Insights into the Plasmodium falciparum schizont phospho-proteome

Microbes and Infection 14 (2012) 811e819 www.elsevier.com/locate/micinf

Insights into the Plasmodium falciparum schizont phospho-proteome Edwin Lasonder a,*,1, Moritz Treeck b,*,1, Mahmood Alam c, Andrew B. Tobin c,* a

Centre for Molecular and Biomolecular Informatics, Radboud University Nijmegen Medical Centre, Geert Grooteplein 28, 6525 GA Nijmegen, The Netherlands b Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA 94305, USA c MRC Toxicology Unit, Lancaster Road, Leicester LE1 9HN, UK Received 16 February 2012; accepted 12 April 2012 Available online 5 May 2012

Abstract It is becoming clear that, as is the case with many human diseases, targeting protein phosphorylation in strategies aimed at developing the next generation of anti-malarials is likely to bear considerable fruit. A major barrier to this development, however, is the paucity of information regarding the role of protein phosphorylation in malaria. A major step has recently been taken in this area with the publication of the first analyses of the phospho-proteome of the most virulent species of human malaria Plasmodium falciparum. Here, we discuss these studies. Ó 2012 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved. Keywords: Plasmodium falciparum; Schizont; Phospho-proteome; Protein kinase; Signalling; Toxoplasma gondii

1. Introduction The optimism engendered by the undoubted success of artemisinin based combination therapies (ACTs) for the treatment for malaria [1,2] has been tempered not only by recent findings that malaria is still responsible for more than 1.2 million deaths a year [3] but also by evidence of the emergence of resistance to ACTs [1,2]. These facts underline the importance of the continued need for a pipeline of novel anti-malaria drug treatments and the need to identify essential processes that are unique to the parasite, where pharmacological targeting would provide a mechanism of killing the parasite without affecting the human host. One such process might be protein phosphorylation, which has proven a successful target in the treatment of at least one human cancer [4] and is considered to have therapeutic potential in a number of human diseases [5,6], but has yet to be fully explored with regard to malaria. That Plasmodium protein kinases might be potential antimalarial targets was first indicated in a seminal study by

* Corresponding authors. E-mail address: [email protected] (E. Lasonder). 1 The authors contributed equally.

Doerig and colleagues who performed an in silico genomic analysis that identified 84 protein kinases in the genome of Plasmodium falciparum [7]. Subsequent studies have supported this early analysis [8,9] and together have established that 85e100 protein kinases constitute the P. falciparum kinome. Considering that the parasite genome consists of nearly 5500 genes its kinome represents 1.5e1.8% of the predicted genome, which is comparable to that of the human kinome which constitutes w2% of the human genome [9,10]. Furthermore, the parasite kinases can largely be assigned to one of the seven established protein kinase groups including the familiar AGC, CMGC and CK1 groupings [7]. Thus, as is the case for the human kinome, the P. falciparum kinome appears to be well adapted to mediating diverse phosphorylation events on a large complement of protein substrates and therefore likely to play a complex and multi-functional role in the biology of the parasite. Despite this broad similarity with the mammalian protein kinases the Plasmodium kinome otherwise shows significant divergence from its mammalian counter part. For example, the parasite kinome contains no recognizable tyrosine kinases, nor MAP-kinase-kinases (STEs) [7e9]. Conversely, the Plasmodium kinome includes kinase families that are absent from mammalian kinomes such as the CamK family, that are more closely related to calcium-sensitive plant kinases [7e9], and

1286-4579/$ - see front matter Ó 2012 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved. doi:10.1016/j.micinf.2012.04.008

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the FIKK family [11]. The significant phylogenetic distance between the malaria and mammalian kinomes is further exemplified by the fact that many of the parasite kinases have no mammalian orthologues. Where mammalian orthologues could be identified, as for example the parasite kinase PfCLK3 that is related to the mammalian pre-mRNA processing kinase hPRP4 [7,9], there are often specific differences in the kinase catalytic domain that indicate the parasite kinase possess different substrate recognition and binding sites together with mechanisms of phosphorylation from that of the mammalian orthologue [9]. It is also common to identify low complexity extensions and insertions in the catalytic domain of parasite kinases that are not found in the orthologous mammalian counter parts. Further divergence is evident in some parasite kinases that uniquely show features of more than one kinase group. For example, PfNEK-1 is a parasite kinase that shows similarities to both the mammalian NIMA kinases and similarities to MAP-kinase-kinases [7,9]. Thus, there appears to be significant divergence between the malaria and mammalian kinomes which has lead to the suggestion that it may be possible to develop protein kinase inhibitors that can be selective for the malarial kinome [12]. That this might be an affective strategy to adopt for the development of anti-malarial therapies has recently been suggested by the identification of 36 P. falciparum protein kinases that are essential for the survival of the parasite at the erythrocytic stage [12]. In a related study on the mouse malaria parasite Plasmodium berghei it was seen that many of the orthologous kinases identified as likely essential in P. falciparum were also essential in the erythrocytic stage of P. berghei [12,13]. These studies would suggest that there is very little redundancy within the parasite kinome and that there exist a large number of potential parasite kinase targets, which, due to phylogenetic divergence from the mammalian kinases, might be selectively inhibited by novel pharmacological entities. Pursuing these targets has however been hampered by the general paucity of information regarding the role of protein phosphorylation in malaria. Whereas a number of individual protein kinases have been examined for example the role of; PfPKG in egress and gametogenesis [14,15], PfCDPK5 in egress [14], the Aurora-related kinases in spindle pole development [16], the NIMA-related kinases in sexual development [17,18], PfPKB in invasion [19,20] and PfPK7 in schizogony [21], PfCDPK4 [22] in gametocytogenesis and PfPKA in anion-channel regulation [23] and in host-cell invasion [24] e a comprehensive assessment of the role played by phosphorylation in the regulation of malaria parasite biology is only slowly emerging. However, the field of protein phosphorylation in malaria has recently been transformed by the application of mass-spectrometry based phospho-proteomic techniques that have allowed for the identification of large portions of the phospho-proteome in P. falciparum. Two such studies have recently been published from the author’s laboratories [12,24] and a third is shortly to be released that reveals thousands of phosphorylation sites on hundreds of parasite proteins in the erythrocytic stage of the P. falciparum. It is

