Heme binding and polymerization by Plasmodium falciparum histidine rich protein II: influence of pH on activity and conformation.

The histidine rich protein II (HRPII) from Plasmodium falciparum has been implicated as a heme polymerase which detoxifies free heme by its polymerization to inactive hemozoin. Histidine-iron center coordination is the dominant mechanism of interaction between the amino acid and heme. The protein also contains aspartate allowing for ionic/coordination interactions between the carboxylate side chain and the heme metal center. The pH profile of heme binding and polymerization shows the possibility of these two types of binding sites being differentiated by pH. Circular dichroism studies of the protein show that pH and heme binding cause a change in conformation above pH 6 implying the involvement of His-His+ transitions. Heme binding at pHs above 6 perturbs HRPII conformation, causing an increase in helicity.     

Hemozoin formation in malaria: a two-step process involving histidine-rich proteins and lipids

Major blood stage antimalarial drugs like chloroquine and artemisinin target the heme detoxification process of the malaria parasite. Hemozoin formation reactions in vitro using the Plasmodium falciparum histidine-rich protein-2 (Pfhrp-2), lipids, and auto-catalysis are slow and could not explain the speed of detoxification needed for parasite survival. Here, we show that malarial hemozoin formation is a coordinated two component process involving both lipids and histidine-rich proteins. Hemozoin formation efficiency in vitro is 1-2% with Pfhrp-2 and 0.25-0.5% with lipids. We added lipids after 9h in a 12h Pfhrp-2 mediated reaction that resulted in sixfold increase in hemozoin formation. However, a lipid mediated reaction in which Pfhrp-2 was added after 9h produced only twofold increase in hemozoin production compared to the reaction with Pfhrp-2 alone. Synthetic peptides corresponding to the Pfhrp-2 heme binding sequences, based on repeats of AHHAAD, neither alone nor in combination with lipids were able to generate hemozoin in vitro. These results indicate that hemozoin formation in malaria parasite involves both the lipids and the scaffolding proteins. Histidine-rich proteins might facilitate hemozoin formation by binding with a large number of heme molecules, and facilitating the dimer formation involving iron-carboxylate bond between two heme molecules, and lipids may then subsequently assist the mechanism of long chain formation, held together by hydrogen bonds or through extensive networking of hydrogen bonds.     

Plasmodium berghei: Uptake and distribution of chloroquine in infected mouse erythrocytes

The capacity of mouse erythrocytes infected with Plasmodium berghei to accumulate chloroquine is developed with maturation of the parasites. This is shown by direct comparison of the early and mature stages, which are separated by density difference. After drug accumulation, infected cells were fractionated by saponin lysis or nitrogen decompression to study the drug distribution. Effectiveness of isolating intact parasites and host components was checked by SDS-polyacrylamide gel electrophoresis and by low leakage of parasite-specific lactate dehydrogenase used as a marker enzyme. At low external drug concentration (~10^?7M), chloroquine is principally accumulated in the parasites. However, at higher drug concentrations (~10^?5and ~10^?3M), the proportion of the drug found in the host cytosol fraction is increased. A small but significant proportion of the drug (<20%) is associated with the host cell membrane. The pellet fraction of the freed parasites, further fractionated by freeze-thaw lysis, contains a major proportion of the drug at low external concentrations. However, the pellet fraction obtained from prolonged sonication of the parasites, which contains the bulk of hemozoin pigment, carries only a small proportion of the drug. This indicates that parasite membrane components may bind most of the drug. As external chloroquine concentration is increased, the proportion of drug in the parasite supernatant increases, some or most of which is probably bound by soluble hemecontaining compounds. However, the presence of chloroquine in the parasite does not affect the partition of heme in particulate and soluble forms.     

