Biochemistry of Plasmodium Mark F. Wiser

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

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

Endocytosis of Host Cytoplasm food vacuole cytostome pinocytosis (rings)

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

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

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 

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

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

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

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

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 ?

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)

Hemozoin =  -Hematin heme  -hematin

 -hematin forms insoluble crystals 'biocrystallization' or 'biomineralization'

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

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

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 ?