clear from these studies that protein phosphorylation plays a diverse role in the regulation of cell processes in the parasite. These global, systems based studies, have allowed us to interrogate the expansive nature of the phospho-proteome in malaria and begin to dissect the role of phosphorylation in the regulation of key parasite processes. Here, we will discuss these global phospho-proteomic studies, describing something of the methodological and analytical approaches used and the major findings from these studies, and the implications of these findings with regard to the regulation of parasite biology and the potential of targeting phosphorylation in anti-malaria therapy. 2. Overview of the P. falciparum schizont phosphoproteomes The three phospho-proteomic datasets reported to date have used very different methods of sample preparation and filtering to assign the phosphorylation sites, which has led to some inconsistency in the reporting of the phosphorylation sites. Thus, Treeck reported 8463 sites in total [25] where as Lasonder reported 2541 [26] and Solyakov 1171 [12]. All the datasets reported have been filtered with high stringency to a False Discovery Rate (FDR) <1%. However, Solyakov et al. further increased the stringency of their data until it reached an FDR of zero, in addition, all spectra that passed these criteria were manually inspected and finally only those sites that were observed in at least two of the six independent biological replicates were reported in the manuscript [12]. This results in the highest confidence that the sites reported actually exist. Lasonder’s dataset contains all sites that have a localization probability of >75%, and that the dataset of Treeck contains 5038 high confident sites (>99% correct localization probability) out of 8463 total reported sites [25], which includes all sites that could not be localized. The implications of these different filtering criteria are further discussed below. The overall distribution of phosphorylated residues (with at least 75% site localization) is 87.5% for serine residues and 12.2% for threonine residues and reflects the general trend from other organisms. Interestingly, tyrosine-phosphorylated residues were identified in all studies in the absence of tyrosine kinases; however, highest confidence sites were identified predominantly in proteins with kinase activity, likely resulting from autophosphorylation events [12] and only few non-kinase proteins with good spectral evidence for correct site localization [25]. Lasonder et al. reports 1.2% phosphotyrosines, but these were not manually inspected and include localization probability scores of 75e100%. The analysis of tyrosine phosphorylation is further discussed in the Treeck et al. highlight section. 3. Experimental challenge of evaluating the P. falciparum phospho-proteomes Broadly, the P. falciparum phospho-proteomes investigated have followed similar protocols. All have taken advantage of

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the fact that the schizont stage of infected red blood cells is readily isolated in quantities that allows for mass-spectrometry based phosphoproteomics [12,25]. Thus, they all focus on this stage in the life cycle although, importantly, the Solyakov study centred on later stage schizonts than that of the Treeck study, a fact that might explain some of the reported differences between the phosphopeptides in these two studies. All three phospho-proteomic studies employed methods that enriched for phosphopeptides contained in tryptic digests of lysates from the schizont stage parasites. Despite these overall similarities there are sufficient differences in the protocols employed by the three laboratories that most likely account for the observed variation in detected phosphorylation sites. These differences not only include the subtle stage of the parasite and enrichment protocols, but also the liquid chromatography upstream of the mass spectrometer, the settings of the mass spectrometers, and the instruments themselves. For example, within an LCeMS/MS run, where peptides are loaded on a reverse phase column and eluted by an acetonitrile gradient in 1e2 h and electrosprayed into the mass spectrometer, the number of peptides that can be measured in a single MS scan is much higher than the number of peptides that can be fragmented by separate MSMS scans within the elution time window. Only the peptides that are fragmented contain the sequence information that is required for identification by database search engine such as Mascot and SEQUEST, where the fragmentation data is matched with theoretical MSMS spectra computed from the protein database composed of P. falciparum and human proteins. This general characteristic of LCeMS/MS affect the reproducibility of low abundant peptides (such as phosphopeptides), and thus multiple measurements usually help to increase the coverage. To overcome the sample complexity limitation properties in LCeMS/MS studies, in the last 5e10 years a wide variety of fractionation strategies and phosphopeptide enrichment procedures have been developed to measure in vivo phosphorylation sites in eukaryotic cells [27e31]. These methods usually comprise of a sample fractionation step prior to phosphopeptide enrichment using Fe3þ-based IMAC or TiO2 beads, where in both cases phosphate groups bind to immobilized metal atoms. The enriched phosphopeptides are subsequently measured by LCeMS/MS with state-of-the-art mass spectrometers. It was difficult to predict beforehand which method would be most suitable for the analysis of isolated P. falciparum proteins, because these methods are still being optimized further and studies that compare the performance of these methods show only 35% overlap of reproducible sites between IMAC and TiO2 [32]. It is also clear that sample amount is a key factor, which ultimately controls the number of phosphorylation sites that can be detected in cellular samples. Milligrams of total protein isolated from eukaryotic cells are typically required as starting material for large-scale phospho-proteome studies that are capable of identifying several thousands of phosphorylation sites [33,34]. The three phospho-proteome studies conducted so far have applied the common strategy [28] to first fractionate the schizont samples before and/or after digestion with trypsin,