Bilirubin inhibits Plasmodium falciparum growth through the generation of reactive oxygen species

Free heme is very toxic because it generates highly reactive hydroxyl radicals ((.)OH) to cause oxidative damage. Detoxification of free heme by the heme oxygenase (HO) system is a very common phenomenon by which free heme is catabolized to form bilirubin as an end product. Interestingly, the malaria parasite, Plasmodium falciparum, lacks an HO system, but it forms hemozoin, mainly to detoxify free heme. Here, we report that bilirubin significantly induces oxidative stress in the parasite as evident from the increased formation of lipid peroxide, decrease in glutathione content, and increased formation of H(2)O(2) and (.)OH. Bilirubin can effectively inhibit hemozoin formation also. Furthermore, results indicate that bilirubin inhibits parasite growth and induces caspase-like protease activity, up-regulates the expression of apoptosis-related protein (Gene ID PFI0450c), and reduces the mitochondrial membrane potential. (.)OH scavengers such as mannitol, as well as the spin trap alpha-phenyl-n-tert-butylnitrone, effectively protect the parasite from bilirubin-induced oxidative stress and growth inhibition. These findings suggest that bilirubin, through the development of oxidative stress, induces P. falciparum cell death and that the malaria parasite lacks an HO system probably to protect itself from bilirubin-induced cell death as a second line of defense.     

Inhibition of hemozoin formation in Plasmodium falciparum trophozoite extracts by heme analogs: possible implication in the resistance to malaria conferred by the beta-thalassemia trait.

BACKGROUND: Human falciparum malaria, caused by the intracellular protozoa Plasmodium falciparum, results in 1-2 million deaths per year. P. falciparum digests host erythrocyte hemoglobin within its food vacuole, resulting in the release of potentially toxic free heme. A parasite-specific heme polymerization activity detoxifies the free heme by cross-linking the heme monomers to form hemozoin or malaria pigment. This biochemical process is the target of the widely successful antimalarial drug chloroquine, which is rapidly losing its effectiveness due to the spread of chloroquine resistance. We have shown that chloroquine resistance is not due to changes in the overall catalytic activity of heme polymerization or its chloroquine sensitivity. Therefore, the heme polymerization activity remains a potential target for novel antimalarials. In this study, we investigated the ability of heme analogs to inhibit heme polymerization and parasite growth in erythrocytes. MATERIALS AND METHODS: Incorporation of radioactive hemin substrate into an insoluble hemozoin pellet was used to determine heme polymerization. Incorporation of radioactive hypoxanthine into the nucleic acid of dividing parasites was used to determine the effects of heme analogs on parasite growth. Microscopic and biochemical measurements were made to determine the extent of heme analog entry into infected erythrocytes. RESULTS: The heme analogs tin protoporphyrin IX (SnPP), zinc protoporphyrin IX (ZnPP), and zinc deuteroporphyrin IX, 2,4 bisglycol (ZnBG) inhibited polymerization at micromolar concentrations (ZnPP << SnPP < ZnBG). However, they did not inhibit parasite growth since they failed to gain access to the site of polymerization, the parasite's food vacuole. Finally, we observed high ZnPP levels in erythrocytes from two patients with beta-thalassemia trait, which may inhibit heme polymerization. CONCLUSIONS: The heme analogs tested were able to inhibit hemozoin formation in Plasmodium falciparum trophozite extracts. The increased ZnPP levels found in thalassemic erythrocytes suggest that these may contribute, at least in part, to the observed antimalarial protection conferred by the beta-thalassemia trait. This finding may lead to the development of new forms of antimalarial therapy.     

Dendrimeric Template of Plasmodium falciparum Histidine Rich Protein II Repeat Motifs Bearing Asp→Asn Mutation Exhibits Heme Binding and β-Hematin Formation

Plasmodium falciparum (Pf) employs a crucial PfHRPII catalyzed reaction that converts toxic heme into hemozoin. Understanding heme polymerization mechanism is the first step for rational design of new drugs, targeting this pathway. Heme binding and hemozoin formation have been ascribed to PfHRPII aspartate carboxylate-heme metal ionic interactions. To investigate, if this ionic interaction is indeed pivotal, we examined the comparative heme binding and β-hematin forming abilities of a wild type dendrimeric peptide BNT1 {harboring the native sequence motif of PfHRPII (AHHAHHAADA)} versus a mutant dendrimeric peptide BNTM {in which ionic Aspartate residues have been replaced by the neutral Asparaginyl residues (AHHAHHAANA)}. UV and IR data reported here reveal that at pH 5, both BNT1 and BNTM exhibit comparable heme binding as well as β-hematin forming abilities, thus questioning the role of PfHRPII aspartate carboxylate-heme metal ionic interactions in heme binding and β-hematin formation. Based on our data and information in the literature we suggest the possible role of weak dispersive interactions like N-H···π and lone-pair···π in heme binding and hemozoin formation.     

