Cardi D , Pozza A , Arnou B , Marchal E , Clausen JD , Andersen JP , Krishna S , Møller JV , le Maire M , Jaxel C .

074395/Z/04/Z Wellcome Trust

	FIGURE 1. Effect of drugs on COS-1 cell-expressed SERCA1a wild type and E255L mutant. The rate of Ca2+-activated ATP hydrolysis was determined at 25 °C in the presence of 4 μm Ca2+ ionophore A23187 in a medium containing 50 mm TES/Tris (pH 7.0), 100 mm KCl, 7 mm MgCl2, 1 mm EGTA, 0.9 mm CaCl2 (giving a free Ca2+ concentration of 3 μm), and 5 mm ATP. The effect of 1.5 μm Tg, 10 μm artemisinin (ART), and 10 μm artemisinin together with 10 μm FeSO4 (ART+Fe2+) were tested on wild type SERCA1a (black bars) and E255L mutant (open bars). The inhibitor was added dissolved in DMSO, giving a final DMSO concentration of 5% (v/v), and turnover rates are shown as a percentage of the turnover rate measured when the same amount of DMSO was added without inhibitor. S.E. values are indicated by the error bars on the columns.
	FIGURE 2. Characteristics of purified SERCA1a E255L mutant; purification and ATPase activity. A, SDS-PAGE followed by Coomassie Blue staining. 7.5 μl of the eluted fraction was loaded (E255L), and to quantify the amount of SERCA-E255L in this sample, 100 and 400 ng of the wild type SERCA (SR) preparation were loaded. M, molecular mass markers. B, Western blot analysis with anti-SERCA1a antibodies. 0.75 μl of the eluted fraction (E255L) and 10 and 40 ng of the SERCA (SR) preparation were loaded. C, ATPase activities at 25 °C of SERCA1a E255L in C12E8 (1 mg/ml) and C12E8/DOPC (0.2/0.05 mg/ml) and inhibition assays with thapsigargin and artemisinin. Purified SERCA1a E255L was diluted to 0.75 μg/ml in the ATPase assay medium. The medium contained also 125 μm Ca2+ and either C12E8 (1 mg/ml) (experiment A) or C12E8/DOPC (0.2/0.05 mg/ml) (experiments B–E), and hydrolytic activity was monitored continuously with a coupled enzyme system by recording NADH oxidation at 340 nm. The reactions were triggered by the addition of 5 mm ATP and stopped by the addition of 750 μm EGTA. The inhibition effect of 1.5 μm Tg was first checked (experiments A and B), and then the effect of artemisinin was tested by preincubation of the protein with 10 μm artemisinin (experiment C) and 10 μm Fe2+ + 10 μm artemisinin (experiment E). The effect of Fe2+ + DMSO was controlled by the addition of 10 μm Fe2+ + 2 μl of DMSO (experiment D). In this and subsequent figures, the initial decrease in NADH absorbance due to a small amount of ADP in our ATP solution was removed.
	FIGURE 3. Expression of PfATP6. A, on minimal medium. Shown is Western blot analysis with anti-PfATP6 antibodies of the level of expression of PfATP6-BAD expressed from the wild type cDNA of Pfatp6 (WT) and the codon-optimized one (CO). 10 μl of each protein solution after the expression assay were loaded, corresponding to 8 and 10 μg of proteins for the WT and CO samples, respectively. 1 and 2 correspond to a signal acquisition of 14 and 2 min, respectively. The arrow indicates the band corresponding to monomeric PfATP6-BAD. B, on rich medium. Shown are analyses of fractions recovered during the membrane preparation by Western blot analysis with avidin peroxidase. Fractions of 2 μg of total proteins of crude extract (CE) and 1 μg of total proteins of LM and soluble fraction (S) were loaded. The arrows indicate the biotinylated proteins endogenous of the yeast S. cerevisiae (acetyl-CoA carboxylase (ACC), 250 kDa; pyruvate carboxylase (PC), 120 kDa; Arc1p protein (Arc1p), 45 kDa). C, analyses of these fractions by Western blot with anti-PfATP6 antibodies. To quantify the amount of PfATP6-BAD in the LM fraction, 5, 10, 20, and 30 ng of purified PfATP6 supplemented with 1 μg of total proteins of light membrane fraction of yeast expressing SERCA1a were loaded.
