Transcript Autophagy and Immunity

Autophagy and Immunity
Qingqing Wang
Institute of Immunology
Zhejiang University School of Medicine
Nov 2, 2012
Autophagy
Phagy = eating/lysosomal degradation
Autophagy = self-eating
Mechanism of breakdown of cytoplasm within the
lysosome
Autophagy: a basic cellular process in eukaryotes
Autophagy
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Highly conserved and regulated process that maintains cellular homeostasis and
protects cells against starvation and microbe invasion
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 species.
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Involved in:
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Tumor suppression
Starvation responses
Preventing premature cell senescence
3 Stages
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Initiation: nutrient starvation,
growth factor-mediated starvation
Execution
Maturation
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.
The physilogical significance of autophagy
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自噬参与代谢应激的保护机制
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细胞自噬是细胞的看家者
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自噬是染色体的守护者
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细胞自噬决定细胞的生死
There are many ways to die…
Programmed cell death
Type I:
caspase mediated
Type II:
autophagy-mediated
Apoptosis
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Programmed cell death
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Type I: caspase mediated
Type II: autophagy-mediated
Entire cell dismantled within
membrane-enclosed vesicles
Taken up by phagocytes, preventing
release of intracellular components
from dying cells
Normal morphogenesis, removing
genetically damaged cells, proper tissue
homeostasis, invading microbes
Mechanism
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Sequential activation of
cysteine proteases
(caspases)
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Caspase 1-related:
1,4,5,13,14
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Cytokine processing and
pro-inflammatory cell
death
Initiator caspases: 2, 8,
9, 10
Effector caspases: 3,6,7
Regulated process
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Extrinsic: stimulation of
Fas or TNFR surface
receptors
Intrinsic: altered
mitochondrial membrane
integrity
Necrosis
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Cell swelling and rupture
Release in intracellular
components
Activation of inflammatory
response
Can be regulated
Can occur in concert with or
instead of apoptosis (e.g. if
apoptosis is blocked)
Pyroptosis
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Caspase-1-dependent cell death
Convert IL-1B and IL-18 to active
forms
Induced by Shigella IpaB and
Salmonella SipB
Autophagy and diseases
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Oncology
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Liver
 Breast
 Colon
 Lung
Beclin-1
Myodegerative
disease
 Cardiac stress
 a-antitrypsin
deficiency
……
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Brain
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Alzheimer
Multiple Sclerosis
Parkinson’s
disease
Hungtingdon’s
disease
Miscellaneous
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Cellular Process
Apoptosis
 Aging
 Growth
……
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Autophagy and immunity
Antigen presentation
 TLR signaling
 Infection
……
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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?
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.
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Localizes to autophagosome-like compartments.
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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?