#19 ApoptosisChapter 9NeeluYadavPhD

Neelu.Yadav@Roswellpark.org

Cell

DeathstimulationorStress

Survival Death

Whycellsdecidetodie? -Stress,harmful,notneeded -Completeditslifespan

FunctionsofPCDduringDevelopment

FuchsandSteller.CellVolume147,Issue42011742-758

Involutionofthemammaryglandafterlactation

CellDeath

ControlledcelldeathUncontrolledcelldeath

Physiologicalcelldeathor Necroticcelldeath(?)Programmedcelldeath (TypeIII)

AutophagiccelldeathApoptoticcelldeath(TypeII) (TypeI)

Caspase-independent Caspase-dependent

Extrinsicpathway Intrinsicpathway(Deathreceptor-mediated)

TypesofCellDeath

Fink and Cookson, Infect Immun. 2005

TypesofCellDeath

EvolutionaryConservationoftheCoreApoptoticMachinery

YaronFuchs,HermannStellerCellVolume147,Issue42011742-758

Whatisthedifferencebetweenkeycelldeathmechanisms

Apoptosis Autophagy Necrosis

-Geneticallyprogrammed Geneticallyprogrammed Not(?)genetically-Extraandintracellular Extraandintracellular Acuteinjury(?)(Extra)-Cellshrinkage Autophagicvacuoles Cytoplasmswelling-Blebbing Blebbing DisruptionofMemb-Organelleintact Sequesteration Disruption-Chromatincondensation Partialcondensation Non-condensed-DNAfragmentation(laddering) Noladdering Noladdering-Phagocytosiswith Phagocytosiswith Releaseofintracellularnoinflammation noinflammation contents&inflammation-Caspase-dependent Caspase-independent Caspase-independent

WhatisApoptosis? -Apredominantformofcelldeath -Asequenceofeventsinvolvingvariousclassofproteins -Geneticallyprogrammedandevolutionarilyconserved

HallmarksofApoptosis

-Intactdeathprogramisrequiredforsuccessfulembryonicdevelopmentandmaintenanceofnormaltissuehomeostasis

-Insufficientorevasionofapoptosismanifestas: -Cancer -Autoimmunity(proliferationofimmunesystemcells thatrecognizeandattackselfantigens) -Viralinfections(removalofvirallyinfectedcellsby apoptosis)

-Whileacceleratedcelldeathisrelatedto: -Neurodegenerativediseases -Immunodeficiency,infertility -Hematologicdiseases

Whyshouldwestudyapoptosis?

MajorPlayersinApoptosis

-Bcl-2familyproteins -Antiapoptotic -Proapoptoticmulti-domainproteins -ProapoptoticBH3-onlydomainprotein

-Caspases -Initiatorcaspases -Executionercaspases

Bcl-2familyproteins

Variouscell-physiologicstressesoperatethroughdifferentproapoptoticproteinstoantagonizeantiapoptoticproteinssuchasBcl2/Bcl-xL.BH3-onlyproteinssensesignalsandgalvanizeaction.

Bcl-2familyproteins

Bcl-2familyproteins-mediatedapoptosis

BAD: Phosphorylation sequesters it in the cytosol

Bcl-2familyproteins-mediatedapoptosis

*Bcl-2 must change conformation (or be ‘activated’) on the mitochondria to inhibit Bax (Dglugosz PJ, EMBO J. 25, 2287, 2006).

*Bcl-2 and Bcl-xL (and other prosurvival members) are the guardians of the mitochondria: they are inactivated when BH3-only proteins juxtapose their BH3 domain to Bcl-2 (protein-protein interaction)..

*These proteins are transcribed into multiple splice variants.

*Bcl-2 is phosphorylated by many apoptotic stimuli. Phosphorylation of Bcl-2 within the flexible loop generally inhibits its anti-apoptotic activity. But it has also been reported that phosphorylation is required for its apoptosis-inhibitory effect.

*Bcl-2 and Bcl-xL can also be cleaved by caspases during apoptosis. Cleaved proteins turns themselves into apoptotic killers.

*Bcl-xL may undergo deamidation: deamidation of Asn imparts susceptibility to apoptosis by disrupting the ability of Bcl-xL to block the proapoptotic activity of BH3-only proteins.

D. Tang

Regulation of Bcl-2 and Bcl-xL: Post-translational modifications

Mitochondria

Mitochondrialrespiratorychain

Apocytochromecandholocytochromec

Newmeyer,D.D.,andFerguson-Miller,S.Cell112,481-490,2003

MitochondrialPorinorPTP(permeabilitytransitionpore)

Newmeyer, D.D., and Ferguson-Miller, S. Cell 112, 481-490, 2003

OMMpermeabilization,PTP,andMitochondrialfission

Casp-3/7 may be involved in feedback cyt. c release

Lakhani SA et al., Science 311, 847, 2006 Adrain and Martin Science 311, 785, 2006

Prodomain Largesubunit Smallsubunit

Linker

p10p20p3-26

Prodomains:Inexecutionercaspases:~3kd Ininitiatorcaspases:10-26kd

Typesofcaspases: -Initiatorcaspases:caspase-2,-8,-9and-10 -ExecutionerCaspases:caspase-3,-6and-7

