Inhibition of Plasmepsin V Activity Demonstrates Its Essential Role in Protein Export, PfEMP1 Display, and Survival of Malaria Parasites
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{"title"=>"Inhibition of Plasmepsin V Activity Demonstrates Its Essential Role in Protein Export, PfEMP1 Display, and Survival of Malaria Parasites", "type"=>"journal", "authors"=>[{"first_name"=>"Brad E.", "last_name"=>"Sleebs", "scopus_author_id"=>"10042470100"}, {"first_name"=>"Sash", "last_name"=>"Lopaticki", "scopus_author_id"=>"6507830490"}, {"first_name"=>"Danushka S.", "last_name"=>"Marapana", "scopus_author_id"=>"36810074500"}, {"first_name"=>"Matthew T.", "last_name"=>"O'Neill", "scopus_author_id"=>"35725876200"}, {"first_name"=>"Pravin", "last_name"=>"Rajasekaran", "scopus_author_id"=>"56373434200"}, {"first_name"=>"Michelle", "last_name"=>"Gazdik", "scopus_author_id"=>"56308671600"}, {"first_name"=>"Svenja", "last_name"=>"Günther", "scopus_author_id"=>"57198020370"}, {"first_name"=>"Lachlan W.", "last_name"=>"Whitehead", "scopus_author_id"=>"23969683900"}, {"first_name"=>"Kym N.", "last_name"=>"Lowes", "scopus_author_id"=>"8538084200"}, {"first_name"=>"Lea", "last_name"=>"Barfod", "scopus_author_id"=>"6506596610"}, {"first_name"=>"Lars", "last_name"=>"Hviid", "scopus_author_id"=>"7005571610"}, {"first_name"=>"Philip J.", "last_name"=>"Shaw", "scopus_author_id"=>"24469250700"}, {"first_name"=>"Anthony N.", "last_name"=>"Hodder", "scopus_author_id"=>"7005675754"}, {"first_name"=>"Brian J.", "last_name"=>"Smith", "scopus_author_id"=>"57200588114"}, {"first_name"=>"Alan F.", "last_name"=>"Cowman", "scopus_author_id"=>"7006258900"}, {"first_name"=>"Justin A.", "last_name"=>"Boddey", "scopus_author_id"=>"6506639761"}], "year"=>2014, "source"=>"PLoS Biology", "identifiers"=>{"pui"=>"373686445", "sgr"=>"84905008960", "issn"=>"15457885", "pmid"=>"24983235", "scopus"=>"2-s2.0-84905008960", "doi"=>"10.1371/journal.pbio.1001897", "isbn"=>"1545-7885 (Electronic)\\r1544-9173 (Linking)"}, "id"=>"db604973-11a4-38e3-9200-0b3096579b79", "abstract"=>"The malaria parasite Plasmodium falciparum exports several hundred proteins into the infected erythrocyte that are involved in cellular remodeling and severe virulence. The export mechanism involves the Plasmodium export element (PEXEL), which is a cleavage site for the parasite protease, Plasmepsin V (PMV). The PMV gene is refractory to deletion, suggesting it is essential, but definitive proof is lacking. Here, we generated a PEXEL-mimetic inhibitor that potently blocks the activity of PMV isolated from P. falciparum and Plasmodium vivax. Assessment of PMV activity in P. falciparum revealed PEXEL cleavage occurs cotranslationaly, similar to signal peptidase. Treatment of P. falciparum-infected erythrocytes with the inhibitor caused dose-dependent inhibition of PEXEL processing as well as protein export, including impaired display of the major virulence adhesin, PfEMP1, on the erythrocyte surface, and cytoadherence. The inhibitor killed parasites at the trophozoite stage and knockdown of PMV enhanced sensitivity to the inhibitor, while overexpression of PMV increased resistance. This provides the first direct evidence that PMV activity is essential for protein export in Plasmodium spp. and for parasite survival in human erythrocytes and validates PMV as an antimalarial drug target.", "link"=>"http://www.mendeley.com/research/inhibition-plasmepsin-v-activity-demonstrates-essential-role-protein-export-pfemp1-display-survival", "reader_count"=>93, "reader_count_by_academic_status"=>{"Professor > Associate Professor"=>5, "Researcher"=>19, "Student > Doctoral Student"=>6, "Student > Ph. D. Student"=>32, "Student > Postgraduate"=>2, "Student > Master"=>10, "Other"=>2, "Student > Bachelor"=>12, "Lecturer"=>2, "Lecturer > Senior Lecturer"=>1, "Professor"=>2}, "reader_count_by_user_role"=>{"Professor > Associate Professor"=>5, "Researcher"=>19, "Student > Doctoral Student"=>6, "Student > Ph. D. Student"=>32, "Student > Postgraduate"=>2, "Student > Master"=>10, "Other"=>2, "Student > Bachelor"=>12, "Lecturer"=>2, "Lecturer > Senior Lecturer"=>1, "Professor"=>2}, "reader_count_by_subject_area"=>{"Unspecified"=>1, "Biochemistry, Genetics and Molecular Biology"=>16, "Medicine and Dentistry"=>10, "Agricultural and Biological Sciences"=>53, "Design"=>1, "Chemistry"=>10, "Social Sciences"=>1, "Economics, Econometrics and Finance"=>1}, "reader_count_by_subdiscipline"=>{"Design"=>{"Design"=>1}, "Medicine and Dentistry"=>{"Medicine and Dentistry"=>10}, "Chemistry"=>{"Chemistry"=>10}, "Social Sciences"=>{"Social Sciences"=>1}, "Economics, Econometrics and Finance"=>{"Economics, Econometrics and Finance"=>1}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>53}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>16}, "Unspecified"=>{"Unspecified"=>1}}, "reader_count_by_country"=>{"Canada"=>1, "Austria"=>1, "Burkina Faso"=>1, "Belgium"=>1, "United States"=>2, "United Kingdom"=>2, "Germany"=>1, "India"=>1}, "group_count"=>4}

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/1576691"], "description"=>"<p>(A) Immunoblot with Rα-PfPMV antibodies shows successful integration of the PMVHA-<i>glmS</i> or -M9 plasmid (M9 is an inactive <i>glmS</i> riboswitch control). The upper band (α-PfPMV blot) in lane 1 (denoted by *) is nonspecific. The same blot is shown below, probed with α-HA antibodies. HSP70 is a loading control. (B) Knockdown of PMV in <i>P. falciparum</i> NF54 following 5 mM GlcN treatment. (Left) 0 h GlcN treatment of trophozoites causes no knockdown. (Center) The 24 h GlcN treatment of trophozoites causes ∼80% knockdown of PMV in subsequent rings compared to “−GlcN.” (Right) 48 h GlcN treatment of trophozoites causes ∼90% knockdown of PMV in subsequent trophozoites compared to “−GlcN.” A small degree of knockdown is seen for M9, indicating GlcN has a minor effect. (C) PMV knockdown by GlcN has no significant effect on parasite growth rate (<i>p</i> = .6250). Trophozoites were treated with 0 mM or 5 mM GlcN and parasitaemia determined 48 h later by flow cytometry. Data are % growth “+GlcN” relative to “−GlcN,” and data are mean ±SEM of a representative of duplicate experiments. (D) PEXEL processing of PfEMP3-GFP in <i>P. falciparum</i> parasites expressing PMVHA-<i>glmS</i> is reduced more by 916 treatment when PMV is knocked down [+GlcN (5 mM for 48 h prior to addition of 916)]. Densitometry shows the ratio of uncleaved to PEXEL-cleaved protein in each lane beneath the blot. Note that PfEMP3-GFP expression is lower in “+GlcN” parasites despite relatively similar HSP70 levels. (E) Dose-response curves of <i>P. falciparum</i> expressing PMVHA-<i>glmS</i> shows parasites have enhanced sensitivity to 916 following PMV knockdown (3.3-fold decrease in EC<sub>50</sub>). Parasitemia was determined 72 h after addition of 916 to ring parasites with or without PMV knockdown (knockdown ring parasites were obtained by adding 6 mM GlcN to trophozoites for 24 h). GlcN and 916 were maintained in the culture medium throughout. (F) Dose-response curves of <i>P. falciparum</i> overexpressing PvPMVHA or a mini PfEMP1HA reporter (miniVarHA) in the presence of 5 nM WR99210 show parasites have increased resistance to 916 when PMV is overexpressed (1.9-fold increase in EC<sub>50</sub>).