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Treatments and New drug targets
Apicoplast What is the apicoplast? Where does it come from? How are proteins targeted to
the organelle? How does the organelle
replicate? What is the function of the
organelle?
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Apicoplast - history Discovery just waiting to happen! “mysterious membrane-bound
organelle” viewed in 1960s. Term coined only in 1995 Four membranes - 1997 Fatty acid synthesis proteins are
targeted to apicoplast 1998
New drug - Human trials Fosmidomycin
Isoprenoid biosynthesis Good tolerance Recrudescence
Apicomplexans have 3 genomes
Band 3: Nuclear genome on chromosomes
Band 2: Small 6 kb mitochondrial genome (linear)
Band 1: 35 kb circular genome Discovered in 1975
CsCl Sample EtBr
Isopycnic Centrifugation (Density gradient separation)
Before centrifugation After centrifugation
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Common apicoplast ancestor ~35 kb circular genome
Toxoplasma Plasmodium Eimeria
Gene synteny Similarity to chloroplast genes 2 large subunit rRNA 2 small subunit rRNA 3 subunits of RNA polymerase Ribosomal proteins Complete set of tRNAs Elongation factor (EF-TS) Chaperone proteins (clp) Nothing related to photosynthetic
machinery Big surprise - no apicoplast DNA in Cryptosporidium, no apicoplast
Subcellular Localization of the 35 kb DNA
How?
Methods
Localization in 1996
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Delayed Death Phenotype Ciprofloxacin
inhibited apicoplast DNA replication (T. gondii)
Treatment resulted in: Loss of apicoplast DNA Cell growth inhibited after 1 round of
cell division
What is a plastid?
General term for membrane-bound organelles found in plants and algae.
Contain DNA, various pigments and various storage products.
Plastid forms are interconvertible. (ie. many fall colors results from transition of chloroplast to chromoplast in deciduous trees)
Originated from an endosymbiotic cyanobacteria.
Apicomplexans synthesize amylopectin! - Highly branched glucose polymer Not water soluble
Undifferentiated colorless
colorless
chlorophyll
Pigments red, yellow
carotenoids
Starch storage
develops in dark
Petals, fruit Roots, seeds, fruit leaves, stems
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Plastid Evolution
Endosymbiotic origin - 1800s Microscopic observations - pigments
Discovery of extrachromosomal DNA Phylogenetic studies Close relationship to cyanobacteria
Primary endosymbiosis Eukaryote + cyanobacterium
Secondary endosymbiosis Eukaryote + algae
Plastid Evolution
A single primary endosymbiotic event gave rise to all present day plastids
Primary plastid account for only a small fraction of plastid biodiversity
At least 3 major secondary endosymbiotic events
Numerous cases of loss of photosynthetic machinery
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Apicoplast Evolution
P r i m a r y E n d o s y m b i o s i s : O R I G I N O F T H E C H L O R O P L A S T
T S e c o n d a r y E n d o s y m b i o s i s : O R I G I N O F T H E A P I C O P L A S
Protein targeting via secretory pathway
signal sequence plastid-targetingdomain
Mature PlastidProtein
B)
A) Domain structure of nuclear-encoded apicoplast proteins
Bipartite signal is required for apicoplast targeting At least a two-step process
Hydrophobic signal peptide - secretory signal Transit peptide - translocation into plastid
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Apicoplast targeted proteins are proteolytically processed
Apicoplast targeting
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Apicoplast/Mitochondria Association
Toxoplasma
Apicoplast
Plasmodium Apicoplast
Mitochondrion
Mitochondrion
Apicoplast/Mitochondria Association Toxoplasma Plasmodium Dinoflagellate
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Plastid Division with Cell Cycle 1
2
3
Toxoplasma divides by Endodyogeny 2 daughter cells inside mother
1 - Interphase Apicoplast is apical to nucleus
2 - S phase Centrioles duplicated DNA replicated and divided
3 - Segregation Associated with spindle poles
4 - Segregation/growth U-shaped structure
5 - Division Centriole association Movement into daughter bud
6 - Nuclear fission 4
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Apicoplast Division
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Apicoplast division
The Power of Genomics Hugely difficult to isolate intact
apicoplasts in pure form. Why?
How do you study the function?
Virtual pathways!
Confirmation!!!!!
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Apicoplast functions - Simplified!
Apicoplast is an essential organelle - delayed death phenotype
Non-photosynthetic!
Transporters: Plant-like
1-deoxy-D-xylulose-5-phosphate DOXP pathway:
Bacteria, chloroplasts
*
*
*
*
An Unusual DNA Polymerase
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Presence of Plastid Fatty acid biosynthesis
Cytosol - animals Plastid
Location of Haem biosynthesis
Close association between plastid and mitochondrion
ALA = aminolevulinic acid Early precursor of haem
biosynthesis Mitochondrial!!!!!