Inhibition of heme sequestration of histidine‐rich protein 2 using multiple epitope‐targeted peptides

Plasmodium falciparum is the most lethal species of malaria. In infected human red blood cells, P. falciparum digests hemoglobin as a nutrient source, liberating cytotoxic free heme in the process. Sequestration and subsequent conversion of this byproduct into hemozoin, an inert biocrystalline heme aggregate, plays a key role in parasite survival. Hemozoin has been a longstanding target of antimalarials such as chloroquine (CQ), which inhibit the biocrystallization of free heme. In this study, we explore heme‐binding interactions with histidine‐rich‐protein 2 (HRP2), a known malarial biomarker and purported player in free heme sequestration. HRP2 is notoriously challenging to target due to its highly repetitious sequence and irregular secondary structure. We started with three protein‐catalyzed capture agents (PCCs) developed against epitopes of HRP2, inclusive of heme‐binding motifs, and explored their ability to inhibit heme:HRP2 complex formation. Cocktails of the individual PCCs exhibit an inhibitory potency similar to CQ, while a covalently linked structure built from two separate PCCs provided considerably increased inhibition relative to CQ. Epitope‐targeted disruption of heme:HRP2 binding is a novel approach towards disrupting P. falciparum‐related hemozoin formation.     

Clotrimazole binds to heme and enhances heme-dependent hemolysis: proposed antimalarial mechanism of clotrimazole.

Two recent studies have demonstrated that clotrimazole, a potent antifungal agent, inhibits the growth of chloroquine-resistant strains of the malaria parasite, Plasmodium falciparum, in vitro. We explored the mechanism of antimalarial activity of clotrimazole in relation to hemoglobin catabolism in the malaria parasite. Because free heme produced from hemoglobin catabolism is highly toxic to the malaria parasite, the parasite protects itself by polymerizing heme into insoluble nontoxic hemozoin or by decomposing heme coupled to reduced glutathione. We have shown that clotrimazole has a high binding affinity for heme in aqueous 40% dimethyl sulfoxide solution (association equilibrium constant: K(a) = 6.54 x 10(8) m(-2)). Even in water, clotrimazole formed a stable and soluble complex with heme and suppressed its aggregation. The results of optical absorption spectroscopy and electron spin resonance spectroscopy revealed that the heme-clotrimazole complex assumes a ferric low spin state (S = 1/2), having two nitrogenous ligands derived from the imidazole moieties of two clotrimazole molecules. Furthermore, we found that the formation of heme-clotrimazole complexes protects heme from degradation by reduced glutathione, and the complex damages the cell membrane more than free heme. The results described herein indicate that the antimalarial activity of clotrimazole might be due to a disturbance of hemoglobin catabolism in the malaria parasite.     

Heme metabolism of Plasmodium is a major antimalarial target

The malarial parasite manifests unique features of heme metabolism. In the intraerythrocyte stage it utilizes the host hemoglobin to generate amino acids for its own protein synthesis, but polymerizes the acquired heme as a mechanism for detoxification. At the same time the parasite synthesizes heme de novo for metabolic use. The heme biosynthetic pathway of the parasite is similar to that of hepatocytes and erythrocytes. However, while the parasite makes its own delta-aminolevulinate (ALA) synthase that is immunochemically different from that of the host, it imports ALA dehydrase and perhaps the subsequent enzymes of the pathway from the host red cell. Many schizonticidal drugs such as chloroquine and artemisinin act by interfering with the heme metabolism of the parasite and there is scope to design new molecules based on the unique features of this metabolic machinery in the parasite.     