</p>", "links"=>[], "tags"=>["cell biology", "Cell physiology", "Membrane trafficking", "Molecular cell biology", "organisms", "protozoans", "Parasitic protozoans", "Malarial parasites", "Plasmodium falciparum", "Plasmodium vivax", "genetics", "Gene function", "Gene identification and analysis", "Molecular genetics", "mutagenesis", "mutation", "Biochemistry", "enzymology", "Enzyme kinetics", "enzymes", "chemical biology", "microbiology", "Medical microbiology", "Parasitology", "Computing methods", "Mathematical computing", "Pathology and laboratory medicine", "pathogenesis", "Host-pathogen interactions", "Infectious diseases", "Infectious disease control", "Parasitic diseases", "malaria", "pharmacology", "Drug research and development", "drug discovery", "chemistry", "mathematics", "knockdown", "overexpression", "modulates"], "article_id"=>1090517, "categories"=>["Biological Sciences"], "users"=>["Brad E. Sleebs", "Sash Lopaticki", "Danushka S. Marapana", "Matthew T. O'Neill", "Pravin Rajasekaran", "Michelle Gazdik", "Svenja Günther", "Lachlan W. Whitehead", "Kym N. Lowes", "Lea Barfod", "Lars Hviid", "Philip J. Shaw", "Anthony N. Hodder", "Brian J. Smith", "Alan F. Cowman", "Justin A. Boddey"], "doi"=>"https://dx.doi.org/10.1371/journal.pbio.1001897.g005", "stats"=>{"downloads"=>6, "page_views"=>9, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_PMV_knockdown_or_overexpression_modulates_sensitivity_to_WEHI_916_/1090517", "title"=>"PMV knockdown or overexpression modulates sensitivity to WEHI-916.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-07-01 03:32:19"}
  • {"files"=>["https://ndownloader.figshare.com/files/1576687"], "description"=>"<p>(A) Nomenclature for each residue in the PEXEL substrate (circles) and each pocket of the PMV active site (semicircles) with respect to the cleavage site (arrow). (B) Compound structures in this study include PMV inhibitor WEHI-916 and control analogs WEHI-024 and WEHI-025. (C) Structural model of PfPMV bound to WEHI-916. Residues forming the S<sub>3</sub> site, Try177, Glu179, and Glu215, form interactions with the guanidine side chain of WEHI-916. The Leu side chain of WEHI-916 packs tightly against the side-chain groups of Ile116, Tyr177, and Val227 in the S<sub>1</sub> site. The statine hydroxyl forms hydrogen bonds with the two catalytic aspartate residues Asp118 and Asp365. (D) Inhibition of PfPMV and PvPMV by WEHI-916 (blue) and weak activities of WEHI-024 (red) and WEHI-025 (brown). The grey box summarizes compound activity against BACE-1 and Cathepsin D (CathD) and lack of toxicity against human HEpG2 cells and Human Foreskin Fibroblasts (HFF).</p>", "links"=>[], "tags"=>["cell biology", "Cell physiology", "Membrane trafficking", "Molecular cell biology", "organisms", "protozoans", "Parasitic protozoans", "Malarial parasites", "Plasmodium falciparum", "Plasmodium vivax", "genetics", "Gene function", "Gene identification and analysis", "Molecular genetics", "mutagenesis", "mutation", "Biochemistry", "enzymology", "Enzyme kinetics", "enzymes", "chemical biology", "microbiology", "Medical microbiology", "Parasitology", "Computing methods", "Mathematical computing", "Pathology and laboratory medicine", "pathogenesis", "Host-pathogen interactions", "Infectious diseases", "Infectious disease control", "Parasitic diseases", "malaria", "pharmacology", "Drug research and development", "drug discovery", "chemistry", "mathematics", "pexel-mimetic", "inhibitor"], "article_id"=>1090513, "categories"=>["Biological Sciences"], "users"=>["Brad E. Sleebs", "Sash Lopaticki", "Danushka S. Marapana", "Matthew T. O'Neill", "Pravin Rajasekaran", "Michelle Gazdik", "Svenja Günther", "Lachlan W. Whitehead", "Kym N. Lowes", "Lea Barfod", "Lars Hviid", "Philip J. Shaw", "Anthony N. Hodder", "Brian J. Smith", "Alan F. Cowman", "Justin A. Boddey"], "doi"=>"https://dx.doi.org/10.1371/journal.pbio.1001897.g002", "stats"=>{"downloads"=>1, "page_views"=>13, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_A_PEXEL_mimetic_inhibitor_of_PMV_/1090513", "title"=>"A PEXEL-mimetic inhibitor of PMV.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-07-01 03:32:19"}
