Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0024530 (malaria)
 44,886 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Serine proteases play crucial roles in erythrocyte invasion by merozoites of the malaria parasite. Plasmodium falciparum subtilisin-like protease-1 (PfSUB-1) is synthesized during maturation of the intraerythrocytic parasite and accumulates in a set of merozoite secretory organelles, suggesting that it may play a role in host cell invasion or post-invasion events. We describe the production, purification, and characterization of recombinant PfSUB-1 and comparison with the authentic protease detectable in parasite extracts. The recombinant protease requires high levels of calcium for optimum activity and has an alkaline pH optimum. Using a series of decapeptide and protein substrates, PfSUB-1 was found to have a relaxed substrate specificity with regard to the P1 position but is unable to efficiently cleave substrates with a P1 leucine residue. Similarly, replacement of a P4 valine with alanine severely reduced cleavage efficiency, whereas its replacement with lysine abolished cleavage. In all respects investigated, the recombinant protease was indistinguishable from parasite-derived enzyme. Three-dimensional homology modeling of the PfSUB-1 catalytic domain based on an alignment with closely related bacterial subtilisins and an orthologue from the rodent malaria Plasmodium yoelii suggests that the protease has at least three potential calcium ion-binding sites, three intramolecular disulfide bridges, and a single free cysteine within the enzyme S1 pocket. A predicted highly polar S1 pocket and a hydrophobic S4 subsite are in broad agreement with the experimentally determined substrate specificity.
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PMID:Expression of recombinant Plasmodium falciparum subtilisin-like protease-1 in insect cells. Characterization, comparison with the parasite protease, and homology modeling. 1205 28

For malaria to be transmitted, the Plasmodium falciparum parasite must invade an erythrocyte and undergo gametocytogenesis. When mature intraerythrocytic gametocytes are taken up in a blood meal by a mosquito they emerge as gametes and, once fertilized, continue to differentiate into infectious sporozoites. One of the major proteins associated with the surface of the parasite during gamete differentiation is Pfs230, a 360 kDa member of a family of P. falciparum proteins that contains a repeated cysteine motif domain. To characterize the role of different regions of Pfs230, the gene was disrupted by targeted integration and clones isolated that expressed distinct sections of Pfs230. Independent clones D1.356 a and b express the first 452 amino acids (aa) of Pfs230 and do not contain a cysteine motif domain, whereas clones D2.850 a and b express the first 950 aa, including the first cysteine motif domain. Although both sets of clones undergo gametogenesis and produce morphologically normal gametes, neither truncated Pfs230 is located on the surface of the gamete. In clones D1.356 a and b, the 452 aa Pfs230 is secreted into the parasitophorous vacuole and released as a soluble protein when the parasite emerges from the erythrocyte as a gamete. In marked contrast, the 950 aa form of Pfs230 expressed by clones D2.850 a and b is sequestered in a novel tubular compartment in the erythrocyte cytoplasm. This sexual-stage tubular intraerythrocytic compartment (STIC) is not recognized by antibodies specific for proteins associated with the parasitophorous vacuole membrane (Pfs16 or Exp-1) or Maurer's clefts (Pfsbp 1 or mAb LWL1) or intraerythrocytic asexual parasite proteins (PfEMP2 or HRP II).
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PMID:Targeting and sequestration of truncated Pfs230 in an intraerythrocytic compartment during Plasmodium falciparum gametocytogenesis. 1206 40