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and then to purify phosphorylated peptides sample with IMAC (Treeck, Solyakov) or TiO2 (Solyakov, Lasonder). The proteomic pipelines for these studies are summarized in Table 1. The phospho-proteome studies applied a diverse repertoire of fractionation techniques (see Table 1) on the protein level (SDS PAGE, HILIC) and peptide level (SCX, SAX), that were applied successfully previously in large-scale phosphopeptide identification studies [31]. The LCeMS/MS measurements were performed using similar nano liquid reverse phase chromatography systems coupled to either the Orbitrap Velos mass spectrometer (Treeck, Solyakov), or the FTICR mass spectrometer. The data processing and peptide validation relied on different analysis software packages, with platform dependent peptide scoring algorithms. Despite these differences, the quality of the identifications can be evaluated by inspection of peptide score distributions (Fig. 1). All three datasets show uniform Gaussian-like score distributions with a minimum score threshold at the left tail of the score distribution. The Gaussian score distributions show that the validation procedures successfully eliminated false positive peptide identifications; otherwise the score distributions would deviate from a mono phasic Gaussian-like distribution for peptides with low scores. The frequency of these peptides would increase and would disturb the Gaussian distribution. In all the three studies this was prevented by applying correct peptide score cutoffs and data filtering procedures, either by manual verification of MSMS spectra, or by automated False Discovery Rate filtering. Without any validation the score distribution plots would show a biphasic curve, as a resultant of the true positive and false positive peptide score distributions [35,36]. The datasets share 450 phosphorylation sites that show slightly higher relative average peptide scores than the relative average peptide scores of the full datasets (Fig. 1), indicative for identification with higher confidence of the shared sites than the other phosphorylation sites. This analysis further indicates that the dataset of Solyakov is of the highest confidence, because the average peptide score of the full dataset is close to that of the 450 sites detected by all three studies. 4. Data filtering On top of the biological and technical differences between samples, alternative data filtering also accounts for some important dichotomies between the datasets and it is important to understand the differences when planning downstream experiments. There are two broad audiences for large-scale phosphoproteome datasets. One group consists of researchers interested in how complex pathways within an organism are regulated by phosphorylation. The second are those scientists interested in a certain phosphorylation event on protein or a group of proteins for which they have a particular interest. It is imperative for both of these groups that the data that is used to assign the phosphorylation sites is of the highest quality. The question, however, arises as to the criteria set for the publication of a phosphorylation site. Currently no general

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Table 1 Summary of three phospho-proteomic pipelines. A schematic overview of the phospho-proteomic workflows of the three studies that investigated in vivo protein phosphorylation in asexual blood stage development.

Sample

Life cycle stage Synchronization Sample isolation

Solyakov

Treeck

Lasonder

Late schizonts 3D7A 36e40 h after sorbitol treatment MACS purification

Late schizonts 3D7 40 h after 2nd sorbitol treatment SuperMACSII purification >6 mg

Early schizonts 3D7 4 h after sorbitol treatment

Amount Fractionation

LCeMS/MS

Data processing

Protein fractionation

Pellet & soluble fraction

1 fraction after lysis in 8 M urea

Peptide fractionation

HILIC separation soluble fraction IMAC followed by TiO2

SCX

Symmetry C18 100 A beads 25 cm 75 mm column LTQ Velos Orbitrap

MagicC18AQ beads 15 cm 100 mm column 7e25% acetonitrile (60 min) LTQ Velos Orbitrap SEQUEST 15 ppm peptide probability >95% ASCORE Phosphopeptide FDR <1% Protein FDR <3%

Phosphopeptide enrichment LC beads LC column Gradient Mass spectrometer Database search engine Peptide mass accuracy Analysis software Validation criteria

Site localization

Mascot þ Xtandem 5 ppm Scaffold 1. Peptide probability >95% 2A. Mascot score > 31, or 2B. Xtandem score > 2.5 3. Manual verification Manual verification

IMAC

Ascore > 19

Percoll sedimentation 8  108 iRBCs þ 1  108 RBCs (w2 mg) A. Pellet & soluble fraction B. 10% SDS PAGE of 50% sample A C. Haemoglobin depletion 50% soluble fraction FASP and SAX: 50% from pellet and from C TiO2 3 mm Reprosil C18 beads 15 cm 100 mm column 7e28% acetonitrile (90e120 min) LTQ-FT ICR MS Mascot 10 ppm MaxQuant Phosphopeptide FDR ¼ 1%

PTM localization prob > 0.75

Abbreviations: False Discovery Rate (FDR); Filter assisted sample preparation (FASP); Hydrophilic Interaction Chromatography (HILIC); Immobilized Metal Affinity Chromatography (IMAC); Liquid Chromatography (LC); Magnetic Cell Sorting (MACS); parts per million (ppm); Post Translational Modification (PTM); Red Blood Cell (RBC); Strong Anion eXchange (SAX); Strong Cation eXchange (SCX).

Fig. 1. Comparison of score distributions across the phospho-proteomes. A. Peptide score distribution of the three phosphorylation datasets, showing the relative peptide score counts with bin sizes of 5 peptide score units, respectively in green (Treeck), red (Lasonder), and blue (Solyakov). Relative scores were obtained by normalization, where the average scores of the individual datasets were set to 1. The average absolute scores were 102.3 (Treeck), 64.0 (Solyakov), and 39.6 (Lasonder) respectively. Different scales in absolute peptide scores were the result of two type of mass spectrometers and scoring algorithms. B. Distribution of relative average peptide scores for the individual datasets from A and for the sub set of 450 phosphorylation sites that were detected by all datasets. Here, the average peptide score of the shared set of 450 sites was set at 1 (grey bar) and compared with the relative peptide scores of the individual dataset, respectively Solyakov (green bar), Lasonder (blue bar) and Treeck (green bar). Absolute average peptide scores of the individual and shared datasets are provided below the Xaxis. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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guidelines for how phosphorylation site data is best represented, although initiatives for standardized publication criteria for classical proteomic datasets have been developed by the human proteome organization (HUPO) and by proteomic journals, e.g. MCP. The lack of such guidelines makes the comparison between datasets complicated. In most cases two types of scores are attached to a phosphorylation site. The peptide identification score (e.g. MASCOT probability score, SEQUEST Xcorr peptide score, a Scaffold peptide score, a peptide posterior error probability (PEP)) is a measure of the confidence of the phosphopeptide identification. This score varies by search algorithm and if special software has been used to consolidate a number of parameters to score a single peptide. Often this score is used to filter the datasets to the desired False Discovery Rate (FDR). A peptide FDR of 1% means that 1 out of 100 peptides within a dataset is a false positive. The second score is the localization confidence score (for example the ASCORE-score [30] and the PTM localization probability score [37]), which measures the correct localization of the phosphorylation site within a peptide. A phosphopeptide with a very good peptide identification score has a high probability of being a true positive, but might still contain several phosphorylatable residues. If the program (or manual inspection) is not able to distinguish between these potential sites based on the spectrum analysed, the localization confidence score will be low, indicating the ambiguity. When filtering large-scale phosphoproteomics datasets most researchers aim for peptide FDR 1e5% and a protein FDR between 1 and 5%. When choosing the FDR, there is a tradeoff between specificity and sensitivity. A very low FDR (w0%) excludes more incorrect peptides from the dataset than an FDR of 1% at the expense of also excluding many correct hits. Whereas for large-scale analysis a higher FDR is often acceptable, for in vivo-follow up experiments the FDR should be set as low as possible without losing too many true positives. This tradeoff between sensitivity and specificity is illustrated by the way the datasets discussed in this study were analysed. Whereas Treeck and Lasonder wanted to include most correct peptides and accept the presence of a small percentage (<1%) of false positives in their dataset, Solyakov et al. decided for the most stringent of all possibilities by including only manually validated phosphorylation sites of all identified spectra (>2500) that passed a certain statistical threshold [12]. However, by using statistical filtering coupled to manual inspection of the spectra resulted in the exclusion of phosphopeptides, where the phosphosite could not be localized. This inevitably means that some valuable information was lost. For example: the apical membrane protein 1 (AMA1) of P. falciparum was found phosphorylated [38] at multiple residues, and phosphorylation is essential for AMA1 function [24]. In a previous study one phosphorylation site could be identified (S610) by in vitro kinase assays, mutagenesis and 2D-chromatography, but the other phosphorylation sites could not be localized. In the mass-spectrometry datasets, one phosphopeptide that contains a phosphorylation site at S588 or S590 was identified, but not unambiguously localized. This

