      • Dame J.B.
      Critical roles for the digestive vacuole plasmepsins of Plasmodium falciparum in vacuolar function.
      ), and knockouts of falcipain-2 are more sensitive to aspartic protease inhibitors (
      • Sijwali P.S.
      • Rosenthal P.J.
      Gene disruption confirms a critical role for the cysteine protease falcipain-2 in hemoglobin hydrolysis by Plasmodium falciparum.
      ), highlighting the redundancy between the two digestive vacuole protease families. Plasmepsins and falcipains are synergistic in biochemical assays of hemoglobin degradation (
      • Goldberg D.E.
      • Slater A.F.G.
      • Cerami A.
      • Henderson G.B.
      Hemoglobin degradation in the malaria parasite Plasmodium falciparum: an ordered process in a unique organelle.
      ,
      • Gluzman I.Y.
      • Francis S.E.
      • Oksman A.
      • Smith C.E.
      • Duffin K.L.
      • Goldberg D.E.
      Order and specificity of the Plasmodium falciparum hemoglobin degradation pathway.
      ,
      • Moon S.U.
      • Kang J.M.
      • Kim T.S.
      • Kong Y.
      • Sohn W.M.
      • Na B.K.
      Plasmodium vivax: collaborative roles for plasmepsin 4 and vivapains in hemoglobin hydrolysis.
      ), and cysteine/aspartic protease inhibitor synergism is observed in culture (
      • Bailly E.
      • Jambou R.
      • Savel J.
      • Jaureguiberry G.
      Plasmodium falciparum: differential sensitivity in vitro to E-64 (cysteine protease inhibitor) and pepstatin A (aspartyl protease inhibitor).
      ,
      • Semenov A.
      • Olson J.E.
      • Rosenthal P.J.
      Antimalarial synergy of cysteine and aspartic protease inhibitors.
      ) as well as in a rodent malaria model (
      • Semenov A.
      • Olson J.E.
      • Rosenthal P.J.
      Antimalarial synergy of cysteine and aspartic protease inhibitors.
      ). A final level of redundancy is that digestive vacuole plasmepsins are activated by falcipains, but if the falcipains are impaired, plasmepsins can autoactivate (
      • Drew M.E.
      • Banerjee R.
      • Uffman E.W.
      • Gilbertson S.
      • Rosenthal P.J.
      • Goldberg D.E.
      Plasmodium food vacuole plasmepsins are activated by falcipains.
      ) (see below). Further proteolysis of globin fragments by the metalloprotease falcilysin (
      • Eggleson K.K.
      • Duffin K.L.
      • Goldberg D.E.
      Identification and characterization of falcilysin, a metallopeptidase involved in hemoglobin catabolism within the malaria parasite Plasmodium falciparum.
      ,
      • Murata C.E.
      • Goldberg D.E.
      Plasmodium falciparum falcilysin: a metalloprotease with dual specificity.
      ), dipeptidyl aminopeptidase 1 (DPAP1) (
      • Klemba M.
      • Gluzman I.
      • Goldberg D.E.
      A Plasmodium falciparum dipeptidyl aminopeptidase I participates in vacuolar hemoglobin degradation.
      ), aminopeptidase P (
      • Ragheb D.
      • Bompiani K.
      • Dalal S.
      • Klemba M.
      Evidence for catalytic roles for Plasmodium falciparum aminopeptidase P in the food vacuole and cytosol.
      ), and aminopeptidase M1 (
      • Ragheb D.
      • Dalal S.
      • Bompiani K.M.
      • Ray W.K.
      • Klemba M.
      Distribution and biochemical properties of an M1-family aminopeptidase in Plasmodium falciparum indicate a role in vacuolar hemoglobin catabolism.
      ) finishes the degradation process (Fig. 3). The digestive vacuole plasmepsins have been proposed to be in a complex with some of the downstream proteases as well as with a heme detoxification protein (HDP) in the digestive vacuole (
      • Chugh M.
      • Sundararaman V.
      • Kumar S.
      • Reddy V.S.
      • Siddiqui W.A.
      • Stuart K.D.
      • Malhotra P.
      Protein complex directs hemoglobin-to-hemozoin formation in Plasmodium falciparum.
      ).
      Curiously, recent reports have associated amplification of PM II and PM III with resistance to the antimalarial drug piperaquine in several lineages of Southeast Asian field isolates (
      • Amato R.
      • Lim P.
      • Miotto O.
      • Amaratunga C.
      • Dek D.
      • Pearson R.D.
      • Almagro-Garcia J.
      • Neal A.T.
      • Sreng S.
      • Suon S.
      • Drury E.
      • Jyothi D.
      • Stalker J.
      • Kwiatkowski D.P.
      • Fairhurst R.M.
      Genetic markers associated with dihydroartemisinin–piperaquine failure in Plasmodium falciparum malaria in Cambodia: a genotype–phenotype association study.
      ,
      • Witkowski B.
      • Duru V.
      • Khim N.
      • Ross L.S.
      • Saintpierre B.
      • Beghain J.
      • Chy S.
      • Kim S.
      • Ke S.
      • Kloeung N.
      • Eam R.
      • Khean C.
      • Ken M.
      • Loch K.
      • Bouillon A.
      • et al.
      A surrogate marker of piperaquine-resistant Plasmodium falciparum malaria: a phenotype–genotype association study.
      ,
      • Bopp S.
      • Magistrado P.
      • Wong W.
      • Schaffner S.F.
      • Mukherjee A.
      • Lim P.
      • Dhorda M.
      • Amaratunga C.
      • Woodrow C.J.
      • Ashley E.A.
      • White N.J.
      • Dondorp A.M.
      • Fairhurst R.M.
      • Ariey F.
      • Menard D.
      • Wirth D.F.
      • Volkman S.K.
      Plasmepsin II–III copy number accounts for bimodal piperaquine resistance among Cambodian Plasmodium falciparum.
      ) Removal of these extra copies of PM II/PM III resensitized parasites to piperaquine, suggesting that this amplification contributes to the resistance phenotype (
      • Bopp S.
      • Magistrado P.
      • Wong W.
      • Schaffner S.F.
      • Mukherjee A.
      • Lim P.
      • Dhorda M.
      • Amaratunga C.
      • Woodrow C.J.
      • Ashley E.A.
      • White N.J.
      • Dondorp A.M.
      • Fairhurst R.M.
      • Ariey F.
      • Menard D.
      • Wirth D.F.
      • Volkman S.K.
      Plasmepsin II–III copy number accounts for bimodal piperaquine resistance among Cambodian Plasmodium falciparum.
      ). Additionally, deletion of PM II or PM III from a laboratory P. falciparum strain sensitized parasites to piperaquine in vitro (
      • Mukherjee A.
      • Gagnon D.
      • Wirth D.F.
      • Richard D.
      Inactivation of plasmepsins 2 and 3 sensitizes Plasmodium falciparum to the antimalarial drug piperaquine.
      ). However, increasing PM II/PM III levels is not sufficient to impart piperaquine resistance in laboratory strains, hinting at a more complex picture, where interplay between PM II/PM III levels and other constituents of a strain's genetic background contributes to phenotypic resistance (
      • Loesbanluechai D.
      • Kotanan N.
      • de Cozar C.
      • Kochakarn T.
      • Ansbro M.R.
      • Chotivanich K.
      • White N.J.
      • Wilairat P.
      • Lee M.C.S.
      • Gamo F.J.
      • Sanz L.M.
      • Chookajorn T.
      • Kümpornsin K.
      Overexpression of plasmepsin II and plasmepsin III does not directly cause reduction in Plasmodium falciparum sensitivity to artesunate, chloroquine and piperaquine.
      ). Consistent with this, polymorphisms in the digestive vacuolar membrane transporter PfCRT are sufficient to impart piperaquine resistance in vitro, and parasite lines with these PfCRT mutations naturally lose PM II/PM III amplification while retaining piperaquine resistance in culture (
      • Ross L.S.
      • Dhingra S.K.
      • Mok S.
      • Yeo T.
      • Wicht K.J.
      • Kümpornsin K.
      • Takala-Harrison S.
      • Witkowski B.
      • Fairhurst R.M.
      • Ariey F.
      • Menard D.
      • Fidock D.A.
      Emerging Southeast Asian PfCRT mutations confer Plasmodium falciparum resistance to the first-line antimalarial piperaquine.
      ).