  • {"files"=>["https://ndownloader.figshare.com/files/1576686"], "description"=>"<p>(A) Structure and size of PMVHA proteins used in this study. Catalytic dyad residues DTG/DSG are shown including Asp to Ala mutations* in red. TM, transmembrane domain. (B) Immunoblot of infected erythrocytes with α-HA antibodies shows expression of PMVHA proteins in <i>P. falciparum</i>. Sizes indicate that the signal peptides were removed (PfPMVHA, 69.1 kDa; PvPMVHA, 60.9 kDa). (C) Immunoblotting of infected erythrocytes with rabbit α-PfPMV antibodies (Rα-PfPMV) validates they are specific for PfPMV. Endogenous PfPMV is the lower band (lanes 1, 3, 4, 5), and the larger band corresponds to 3× HA-tagged PfPMV (lanes 2, 4). Aldolase is a loading control in (B) and (C) and shows slight overloading of some lanes compared to others. (D, Top) Immunofluorescence micrographs show rabbit α-PfPMV antibodies (Rα-PfPMV, green) label PfPMV in the ER. Colocalizations were performed with mouse α-PfPMV antibodies (Mα-PfPMV, red), shown previously to label PMV in the ER <a href=\"http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001897#pbio.1001897-Klemba1\" target=\"_blank\">[16]</a>. (Middle) α-HA antibodies (red) label PfPMVHA (Top) and PvPMVHA (Bottom) in the parasite ER. (Bottom) α-HA antibodies (red) label PvPMVHA in the ER, as shown by clocalization with ERC (green). (E) Immunopurified PfPMVHA and PvPMVHA cleave KAHRP peptides containing the PEXEL sequence RTLAQ but not peptides containing point mutations R>K, L>I, or RL>A. Pf and Pv PMVmutHA proteins with catalytic D>A mutations did not cleave the KAHRP RTLAQ peptide. (F) Overexpression of PfPMVmutHA from episomes in <i>P. falciparum</i> 3D7 impairs growth relative to expression of a similar episomal construct encoding a mini PfEMP1HA reporter (miniVarHA). Parasites expressing episomes were selected on 5 nM WR99210 (WR). Two starting inocula were used in triplicate wells, and parasitaemia was determined at 72 h. *<i>p</i><.0001 (<i>t</i> test). Data are mean ± SEM from duplicate experiments.</p>", "links"=>[], "tags"=>["cell biology", "Cell physiology", "Membrane trafficking", "Molecular cell biology", "organisms", "protozoans", "Parasitic protozoans", "Malarial parasites", "Plasmodium falciparum", "Plasmodium vivax", "genetics", "Gene function", "Gene identification and analysis", "Molecular genetics", "mutagenesis", "mutation", "Biochemistry", "enzymology", "Enzyme kinetics", "enzymes", "chemical biology", "microbiology", "Medical microbiology", "Parasitology", "Computing methods", "Mathematical computing", "Pathology and laboratory medicine", "pathogenesis", "Host-pathogen interactions", "Infectious diseases", "Infectious disease control", "Parasitic diseases", "malaria", "pharmacology", "Drug research and development", "drug discovery", "chemistry", "mathematics"], "article_id"=>1090512, "categories"=>["Biological Sciences"], "users"=>["Brad E. Sleebs", "Sash Lopaticki", "Danushka S. Marapana", "Matthew T. O'Neill", "Pravin Rajasekaran", "Michelle Gazdik", "Svenja Günther", "Lachlan W. Whitehead", "Kym N. Lowes", "Lea Barfod", "Lars Hviid", "Philip J. Shaw", "Anthony N. Hodder", "Brian J. Smith", "Alan F. Cowman", "Justin A. Boddey"], "doi"=>"https://dx.doi.org/10.1371/journal.pbio.1001897.g001", "stats"=>{"downloads"=>7, "page_views"=>7, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_PMV_conservation_and_expression_/1090512", "title"=>"PMV conservation and expression.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-07-01 03:32:19"}
