MeBOP Module 2 Introduction
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1 MeBOP Module 2 Introduction
2 Karine Frenal: The expert of Toxo glideosome and IMC Damien Jacot: Expert in about everything
3 Sunil Kumar Dogga: TgAsp3 Budhaditya Mukherjee: Biochemistry
4 Apicomplexans are human and animal pathogens Piroplasmida Theileriidiae Theileria East coast fever Babesiidae Babesia Haemosporida Plasmodium spp Malaria Apicomplexa Coccidia Eimeriida Eimeriidae Eimeria Coccidiosis Neospora Sarcocystidae Toxoplasma Toxoplasmosis Sarcocystis Cryptosporidiidae Cryptosporidium spp cryptosporidiosis
5 Malaria Caused by - Plasmodium falciparum - P. vivax - P. malariae - P. ovale - P. knowlesi Malaria - Fever - Anaemia - Metabolic dysfunctions : acidosis, hypoglycemia At risk - More than 40% of the world population Deaths - Around 0.7 million per year
6 Plasmodium falciparum life cycle oocyste' ookinete'
7 Plasmodium falciparum erythrocytic stages Feeding phase Replication phase Egress & Invasion phase
8 Plasmodium falciparum erythrocytic stages 0!5h%Rings% 5!10h%%%%%%%%%%%%%%%%%%%%%%%%%%%%% 10!20h%%%%%%%%%%%%%%%%%%%%%%%%% Early%trophozoite% Mid!Late%Trophozoite% >40h%Schizont%
9 Apicomplexan invasive tachyzoite and merozoite Micronemes Rhoptries Inner Membrane Complex (IMC) Dense granules Adapted from Frenal K et al (2013) Traffic
10 Toxoplasma gondii Model organism Easy genetics good looking Easy to grow Plasmodium berghei Mouse malaria Easy genetics In vivo only Full life cycle accessible Plasmodium falciparum Human malaria Tricky genetics Relevant
11 Aspartic endopeptidases (ASP/PM)! Present in all eukaryotes! Broad range of roles : - protein degradation - enzyme maturation - signal transduction - virulence factors! Use Asp residues in the motifs DTG or DSG! Pro-region inactivates enzyme! Proteolytic maturation leads to activation
12 Mode of action Activation with water hydroxyl-anion formation Peptidic bond lysis
13 Apicomplexan ASPs follow 6 distinct groups Protein*export* PV/PVM* PMVIII' TgASP4' TgASP2' PMVI' PMVII' TgASP5' PMV' D' F' B' C' E' TgASP6' TgASP7' A' TgASP3' TgASP1' PM1,2,4,HAP' PMX' PMIX' Plasmodium*specific* Hemoglobin*degrada4on*
14 Plasmodium falciparum Plasmepsins 10 aspartic proteases: PfPMI-PfPMX 7 expressed during the erythrocytic stages Food vacuole Hemoglobin degradation ER Export? Expressed during sexual stages
15 Hemoglobin degradation Goldberg, PNAS A massive catabolic process. - Consumes 75% of the infected cell Hb, which provides an important source of amino acids for the parasite growth and maturation - In an acidic food vacuole - Catalyzed by four aspartic proteases (plasmepsins), three cysteine proteases (falcipains) and one metalloprotease (falcilicin) " Drug target?
16 The way you cut your meat reflects the way you live Confucius endocytosis Hemoglobin Hemoglobin Oxyhemoglobin Globin Ferriprotoporphyrin IX Large peptides Small peptides Plasmepsins aspartic-proteases Falcipains cysteine-proteases Falcilysin metallopeptidase Aminopeptidase metallopeptidase Transporter? Amino acids
17 The way you cut your meat reflects the way you live Confucius endocytosis Hemoglobin Hemoglobin Oxyhemoglobin Toxic' Globin Ferriprotoporphyrin IX Polymerization Large peptides Small peptides Hemozoin Non'toxic' Transporter? Amino acids
18 Plasmodium falciparum Plasmepsins 10 aspartic proteases: PfPMI-PfPMX 7 expressed during the erythrocytic stages Food vacuole Hemoglobin degradation ER Export? Expressed during sexual stages
19 Aspartyl proteases implicated in protein export in P. falciparum Maurer s cleft Red Blood Cell cytosol PV PEXEL/HT motif R/KxLxE/Q/D PfPMV - Plasmepsin V Boddey et al, Nature, 2010 Russo et al, Nature, 2010 PMV Nucleus ER Parasite Homologue in T. gondii RxLxE/D TgASP5 - Aspartyl Protease 5 Hsiao et al, Traffic, 2013 Curt-Varesano et al, Cell microbial, 2015 Hammoudi et al, PLoS pathogens, 2015 Coffey et al, elife, 2015
20 Plasmepsins expression throughout the life cycle PMI PMVI PMII PMV PMVII PMIX PMIII/HAP PMIV PMVIII PMX RPKM Ring Early Trophozoite Late Trophozoite Schizont Gametocyte II Gametocyte V Ookinete
21 Plasmodium falciparum Plasmepsins KO viable ' ' ' ' Phenotype None None None None ' Asexual growth???? ' '? Oocyst None?
22 PMIX localizes at the apical end of merozoites PM9-Ty AMA1 PM9-Ty CyRPA
23 PfPMIX-Ty-Lox expression/excision PfPMIX expected size 74 kda PRF
24 PMIX is critical for blood stages development 40% 35% 30% PM9LoxP%+%Rapa% WT%+%Rapa% PM9LoxP% Parasitemia'%' 25% 20% 15% 10% +%Rapa% 5% 0% 0% 1% 2% 3% 4% 5% 6% 7% Days'
25 TgASP3' PMIX' Toxoplasma gondii ASPs PMX' 331!351% 167!187% 340!360% 131!151% 500!521% 155!175% 269!290%
26 Asp3 is a post-golgi resident protease Asp3%3Ty ( 5 UTR' Modified%locus% 3Ty'tag ' SAG1'3 UTR' HXGPRT' 3 UTR' TgASP3LTy' αlmlc1' Merge' TgASP3LTy' GRASPLGFP' Merge'
27 Tet-inducible knock-down of ASP3 ASP3L3Ty' +'ATc' ASP3L3Ty' 130' 100' 70' Pro' 55' 40' Mature' Asp3L3Ty' LATC' +ATC' αty' αcatalase' DTGS% FLAP' DSGT% 30% 298% 489% 3Ty% DTGS% FLAP' DSGT% 298% 489% 3Ty% GAP45LTy'
28 TgAsp3 is critical for Toxo lytic cycle -ATC +ATc Gliding' moulity' AYachment' Invasion' Egress' Intracellular' growth'
29 hydroxyethylamine scaffold-based drug 49c Low%IC50%at%72%hr% High%IC50%at%24hr%
30 hydroxy-ethyl-amine scaffold-based drug 49c IC 50 (24 hours) >500 nm, IC 50 (72 hours) 0.6 nm Ciana et al Peptidomimetic inhibitor of aspartic proteases - Designed to target Plasmodium food vacuole aspartyl proteases - slow acting drug and dropped
31 Compound 49c efficiently blocks Toxo lytic cycle IC nm (4 days) CTL' 1000'nM' 500'nM' 250'nM' 125'nM' 63'nM' 31'nM' 16'nM' 8'nM' 4'nM' 2'nM' 1'µM'PYR'
32 What we want! - Functional characterization of PfPMIX - Functional characterization of TgAsp3 - Molecular targets of compound 49c
Biochimica et Biophysica Acta xxx (2011) xxx xxx. Contents lists available at ScienceDirect. Biochimica et Biophysica Acta
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