Download Wk7- Autophagy

Autophagy
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Autophagosome: A double membrane bound
compartment that engulfs cytosol and
degrades the cytoplasmic contents.
Large: 400-1500 nm
May originate from ER or from fusion of
lipid-containing vesicles that form
‘sequestration crescent’.
Autophagic pathways
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Plants and animals
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Bacteria, viruses, parasites
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Highly regulated processes
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Stages: induction, execution, maturation
Autophagy genes
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Based on S. cerevisiae yeast studies:
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ATG in yeast, homologs in other spp.
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Involved in:
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Tumor suppression
Starvation responses
Preventing premature cell senescence
Role of autophagy
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Does autophagy benefit the host as a
defense mechanism?
Does autophagy benefit the microbe by
facilitating survival and replication?
Or both?
Role of autophagy
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A host response to degrade intracellular
pathogens?
Hepatic pathogen purging
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10 weeks: 75% of chimpanzee liver cells
contained hepatitis B viral proteins.
20 weeks: chimpanzees were virus free,
with no evidence of extensive cell death!
Role of autophagy
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A microbe strategy for enhanced persistence
and intracellular replication?
Murine hepatitis virus (MHV):
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Cellular markers on viral membranes were
consistent with autophagosomal markers.
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ATG5 mutant had 1000-fold decrease in viral yield.
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Plasmid expressing ATG5 restored viral yield.
Induction
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TOR (Target Of Rapamycin) kinase
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Inhibits autophagy when phosphorylated.
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Also regulates protein and amino acid synthesis.
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Rapamycin and nutrient starvation
dephosphorylate TOR, inducing autophagy.
Tamoxifen induces autophagy too (TOR?)
Induction
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Trimeric G proteins
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High amino acids inactivate G proteins, so
aa depletion may induce autophagy via G
proteins.
PI3Ks (Phosphatidylinositol-3-kinases)
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Essential for starvation induced autophagy.
3-MA, wortmannin, LY294002 target PI3Ks,
and result in inhibition of autophagy.
Execution
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Covalent linkage of Atg5 and Atg12
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Covalent lipidation of Atg8
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Enzymes Atg3, Atg7, and Atg10 are
homologs of ubiquitylation enzymes but
are used to modify pathway
components instead of labeling them
for degradation.
Maturation
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GTPases (Rab24) mediate vesicle fusion.
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Intermediate autophagosomes
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Fuse with endosomal vesicles.
Acquire LAMP, accumulate DAMP proteins.
Mature autolysosomes
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Fuse with lysosomes.
Acquire cathepsins and acid phosphatases.
Detection methods
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Microscopy
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Biochemistry
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Electron microscopy – ultrastructure,
morphology, volumetrics, staining.
Enzyme activity assays.
Radioactive degradation studies.
Marker studies
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Autophagosomal or organelle markers.
Fluorescence or immunodetection.
Bacterial susceptibility
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Astragalus-Mesorhizobium:
Bacteria differentiate within
membrane compartments
until they can fix nitrogen
and establish symbiosis.
Nutrient starvation:
bacterial degradation
observed. Autophagy?
http://www.bioscience.drexel.edu/Homepage/immunology/presentations/group6/Aintro.htm
Bacterial susceptibility
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Rickettsiae conorii
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Sensitive to NO produced by IFN, TNF-α.
Correlations between autophagosome-like
structures and bacterial degradation.
Are autophagosomes destroying bacteria
or just cleaning up after bacteria are killed?
Bacterial susceptibility
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Listeria monocytogenes
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Enter host cells by phagocytosis, escape
from phagosomes, multiply in cytoplasm.
ActA (actin) mutants are engulfed in
autophagosome-like compartments.
Wortmannin reduces bacterial entry into
autophagosomes.
Nutrient depletion increases bacterial entry
into autophagosomes.
http://www.rapidmicrobiology.com/news/603h48.php
Bacterial subversion
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Porphyromonas gingivalis
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Infects human coronary artery endothelial
cells.
Localizes to autophagosome-like
compartments.
Wortmannin: compartments resembled
lysosomes and acquired cathepsin earlier.
Bacterial survival decreased.
http://www.pgingivalis.org/ATCC33277(1).htm
Bacterial subversion
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Brucella abortus
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Endosomal uptake, then autophagosomes.
Wortmannin reduced survival, cell
starvation increased survival.
VirB mutants have Type IV secretion
mutation that inhibits intracellular transport
and growth. Mutants are localized to
membrane compartments that acquire
cathepsin earlier, resembling lysosomes.
Bacterial subversion
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Legionella pneumophila
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Replicates in autophagosome-like compartments
in macrophages.
Dot/icm mutants are defective in Type IV secretion
involved with organelle trafficking or intracellular
multiplication. Mutants localized to lysosomal-like
vesicles, not autophagosomes.
‘Pregnant pause’ model says that autophagosome
maturation is delayed to allow for pathogen
development.
Bacterial subversion
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Coxiella burnetti
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Replicates within autophagosome-like
acidic vesicles.
Rab7 mutants had altered size and
numbers of vesicles containing bacteria.
Dot/icm homologs in Coxiella were able to
return Dot/icm deficient Legionella to a wild
phenotype.
Viral susceptibility
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Herpes-virus
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PKR kinase – phosphorylates eukaryotic
translation-initiation factor eIF2α to inhibit and
deregulate cellular translation.
PKR can also induce autophagy, apoptosis, and
activate NF-KB.
ICP34.5 – produced by herpes simplex virus 1 to
antagonize PKR function by dephosphorylating
eIF2α.
Viral subversion
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Positive-strand RNA viruses
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Poliovirus, murine hepatitis virus (MHV), equine
arterivirus (EAV), SARS human corona virus.
Require membranes for replication.
Autophagosomes are induced during infection,
but are they part of the viral replication process
or the host response to eliminate pathogens?
TIP OF THE
ICEBERG?