Proteins interacting with PfEMP1 in P. falciparum infected erythrocyte

During infection in humans, P. falciparum invades and refurbishes the red blood cells (RBCs) in order to persist and proliferate. Parasite survival depends on expression of a parasite encoded cytoadherence ligand at the surface of the infected RBC called PfEMP1 (P. falciparum erythrocyte membrane protein 1). PfEMP1 comprises a N-terminal extracellular part, which mediates binding to receptors at the host endothelium. It also contains a C-terminal part which is conserved among all PfEMP1 molecules. This acidic terminal segment (ATS) anchors PfEMP1 to the membrane of infected RBCs and has a role for trafficking. To bring proteins to the RBC surface, P. falciparum builds its own trafficking machinery de novo since the erythrocytes do not contain secretory organelles. These new structures include parasite derived compartments called Maurer's clefts (MCs) located at the RBC periphery. The resident MCs protein MAHRP1 (membrane-associated histidine-rich protein 1) has been shown to be critical for PfEMP1 trafficking. A MAHRP1 KO strain had no PfEMP1 on the RBC surface but accumulated PfEMP1 within the parasite. MAHRP1 is a 28.9kDa protein with 249 amino acids containing a N-terminal, a transmembrane, and a C-terminal domain comprising histidine-rich repeats. To re-establish PfEMP1 trafficking, the MAHRP1 KO parasite line was complemented with various truncated HA-tagged fragments of MAHRP1. Immunofluorescent assays (IFAs) showed that the Cterminal domain with the histidine-rich repeats was not essential for PfEMP1 trafficking. ${MAHRP1}_{1-130}$ and ${MAHRP1}_{1-169}$ were exported to MCs and restored trafficking of PfEMP1. Shorter MAHRP1 fragments were not exported to MCs and PfEMP1 transport remained impaired. New transfection constructs were designed including the C-terminal part to identify important domain(s) of MAHRP1 for PfEMP1 trafficking. To identify other proteins interacting with PfEMP1, the ATS domain of PfEMP1 was recombinantly expressed and used in pull-down experiments. In these experiments using parasite lysates, PF14_0377 was identified as a potential interaction partner. This protein has been described as putative, vesicular-associated membrane protein. To subsequently assess the interaction(s) and the localization(s), PF14_0377 was cloned and transfected in 3D7 for IFAs. Due to time restrictions all these new transfectants could not be analyzed completely

An essential Aurora-related kinase transiently associates with spindle pole bodies during Plasmodium falciparum erythrocytic schizogony

Aurora kinases compose a family of conserved Ser/Thr protein kinases playing essential roles in eukaryotic cell division. To date, Aurora homologues remain uncharacterized in the protozoan phylum Apicomplexa. In malaria parasites, the characterization of Aurora kinases may help understand the cell cycle control during erythrocytic schizogony where asynchronous nuclear divisions occur. In this study, we revisited the kinome of Plasmodium falciparum and identified three Aurora-related kinases, Pfark-1, -2, -3. Among these, Pfark-1 is highly conserved in malaria parasites and also appears to be conserved across Apicomplexa. By tagging the endogenous Pfark-1 gene with the green fluorescent protein (GFP) in live parasites, we show that the Pfark-1-GFP protein forms paired dots associated with only a subset of nuclei within individual schizonts. Immunofluorescence analysis using an anti-a-tubulin antibody strongly suggests a recruitment of Pfark-1 at duplicated spindle pole bodies at the entry of the M phase of the cell cycle. Unsuccessful attempts at disrupting the Pfark-1 gene with a knockout construct further indicate that Pfark-1 is required for parasite growth in red blood cells. Our study provides new insights into the cell cycle control of malaria parasites and reports the importance of Aurora kinases as potential targets for new antimalarials.

Wiley-Blackwell2011

Structural characterisation and membrane insertion of M13 procoat, M13 coat and Pf3 coat proteins

