#19 apoptosis chapter 9 - roswell park cancer institute at d331 (p22) caspase-9 cleavage cleavage at...
TRANSCRIPT
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