Presentation on theme: "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."— Presentation transcript:
1 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 conversionsAll 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 constructiveWhy 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 parasiteToday will focus on proteins and amino acids. Central importance and many known antimalarials affect protein metabolism.
2 Sources of Amino Acids De Novo Synthesis Host Plasma CO2 fixation (ala, asp, glu)little incorporated into proteinHost Plasma uptake of all amino acidsin vitro growth requires ile, met, cys, gln, gluDigestion of Host HemoglobinThe 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 Hbto remind you, Hb is …… next slide
3 Hemoglobin 95% of total erythrocyte protein very abundant (>300 mg/ml or approximately 5 mM)60-80% is degraded during erythrocytic stage110 g (of 750 total) is consumed in 48 hrs at 20% parasitemiaHb is major protein in erythrocyteParasite degrades a large proportion.Host mass is converted into parasite mass.How is this accomplished? …..
4 Endocytosis of Host Cytoplasm cytostomefood vacuoleAn early step in the digestion of Hb is its endocytocispinocytosis during ring stagespecialized organelle called cytostome in later stagesPVM membrane is broken downHemoglobin is digested within vacuole called food vacuoleLate in trophozoite stage the small food vacuoles coalesce into large food vacuolemalaria pigment, or hemozoin, is the waste product from Hb digestionWhat is this FV? ….pinocytosis (rings)
5 The Food Vacuole A Specialized Lysosome ATPhemoglobin digestionH+(pH 5-5.4)ADPFood Vacuole Proteasesplasmepsins I & II (acid)falcipains I - III (thiol)falcilysin (metallo)Absent:other acid hydrolasesEndocyticPathwayparasitecytoplasmSeveral distinct proteases have been identified in FVFv is analogous to lysosomeacid compartment with hydrolasespart of endocytic pathwaynoted differences are lack of other hydrolases (not needed because of erythrocyte)proteases are enzymes that break down proteins into peptides or amino acidsthis can be illustrated in the following …
6 Proteases Mediate the Catabolism of Proteins proteases (aka peptidases) break the peptide bonds that hold amino acids togetherexopeptidases remove amino acids sequentially from either N- or C-terminusendopeptidases cleave between ‘specific’ residues within polypeptide chainProteases are enzymes that hydrolyze peptide bondsendo vs. exoWhat about the proteases in food vacuole?all are endolets look at these proteases in regards to the digestion of Hb ….
7 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’conservedimportant for tetramer stabilitythe large globin fragments dissociateheme is releasedglobin fragments are susceptible to further proteolysisa-F33/L34íNative Hb is a globular protein made of 4 subunits--relative resistance to proteolysisspecific cleavage of Hb in hinge region results in protein falling apartThis will expose more protease sites
8 Hemoglobin Digestion is an Ordered Process +hemelarge globin fragmentssmall fragments(6-8 amino acids)plasmepsinfalcipainmedium fragments(20 amino acids)falcilysinexopeptidase?free amino acids?The release of the globin fragments exposes more protease sites for the continued digestion of the polypeptidesfalcipains 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 residuesHow to get to amino acids?Missing activity possible, but let look at another area ….
9 ABC Transporter Super Family large and ubiquitous gene familydefined by ATP-Binding Cassetteaka Multi-Drug Resistance (MDR)transport is usually specific for particular types of substratesPfmdr-1 protein localized to food vacuolePfmdr-1 complements yeast ste6 geneste6 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.
10 The Food Vacuole A Specialized Lysosome ATPhemoglobinH+plasmepsinglobinfragmentsADPheme+aminoacidsfalcipainplasmepsinfalcilysinABC transporter associated with food vacuoleamino-peptidase activities in parasite cytoplasmamino-peptidaseProbable 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 toxicATPPfmdr-1?small fragments(6-8 amino acids)ADP
11 Free Heme is Toxic heme destabilizes and lyses membranes hydrolases released into parasite cytoplasmparasite diesPossible Detoxification Mechanismsheme hemazoin (malaria pigment)H2O2 mediated degradationGSH mediated degradationheme oxygenase (P.b. and P.k. only)Free heme is toxicdestabilizes membranes leading to cell deathdetoxified by several possible mechanisms—predominant is formation of hemozoin or pigmentPolymerized heme is the malarial pigment (=hemazoin)waste product that is encapsulated in residual body after merozite budding. Found deposited in the tissues--well known phenomenonwhat is hemazoin?
12 Hemazoin = b-Hematin b-hematin heme X-ray crystallography and spectroscopic analysis indicates that hemozoin has the same structure as b-hematinb-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 moleculesb-hematinheme
13 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'
14 Pigment Formation biocrystallization mechanism unknown beta-hematin can form spontaneously (harsh conditions)histidine-rich proteins or lipids can promote the processheme biocrystallization inhibited by chloroquine and other anti-malarialsHemozoin 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 ...
15 The Food Vacuole A Specialized Lysosome ATPhemoglobinH+Fe2+plasmepsinO2globinfragmentsADPFe3+heme+aminoacids-O2 O2falcipainplasmepsinfalcilysin?iron oxidized after release from Hboxidation promotes formation of ROIoxidative stressamino-peptidaseThe release of free heme also leads to ROI which place additional stress on the parasite.hemazoinATPsmall fragments(6-8 amino acids)Pfmdr-1?ADP
16 The Food Vacuole A Specialized Lysosome ATPhemoglobinH+Fe2+plasmepsinO2globinfragmentsADPFe3+heme+aminoacids-O2 O2falcipainplasmepsinfalcilysinsuperoxidedismutase??amino-peptidaseSOD 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:H2O2hemazoincatalase?ATPsmall fragments(6-8 amino acids)Pfmdr-1?H2O + O2ADP