      Structure and mechanism

      The structures of all four P. falciparum digestive vacuole plasmepsins and the singleton PM IV from several other species have been elucidated (
      • Silva A.M.
      • Lee A.Y.
      • Gulnik S.V.
      • Maier P.
      • Collins J.
      • Bhat T.N.
      • Collins P.J.
      • Cachau R.E.
      • Luker K.E.
      • Gluzman I.Y.
      • Francis S.E.
      • Oksman A.
      • Goldberg D.E.
      • Erickson J.W.
      Structure and inhibition of plasmepsin II, a hemoglobin-degrading enzyme from Plasmodium falciparum.
      ,
      • Asojo O.A.
      • Gulnik S.V.
      • Afonina E.
      • Yu B.
      • Ellman J.A.
      • Haque T.S.
      • Silva A.M.
      Novel uncomplexed and complexed structures of plasmepsin II, an aspartic protease from Plasmodium falciparum.
      ,
      • Bernstein N.K.
      • Cherney M.M.
      • Yowell C.A.
      • Dame J.B.
      • James M.N.G.
      Structural insights into the activation of P. vivax plasmepsin.
      ,
      • Clemente J.C.
      • Govindasamy L.
      • Madabushi A.
      • Fisher S.Z.
      • Moose R.E.
      • Yowell C.A.
      • Hidaka K.
      • Kimura T.
      • Hayashi Y.
      • Kiso Y.
      • Agbandje-McKenna M.
      • Dame J.B.
      • Dunn B.M.
      • McKenna R.
      Structure of the aspartic protease plasmepsin 4 from the malarial parasite Plasmodium malariae bound to an allophenylnorstatine-based inhibitor.
      ,
      • Madabushi A.
      • Chakraborty S.
      • Fisher S.Z.
      • Clemente J.C.
      • Yowell C.
      • Agbandje-McKenna M.
      • Dame J.B.
      • Dunn B.M.
      • McKenna R.
      Crystallization and preliminary X-ray analysis of the aspartic protease plasmepsin 4 from the malarial parasite Plasmodium malariae.
      ,
      • Bhaumik P.
      • Gustchina A.
      • Wlodawer A.
      Structural studies of vacuolar plasmepsins.
      ,
      • Bhaumik P.
      • Xiao H.
      • Parr C.L.
      • Kiso Y.
      • Gustchina A.
      • Yada R.Y.
      • Wlodawer A.
      Crystal structures of the histo-aspartic protease (HAP) from Plasmodium falciparum.
      ,
      • Bhaumik P.
      • Horimoto Y.
      • Xiao H.
      • Miura T.
      • Hidaka K.
      • Kiso Y.
      • Wlodawer A.
      • Yada R.Y.
      • Gustchina A.
      Crystal structures of the free and inhibited forms of plasmepsin I (PMI) from Plasmodium falciparum.
      ). Each has the typical bilobal pepsin-family aspartic protease fold. These plasmepsins are capable of forming dimers in crystalline form and in solution (
      • Liu J.
      • Istvan E.S.
      • Goldberg D.E.
      Hemoglobin-degrading plasmepsin II is active as a monomer.
      ,
      • Xiao H.
      • Briere L.A.K.
      • Dunn S.D.
      • Yada R.Y.
      Characterization of the monomer-dimer equilibrium of recombinant histo-aspartic protease from Plasmodium falciparum.
      ), although it is the monomer that has been shown to be active. The PM III dimer has a loop from one of the subunits that intrudes into the second subunit active site, where it coordinates a zinc ion via Asp215 and His32 (
      • Bhaumik P.
      • Xiao H.
      • Parr C.L.
      • Kiso Y.
      • Gustchina A.
      • Yada R.Y.
      • Wlodawer A.
      Crystal structures of the histo-aspartic protease (HAP) from Plasmodium falciparum.
      ). The canonical Asp215–Thr218 hydrogen bond is disrupted by the zinc coordination.
      PM I, II, and IV appear to function as typical aspartic proteases, with their two aspartates participating in acid-base catalytic activation of a water molecule through a tetrahedral transition state (
      • Friedman R.
      • Caflisch A.
      The protonation state of the catalytic aspartates in plasmepsin II.
      ,
      • Xie D.
      • Gulnik S.
      • Collins L.
      • Gustchina E.
      • Suvorov L.
      • Erickson J.W.
      Dissection of the pH dependence of inhibitor binding energies for an aspartic protease: direct measurement of the protonation states of the catalytic aspartic acid residues.
      ), but in contrast, the mechanism of PM III (Fig. 4A) is not fully understood (
      • Bhaumik P.
      • Gustchina A.
      • Wlodawer A.
      Structural studies of vacuolar plasmepsins.
      ). In PM III, Asp215 appears to function as a catalytic base, activating the water to form the hydrolytic nucleophile (
      • Bhaumik P.
      • Xiao H.
      • Parr C.L.
      • Kiso Y.
      • Gustchina A.
      • Yada R.Y.
      • Wlodawer A.
      Crystal structures of the histo-aspartic protease (HAP) from Plasmodium falciparum.
      ). It is not clear whether His32 is involved in catalysis. It could be the catalytic acid for resolution of the tetrahedral intermediate typical of aspartic protease action, in analogy to the standard aspartate in this position. The positioning of the statine hydroxyl between Asp215 and His32 in the transition-state inhibitor pepstatin-bound structure is supportive of this concept (
      • Bhaumik P.
      • Xiao H.
      • Parr C.L.
      • Kiso Y.
      • Gustchina A.
      • Yada R.Y.
      • Wlodawer A.
      Crystal structures of the histo-aspartic protease (HAP) from Plasmodium falciparum.
      ). A H32A mutant of recombinant PM III retained activity, however (
      • Parr C.L.
      • Tanaka T.
      • Xiao H.
      • Yada R.Y.
      The catalytic significance of the proposed active site residues in Plasmodium falciparum histoaspartic protease.
      ). It has been suggested that PM III could be a serine protease, using Ser35 as the nucleophile (
      • Andreeva N.
      • Bogdanovich P.
      • Kashparov I.
      • Popov M.
      • Stengach M.
      Is histoaspartic protease a serine protease with a pepsin-like fold?.
      ), but a S35A mutant retained activity (
      • Parr C.L.
      • Tanaka T.
      • Xiao H.
      • Yada R.Y.
      The catalytic significance of the proposed active site residues in Plasmodium falciparum histoaspartic protease.
      ), and the pepstatin active site interactions in the structure look more consistent with an aspartic protease mechanism (
      • Bhaumik P.
      • Xiao H.
      • Parr C.L.
      • Kiso Y.
      • Gustchina A.
      • Yada R.Y.
      • Wlodawer A.
      Crystal structures of the histo-aspartic protease (HAP) from Plasmodium falciparum.
      ). Concerning the possibility that it could be a zinc metalloprotease, native enzyme was not inhibited by EDTA (
      • Banerjee R.
      • Liu J.
      • Beatty W.
      • Pelosof L.
      • Klemba M.
      • Goldberg D.E.
      Four plasmepsins are active in the Plasmodium falciparum food vacuole, including a protease with an active-site histidine.
      ), and only the dimeric apoenzyme structure had active site zinc (
      • Bhaumik P.
      • Xiao H.
      • Parr C.L.
      • Kiso Y.
      • Gustchina A.
      • Yada R.Y.
      • Wlodawer A.
      Crystal structures of the histo-aspartic protease (HAP) from Plasmodium falciparum.
      ), whereas it is the monomer that is active (
      • Xiao H.
      • Briere L.A.K.
      • Dunn S.D.
      • Yada R.Y.
      Characterization of the monomer-dimer equilibrium of recombinant histo-aspartic protease from Plasmodium falciparum.
      ). A proposal was put forth that Asp215 acts as an acid as well as a base, but this model was based on a profound stabilization (several orders of magnitude) by His32 (
      • Bjelic S.
      • Åqvist J.
      Computational prediction of structure, substrate binding mode, mechanism, and rate for a malaria protease with a novel type of active site.
      ). Perhaps a different residue could be the stabilizer; Lys78 has been suggested (
      • Parr C.L.
      • Tanaka T.
      • Xiao H.
      • Yada R.Y.
      The catalytic significance of the proposed active site residues in Plasmodium falciparum histoaspartic protease.
      ). Weighing on this debate is the fact that the properties of native enzyme and the recombinant enzyme used for mutagenesis studies are very different. PM III was originally isolated from digestive vacuoles, had a sharp pH optimum of 5.5, and was well-inhibited by pepstatin (
      • Banerjee R.
      • Liu J.
      • Beatty W.
      • Pelosof L.
      • Klemba M.
      • Goldberg D.E.
      Four plasmepsins are active in the Plasmodium falciparum food vacuole, including a protease with an active-site histidine.
      ). Three different isolation procedures gave similar results, and there was no detectable contamination by other plasmepsins (although trace contamination by a plasmepsin or another protease cannot be completely excluded). Recombinant protein expressed in Escherichia coli had similar properties (
      • Xiao H.
      • Sinkovits A.F.
      • Bryksa B.C.
      • Ogawa M.
      • Yada R.Y.
      Recombinant expression and partial characterization of an active soluble histo-aspartic protease from Plasmodium falciparum.
      ). Later, recombinant protein was again expressed in E. coli for analysis of site-directed mutants mentioned above but had a broad pH optimum from 5 to 8.5, was poorly inhibited by pepstatin, and was potently inhibited by phenylmethylsulfonyl fluoride (
      • Parr C.L.
      • Tanaka T.
      • Xiao H.
      • Yada R.Y.
      The catalytic significance of the proposed active site residues in Plasmodium falciparum histoaspartic protease.
      ). A H215A mutant had no activity, but the S35A and H32A mutants were similar to WT. It is hard to reconcile these later results with the earlier data, unless there was a contaminating activity present in some of the samples, perhaps from the enterokinase used to generate mature recombinant enzyme. The existing data do not yet form a coherent picture of the mechanism of action of this unusual aspartic protease ortholog.
      Figure thumbnail gr4
      Figure 4A, crystal structure of PM III complexed with pepstatin. Shown is a ribbon structure (blue) with Asp215 and His32 highlighted in yellow with red and blue heteroatoms. Pepstatin is in green with red oxygens. The figure was constructed from PDB entry 3FNT. B, crystal structure of PM II (blue) with the B helix of the hemoglobin α chain modeled in orange. The α33–34 cleavage site is green; the helix-interacting loop is magenta; from PDB entry 1PSE. Created using PyMOL Molecular Graphics System, Version 2.3.

      Specificity

      An extensive series of studies has assessed specificity of cleavage of hemoglobin or synthetic peptides using native and recombinant digestive vacuole plasmepsins. The four P. falciparum enzymes and the single enzyme from the three other human parasite species (Plasmodium ovale, Plasmodium vivax, and Plasmodium malariae) as well as enzyme from rodent species (P. berghei and Plasmodium chabaudi) have been studied (
      • Dame J.B.
      • Yowell C.A.
      • Omara-Opyene L.
      • Carlton J.M.
      • Cooper R.A.
      • Li T.
      Plasmepsin 4, the food vacuole aspartic proteinase found in all Plasmodium spp. infecting man.
      ,
      • Banerjee R.
      • Liu J.
      • Beatty W.
      • Pelosof L.
      • Klemba M.
      • Goldberg D.E.
      Four plasmepsins are active in the Plasmodium falciparum food vacuole, including a protease with an active-site histidine.
      ,
      • Hill J.
      • Tyas L.
      • Phylip L.H.
      • Kay J.
      • Dunn B.M.
      • Berry C.
      High level expression and characterisation of Plasmepsin II, an aspartic proteinase from Plasmodium falciparum.
      ,
      • Luker K.E.
      • Francis S.E.
      • Gluzman I.Y.
      • Goldberg D.E.
      Kinetic analysis of plasmepsins i and II, aspartic proteases of the plasmodium falciparum digestive vacuole.
      ,
      • Moon R.P.
      • Tyas L.
      • Certa U.
      • Rupp K.
      • Bur D.
      • Jacquet C.
      • Matile H.
      • Loetscher H.
      • Grueninger-Leitch F.
      • Kay J.
      • Dunn B.M.
      • Berry C.
      • Ridley R.G.
      Expression and characterisation of plasmepsin I from Plasmodium falciparum.
      ,
      • Westling J.
      • Yowell C.A.
      • Majer P.
      • Erickson J.W.
      • Dame J.B.
      • Dunn B.M.
      Plasmodium falciparumP. vivaxP. malariae: a comparison of the active site properties of plasmepsins cloned and expressed from three different species of the malaria parasite.
      ,
      • Tyas L.
      • Gluzman I.
      • Moon R.P.R.P.
      • Rupp K.
      • Westling J.
      • Ridley R.G.R.G.
      • Kay J.
      • Goldberg D.E.D.E.
      • Berry C.
      Naturally-occurring and recombinant forms of the aspartic proteinases plasmepsins I and II from the human malaria parasite Plasmodium falciparum.
      ,
      • Westling J.
      • Cipullo P.
      • Hung S.-H.
      • Saft H.
      • Dame J.B.
      • Dunn B.M.
      Active site specificity of plasmepsin II.
      ,
      • Humphreys M.J.
      • Moon R.P.
      • Klinder A.
      • Fowler S.D.
      • Rupp K.
      • Bur D.
      • Ridley R.G.
      • Berry C.
      The aspartic proteinase from the rodent parasite Plasmodium berghei as a potential model for plasmepsins from the human malaria parasite, Plasmodium falciparum.
      ,
      • Wyatt D.M.
      • Berry C.
      Activity and inhibition of plasmepsin IV, a new aspartic proteinase from the malaria parasite, Plasmodium falciparum.
      ,
      • Siripurkpong P.
      • Yuvaniyama J.
      • Wilairat P.
      • Goldberg D.E.
      Active site contribution to specificity of the aspartic proteases plasmepsins I and II.
      ,
      • Li T.
      • Yowell C.A.
      • Beyer B.B.
      • Hung S.H.
      • Westling J.
      • Lam M.T.
      • Dunn B.M.
      • Dame J.B.
      Recombinant expression and enzymatic subsite characterization of plasmepsin 4 from the four Plasmodium species infecting man.
      ,
      • Beyer B.B.
      • Johnson J.V.
      • Chung A.Y.
      • Li T.
      • Madabushi A.
      • Agbandje-McKenna M.
      • McKenna R.
      • Dame J.B.
      • Dunn B.M.
      Active-site specificity of digestive aspartic peptidases from the four species of Plasmodium that infect humans using chromogenic combinatorial peptide libraries.
      ,
      • Martins T.M.
      • Domingos A.
      • Berry C.
      • Wyatt D.M.
      The activity and inhibition of the food vacuole plasmepsin from the rodent malaria parasite Plasmodium chabaudi.
      ,
      • Xiao H.
      • Tanaka T.
      • Ogawa M.
      • Yada R.Y.
      Expression and enzymatic characterization of the soluble recombinant plasmepsin I from Plasmodium falciparum.
      ,
      • Liu P.
      • Marzahn M.R.
      • Robbins A.H.
      • Gutiérrez-de-Terán H.
      • Rodríguez D.
      • McClung S.H.
      • Stevens Jr., S.M.
      • Yowell C.A.
      • Dame J.B.
      • McKenna R.
      • Dunn B.M.
      Recombinant plasmepsin 1 from the human malaria parasite Plasmodium falciparum: enzymatic characterization, active site inhibitor design, and structural analysis.
      ,
      • Liu P.
      • Robbins A.H.
      • Marzahn M.R.
      • McClung S.H.
      • Yowell C.A.
      • Stevens Jr., S.M.
      • Dame J.B.
      • Dunn B.M.
      Enzymatic characterization of recombinant food vacuole plasmepsin 4 from the rodent malaria parasite plasmodium berghei.
      ). All four P. falciparum enzymes are capable of degrading hemoglobin and function at the acidic pH of the digestive vacuole (
      • Banerjee R.
      • Liu J.
      • Beatty W.
      • Pelosof L.
      • Klemba M.
      • Goldberg D.E.
      Four plasmepsins are active in the Plasmodium falciparum food vacuole, including a protease with an active-site histidine.
      ). Other degradative proteases such as cathepsin E are not able to degrade native hemoglobin, so this is a special property of the digestive vacuole plasmepsins (
      • Istvan E.S.
      • Goldberg D.E.
      Distal substrate interactions enhance plasmepsin activity.
      ). There are minor differences between species, but activity on synthetic peptide libraries shows a general preference for large hydrophobic residues proximal to the cleavage site on the P (N-terminal) and P′ (C-terminal) sides, especially at the P1 and P3 positions, with somewhat broader tolerance at P1′ and P3′ and wide latitude at P2 and P2′. On native hemoglobin, time course studies using native PM I and PM II revealed an initial cleavage of hemoglobin (α2β2 tetramer) on the α chain between Phe33 and Leu34 (
      • Goldberg D.E.
      • Slater A.F.G.
      • Beavis R.
      • Chait B.
      • Cerami A.
      • Henderson G.B.
      Hemoglobin degradation in the human malaria pathogen Plasmodium falciparum: a catabolic pathway initiated by a specific aspartic protease.
      ,
      • Gluzman I.Y.
      • Francis S.E.
      • Oksman A.
      • Smith C.E.
      • Duffin K.L.
      • Goldberg D.E.
      Order and specificity of the Plasmodium falciparum hemoglobin degradation pathway.
      ). Proteolysis proceeded with cleavages at a number of sites on both chains. Specificity of cleavage correlated well with the data just mentioned for synthetic peptides. Experiments using peptides corresponding to the scissile α-helix (helix B) revealed enhanced cleavage by PM II when the helix was extended N-terminally and could then interact with a loop on the PM II molecule (
      • Istvan E.S.
      • Goldberg D.E.
      Distal substrate interactions enhance plasmepsin activity.
      ). A model was put forth wherein the plasmepsin recognizes the beginning of the B helix on the α chain of hemoglobin and perches, waiting for the helix to breathe. The B helix is the weakest link on the protein because it has two helix-disrupting glycines. The protease is in position to cleave the 33–34 peptide bond as soon as the helix opens, helping unravel the tightly wound hemoglobin molecule so that further proteolysis can proceed (Fig. 4B).

