CASPASE ACTIVATION& APOPTOSISNote: This poster conveys a
general overview and should be considered neither comprehensive nor
denitive. The details of the process are understood to be subject
to interpretation. R&D Systems, Inc. 2010R&D Systems,
Inc.|1-800-343-7475|www.RnDSystems.comExtrinsic & Intrinsic
Pathways of Caspase ActivationCaspases are a family of
aspartate-specic, cysteine proteases that serve as the primary
mediators of apoptosis. Mammalian caspases can be subdivided into
three functional groups, apoptotic initiator caspases (Caspase-2,
-8, -9, -10), apoptotic eector caspases (Caspase-3, -6, -7), and
caspases involved in inammatory cytokine processing (Caspase-1, -4,
-5, 11, and -12L/12S). All caspases are synthesized as inactive
zymogens containing a variable length pro-domain, followed by a
large (20 kDa) and a small (10 kDa) subunit. Apoptotic caspases are
activated upon the receipt of either an extrinsic or an intrinsic
death signal. The extrinsic pathway (green arrows) is initiated by
ligand binding to cell surface death receptors (TNF RI, Fas/CD95,
DR3, TRAIL R1/DR4, TRAIL R2/DR5) followed by receptor
oligomerization and cleavage of Pro-caspase-8 and -10. Activation
of Caspase-8 and Caspase-10 results in the cleavage ofBID and
downstream eector caspases. The intrinsic pathway of caspase
activation (purple arrows) is initiated by events such as DNA
damage, growth factor withdrawal, or loss of contact with the
extracellular matrix. These events lead to changes in the integrity
of the mitochondrial mem-brane that result in the release of
pro-apoptotic proteins including Cytochrome c, Smac/Diablo,
HTRA2/Omi, Apoptosis-Inducing Factor (AIF), and Endonuclease G.
Mitochondrial membrane permeability is regulated by the Bcl-2
family of proteins. The balance between pro- and anti-apoptotic
family members determines whether or not a cell will undergo
apoptosis. In healthy cells, Bcl-2 and Bcl-xL inhibit apoptosis by
binding to the pro-apoptotic Bax and BAK proteins. Bad is also
phos-phorylated and sequestered in the cytoplasm by the 14-3-3
protein in these cells. If cytoplasmic levels of free BAD increase,
Bcl-2 and Bcl-xL bind to Bad and release Bax and BAK. Bax and BAK,
or processed forms of these proteins, can then insert into the
mitochondrial membrane, compromising its integrity. Initiator
caspases are activated in three distinct protein complexes, the
death-inducing signaling complex (DISC; Caspase-8 and -10), the
apoptosome (Caspase-9), and the PIDDosome (Caspase-2). The DISC is
formed following ligand binding and death receptor oligomerization.
Pro-caspase-8 and Pro-caspase-10 are recruited to the death
receptors through their interactions with the adaptor protein,
FADD. This interaction is mediated by their shared death eector
domains (DED). Clustering of pro-caspases near the death receptors
results in their cleavage and activation. In contrast,
Pro-caspase-9 is activated following an intrinsic change associated
with the release of Cytochrome c from the mitochondria. In the
cytoplasm, Cytochrome c interacts with APAF-1, which recruits
Pro-caspase-9 by way of its caspase recruitment domain (CARD) to
form the apoptosome. Formation of the apoptosome leads to the
cleavage and activation of Caspase-9. Intrinsic cellular changes
can also lead to the activation of Caspase-2. Following DNA damage,
p53 induces the expression of p53-induced protein with a death
domain (PIDD), which associates with the CRADD/RAIDD adaptor
protein and Pro-caspase-2 to form the PIDDosome. The association
between CRADD/RAIDD and PIDD is mediated by their shared death
domains (DD), while CARD domains mediate the interaction between
CRADD/RAIDD and Pro-caspase-2. Formation of the PIDDosome leads to
the cleavage of Pro-caspase-2. Autocatalytic cleavage of the
initiator pro-caspases occurs at aspartic acid residues located
after the pro-domain, and in between the large and the small
subunits. Upon cleavage, mature caspases form a proteolytically
active heterotetramer consisting of two small and two large
subunits. Once activated, initiator caspases cleave downstream
eector caspases that promote the ordered disassembly of the cell by
targeting a number of critical cellular proteins including
structural proteins, DNA repair proteins, and proteins involved in
signal transduction pathways. R&D Systems,
Inc.|1-800-343-7475|www.RnDSystems.comfunctional groups, apoptotic
initiator caspases (Caspase-2, -8, -9, -10), apoptotic eector
caspases (Caspase-3, -6, -7), andnammatory cytokine processing
(Caspase-1, -4, -5, 11, and -12L/12S). All caspases are synthesized
as inactive zymogens length pro-domain, followed by a large (20
kDa) and a small (10 kDa) subunit.re activated upon the receipt of
either an extrinsic or an intrinsic death signal. The extrinsicws)
is initiated by ligand binding to cell surface death receptors (TNF
RI, Fas/CD95, DR3,R2/DR5) followed by receptor oligomerization and
cleavage of Pro-caspase-8 and -10.-8 and Caspase-10 results in the
cleavage ofBID and downstream eectorc pathway of caspase activation
(purple arrows) is initiated by events such as factor withdrawal,
or loss of contact with the extracellular matrix. Thesees in the
integrity of the mitochondrial mem-e release of s including Diablo,
is-and rane permeability is regulated by thens. The balance between
pro- and members determines whether or o apoptosis. In healthy
cells, t apoptosis by binding to the d BAK proteins. Bad is also
phos-estered in the cytoplasm by the 14-3-3 If cytoplasmic levels
of free BAD increase, Bcl-2 and release Bax and BAK. Bax and BAK,
or processed ns, can then insert into the mitochondrial
membrane,grity. activated in three distinct protein complexes, the
death-mplex (DISC; Caspase-8 and -10), the apoptosome PIDDosome
(Caspase-2). The DISC is formed ing and death receptor
oligomerization. o-caspase-10 are recruited to the deatheir
interactions with the adaptor teraction is mediated by ector
domains (DED).pases near the ts in their on. In -9 is n e
mitochondria. In the cytoplasm, Cytochrome c interacts with APAF-1,
which 9 by way of its caspase recruitment domain (CARD) to form the
apoptosome. ptosome leads to the cleavage and activation of
Caspase-9. Intrinsic cellular to the activation of Caspase-2.
