PLoS One. 2015 Nov 5;10(11):e0140994.
A Putative Non-Canonical Ras-Like GTPase from P. falciparum: Chemical Properties and Characterization of the Protein.
Annette Kaiser1*, Barbara Langer1,2, Jude Przyborski3, David Kersting1, Mirko Krüger1
1 Medical Research Centre, Institute for Pharmacogenetics, University Duisburg-Essen,
Hufelandstrasse 55, 45147 Essen, Germany
2Institute for Pharmacology, Hufelandstrasse 55,45147 Essen, Germany
3Department of Parasitology, FB Biology, Phillipps University Marburg, Karl-von Frisch-Strasse 8, 34043 Marburg, Germany
*Email Correspondence to author at present:firstname.lastname@example.org
During its development the malaria parasite P. falciparum has to adapt to various different environmental contexts. Key cellular mechanisms involving G-protein coupled signal transduction chains are assumed to act at these interfaces. Heterotrimeric G-proteins are absent in Plasmodium. We here describe the first cloning and expression of a putative, non-canonical Ras-like G protein (acronym PfG) from Plasmodium. PfG reveals an open reading frame of 2736 bp encoding a protein of 912 amino acids with a theoretical pI of 8.68 and a molecular weight of 108.57 kDa. Transcript levels and expression are significantly increased in the erythrocytic phase in particular during schizont and gametocyte formation. Most notably, PfG has GTP binding capacity and GTPase activity due to an EngA2 domain present in small Ras-like GTPases in a variety of Bacillus species and Mycobacteria. By contrast, plasmodial PfG is divergent from any human alpha-subunit. PfG was expressed in E. coli as a histidine-tagged fusion protein and was stable only for 3.5 hours. Purification was only possible under native conditions by Nickel-chelate chromatography and subsequent separation by Blue Native PAGE. Binding of a fluorescent GTP analogue BODIPY® FL guanosine 5’O-(thiotriphosphate) was determined by fluorescence emission. Mastoparan stimulated GTP binding in the presence of Mg2+. GTPase activity was determined colorimetrically. Activity expressed as absolute fluorescence was 50% higher for the human paralogue than the activity of the parasitic enzyme. The PfG protein is expressed in the erythrocytic stages and binds GTP after immunoprecipitation. Immunofluorescence using specific antiserum suggests that PfG localizes to the parasite cytosol. The current data suggest that the putitative, Ras-like G-protein might be involved in a non-canonical signaling pathway in Plasmodium. Research on the function of PfG with respect to pathogenesis and antimalarial chemotherapy is currently under way.
One of the main functions of Ras proteins can be attributed to serum-independent cell proliferation in higher eukaryotes. In contrast, in lower eukaryotes like C. elegans Ras-proteins are involved in differentiation processes and determine the fate of a cell type. In the fission yeast Ras is involved in the control of cAMP levels during vegetative growth while it functions as a signal transducer in Drosophila. The mammalian Ras-GTPase family currently contains 13 members with considerable homology in their effector region.
