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Characterization of a conserved rhoptry-associated leucine zipper-like protein in the malaria parasite Plasmodium falciparum.

ABSTRACT: One of the key processes in the pathobiology of the malaria parasite is the invasion and subsequent modification of the human erythrocyte. In this complex process, an unknown number of parasite proteins are involved, some of which are leading vaccine candidates. The majority of the proteins that play pivotal roles in invasion are either stored in the apical secretory organelles or located on the surface of the merozoite, the invasive stage of the parasite. Using transcriptional and structural features of these known proteins, we performed a genomewide search that identified 49 hypothetical proteins with a high probability of being located on the surface of the merozoite or in the secretory organelles. Of these candidates, we characterized a novel leucine zipper-like protein in Plasmodium falciparum that is conserved in Plasmodium spp. This protein is expressed in late blood stages and localizes to the rhoptries of the parasite. We demonstrate that this Plasmodium sp.-specific protein has a high degree of conservation within field isolates and that it is refractory to gene knockout attempts and thus might play an important role in invasion.

SUBMITTER: Haase S

PROVIDER: S-EPMC2258820 | biostudies-other | 2008 Mar

REPOSITORIES: biostudies-other

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Characterization of a conserved rhoptry-associated leucine zipper-like protein in the malaria parasite Plasmodium falciparum.

Haase Silvia S Cabrera Ana A Langer Christine C Treeck Moritz M Struck Nicole N Herrmann Susann S Jansen Pascal W PW Bruchhaus Iris I Bachmann Anna A Dias Suzana S Cowman Alan F AF Stunnenberg Hendrik G HG Spielmann Tobias T Gilberger Tim-Wolf TW

Infection and immunity 20080103 3

One of the key processes in the pathobiology of the malaria parasite is the invasion and subsequent modification of the human erythrocyte. In this complex process, an unknown number of parasite proteins are involved, some of which are leading vaccine candidates. The majority of the proteins that play pivotal roles in invasion are either stored in the apical secretory organelles or located on the surface of the merozoite, the invasive stage of the parasite. Using transcriptional and structural fe ...[more]

PMID: 18174339

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Project description:BackgroundMalaria continues to be one of the most severe global infectious diseases, responsible for 1-2 million deaths yearly. The rapid evolution and spread of drug resistance in parasites has led to an urgent need for the development of novel antimalarial targets. Proteases are a group of enzymes that play essential roles in parasite growth and invasion. The possibility of designing specific inhibitors for proteases makes them promising drug targets. Previously, combining a comparative genomics approach and a machine learning approach, we identified the complement of proteases (degradome) in the malaria parasite Plasmodium falciparum and its sibling species 123, providing a catalog of targets for functional characterization and rational inhibitor design. Network analysis represents another route to revealing the role of proteins in the biology of parasites and we use this approach here to expand our understanding of the systems involving the proteases of P. falciparum.ResultsWe investigated the roles of proteases in the parasite life cycle by constructing a network using protein-protein association data from the STRING database 4, and analyzing these data, in conjunction with the data from protein-protein interaction assays using the yeast 2-hybrid (Y2H) system 5, blood stage microarray experiments 678, proteomics 9101112, literature text mining, and sequence homology analysis. Seventy-seven (77) out of 124 predicted proteases were associated with at least one other protein, constituting 2,431 protein-protein interactions (PPIs). These proteases appear to play diverse roles in metabolism, cell cycle regulation, invasion and infection. Their degrees of connectivity (i.e., connections to other proteins), range from one to 143. The largest protease-associated sub-network is the ubiquitin-proteasome system which is crucial for protein recycling and stress response. Proteases are also implicated in heat shock response, signal peptide processing, cell cycle progression, transcriptional regulation, and signal transduction networks.ConclusionsOur network analysis of proteases from P. falciparum uses a so-called guilt-by-association approach to extract sets of proteins from the proteome that are candidates for further study. Novel protease targets and previously unrecognized members of the protease-associated sub-systems provide new insights into the mechanisms underlying parasitism, pathogenesis and virulence.

Project description:Cyclosporin (Cs) A has pronounced antimalarial activity in vitro and in vivo. In other organisms, the drug is thought to exert its effects either by inhibiting the peptidylprolyl cis/trans isomerase activity of cyclophilin (CyP) or by forming a CyP-CsA complex that inhibits the phosphatase activity of calcineurin. We have cloned and overexpressed in Escherichia coli a gene encoding a CyP from Plasmodium falciparum (PfCyP19) that is located on chromosome 3. The sequence of PfCyP19 shows remarkable sequence identity with human CyPA and, unlike the two previously identified CyPs from P. falciparum, PfCyP19 has no signal peptide or N-terminal sequence extension, suggesting a cytosolic localization. All the residues implicated in the recognition of the synthetic substrate N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide are conserved, resulting in characteristically high Michaelis-Menten and specificity constants (Km>>120 microM, kcat/Km=1.2x10(7) M-1.s-1 respectively). As the first line in the functional characterization of this enzyme, we have assessed its binding affinity for CsA. In accordance with its tryptophan-containing CsA-binding domain, PfCyP19 binds CsA with high affinity (Kd=13 nM, Ki=6.9 nM). Twelve CsA analogues were also found to possess Ki values similar to CsA, with the notable exceptions of Val2-Cs (Ki=218 nM) and Thr2-Cs (Ki=690 nM). The immunosuppressants rapamycin and FK506 were inactive as inhibitors, consistent with other members of the CyP family of rotamases. The CsA analogues were also assessed as inhibitors of P. falciparum growth in vitro. Although their antimalarial activity did not correlate with inhibition of enzyme activity, residues 3 and 4 of CsA appeared to be important for inhibition of parasite growth and residues 1 and 2 for PfCyP19 inhibition. We propose that a malarial CyP-CsA complex presents residues 3 and 4 as part of an 'effector surface' for recognition by a downstream target, similar to the proposed mechanism for T-cell immunosuppression.

Dataset Information

Characterization of a conserved rhoptry-associated leucine zipper-like protein in the malaria parasite Plasmodium falciparum.

Publications

Characterization of a conserved rhoptry-associated leucine zipper-like protein in the malaria parasite Plasmodium falciparum.

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