Following DNA damage, p53 induces the uced protein with a death
domain (PIDD), which associates with theor protein and
Pro-caspase-2 to form the PIDDosome. The associationDD and PIDD is
mediated by their shared death domains (DD), while te the
interaction between CRADD/RAIDD and Pro-caspase-2.Dosome leads to
the cleavage of Pro-caspase-2. e of the initiator pro-caspases
occurs at aspartic acid residuesdomain, and in between the large
and the small subunits.re caspases form a proteolytically active
heterotetramerll and two large subunits. Once activated,
initiatornstream eector caspases that promote the ordered ll b b f
l ll l l dp53 p53DNA DAMAGENUCLEUSx xx xxp53PCaspase
RecruitmentDomain (CARD)Cytochrome cBIDSMAC/DiabloHTRA2/OmiEndo
GAIFBIDCaspase-2Cytochrome cCytochrome cCytochrome cCytochrome
ctBIDtBIDtBIDtBIDtBIDtBIDtBIDtBIDtBIDtBID Bcl-2Bcl-2Bcl-2Bcl-2
Bcl-2BaxBaxBaxBaxBadBadBadBadBadBadBadBaxBAKBAKBAKBAKBAKBaxBaxCaspase-8,
-10Caspase-9Caspase-3TRAF-2TRAF-2TRADDTRADDTRADDFADDFADD
FADDTRADDRIP1RIP1DEDDDDeath Domain (DD)Death Eector Domain
(DED)Caspase-7Caspase-6IAPsIAPsHSPsTarget Molecules (e.g.
Actin,Nuclear lamins,ICAD,
PARP)Pro-caspase-7Pro-caspase-6Pro-caspase-9ApoptosomeAPAF-1DNA
FRAGMENTATIONPro-apoptotic: Bad PUMA Bax PIDD BID others
NoxaPro-caspase-3PIDDosome14-3-3Bad14-3-3FADD FADDTNF-TNF RIFas
LigandTRAILTWEAKDR3 (or another TWEAK R)TRAIL R1TRAIL
R2Fas/CD95FLIPPro-caspase-8, -10Pro-caspase-8, -10 Pro-caspase-8,
-10Pro-caspase-8, -10Pro-caspase-8, -10Pro-caspase-8,
-10PIDDDDCRADD/RAIDDDDCARDPro-caspase-2Anti-apoptotic: 14-3-3
p21othersPPPtBIDBcl-xLBcl-xLBAKBcl-xLBcl-xLPPPBcl-xLARCARCMAMMALIAN
CASPASE DOMAINS & CLEAVAGE SITESCASPASE CLEAVAGE &
ACTIVATIONAsp-x Asp-xPro-DomainPro-DomainLarge Subunit (p20) Small
Subunit (p10) chain chainINFLAMMATORY CASPASESAPOPTOTIC
CASPASES1419Caspase-12S CARDCaspase-12L CARD1251373Caspase-11
CARD1INITIATOR CASPASESEFFECTOR CASPASESPro-Domain chain
chainProteolytic cleavageHeterotetramerFormation chain chain
chainActive caspaseCASPASE ACTIVATING COMPLEXESDISC INTERACTING
DOMAINSINTERACTING DOMAINSINTERACTING DOMAINSINTERACTING
DOMAINSINHIBITORS OF COMPLEX FORMATIONINHIBITORS OF COMPLEX
FORMATIONPIDDOSOMEINFLAMMASOMEDDDD/CARDCARDPyrinPyrin/CARDCARDPIDDCRADD/RAIDDPro-caspase-2NALP3,
NALP1 or IPAFASCPro-caspase-1DDDD/DEDDEDFLIP ARCFas
LigandFas/CD95FADDPro-caspase-8,-10DED DDHSP27APAF-1HSP70
HSP27APOPTOSOMECARDCARDCytochrome cCytochrome
cAPAF-1Pro-caspase-9Intrinsic PathwayExtrinsic PathwayPyrin
DomainDeath Domain (DD)Death Eector Domain (DED)Caspase Recruitment
Domain (CARD)DOMAIN KEYCaspase-723 198303 1Caspase-1 CARD119 297
317404 1Caspase-328 175277 1Caspase-10 DED DED219 415521
1194Caspase-623 179293 1Caspase-9 CARD138 315 331416 1Caspase-8 DED
DED216 374 385479 1Caspase-2 CARD1521316 331435Caspase-4 CARD93 270
290377 1Caspase-5 CARD134 311 331418 1
