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Biochemistry of Plasmodium

Biochemistry of Plasmodium

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Biochemistry of Plasmodium

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

  2. 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

  3. 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

  4. Endocytosis of Host Cytoplasm cytostome food vacuole pinocytosis (rings)

  5. The Food VacuoleA Specialized Lysosome ATP hemoglobin digestion H+ (pH 5-5.4) ADP • Food Vacuole Proteases • plasmepsins I - IV (acid) • falcipains I - III (thiol) • falcilysin (metallo) • Absent: • other acid hydrolases Endocytic Pathway parasite cytoplasm

  6. 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

  7. Initial plasmepsin cleavage is specificand 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 a-F33/L34 í

  8. hemoglobin amino acids plasmepsin aminopeptidase large globin fragments heme + di-peptides falcipain plasmepsin medium fragments (~20 amino acids) small peptides (6-8 amino acids) falcilysin aminopeptidase amino acids Hemoglobin Digestion is an Ordered Process dipeptidyl aminopeptidase

  9. 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

  10. 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

  11. ATP H+ ADP The Food VacuoleA Specialized Lysosome hemoglobin proteins plasmepsin globin fragments heme + amino acids falcipain plasmepsin falcilysin • ABC transporter associated with food vacuole • amino-peptidase activities in parasite cytoplasm amino- peptidase ATP Pfmdr-1? amino acids small fragments (6-8 amino acids) ? ADP

  12. 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)

  13. Hemozoin = b-Hematin b-hematin heme

  14. b-hematin forms insoluble crystals 'biocrystallization' or 'biomineralization'

  15. 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

  16. The Food VacuoleA Specialized Lysosome ATP hemoglobin H+ plasmepsin Fe2+ O2 ADP globin fragments Fe3+ heme + amino acids -O2 O2 falcipain plasmepsin falcilysin ? • iron oxidized after release from Hb • oxidation promotes formation of ROI • oxidative stress amino- peptidase hemozoin ATP small fragments (6-8 amino acids) Pfmdr-1? ADP

  17. The Food VacuoleA Specialized Lysosome ATP hemoglobin H+ plasmepsin Fe2+ O2 ADP globin fragments Fe3+ heme + amino acids -O2 O2 falcipain plasmepsin falcilysin ? superoxide dismutase? amino- peptidase H2O2 hemozoin ATP catalase? small fragments (6-8 amino acids) amino acids Pfmdr-1? H2O + O2 ADP