Malaria is one of the deadliest human diseases and efforts to control it have been difficult due to the protozoan parasites' complex biology. Malaria merozoite invasion of erythrocytes is an essential part of blood-stage infections. The invasion process is mediated by numerous parasite molecules, such as EBA-175, a member of the ebl family of erythrocyte binding proteins. We have identified maebl, an ebl paralogue, in Plasmodium falciparum and found it highly conserved with its orthologues in P. yoelii and P. berghei, but distinct from other Plasmodium ebl. Importantly, the putative MAEBL ligand domains are highly conserved and are similar to AMA-1, but not the consensus DBL ligand domains present in all other ebl. In mature merozoites, MAEBL localized with rhoptry proteins (RhopH2, RAP-1), including surface localization with RhopH2, but not microneme proteins (EBA-175, BAEBL). MAEBL appears as proteolytically processed fragments in P. falciparum parasites. The amino cysteine-rich ligand domains were present primarily in culture supernatants, while the carboxyl cysteine-rich domain adjacent to the transmembrane domain was preferentially isolated from Triton X-100 extracted fractions. These data indicate that the primary structure of maebl is highly conserved among Plasmodium species, while its characteristics demonstrate a function unique among the ebl proteins.
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PMID:Plasmodium falciparum MAEBL is a unique member of the ebl family. 1207 68

Malaria parasites invade erythrocytes in a process mediated by a series of molecular interactions. Invasion of human erythrocytes by Plasmodium vivax is dependent upon the presence of a single receptor, but P. falciparum, as well as some other species, exhibits the ability to utilize multiple alternative invasion pathways. Conserved cysteine-rich domains play important roles at critical times during this invasion process and at other stages in the life cycle of malaria parasites. Duffy-binding-like (DBL) domains, expressed as a part of the erythrocyte-binding proteins (DBL-EBP), are such essential cysteine-rich ligands that recognize specific host cell surface receptors. DBL-EBP, which are products of the erythrocyte-binding-like (ebl) gene family, act as critical determinants of erythrocyte specificity and are the best-defined ligands from invasive stages of malaria parasites. The ebl genes include the P. falciparum erythrocyte-binding antigen-175 (EBA-175) and P. vivax Duffy-binding protein. DBL domains also mediate cytoadherence as a part of the variant erythrocytic membrane protein-1 (PfEMP-1) antigens expressed from var genes on the surface of P. falciparum-infected erythrocytes. A paralogue of the ebl family is the malarial ligand MAEBL, which has a chimeric structure where the DBL domain is functionally replaced with a distinct cysteine-rich erythrocyte-binding domain with similarity to the apical membrane antigen-1 (AMA-1) ligand domain. The Plasmodium AMA-1 ligand domain, which encompasses the extracellular cysteine domains 1 and 2 and is well conserved in a Toxoplasma gondii AMA-1, has erythrocyte-binding activity distinct from that of MAEBL. These important families of Plasmodium molecules (DBL-EBP, PfEMP-1, MAEBL, AMA-1) are interrelated through the MAEBL. Because MAEBL and the other ebl products have the characteristics expected of homologous ligands involved in equivalent alternative invasion pathways to each other, we sought to better understand their roles during invasion by determining their relative origins in the Plasmodium genome. An analysis of their multiple cysteine-rich domains permitted a unique insight into the evolutionary development of PLASMODIUM: Our data indicate that maebl, ama-1, and ebl genes have ancient origins which predate Plasmodium speciation. The maebl evolved as a single locus, including its unique chimeric structure, in each Plasmodium species, in parallel with the ama-1 and the ebl genes families. The ancient character of maebl, along with its different expression characteristics suggests that MAEBL is unique and does not play an alternative role in invasion to ebl products such as EBA-175. The multiple P. falciparum ebl paralogues that express DBL domains, which have occurred by duplication and diversification, potentially do provide multiple functionally equivalent ligands to EBA-175 for alternative invasion pathways.
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PMID:Evolutionary relationships of conserved cysteine-rich motifs in adhesive molecules of malaria parasites. 1208 32

