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We report the proteomes of four life-cycle stages of the Apicomplexan parasite Eimeria tenella. A total of 1868 proteins were identified, with 630, 699, 845 and 1532 found in early oocysts (unsporulated), late oocysts (sporulated), sporozoites and second-generation merozoites, respectively. A multidimensional protein identification technology shotgun approach identified 812 sporozoites, 1528 merozoites and all of the oocyst proteins, whereas 2-D gel proteomics identified 230 sporozoites and 98 merozoite proteins. Comparing the invasive stages, we find moving junction components RON2 in both, whereas AMA-1 and RON4 are found only in merozoites and AMA-2 and RON5 are only found in sporozoites, suggesting stage-specific moving junction proteins. During early oocyst to sporozoite development, refractile body and most "glideosome" proteins are found throughout, whereas microneme and most rhoptry proteins are only found after sporulation. Quantitative analysis indicates glycolysis and gluconeogenesis are the most abundant metabolic groups detected in all stages. The mannitol cycle "off shoot" of glycolysis was not detected in merozoites but was well represented in the other stages. However, in merozoites we find more protein associated with oxidative phosphorylation, suggesting a metabolic shift mobilising greater energy production. We find a greater abundance of protein linked to transcription, protein synthesis and cell cycle in merozoites than in sporozoites, which may be residual protein from the preceding massive replication during schizogony.

Secretion of microneme proteins is essential to Plasmodium invasion but the molecular composition of these secretory organelles remains poorly defined. Here, we describe the first Plasmodium microneme proteome. Purification of micronemes by subcellular fractionation from cultured ookinetes was confirmed by enrichment of known micronemal proteins and electron microscopy. Quantitation of electron micrographs showed >14-fold microneme enrichment compared to the intact ookinete, such that micronemes comprised 85% of the identifiable organelles in the fraction. Gel LC-MS/MS of the most abundant protein constituents of the fraction identified three known micronemal proteins chitinase, CTRP, SOAP, together with protein disulphide isomerase (PDI) and HSP70. Highly sensitive MudPIT shotgun proteomics described a total of 345 proteins in the fraction. M1 aminopeptidase and PDI, the former a recognised target of drug development, were both shown to have a micronemal location by IFA. We further identified numerous proteins with established vesicle trafficking and signaling functions consistent with micronemes being part of a regulated secretory pathway. Previously uncharacterised proteins comprise the largest functional group of the microneme proteome and will include secreted proteins important to invasion.

The malaria parasite Plasmodium falciparum possesses four unique selenoproteins (PfSel1-PfSel4) which are likely to represent important components of the redox-regulatory network of this infectious agent. So far these proteins have only been characterized in silico. The aim of the present study was to gain further insight into the structural, biochemical, and functional properties of P. falciparum selenoproteins.Using (75)Se labeling in P. falciparum cell culture, the presence of selenoproteins in the parasite could be verified for the first time. Bioinformatic analyses indicated distant relatedness between the Plasmodium proteins and selenoproteins described in other organisms, namely between PfSel1 and SelK, PfSel2 and SelT, and between PfSel4 and SelS. For PfSel3 no remarkable similarities with proteins from other organisms were identified. All four proteins were recombinantly produced in Escherichia coli as UGA→UGU (selenocysteine→cysteine) mutants. Using green fluorescent protein (GFP)-fusion proteins and immunofluorescence, the subcellular localization of the four selenoprotein mutants was studied. PfSel1, PfSel2, and PfSel4 localized to the endoplasmic reticulum whereas PfSel3 was visualized in the nucleus and/or the apicoplast. Functional assays support the roles of PfSel1 and PfSel4 in cellular redox reactions. Transcriptional profiles of the four selenoproteins, and proteins involved in selenoprotein biosynthesis, indicate that their expression is regulated via the availability of selenium and via oxidative and nitrosative stress.In this study the presence of selenoproteins in Plasmodium has been proven for the first time; the subcellular localization of the proteins and their relatedness to known selenoproteins have been systematically studied, and recombinant proteins as well as information on regulation of transcript levels have been obtained.Taken together, our data enhance our understanding of the functional role of selenoproteins in Plasmodium.