Malaria is caused by the genus Plasmodium. After infection of the human host malaria parasites go through multiple developmental stages where they face different environments i.e. the liver, human erythrocytes and finally the mosquito. Intracellular signalling is responsible to adapt to these environmental changes and requires cyclic nucleotides, calcium and phospholipid derived molecules which in turn transfer the signal to second messengers. The Plasmodium genome has orthologues of the cyclic nucleotide signaling pathway (Fig.1) i.e. two predicted adenylate and guanylate cyclases, four cyclic nucleotide phoshodiesterases, a cGMP-dependent protein kinase and both catalytic and regulatory subunits of a cAMP-dependent protein kinase. However, a major gap in our knowledge is the absence of cell-surface receptors and heterotrimeric G-proteins that trigger cyclic nucleotide signalling (Fig.1). The fact that there are currently three database entries in the Plasmodium database (PlasmoDB) i.e. two encoding for serpentine receptors and one encoding for a G-protein coupled receptor from P. falciparum, P. reichenowi and P. vivax Salvador strain-1 encouraged us to screen for an orthologue of a heterotrimeric G-protein. Moreover, our screening was challenged by previous findings that the erythrocyte β2-adrenergic receptor and heterotrimeric guanine nucleotide–binding protein (Gαs) regulate the entry of the human malaria parasite Plasmodium falciparum into the erythrocyte. Our results clearly demonstrate that heterotrimeric G-proteins are absent in Plasmodium. Instead, we identified a Ras-like GTPase (Acronym PfG) with still unknown function in the infection process. PfG encodes a protein of 911 amino acids with characteristic domains like an EngA2 domain (amino acid position 730 to 800) and a KH-domain (805-875) at the C-terminus. Both domains are depicted in Fig.2. PfG has 72% amino acid identity to EngA proteins which represent Ras-like GTPases in pathogenic bacteria like Mycoplasma mobile a grampositive Eubacterium and Bacillus subtilis. In this context, the fact that PfG has significant GTPase activity can be attributed to the EngA2 domain. Five G-Box binding motifs and a separate Mg binding site are present in PfG. These binding sites support our data that GTP binding to PfG can be costimulated by Mg2+ and mastoparan, a peptide toxin from the wasp venom mimicking the G-protein coupled receptor. PfG displays significant homology to the rodent malaria parasites paralogues but cannot be classified within any of the 19 characterized G-alpha subunits from the human host. We therefore conclude that PfG represents a non-canonical G-protein.
Fig.1: Part A) A hypothetical GPCR-coupled pathway in Plasmodium. An unknown ligand may bind to a non-canonical GPCR-like structure. Currently, three PlasmoDB database entries for putative GPCRs exist in Plasmodium with no proof of principle. There is no evidence for the occurrence of heterotrimeric G-proteins in Plasmodium. However, there is evidence that non- canonical G-proteins of the Ras-type exist in Plasmodium. cAMP or cGMP is generated by either adenylate or guanylate cylases which were identified in Plasmodium. The signal is transferred by second messengers (cAMP or cGMP) to the cyclic-nucleotide dependent kinases (protein kinase A, protein kinase G) and calcium/phospholipid-dependent kinases (protein kinase C) which form the AGC group (right part of the figure). The PKA catalytic and regulatory subunits-have been characterized in P. falciparum i.e. PfPKAr and PfPKAc. A potential anchor protein (AKAP) has been recently identified which differs significantly to its human counterpart.
Fig.1 Part B: A putative Gq-signaling pathway involving protein kinase B in Plasmodium. A possible, hormone-like signal stimulates the Gq- subunit of the receptor and in consequence phospholipase C (PLC). PLC hydrolyses phoshoinositol-diphosphate (PIP2) to inositol and diacylglycerol (DAG). Ca2+-ions are also involved in stimulation of PKC. This pathway maybe also hypothesized for Plasmodium.
Our recent findings suggest that it seems to be half-truth that the invasion of P. falciparum is only dependent on erythrocyte G-protein-coupled receptor signaling and the G-alphas subunit in the erythrocyte membrane. This notion is further supported by the fact that the ß-blocker propanolol reduced parasitemia to only 30% due to down regulation of Gs signaling . The drug particularly affected the development of schizonts. Thus it seems likely that there may be alternative pathways in the parasite which are not controlled by erythrocyte G-protein-coupled receptor signaling. Hitherto, the role of PfG in malaria infection remains to be explained. A significant increase of PfG transcript levels was detected in schizonts and gametocytes. In the near future a loss of function mutant obtained by novel genome editing techniques i.e. the RNA-guided CRISPR (clustered regularly interspaced short palindromic repeats- the nuclease Cas (CRISPR-associated proteins) system or by homologous recombination will delineate its role in parasite infection.
Our understanding of a non-canonical G-protein coupled pathway is rudimentary but it might be a major groove in finding suitable drug targets. The recent results demonstrate that PfG might be of further interest for an evaluation as a drug target. There is a mammalian Ras-GTPase inhibitor 2-Cyano-N-octyl-3-(1-(3-dimethylaminopropyl)-1H-indol-3-yl)-acrylamide which might also function as a suitable lead structure for an in vitro assay in Plasmodium and in an in vivo rodent, humanized mouse model. PfG cannot be classified within the mammalian Gα subunits and neither a ß-subunit nor a gamma subunit cannot be identified within the PlasmoDB database. These properties might be exploited in the drug discovery process for selective inhibition of the parasitic protein.
