Biochemistry of Plasmodium Mark F. Wiser http://www.tulane.edu/~wiser/malaria/fv.html

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<ul><li> Slide 1 </li> <li> Biochemistry of Plasmodium Mark F. Wiser http://www.tulane.edu/~wiser/malaria/fv.html </li> <li> Slide 2 </li> <li> Slide 3 </li> <li> Sources of Amino Acids De Novo Synthesis CO 2 fixation (ala, asp, glu) little incorporated into protein Host Plasma uptake of all amino acids in vitro growth requires ile, met, cys, gln, glu Digestion of Host Hemoglobin </li> <li> Slide 4 </li> <li> Hemoglobin 95% of total erythrocyte protein very abundant (340 mg/ml or approximately 5 mM) 60-80% is degraded during erythrocytic stage 110 g (of 750 total) is consumed in 48 hrs at 20% parasitemia </li> <li> Slide 5 </li> <li> Endocytosis of Host Cytoplasm food vacuole cytostome pinocytosis (rings) </li> <li> Slide 6 </li> <li> The Food Vacuole A Specialized Lysosome ATP ADP H+H+ (pH 5-5.4) Food Vacuole Proteases plasmepsins I - IV (acid) falcipains I - III (thiol) falcilysin (metallo) Absent: other acid hydrolases except acid phosphatase hemoglobin digestion parasite cytoplasm Endocytic Pathway </li> <li> Slide 7 </li> <li> Proteases Mediate the Catabolism of Proteins proteases (aka peptidases) break the peptide bonds that hold amino acids together exopeptidases remove amino acids sequentially from either N- or C-terminus endopeptidases cleave between specific residues within polypeptide chain </li> <li> Slide 8 </li> <li> Initial plasmepsin cleavage is specific and leads to a destabilization of hemoglobin native Hb is cleaved between Phe-33 and Leu-34 ( chains) hinge region conserved important for tetramer stability the large globin fragments dissociate heme is released globin fragments are susceptible to further proteolysis F33/L34 </li> <li> Slide 9 </li> <li> Hemoglobin Digestion is an Ordered Process dipeptidyl aminopeptidase aminopeptidase hemoglobin + heme large globin fragments small peptides (6-8 amino acids) plasmepsin falcipain plasmepsin medium fragments (~20 amino acids) falcilysin amino acids aminopeptidase di-peptides </li> <li> Slide 10 </li> <li> Membrane Transport Channel proteins (hydrophilic pores) Carrier proteins substrate specific most require energy ATPase or gradients 6 amino acid transporters identified in Plasmodium genome (location?) PfMDR-1 and PfCRT located on food vacuole membrane </li> <li> Slide 11 </li> <li> PfMDR-1 Member of ABC (ATP-binding cassette) transporter super family Associated with drug resistance (MDR = multi-drug resistance) Capable of peptide transport complements yeast ste6 gene (transporter of yeast peptide mating factor) However, imports solutes (including drugs) into food vacuole </li> <li> Slide 12 </li> <li> PfCRT Member of DMT (drug/metabolite transporter) super family Associated with chloroquine resistance (CRT = chloroquine resistance transporter) Exports chloroquine and other drugs from the food vacuole Peptides can block drug export </li> <li> Slide 13 </li> <li> The Food Vacuole A Specialized Lysosome hemoglobin +heme globin fragments small fragments (6-8 amino acids) ATP ADP ATP ADP H+H+ plasmepsin falcipain plasmepsin falcilysin PfCRT? amino acids amino- peptidase transporter s associated with food vacuole amino-peptidase activities in parasite cytoplasm proteins amino acids ? </li> <li> Slide 14 </li> <li> heme destabilizes and lyses membranes hydrolases released into parasite cytoplasm parasite dies Free Heme is Toxic Possible Detoxification Mechanisms heme hemozoin (malaria pigment) H 2 O 2 mediated degradation GSH mediated degradation heme oxygenase (P.b. and P.k. only) </li> <li> Slide 15 </li> <li> Hemozoin = -Hematin heme -hematin </li> <li> Slide 16 </li> <li> -hematin forms insoluble crystals 'biocrystallization' or 'biomineralization' </li> <li> Slide 17 </li> <li> Pigment Formation biocrystallization mechanism unknown -hematin can form spontaneously (harsh conditions) lipid bodies can promote the process derived from PVM potential heme detoxification protein recently described unique to Plasmodium species binds 2-3 heme groups with high affinity (80 nM) exported to host cytoplasm and taken up into food vacuole heme biocrystallization inhibited by chloroquine and other anti-malarials </li> <li> Slide 18 </li> <li> The Food Vacuole A Specialized Lysosome hemoglobin + heme globin fragments small fragments (6-8 amino acids) hemozoin ATP ADP ATP ADP H+H+ Fe 3+ Fe 2+ O2O2 - O 2 O 2 ? plasmepsin falcipain plasmepsin falcilysin amino acids amino- peptidase iron oxidized after release from Hb oxidation promotes formation of ROS oxidative stress </li> <li> Slide 19 </li> <li> The Food Vacuole A Specialized Lysosome hemoglobin + heme globin fragments small fragments (6-8 amino acids) hemozoin ATP ADP ATP ADP H+H+ Fe 3+ Fe 2+ O2O2 H2O2H2O2 H 2 O + O 2 - O 2 O 2 superoxide dismutase? catalase? plasmepsin falcipain plasmepsin falcilysin ? amino acids amino- peptidase amino acids ? </li> </ul>