  • {"files"=>["https://ndownloader.figshare.com/files/1576695", "https://ndownloader.figshare.com/files/1576696", "https://ndownloader.figshare.com/files/1576697", "https://ndownloader.figshare.com/files/1576698", "https://ndownloader.figshare.com/files/1576699", "https://ndownloader.figshare.com/files/1576700", "https://ndownloader.figshare.com/files/1576701", "https://ndownloader.figshare.com/files/1576702"], "description"=>"<div><p>The malaria parasite <i>Plasmodium falciparum</i> exports several hundred proteins into the infected erythrocyte that are involved in cellular remodeling and severe virulence. The export mechanism involves the <i>Plasmodium</i> export element (PEXEL), which is a cleavage site for the parasite protease, Plasmepsin V (PMV). The <i>PMV</i> gene is refractory to deletion, suggesting it is essential, but definitive proof is lacking. Here, we generated a PEXEL-mimetic inhibitor that potently blocks the activity of PMV isolated from <i>P. falciparum</i> and <i>Plasmodium vivax</i>. Assessment of PMV activity in <i>P. falciparum</i> revealed PEXEL cleavage occurs cotranslationaly, similar to signal peptidase. Treatment of <i>P. falciparum</i>–infected erythrocytes with the inhibitor caused dose-dependent inhibition of PEXEL processing as well as protein export, including impaired display of the major virulence adhesin, PfEMP1, on the erythrocyte surface, and cytoadherence. The inhibitor killed parasites at the trophozoite stage and knockdown of PMV enhanced sensitivity to the inhibitor, while overexpression of PMV increased resistance. This provides the first direct evidence that PMV activity is essential for protein export in <i>Plasmodium</i> spp. and for parasite survival in human erythrocytes and validates PMV as an antimalarial drug target.</p></div>", "links"=>[], "tags"=>["cell biology", "Cell physiology", "Membrane trafficking", "Molecular cell biology", "organisms", "protozoans", "Parasitic protozoans", "Malarial parasites", "Plasmodium falciparum", "Plasmodium vivax", "genetics", "Gene function", "Gene identification and analysis", "Molecular genetics", "mutagenesis", "mutation", "Biochemistry", "enzymology", "Enzyme kinetics", "enzymes", "chemical biology", "microbiology", "Medical microbiology", "Parasitology", "Computing methods", "Mathematical computing", "Pathology and laboratory medicine", "pathogenesis", "Host-pathogen interactions", "Infectious diseases", "Infectious disease control", "Parasitic diseases", "malaria", "pharmacology", "Drug research and development", "drug discovery", "chemistry", "mathematics", "plasmepsin", "demonstrates", "pfemp1"], "article_id"=>1090521, "categories"=>["Biological Sciences"], "users"=>["Brad E. Sleebs", "Sash Lopaticki", "Danushka S. Marapana", "Matthew T. O'Neill", "Pravin Rajasekaran", "Michelle Gazdik", "Svenja Günther", "Lachlan W. Whitehead", "Kym N. Lowes", "Lea Barfod", "Lars Hviid", "Philip J. Shaw", "Anthony N. Hodder", "Brian J. Smith", "Alan F. Cowman", "Justin A. Boddey"], "doi"=>[nil, nil, nil, nil, nil, nil, nil, nil], "stats"=>{"downloads"=>112, "page_views"=>55, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/Inhibition_of_Plasmepsin_V_Activity_Demonstrates_Its_Essential_Role_in_Protein_Export_PfEMP1_Display_and_Survival_of_Malaria_Parasites/1090521", "title"=>"Inhibition of Plasmepsin V Activity Demonstrates Its Essential Role in Protein Export, PfEMP1 Display, and Survival of Malaria Parasites", "pos_in_sequence"=>0, "defined_type"=>4, "published_date"=>"2014-07-01 03:32:19"}