      Biosynthesis

      Biosynthesis of digestive vacuole plasmepsins follows a circuitous route. The enzymes are made as transmembrane zymogens that traverse the secretory system, reach the parasite surface, are internalized via the cytostome along with their substrate hemoglobin, get delivered to the digestive vacuole membrane, and are then cleaved, releasing the mature enzyme into the vacuolar lumen (
      • Francis S.E.
      • Banerjee R.
      • Goldberg D.E.
      Biosynthesis and maturation of the malaria aspartic hemoglobinases plasmepsins I and II.
      ,
      • Banerjee R.
      • Francis S.E.
      • Goldberg D.E.
      Food vacuole plasmepsins are processed at a conserved site by an acidic convertase activity in Plasmodium falciparum.
      ,
      • Klemba M.
      • Beatty W.
      • Gluzman I.
      • Goldberg D.E.
      Trafficking of plasmepsin II to the food vacuole of the malaria parasite Plasmodium falciparum.
      ) (Fig. 3C).
      Maturation is carried out by the digestive vacuole cysteine proteases falcipain-2 and -3 (
      • Drew M.E.
      • Banerjee R.
      • Uffman E.W.
      • Gilbertson S.
      • Rosenthal P.J.
      • Goldberg D.E.
      Plasmodium food vacuole plasmepsins are activated by falcipains.
      ). If those enzymes are inhibited, plasmepsin autoprocessing takes place, albeit at a slower rate and at a site 1 amino acid upstream from the normal cleavage site. Structures of P. falciparum PM II, PM III, and PM IV proenzymes as well as those of P. vivax PM IV have been solved (
      • Bernstein N.K.
      • Cherney M.M.
      • Yowell C.A.
      • Dame J.B.
      • James M.N.G.
      Structural insights into the activation of P. vivax plasmepsin.
      ,
      • Bernstein N.K.
      • Cherney M.M.
      • Loetscher H.
      • Ridley R.G.
      • James M.N.G.
      Crystal structure of the novel aspartic proteinase zymogen proplasmepsin II from Plasmodium falciparum.
      ,
      • Bhaumik P.
      • Xiao H.
      • Hidaka K.
      • Gustchina A.
      • Kiso Y.
      • Yada R.Y.
      • Wlodawer A.
      Structural insights into the activation and inhibition of histo-aspartic protease from Plasmodium falciparum.
      ,
      • Recacha R.
      • Jaudzems K.
      • Akopjana I.
      • Jirgensons A.
      • Tars K.
      Crystal structure of Plasmodium falciparum proplasmepsin IV: the plasticity of proplasmepsins.
      ). The proenzyme of each is inactive because the propiece pushes apart the N- and C-terminal lobes of the protease, keeping the catalytic residues too far apart for activity. This differs from mammalian aspartic protease zymogens, for which the propiece binds to the substrate cleft to maintain inactivity until pH-dependent autoactivation can take place.

      Key questions

      • What is the mechanism of PM III? Is His32 involved in catalysis? Is PM III even an active enzyme?
      • Systems biology of hemoglobin degradation: How do cleavages made by each plasmepsin and by other proteases synergize? Why maintain four plasmepsins in P. falciparum? Why do some parasites amplify the digestive vacuole plasmepsin gene region even further?
      • When is heme released during hemoglobin proteolysis, and how is this orchestrated with hemozoin formation?

      Effector protein export: Plasmepsin V

      After invasion, the parasite exports hundreds of proteins into the host RBC. These exported effectors enact a dramatic program of host modification, reconstituting a complex trafficking system in the RBC cytosol and altering the host cell's rigidity, nutrient permeability, and endothelial binding properties (
      • Spillman N.J.
      • Beck J.R.
      • Goldberg D.E.
      Protein export into malaria parasite–infected erythrocytes: mechanisms and functional consequences.
      ,
      • Matthews K.M.
      • Pitman E.L.
      • de Koning-Ward T.F.
      Illuminating how malaria parasites export proteins into host erythrocytes.
      ). A significant portion of this export program appears dedicated to trafficking families of variable membrane adhesins to the RBC surface, creating adhesin-rich protrusions called “knobs” that mediate binding to the vascular endothelium (Fig. 5) (
      • Maier A.G.
      • Rug M.
      • O'Neill M.T.
      • Brown M.
      • Chakravorty S.
      • Szestak T.
      • Chesson J.
      • Wu Y.
      • Hughes K.
      • Coppel R.L.
      • Newbold C.
      • Beeson J.G.
      • Craig A.
      • Crabb B.S.
      • Cowman A.F.
      Exported proteins required for virulence and rigidity of Plasmodium falciparum-infected human erythrocytes.
      ). In P. falciparum, around 400 predicted gene products are annotated as likely to be exported (
      • Boddey J.A.
      • Carvalho T.G.
      • Hodder A.N.
      • Sargeant T.J.
      • Sleebs B.E.
      • Marapana D.
      • Lopaticki S.
      • Nebl T.
      • Cowman A.F.
      Role of plasmepsin V in export of diverse protein families from the Plasmodium falciparum exportome.
      ,
      • Heiber A.
      • Kruse F.
      • Pick C.
      • Grüring C.
      • Flemming S.
      • Oberli A.
      • Schoeler H.
      • Retzlaff S.
      • Mesén-Ramírez P.
      • Hiss J.A.
      • Kadekoppala M.
      • Hecht L.
      • Holder A.A.
      • Gilberger T.-W.
      • Spielmann T.
      Identification of new PNEPs indicates a substantial non-PEXEL exportome and underpins common features in Plasmodium falciparum protein export.
      ). Nearly 200 belong to three families of membrane adhesins (∼120 rifins, 50 PfEMP1s, and 30 stevors) (
      • Boddey J.A.
      • Carvalho T.G.
      • Hodder A.N.
      • Sargeant T.J.
      • Sleebs B.E.
      • Marapana D.
      • Lopaticki S.
      • Nebl T.
      • Cowman A.F.
      Role of plasmepsin V in export of diverse protein families from the Plasmodium falciparum exportome.
      ,
      • Sargeant T.J.
      • Marti M.
      • Caler E.
      • Carlton J.M.
      • Simpson K.
      • Speed T.P.
      • Cowman A.F.
      Lineage-specific expansion of proteins exported to erythrocytes in malaria parasites.
      ). Many of the remaining predicted proteins have no clear function and no homology to genes outside Plasmodium. We are still just beginning to understand the mechanisms by which Plasmodium manipulates its host through its exported proteins.
      Figure thumbnail gr5
      Figure 5Biosynthesis and trafficking of exported proteins. Proteins are synthesized in the ER and traverse the secretory system (green) to the parasitophorous vacuole (blue), where they are recognized by the PTEX translocon and exported into the RBC (pink). In the RBC, effectors can be soluble, can reside in the Golgi-like Maurer's clefts that are established by the parasite in the host cell, can be vesicular, or can go to the RBC surface, forming nutrient acquisition channels (PSAC) or clustering variant surface cytoadhesins in knobs at the RBC surface.

