biochemistry of plasmodium mark f. wiser wiser/malaria/fv.html
TRANSCRIPT
Biochemistry of Plasmodium
Mark F. Wiser
http://www.tulane.edu/~wiser/malaria/fv.html
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 (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 VacuoleA 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
except acid phosphatase
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
dipeptidyl aminopeptidase
aminopeptidase
hemoglobin
+ hemelarge globin fragments
small peptides(6-8 amino acids)
plasmepsin
falcipainplasmepsin
medium fragments(~20 amino acids) falcilysin
amino acids
amino acidsaminopeptidase
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 VacuoleA Specialized Lysosome
hemoglobin
+heme globinfragments
small fragments(6-8 amino acids)
ATP
ADP
ATP
ADP
H+ plasmepsin
falcipain
plasmepsin
falcilysin
PfCRT?
aminoacids
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)• H2O2 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 VacuoleA Specialized Lysosome
hemoglobin
+heme globinfragments
small fragments(6-8 amino acids)
hemozoin
ATP
ADP
ATP
ADP
H+
Fe3+
Fe2+O2
-O2 O2
?
plasmepsin
falcipain
plasmepsin
falcilysin
aminoacids
amino-peptidase
iron oxidized after release from Hb
oxidation promotes formation of ROS
oxidative stress
The Food VacuoleA Specialized Lysosome
hemoglobin
+heme globinfragments
small fragments(6-8 amino acids)
hemozoin
ATP
ADP
ATP
ADP
H+
Fe3+
Fe2+O2
H2O2
H2O + O2
-O2 O2
superoxidedismutase?
catalase?
plasmepsin
falcipain
plasmepsin
falcilysin
?
aminoacids
amino-peptidase
amino acids
?