Membrane proteins fulfill many central functions in the biological membrane. The insertion process of these proteins and their structure, which are intimately linked to their function, are not yet well understood. As a model we studied three proteins reconstituted into lipid bilayers (which serve as a model for the biological membrane): the major coat proteins of phages Pf3 and M13 and the precursor of the latter, M13 procoat protein, which spans the inner membrane two times and serves as a model for a multi-spanning membrane protein. Whereas Pf3 coat protein can insert into the membrane in its mature form, M13 coat protein needs the signal sequence present in the precursor to insert. A new, simple and efficient purification method has been developed for M13 procoat, M13 and Pf3 coat proteins. Homogenous preparations were obtained in isopropanol/0.l% TFA, where M13 coat protein is found to be dissolved in a monomeric form, and the two other proteins as dimers. The conformations of these particular proteins in different environments have been determined by circular dichroism and infrared spectroscopy. In organic solvents, the proteins adopt a conformation with an average helix content of 90%. In lipid bilayers composed of phospatidylcholine and phosphatidylglycerol lipids, the average helix content is 50% for M13 procoat protein, 60% for M13 coat protein and 75% for Pf3 coat protein. In order to characterize the orientational distribution the orientational order parameter Sα of the protein helices in planar lipid bilayers have been determined by polarized infrared measurements in the amide I spectral range. The helices of the three proteins are oriented preferentially parallel to the membrane normal, with Sα = 0.63 for M13 procoat protein, Sα = 0.58 for Pf3 coat protein and a distinctly higher value of Sα = 0.81 for M13 coat protein. The topology of M13 and Pf3 coat proteins was investigated using limited proteolysis in lipid vesicles with proteinase K combined with mass spectroscopy to analyze the lytic fragments. The C-terminal part of both proteins was found to be inaccessible to proteinase K, only 14 (Pf3) or 15 (M13) residues on the N-terminal were protease accessible from the outside of the vesicles. Furthermore, the standard free energy change, ΔGo, of a membrane-inserting protein with a leader sequence has been determined experimentally for the first time, using M13 procoat protein as an example. The partition coefficient for the distribution of this protein between the aqueous phase and the membrane phase of preformed lipid vesicles yielded a value of Γ = 6.5×105 M-1, corresponding to a ΔGo of -10.4 kcal/mol, based on measurements of the fluorescence energy transfer between the intrinsic tryptophan of the protein and a suitably labeled lipid membrane of POPC. For comparison, the partition coefficient of the M13 coat protein between the aqueous and the POPC lipid bilayer phase was determined to be distinctly lower: Γ = 1×105 M-1 (ΔGo = -9.3 kcal/mol). Proteinase K digestion experiments have been performed, showing that 20% of the procoat protein bound to lipid vesicles spontaneously integrate in a transbilayer form, whereas 80% remain inserted in the interfacial membrane region. By taking together these results, an upper limit for the free energy change of the transmembrane insertion of procoat protein was estimated to be -14.8 kcal/mol. In order to distinguish further the contribution arising from insertion of the procoat protein into the membrane interfacial region from that due to transmembrane insertion, the partition coefficient of a mutant procoat protein OM30R (which contains a positively charged amino acid in its mature hydrophobic segment (exchange of a Val to an Arg residue at position 30)) was determined, yielding Γ = 0.3×105 M-1 (ΔGo = -8.6 kcal/mol). Previously reported in vivo experiments have shown that the OM30R mutant protein is not translocated across Escherichia coli inner membrane, but only binds to the inner surface. The results presented here indicate that although the insertion of the procoat protein into the interfacial region of the lipid bilayer contributes the major part to ΔGo, it is the final energy gain of the interaction of the hydrophobic portions of the folded pre-protein with the lipid chains which drives the transmembrane insertion of the M13 procoat protein. Neither the leader sequence nor the mature coat protein alone yields this free energy gain. For the different proteins investigated here, spontaneous membrane insertion occurs only for fluid lipid bilayers, but not for membranes in the crystalline lipid phase. Furthermore, by using lipid bilayers with negative membrane surface charges, it was shown that both procoat and coat proteins are electrostatically attracted to the surface of the lipid membrane, though only to a small extent, with apparent partition coefficients of the same order of magnitude as for the phosphatidylcholine lipid membrane. This work has yielded new information about the structure and the insertion of the phage coat proteins. The new methods developed in this work might be of general applicability to study the structural dynamics of membrane protein assembly.

EPFL1996

Developing a Reconciled Genome-Scale Model of the Malaria Parasite Plasmodium falciparum and its Thermodynamics-based Flux Balance Analysis

Meriç Ataman, Vassily Hatzimanikatis, Dominique Soldati-Favre, Stepan Tymoshenko

Understanding the metabolism of the malaria parasites is of high priority to identify novel enzymatic drug targets and face the rise in drug resistant parasites. Based on the knowledge of metabolic functions and molecular interactions, we can reconstruct genome-scale models (GEMs) to study the metabolic behavior of the organism as a whole. In this work, we have developed a reconciled GEM of Plasmodium falciparum, the deadliest of the malaria parasites. To accomplish this, we firstly reconstructed the metabolic network of P. falciparum using the RAVEN toolbox. Then, we curated the biochemistry of the metabolic network by integrating information from two previously published GEMs (Plata et al. and by Hutmacher et al., 2010) and from the experimental characterization of enzymatic activity in the malaria parasites. Using a gap-filling approach, we hypothesized which enzymatic reactions are required to fulfill certain metabolic capabilities, such as anaerobic lactate production from glucose. We then added thermodynamic constraints and three sets of metabolomics data, which were measured during infection of the red blood cells, and we performed Thermodynamics-based Flux Balance Analysis. With our in silico approach, we have identified enzymes that are essential for the proliferation of the parasites at the blood stage. Some of these enzymes were reported to be essential in the literature, but previous computational studies had failed to identify them as essential. Our GEM of P. falciparum is a valuable tool for understanding the metabolism of the malaria parasite, for the integration of omics data, and for the identification of novel antimalarial drug targets.

2015

Structural characterisation and membrane insertion of M13 procoat, M13 coat and Pf3 coat proteins

EPFL1996