SUMMARYHere we discuss proteomic analyses of whole cell preparations of the mosquito stages of malaria parasite development (i.e. gametocytes, microgamete, ookinete, oocyst and sporozoite) of Plasmodium berghei. We also include critiques of the proteomes of two cell fractions from the purified ookinete, namely the micronemes and cell surface. Whereas we summarise key biological interpretations of the data, we also try to identify key methodological constraints we have met, only some of which we were able to resolve. Recognising the need to translate the potential of current genome sequencing into functional understanding, we report our efforts to develop more powerful combinations of methods for the in silico prediction of protein function and location. We have applied this analysis to the proteome of the male gamete, a cell whose very simple structural organisation facilitated interpretation of data. Some of the in silico predictions made have now been supported by ongoing protein tagging and genetic knockout studies. We hope this discussion may assist future studies.

In eukaryotic cells, the ubiquitin-proteasome system as a key regulator of protein quality control is an excellent drug target. We therefore aimed to analyze the 26S proteasome complex in the malaria parasite Plasmodium falciparum, which still threatens almost half of the world’s population. First, we established an affinity purification protocol allowing for the isolation of functional 26S proteasome complexes from the parasite. Subunit composition of the proteasome and component stoichiometry were studied and physiologic interacting partners were identified via in situ protein crosslinking. Furthermore, intrinsic ubiquitin receptors of the plasmodial proteasome were determined and their roles in proteasomal substrate recognition were analyzed. Notably, PfUSP14 was characterized as a proteasome-associated deubiquitinase resulting in the concept that targeting proteasomal deubiquitinating activity in P. falciparum may represent a promising antimalarial strategy. The data provide insights into a profound network orchestrated by the plasmodial proteasome and identified novel drug target candidates in the ubiquitin-proteasome system.

Thrombomodulin (TM) forms a 1:1 complex with thrombin. Whereas thrombin alone cleaves fibrinogen to make the fibrin clot, the thrombin-TM complex cleaves protein C to initiate the anticoagulant pathway. The fourth and fifth EGF-like domains of TM together form the minimal fragment with anticoagulant cofactor activity. A short linker connects the fourth and fifth EGF-like domains of TM, and Met 388 in the middle of the linker interacts with both domains. Several different structures of TMEGF45 variants are now available, and these show that mutation of Met 388 alters the structure of the fifth domain, as well as the connectivity of the two domains. To probe this phenomenon more thoroughly, NMR backbone dynamics experiments have been carried out on the individual fourth and fifth domains as well as on the wild type, the Met 388 Leu mutant, and the variant in which Met 388 is oxidized. The results presented here show that changes at Met 388 cause significant changes in backbone dynamics in both the fourth and fifth EGF-like domains of TM. Backbone dynamics within the small loop of the fourth domain Tyr 358 correlate with anticoagulant cofactor activity. Backbone dynamics of the thrombin-binding residues Tyr 413 and Ile 414 are inversely correlated with thrombin binding. The preordering of the backbone of Tyr 413 and Ile 414 only occurs in the two-domain fragments, revealing a role for the fourth domain in thrombin binding as well as in anticoagulant cofactor activity.

Thrombomodulin (TM) is an endothelial cell surface glycoprotein that is responsible for switching the catalytic activity of thrombin away from fibrinogen cleavage (pro-coagulant) and towards protein C cleavage (anticoagulant). Although TM is a large protein, only the fourth and fifth epidermal growth factor-like (EGF-like) domains are required for anticoagulant function. These two domains must work together, and the linker between the two domains contains a single methionine residue, Met 388. Oxidation of Met 388 is deleterious for TM activity. Structural studies, both X-ray and NMR, of wild type and variants at position 388 show that Met 388 provides a key linkage between the two domains. Oxidation of the methionine has consequences for the structure of the fifth domain, which binds to thrombin. Oxidation also appears to disrupt the interdomain contacts resulting in structural and dynamic changes. The functional consequences of oxidation of Met 388 include decreased anticoagulant activity. Oxidative stress from several causes is reflected in lower serum levels of activated protein C and a higher thrombotic tendency, and this is thought to be linked to the oxidation of Met 388 in TM. Thus, TM structure and function are altered in a subtle but functionally critical way upon oxidation of Met 388.

