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