  • {"files"=>["https://ndownloader.figshare.com/files/1576692"], "description"=>"<p>(A) (Top) Immunofluorescent micrographs show Hyp8-GFP is exported and localizes within puncta in the infected erythrocyte. (Middle) Hyp8-HA localizes within SBP1-containing MCs. (Right) Immunoelectron microscopy shows Hyp8-HA localization at MCs. Scale bar is 100 nm. (B) Maximum intensity projection micrographs showing export of Hyp8-GFP to MCs and secretion of EXP2 to the parasitophorous vacuole membrane following treatment with DMSO or 916 (50 µM). Puncta of nonexported GFP within the parasite and vacuole is shown (arrows). (C) GFP intensity in MCs (outside EXP2 signal) and within the parasite and parasitophorous vacuole (inside EXP2 signal) was quantified following drug treatment (see <a href=\"http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001897#s4\" target=\"_blank\">Materials and Methods</a>) and is presented as a ratio: exported = ratio outside/inside EXP2. The number of infected cells counted (<i>n</i>) is shown. (D) GFP within the parasite and parasitophorous vacuole (inside EXP2 signal) was quantified across treatments and is presented as a ratio: nonexported = ratio inside/outside EXP2. (E) EXP2 intensity in the parasitophorous vacuole membrane (red) was quantified across treatments and is presented as average (threshold) signal. (F) The number of GFP-positive MCs per infected erythrocyte was quantified across treatments. Error bars represent the mean ±SEM, and <i>p</i> values were determined by ANOVA in (C–F). (G) Surface-exposed PfEMP1 (VAR2CSA) on infected erythrocytes was measured following inhibitor treatment by FACS using monoclonal human PAM1.4 serum <a href=\"http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001897#pbio.1001897-Barfod1\" target=\"_blank\">[30]</a>. Geometric mean fluorescence of >100,000 cells per condition are shown relative to no treatment. Parasites received inhibitor at 15 µM for 23 h (15 µM) or 50 µM for 12 h followed by a reduction to 15 µM for 11 h (50>15 µM). Shown is a single representative of duplicate experiments. Raw FACS data are presented in <a href=\"http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001897#pbio.1001897.s007\" target=\"_blank\">Figure S7</a>. (H) Adhesion of infected red blood cells (iRBCs) to chondroitin sulfate A (CSA) under static conditions. Adherent iRBCs were counted in ten 0.28 mm<sup>2</sup> fields of view per sample from triplicate samples and are shown as number of iRBCs per mm<sup>2</sup>. Shown is a single representative of duplicate experiments. Data represent mean ±SEM. 95% confidence intervals are shown (grey dashed lines). 916 significantly reduced adhesion to CSA <i>p</i><.0001.</p>", "links"=>[], "tags"=>["cell biology", "Cell physiology", "Membrane trafficking", "Molecular cell biology", "organisms", "protozoans", "Parasitic protozoans", "Malarial parasites", "Plasmodium falciparum", "Plasmodium vivax", "genetics", "Gene function", "Gene identification and analysis", "Molecular genetics", "mutagenesis", "mutation", "Biochemistry", "enzymology", "Enzyme kinetics", "enzymes", "chemical biology", "microbiology", "Medical microbiology", "Parasitology", "Computing methods", "Mathematical computing", "Pathology and laboratory medicine", "pathogenesis", "Host-pathogen interactions", "Infectious diseases", "Infectious disease control", "Parasitic diseases", "malaria", "pharmacology", "Drug research and development", "drug discovery", "chemistry", "mathematics", "inhibition", "impairs", "pfemp1"], "article_id"=>1090518, "categories"=>["Biological Sciences"], "users"=>["Brad E. Sleebs", "Sash Lopaticki", "Danushka S. Marapana", "Matthew T. O'Neill", "Pravin Rajasekaran", "Michelle Gazdik", "Svenja Günther", "Lachlan W. Whitehead", "Kym N. Lowes", "Lea Barfod", "Lars Hviid", "Philip J. Shaw", "Anthony N. Hodder", "Brian J. Smith", "Alan F. Cowman", "Justin A. Boddey"], "doi"=>"https://dx.doi.org/10.1371/journal.pbio.1001897.g006", "stats"=>{"downloads"=>4, "page_views"=>20, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_PMV_inhibition_impairs_protein_export_PfEMP1_display_and_cytoadherence_/1090518", "title"=>"PMV inhibition impairs protein export, PfEMP1 display, and cytoadherence.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-07-01 03:32:19"}
