sources of amino acids de novo synthesis co 2 fixation (ala, asp, glu) little incorporated into...

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