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phosphopeptide (which was included as an exception in the Solyakov dataset, because of the information given above) would not have been included in datasets where ambiguous sites were to be omitted, but now provide very important evidence for identifying the second phosphorylation site. Until a common way is established to present and filter phospho-proteome data from large-scale studies, the researcher interested in the sites can not blindly take all presented sites for granted as true hits and it is important to understand the quality of the dataset presented. As with largescale datasets in general that are filtered mainly by statistical measures, further assessment should be performed, however, this is not a trivial task, as ideally multiple parameters should be used. The best way is looking at the primary scores attached to each peptide that is given by the database search program used (e.g. MASCOT, SEQUEST, Scaffold), however this requires knowledge about how these scores are interpreted. An additional measure is to look in the published datasets and see how many times a peptide has been identified (¼spectral counts). In general, peptides with more identifications are more likely to be correct. However, very low abundance phosphopeptides might be identified only once and for such peptides looking at the associated primary score is the only way of gaining confidence in the hit. Also looking for a specific phosphorylation site in Toxoplasma gondii, or other orthologous proteins might give further evidence about the presence of this phosphorylation site. For example, the tyrosine phosphorylation sites reported for GSK-3 or DYRK kinase are highly conserved throughout widely different species [39]. Finally, the authors of the herein discussed manuscripts are able to help interpretation of their massspectrometry data for further downstream analysis. 5. Highlighted biological findings 5.1. Treeck et al.: comparative analysis of the two related apicomplexan parasites P. falciparum and T. gondii The comparison of these two related parasites allowed one to build confidence in some unexpected findings. First of all, the presence of w2% phosphorylated tyrosines in P. falciparum and T. gondii datasets was surprising due to the absence of classical tyrosine kinases in both parasites. Close inspection of the datasets revealed a substantially higher False Discovery Rate for phosphotyrosine, and to a lesser extent for phosphothreonine, than for phosphoserine. Filtering the whole dataset on a site-specific level (that is calculating the FDR for all pY, pT and pY individually), they found tyrosine phosphorylated in at most 0.25%e0.5% of the phospho-proteome of either parasite. Importantly, when applying the filtering method to the host phospho-proteome that was also measured but excluded from the datasets, a substantially larger phosphotyrosine fraction was identified showing true biological differences between the parasites and the host. The reason for this unequal distribution of false positives is very likely an equal distribution of false positive hits according to the

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frequency of each amino acid in the proteome. Manual inspection of a subset of the remaining phosphotyrosines showed that most of the spectra with good evidence for tyrosine-phosphorylation derived from proteins with kinase domains, many of which have been shown to be able to autophosphorylate (see also Solyakov highlights). Whether the remainder phosphotyrosines are important signalling nodes, result from promiscuous kinase activity, or belong to false positives remains to be answered. To engage in more broad analysis of the phospho-proteome, Treeck et al. also analysed the non-phosphorylated proteome and used this data to identify phosphorylation motifs and the distribution of phosphorylated proteins in the total proteome. For the phosphorylation motif-analysis their data was compared to a mouse phospho-proteome and proteome datasets that were generated using the same methodology. This provided evidence that P. falciparum contains unusual phosphorylation motifs, presumably driven by the A:T rich genome and the resulting unusual amino acid composition of its proteome. Despite very high conservation of phosphorylation motifs overall and the evolutionary forces acting on their conservation, the pressure to maintain an A:T rich genome seems stronger. This results in a very low usage of proline directed phosphorylation motifs and the evolution of a high number w25% of novel phosphorylation motifs, some of which are made of amino acids that are depleted in human kinase phosphorylation motifs. This might relate to a change in the structure in the substrate-binding region of such kinases that can potentially be exploited to design highly P. falciparum specific kinase inhibitors. Comparative analyses of functional proteins groups showed that in both parasites the trends are the same, with most proteins being phosphoproteins that are intracellular and few that are on the surface of the parasite. Analysis of the kinome identified kinases to be overrepresented in the phosphoproteins group and among the kinases identified were highly conserved tyrosine phosphorylation motifs that are crucial for kinase activity. Interestingly, a substantial fraction of proteins that are exported/secreted from the parasite into the parasitophorous vacuole and/or the host-cell cytoplasm have been identified, which suggests regulation of secreted parasite and/or host proteins to the parasite’s advantage [40,41]. Using a differential phospho-proteome analysis, where Toxoplasma parasites were purified and all material from the host as well as parasite proteins that have been injected/exported into the host cell have been removed identified that indeed, a number of parasite proteins are only phosphorylated after being transferred into the host cell. The presence of a number of kinases known, or suspected to be transferred into the host cell is highly suggestive of parasite kinases being a major force in regulating the secretome. The presence of exported P. falciparum kinases (FIKK kinases) [7,11,42] strongly suggests a similar mechanism in P. falciparum although experimental evidence is still missing, but the potential to use knockout lines to analyse phosphorylation caused by these kinases seems within reach.