Oxidation of Met 388, one of the three linker residues connecting the fourth and fifth EGF- like domains of thrombomodulin (TM), is deleterious for TM activity. An NMR structure of the smallest active fragment of TM (TMEGF45) and a crystal structure of a larger fragment (TMEGF456) bound to thrombin both show that Met 388 is packed into the fifth domain. Using multidimensional NMR, we have solved the structure of TMEGF45 in which Met 388 is oxidized (TMEGF45ox) and the structure of TMEGF45 in which Met 388 is mutated to Leu (TMEGF45ML). Comparison of the structures shows that the fifth domain has a somewhat different structure depending on the residue at position 388, and several of the thrombin-binding residues are packed into the fifth domain in the oxidized protein while they are exposed and free to interact with thrombin in the native structure and the Met-Leu mutant. This observation is consistent with kinetic measurements showing that the Km for TMEGF45ox binding to thrombin is 3.3-fold higher than for the native protein. Most importantly, the connection between the two domains, as indicated by interdomain NOEs, appears to be essential for activity. In the TMEGF45ox structure which has a reduced kcat for protein C activation by the thrombin-TMEGF45ox complex, interaction between the two domains is lost. Conversely, a tighter connection is observed between the two domains in TMEGF45ML, which has a higher kcat for protein C activation by the thrombin- TMEGF45ML complex. ‡ These two authors contributed equally. 1 Abbreviations: thrombomodulin, TM; epidermal growth factor, EGF; trifluoroacetic acid, TFA; nuclear magnetic resonance, NMR; N-acetylglucosamine, GlcNac; TMEGF45, the fragment of thrombo- modulin containing the fourth and fifth EGF-like domains; TMEGF45ox, the fragment of thrombomodulin containing the fourth and fifth EGF- like domains and Met388 oxidized; TMEGF45ML, the fragment of thrombomodulin containing the fourth and fifth EGF-like domains but with Met 388 substituted for Leu; heteronuclear nuclear Overhauser effects, hNOEs.

Here, we establish a methodology for large-scale quantitative proteomics using SIL (stable isotope labeling) to examine protein expression changes in trophozoite stages of the malaria parasite Plasmodium falciparum following drug treatment. For this purpose, exposure to (13)C6 (15)N1-isoleucine was optimized in order to obtain 99% atomic enrichment. Proteome fractionation with anion exchange chromatography was used to reduce sample complexity and increase quantitative coverage of protein expression. Tryptic peptides of subfractions were subjected to SCX/RP separation, measured by LC-MS/MS, and quantified using the software tool Census. In drug-treated parasites, we identified a total number of 1,253 proteins, thus increasing the overall number of proteins so far identified in the trophozoite stage by 30% in the previous literature. A relative quantification was obtained for more than 800 proteins. About 5% of proteins showed a clear up- or downregulation upon drug treatment.

Aims: Due to its life in different hosts and environments, the human malaria parasite Plasmodium falciparum is exposed to oxidative and nitrosative challenges. Nitric oxide (NO) and NO-derived reactive nitrogen species can constitute nitrosative stress and play a major role in NO-related signaling. However, the mode of action of NO and its targets in P. falciparum have hardly been characterized. Protein S-nitrosylation (SNO), a posttranslational modification of protein cysteine thiols, has emerged as a principal mechanism by which NO exerts diverse biological effects. Despite its potential importance, SNO has hardly been studied in human malaria parasites. Using a biotin-switch approach coupled to mass spectrometry, we systemically studied SNO in P. falciparum cell extracts. Results: We identified 319 potential targets of SNO that are widely distributed throughout various cellular pathways. Glycolysis in the parasite was found to be a major target, with glyceraldehyde-3-phosphate dehydrogenase being strongly inhibited by S-nitrosylation of its active site cysteine. Furthermore, we show that P. falciparum thioredoxin 1 (PfTrx1) can be S-nitrosylated at its nonactive site cysteine (Cys43). Mechanistic studies indicate that PfTrx1 possesses both denitrosylating and transnitrosylating activities mediated by its active site cysteines and Cys43, respectively. Innovation: This work provides first insights into the S-nitrosoproteome of P. falciparum and suggests that the malaria parasite employs the thioredoxin system to deal with nitrosative challenges. Conclusion: Our results indicate that SNO may influence a variety of metabolic processes in P. falciparum and contribute to our understanding of NO-related signaling processes and cytotoxicity in the parasites. Antioxid. Redox Signal. 20, 2923–2935.

Plasmodium falciparum is the cause of human malaria and is one of two malaria parasites known to have drug resistance. Since there are no preventative vaccinations against malaria, the control of this disease is heavily dependent upon the use of antimalarial drugs. Antimalarial drugs, such as methylene blue, are effective therapies against human malaria. At a specific concentration, methylene blue has been shown to be a selective inhibitor of the parasite's glutathione reductase (PfGR).Glutathione reductase is an important target when studying malaria drug resistance because it is a flavoenzyme that regenerates glutathione, which is an essential protein for antioxidant defense against cell damage. Methylene blue is also a substrate that is reduced by gluthathione reductase to produce leucoMB. This is then spontaneously oxidized by molecular oxygen to form methylene blue again. During this process, reactive oxygen species, such as hydrogen peroxide and superoxide form. These act as recycling catalysts against infectious organisms. Due to PfGR's central position in redox control, it is ranked number one as an antimalarial drug target. The goal of this research is to study the interface between methylene blue and the putative protein target, glutathione reductase, in order to understand the drug action mechanism. PfGR was expressed and purified and hydrogen-deuterium exchange (HDX) will be used to map the drug-protein interface. We will present the mass spectrometry data for the solvent exposed peptides after digestion with pepsin when PfGR is complexed with methylene blue, which will allow us to narrow down the active site.