	FIGURE 4. Purification of PfATP6 on streptavidin-Sepharose. A, Western blot analysis with avidin peroxidase of the washing, the solubilization, and the purification steps before thrombin cleavage of PfATP6. 0.5 μl of each fraction was loaded (corresponding to 5 μg of total proteins in the LM lane, for example). In order to remove soluble biotinylated proteins (acetyl-CoA carboxylase (ACC), 250 kDa; pyruvate carboxylase (PC), 120 kDa; Arc1p protein (Arc1p), 45 kDa), LM were diluted in washing buffer at 10 mg/ml and centrifuged. The resulting supernatant (WS) was discarded, and the pellet was resuspended in solubilization buffer (WP) with the same volume as the LM fraction and centrifuged. DDM was added, and solubilization was performed as described under “Experimental Procedures.” The supernatant corresponding to the solubilized fraction (SF) was added to the resin, and the flow-through fraction (FT) was removed. The resin was then washed with high salt buffer (HS) and low salt buffer (LS). B, analyses of the resin and the eluted fractions by SDS-PAGE followed by Coomassie Blue staining. Except for the molecular mass marker (M) and the SERCA (SR) fractions, 7.5 μl of each fraction were loaded. To check the action of thrombin in the cleavage of PfATP6-BAD (% of cleavage) and the efficiency of elution, the resin before (R) and after the first incubation with thrombin (about 60% of cleavage, R1) and after the second one (about 90% of cleavage, R2) and also the resin after elution (R*) were analyzed. To quantify the amount of PfATP6 recovered after the first (E1) and the second (E2) elution, 100, 200, and 400 ng of the wild type SERCA (SR) preparation were loaded. The arrows indicate potential nonspecific cleavages of PfATP6. C, analyses of the same fractions as in B by Western blot with anti-PfATP6 antibodies. Only 0.75 μl were loaded in each lane. Cleavage products of PfATP6 are hardly detectable (indicated by the arrows).
	FIGURE 5. ATPase activities of purified PfATP6 protein and effect of artemisinin drugs. A, activity was measured at 25 °C and pH 7.5 with 10 μg of proteins in 2 ml of assay buffer (see legend to Fig. 1). The reaction was triggered by the addition of 5 mm ATP into the medium and stopped by the addition of a final concentration of 750 μm EGTA. The reactions were carried out in the presence of C12E8 (0.2 mg/ml), C12E8/DOPC (0.2/0.05 mg/ml), and C12E8/lipid mixture (0.2/0.05 mg/ml). B, the reactions were performed with 5 μg/ml purified PfATP6 at 25 °C in C12E8/DOPC (0.2/0.05 mg/ml). Artemisinins were added with 2 μl of a solution at 10 mm prepared in DMSO. The effect of various artemisinins was tested by the addition of 10 μm (final concentrations) of the drugs during the ATP hydrolysis of the enzyme.
	FIGURE 6. Effect of inhibitors of SERCA-type proteins on the ATPase activity of PfATP6. The reactions were performed with 5 μg/ml purified PfATP6 at 25 °C in C12E8/DOPC (0.2/0.05 mg/ml). They were triggered by the addition of 5 mm ATP and stopped by the addition of 750 μm EGTA. Inhibitors, diluted in DMSO, were added with 2 μl of the corresponding mother solution. First, the effect of DMSO on the activity of PfATP6 was monitored after the addition of 2 μl (DMSO 0.1%) and 4 μl (DMSO 0.6%) to PfATP6. Then the inhibitions of the enzyme by thapsigargin (45 μm), BHQ (20 μm), and CPA (3 μm) were tested.
	FIGURE 7. Summary of the effect of specific inhibitors of SERCA and artemisinin drugs on the ATPase activities of PfATP6 measured as described in the legends to Figs. 5 and 6. Each bar represents a minimum of three experiments ± S.D. (error bars).