Despite the global analysis of the phospho-proteome data, a direct comparison of the phosphorylation sites between the two parasites (P. falciparum and T. gondii) also helps growing confidence in phosphorylation sites and phosphorylated residues. Many phosphorylated residues were identified that are found in both parasites within homologues regions of orthologous proteins. For example, in several rhomboid proteases exclusively N-terminal phosphorylation sites have been identified in P. falciparum and the T. gondii orthologues. This could indicate a regulatory mechanism of these proteases yet unknown in other organisms. 5.2. Lasonder et al.: identification of signalling pathways in P. falciparum The objective of the study of Lasonder et al. was to identify prominent signalling pathways that are active in asexual blood stage development, and to examine the Plasmodium biology associated with the phospho-proteome data of early schizonts. Understanding the signalling pathways that are involved in the regulation mechanisms controlling P. falciparum development is a major challenge in malaria research. This cannot be achieved by bioinformatics predictions using kinome and phospho-proteome data from other species, since the majority of the protein kinase sequences from Plasmodium parasites are divergent from their mammalian counterparts. However, this drawback has the advantage that in principle it enables selective inhibition of Plasmodium signalling pathways by small molecules, which underscores the need to understand how these pathways function in blood stage development. At first, the generated phospho-proteome data was analysed by computational approaches using generic and dedicated tools to analyse phospho-proteome data. This included functional enrichment analyses relative to the full P. falciparum proteome (5500 proteins) with Gene Ontology and protein domain data, phospho-motif analysis within a 13 amino acid window surrounding the phosphorylation site by the MotifX tool [43], phospho interactome network clustering by the molecular complex detection (MCODE) algorithm [44] and signal pathways analysis. Taken together, these bioinformatic analyses revealed that protein phosphorylation in P. falciparum is widespread, like in other species, and influences biological processes ranging from basic metabolic processes, to regulatory processes involved in cell communication, cell cycle, cell proliferation, and parasite-specific processes such as trafficking, locomotion and invasion. These findings are in agreement with the massive cellular multiplication events that take place during schizont development, where up to 22 new merozoites inside RBCs are formed before rupture. Prominent roles for cAMP-dependent Protein Kinase A (PKA)- and phosphatidylinositol-signalling were identified following the bioinformatic analyses. Most key enzymes in the inositol pathway were phosphorylated, which indicates crosstalk with other protein kinases (such as PKA) and additional levels of regulation that together co-regulate different biological processes. The bioinformatics analyses showed further, that cAMPePKA signalling is highly active in schizonts, and is

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involved in a wide variety of processes, for example in motility. This finding in particular, was verified experimentally using an in vitro kinase assay and resulted in the identification of three novel PKA substrates; namely, Myosin A, Glideosome Associated Protein 45 (GAP45) and calcium dependent protein kinase 1 (CDPK1) that are associated with the glideosome motor complex. Hence, besides the established role for CDPK1 in the motor complex, this study suggests the coinvolvement of PKA, further implicating PKA-mediated signalling as an important regulator of host-cell invasion. 5.3. Solyakov et al.: determination of the essential protein kinases and phospho-proteome of P. falciparum The Doerig and Tobin laboratories combined efforts to identify which of the eukaryotic protein kinases within the P. falciparum kinome are likely essential for the survival of the parasite in cell culture and link this with phospho-proteomic analysis of the schizont stage of the parasite. They found that there was a remarkable lack of redundancy in the P. falciparum kinome with 36 of the 65 ePKs being essential for parasite survival. This was established using a reverse genetics approach, where firstly the protein kinase gene locus was targeted with a knockout targeting vector. Lack of integration of the targeting vector was attributed to the likelihood that the protein kinase was essential. To test that the lack of integration was not due to the locus being refractive to gene targeting the locus was targeted with a vector that placed an epitope tag inframe with the protein kinase coding sequence. In further cases, integration of the targeting vector was observed only following complementation with episomally expressed protein kinase. Thus, this study provided evidence for an abundance of essential protein kinases in P. falciparum and thereby a wealth of potential anti-malarial targets. The phospho-proteome itself revealed the potential for phosphorylation to regulate a huge array of parasite processes including house keeping functions such as DNA replication, transcription, translation and metabolism as well as parasitespecific processes such as invasion and cyto-adhesion. Interestingly, this study (and subsequent unpublished work) discovered very little evidence for tyrosine phosphorylation. Only in specialized cases, as seen for autophosphorylation of the protein kinases PfGSK3 and PfCLK3, did this study observe clear evidence for tyrosine phosphorylation. Where peptide scores revealed a high likelihood of tyrosine phosphorylated peptides manual inspection determined there to be ambiguity in the MS/MS data and often revealed a neighbouring serine that might be phosphorylated. It would seem that tyrosine phosphorylation might be very low abundance in P. falciparum consistent with the lack of a tyrosine kinase family in the genome. One particular feature of the Solyakov study was the demonstration that at least 23 parasite protein kinases were themselves phosphoproteins. A number of these phosphorylation sites were determined to be either within the activation loop of the kinase, or within one of the eleven kinase domains. This very much indicates that, as is the case in the mammalian