The downstream part of the cyclic nucleotide signaling pathway in Plasmodium resembles that of other eukaryotes . It involves soluble cAMP or cGMP as a second messenger molecule in Plasmodium. Reversible protein phosphorylations by protein kinases are a conserved mode of protein modification in the parasite. The protein kinase A comprises two regulatory subunits PfKAc and PfKAr (Fig.1) in Plasmodium. The most significant difference is the lack of a phosphorylation at a key site in protein kinase A implying that the protein is not so tightly regulated as its human counterpart. Target evaluation of the identified kinases just started for antimalarial intervention. A series of isoquinolines has been tested as lead compounds to inhibit the PfKA regulatory subunits. Although many had a growth limiting impact on chloroquine-resistant and sensitive P. falciparum in vitro cultures they did not inhibit PfKA. In sum, a selective inhibitor has to be identified for PfKAc and PfKAr.
Mammalian protein kinase G inhibitors i.e. coccidial inhibitors 2,3-diarylpyrrole and imidazopyrimidines were tested in a transgenic Plasmodium berghei line expressing the P. falciparum cGMP-dependent protein kinase (PfKG) paralogue. Both compounds showed the same in vivo efficacy against the mammalian and parasitic enzymes. Hitherto, screenings for a Gq-mediated signaling of protein kinase C (PfKC) have not been successful in Plasmodium. However, a serine kinase paralogue, protein kinase B as an important member of the phosphatidylinositol 3-kinase-dependent signaling pathway was recently isolated from Plasmodium. Inhibition of protein kinase B was indirectly conducted by calmodulin inhibitors or phospholipase C inhibitors. G-protein-coupled receptor kinases (GRKs) have been originally discovered for their role in receptor phosphorylation. However, recent studies demonstrated a broader function including phosphorylation of cytosolic substrates involved in signaling pathways. The latter role pinpoints a role in pathophysiological processes like inflammation. Thus it seems likely that the parasite needs a network of kinases to invade the human erythrocyte and in each state of its development.
Since Plasmodia are no exception with respect to cyclic nucleotide signaling pathways operating through phosphosignaling our focus will be directed towards the first part of the pathway with the identification of a putative,non-canonical receptor and its unknown ligand. Future experiments will unreveal whether PfG is capable of binding to a putative receptor.
Fig.2: Schematic representation of the putitative characteristic domains of the PfG protein from Plasmodium: At the C-terminus a KH-like domain (blue colour, amino acid position 805-875, blue arrow) of the EngA subfamily of essential bacterial GTPases with two adjacent GTPase domains is located. The EngA2 domain covers amino acid positions 730-800 (blue arrow). However, the GTPase domain elements for binding ribonucleoproteins are missing. A small GTP-binding domain respresents a Ras-like protein which is similar to the DER protein in E. coli responsible for cell viability and also present in Neisseria gonorrhea and Thermotoga maritima. This EngA2 subfamily CD represents the second GTPase domain of EngA and its orthologs, which are composed of two adjacent GTPase domains. Since the sequences of the two domains are more similar to each other than to other GTPases, it is likely that an ancient gene duplication, rather than a fusion of evolutionarily distinct GTPases, gave rise to this family. However, the exact function of these proteins has not been elucidated. The GTP/Mg-binding site is marked by a blue arrow and responsible for the chemical binding of GTP and Mg2+. Five G Box motifs (marked by triangles] for GTP-binding are present within the EngA2 domain including a switch region II which is a surface loop undergoing conformational change upon GTP-binding. The G1 box motif: GXXXXGK[T/S] is a signature motif of a phosphate-binding loop. G2 box motif: T Thr is conserved throughout the superfamily, but surrounding residues are conserved within families. G3 box motif: DXXG overlaps the Switch II region, which includes the Walker B motif. G4 box has the motif: [N/T]KXD and the G5 box the motif: [C/S]A[K/L/T].
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