Heme-binding properties of heme detoxification protein from Plasmodium falciparum

The heme detoxification protein of the malaria parasite Plasmodium falciparum is involved in the formation of hemozoin, an insoluble crystalline form of heme. Although the disruption of hemozoin formation is the most widely used strategy for controlling the malaria parasite, the heme-binding properties of heme detoxification protein are poorly characterized. In this study, we established a method for the expression and purification of the non-tagged protein and characterized heme-binding properties. The spectroscopic features of non-tagged protein differ from those of the His-tagged protein, suggesting that the artificial tag interferes with the properties of the recombinant protein. The purified recombinant non-tagged heme detoxification protein had two heme-binding sites and exhibited a spectrum typical of heme proteins. A mechanism for hemozoin formation is proposed.     

Genesis of and trafficking to the Maurer's clefts of Plasmodium falciparum-infected erythrocytes

Malaria parasites export proteins beyond their own plasma membrane to locations in the red blood cells in which they reside. Maurer's clefts are parasite-derived structures within the host cell cytoplasm that are thought to function as a sorting compartment between the parasite and the erythrocyte membrane. However, the genesis of this compartment and the signals directing proteins to the Maurer's clefts are not known. We have generated Plasmodium falciparum-infected erythrocytes expressing green fluorescent protein (GFP) chimeras of a Maurer's cleft resident protein, the membrane-associated histidine-rich protein 1 (MAHRP1). Chimeras of full-length MAHRP1 or fragments containing part of the N-terminal domain and the transmembrane domain are successfully delivered to Maurer's clefts. Other fragments remain trapped within the parasite. Fluorescence photobleaching and time-lapse imaging techniques indicate that MAHRP1-GFP is initially trafficked to isolated subdomains in the parasitophorous vacuole membrane that appear to represent nascent Maurer's clefts. The data suggest that the Maurer's clefts bud from the parasitophorous vacuole membrane and diffuse within the erythrocyte cytoplasm before taking up residence at the cell periphery.     

An assay procedure to investigate the transformation of toxic heme into inert hemozoin via plasmodial heme detoxification protein

Most antimalarial therapeutics, including chloroquine and artemisinin, induce free heme-mediated toxicity in Plasmodium. This cytotoxic heme is produced as a by-product during the large-scale digestion of host hemoglobin. Conversion of this host-derived heme into inert crystalline hemozoin is the only defense mechanism in Plasmodium against heme-induced cytotoxicity. Heme detoxification protein (HDP), a highly conserved plasmodial protein, is reported to be the most efficient biological mediator for heme to hemozoin transformation. Despite its significance, HDP has never been extensively studied for heme transformation into hemozoin. Therefore, we wish to develop a method to study the HDP-mediated transformation of heme into hemozoin. We have adopted, modified, and optimized the pyridine hemochrome assay to study HDP catalysis and use substrate and time kinetics to study the HDP-mediated transformation of heme into hemozoin. In contrast to the previously reported assay for HDP, we found that the new assay is more precise, accurate, and handy, making it more suitable for kinetic studies. HDP-mediated transformation of heme into hemozoin is not a single-step process, and involves a transient intermediate, most likely a cyclic heme dimer. The kinetics and the manner of HDP-mediated hemozoin production are dependent on the substrate concentration, and a small fraction of substrate remains untransformed to hemozoin irrespective of reaction time. Combining HDP as a catalyst and the pyridine hemochrome assay will facilitate the efficient screening of future antimalarials.     

The Toxoplasma gondii protein ROP2 mediates host organelle association with the parasitophorous vacuole membrane.

