Today I will be talking about the biochemistry of the malaria parasite. Biochemistry is the study of the interconversion of molecules as depicted by this figure with each dot representing a distinct chemical and the lines representing enzymes that are responsible for the conversions All organisms are made of macromolecules. These macromolecules are composed of subunits. (Table) Living and growing organisms require these building blocks. Nutrients are acquired from the environment and converted into substances or energy needed by the organism. Catabolism is destructive. Anabolism is constructive Why study biochemistry of Plasmodium? It will be similar to other organisms. This question will be the focus of the second lecture on drug action. Namely, biochemical differences can be exploited. For example, a pathogen may have unique pathways not found in the host. Or a pathway may be more important in the pathogen than the host. Or the pathway is equally important, but drugs specific for enzymes of the pathogen are available. Parasite has huge demand for precursors, especially DNA. Antifolates block synthesis of nucleotides and inhibit the parasite Today will focus on proteins and amino acids. Central importance and many known antimalarials affect protein metabolism.

Sources of Amino Acids De Novo Synthesis Host Plasma 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 The parasite acquires amino acids by three different means. De novo synthesis: CO2 fixation has been demonstrated, but little is incorporated into proteins. Probably functions as part of other metabolic pathways. Host plasma: amino acids are taken up at increased rates by infected erythrocyte, many are incorporated into protein. Some are absolutely required. Major source is from the digestion of host Hb to remind you, Hb is …… next slide

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 Hb is major protein in erythrocyte Parasite degrades a large proportion. Host mass is converted into parasite mass. How is this accomplished? …..

Endocytosis of Host Cytoplasm cytostome food vacuole An early step in the digestion of Hb is its endocytocis pinocytosis during ring stage specialized organelle called cytostome in later stages PVM membrane is broken down Hemoglobin is digested within vacuole called food vacuole Late in trophozoite stage the small food vacuoles coalesce into large food vacuole malaria pigment, or hemozoin, is the waste product from Hb digestion What is this FV? …. pinocytosis (rings)

The Food Vacuole A Specialized Lysosome ATP hemoglobin digestion H+ (pH 5-5.4) ADP Food Vacuole Proteases plasmepsins I & II (acid) falcipains I - III (thiol) falcilysin (metallo) Absent: other acid hydrolases Endocytic Pathway parasite cytoplasm Several distinct proteases have been identified in FV Fv is analogous to lysosome acid compartment with hydrolases part of endocytic pathway noted differences are lack of other hydrolases (not needed because of erythrocyte) proteases are enzymes that break down proteins into peptides or amino acids this can be illustrated in the following …

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 Proteases are enzymes that hydrolyze peptide bonds endo vs. exo What about the proteases in food vacuole? all are endo lets look at these proteases in regards to the digestion of Hb ….

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 Native Hb is a globular protein made of 4 subunits--relative resistance to proteolysis specific cleavage of Hb in hinge region results in protein falling apart This will expose more protease sites

Hemoglobin Digestion is an Ordered Process + heme large globin fragments small fragments (6-8 amino acids) plasmepsin falcipain medium fragments (20 amino acids) falcilysin exopeptidase? free amino acids? The release of the globin fragments exposes more protease sites for the continued digestion of the polypeptides falcipains and plasmepsins will act at numerous places (globin chain approximately. 140 residues) falcilysin does not efficiently digest fragment larger that 20 residues. Probably responsible for going down to fragments of 6-8 residues How to get to amino acids? Missing activity possible, but let look at another area ….

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) Drug resistance is an important problem in malaria. Some drug resistance in other systems (eg., bacteria, cancer) due to MDR genes. These are know called ABC transporters. One mdr gene in plasmodium might be a peptide transporter in the food vacuole.

The Food Vacuole A Specialized Lysosome ATP hemoglobin H+ plasmepsin globin fragments ADP heme + amino acids falcipain plasmepsin falcilysin ABC transporter associated with food vacuole amino-peptidase activities in parasite cytoplasm amino- peptidase Probable scenario is that the final conversion to amino acids is carried out in parasite cytoplasm. These amino acids are then available for protein synthesis and other metabolic pathways. Some data on specific inhibitors of proteases as effective anti-malarias. Parasite would starve to death. There are some problems though--heme lyses membranes and therefore is toxic ATP Pfmdr-1? small fragments (6-8 amino acids) ADP

Free Heme is Toxic heme destabilizes and lyses membranes hydrolases released into parasite cytoplasm parasite dies Possible Detoxification Mechanisms heme  hemazoin (malaria pigment) H2O2 mediated degradation GSH mediated degradation heme oxygenase (P.b. and P.k. only) Free heme is toxic destabilizes membranes leading to cell death detoxified by several possible mechanisms—predominant is formation of hemozoin or pigment Polymerized heme is the malarial pigment (=hemazoin) waste product that is encapsulated in residual body after merozite budding. Found deposited in the tissues--well known phenomenon what is hemazoin?

Hemazoin = b-Hematin b-hematin heme X-ray crystallography and spectroscopic analysis indicates that hemozoin has the same structure as b-hematin b-hematin is a heme dimer formed via reciprocal covalent bonds between carboxylic acid groups on the protoporphyrin-IX ring and the iron atoms of two heme molecules b-hematin heme

b-hematin forms insoluble crystals These dimers interact through hydrogen bonds to form crystals of hemozoin. Therefore, pigment formation is best described as a biocrystalization, or biomineralization, process '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 Hemozoin can be formed chemically. But under harsh conditions. Parasite proteins likely involved. But none identified. Histidine rich proteins and lipids have been implicated. Polymerization inhibited by CQ and other 4-aminoquinolines. CQ kills by build of toxic free heme. The updated Fv now looks like this ...

The Food Vacuole A Specialized Lysosome ATP hemoglobin H+ Fe2+ plasmepsin O2 globin fragments ADP Fe3+ heme + amino acids -O2 O2 falcipain plasmepsin falcilysin ? iron oxidized after release from Hb oxidation promotes formation of ROI oxidative stress amino- peptidase The release of free heme also leads to ROI which place additional stress on the parasite. hemazoin ATP small fragments (6-8 amino acids) Pfmdr-1? ADP

The Food Vacuole A Specialized Lysosome ATP hemoglobin H+ Fe2+ plasmepsin O2 globin fragments ADP Fe3+ heme + amino acids -O2 O2 falcipain plasmepsin falcilysin superoxide dismutase? ? amino- peptidase SOD and catalase, possibly derived from host may help protect against oxygen stress. Some genetic conditions place additional oxygen stress as well as some antimalarials. Summary: H2O2 hemazoin catalase? ATP small fragments (6-8 amino acids) Pfmdr-1? H2O + O2 ADP