Cysteine proteases of the malaria parasite Plasmodium falciparum, known as falcipains, are promising targets for antimalarial chemotherapy. We evaluated cultured parasites for the stage-specific expression of cysteine proteases and sensitivity to cysteine protease inhibitors. Protease activity and inhibitor sensitivity varied markedly over time. Cysteine protease activity was greatest in early trophozoites, while sensitivity to cysteine protease inhibitors was greatest in mature trophozoites. Our results indicate the importance of considering the stage-specific effects of antimalarials and are consistent with the conclusion that the principal antimalarial activity of cysteine protease inhibitors is due to a block in hemoglobin hydrolysis.
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PMID:Stage-specific antimalarial activity of cysteine protease inhibitors. 1210 50

New drugs to treat malaria are urgently needed. Cysteine proteases of malaria parasites offer potential new chemotherapeutic targets. Cysteine protease inhibitors block parasite hemoglobin hydrolysis and development, indicating that cysteine proteases play a key role in hemoglobin degradation, a necessary function of erythrocytic trophozoites. These inhibitors also block the rupture of erythrocytes by mature parasites, suggesting an additional role for cysteine proteases in the hydrolysis of erythrocyte cytoskeletal proteins. Recent studies have shown that the repertoire of cysteine proteases of malaria parasites is larger than was previously realized. Plasmodium falciparum, the most virulent human malaria parasite, expresses three papain-family cysteine proteases, known as falcipains. All three proteases are expressed by trophozoites and hydrolyze hemoglobin at acidic pH, suggesting roles in this process. Falcipain-2 also hydrolyzes ankyrin at neutral pH, suggesting additional activity against erythrocyte cytoskeletal targets. Multiple orthologs of the falcipains have been identified in other plasmodial species. Analysis of orthologs from animal model rodent parasites identified similar features, but some noteworthy biochemical differences between the cysteine proteases. These differences must be taken into account in interpreting in vivo experiments. A number of small molecule cysteine protease inhibitors blocked parasite hemoglobin hydrolysis and development, and inhibitory effects against parasites generally correlated with inhibition of falcipain-2. Some compounds also cured mice infected with otherwise lethal malaria infections. Current research priorities are to better characterize the biological roles and biochemical features of the falcipains. In addition, efforts to identify optimal falcipain inhibitors as antimalarials are underway.
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PMID:Cysteine proteases of malaria parasites: targets for chemotherapy. 1213 97

Malaria parasites make specific receptor-ligand interactions to invade erythrocytes. A 175 kDa Plasmodium falciparum erythrocyte binding antigen (EBA-175) binds sialic acid residues on glycophorin A during invasion of human erythrocytes. The receptor-binding domain of EBA-175 lies in a conserved, amino-terminal, cysteine-rich region, region F2 of EBA-175 (PfF2), that is homologous to the binding domains of other erythrocyte binding proteins such as Plasmodium vivax Duffy binding protein. We have developed methods to produce recombinant PfF2 in its functional form. Recombinant PfF2 was expressed in Escherichia coli, purified from inclusion bodies, renatured by oxidative refolding and purified to homogeneity by ion-exchange and gel filtration chromatography. Refolded PfF2 has been characterized using biochemical and biophysical methods and shown to be pure, homogenous and functional in that it binds human erythrocytes with specificity. Immunization with refolded PfF2 yields high titre antibodies that efficiently inhibit P. falciparum invasion of erythrocytes in vitro. Importantly, antibodies raised against PfF2 block invasion by a P. falciparum field isolate that invades erythrocytes using multiple pathways. These observations support the development of recombinant PfF2 as a vaccine candidate for P. falciparum malaria.
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PMID:Bacterially expressed and refolded receptor binding domain of Plasmodium falciparum EBA-175 elicits invasion inhibitory antibodies. 1216 86