Toxoplasma gondii replicates within a specialized vacuole surrounded by the parasitophorous vacuole membrane (PVM). The PVM forms intimate interactions with host mitochondria and endoplasmic reticulum (ER) in a process termed PVM-organelle association. In this study we identify a likely mediator of this process, the parasite protein ROP2. ROP2, which is localized to the PVM, is secreted from anterior organelles termed rhoptries during parasite invasion into host cells. The NH(2)-terminal domain of ROP2 (ROP2hc) within the PVM is exposed to the host cell cytosol, and has characteristics of a mitochondrial targeting signal. In in vitro assays, ROP2hc is partially translocated into the mitochondrial outer membrane and behaves like an integral membrane protein. Although ROP2hc does not translocate across the ER membrane, it does exhibit carbonate-resistant binding to this organelle. In vivo, ROP2hc expressed as a soluble fragment in the cytosol of uninfected cells associates with both mitochondria and ER. The 30-amino acid (aa) NH(2)-terminal sequence of ROP2hc, when fused to green fluorescent protein (GFP), is sufficient for mitochondrial targeting. Deletion of the 30-aa NH(2)-terminal signal from ROP2hc results in robust localization of the truncated protein to the ER. These results demonstrate a new mechanism for tight association of different membrane-bound organelles within the cell cytoplasm.     

Import of host delta-aminolevulinate dehydratase into the malarial parasite: identification of a new drug target

The parasite Plasmodium berghei imports the enzyme delta-aminolevulinate dehydratase (ALAD), and perhaps the subsequent enzymes of the pathway from the host red blood cell to sustain heme synthesis. Here we have studied the mechanism of this import. A 65-kDa protein on the P. berghei membrane specifically bound to mouse red blood cell ALAD, and a 93-amino-acid fragment (ALAD-DeltaNC) of the host erythrocyte ALAD was able to compete with the full-length enzyme for binding to the P. berghei membrane. ALAD-DeltaNC was taken up by the infected red blood cell when added to a culture of P. falciparum and this led to a substantial decrease in ALAD protein and enzyme activity and, subsequently, heme synthesis in the parasite, resulting in its death.     

Plasmodium falciparum SURFIN4.1 forms an intermediate complex with PTEX components and Pf113 during export to the red blood cell

Plasmodium falciparum malaria parasites export several hundred proteins to the cytoplasm of infected red blood cells (RBCs) to modify the cell environment suitable for their growth. A Plasmodium translocon of exported proteins (PTEX) is necessary for both soluble and integral membrane proteins to cross the parasitophorous vacuole (PV) membrane surrounding the parasite inside the RBC. However, the molecular composition of the translocation complex for integral membrane proteins is not fully characterized, especially at the parasite plasma membrane. To examine the translocation complex, here we used mini-SURFIN_4.1, consisting of a short N-terminal region, a transmembrane region, and a cytoplasmic region of an exported integral membrane protein SURFIN_4.1. We found that mini-SURFIN_4.1 forms a translocation intermediate complex with core PTEX components, EXP2, HSP101, and PTEX150. We also found that several proteins are exposed to the PV space, including Pf113, an uncharacterized PTEX-associated protein. We determined that Pf113 localizes in dense granules at the merozoite stage and on the parasite periphery after RBC invasion. Using an inducible translocon-clogged mini-SURFIN_4.1, we found that a stable translocation intermediate complex forms at the parasite plasma membrane and contains EXP2 and a processed form of Pf113. These results suggest a potential role of Pf113 for the translocation step of mini-SURFIN_4.1, providing further insights into the translocation mechanisms for parasite integral membrane proteins.     

Spectroscopic characterization of the heme-binding sites in Plasmodium falciparum histidine-rich protein 2