  • {"files"=>["https://ndownloader.figshare.com/files/1576689"], "description"=>"<p>(A) Dose-response curves of <i>P. falciparum</i> 3D7 in the presence of 916, 024, or 025. EC<sub>50</sub> values are shown. (B) Parasitemia measured at 72 h (<i>y</i>-axis) following drug treatment at rings (30 min postinvasion) and replacement of the medium with inhibitor-free medium (wash-out) at the time intervals shown (<i>x</i>-axis). (C) Parasitemia at 72 h (<i>y</i>-axis) after replacement of inhibitor-free medium with media containing compounds at the intervals shown (<i>x</i>-axis). Parasitemia was determined by FACS in (A–C) and is relative to DMSO treatment in (B) and (C). Concentrations are as follows: 916, 024, 025 (15 µM); CQ, chloroquine (150 ng/ml); ART, artemisinin (100 ng/ml). Error bars in (A–C) are mean ±SEM from duplicate experiments. (D) Light micrographs of Giemsa-stained parasites 16 and 32 h after drug treatment at early rings (15 µM). 916-treated parasites failed to develop into trophozoites and did not recover. Ring parasites treated with E-64 (10 µM) <a href=\"http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001897#pbio.1001897-terKuile1\" target=\"_blank\">[22]</a> contained swollen food vacuoles (arrow) due to inhibition of proteases involved in hemoglobin degradation; however, treatment with DMSO, 916, 024, or 025 did not cause swelling. Swelling was quantified using 500 infected cells per condition in duplicate. Scale bar is 6 µm.</p>", "links"=>[], "tags"=>["cell biology", "Cell physiology", "Membrane trafficking", "Molecular cell biology", "organisms", "protozoans", "Parasitic protozoans", "Malarial parasites", "Plasmodium falciparum", "Plasmodium vivax", "genetics", "Gene function", "Gene identification and analysis", "Molecular genetics", "mutagenesis", "mutation", "Biochemistry", "enzymology", "Enzyme kinetics", "enzymes", "chemical biology", "microbiology", "Medical microbiology", "Parasitology", "Computing methods", "Mathematical computing", "Pathology and laboratory medicine", "pathogenesis", "Host-pathogen interactions", "Infectious diseases", "Infectious disease control", "Parasitic diseases", "malaria", "pharmacology", "Drug research and development", "drug discovery", "chemistry", "mathematics", "lethal"], "article_id"=>1090515, "categories"=>["Biological Sciences"], "users"=>["Brad E. Sleebs", "Sash Lopaticki", "Danushka S. Marapana", "Matthew T. O'Neill", "Pravin Rajasekaran", "Michelle Gazdik", "Svenja Günther", "Lachlan W. Whitehead", "Kym N. Lowes", "Lea Barfod", "Lars Hviid", "Philip J. Shaw", "Anthony N. Hodder", "Brian J. Smith", "Alan F. Cowman", "Justin A. Boddey"], "doi"=>"https://dx.doi.org/10.1371/journal.pbio.1001897.g004", "stats"=>{"downloads"=>0, "page_views"=>13, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_WEHI_916_is_lethal_to_P_falciparum_3D7_/1090515", "title"=>"WEHI-916 is lethal to <i>P. falciparum</i> 3D7.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-07-01 03:32:19"}
  • {"files"=>["https://ndownloader.figshare.com/files/1576688"], "description"=>"<p>(A) Immunoblotting with α-GFP antibodies shows dose-dependent inhibition of PEXEL cleavage of PfEMP3-GFP in parasites after 5 h 916 treatment at the indicated concentrations. Uncleaved protein (black arrow), PEXEL-cleaved protein (blue arrow), and degraded chimera in food vacuole (GFP only) are labeled. PfEMP3-GFP R>A PEXEL mutant is shown as a size control. (B) Immunoblot shows time-dependent inhibition of PEXEL cleavage in parasites. HSP70 is a loading control in (A) and (B) and densitometry of the uncleaved band in each lane is shown below the blots in (A) and (B). (C) Immunoblot shows 916 treatment (20 and 50 µM for 5 h) causes accumulation of uncleaved PfEMP3-GFP and KAHRP-GFP (black arrow), but 