Cysteine proteases of Plasmodium falciparum, known as falcipains, have been identified as haemoglobinases and potential drug targets. As anti-malarial drug discovery requires the analysis of non-primate malaria, genes encoding related cysteine proteases of the rodent malaria parasites P. vinckei (vinckepain-2) and P. berghei (berghepain-2) were characterized. These genes encoded fairly typical papain-family proteases, but they contained an unusual substitution of Gly23 with Ala (papain numbering system). Vinckepain-2 was expressed in Escherichia coli, solubilized, refolded and autoprocessed to an active enzyme. The protease shared important features with the falcipains, including an acidic pH optimum, preference for reducing conditions, optimal cleavage of peptide substrates with P2 Leu and ready hydrolysis of haemoglobin. However, key differences between the plasmodial proteases were identified. In particular, vinckepain-2 showed very different kinetics against many substrates and an unusual preference for peptide substrates with P1 Gly. Replacement of Ala23 with Gly remarkably altered vinckepain-2, including loss of the P1 Gly substrate preference, markedly increased catalytic activity ( k cat/ K m increased approx. 100-fold) and more rapid autohydrolysis. The present study identifies key animal-model parasite targets. It indicates that drug discovery studies must take into account important differences between plasmodial proteases and sheds light on the critical role of amino acid 23 in catalysis by papain-family proteases.
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PMID:Critical role of amino acid 23 in mediating activity and specificity of vinckepain-2, a papain-family cysteine protease of rodent malaria parasites. 1216 96

The genome of the malaria parasite, Plasmodium falciparum, appears to contain the proteins necessary for a Type II dissociated fatty acid biosynthetic system. Here we report the functional characterization of two proteins from this system. Purified recombinant acyl carrier protein (ACP) and beta-ketoacyl-ACP synthase III (KASIII) from P. falciparum are soluble and active in a truncated form. Malarial ACP is activated by the addition of a 4'-phosphopantetheine prosthetic group derived from coenzyme A, generating holo-PfACP. Holo-PfACP is an effective substrate for the transacylase activity of PfKASIII, but substitution of a key active site cysteine in PfKASIII to alanine or serine abolishes enzymatic activity. During the schizont stage of parasite development, there is a significant up-regulation of the mRNAs corresponding to these proteins, indicating an important metabolic requirement for fatty acids during this stage.
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PMID:Functional characterization of the acyl carrier protein (PfACP) and beta-ketoacyl ACP synthase III (PfKASIII) from Plasmodium falciparum. 1227 Jun 24

Apical membrane antigen-1 (AMA1) is a prime vaccine candidate for inclusion in a vaccine against malaria. It is known that the disulphide bond stabilised conformation of this antigen is important for eliciting a protective antibody response, however little is known about the epitopes within this molecule that are targeted by the immune response. We have used a peptide approach for the identification and characterisation of such regions. In this study, the in vitro refolded, recombinant ectodomain of AMA1 from the D strain of Plasmodium chabaudi adami, was digested with trypsin and individual peptide fragments examined for antigenic activity. We found that a tryptic fragment, which was derived from a loop-like structure within the putative domain I of the intact AMA1 molecule, was highly reactive with antibodies from the sera of hyperimmune mice. Two different synthetic peptide constructs incorporating this antigenically active fragment were assembled. The first consisted of two separate peptide chains which were linked through a disulphide bond formed using chemo-selective chemistry. A larger 45-mer loop peptide, generated by the oxidation of two cysteine residues close to the N- and C-termini of the 45-mer, represented the complete loop structure and incorporated the tryptic fragment. Each peptide construct was also able to elicit production of high titres of antibodies in mice and furthermore, the 45-residue loop peptide elicited antibodies capable of binding to AMA1 with titres comparable to those present in a mouse which had recovered from multiple exposures to P. chabaudi adami parasites. Passive immunisation with anti-loop antibodies did not suppress the development of parasitaemia in mice challenged with P. chabaudi adami suggesting that although highly immunogenic, the peptides represented inadequate or inappropriate epitopes for vaccination purposes.
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PMID:Identification of antigenically active tryptic fragments of apical membrane antigen-1 (AMA1) of Plasmodium chabaudi malaria: strategies for assembly of immunologically active peptides. 1229 93


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