	FIGURE 8. Effect of artemisone and thapsigargin on the ATPase activity of relipidated PfATP6. A, freeze-fracture electron microscopy analysis of relipidated PfATP6 with egg yolk phosphatidylcholine. Bar, 100 nm. The sample is heterogenous and contains different lipid structures: vesicles with one membrane protein (1), large membranous structures with dispersed proteins (2), multilamellar vesicles (3), and vesicles with membrane protein clusters (4). Each arrow shows one molecule of PfATP6. B, ATPase activities performed with 1.75 μg/ml reconstituted PfATP6 at 37 °C. They were triggered by successive additions of 5 mm ATP and 100 μm Ca2+ and stopped by the addition of 750 μm EGTA. Artemisone was added with 2 μl of solutions at 10 or 100 mm prepared in DMSO. The effects of artemisone were tested by preincubation of the protein in 150 μm EGTA with 10 μm artemisone (experiment B), 10 μm Fe2+ + 2 μl of DMSO (experiment C), 10 μm Fe2+ + 10 μm artemisone (experiment D), or 10 μm Fe2+ + 100 μm artemisone (experiment E) before starting the reaction. The effect of 15 μm thapsigargin (experiment F) was tested in the same conditions. The effect of DMSO and Fe2+ + DMSO was controlled by the addition of 2 μl of DMSO (experiment A) and 10 μm Fe2+ + 2 μl DMSO (experiment C) to the protein before starting the reaction.

Asawamahasakda, Reaction of antimalarial endoperoxides with specific parasite proteins. 1994, Pubmed

Asawamahasakda, Reaction of antimalarial endoperoxides with specific parasite proteins. 1994, Pubmed
Avery, Structure-activity relationships of the antimalarial agent artemisinin. 1. Synthesis and comparative molecular field analysis of C-9 analogs of artemisinin and 10-deoxoartemisinin. 1993, Pubmed
Baginski, Microdetermination of inorganic phosphate, phospholipids, and total phosphate in biologic materials. 1967, Pubmed
Bhisutthibhan, The Plasmodium falciparum translationally controlled tumor protein homolog and its reaction with the antimalarial drug artemisinin. 1998, Pubmed
Burk, Antimalarial artemisinin drugs induce cytochrome P450 and MDR1 expression by activation of xenosensors pregnane X receptor and constitutive androstane receptor. 2005, Pubmed
Centeno, Expression of the sarcoplasmic reticulum Ca(2+)-ATPase in yeast. 1994, Pubmed
Champeil, Interaction of magnesium and inorganic phosphate with calcium-deprived sarcoplasmic reticulum adenosinetriphosphatase as reflected by organic solvent induced perturbation. 1985, Pubmed
Chen, High-efficiency transformation of mammalian cells by plasmid DNA. 1987, Pubmed
Clausen, Roles of Leu249, Lys252, and Leu253 in membrane segment M3 of sarcoplasmic reticulum Ca2+-ATPase in control of Ca2+ migration and long-range intramolecular communication. 2003, Pubmed
Cojean, Resistance to dihydroartemisinin. 2006, Pubmed
Dondorp, Artemisinin resistance in Plasmodium falciparum malaria. 2009, Pubmed
Eckstein-Ludwig, Artemisinins target the SERCA of Plasmodium falciparum. 2003, Pubmed , Xenbase
Falson, The cytoplasmic loop between putative transmembrane segments 6 and 7 in sarcoplasmic reticulum Ca2+-ATPase binds Ca2+ and is functionally important. 1997, Pubmed
Ferreira, Plasmodium falciparum from Pará state (Brazil) shows satisfactory in vitro response to artemisinin derivatives and absence of the S769N mutation in the SERCA-type PfATPase6. 2008, Pubmed
Fidock, Recent highlights in antimalarial drug resistance and chemotherapy research. 2008, Pubmed
Garah, Docking studies of structurally diverse antimalarial drugs targeting PfATP6: no correlation between in silico binding affinity and in vitro antimalarial activity. 2009, Pubmed
