Biochemistry of Plasmodium

Biochemistry of Plasmodium
Mark F. Wiser
http://www.tulane.edu/~wiser/malaria/
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 (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+
(pH 5-5.4)
Food Vacuole Proteases
plasmepsins I - IV (acid)
falcipains I - III (thiol)
falcilysin (metallo)

Absent:
other acid hydrolases
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
a-F33/L34
í
Hemoglobin Digestion is an Ordered Process
dipeptidyl aminopeptidase
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
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
The Food Vacuole
A Specialized Lysosome
hemoglobin
+
heme
globin
fragments
small fragments
(6-8 amino acids)
ATP
ADP
plasmepsin
falcipain
plasmepsin
falcilysin
Pfmdr-1?
amino
acids
amino-
peptidase
ABC transporter 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)
H2O2 mediated degradation
GSH mediated degradation
heme oxygenase (P.b. and P.k. only)
Hemozoin = b-Hematin
heme
b-hematin
b-hematin forms insoluble crystals
`biocrystallization` or `biomineralization`
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
The Food Vacuole
A Specialized Lysosome
hemoglobin
+
heme
globin
fragments
small fragments
(6-8 amino acids)
hemozoin
ATP
ADP
ATP
ADP
H+
Fe3+
Fe2+
O2
-O2 O2
?
plasmepsin
falcipain
plasmepsin
falcilysin
Pfmdr-1?
amino
acids
amino-
peptidase
iron oxidized after release from Hb
oxidation promotes formation of ROI
oxidative stress
The Food Vacuole
A Specialized Lysosome
hemoglobin
+
heme
globin
fragments
small fragments
(6-8 amino acids)
hemozoin
ATP
ADP
ATP
ADP
H+
Fe3+
Fe2+
O2
H2O2
H2O + O2
-O2 O2
superoxide
dismutase?
catalase?
?
plasmepsin
falcipain
plasmepsin
falcilysin
Pfmdr-1?
amino
acids
amino-
peptidase
amino acids