      Discovery

      Initial evidence of aspartic proteases acting on processes other than hemoglobin degradation came nearly 2 decades ago, when antibodies raised against newly annotated aspartic proteases labeled parasites outside the digestive vacuole (
      • Banerjee R.
      • Liu J.
      • Beatty W.
      • Pelosof L.
      • Klemba M.
      • Goldberg D.E.
      Four plasmepsins are active in the Plasmodium falciparum food vacuole, including a protease with an active-site histidine.
      ). PM V was characterized as a constitutively expressed endoplasmic reticulum (ER)-resident integral membrane protein (
      • Klemba M.
      • Goldberg D.E.
      Characterization of plasmepsin V, a membrane-bound aspartic protease homolog in the endoplasmic reticulum of Plasmodium falciparum.
      ). In 2004, the Haldar and Cowman groups independently discovered that many proteins destined for export contain a pentameric amino acid motif ∼15–30 amino acids downstream from the signal sequence, termed the Plasmodium export element (PEXEL) with the consensus sequence RXLX(E/Q/D) (
      • Hiller N.L.
      • Bhattacharjee S.
      • van Ooij C.
      • Liolios K.
      • Harrison T.
      • Lopez-Estraño C.
      • Haldar K.
      A host-targeting signal in virulence proteins reveals a secretome in malarial infection.
      ,
      • Marti M.
      • Good R.T.
      • Rug M.
      • Knuepfer E.
      • Cowman A.F.
      Targeting malaria virulence and remodeling proteins to the host erythrocyte.
      ). This element was shown to be necessary and sufficient to export fluorescent reporters. In 2008, Chang et al. (
      • Chang H.H.
      • Falick A.M.
      • Carlton P.M.
      • Sedat J.W.
      • DeRisi J.L.
      • Marletta M.A.
      N-terminal processing of proteins exported by malaria parasites.
      ) reported that exported proteins are cleaved in the ER, after the conserved Leu in the PEXEL sequence. The advent of tools for reverse genetics in P. falciparum enabled the assignment of the PEXEL-processing function to PM V (
      • Russo I.
      • Babbitt S.
      • Muralidharan V.
      • Butler T.
      • Oksman A.
      • Goldberg D.E.
      Plasmepsin V licenses Plasmodium proteins for export into the host erythrocyte.
      ,
      • Boddey J.A.
      • Hodder A.N.
      • Günther S.
      • Gilson P.R.
      • Patsiouras H.
      • Kapp E.A.
      • Pearce J.A.
      • de Koning-Ward T.F.
      • Simpson R.J.
      • Crabb B.S.
      • Cowman A.F.
      An aspartyl protease directs malaria effector proteins to the host cell.
      ). Development of peptidomimetic inhibitors of PM V has driven further discovery, validating PM V as essential for parasite survival in RBCs during the asexual cycle and gametocytogenesis (
      • Sleebs B.E.
      • Lopaticki S.
      • Marapana D.S.
      • O'Neill M.T.
      • Rajasekaran P.
      • Gazdik M.
      • Günther S.
      • Whitehead L.W.
      • Lowes K.N.
      • Barfod L.
      • Hviid L.
      • Shaw P.J.
      • Hodder A.N.
      • Smith B.J.
      • Cowman A.F.
      • Boddey J.A.
      Inhibition of Plasmepsin V activity demonstrates its essential role in protein export, PfEMP1 display, and survival of malaria parasites.
      ,
      • Sleebs B.E.
      • Gazdik M.
      • O'Neill M.T.
      • Rajasekaran P.
      • Lopaticki S.
      • Lackovic K.
      • Lowes K.
      • Smith B.J.
      • Cowman A.F.
      • Boddey J.A.
      Transition state mimetics of the Plasmodium export element are potent inhibitors of Plasmepsin V from P. falciparumP. vivax.
      ,
      • Jennison C.
      • Lucantoni L.
      • O'Neill M.T.
      • McConville R.
      • Erickson S.M.
      • Cowman A.F.
      • Sleebs B.E.
      • Avery V.M.
      • Boddey J.A.
      • O'Neill M.T.
      • McConville R.
      • Erickson S.M.
      • Cowman A.F.
      • Sleebs B.E.
      • Avery V.M.
      • Boddey J.A.
      Inhibition of plasmepsin V activity blocks Plasmodium falciparum gametocytogenesis and transmission to mosquitoes.
      ). In addition to uncovering novel parasite biology, these inhibitors have enabled the determination of a high-resolution PM V structure, opening the door to biochemical and pharmacological investigation of this essential enzyme (
      • Hodder A.N.
      • Sleebs B.E.
      • Czabotar P.E.
      • Gazdik M.
      • Xu Y.
      • O'Neill M.T.
      • Lopaticki S.
      • Nebl T.
      • Triglia T.
      • Smith B.J.
      • Lowes K.
      • Boddey J.A.
      • Cowman A.F.
      Structural basis for plasmepsin V inhibition that blocks export of malaria proteins to human erythrocytes.
      ). A number of tools have now been turned toward the study of PM V, including inducible Di-Cre excision (
      • Boonyalai N.
      • Collins C.R.
      • Hackett F.
      • Withers-Martinez C.
      • Blackman M.J.
      Essentiality of Plasmodium falciparum plasmepsin V.
      ) and post-transcriptional depletion (
      • Sleebs B.E.
      • Lopaticki S.
      • Marapana D.S.
      • O'Neill M.T.
      • Rajasekaran P.
      • Gazdik M.
      • Günther S.
      • Whitehead L.W.
      • Lowes K.N.
      • Barfod L.
      • Hviid L.
      • Shaw P.J.
      • Hodder A.N.
      • Smith B.J.
      • Cowman A.F.
      • Boddey J.A.
      Inhibition of Plasmepsin V activity demonstrates its essential role in protein export, PfEMP1 display, and survival of malaria parasites.
      ,
      • Gambini L.
      • Rizzi L.
      • Pedretti A.
      • Taglialatela-Scafati O.
      • Carucci M.
      • Pancotti A.
      • Galli C.
      • Read M.
      • Giurisato E.
      • Romeo S.
      • Russo I.
      Picomolar inhibition of plasmepsin V, an essential malaria protease, achieved exploiting the prime region.
      ,
      • Polino A.J.
      • Nasamu A.S.
      • Niles J.C.
      • Goldberg D.E.
      Assessment of biological role and insight into druggability of the Plasmodium falciparum protease plasmepsin V.
      ), as well as expression, purification, and activity assessment of PM V expressed from E. coli (
      • Boddey J.A.
      • Hodder A.N.
      • Günther S.
      • Gilson P.R.
      • Patsiouras H.
      • Kapp E.A.
      • Pearce J.A.
      • de Koning-Ward T.F.
      • Simpson R.J.
      • Crabb B.S.
      • Cowman A.F.
      An aspartyl protease directs malaria effector proteins to the host cell.
      ,
      • Xiao H.
      • Bryksa B.C.
      • Bhaumik P.
      • Gustchina A.
      • Kiso Y.
      • Yao S.Q.
      • Wlodawer A.
      • Yada R.Y.
      The zymogen of plasmepsin v from Plasmodium falciparum is enzymatically active.
      ,
      • Boonyalai N.
      • Sittikul P.
      • Yuvaniyama J.
      Plasmodium falciparum plasmepsin V (PfPMV): insights into recombinant expression, substrate specificity and active site structure.
      ,
      • Loymunkong C.
      • Sittikul P.
      • Songtawee N.
      • Wongpanya R.
      • Boonyalai N.
      Yield improvement and enzymatic dissection of Plasmodium falciparum plasmepsin V.
      ,
      • Sittikul P.
      • Songtawee N.
      • Kongkathip N.
      • Boonyalai N.
      In vitroin silico studies of naphthoquinones and peptidomimetics toward Plasmodium falciparum plasmepsin V.
      ), from insect cells (
      • Hodder A.N.
      • Sleebs B.E.
      • Czabotar P.E.
      • Gazdik M.
      • Xu Y.
      • O'Neill M.T.
      • Lopaticki S.
      • Nebl T.
      • Triglia T.
      • Smith B.J.
      • Lowes K.
      • Boddey J.A.
      • Cowman A.F.
      Structural basis for plasmepsin V inhibition that blocks export of malaria proteins to human erythrocytes.
      ), and from parasite culture (
      • Russo I.
      • Babbitt S.
      • Muralidharan V.
      • Butler T.
      • Oksman A.
      • Goldberg D.E.
      Plasmepsin V licenses Plasmodium proteins for export into the host erythrocyte.
      ,
      • Boddey J.A.
      • Hodder A.N.
      • Günther S.
      • Gilson P.R.
      • Patsiouras H.
      • Kapp E.A.
      • Pearce J.A.
      • de Koning-Ward T.F.
      • Simpson R.J.
      • Crabb B.S.
      • Cowman A.F.
      An aspartyl protease directs malaria effector proteins to the host cell.
      ). This has enabled rapid progress in our knowledge of this enzyme and its development as a potential antimalarial target.