Gietz, Studies on the transformation of intact yeast cells by the LiAc/SS-DNA/PEG procedure. 1995, Pubmed
Golenser, Current perspectives on the mechanism of action of artemisinins. 2006, Pubmed
Gu, Uptake of [3H] dihydroartemisinine by erythrocytes infected with Plasmodium falciparum in vitro. 1984, Pubmed
Hartwig, Accumulation of artemisinin trioxane derivatives within neutral lipids of Plasmodium falciparum malaria parasites is endoperoxide-dependent. 2009, Pubmed
Haviv, Stabilization of Na(+),K(+)-ATPase purified from Pichia pastoris membranes by specific interactions with lipids. 2007, Pubmed
Haynes, Artemisone--a highly active antimalarial drug of the artemisinin class. 2006, Pubmed
Hedfalk, Production, characterization and crystallization of the Plasmodium falciparum aquaporin. 2008, Pubmed
Hoppe, Antimalarial quinolines and artemisinin inhibit endocytosis in Plasmodium falciparum. 2004, Pubmed
Ittarat, Effect of dihydroartemisinin on the antioxidant capacity of P. falciparum-infected erythrocytes. 2003, Pubmed
Jambou, Resistance of Plasmodium falciparum field isolates to in-vitro artemether and point mutations of the SERCA-type PfATPase6. 2005, Pubmed
Jidenko, Crystallization of a mammalian membrane protein overexpressed in Saccharomyces cerevisiae. 2005, Pubmed
Jidenko, Expression in yeast and purification of a membrane protein, SERCA1a, using a biotinylated acceptor domain. 2006, Pubmed
Juul, Do transmembrane segments in proteolyzed sarcoplasmic reticulum Ca(2+)-ATPase retain their functional Ca2+ binding properties after removal of cytoplasmic fragments by proteinase K? 1995, Pubmed
Kaufman, The phosphorylation state of eucaryotic initiation factor 2 alters translational efficiency of specific mRNAs. 1989, Pubmed
Kimura, Cloning of a Ca(2+)-ATPase gene of Plasmodium falciparum and comparison with vertebrate Ca(2+)-ATPases. 1993, Pubmed
Kombila, Light microscopic changes in Plasmodium falciparum from Gabonese children treated with artemether. 1997, Pubmed
Krishna, Artemisinins: their growing importance in medicine. 2008, Pubmed
Krishna, Expression and functional characterization of a Plasmodium falciparum Ca2+-ATPase (PfATP4) belonging to a subclass unique to apicomplexan organisms. 2001, Pubmed , Xenbase
Kumar, Stage-specific gametocytocidal effect in vitro of the antimalaria drug qinghaosu on Plasmodium falciparum. 1990, Pubmed
Lenoir, Overproduction in yeast and rapid and efficient purification of the rabbit SERCA1a Ca(2+)-ATPase. 2002, Pubmed
Lenoir, Functional properties of sarcoplasmic reticulum Ca(2+)-ATPase after proteolytic cleavage at Leu119-Lys120, close to the A-domain. 2004, Pubmed
Li, Yeast model uncovers dual roles of mitochondria in action of artemisinin. 2005, Pubmed
Lund, Detergent structure and associated lipid as determinants in the stabilization of solubilized Ca2+-ATPase from sarcoplasmic reticulum. 1989, Pubmed
Marchand, Crystal structure of D351A and P312A mutant forms of the mammalian sarcoplasmic reticulum Ca(2+) -ATPase reveals key events in phosphorylation and Ca(2+) release. 2008, Pubmed
Maruyama, Mutation of aspartic acid-351, lysine-352, and lysine-515 alters the Ca2+ transport activity of the Ca2+-ATPase expressed in COS-1 cells. 1988, Pubmed
Meshnick, Artemisinin and the antimalarial endoperoxides: from herbal remedy to targeted chemotherapy. 1996, Pubmed
Montigny, Fe2+ -catalyzed oxidative cleavages of Ca2+ -ATPase reveal novel features of its pumping mechanism. 2004, Pubmed
Møller, Enzyme kinetics and substrate stabilization of detergent-solubilized and membraneous (Ca2+ + Mg2+)-activated ATPase from sarcoplasmic reticulum. Effect of protein-protein interactions. 1980, Pubmed
