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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

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  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

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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

X

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).