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kinome, protein kinases in malaria are organized into kinase cascades, where up-stream kinases are responsible for the phosphorylation and regulation other kinases. The nature of the relationship between the kinases has yet to be determined, but the knowledge of specific phosphorylation site information will now help to unravel such interactions and their functional relevance. Among the protein kinases phosphorylated within the activation loop were PfGSK3 and the CDK-like kinase PfCLK3; these kinases are however phosphorylated on tyrosine [12]. Phosphorylation of PfGSK3 occurred as a cisautophosphorylation event at Y229, analogous to Y279 and Y216 on mammalian GSK3a/b, [45,46]. Similarly, tyrosine phosphorylation of the PfCLK3, a serine/threonine kinase related to the pre-mRNA processing kinase hPRP4 found in higher eukaryotes [7], proceeds in a manner consistent with this kinase being a member of the dual-specificity tyrosine phosphorylated-regulated kinase (DYRK) family [7,47]. Members of the DYRK family are distinguished by cisautophosphorylation within the activation loop, which occurs at an active transient intermediate during translation [46]. Solyakov et al. demonstrated that the site of autophosphorylation was Y526 within a TSY motif in the activation loop that when mutated significantly reduced the serine/threonine kinase activity of the recombinant enzyme [12], demonstrating that tyrosine phosphorylation of PfCLK3 is essential for full kinase activity. These studies on PfGSK3 and PfCLK3 provide a mechanism by which tyrosine phosphorylation can occur in Plasmodium, even in the absence of a tyrosine kinase family, and have an impact on the biology of the parasite. 6. Conclusions and future prospective The publications of 3 phospho-proteome datasets for P. falciparum represent a milestone for signalling cascade analysis in the field of malaria research. Now researchers can start targeted analysis of phosphorylation events within the parasites that goes beyond conserved phosphorylation sites known from other organisms. This will identify yet unknown pathways that ultimately lead to a better understanding of the parasite’s biology and reveal potential novel drug targets. The large-scale efforts to target the malaria kinome using a knockout strategy, or the use of pharmaceutical approaches to perturb phosphorylation-mediated signalling pathways in malaria parasites will allow systems-biology approaches to identify the underlying networks ultimately allowing predictions on how different pathways are interconnected. This will undoubtedly require the development of quantitative tools to monitor phosphorylation sites. Quantitative MS-methods [27,29,31] like SILAC (stable isotope labelling in cell culture), chemical-labelling (like iTRAQ, TMT-tagging or dimethyl-labelling, O18), label-free quantification or spectral counting might provide the basis for such efforts. SILAC labelling using Isoleucine has been established for P. falciparum [48,49], chemical labelling (iTRAQ) has been used for proteome expression analysis in Plasmodium [50] and spectral counting was applied by Bowyer et al. [51] and Silvestrini

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et al. [52]. Finally, an elegant approach to monitor phosphorylation site occupancy in a targeted way based on known phosphorylation site information would be SRM (selective reaction monitoring) [53]. However, studies from Yeast [54] indicate that only abundant proteins with a high proportion of the phosphorylated isoform can be reproducibly identified without prior phosphopeptide enrichment, diminishing the prospects of monitoring phosphorylation sites in non-enriched samples using this method (Christina Ludwig and Ruedi Aebersold, personal communication). Also it will be pertinent for further studies to establish protocols to remove large parts of the host-cell proteins without pertubating signalling pathways. Reproducible identification of low abundant parasite phosphopeptides is clearly linked to sample complexity [25] which is dictated by the amount of host-cell proteins in the intracellular parasites. Quantitative studies will inevitably result in greater insights into the in vivo phosphorylation networks and the impact that phosphorylation plays on the essential processes in the parasite. Layering this information onto drug discovery effects will allow for the full assessment of the potential of targeting protein kinases in malaria. Interestingly, the outcome of many of the anti-malarial screens conducted by the pharmaceutical companies has been the identification of many hundreds of compounds that either have protein kinase inhibitory activity, or that have chemoforms consistent with protein kinase inhibitors. Whereas abundance of these proteins may relate to the bias nature of some of the compound libraries, it nevertheless provides encouragement that protein kinases might make potentially important therapeutic targets. Certainly conducting system-based approaches aimed at dissecting the nature of the phospho proteome and kinome as discussed here will go a long way to not only defining the biological mechanisms and regulatory processes in malaria, but also they will be essential in addressing the question of whether protein kinases can be targeted in therapeutic intervention in malaria.

[4] [5] [6] [7]

[8] [9]

[10]

[11]

[12]

[13]

[14]

[15]

[16]

Acknowledgements [17]

The work carried for the Lasonder study was supported by MALSIG and EVIMALAR. MT was supported in parts by the German Research Foundation, the American Heart Foundation and the NIH (AI09763301). The work by MA and ABT is supported by the Wellcome Trust (090313). We thank Christian Doerig, John Sanders and Josh Elias for critical reading and input and Christina Ludwig and Ruedi Aebersold for sharing unpublished results. References [1] T.N. Wells, E.M. Poll, When is enough enough? The need for a robust pipeline of high-quality antimalarials, Discov. Med. 9 (2010) 389e398. [2] J.N. Burrows, K. Chibale, T.N. Wells, The state of the art in anti-malarial drug discovery and development, Curr. Top. Med. Chem. 11 (2011) 1226e1254. [3] C.J. Murray, L.C. Rosenfeld, S.S. Lim, K.G. Andrews, K.J. Foreman, D. Haring, N. Fullman, M. Naghavi, R. Lozano, A.D. Lopez, Global

[18]

[19]

[20]

[21]

[22]

