MACRO-MOLECULE
BUILDINGBLOCK
MAJORFUNCTION
DNA Genetic Material
RNANucleo-tides Template for
Protein synthesis
ProteinsAminoacids
Cell Structure andFunction
LipidFattyacids
MembraneComponent
Carbo-hydrates
SugarsEnergyProduction
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
Hemoglobin 95% of total erythrocyte protein very abundant (>300 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 VacuoleA Specialized Lysosome
ATP
ADP
H+
(pH 5-5.4)
Food Vacuole Proteases• plasmepsins I & II (acid)• falcipains I - III (thiol)• falcilysin (metallo)
Absent:• other acid hydrolases
hemoglobin digestion
parasitecytoplasm
EndocyticPathway
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 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
F33/L34
Hemoglobin Digestion is an Ordered Process
• exopeptidase?• free amino acids?
hemoglobin
+ hemelarge globin fragments
small fragments(6-8 amino acids)
plasmepsin
falcipainplasmepsin
medium fragments(20 amino acids) falcilysin
ABC Transporter Super Family
• large and ubiquitous gene family• defined by ATP-Binding Cassette• aka Multi-Drug Resistance (MDR)
• transport is usually specific for particular types of substrates• Pfmdr-1 protein localized to food vacuole• Pfmdr-1 complements yeast ste6 gene• ste6 transports a-type mating factor
(12 residue peptide)
The Food VacuoleA Specialized Lysosome
hemoglobin
+heme globinfragments
small fragments(6-8 amino acids)
ATP
ADP
ATP
ADP
H+plasmepsin
falcipain
plasmepsin
falcilysin
Pfmdr-1?
aminoacids
amino-peptidase
ABC transporter associated with food vacuole
amino-peptidase activities in parasite cytoplasm
• heme destabilizes and lyses membranes• hydrolases released into
parasite cytoplasm• parasite dies
Free Heme is Toxic
Possible Detoxification Mechanisms• heme hemazoin (malaria pigment)• H2O2 mediated degradation• GSH mediated degradation• heme oxygenase (P.b. and P.k. only)
Hemazoin = -Hematin
heme -hematin
-hematin forms insoluble crystals
'biocrystallization' or 'biomineralization'
Pigment Formation• biocrystallization mechanism
unknown• beta-hematin can form
spontaneously (harsh conditions)• histidine-rich proteins or lipids can
promote the process• heme biocrystallization inhibited
by chloroquine and other anti-malarials
The Food VacuoleA Specialized Lysosome
hemoglobin
+hemeglobin
fragments
small fragments(6-8 amino acids)
hemazoinATP
ADP
ATP
ADP
H+
Fe3+
Fe2+
O2
-O2 O2
?
plasmepsin
falcipain
plasmepsin
falcilysin
Pfmdr-1?
aminoacids
amino-peptidase
iron oxidized after release from Hb
oxidation promotes formation of ROI oxidative stress
The Food VacuoleA Specialized Lysosome
hemoglobin
+hemeglobin
fragments
small fragments(6-8 amino acids)
hemazoinATP
ADP
ATP
ADP
H+
Fe3+
Fe2+
O2
H2O2
H2O + O2
-O2 O2superoxidedismutase?
catalase?
?
plasmepsin
falcipain
plasmepsin
falcilysin
Pfmdr-1?
aminoacids
amino-peptidase