Nagamune, Comparative genomic and phylogenetic analyses of calcium ATPases and calcium-regulated proteins in the apicomplexa. 2006, Pubmed
Nagamune, Artemisinin-resistant mutants of Toxoplasma gondii have altered calcium homeostasis. 2007, Pubmed
Nagamune, Artemisinin induces calcium-dependent protein secretion in the protozoan parasite Toxoplasma gondii. 2007, Pubmed
O'Neill, The therapeutic potential of semi-synthetic artemisinin and synthetic endoperoxide antimalarial agents. 2005, Pubmed
Olliaro, Possible modes of action of the artemisinin-type compounds. 2001, Pubmed
Pompon, Yeast expression of animal and plant P450s in optimized redox environments. 1996, Pubmed
Riganti, Artemisinin induces doxorubicin resistance in human colon cancer cells via calcium-dependent activation of HIF-1alpha and P-glycoprotein overexpression. 2009, Pubmed
Rigaud, Bio-Beads: an efficient strategy for two-dimensional crystallization of membrane proteins. 1997, Pubmed
Sagara, Inhibition of the sarcoplasmic reticulum Ca2+ transport ATPase by thapsigargin at subnanomolar concentrations. 1991, Pubmed
Sharp, The codon Adaptation Index--a measure of directional synonymous codon usage bias, and its potential applications. 1987, Pubmed
Smith, Measurement of protein using bicinchoninic acid. 1985, Pubmed
Sorensen, Mutation Lys758 --> Ile of the sarcoplasmic reticulum Ca2+-ATPase enhances dephosphorylation of E2P and inhibits the E2 to E1Ca2 transition. 1997, Pubmed
Soulié, Treatment with crystalline ultra-pure urea reduces the aggregation of integral membrane proteins without inhibiting N-terminal sequencing. 1998, Pubmed
Stockwin, Artemisinin dimer anticancer activity correlates with heme-catalyzed reactive oxygen species generation and endoplasmic reticulum stress induction. 2009, Pubmed
Tan, Functional reconstitution of purified chloroquine resistance membrane transporter expressed in yeast. 2006, Pubmed
Thastrup, Thapsigargin, a tumor promoter, discharges intracellular Ca2+ stores by specific inhibition of the endoplasmic reticulum Ca2(+)-ATPase. 1990, Pubmed
Toovey, Effect of artemisinins and amino alcohol partner antimalarials on mammalian sarcoendoplasmic reticulum calcium adenosine triphosphatase activity. 2008, Pubmed
Toyoshima, Structural changes in the calcium pump accompanying the dissociation of calcium. 2002, Pubmed
Uhlemann, A single amino acid residue can determine the sensitivity of SERCAs to artemisinins. 2005, Pubmed , Xenbase
Varotti, Plasmodium falciparum malaria parasites display a THG-sensitive Ca2+ pool. 2003, Pubmed
Vial, Phospholipids in parasitic protozoa. 2003, Pubmed
Vilsen, Functional consequences of alterations to amino acids located in the hinge domain of the Ca(2+)-ATPase of sarcoplasmic reticulum. 1991, Pubmed
Vyas, Carrier-mediated partitioning of artemisinin into Plasmodium falciparum-infected erythrocytes. 2002, Pubmed
White, Qinghaosu (artemisinin): the price of success. 2008, Pubmed
Witting, Reaction of human myoglobin and nitric oxide. Heme iron or protein sulfhydryl (s) nitrosation dependence on the absence or presence of oxygen. 2001, Pubmed
Woodrow, Artemisinins. 2005, Pubmed
Zhang, No PfATPase6 S769N mutation found in Plasmodium falciparum isolates from China. 2008, Pubmed
Zhang, Analysis of the antimalarial drug resistance protein Pfcrt expressed in yeast. 2002, Pubmed
de Pilla Varotti, Synthesis, antimalarial activity, and intracellular targets of MEFAS, a new hybrid compound derived from mefloquine and artesunate. 2008, Pubmed
ter Kuile, Plasmodium falciparum: in vitro studies of the pharmacodynamic properties of drugs used for the treatment of severe malaria. 1993, Pubmed