      Function

      PEXEL-containing proteins are translated into the ER, immediately processed by PM V after the PEXEL Leu, and then acetylated at the new N terminus by an unknown N-acetyltransferase (
      • Chang H.H.
      • Falick A.M.
      • Carlton P.M.
      • Sedat J.W.
      • DeRisi J.L.
      • Marletta M.A.
      N-terminal processing of proteins exported by malaria parasites.
      ,
      • Russo I.
      • Babbitt S.
      • Muralidharan V.
      • Butler T.
      • Oksman A.
      • Goldberg D.E.
      Plasmepsin V licenses Plasmodium proteins for export into the host erythrocyte.
      ,
      • Boddey J.A.
      • Hodder A.N.
      • Günther S.
      • Gilson P.R.
      • Patsiouras H.
      • Kapp E.A.
      • Pearce J.A.
      • de Koning-Ward T.F.
      • Simpson R.J.
      • Crabb B.S.
      • Cowman A.F.
      An aspartyl protease directs malaria effector proteins to the host cell.
      ,
      • Boddey J.A.
      • Moritz R.L.
      • Simpson R.J.
      • Cowman A.F.
      Role of the Plasmodium export element in trafficking parasite proteins to the infected erythrocyte.
      ,
      • Osborne A.R.
      • Speicher K.D.
      • Tamez P.A.
      • Bhattacharjee S.
      • Speicher D.W.
      • Haldar K.
      The host targeting motif in exported Plasmodium proteins is cleaved in the parasite endoplasmic reticulum.
      ). Preventing PEXEL processing by mutating PEXEL or depleting/inhibiting PM V blocks protein export, indicating that PM V serves as a gatekeeper for the export pathway (
      • Hiller N.L.
      • Bhattacharjee S.
      • van Ooij C.
      • Liolios K.
      • Harrison T.
      • Lopez-Estraño C.
      • Haldar K.
      A host-targeting signal in virulence proteins reveals a secretome in malarial infection.
      ,
      • Marti M.
      • Good R.T.
      • Rug M.
      • Knuepfer E.
      • Cowman A.F.
      Targeting malaria virulence and remodeling proteins to the host erythrocyte.
      ,
      • Sleebs B.E.
      • Lopaticki S.
      • Marapana D.S.
      • O'Neill M.T.
      • Rajasekaran P.
      • Gazdik M.
      • Günther S.
      • Whitehead L.W.
      • Lowes K.N.
      • Barfod L.
      • Hviid L.
      • Shaw P.J.
      • Hodder A.N.
      • Smith B.J.
      • Cowman A.F.
      • Boddey J.A.
      Inhibition of Plasmepsin V activity demonstrates its essential role in protein export, PfEMP1 display, and survival of malaria parasites.
      ,
      • Polino A.J.
      • Nasamu A.S.
      • Niles J.C.
      • Goldberg D.E.
      Assessment of biological role and insight into druggability of the Plasmodium falciparum protease plasmepsin V.
      ). Processed PEXEL proteins are secreted into the parasitophorous vacuole and then exported across the vacuolar membrane into the host cell via the Plasmodium translocon for exported proteins (PTEX) (
      • Elsworth B.
      • Matthews K.
      • Nie C.Q.
      • Kalanon M.
      • Charnaud S.C.
      • Sanders P.R.
      • Chisholm S.A.
      • Counihan N.A.
      • Shaw P.J.
      • Pino P.
      • Chan J.-A.
      • Azevedo M.F.
      • Rogerson S.J.
      • Beeson J.G.
      • Crabb B.S.
      • et al.
      PTEX is an essential nexus for protein export in malaria parasites.
      ,
      • Beck J.R.
      • Muralidharan V.
      • Oksman A.
      • Goldberg D.E.
      PTEX component HSP101 mediates export of diverse malaria effectors into host erythrocytes.
      ,
      • de Koning-Ward T.F.
      • Gilson P.R.
      • Boddey J.A.
      • Rug M.
      • Smith B.J.
      • Papenfuss A.T.
      • Sanders P.R.
      • Lundie R.J.
      • Maier A.G.
      • Cowman A.F.
      • Crabb B.S.
      A newly discovered protein export machine in malaria parasites.
      ,
      • Ho C.-M.
      • Beck J.R.
      • Lai M.
      • Cui Y.
      • Goldberg D.E.
      • Egea P.F.
      • Zhou Z.H.
      Malaria parasite translocon structure and mechanism of effector export.
      ) (Fig. 5). A number of proteins lack PEXEL yet are still exported, termed PEXEL-negative exported proteins (PNEPs). Their N termini are believed to confer export competency similarly to that of the mature PEXEL N terminus (
      • Grüring C.
      • Heiber A.
      • Kruse F.
      • Flemming S.
      • Franci G.
      • Colombo S.F.
      • Fasana E.
      • Schoeler H.
      • Borgese N.
      • Stunnenberg H.G.
      • Przyborski J.M.
      • Gilberger T.-W.
      • Spielmann T.
      Uncovering common principles in protein export of malaria parasites.
      ). Most known PNEPs are involved in trafficking adhesins to the RBC surface or are adhesins themselves.
      PM V is not the only gatekeeper to protein export; several mutated PEXEL reporters are correctly cleaved by PM V but still retained in the parasitophorous vacuole (
      • Boddey J.A.
      • Carvalho T.G.
      • Hodder A.N.
      • Sargeant T.J.
      • Sleebs B.E.
      • Marapana D.
      • Lopaticki S.
      • Nebl T.
      • Cowman A.F.
      Role of plasmepsin V in export of diverse protein families from the Plasmodium falciparum exportome.
      ,
      • Osborne A.R.
      • Speicher K.D.
      • Tamez P.A.
      • Bhattacharjee S.
      • Speicher D.W.
      • Haldar K.
      The host targeting motif in exported Plasmodium proteins is cleaved in the parasite endoplasmic reticulum.
      ). Two non-mutually exclusive models have been put forward to explain this. First, export-destined proteins may contain trafficking information in addition to PEXEL that targets them for export. This information would have to be C-terminal to PEXEL, as the N-terminal sequence is removed by PM V. Exported reporter studies have greatly limited the possible locations of such information. The N terminus of PEXEL proteins, containing the PEXEL motif followed by just 11 amino acids, supports export of a GFP fusion (
      • Hiller N.L.
      • Bhattacharjee S.
      • van Ooij C.
      • Liolios K.
      • Harrison T.
      • Lopez-Estraño C.
      • Haldar K.
      A host-targeting signal in virulence proteins reveals a secretome in malarial infection.
      ,
      • Marti M.
      • Good R.T.
      • Rug M.
      • Knuepfer E.
      • Cowman A.F.
      Targeting malaria virulence and remodeling proteins to the host erythrocyte.
      ). Following PEXEL cleavage, the only detectable sequence conservation is the P2′ residue (largely restricted to Gln, Glu, or Asp). Residues beyond P2′ are important for export, as an inserted PEXEL was not sufficient to re-target the normally vacuole-resident SERA5 to the RBC, but if the subsequent 18 amino acids from the PEXEL protein PfEMP3 were added, export was restored (
      • Boddey J.A.
      • Carvalho T.G.
      • Hodder A.N.
      • Sargeant T.J.
      • Sleebs B.E.
      • Marapana D.
      • Lopaticki S.
      • Nebl T.
      • Cowman A.F.
      Role of plasmepsin V in export of diverse protein families from the Plasmodium falciparum exportome.
      ). However, the properties of these sequences that support export have remained elusive; they have no obvious conserved sequence, structure, or biochemical properties; and Boddey et al. (
      • Boddey J.A.
      • Carvalho T.G.
      • Hodder A.N.
      • Sargeant T.J.
      • Sleebs B.E.
      • Marapana D.
      • Lopaticki S.
      • Nebl T.
      • Cowman A.F.
      Role of plasmepsin V in export of diverse protein families from the Plasmodium falciparum exportome.
      ) reported the surprising finding that even replacing the post-P2′ amino acids with all Ala supports protein export, leaving us to wonder what signaling information this sequence could contain. A variant of this model would be that secreted proteins bind HSP101 and get exported by default, unless they are too tightly folded (
      • Matthews K.M.
      • Kalanon M.
      • de Koning-Ward T.F.
      Uncoupling the threading and unfoldase actions of Plasmodium HSP101 reveals differences in export between soluble and insoluble proteins.
      ) or contain an N-terminal sequence that prevents chaperone recognition.
      The second model posits that following PEXEL cleavage, PM V hands off export-destined cargo to an ER chaperone that ushers it through the export pathway (
      • Russo I.
      • Babbitt S.
      • Muralidharan V.
      • Butler T.
      • Oksman A.
      • Goldberg D.E.
      Plasmepsin V licenses Plasmodium proteins for export into the host erythrocyte.
      ). In this model, PM V is required not just for cleavage, but also for some downstream steps. Tests of this model were attempted with constructs that would create a post-PEXEL N terminus independent of PM V using either an engineered signal peptidase or a fused viral protease; these gave discordant results, with the signal peptidase-generated N terminus unable to support export, whereas the viral protease-generated N terminus did support export (
      • Boddey J.A.
      • Hodder A.N.
      • Günther S.
      • Gilson P.R.
      • Patsiouras H.
      • Kapp E.A.
      • Pearce J.A.
      • de Koning-Ward T.F.
      • Simpson R.J.
      • Crabb B.S.
      • Cowman A.F.
      An aspartyl protease directs malaria effector proteins to the host cell.
      ,
      • Tarr S.J.
      • Cryar A.
      • Thalassinos K.
      • Haldar K.
      • Osborne A.R.
      The C-terminal portion of the cleaved HT motif is necessary and sufficient to mediate export of proteins from the malaria parasite into its host cell.
      ).
      Another avenue for elucidating the post-PM V path is to determine its interacting partners, a task that was carefully undertaken by Danushka et al. (
      • Marapana D.S.
      • Dagley L.F.
      • Sandow J.J.
      • Nebl T.
      • Triglia T.
      • Pasternak M.
      • Dickerman B.K.
      • Crabb B.S.
      • Gilson P.R.
      • Webb A.I.
      • Boddey J.A.
      • Cowman A.F.
      Plasmepsin V cleaves malaria effector proteins in a distinct endoplasmic reticulum translocation interactome for export to the erythrocyte.
      ). They found that PM V interacts with a noncatalytic component of the signal peptidase complex SPC25 and, if cross-linked, with the ER translocon Sec61, its accessory proteins for post-translational import Sec62/63, as well as signal peptidase. They posited that secretory proteins follow one of two distinct paths: either they are cleaved by the canonical signal peptidase complex, or they are recognized and cleaved by a distinct signal-processing complex consisting of PM V and SPC25 (
      • Marapana D.S.
      • Dagley L.F.
      • Sandow J.J.
      • Nebl T.
      • Triglia T.
      • Pasternak M.
      • Dickerman B.K.
      • Crabb B.S.
      • Gilson P.R.
      • Webb A.I.
      • Boddey J.A.
      • Cowman A.F.
      Plasmepsin V cleaves malaria effector proteins in a distinct endoplasmic reticulum translocation interactome for export to the erythrocyte.
      ) (Fig. 6). Perhaps there are chaperones that recognize this complex selectively, although their identity is not apparent at this time. A variant of the chaperone hand-off model is that PM V could be in a subregion of the ER with a direct route to the PTEX translocon at the PVM (
      • Boddey J.A.
      • Hodder A.N.
      • Günther S.
      • Gilson P.R.
      • Patsiouras H.
      • Kapp E.A.
      • Pearce J.A.
      • de Koning-Ward T.F.
      • Simpson R.J.
      • Crabb B.S.
      • Cowman A.F.
      An aspartyl protease directs malaria effector proteins to the host cell.
      ,
      • Goldberg D.E.
      • Cowman A.F.
      Moving in and renovating: exporting proteins from Plasmodium into host erythrocytes.
      ). Subregions of the PVM have recently been described, with each region hosting distinct secretory proteins (
      • Garten M.
      • Beck J.R.
      • Roth R.
      • Tenkova-Heuser T.
      • Heuser J.
      • Bleck C.K.E.
      • Goldberg D.E.
      • Zimmerberg J.
      Contacting domains that segregate lipid from solute transporters in malaria parasites.
      ,
      • Nessel T.
      • Beck J.M.
      • Rayatpisheh S.
      • Jami-Alahmadi Y.
      • Wohlschlegel J.A.
      • Goldberg D.E.
      • Beck J.R.
      EXP1 is required for organization of EXP2 in the intraerythrocytic malaria parasite vacuole.
      ). However, there is no information yet about whether these PVM subregions are fed by distinct regions of the ER.
      Figure thumbnail gr6
      Figure 6Model for signal processing in the ER. Secretory proteins are cleaved immediately after translation either by the canonical signal peptidase (SPC21) complex (left) or a noncanonical PM V–processing complex (right). NAT, putative N-acetyltransferase.
      There is some evidence that PM V additionally functions as a secretory maturase for dense granule proteins. Dense granules are small secretory vesicles that discharge shortly after parasite invasion into the parasitophorous vacuole surrounding the parasite. In addition to the canonical PEXEL, PM V cleaves the “relaxed PEXEL” (RXLXXE) of the dense granule protein RESA (
      • Boddey J.A.
      • Carvalho T.G.
      • Hodder A.N.
      • Sargeant T.J.
      • Sleebs B.E.
      • Marapana D.
      • Lopaticki S.
      • Nebl T.
      • Cowman A.F.
      Role of plasmepsin V in export of diverse protein families from the Plasmodium falciparum exportome.
      ,
      • Polino A.J.
      • Nasamu A.S.
      • Niles J.C.
      • Goldberg D.E.
      Assessment of biological role and insight into druggability of the Plasmodium falciparum protease plasmepsin V.
      ). Following PEXEL cleavage, RESA is diverted to the dense granules of the forming daughter merozoites. When these merozoites re-invade new RBCs, RESA is secreted into the vacuolar lumen and rapidly exported via PTEX into the host cell (
      • Elsworth B.
      • Matthews K.
      • Nie C.Q.
      • Kalanon M.
      • Charnaud S.C.
      • Sanders P.R.
      • Chisholm S.A.
      • Counihan N.A.
      • Shaw P.J.
      • Pino P.
      • Chan J.-A.
      • Azevedo M.F.
      • Rogerson S.J.
      • Beeson J.G.
      • Crabb B.S.
      • et al.
      PTEX is an essential nexus for protein export in malaria parasites.
      ,
      • Beck J.R.
      • Muralidharan V.
      • Oksman A.
      • Goldberg D.E.
      PTEX component HSP101 mediates export of diverse malaria effectors into host erythrocytes.
      ). Inhibition of PM V shortly before egress is rapidly deleterious in newly invaded ring-stage parasites (
      • Polino A.J.
      • Nasamu A.S.
      • Niles J.C.
      • Goldberg D.E.
      Assessment of biological role and insight into druggability of the Plasmodium falciparum protease plasmepsin V.
      ). RESA itself is nonessential, and whether PM V cleaves additional dense granule proteins is not yet clear. In T. gondii, the PM V ortholog ASP5 cleaves dense granule proteins at a PEXEL-like motif (
      • Curt-Varesano A.
      • Braun L.
      • Ranquet C.
      • Hakimi M.-A.A.
      • Bougdour A.
      The aspartyl protease TgASP5 mediates the export of the Toxoplasma GRA16 and GRA24 effectors into host cells.
      ,
      • Hammoudi P.-M.
      • Jacot D.
      • Mueller C.
      • Di Cristina M.
      • Dogga S.K.
      • Marq J.-B.
      • Romano J.
      • Tosetti N.
      • Dubrot J.
      • Emre Y.
      • Lunghi M.
      • Coppens I.
      • Yamamoto M.
      • Sojka D.
      • Pino P.
      • Soldati-Favre D.
      Fundamental roles of the Golgi-associated Toxoplasma aspartyl protease, ASP5, at the host-parasite interface.
      ,
      • Coffey M.J.
      • Dagley L.F.
      • Seizova S.
      • Kapp E.A.
      • Infusini G.
      • Roos D.S.
      • Boddey J.A.
      • Webb A.I.
      • Tonkin C.J.
      Aspartyl protease 5 matures dense granule proteins that reside at the host-parasite interface in Toxoplasma gondii.
      ). When ASP5 processing is blocked, substrates still traffic to the dense granules but are unable to fulfill their roles in modifying the parasitophorous vacuole or host cell (
      • Hammoudi P.-M.
      • Jacot D.
      • Mueller C.
      • Di Cristina M.
      • Dogga S.K.
      • Marq J.-B.
      • Romano J.
      • Tosetti N.
      • Dubrot J.
      • Emre Y.
      • Lunghi M.
      • Coppens I.
      • Yamamoto M.
      • Sojka D.
      • Pino P.
      • Soldati-Favre D.
      Fundamental roles of the Golgi-associated Toxoplasma aspartyl protease, ASP5, at the host-parasite interface.
      ). If PM V does act as a dense granule protein maturase in both Toxoplasma and Plasmodium, perhaps this is its ancestral function, a concept that would be especially interesting given recent findings on the translocon, PTEX. This complex is made up of three core components: an AAA+-ATPase, Hsp101; a channel-forming protein, Exp2; and an adaptor protein, PTEX150 (
      • de Koning-Ward T.F.
      • Gilson P.R.
      • Boddey J.A.
      • Rug M.
      • Smith B.J.
      • Papenfuss A.T.
      • Sanders P.R.
      • Lundie R.J.
      • Maier A.G.
      • Cowman A.F.
      • Crabb B.S.
      A newly discovered protein export machine in malaria parasites.
      ). Hsp101 is a ubiquitous chaperone that has been co-opted by Plasmodium to unfold and thread proteins through the translocon. Exp2, in Plasmodium and Toxoplasma, forms a nutrient pore to get essential substances such as glucose and amino acids from the host cell (
      • Garten M.
      • Nasamu A.S.
      • Niles J.C.
      • Zimmerberg J.
      • Goldberg D.E.
      • Beck J.R.
      EXP2 is a nutrient-permeable channel in the vacuolar membrane of Plasmodium and is essential for protein export via PTEX.
      ,
      • Gold D.A.
      • Kaplan A.D.
      • Lis A.
      • Bett G.C.L.
      • Rosowski E.E.
      • Cirelli K.M.
      • Bougdour A.
      • Sidik S.M.
      • Beck J.R.
      • Lourido S.
      • Egea P.F.
      • Bradley P.J.
      • Hakimi M.-A.
      • Rasmusson R.L.
      • Saeij J.P.J.
      The Toxoplasma dense granule proteins GRA17 and GRA23 mediate the movement of small molecules between the host and the parasitophorous vacuole.
      ). This protein has also been deployed by Plasmodium for a second function—protein export. To dock Hsp101 to the Exp2 pore for protein export, the organism has come up with a novel adapter, PTEX150 (
      • Ho C.-M.
      • Beck J.R.
      • Lai M.
      • Cui Y.
      • Goldberg D.E.
      • Egea P.F.
      • Zhou Z.H.
      Malaria parasite translocon structure and mechanism of effector export.
      ). Thus, we suggest that Plasmodium has cobbled together a protein export system from mostly preexisting components, and further, it has recruited another preexisting protein, PM V, to prepare proteins for export. It is not at all clear why most exported proteins need to be made as preproteins and processed by PM V co-translationally unless the cleavage motif (PEXEL) is an important component of the export recognition system.