Coevolution of the malarial parasite and its human host has resulted in a complex network of interactions contributing to the homeodynamics of the host-parasite unit. As a rapidly growing and multiplying organism, Plasmodium falciparum depends on an adequate antioxidant defense system that is efficient despite the absence of genuine catalase and glutathione peroxidase. Using different experimental approaches, we demonstrate that P. falciparum imports the human redox-active protein peroxiredoxin 2 (hPrx-2, hTPx1) into its cytosol. As shown by confocal microscopy and immunogold electron microscopy, hPrx-2 is also present in the Maurer's clefts, organelles that are described as being involved in parasite protein export. Enzyme kinetic analyses prove that hPrx-2 accepts Plasmodium cytosolic thioredoxin 1 as a reducing substrate. hPrx-2 accounts for roughly 50% of thioredoxin peroxidase activity in parasite extracts, thus indicating a functional role of hPrx-2 as an enzymatic scavenger of peroxides in the parasite. Under chloroquine treatment, a drug promoting oxidative stress, the abundance of hPrx-2 in the parasite increases significantly. P. falciparum has adapted to adopt the hPrx-2, thereby using the host protein for its own purposes.

Tropical malaria caused by the protozoan Plasmodium falciparum is responsible for more than 200 million clinical episodes and at least 660,000 human deaths annually. Parasite resistance increases continuously against the presently available drugs. To further understand the biochemical pathways involved in the mechanisms of drug resistance, a quantitative proteomic approach has been developed using stable isotope labeling by amino acids in cell culture (SILAC). In contrast to previous studies with higher drug concentrations, three different sub-lethal dosage amounts of chloroquine were used to treat parasite cultures. The protein abundance was quantified and a list of proteins and their related pathways was collected. Programmed Cell Death (Apoptosis) is one of the control mechanisms used by higher multicellular animals that are crucial for the continuing development of the surviving cells. Surprisingly the causative agent of malaria, a protozoan parasite, shows signs of this programmed “suicide”. DNA fragmentation assays and morphological characterization have been carried out under similar sub-lethal drug concentrations conditions as the proteome samples pointing to an apoptotic phenotype. The proteomic data point to some of the players that might be involved. An up-regulation of cytochrome C suggests it is a protein involved in the apoptosis pathway. We hypothesize that cytochrome C is involved in a pathway similar to the one found in another unicellular organism, yeast, where after binding a target protein a proteolytic cascade is turned on. In order to test this working hypothesis an assay was developed to confirm the involvement of cytochrome C in proteolysis of downstream targets using a fluorogenic substrate.

A proteomics analysis identifies one third of the predicted Toxoplasma gondii proteins and integrates proteomics and genomics data to refine genome annotation., Background Although the genomes of many of the most important human and animal pathogens have now been sequenced, our understanding of the actual proteins expressed by these genomes and how well they predict protein sequence and expression is still deficient. We have used three complementary approaches (two-dimensional electrophoresis, gel-liquid chromatography linked tandem mass spectrometry and MudPIT) to analyze the proteome of Toxoplasma gondii, a parasite of medical and veterinary significance, and have developed a public repository for these data within ToxoDB, making for the first time proteomics data an integral part of this key genome resource. Results The draft genome for Toxoplasma predicts around 8,000 genes with varying degrees of confidence. Our data demonstrate how proteomics can inform these predictions and help discover new genes. We have identified nearly one-third (2,252) of all the predicted proteins, with 2,477 intron-spanning peptides providing supporting evidence for correct splice site annotation. Functional predictions for each protein and key pathways were determined from the proteome. Importantly, we show evidence for many proteins that match alternative gene models, or previously unpredicted genes. For example, approximately 15% of peptides matched more convincingly to alternative gene models. We also compared our data with existing transcriptional data in which we highlight apparent discrepancies between gene transcription and protein expression. Conclusion Our data demonstrate the importance of protein data in expression profiling experiments and highlight the necessity of integrating proteomic with genomic data so that iterative refinements of both annotation and expression models are possible.