Biochemistry of Plasmodium Mark F. Wiser http://www.tulane.edu/~wiser/malaria/fv.html

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 16% of amino acids derived from hemoglobin are incorporated into parasite proteins

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 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 - IV (acid) falcipains I - III (thiol) falcilysin (metallo) Absent: other acid hydrolases except acid phosphatase 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 aminopeptidase hemoglobin + heme large globin fragments small peptides (6-8 amino acids) plasmepsin falcipain medium fragments (~20 amino acids) falcilysin amino acids di-peptides dipeptidyl aminopeptidase 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 These peptides are then further degraded to amino acids However, not complete due to specificity of proteases Final result is mixture of amino acids and peptides So how do these get exported into host cytoplasm where they are need for further metabolism and protein synthesis? ... End-products are small peptides, dipeptides, and amino acids

Membrane Transport Hydrophilic solutes require transporters (ie, carrier proteins) substrate specific most require energy ATPase or gradients (eg, H+) 6 amino acid transporters identified in Plasmodium genome (location?) PfMDR-1 and PfCRT located on food vacuole membrane Two ways in which small molecules are translocted across membranes: channels and carriers For amino acids carriers are most important Generally substrate specific and require energy In the genome there are 6 potential amino acid transporters, but none have been further characterized ABC transporters are also important ....

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, PfMDR-1 imports solutes (including drugs) into food vacuole

PfCRT Member of DMT (drug/metabolite transporter) super family H+-coupled polyspecific nutrient exporter including amino acids and peptides Associated with chloroquine resistance (CRT = chloroquine resistance transporter) identified through genetic linkage studies Exports chloroquine and other drugs from the food vacuole

heme is by-product of hemoglobin catabolism PfCRT is probable transporter amino-peptidase activity in cytoplasm

Free Heme is Toxic heme destabilizes and lyses membranes parasite dies hydrolytic enzymes released into parasite cytoplasm parasite dies Possible Detoxification Mechanisms heme  hemozoin (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?

Hemozoin = b-Hematin heme b-hematin dimer formation 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 heme b-hematin

b-hematin forms insoluble crystals biocrystallization These dimers interact through hydrogen bonds to form crystals of hemozoin. Therefore, pigment formation is best described as a biocrystalization, or biomineralization, process hemozoin can form spontaneously under harsh conditions (non-biological)

Pigment Formation biocrystallization mechanism unknown lipid bodies can promote the process possibly derived from PVM potential heme detoxification protein (HDP) unique to Plasmodium species exported to host cytoplasm and taken up into food vacuole binds 2 heme groups with high affinity (80 nM) promoting b-hematin dimer formation dimer seeds biocrystallization reaction? 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 -lipids? -detox protein? multiple proteases iron oxidized after release from Hb oxidation promotes formation of ROS oxidative stress The release of free heme also leads to ROI which place additional stress on the parasite. hemozoin amino- peptidase small peptides di-amino acids amino acids PfCRT H+

The Food Vacuole A Specialized Lysosome ATP hemoglobin H+ Fe2+ plasmepsin O2 globin fragments ADP Fe3+ heme + amino acids -O2 O2 -lipids? -detox protein? superoxide dismutase? multiple proteases 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 hemozoin amino- peptidase catalase? peroxidase? small peptides di-amino acids amino acids PfCRT H2O + O2 H+ parasite has SOD and peroxidases, but no catalase location?

Summary Food vacuole is lysosome-like organelle Digestion of hemoglobin and detoxification of heme are primary metabolic activities Crucial organelle for the parasite and provides many potential drug targets