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Seamus MartinTrinity College Dublin
Ireland
ApoptosisBasic mechanisms
Some
Cell division and cell death are two halves of the same coin
The body needs to balancecell division with cell deaththroughout life
cell deathcell division
How do cells die?
By accident or design?
The cell does not ‘choose’ to die
or participate in its own death
The cell is an ‘active participant’ in the decision to die and can set this in motion
The cell is ‘condemned’ to die by its cellular peers and is executed by a specially appointed cell
Murder
Suicide
Execution
Similar, but different……
Apoptosis
Necrosis
•Cell does not ‘choose’ to dieSevere trauma (rupture, compression)Toxins (e.g. large amounts of alcohol)Extremes of heat or cold (burns)Radiation (high dose)
Basically, nasty stuff ……
Cell murder: necrosis
Very unfriendly…kills neighbouring cellsRelease of ‘danger’ signals
Cell murder: necrosisCell murder: necrosis
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H2O2
Necrosis: uncontrolled cellNecrosis: uncontrolled cell lysislysis
•Cell ‘chooses’ to dieNatural ‘triggers’ made by the body itself
e.g. death ligands, cytokine deprivationBut, also provoked by mild damage or injury
If a cell has to die ……this is the way to do it!
Cell Suicide
Neighbours are not affected and may eat the dead ce ll
Cell Suicide: Apoptosis
John Kerr, Andrew Wyllie, Alistair Currie
Professor of Greek suggests‘apoptosis’ to drop or fall away
Why apoptosis?Why apoptosis?
•Cells Cells round upround up and detach and detach from their from their neighboursneighbours
••Membrane blebsMembrane blebs appear and appear and apoptotic bodies are producedapoptotic bodies are produced
••Plasma membrane alterationsPlasma membrane alterations trigger trigger engulfment by phagocytes (e.g. PS exposure)engulfment by phagocytes (e.g. PS exposure)
••Minimal production of proMinimal production of pro --inflammatory cytokines, inflammatory cytokines, neighbouring neighbouring cells are spared collateral damagecells are spared collateral damage
Dramatic reorganization of the cellular architecture occurs during apoptosis
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Cell suicide: apoptosisCell suicide: apoptosis
Cell Execution: involuntary apoptosis
•Cell is behaving in an anti-social manner and is a threat to the cellular ‘society’.
•Might be a cancer in the making or Infected with a virus.
Natural Killer cells & cytotoxic T cells carry out the ‘hit’
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Cell Execution: involuntary apoptosis
Natural Killer cells
Necrosis
Apoptosis
DevelopmentalFormation of digitsCavitationDevelopment of the Neural
& Immune systemsHomeostatic
Immune SystemLiverGI Tract
Response to stress, damage, infectionGeneral cell stressDNA DamageViral Infection
Apoptosis: an efficient disposal mechanismfor aged, defective or superfluous cells
Limb development
Developmentalsculpting
So how do cells die by apoptosis?
•what happens within the cell?
•are there genes that regulate apoptosis?
•how does it all work?
•can we control cell death?
The worm turns
Bob Horvitz, MIT
Nobel Prize for Physiology or Medicine 2002
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Nematode wormsFree living in soil
~1mm long
Sydney Brenner, LMB, Cambridge, UK
C. elegans
1090cells born, 131die during development
Cells die:same timesame placeevery time
Development in the nematode
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normalembryo
mutantembryo
Mutants with:•Too little cell death•Too muchcell death•Normal death but delayed burial
Cell death is under genetic control
Cell death defective (ced) mutants
A genetic blueprint for cell death in the worm
ced-3
ced-4 Death
ced-9
egl-1
CED-9 CED-4
CED-3
Inactive Active
CED-3
ATP
CED-4
EGL-1
The worm apoptosome is built upon a CED-4 tetramer
Cell Death
How does CED-3 kill?
Worm humanCED-3 Caspases (12)CED-4 Apaf-1 (1)CED-9/EGL-1 Bcl-2 family (18)
Man is basically a complicated worm
Similar but different…………
Apoptosis regulation Circa 1990
TriggersCytotoxic drugs, radiation
DNA fragmentation
Apoptosis
We have absolutely no idea…… ..