malaria mortality between 1980 and 2010: a systematic analysis, Lancet 379 (2012) 413e431. L.N. Johnson, Protein kinase inhibitors: contributions from structure to clinical compounds, Q. Rev. Biophys. 42 (2009) 1e40. L.N. Johnson, M.E. Noble, D.J. Owen, Active and inactive protein kinases: structural basis for regulation, Cell 85 (1996) 149e158. J. Zhang, P.L. Yang, N.S. Gray, Targeting cancer with small molecule kinase inhibitors, Nat. Rev. Cancer 9 (2009) 28e39. P. Ward, L. Equinet, J. Packer, C. Doerig, Protein kinases of the human malaria parasite Plasmodium falciparum: the kinome of a divergent eukaryote, BMC Genomics 5 (2004) 79. Anamika, N. Srinivasan, A. Krupa, A genomic perspective of protein kinases in Plasmodium falciparum, Proteins 58 (2005) 180e189. E. Talevich, A. Mirza, N. Kannan, Structural and evolutionary divergence of eukaryotic protein kinases in Apicomplexa, BMC Evol. Biol. 11 (2011) 321. C. Doerig, O. Billker, T. Haystead, P. Sharma, A.B. Tobin, N.C. Waters, Protein kinases of malaria parasites: an update, Trends Parasitol. 24 (2008) 570e577. A.G. Schneider, O. Mercereau-Puijalon, A new Apicomplexa-specific protein kinase family: multiple members in Plasmodium falciparum, all with an export signature, BMC Genomics 6 (2005) 30. L. Solyakov, J. Halbert, M.M. Alam, J.P. Semblat, D. Dorin-Semblat, L. Reininger, A.R. Bottrill, S. Mistry, A. Abdi, C. Fennell, Z. Holland, C. Demarta, Y. Bouza, A. Sicard, M.P. Nivez, S. Eschenlauer, T. Lama, D.C. Thomas, P. Sharma, S. Agarwal, S. Kern, G. Pradel, M. Graciotti, A.B. Tobin, C. Doerig, Global kinomic and phospho-proteomic analyses of the human malaria parasite Plasmodium falciparum, Nat. Commun. 2 (2011) 565. R. Tewari, U. Straschil, A. Bateman, U. Bohme, I. Cherevach, P. Gong, A. Pain, O. Billker, The systematic functional analysis of Plasmodium protein kinases identifies essential regulators of mosquito transmission, Cell Host Microbe 8 (2010) 377e387. J.D. Dvorin, D.C. Martyn, S.D. Patel, J.S. Grimley, C.R. Collins, C.S. Hopp, A.T. Bright, S. Westenberger, E. Winzeler, M.J. Blackman, D.A. Baker, T.J. Wandless, M.T. Duraisingh, A plant-like kinase in Plasmodium falciparum regulates parasite egress from erythrocytes, Science 328 (2010) 910e912. L. McRobert, C.J. Taylor, W. Deng, Q.L. Fivelman, R.M. Cummings, S.D. Polley, O. Billker, D.A. Baker, Gametogenesis in malaria parasites is mediated by the cGMP-dependent protein kinase, PLoS Biol. 6 (2008) e139. L. Reininger, J.M. Wilkes, H. Bourgade, D. Miranda-Saavedra, C. Doerig, An essential Aurora-related kinase transiently associates with spindle pole bodies during Plasmodium falciparum erythrocytic schizogony, Mol. Microbiol. 79 (2011) 205e221. D. Dorin-Semblat, S. Schmitt, J.P. Semblat, A. Sicard, L. Reininger, D. Goldring, S. Patterson, N. Quashie, D. Chakrabarti, L. Meijer, C. Doerig, Plasmodium falciparum NIMA-related kinase Pfnek-1: sex specificity and assessment of essentiality for the erythrocytic asexual cycle, Microbiology 157 (2011) 2785e2794. L. Reininger, R. Tewari, C. Fennell, Z. Holland, D. Goldring, L. RanfordCartwright, O. Billker, C. Doerig, An essential role for the Plasmodium Nek-2 Nima-related protein kinase in the sexual development of malaria parasites, J. Biol. Chem. 284 (2009) 20858e20868. A. Vaid, P. Sharma, PfPKB, a protein kinase B-like enzyme from Plasmodium falciparum: II. Identification of calcium/calmodulin as its upstream activator and dissection of a novel signaling pathway, J. Biol. Chem. 281 (2006) 27126e27133. A. Vaid, D.C. Thomas, P. Sharma, Role of Ca2þ/calmodulin-PfPKB signaling pathway in erythrocyte invasion by Plasmodium falciparum, J. Biol. Chem. 283 (2008) 5589e5597. D. Dorin-Semblat, A. Sicard, C. Doerig, L. Ranford-Cartwright, Disruption of the PfPK7 gene impairs schizogony and sporogony in the human malaria parasite Plasmodium falciparum, Eukaryot. Cell 7 (2008) 279e285. R. Ranjan, A. Ahmed, S. Gourinath, P. Sharma, Dissection of mechanisms involved in the regulation of Plasmodium falciparum calciumdependent protein kinase 4, J. Biol. Chem. 284 (2009) 15267e15276.