Apicomplexan Biochemistry Basics Toxoplasma
Good cell biology model Genome sequencing not completed Virtual pathways
Cryptosporidium The “strange one” Genome sequence completed Virtual pathways
Plasmodium Best biochemistry data Numerous studies over many years Genome sequencing completed!
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Glycolysis - Plasmodium
Lactate
Pentose Pathway
Pyruvate
PEP
Glucose-6-P
Pyruvate Metabolism
Erythrocytic stage Glucose is major substrate
Infected RBC’s utilize 75% more glucose than uninfected
Lactate is major end product 85% of glucose lactate
High glycolytic rate Enzyme utilize ATP (not PPi) Overall 2 ATP produced for
every glucose utilized
Role of mitochondrion???
Pentose Phosphate Pathway
Salvage pathway
Essential biosynthetic
pathway Shikimate pathway
Glycolytic intermediate is utilized (G-6-P)
Reducing equivalents generated
Products utilized for Nucleotide metabolism
Products utilized for shikimate pathway
Regenerates some glycolytic intermediates
*
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Shikimate Pathway
Important pathway that is absent from animals! Folate synthesis Aromatic amino acid
synthesis Essential for apicomplexan
parasites Large body of data on
biochemistry Bacteria, fungi, plants
Glycolysis
PPP
Folate Synthesis
Ubiquinone Synthesis
Tryptophan Synthesis
Mitochondrial Function Simplified View???
Historically thought to be quiescent
Few if any cristae Developmental changes
Asexual stages Single mitochondrion tubular
network Gametocytes
Multiple organelles Basics - Asexual stage
Cytosolic glycolysis Complete complement of TCA
cycle enzymes Electron transport subunits Alternative oxidase
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Mito Oxidative Phosphorylation Apicomplexans
Trypanosomatids
Cryptosporidium
Toxoplasma
Plasmodium
Trypanosoma Bloodstream form
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Puzzle of Pyruvate Metabolism
Acetyl-CoA
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Pyruvate Dehydrogenase Complex Multienzyme complex (PDC)
E1 - pyruvate dehydrogenase E2 - dihyrolipoyl transacteylase E3 - dihydrolipoyl dehydrogenase
Convert Pyruvate to Acetyl-CoA, generate reducing equivalents
Typically the regulated step committing to TCA cycle
PDC is absent from mitochondria PDC is present in apicoplast E3 subunits present in mitochondria
Endocytosis of Host Cytoplasm
Pinocytosis Cytostome Endocytic pathway Food Vacuole
Hemoglobin digestion
• heme destabilizes and lyses membranes • hydrolases released into
parasite cytoplasm • parasite dies
Heme β-hematin
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The Plasmodium Food Vacuole
hemoglobin
+ heme globin
fragments
small fragments (6-8 amino acids)
hemozoin
ATP
ADP
ATP
ADP
H+
Fe3+
Fe2+ O2
H2O2
H2O + O2
-O2 •O2 superoxide dismutase?
catalase?
?
plasmepsin
falcipain
plasmepsin
falcilysin
Pfmdr-1?
amino acids
A specialized lysosome - acidic compartment
Fatty Acid Synthesis Iterative elongation of
acyl chains Growth of chain by 2 C
Type I (Eukaryotic) Multiple enzymatic
activities on a single large multifunctional protein
Type II (Prokaryotic) Each activity is on a
separate polypeptide
Dr. Kim Paul
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Inhibition of Type II FAS
Initially thought: no FAS Type II - prokaryotic
Separate subunits ACC - acetyl CoA carboxylase
Aryloxyphenoxyproprionate herbicides (FOPS)
ACP - acyl carrier protein KAS - keto-acyl ACP synthase
Thiolactomycin Cerulenin
ENR - enoyl reductase Triclosan
The Good, the Bad, the Ugly
What purpose do Triclosan and the Copolymer serve?
Colgate Total is the only toothpaste to contain triclosan with a copolymer. Triclosan is an effective antibacterial ingredient, which is effective in controlling a wide range of bacteria. The copolymer is used in the formulation to prolong retention of triclosan on oral hard and soft tissues and in plaque. Without the copolymer, triclosan would be rapidly lost from the mouth, reducing its clinical effect.
Bottom line: (numerous studies) Antibacterial ingredient triclosan degrades rapidly
when exposed to chlorinated tap water, producing potentially toxic byproducts (chloroform).
The really ugly When flushed into water systems, and exposed to ultra violet radiation, triclosan converts to a dioxin. The amount of dioxins created is small, however, it's an extremely toxic and stable chemical that persists in the environment and is eliminated slowly from the body.
Sewage sludge - commonly used on farmer’s fields
Isoniazid
1st line anti-tuberculosisInhibits mycolic acid synthesis (mycobacterial cell wall)
Drug resistance is a big problem. Never use Isoniazid alone.
Mycolic acids consist of long-chainalpha-alkyl-beta-hydroxy fatty acids that are
produced by successive rounds of elongation catalyzed by a type II fatty acid synthase (FAS-II).