(H2a, H2b)2 octamer
H1Endonuclease cuts wherever it can
Apoptosis regulation Circa 1995
DNA fragmentation(although we don’t really care about this anymore)
Apoptosis
Caspases(Known as ICE/CED-3 proteases then)
PARP, Fodrin(All caspase substrates are really ‘special’ and mag ical)
Triggers
Apoptosis regulation Circa 2000
Triggers
Apoptosis
Caspase cascade
Many substrates(not looking so magical now )
Cytochrome c releaseis important in mammals
(weird but true)
Intrinsic
Apoptosis regulation
BH3-onlyproteins
Circa 2007
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CaspasesCaspases
Apoptosis is coordinated by caspases
•Caspases are Cysteine Asp artic acid specific prote ases
•Normally dormant, activated by death triggers
ATime (min): 0 15 30 60 90 120 0 15 30 60 90 120
Untreated Cytochrome c
24 17
Mr (kDa)36
Caspase-3
45Caspase-1
Caspase activation occurs at the onset of apoptosis
Inactive active
A subset of caspases coordinate apoptosis
Substrates
IL-1β, IL-18, IL-33?
???
???
Hundreds (www.casbah.ie )
Bid, RIP, Caspase-3, -7
Caspase-3, -7
Effector
Initiator
???
Lamin A
Similar to casp-8
Partial redundancy with casp-3
ICAD/DFF45
Lamins FodrinVimentinActin
Multiple kinasesBid (Cyt c release)Gelsolin (severs actin)
Caspases cleave key structural and regulatory proteins during apoptosis
Special substrates?
Identification of proteins that relocate on protein maps Identification of proteins that relocate on protein maps using MALDIusing MALDI --TOF Mass spectrometryTOF Mass spectrometry
Analyse protein composition by 2D gel separation
Generating proteomic maps of apoptosis
ApoptoticViable
= caspase substrates = cleavage fragments
Hundreds of proteins are cleaved by mammalian caspases
Control Apoptotic
Control Apoptotic
VimentinVimentin
CleavedCleavedVimentinVimentin
CaspaseCaspase --3 depletion abolishes majority of alterations to the proteome3 depletion abolishes majority of alterations to the proteome
400 mammaliancaspase substratespublished to date
•Approx. >1000 substrates
Death by a thousand cuts?
www.casbah.ie
How do caspases become activated?
3 major pathways
(I) Extrinsic: death receptors(II) Intrinsic: via mitochondrial permeabilization(III) Granule-dependent: granzyme B from CTL/NKs
CTL/NK cells
(1) Extrinsic
(2) Intrinsic
(3) Granzyme B
Caspase activation cascades
BH3-only proteins
Bid Bim
Puma
Noxa
Bmf
Bik
HrkBad
Bax Bak
Bcl-w
A1
Mcl-1Bcl-2
Bcl-xL
Bcl-b
Bax Bak
BH3BH3--only proteins promoteonly proteins promote Cyt Cyt c releasec releasevia via BaxBax //Bak oligomerizationBak oligomerization
Bcl-2-like(anti-apoptotic)
BH3-only(pro-apoptotic)
Bax-like(pro-apoptotic)
Direct activators:Bid & BimSensitizers:Bad, Puma, NoxaBmf, Bik, Hrk
Intrinsic pathway
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Mammalian
Apaf-1
Caspase-9
Caspase-9
Cyt cdATP
Apaf-1
Cytochrome c release promotesassembly of the apoptosome
Cell Death
Caspase cascade
BH3BH3--only proteins are activated in a only proteins are activated in a stimulusstimulus --specific mannerspecific manner
Bid
Bim
Puma
NoxaBmfBik
HrkBad
Growth factordeprivation
Antigen receptorstimulation
Granzyme B
Death receptorsProteasome
inhibitionp53
BclBcl --2 family protein 2 family protein interactions are selectiveinteractions are selective
BH3BH3--only activationonly activation Induces interactionInduces interaction
Bid Bim Puma
NoxaBmf
Bik Hrk
Bad
Bcl-w A1 Mcl-1Bcl-2 Bcl-xL
Interaction induces Interaction induces catastrophic consequencescatastrophic consequences
BH3BH3BH1BH1
BH2BH2BadBad
BclBcl --ww
Mitochondrial Cyt c release representsthe point of no return
Intervention downstream of Cyt c release is typically f utile
Caspases are required for apoptosisbut not death per se
Caspase-independent
cell death (necrosis)
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Actinomycin D-induced apoptosis
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Actinomycin D + zVAD-fmk
Caspase inhibitiors block typical features of apopt osis
Summary
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Rebecca Taylor
Sean Cullen
AlexLuthiColin
AdrainPetrina Delivani
Clare Sheridan
PatrickDuriez
GabiBrumatti
Rosi Soellner
Supported by
Martin Lab, Trinity College Dublin
••AlexanderAlexander LLüüthithi••Sean CullenSean Cullen••Petrina DelivaniPetrina Delivani••Clare SheridanClare Sheridan••Susan LogueSusan Logue
••GabiGabi BrumattiBrumatti••John WalshJohn Walsh••InnaInna AfoninaAfonina••Jill CaseyJill Casey