E. Lasonder et al. / Microbes and Infection 14 (2012) 811e819 [23] A. Merckx, G. Bouyer, S.L. Thomas, G. Langsley, S. Egee, Anion channels in Plasmodium-falciparum-infected erythrocytes and protein kinase A, Trends Parasitol. 25 (2009) 139e144. [24] K. Leykauf, M. Treeck, P.R. Gilson, T. Nebl, T. Braulke, A.F. Cowman, T.W. Gilberger, B.S. Crabb, Protein kinase a dependent phosphorylation of apical membrane antigen 1 plays an important role in erythrocyte invasion by the malaria parasite, PLoS Pathog. 6 (2010) e1000941. [25] M. Treeck, J.L. Sanders, J.E. Elias, J.C. Boothroyd, The phosphoproteomes of Plasmodium falciparum and Toxoplasma gondii reveal unusual adaptations within and beyond the parasites’ boundaries, Cell Host Microbe 10 (2011) 410e419. [26] E. Lasonder, J.L. Green, G. Camarda, H. Talabani, A.A. Holder, G. Langsley, P. Alano, The phosphoproteome of P. falciparum reveals extensive phosphatidylinositol and cAMP-Protein Kinase A signalling during blood stage development. Mol. Cell. Prot., submitted for publication. [27] A. Nita-Lazar, H. Saito-Benz, F.M. White, Quantitative phosphoproteomics by mass spectrometry: past, present, and future, Proteomics 8 (2008) 4433e4443. [28] R. Sopko, B.J. Andrews, Linking the kinome and phosphorylome e a comprehensive review of approaches to find kinase targets, Mol. Biosyst. 4 (2008) 920e933. [29] T.B. Schreiber, N. Mausbacher, S.B. Breitkopf, K. Grundner-Culemann, H. Daub, Quantitative phosphoproteomics e an emerging key technology in signal-transduction research, Proteomics 8 (2008) 4416e4432. [30] S.A. Beausoleil, J. Villen, S.A. Gerber, J. Rush, S.P. Gygi, A probabilitybased approach for high-throughput protein phosphorylation analysis and site localization, Nat. Biotechnol. 24 (2006) 1285e1292. [31] B. Macek, M. Mann, J.V. Olsen, Global and site-specific quantitative phosphoproteomics: principles and applications, Annu. Rev. Pharmacol. Toxicol. 49 (2009) 199e221. [32] B. Bodenmiller, L.N. Mueller, M. Mueller, B. Domon, R. Aebersold, Reproducible isolation of distinct, overlapping segments of the phosphoproteome, Nat. Methods 4 (2007) 231e237. [33] E.L. Huttlin, M.P. Jedrychowski, J.E. Elias, T. Goswami, R. Rad, S.A. Beausoleil, J. Villen, W. Haas, M.E. Sowa, S.P. Gygi, A tissuespecific atlas of mouse protein phosphorylation and expression, Cell 143 (2010) 1174e1189. [34] K.T. Rigbolt, T.A. Prokhorova, V. Akimov, J. Henningsen, P.T. Johansen, I. Kratchmarova, M. Kassem, M. Mann, J.V. Olsen, B. Blagoev, Systemwide temporal characterization of the proteome and phosphoproteome of human embryonic stem cell differentiation, Sci. Signal. 4 (2011) rs3. [35] J.E. Elias, S.P. Gygi, Target-decoy search strategy for increased confidence in large-scale protein identifications by mass spectrometry, Nat. Methods 4 (2007) 207e214. [36] A.I. Nesvizhskii, O. Vitek, R. Aebersold, Analysis and validation of proteomic data generated by tandem mass spectrometry, Nat. Methods 4 (2007) 787e797. [37] J.V. Olsen, M. Mann, Improved peptide identification in proteomics by two consecutive stages of mass spectrometric fragmentation, Proc. Natl. Acad. Sci. U. S. A. 101 (2004) 13417e13422.

819

[38] M. Treeck, S. Zacherl, S. Herrmann, A. Cabrera, M. Kono, N.S. Struck, K. Engelberg, S. Haase, F. Frischknecht, K. Miura, T. Spielmann, T.W. Gilberger, Functional analysis of the leading malaria vaccine candidate AMA-1 reveals an essential role for the cytoplasmic domain in the invasion process, PLoS Pathog. 5 (2009) e1000322. [39] C.S. Tan, A. Pasculescu, W.A. Lim, T. Pawson, G.D. Bader, R. Linding, Positive selection of tyrosine loss in metazoan evolution, Science 325 (2009) 1686e1688. [40] J.C. Boothroyd, J.F. Dubremetz, Kiss and spit: the dual roles of Toxoplasma rhoptries, Nat. Rev. Microbiol. 6 (2008) 79e88. [41] M.L. Reese, J.P. Boyle, Virulence without catalysis: how can a pseudokinase affect host cell signaling? Trends Parasitol. 28 (2012) 53e57. [42] M.C. Nunes, J.P. Goldring, C. Doerig, A. Scherf, A novel protein kinase family in Plasmodium falciparum is differentially transcribed and secreted to various cellular compartments of the host cell, Mol. Microbiol. 63 (2007) 391e403. [43] D. Schwartz, S.P. Gygi, An iterative statistical approach to the identification of protein phosphorylation motifs from large-scale data sets, Nat. Biotechnol. 23 (2005) 1391e1398. [44] G.D. Bader, C.W. Hogue, An automated method for finding molecular complexes in large protein interaction networks, BMC Bioinformatics 4 (2003) 2. [45] A. Cole, S. Frame, P. Cohen, Further evidence that the tyrosine phosphorylation of glycogen synthase kinase-3 (GSK3) in mammalian cells is an autophosphorylation event, Biochem. J. 377 (2004) 249e255. [46] P.A. Lochhead, Protein kinase activation loop autophosphorylation in cis: overcoming a Catch-22 situation, Sci. Signal. 2 (2009) pe4. [47] S. Aranda, A. Laguna, S. de la Luna, DYRK family of protein kinases: evolutionary relationships, biochemical properties, and functional roles, FASEB J. 25 (2011) 449e462. [48] N. Nirmalan, P.F. Sims, J.E. Hyde, Quantitative proteomics of the human malaria parasite Plasmodium falciparum and its application to studies of development and inhibition, Mol. Microbiol. 52 (2004) 1187e1199. [49] J.H. Prieto, S. Koncarevic, S.K. Park, J. Yates 3rd, K. Becker, Large-scale differential proteome analysis in Plasmodium falciparum under drug treatment, PLoS One 3 (2008) e4098. [50] C. Kuss, C.S. Gan, K. Gunalan, Z. Bozdech, S.K. Sze, P.R. Preiser, Quantitative proteomics reveals new insights into erythrocyte invasion by Plasmodium falciparum, Mol. Cell. Proteomics 11 (2012) M111010645. [51] P.W. Bowyer, G.M. Simon, B.F. Cravatt, M. Bogyo, Global profiling of proteolysis during rupture of Plasmodium falciparum from the host erythrocyte, Mol. Cell. Proteomics 10 (2011) M110001636. [52] F. Silvestrini, E. Lasonder, A. Olivieri, G. Camarda, B. van Schaijk, M. Sanchez, S. Younis Younis, R. Sauerwein, P. Alano, Protein export marks the early phase of gametocytogenesis of the human malaria parasite Plasmodium falciparum, Mol. Cell. Proteomics 9 (2010) 1437e1448. [53] V. Lange, P. Picotti, B. Domon, R. Aebersold, Selected reaction monitoring for quantitative proteomics: a tutorial, Mol. Syst. Biol. 4 (2008) 222. [54] A.P. Oliveira, C. Ludwig, P. Picotti, R. Aebersold, U. Sauer, Regulation of yeast central metabolism by enzyme phosphorylation, PLoS Biol., submitted for publication.