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

?