      Specificity

      Unlike the digestive vacuole proteases, PM V has a highly specific recognition sequence, cleaving only after the conserved Leu of the consensus PEXEL sequence RXLX(E/Q/D) (
      • Chang H.H.
      • Falick A.M.
      • Carlton P.M.
      • Sedat J.W.
      • DeRisi J.L.
      • Marletta M.A.
      N-terminal processing of proteins exported by malaria parasites.
      ,
      • Boddey J.A.
      • Moritz R.L.
      • Simpson R.J.
      • Cowman A.F.
      Role of the Plasmodium export element in trafficking parasite proteins to the infected erythrocyte.
      ,
      • Osborne A.R.
      • Speicher K.D.
      • Tamez P.A.
      • Bhattacharjee S.
      • Speicher D.W.
      • Haldar K.
      The host targeting motif in exported Plasmodium proteins is cleaved in the parasite endoplasmic reticulum.
      ). Mutation of the highly conserved PEXEL residue P1 or P3 ablates cleavage (with the notable exception of P1 Ile, which can inconsistently support some PM V cleavage) (
      • Boddey J.A.
      • Carvalho T.G.
      • Hodder A.N.
      • Sargeant T.J.
      • Sleebs B.E.
      • Marapana D.
      • Lopaticki S.
      • Nebl T.
      • Cowman A.F.
      Role of plasmepsin V in export of diverse protein families from the Plasmodium falciparum exportome.
      ,
      • Hiller N.L.
      • Bhattacharjee S.
      • van Ooij C.
      • Liolios K.
      • Harrison T.
      • Lopez-Estraño C.
      • Haldar K.
      A host-targeting signal in virulence proteins reveals a secretome in malarial infection.
      ,
      • Marti M.
      • Good R.T.
      • Rug M.
      • Knuepfer E.
      • Cowman A.F.
      Targeting malaria virulence and remodeling proteins to the host erythrocyte.
      ,
      • Schulze J.
      • Kwiatkowski M.
      • Borner J.
      • Schlüter H.
      • Bruchhaus I.
      • Burmester T.
      • Spielmann T.
      • Pick C.
      The Plasmodium falciparum exportome contains non-canonical PEXEL/HT proteins.
      ). PM V does not cleave the PNEPs SBP1, PfEMP1, and REX2 (
      • Boddey J.A.
      • Carvalho T.G.
      • Hodder A.N.
      • Sargeant T.J.
      • Sleebs B.E.
      • Marapana D.
      • Lopaticki S.
      • Nebl T.
      • Cowman A.F.
      Role of plasmepsin V in export of diverse protein families from the Plasmodium falciparum exportome.
      ) or a fluorogenic globin-derived peptide cleaved by PM I–IV (
      • Klemba M.
      • Goldberg D.E.
      Characterization of plasmepsin V, a membrane-bound aspartic protease homolog in the endoplasmic reticulum of Plasmodium falciparum.
      ). Functional PEXELs are found 15–30 amino acids downstream of the signal sequence (
      • Hiss J.A.
      • Przyborski J.M.
      • Schwarte F.
      • Lingelbach K.
      • Schneider G.
      The Plasmodium export element revisited.
      ); however, whether this location is required for PM V cleavage has not been reported.

      Structure

      The structure of the P. vivax PM V bound to a peptidomimetic inhibitor has been determined and reveals that PM V largely resembles the digestive vacuole plasmepsins, with a few notable differences (
      • Hodder A.N.
      • Sleebs B.E.
      • Czabotar P.E.
      • Gazdik M.
      • Xu Y.
      • O'Neill M.T.
      • Lopaticki S.
      • Nebl T.
      • Triglia T.
      • Smith B.J.
      • Lowes K.
      • Boddey J.A.
      • Cowman A.F.
      Structural basis for plasmepsin V inhibition that blocks export of malaria proteins to human erythrocytes.
      ) (Fig. 7). Like PM I-IV, PM V is produced with a poorly conserved N-terminal extension speculated to be a self-inhibiting prodomain. However, processing of PM V has not been detected (
      • Klemba M.
      • Goldberg D.E.
      Characterization of plasmepsin V, a membrane-bound aspartic protease homolog in the endoplasmic reticulum of Plasmodium falciparum.
      ), and the full-length recombinant protein has been reported to be active (
      • Xiao H.
      • Bryksa B.C.
      • Bhaumik P.
      • Gustchina A.
      • Kiso Y.
      • Yao S.Q.
      • Wlodawer A.
      • Yada R.Y.
      The zymogen of plasmepsin v from Plasmodium falciparum is enzymatically active.
      ,
      • Boonyalai N.
      • Sittikul P.
      • Yuvaniyama J.
      Plasmodium falciparum plasmepsin V (PfPMV): insights into recombinant expression, substrate specificity and active site structure.
      ). As in other plasmepsins, the PM V active site is covered by a flap that may serve to regulate substrate access. The 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+ (
      • Xiao H.
      • Bryksa B.C.
      • Bhaumik P.
      • Gustchina A.
      • Kiso Y.
      • Yao S.Q.
      • Wlodawer A.
      • Yada R.Y.
      The zymogen of plasmepsin v from Plasmodium falciparum is enzymatically active.
      ,
      • Loymunkong C.
      • Sittikul P.
      • Songtawee N.
      • Wongpanya R.
      • Boonyalai N.
      Yield improvement and enzymatic dissection of Plasmodium falciparum plasmepsin V.
      ,
      • Sittikul P.
      • Songtawee N.
      • Kongkathip N.
      • Boonyalai N.
      In vitroin silico studies of naphthoquinones and peptidomimetics toward Plasmodium falciparum plasmepsin V.
      ). Connected directly to the flap is an insert made up of two cysteine pairs folded into a cloverleaf-like structure called a “nepenthesin insert,” which it shares with Nep1 from the pitcher plant Nepenthesia. The nepenthesin insert is present in PM V across the genus, but absent from other plasmepsins. Its location adjoined to the flap over the active site tempts us to speculate that the nepenthesin insert may respond to some environmental condition or binding partner and regulate access to the active site. Alternatively, the nepenthesin insert could be a set distance from the active site and be involved in measuring distance from the N-end of potential substrates (
      • Goldberg D.E.
      Plasmepsin V shows its carnivorous side.
      ). No clear hint comes from its ortholog Nep1, a broad-substrate digestive protease present in the low pH of the pitcher plant lumen (
      • Athauda S.B.P.
      • Matsumoto K.
      • Rajapakshe S.
      • Kuribayashi M.
      • Kojima M.
      • Kubomura-Yoshida N.
      • Iwamatsu A.
      • Shibata C.
      • Inoue H.
      • Takahashi K.
      Enzymic and structural characterization of nepenthesin, a unique member of a novel subfamily of aspartic proteinases.
      ). Another unusual feature is a helix-turn-helix motif, again not present in other plasmepsins. Whereas this is often a nucleic acid-interacting motif, it is not clear what purpose such a motif might have in the ER lumen, where all but the nonessential C-terminal tail of PM V is found (
      • Russo I.
      • Babbitt S.
      • Muralidharan V.
      • Butler T.
      • Oksman A.
      • Goldberg D.E.
      Plasmepsin V licenses Plasmodium proteins for export into the host erythrocyte.
      ,
      • Tarr S.J.
      • Osborne A.R.
      Experimental determination of the membrane topology of the Plasmodium protease plasmepsin V.
      ). 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 (
      • Rawat M.
      • Vijay S.
      • Gupta Y.
      • Tiwari P.K.
      • Sharma A.
      Imperfect duplicate insertions type of mutations in plasmepsin V modulates binding properties of PEXEL motifs of export proteins in Indian Plasmodium vivax.
      ,
      • Sappakhaw K.
      • Takasila R.
      • Sittikul P.
      • Wattana-Amorn P.
      • Assavalapsakul W.
      • Boonyalai N.
      Biochemical characterization of plasmepsin V from Plasmodium vivax Thailand isolates: substrate specificity and enzyme inhibition.
      ); mutagenesis studies on recombinant enzyme have not yet revealed a function for this structure (
      • Sappakhaw K.
      • Takasila R.
      • Sittikul P.
      • Wattana-Amorn P.
      • Assavalapsakul W.
      • Boonyalai N.
      Biochemical characterization of plasmepsin V from Plasmodium vivax Thailand isolates: substrate specificity and enzyme inhibition.
      ). Further work has the potential to both uncover novel parasite biology and also unearth secrets of biochemistry broadly applicable to other enzymes outside of Plasmodium.
      Figure thumbnail gr7
      Figure 7Crystal structure of PM V with the inhibitor WEHI-842 bound (green). Highlighted are the flap over the active site (magenta), nepenthesin loop (yellow, with Cys-Cys bonds in red), and helix-turn-helix (orange); from PDB entry 4ZL4.

      Key questions

      • What do signature features of PM V do (helix-turn-helix motif, nepenthesin loop, unpaired cysteine in the flap)?
      • What is the basis of exported protein targeting, and how is PM V involved? Is N-acetylation a required part of this, and is it coordinated with PM V action?
      • What are the essential PM V substrates required for parasitophorous vacuole establishment?

      Transmission-stage plasmepsins: Plasmepsins VI, VII, and VIII

      PM VI, VII, and VIII are expressed only in transmission-stage parasites and have roles in midgut sporozoite development and function (
      • Banerjee R.
      • Liu J.
      • Beatty W.
      • Pelosof L.
      • Klemba M.
      • Goldberg D.E.
      Four plasmepsins are active in the Plasmodium falciparum food vacuole, including a protease with an active-site histidine.
      ). They have been studied mostly in the rodent malaria parasite P. berghei, where each has been disrupted with no effect in the RBC cycle. PM VI knockout parasites produce oocysts that fail to develop into sporozoites, resulting in a transmission block (
      • Ecker A.
      • Bushell E.S.C.
      • Tewari R.
      • Sinden R.E.
      Reverse genetics screen identifies six proteins important for malaria development in the mosquito.
      ). PM VII can be detected in the cytoplasm of P. falciparum zygotes and ookinetes, but its function is not known (
      • Li F.
      • Bounkeua V.
      • Pettersen K.
      • Vinetz J.M.
      Plasmodium falciparum ookinete expression of plasmepsin VII and plasmepsin X.
      ). The protein is not present in P. falciparum gametocytes, suggesting that it is produced after fertilization. Knockout has no effect on any stage of the P. berghei life cycle (
      • Mastan B.S.
      • Kumari A.
      • Gupta D.
      • Mishra S.
      • Kumar K.A.
      Gene disruption reveals a dispensable role for Plasmepsin VII in the Plasmodium berghei life cycle.
      ). PM VIII knockout parasites form oocysts in the mosquito midgut (
      • Mastan B.S.
      • Narwal S.K.
      • Dey S.
      • Kumar K.A.
      • Mishra S.
      Plasmodium berghei plasmepsin VIII is essential for sporozoite gliding motility.
      ). However, there is a drastic decrease in the number of salivary gland and hemolymph sporozoites when PM VIII is absent, due to a defect in egress from oocysts. The few sporozoites produced have a defect in gliding motility. Consequently, transmission of these parasites to the host is blocked. The egress phenotype mirrors that seen with PM X in intraerythrocytic parasites (see below), but mechanistic details for PM VIII function remain to be elucidated. More work is needed to understand the function of these important enzymes.

      Key questions

      • What are the specific roles and substrates of these proteases?
      • Do the inhibitors being developed against other plasmepsins also target the transmission-stage plasmepsins?
      • What is the role of other plasmepsins, such as PM X, in the transmission stages?