CaspaseStructure:

Caspaseprodomains:

D.Chandra

Caspases

Caspases

Caspases:Executionercaspases

Initiatorcaspases

Mol.Cell9,459-470,2002

1 479 aa

D374D385

D216

Prodomain (P25) Large subunit (P18)

P55/53DED DED

Small subunit (P12)

P10 subunit

Autocleavage at D385

P43/41

Caspase-8 Cleavage

Caspase-8 Cleavage

1 416 aa

D315D330

D130 (casp-3 cleavage site)

CARD domain (P16) Large subunit (P20)

P46CARD

Small subunit (P12)

P10 subunit

Autocleavage at D330

Large subunit + prodomain (P35/P37)

Cleavage at D331 (P22)

Caspase-9Cleavage

Cleavage at D130 = LS + SS (P30)*Prodomain = ~P15

QACGG

A.Caspasecleavage B.Substratecleavage

C.Inhibitoreffects

Apoptosis:Caspase-9Cleavage

LedgerwoodandMorisonClinCancerRes2009

Apoptosis: Caspase-3 activation

A.Caspasecleavage B.Substratecleavage

C.Inhibitoreffects

Caspasesubstrates

Proteosomecomponents(MolCell14,81-93,2004)MitochondrialcomplexIp75subunit(Cell117,773-786,2004)

IAPs

Shi,Y.Mol.Cell,9,459-470,2002

IAPsfunctionascriticalprosurvivalmolecules

*IAPs(especiallyXIAP)aregenerallyoverexpreessed inmultipletypesofcancercells

Smac(secondmitochondrial activatorofcaspases)

apoptoticstimulation

Shi,Y.Mol.Cell,9,459-470,2002

MechanismsofCaspaseActivationandInhibition

Knownapoptoticpathwaystoactivatecaspases

-Intrinsicpathway -Extrinsicpathwayordeath-receptorpathway -Otheralternativepathways -Caspase-independentapoptosis

ExtrinsicApoptoticpathway(deathreceptor)

Crosstalkbetweentwopathways

Howtodetectapoptosis?

-Cellmorphologybyusingmicroscopy-CytochromecreleasebyimmunolabellingandWestern-WesternBlotforcaspasesandtheirsubstrate-DAPIandAnnexin-V-staining-Detectionofapoptosomeusinggelfiltration

GM701/BMD188 (40 µM)

-Actin

-Holocytochrom

-Holocytochrom

Cytosol

Mito

0 30 � 1h 2h 4

A

Immunolabeling,subcellularfractionationandWesternblot

Cellshomogenized

Differentialcentrifugation

Cytosol Mitochondria

SDS-PAGEandWestern

CaspasecleavageandDAPIlabeling

DetectionofApoptosome

Whathappenstotheapoptoticcells?

Whathappenstotheapoptoticcells?

ApoptosisandTumorigenesis

1) Upregulation of Bcl2, BclxL, IAPs, Flip etc.

2) Downregulation/ mutation of Bax, Bak, BH3-only protein, Death-receptors, Apaf-1 etc.

Apoptosisbasedstrategiesforcancertherapy

Exploitingapoptoticmachineryforcancertherapy

ImportanceofBH3-onlyproteinmimeticsincancertherapy

TakeHomemessagefromapoptosis: -Apredominantformofcelldeathfromwormtomammals -Bcl-2familyproteinsmakedecisiontodieorlive

-Caspasesarethemainsoldiersinthebattlefieldtoexecuteapoptosis

-Mitochondrionisthecenterpointforallactivities

-TargetingBcl-2familyproteinshaveenormouspotentialincancertherapy

-FindingN-terminalsmac/DIABLOmimeticswillcounterIAPs

-ApplicationsofdeathligandssuchasTNF-α, TRAILetc.

Otherapoptosis/survivalfunctionsofBcl2-familyproteinsandcaspases

• Bcl-2andBcl-xLcanalsobecleavedbycaspasesduringapoptosisandthiscleavedfragmentsfunctionasproapoptoticproteins.

• Theseproteinsalsoundergoposttranslationalmodifications,whichcanalsoimpactonapoptosissensitivity.

• Pro-apoptoticBH3-onlyproteinssuchasBad,Bid,Puma,Noxahavealsobeenshowntopossessprosurvivalroles

• Caspase-3activationisinvolvedincancercellinvasion

• Caspase-8isrequiredbyNF-kBactivationandpromotescellmotility

Caspase-independentcelldeath

TaitandGreen,Oncogene2008

Autophagy

•FirstrecognizedunderEMearly1960s.

•Alsocalledmacroautophagy:Self-eatingtosurvive.

•Auniqueformofmembranetraffickinginwhichmembranecompartments(autophagosomes)engulfbothorganellesandcytosolicmacromoleculesanddeliverthemtothelysosomefordegradation.

YoshimoriTetal.,Cell128,833-836,2007QuXetal.,Cell128,931-46,2007

Autophagy

MartinLotz,etal.NatRevRheumatol.2011

Autophagy

SagarB.Kudchodkar,etal.RevMedVirol.2009

Autophagy

Mitophagy

Greenetal.,Science2011

AutophagyandCancer

GutianXiao.CytokineGrowthFactorRev.2007