024 and 025 have no effect. R>A PEXEL mutant size controls are shown. Signal peptide-cleaved species of KAHRP-GFP, but not PfEMP3-GFP, can be seen (red arrow). (D) Immunoblotting shows no defect (black arrow indicates the predicted size of uncleaved protein; ∼30 kDa) in signal peptide cleavage (red arrow; 27 kDa) of SERA5s-GFP, which lacks a PEXEL, following treatment with 916, 024, or 025 (20 µM for 5 h). Aldolase was a loading control in (C) and (D). (E) <sup>35</sup>S-Methionine/Cysteine labeling of PfEMP3-GFP in parasites reveals the rapid rate of translation, ER import, and N-terminal processing in <i>P. falciparum</i>. Uncleaved (black arrow), signal peptide-cleaved (red arrow), PEXEL-cleaved (blue arrow), and GFP-only (food vacuole) species are shown. Densitometry of each band per lane (colored traces) is shown. Uncleaved (Un, black), signal peptide-cleaved (SP, red), and PEXEL-cleaved (PEX, blue) bands are shown as percentage of total intensity per lane. Signal peptide-cleaved PfEMP3-GFP can be seen (red arrow, trace). (F) The experiment in (E) was performed after 916 treatment (20 µM for 5 h). Uncleaved protein was most abundant. Note low intensity of bands indicated by red and blue arrows and delay in their appearance compared to (E). (G) Densitometry showing the ratio of PEXEL-cleaved to -uncleaved PfEMP3-GFP with or without 916 treatment (determined using 15 min lanes in (E) and (F)).</p>", "links"=>[], "tags"=>["cell biology", "Cell physiology", "Membrane trafficking", "Molecular cell biology", "organisms", "protozoans", "Parasitic protozoans", "Malarial parasites", "Plasmodium falciparum", "Plasmodium vivax", "genetics", "Gene function", "Gene identification and analysis", "Molecular genetics", "mutagenesis", "mutation", "Biochemistry", "enzymology", "Enzyme kinetics", "enzymes", "chemical biology", "microbiology", "Medical microbiology", "Parasitology", "Computing methods", "Mathematical computing", "Pathology and laboratory medicine", "pathogenesis", "Host-pathogen interactions", "Infectious diseases", "Infectious disease control", "Parasitic diseases", "malaria", "pharmacology", "Drug research and development", "drug discovery", "chemistry", "mathematics", "blocks", "pexel", "cleavage"], "article_id"=>1090514, "categories"=>["Biological Sciences"], "users"=>["Brad E. Sleebs", "Sash Lopaticki", "Danushka S. Marapana", "Matthew T. O'Neill", "Pravin Rajasekaran", "Michelle Gazdik", "Svenja Günther", "Lachlan W. Whitehead", "Kym N. Lowes", "Lea Barfod", "Lars Hviid", "Philip J. Shaw", "Anthony N. Hodder", "Brian J. Smith", "Alan F. Cowman", "Justin A. Boddey"], "doi"=>"https://dx.doi.org/10.1371/journal.pbio.1001897.g003", "stats"=>{"downloads"=>0, "page_views"=>19, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_WEHI_916_blocks_PEXEL_cleavage_in_P_falciparum_/1090514", "title"=>"WEHI-916 blocks PEXEL cleavage in <i>P. falciparum</i>.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-07-01 03:32:19"}

PMC Usage Stats | Further Information

  • {"unique-ip"=>"53", "full-text"=>"48", "pdf"=>"24", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"38", "supp-data"=>"4", "cited-by"=>"0", "year"=>"2014", "month"=>"7"}
  • {"unique-ip"=>"22", "full-text"=>"18", "pdf"=>"12", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"5", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2014", "month"=>"8"}
  • {"unique-ip"=>"26", "full-text"=>"20", "pdf"=>"12", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"14", "supp-data"=>"6", "cited-by"=>"0", "year"=>"2014", "month"=>"9"}
  • {"unique-ip"=>"35", "full-text"=>"63", "pdf"=>"13", "abstract"=>"1", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"8", "supp-data"=>"2", "cited-by"=>"0", "year"=>"2014", "month"=>"10"}
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