      Egress/invasion plasmepsins: Plasmepsins IX and X

      An obvious consequence of the malaria parasite's intraerythrocytic lifestyle is that it needs to get into the host cell and, when it is finished replicating and dividing, needs to get back out (Fig. 8A). Invasion is a multistep process (
      • Gilson P.R.
      • Crabb B.S.
      Morphology and kinetics of the three distinct phases of red blood cell invasion by Plasmodium falciparum merozoites.
      ,
      • Cowman A.F.
      • Tonkin C.J.
      • Tham W.H.
      • Duraisingh M.T.
      The molecular basis of erythrocyte invasion by malaria parasites.
      ) involving initial low-affinity recognition of the red blood cell by the parasite, reorientation of the invasive merozoite, and discharge of secretory organelles. This allows high-affinity binding to erythrocyte surface proteins and to parasite proteins that the organism has secreted onto the host cell membrane. This is followed by erythrocyte cytoskeletal reorganization and signaling cascades, tight junction formation, actin-mediated entry, and finally resealing once inside. The whole invasion process takes less than a minute. Egress is also a complex phenomenon (
      • Hale V.L.
      • Watermeyer J.M.
      • Hackett F.
      • Vizcay-Barrena G.
      • van Ooij C.
      • Thomas J.A.
      • Spink M.C.
      • Harkiolaki M.
      • Duke E.
      • Fleck R.A.
      • Blackman M.J.
      • Saibil H.R.
      Parasitophorous vacuole poration precedes its rupture and rapid host erythrocyte cytoskeleton collapse in Plasmodium falciparum egress.
      ,
      • Thomas J.A.
      • Tan M.S.Y.
      • Bisson C.
      • Borg A.
      • Umrekar T.R.
      • Hackett F.
      • Hale V.L.
      • Vizcay-Barrena G.
      • Fleck R.A.
      • Snijders A.P.
      • Saibil H.R.
      • Blackman M.J.
      A protease cascade regulates release of the human malaria parasite Plasmodium falciparum from host red blood cells.
      ,
      • Glushakova S.
      • Beck J.R.
      • Garten M.
      • Busse B.L.
      • Nasamu A.S.
      • Tenkova-Heuser T.
      • Heuser J.
      • Goldberg D.E.
      • Zimmerberg J.
      Rounding precedes rupture and breakdown of vacuolar membranes minutes before malaria parasite egress from erythrocytes.
      ). The parasite must porate and disrupt the surrounding parasitophorous vacuolar membrane, disassemble the host cell cytoskeleton, porate, and finally rupture the erythrocyte membrane, which it does explosively, releasing new infective merozoites into the circulation. The egress process takes about 10 min. Proteases play key roles in egress and invasion; in invasion, proteolytic processing is essential for invasion protein function; in egress, there is a proteolytic cascade, triggered by the cGMP-dependent discharge of proteases into the parasitophorous vacuole (
      • Thomas J.A.
      • Tan M.S.Y.
      • Bisson C.
      • Borg A.
      • Umrekar T.R.
      • Hackett F.
      • Hale V.L.
      • Vizcay-Barrena G.
      • Fleck R.A.
      • Snijders A.P.
      • Saibil H.R.
      • Blackman M.J.
      A protease cascade regulates release of the human malaria parasite Plasmodium falciparum from host red blood cells.
      ). Recent evidence suggests that aspartic proteases play important roles in both entry and exit from the erythrocyte.
      Figure thumbnail gr8
      Figure 8Plasmodium invasion and egress. A, an infective merozoite (top left) attaches to an RBC, reorients so that its apical end is in contact with the host cell, and invades. It develops, replicates, and divides, forming a schizont (bottom right). When ready to egress, it disrupts the PVM and then the RBC membrane, and finally merozoites rupture out of the RBC explosively (bottom left). B, schematic of a merozoite, showing secretory organelles whose function is influenced by plasmepsins. Exonemes discharge to initiate egress. Then micronemes discharge to secrete RBC adhesion ligands onto the cell surface. Next, rhoptries discharge to prepare the parasite and host cell for invasion and to condition the PVM. Finally, dense granules discharge to effect maturation of the parasitophorous vacuole.
      PM IX is expressed in late-stage schizonts and is localized to rhoptries (Fig. 8B), secretory organelles that contain adhesins for erythrocyte invasion and proteins for setting up the PVM upon invasion (
      • Nasamu A.S.
      • Glushakova S.
      • Russo I.
      • Vaupel B.
      • Oksman A.
      • Kim A.S.
      • Fremont D.H.
      • Tolia N.
      • Beck J.R.
      • Meyers M.J.
      • Niles J.C.
      • Zimmerberg J.
      • Goldberg D.E.
      Plasmepsins IX and X are essential and druggable mediators of malaria parasite egress and invasion.
      ). This organelle is highly segregated, with the invasion ligands at the apical end (neck) and other proteins at the basal end (bulb). Upon depletion/disruption of P. falciparum PM IX, invasion of red blood cells is severely compromised (
      • Nasamu A.S.
      • Glushakova S.
      • Russo I.
      • Vaupel B.
      • Oksman A.
      • Kim A.S.
      • Fremont D.H.
      • Tolia N.
      • Beck J.R.
      • Meyers M.J.
      • Niles J.C.
      • Zimmerberg J.
      • Goldberg D.E.
      Plasmepsins IX and X are essential and druggable mediators of malaria parasite egress and invasion.
      ,
      • Pino P.
      • Caldelari R.
      • Mukherjee B.
      • Vahokoski J.
      • Klages N.
      • Maco B.
      • Collins C.R.
      • Blackman M.J.
      • Kursula I.
      • Heussler V.
      • Brochet M.
      • Soldati-Favre D.
      A multistage antimalarial targets the plasmepsins IX and X essential for invasion and egress.
      ). Depletion/disruption of PM IX also blocks cleavage of the rhoptry-associated protein 1 (RAP1) and the apical sushi protein (ASP). Peptides corresponding to the cleavage sites of both proteins are cleaved by recombinant PM IX. The activity was observed at pH 6.4 (
      • Pino P.
      • Caldelari R.
      • Mukherjee B.
      • Vahokoski J.
      • Klages N.
      • Maco B.
      • Collins C.R.
      • Blackman M.J.
      • Kursula I.
      • Heussler V.
      • Brochet M.
      • Soldati-Favre D.
      A multistage antimalarial targets the plasmepsins IX and X essential for invasion and egress.
      ). RON3, a rhoptry protein inserted into the PVM during or just after invasion, is also thought to be a PM IX substrate from inhibitor studies (
      • Favuzza P.
      • de Lera Ruiz M.
      • Thompson J.K.
      • Triglia T.
      • Ngo A.
      • Steel R.W.J.
      • Vavrek M.
      • Christensen J.
      • Boyce C.
      • Guo Z.
      • Hu M.
      • Khan T.
      • Murgolo N.
      • Zhao L.
      • Penington J.S.
      • et al.
      Dual plasmepsin-targeting antimalarial agents disrupt multiple stages of the malaria parasite life cycle.
      ). Rhoptry morphogenesis and attachment defects were observed in the PM IX-depleted strain (
      • Nasamu A.S.
      • Glushakova S.
      • Russo I.
      • Vaupel B.
      • Oksman A.
      • Kim A.S.
      • Fremont D.H.
      • Tolia N.
      • Beck J.R.
      • Meyers M.J.
      • Niles J.C.
      • Zimmerberg J.
      • Goldberg D.E.
      Plasmepsins IX and X are essential and druggable mediators of malaria parasite egress and invasion.
      ). These defects presumably explain the invasion phenotype observed. The substrates of PM IX responsible for the phenotype are still not known, because RAP1 is not essential (
      • Baldi D.L.
      • Andrewsm K.T.
      • Waller R.F.
      • Roos D.S.
      • Howard R.F.
      • Crabb B.S.
      • Cowman A.F.
      RAP1 controls rhoptry targeting of RAP2 in the malaria parasite Plasmodium falciparum.
      ) and ASP is predicted to be nonessential by a piggy bac transposon mutagenesis screen (
      • Zhang M.
      • Wang C.
      • Otto T.D.
      • Oberstaller J.
      • Liao X.
      • Adapa S.R.
      • Udenze K.
      • Bronner I.F.
      • Casandra D.
      • Mayho M.
      • Brown J.
      • Li S.
      • Swanson J.
      • Rayner J.C.
      • Jiang R.H.Y.
      • Adams J.H.
      Uncovering the essential genes of the human malaria parasite Plasmodium falciparum by saturation mutagenesis.
      ). Maturation of PM IX appears to be autocatalytic (
      • Favuzza P.
      • de Lera Ruiz M.
      • Thompson J.K.
      • Triglia T.
      • Ngo A.
      • Steel R.W.J.
      • Vavrek M.
      • Christensen J.
      • Boyce C.
      • Guo Z.
      • Hu M.
      • Khan T.
      • Murgolo N.
      • Zhao L.
      • Penington J.S.
      • et al.
      Dual plasmepsin-targeting antimalarial agents disrupt multiple stages of the malaria parasite life cycle.
      ).
      PM X is involved in both egress and invasion (
      • Nasamu A.S.
      • Glushakova S.
      • Russo I.
      • Vaupel B.
      • Oksman A.
      • Kim A.S.
      • Fremont D.H.
      • Tolia N.
      • Beck J.R.
      • Meyers M.J.
      • Niles J.C.
      • Zimmerberg J.
      • Goldberg D.E.
      Plasmepsins IX and X are essential and druggable mediators of malaria parasite egress and invasion.
      ,
      • Pino P.
      • Caldelari R.
      • Mukherjee B.
      • Vahokoski J.
      • Klages N.
      • Maco B.
      • Collins C.R.
      • Blackman M.J.
      • Kursula I.
      • Heussler V.
      • Brochet M.
      • Soldati-Favre D.
      A multistage antimalarial targets the plasmepsins IX and X essential for invasion and egress.
      ,
      • Favuzza P.
      • de Lera Ruiz M.
      • Thompson J.K.
      • Triglia T.
      • Ngo A.
      • Steel R.W.J.
      • Vavrek M.
      • Christensen J.
      • Boyce C.
      • Guo Z.
      • Hu M.
      • Khan T.
      • Murgolo N.
      • Zhao L.
      • Penington J.S.
      • et al.
      Dual plasmepsin-targeting antimalarial agents disrupt multiple stages of the malaria parasite life cycle.
      ). In asexual P. falciparum parasites, PM X is localized to small oval-shaped vesicles called exonemes (
      • Nasamu A.S.
      • Glushakova S.
      • Russo I.
      • Vaupel B.
      • Oksman A.
      • Kim A.S.
      • Fremont D.H.
      • Tolia N.
      • Beck J.R.
      • Meyers M.J.
      • Niles J.C.
      • Zimmerberg J.
      • Goldberg D.E.
      Plasmepsins IX and X are essential and druggable mediators of malaria parasite egress and invasion.
      ). The other protein that has been localized to this organelle is the serine protease subtilisin-like protease 1 (SUB1). PM X processes SUB1 to activate it. Knockdown or chemical inhibition of PM X results in the accumulation of SUB1 precursor (
      • Nasamu A.S.
      • Glushakova S.
      • Russo I.
      • Vaupel B.
      • Oksman A.
      • Kim A.S.
      • Fremont D.H.
      • Tolia N.
      • Beck J.R.
      • Meyers M.J.
      • Niles J.C.
      • Zimmerberg J.
      • Goldberg D.E.
      Plasmepsins IX and X are essential and druggable mediators of malaria parasite egress and invasion.
      ,
      • Pino P.
      • Caldelari R.
      • Mukherjee B.
      • Vahokoski J.
      • Klages N.
      • Maco B.
      • Collins C.R.
      • Blackman M.J.
      • Kursula I.
      • Heussler V.
      • Brochet M.
      • Soldati-Favre D.
      A multistage antimalarial targets the plasmepsins IX and X essential for invasion and egress.
      ,
      • Favuzza P.
      • de Lera Ruiz M.
      • Thompson J.K.
      • Triglia T.
      • Ngo A.
      • Steel R.W.J.
      • Vavrek M.
      • Christensen J.
      • Boyce C.
      • Guo Z.
      • Hu M.
      • Khan T.
      • Murgolo N.
      • Zhao L.
      • Penington J.S.
      • et al.
      Dual plasmepsin-targeting antimalarial agents disrupt multiple stages of the malaria parasite life cycle.
      ). Mature SUB1 is required for the degradation of both the parasitophorous vacuole and red cell membranes to allow dissemination of merozoites from a mother schizont. To initiate the egress cascade, SUB1 activates cysteine proteases called SERAs and merozoite surface proteins called MSPs (
      • Thomas J.A.
      • Tan M.S.Y.
      • Bisson C.
      • Borg A.
      • Umrekar T.R.
      • Hackett F.
      • Hale V.L.
      • Vizcay-Barrena G.
      • Fleck R.A.
      • Snijders A.P.
      • Saibil H.R.
      • Blackman M.J.
      A protease cascade regulates release of the human malaria parasite Plasmodium falciparum from host red blood cells.
      ). Full block of PM X traps parasites within the PVM, whereas partial block allows egress from this membrane but prevents escape from the RBC membrane. Presumably, a higher level of activated SUB1 is required for its effects on the erythrocyte. PM X also processes the surface adhesin sheddase SUB2 as well as erythrocyte binding-like (EBL) and Rh family members that are all involved in invasion (
      • Favuzza P.
      • de Lera Ruiz M.
      • Thompson J.K.
      • Triglia T.
      • Ngo A.
      • Steel R.W.J.
      • Vavrek M.
      • Christensen J.
      • Boyce C.
      • Guo Z.
      • Hu M.
      • Khan T.
      • Murgolo N.
      • Zhao L.
      • Penington J.S.
      • et al.
      Dual plasmepsin-targeting antimalarial agents disrupt multiple stages of the malaria parasite life cycle.
      ). In sexual stages of P. falciparum, PM X is expressed in gametes, zygotes, and ookinetes and in P. berghei, it is implicated by inhibitor studies in egress from gametocytes and mosquito midgut invasion (
      • Li F.
      • Bounkeua V.
      • Pettersen K.
      • Vinetz J.M.
      Plasmodium falciparum ookinete expression of plasmepsin VII and plasmepsin X.
      ,
      • Pino P.
      • Caldelari R.
      • Mukherjee B.
      • Vahokoski J.
      • Klages N.
      • Maco B.
      • Collins C.R.
      • Blackman M.J.
      • Kursula I.
      • Heussler V.
      • Brochet M.
      • Soldati-Favre D.
      A multistage antimalarial targets the plasmepsins IX and X essential for invasion and egress.
      ). PM X processes the midgut invasion protein cell-traversal protein for ookinetes and sporozoites (CelTOS) (
      • Pino P.
      • Caldelari R.
      • Mukherjee B.
      • Vahokoski J.
      • Klages N.
      • Maco B.
      • Collins C.R.
      • Blackman M.J.
      • Kursula I.
      • Heussler V.
      • Brochet M.
      • Soldati-Favre D.
      A multistage antimalarial targets the plasmepsins IX and X essential for invasion and egress.
      ). Inhibitors also prevent progression from the liver to erythrocytes, although different compounds either block merosome formation (hence egress of schizonts in hepatocytes (
      • Pino P.
      • Caldelari R.
      • Mukherjee B.
      • Vahokoski J.
      • Klages N.
      • Maco B.
      • Collins C.R.
      • Blackman M.J.
      • Kursula I.
      • Heussler V.
      • Brochet M.
      • Soldati-Favre D.
      A multistage antimalarial targets the plasmepsins IX and X essential for invasion and egress.
      )) or have an effect on merozoite competence (
      • Favuzza P.
      • de Lera Ruiz M.
      • Thompson J.K.
      • Triglia T.
      • Ngo A.
      • Steel R.W.J.
      • Vavrek M.
      • Christensen J.
      • Boyce C.
      • Guo Z.
      • Hu M.
      • Khan T.
      • Murgolo N.
      • Zhao L.
      • Penington J.S.
      • et al.
      Dual plasmepsin-targeting antimalarial agents disrupt multiple stages of the malaria parasite life cycle.
      ). It is worth noting that compounds used in these studies inhibit both PM IX and PM X. PM IX is not expressed in liver or sexual stages, however, suggesting that action may be specific to PM X in these portions of the life cycle, although some effects through action on another aspartic protease may be suggested by the fact that different small molecules have been reported to have somewhat different effects on mosquito and liver-stage parasites.
      Recombinant PM X is active at pH 5.5 and can cleave a variety of synthetic peptide substrates, including ones based on SUB1, EBA-175, EBA-181, and Rh2 (
      • Nasamu A.S.
      • Glushakova S.
      • Russo I.
      • Vaupel B.
      • Oksman A.
      • Kim A.S.
      • Fremont D.H.
      • Tolia N.
      • Beck J.R.
      • Meyers M.J.
      • Niles J.C.
      • Zimmerberg J.
      • Goldberg D.E.
      Plasmepsins IX and X are essential and druggable mediators of malaria parasite egress and invasion.
      ,
      • Pino P.
      • Caldelari R.
      • Mukherjee B.
      • Vahokoski J.
      • Klages N.
      • Maco B.
      • Collins C.R.
      • Blackman M.J.
      • Kursula I.
      • Heussler V.
      • Brochet M.
      • Soldati-Favre D.
      A multistage antimalarial targets the plasmepsins IX and X essential for invasion and egress.
      ,
      • Favuzza P.
      • de Lera Ruiz M.
      • Thompson J.K.
      • Triglia T.
      • Ngo A.
      • Steel R.W.J.
      • Vavrek M.
      • Christensen J.
      • Boyce C.
      • Guo Z.
      • Hu M.
      • Khan T.
      • Murgolo N.
      • Zhao L.
      • Penington J.S.
      • et al.
      Dual plasmepsin-targeting antimalarial agents disrupt multiple stages of the malaria parasite life cycle.
      ). It has a strong preference for cleavage between two hydrophobic residues, not dissimilar from the digestive vacuole plasmepsins, and prefers a polar P2 residue and Glu, Gln, or Asp for P2′ (
      • Favuzza P.
      • de Lera Ruiz M.
      • Thompson J.K.
      • Triglia T.
      • Ngo A.
      • Steel R.W.J.
      • Vavrek M.
      • Christensen J.
      • Boyce C.
      • Guo Z.
      • Hu M.
      • Khan T.
      • Murgolo N.
      • Zhao L.
      • Penington J.S.
      • et al.
      Dual plasmepsin-targeting antimalarial agents disrupt multiple stages of the malaria parasite life cycle.
      ). PM X can cleave PM IX substrates, suggesting overlapping specificity (
      • Favuzza P.
      • de Lera Ruiz M.
      • Thompson J.K.
      • Triglia T.
      • Ngo A.
      • Steel R.W.J.
      • Vavrek M.
      • Christensen J.
      • Boyce C.
      • Guo Z.
      • Hu M.
      • Khan T.
      • Murgolo N.
      • Zhao L.
      • Penington J.S.
      • et al.
      Dual plasmepsin-targeting antimalarial agents disrupt multiple stages of the malaria parasite life cycle.
      ). PM X is autocatalytic, but in parasites not all of the PM X gets processed; they retain some as proenzyme (66 kDa) or as active 51- and 44-kDa versions (
      • Nasamu A.S.
      • Glushakova S.
      • Russo I.
      • Vaupel B.
      • Oksman A.
      • Kim A.S.
      • Fremont D.H.
      • Tolia N.
      • Beck J.R.
      • Meyers M.J.
      • Niles J.C.
      • Zimmerberg J.
      • Goldberg D.E.
      Plasmepsins IX and X are essential and druggable mediators of malaria parasite egress and invasion.
      ,
      • Pino P.
      • Caldelari R.
      • Mukherjee B.
      • Vahokoski J.
      • Klages N.
      • Maco B.
      • Collins C.R.
      • Blackman M.J.
      • Kursula I.
      • Heussler V.
      • Brochet M.
      • Soldati-Favre D.
      A multistage antimalarial targets the plasmepsins IX and X essential for invasion and egress.
      ,
      • Favuzza P.
      • de Lera Ruiz M.
      • Thompson J.K.
      • Triglia T.
      • Ngo A.
      • Steel R.W.J.
      • Vavrek M.
      • Christensen J.
      • Boyce C.
      • Guo Z.
      • Hu M.
      • Khan T.
      • Murgolo N.
      • Zhao L.
      • Penington J.S.
      • et al.
      Dual plasmepsin-targeting antimalarial agents disrupt multiple stages of the malaria parasite life cycle.
      ). An ER calcium-binding protein called PfERC is required for PM X maturation in an unknown fashion (
      • Fierro M.A.
      • Asady B.
      • Brooks C.F.
      • Cobb D.W.
      • Villegas A.
      • Moreno S.N.J.
      • Muralidharan V.
      An endoplasmic reticulum CREC family protein regulates the egress proteolytic cascade in malaria parasites.
      ).
      In Toxoplasma gondii, one aspartic protease called ASP3 is closely related to both PM IX and PM X (
      • Shea M.
      • Jäkle U.
      • Liu Q.
      • Berry C.
      • Joiner K.A.
      • Soldati-Favre D.
      A family of aspartic proteases and a novel, dynamic and cell-cycle-dependent protease localization in the secretory pathway of Toxoplasma gondii.
      ). It is localized in an endosomal compartment (post-Golgi). This protein processes microneme and rhoptry proteins and is implicated in egress and invasion and hence is thought of as a PM IX and PM X chimera (
      • Dogga S.K.
      • Mukherjee B.
      • Jacot D.
      • Kockmann T.
      • Molino L.
      • Hammoudi P.-M.
      • Hartkoorn R.C.
      • Hehl A.B.
      • Soldati-Favre D.
      A druggable secretory protein maturase of Toxoplasma essential for invasion and egress.
      ). Mutations in the conserved Phe344 confer resistance to the small-molecule inhibitor 49c in cultured Toxoplasma and for isolated ASP3, PM IX, or PM X (
      • Mukherjee B.
      • Tessaro F.
      • Vahokoski J.
      • Kursula I.
      • Marq J.B.
      • Scapozza L.
      • Soldati-Favre D.
      Modeling and resistant alleles explain the selectivity of antimalarial compound 49c towards apicomplexan aspartyl proteases.
      ). The cleavage sites for ASP3 substrates are similar to the peptide cleavage sites for the PM IX and PM X that were tested, adding to the concept of their relatedness and similar roles in the two organisms. PM V, IX, and X all share a function as secretory organelle protein maturases (Fig. 8B).