Proteolysis of hemoglobin provides an essential nutrient source for the malaria parasite Plasmodium falciparum during the intraerythrocytic stage of the parasite's lifecycle. Detoxification of the liberated heme occurs through a unique heme polymerization pathway, leading to the formation of hemozoin. Heme polymerization has been demonstrated in the presence of P. falciparum histidine-rich protein 2 (PfHRP2) [Sullivan, D. J., Gluzman, I. Y., and Goldberg, D. E. (1996) Science 271, 219-221]; however, the molecular role that PfHRP2 plays in this polymerization is currently unknown. PfHRP2 is a 30 kDa protein composed of several His-His-Ala-His-His-Ala-Ala-Asp repeats and is present in the parasite food vacuole, the site of hemoglobin degradation and heme polymerization. We found that, at pH 7.0, PfHRP2 forms a saturable complex with heme, with a PfHRP2 to heme stoichiometry of 1:50. Spectroscopic characterization of heme binding by electronic absorption, resonance Raman, and EPR has shown that bound hemes share remarkably similar heme environments as >95% of all bound hemes are six-coordinate, low-spin, and bis-histidyl ligated. The PfHRP2-ferric heme complex at pH 5.5 (pH of the food vacuole) has the same heme spin state and coordination as observed at pH 7.0; however, polymerization occurs as heme saturation is approached. Therefore, formation of a PfHRP2-heme complex appears to be a requisite step in the formation of hemozoin.     

SEC18/NSF-independent, protein-sorting pathway from the yeast cortical ER to the plasma membrane

Classic studies of temperature-sensitive secretory (sec) mutants have demonstrated that secreted and plasma membrane proteins follow a common SEC pathway via the endoplasmic reticulum (ER), Golgi apparatus, and secretory vesicles to the cell periphery. The yeast protein Ist2p, which is synthesized from a localized mRNA, travels from the ER to the plasma membrane via a novel route that operates independently of the formation of coat protein complex II-coated vesicles. In this study, we show that the COOH-terminal domain of Ist2p is necessary and sufficient to mediate SEC18-independent sorting when it is positioned at the COOH terminus of different integral membrane proteins and exposed to the cytoplasm. This domain functions as a dominant plasma membrane localization determinant that overrides other protein sorting signals. Based on these observations, we suggest a local synthesis of Ist2p at cortical ER sites, from where the protein is sorted by a novel mechanism to the plasma membrane.     

A non-radiolabeled heme-GSH interaction test for the screening of antimalarial compounds

Intraerythrocytic Plasmodium produces large amounts of toxic heme during the digestion of hemoglobin, a parasite specific pathway. Heme is then partially biocristallized into hemozoin and mostly detoxified by reduced glutathione. We proposed an in vitro micro assay to test the ability of drugs to inhibit heme-glutathione dependent degradation. As glutathione and o-phthalaldehyde form a fluorescent adduct, we followed the extinction of the fluorescent signal when heme was added with or without antimalarial compounds. In this assay, 50 microM of amodiaquine, arthemether, chloroquine, methylene blue, mefloquine and quinine inhibited the interaction between glutathione (50 microM) and heme (50 microM), while atovaquone did not. Consequently, this test could detect drugs that can inhibit heme-GSH degradation in a fast, simple and specific way, making it suitable for high throughput screening of potential antimalarials.     

Formation of the food vacuole in Plasmodium falciparum: a potential role for the 19 kDa fragment of merozoite surface protein 1 (MSP1(19))

Plasmodium falciparum Merozoite Surface Protein 1 (MSP1) is synthesized during schizogony as a 195-kDa precursor that is processed into four fragments on the parasite surface. Following a second proteolytic cleavage during merozoite invasion of the red blood cell, most of the protein is shed from the surface except for the C-terminal 19-kDa fragment (MSP1(19)), which is still attached to the merozoite via its GPI-anchor. We have examined the fate of MSP1(19) during the parasite's subsequent intracellular development using immunochemical analysis of metabolically labeled MSP1(19), fluorescence imaging, and immuno-electronmicroscopy. Our data show that MSP1(19) remains intact and persists to the end of the intracellular cycle. This protein is the first marker for the biogenesis of the food vacuole; it is rapidly endocytosed into small vacuoles in the ring stage, which coalesce to form the single food vacuole containing hemozoin, and persists into the discarded residual body. The food vacuole is marked by the presence of both MSP1(19) and the chloroquine resistance transporter (CRT) as components of the vacuolar membrane. Newly synthesized MSP1 is excluded from the vacuole. This behavior indicates that MSP1(19) does not simply follow a classical lysosome-like clearance pathway, instead, it may play a significant role in the biogenesis and function of the food vacuole throughout the intra-erythrocytic phase.