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Biochemistry of Plasmodium Mark F. Wiser Downloaded from www.pharmacy123.blogfa.com MACROMOLECULE DNA RNA BUILDING MAJOR BLOCK FUNCTION Nucleotides Genetic Material Template for Protein synthesis Proteins Amino acids Cell Structure and Function Lipid Fatty acids Membrane Component Carbohydrates Sugars Energy Production Downloaded from www.pharmacy123.blogfa.com Sources of Amino Acids De Novo Synthesis CO2 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 Downloaded from www.pharmacy123.blogfa.com 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 Downloaded from www.pharmacy123.blogfa.com Endocytosis of Host Cytoplasm cytostome food vacuole pinocytosis (rings) Downloaded from www.pharmacy123.blogfa.com The Food Vacuole A Specialized Lysosome ATP hemoglobin digestion Endocytic Pathway H+ (pH 5-5.4) Food Vacuole Proteases • plasmepsins I - IV (acid) • falcipains I - III (thiol) • falcilysin (metallo) Absent: • other acid hydrolases Downloaded from www.pharmacy123.blogfa.com ADP parasite cytoplasm 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 Downloaded from www.pharmacy123.blogfa.com 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 -F33/L34 • the large globin fragments dissociate – heme is released • globin fragments are susceptible to further proteolysis Downloaded from www.pharmacy123.blogfa.com Hemoglobin Digestion is an Ordered Process hemoglobin amino acids plasmepsin large globin + heme fragments aminopeptidase di-peptides falcipain plasmepsin dipeptidyl aminopeptidase medium fragments small peptides (~20 amino acids) falcilysin (6-8 amino acids) aminopeptidase amino acids Downloaded from www.pharmacy123.blogfa.com 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?) • ABC transporters also important for amino acid transport Downloaded from www.pharmacy123.blogfa.com ABC Transporter Super Family • large and ubiquitous gene family • defined by ATP-Binding Cassette • transports a wide variety of substrates • aka Multi-Drug Resistance (MDR) • Pfmdr-1 protein localized to food vacuole • Pfmdr-1 complements yeast ste6 gene • ste6 exports mating factor • 12 residue peptide Downloaded from www.pharmacy123.blogfa.com The Food Vacuole A Specialized Lysosome ATP hemoglobin H+ proteins plasmepsin globin heme + fragments ADP ABC transporter associated with food vacuole amino-peptidase activities in parasite cytoplasm ? amino acids falcipain amino acids plasmepsin aminopeptidase falcilysin ATP small fragments (6-8 amino acids) Downloaded from www.pharmacy123.blogfa.com Pfmdr-1? ADP Free Heme is Toxic • heme destabilizes and lyses membranes • hydrolases released into parasite cytoplasm • parasite dies Possible Detoxification Mechanisms • heme hemozoin (malaria pigment) • H2O2 mediated degradation • GSH mediated degradation • heme oxygenase (P.b. and P.k. only) Downloaded from www.pharmacy123.blogfa.com Hemozoin = b-Hematin heme b-hematin Downloaded from www.pharmacy123.blogfa.com b-hematin forms insoluble crystals 'biocrystallization' or 'biomineralization' Downloaded from www.pharmacy123.blogfa.com Pigment Formation • biocrystallization mechanism unknown • b-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 Downloaded from www.pharmacy123.blogfa.com The Food Vacuole A Specialized Lysosome ATP hemoglobin O2 Fe3+ -O O 2 2 Fe2+ plasmepsin globin heme + fragments falcipain ? iron oxidized after release from Hb oxidation promotes formation of ROI oxidative stress H+ plasmepsin hemozoin falcilysin ADP amino acids aminopeptidase ATP small fragments Pfmdr-1? (6-8 amino acids) ADP Downloaded from www.pharmacy123.blogfa.com The Food Vacuole A Specialized Lysosome ATP hemoglobin O2 Fe3+ -O O 2 2 Fe2+ superoxide dismutase? H2O2 H+ plasmepsin globin heme + fragments ? falcipain hemozoin plasmepsin falcilysin catalase? H2O + O2 amino acids ADP amino acids aminopeptidase ATP small fragments Pfmdr-1? (6-8 amino acids) ADP Downloaded from www.pharmacy123.blogfa.com
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