      Key questions

      • What are the key substrates of PM IX required for rhoptry morphogenesis?
      • How does PM X cleave and activate the SUB1 precursor that is bound to its propiece?
      • Where does PM X interact with and process some microneme and rhoptry proteins? It has only been found in exonemes thus far.

      Other aspartic proteases

      Four other putative aspartic proteases are encoded in the Plasmodium genome but were not appreciated when the plasmepsins were named (
      • Weissbach T.
      • Golzmann A.
      • Bennink S.
      • Pradel G.
      • Julius Ngwa C.
      Transcript and protein expression analysis of proteases in the blood stages of Plasmodium falciparum.
      ). MiGS is most homologous to PM V but is expressed in male gametocytes and microgametes (
      • Tachibana M.
      • Ishino T.
      • Takashima E.
      • Tsuboi T.
      • Torii M.
      A male gametocyte osmiophilic body and microgamete surface protein of the rodent malaria parasite Plasmodium yoelii (PyMiGS) plays a critical role in male osmiophilic body formation and exflagellation.
      ,
      • Kehrer J.
      • Frischknecht F.
      • Mair G.R.
      Proteomic analysis of the plasmodium berghei gametocyte egressome and vesicular bioid of osmiophilic body proteins identifies merozoite trap-like protein (MTRAP) as an essential factor for parasite transmission.
      ). It appears to play a role in formation of the secretory osmiophilic bodies of male gametocytes. After secretion onto the surface, it also plays a role in exflagellation of male microgametes that go on to fertilize the female gametes. Like the other plasmepsins, it is predicted to have a classical pepsin-like fold; however, one of the catalytic sites has clearly undergone substantial mutation, with the conserved DTGS mutated to LTNS. Whether MiGS folds in an unexpected way to bring another catalytic residue into the active site, acts as a homodimer, or acts as a pseudoprotease is unclear.
      Ddi1 (DNA damage-inducible protease 1) is another aspartic protease with a Plasmodium ortholog. In other eukaryotes, it is important for the cell cycle through ubiquitin/proteasome interactions (
      • Gabriely G.
      • Kama R.
      • Gelin-Licht R.
      • Gerst J.E.
      Different domains of the UBL-UBA ubiquitin receptor, Ddi1/Vsm1, are involved in its multiple cellular roles.
      ). It has only one catalytic aspartate and homodimerizes to form the active enzyme. In malaria parasites, all that is known is that its gene is refractory to knockout in P. berghei and P. falciparum (
      • Zhang M.
      • Wang C.
      • Otto T.D.
      • Oberstaller J.
      • Liao X.
      • Adapa S.R.
      • Udenze K.
      • Bronner I.F.
      • Casandra D.
      • Mayho M.
      • Brown J.
      • Li S.
      • Swanson J.
      • Rayner J.C.
      • Jiang R.H.Y.
      • Adams J.H.
      Uncovering the essential genes of the human malaria parasite Plasmodium falciparum by saturation mutagenesis.
      ,
      • Bushell E.
      • Gomes A.R.
      • Sanderson T.
      • Anar B.
      • Girling G.
      • Herd C.
      • Metcalf T.
      • Modrzynska K.
      • Schwach F.
      • Martin R.E.
      • Mather M.W.
      • McFadden G.I.
      • Parts L.
      • Rutledge G.G.
      • Vaidya A.B.
      • et al.
      Functional profiling of a Plasmodium genome reveals an abundance of essential genes.
      ,
      • Onchieku N.M.
      • Mogire R.
      • Ndung'u L.
      • Mwitari P.
      • Kimani F.
      • Matoke-Muhia D.
      • Kiboi D.
      • Magoma G.
      Deciphering the targets of retroviral protease inhibitors in Plasmodium berghei.
      ).
      SPP is an intramembrane aspartic protease that processes signal peptides after release from secretory proteins in the ER. Early reports based on antibody and inhibitor studies suggested that the Plasmodium ortholog of SPP might be a micronemal protein involved in invasion (
      • Li X.
      • Chen H.
      • Oh S.S.
      • Chishti A.H.
      A presenilin-like protease associated with Plasmodium falciparum micronemes is involved in erythrocyte invasion.
      ,
      • Li X.
      • Chen H.
      • Bahamontes-Rosa N.
      • Kun J.F.J.
      • Traore B.
      • Crompton P.D.
      • Chishti A.H.
      Plasmodium falciparum signal peptide peptidase is a promising drug target against blood stage malaria.
      ). Further reports, however, instead