RnDSy-lu-2945 Caspase Activation & Apoptosis Extrinsic & Intrinsic Pathways of Caspase Activation CASPASE CLEAVAGE & ACTIVATION Caspases are a family of aspartate-specific, cysteine proteases that serve as the primary mediators of Pro-Domain Large Subunit (p20) Small Subunit (p10) apoptosis. Mammalian caspases can be subdivided into three functional groups, apoptotic initiator TRAIL Pro-Domain α chain β chain caspases (Caspase-2, -8, -9, -10), apoptotic effector caspases (Caspase-3, -6, -7), and caspases involved Asp-x Asp-x Proteolytic cleavage in inflammatory cytokine processing (Caspase-1, -4, -5, 11, and -12L/12S). All caspases are synthesized α chain as inactive zymogens containing a variable length pro-domain, followed by a large (20 kDa) and a small Fas Ligand TRAIL R1 Pro-Domain β chain TRAIL R2 Heterotetramer Formation (10 kDa) subunit. FADD FADD α chain α chain Pro-caspase-8, -10 Active caspase Pro-caspase-8, -10 TWEAK β chain Apoptotic caspases are activated upon the receipt of either an extrinsic or an intrinsic death signal. The Fas/CD95 extrinsic pathway (green arrows) is initiated by ligand binding to cell surface death receptors (TNF RI, Fas/ MAMMALIAN CASPASE DOMAINS & CLEAVAGE SITES FADD FADD TNF-α APOPTOTIC CASPASES CD95, DR3, TRAIL R1/DR4, TRAIL R2/DR5) followed by receptor oligomerization and cleavage of Pro- Extrinsic Pathway DR3 (or another TWEAK R) INITIATOR CASPASES 152 316 331 Pro-caspase-8, -10 Pro-caspase-8, -10 1 435 caspase-8 and -10. Activation of Caspase-8 and Caspase-10 results in the cleavage of BID and Caspase-2 CARD TRADD FLIP TRADD downstream effector caspases. The intrinsic pathway of caspase activation (purple arrows) is initiated by TNF RI ARC 216 374 385 FADD TRAF-2 1 479 Caspase-8, -10 Caspase-8 DED DED RIP1 events such as DNA damage, growth factor withdrawal, or loss of contact with the extracellular matrix. TRADD BID Pro-caspase-8, -10 DD RIP1 TRADD DED 138 315 331 tBID 1 416 These events lead to changes in the integrity of the mitochondrial membrane that result in the release of TRAF-2 Caspase-9 CARD Pro-caspase-8, -10 BAK Cytochrome c pro-apoptotic proteins including Cytochrome c, Smac/Diablo, HTRA2/Omi, Apoptosis-Inducing Factor (AIF), Death Domain (DD) 219 415 SMAC/Diablo 1 521 Death Effector Domain (DED) Caspase-9 IAPs Caspase-10 DED DED and Endonuclease G. tBID HTRA2/Omi IAPs tBID tBID tBID 14-3-3 Bad Bad BAK BAK Bcl-2 Bax 28 175 P P EFFECTOR CASPASES 1 277 P Caspase-3 Mitochondrial membrane permeability is regulated by the Bcl-2 family of proteins. The balance between Bad Bcl-2 Bcl-xL Pro-caspase-3 23 179 194 Caspase Recruitment 1 293 BAK Bcl-xL Caspase-3 pro- and anti-apoptotic family members determines whether or not a cell will undergo apoptosis. In healthy Domain (CARD) Target Molecules Caspase-6 APAF-1 (e.g. Actin, 23 198 tBID Pro-caspase-9 Nuclear lamins, 1 303 cells, Bcl-2 and Bcl-xL inhibit apoptosis by binding to the pro-apoptotic Bax and BAK proteins. Bad is also Bax Pro-caspase-7 Bad Bcl-xL Apoptosome ICAD, PARP) Caspase-7 BAK Caspase-7 BAK tBID Cytochrome c phosphorylated and sequestered in the cytoplasm by the 14-3-3 protein in these cells. If cytoplasmic levels tBID Bcl-2 INFLAMMATORY CASPASES Bcl-xL 119 297 317 Bax 1 404 Bcl-x tBID of free BAD increase, Bcl-2 and Bcl-xL bind to Bad and release Bax and BAK. Bax and BAK, or processed L Bcl-2 Bcl-2 Caspase-1 CARD Cytochrome c Bad tBID Pro-caspase-6 Bax Bax Caspase-6 93 270 290 forms of these proteins, can then insert into the mitochondrial membrane, compromising its integrity. Bad Cytochrome c Cytochrome c 1 377 tBID Caspase-4 CARD Bax AIF HSPs 134 311 331 Bad 1 418 Bax BID Caspase-5 CARD Initiator caspases are activated in three distinct protein complexes, the death-inducing signaling complex Endo G 1 373 (DISC; Caspase-8 and -10), the apoptosome (Caspase-9), and the PIDDosome (Caspase-2). The DISC is 14-3-3 ARC Caspase-2 Caspase-11 CARD P P 1 125 formed following ligand binding and death receptor oligomerization. Pro-caspase-8 and Pro-caspase-10 P PIDDosome Caspase-12S CARD Bad DNA FRAGMENTATION 1 419 Caspase-12L CARD are recruited to the death receptors through their interactions with the adaptor protein, FADD. This Pro-caspase-2 CASPASE ACTIVATING COMPLEXES interaction is mediated by their shared death effector domains (DED). Clustering of pro-caspases near the CRADD/RAIDD DISC INTERACTING DOMAINS INHIBITORS OF COMPLEX FORMATION death receptors results in their cleavage and activation. In contrast, Pro-caspase-9 is activated following CARD Fas Ligand DD DNA DAMAGE an intrinsic change associated with the release of Cytochrome c from the mitochondria. In the cytoplasm, x x x Fas/CD95 DD PIDD x DD x FADD DD/DED Cytochrome c interacts with APAF-1, which recruits Pro-caspase-9 by way of its caspase recruitment P Pro-apoptotic: Anti-apoptotic: p53 p53 p53 • Bad • PUMA • 14-3-3 Pro-caspase-8,-10 DED domain (CARD) to form the apoptosome. Formation of the apoptosome leads to the cleavage and • Bax • PIDD • p21 DED DD • BID others others FLIP ARC activation of Caspase-9. Intrinsic cellular changes can also lead to the activation of Caspase-2. Following • Noxa APOPTOSOME INTERACTING DOMAINS INHIBITORS OF COMPLEX FORMATION DNA damage, p53 induces the expression of p53-induced protein with a death domain (PIDD), which Intrinsic Pathway HSP27 Cytochrome c HSP70 HSP27 associates with the CRADD/RAIDD adaptor protein and Pro-caspase-2 to form the PIDDosome. The Cytochrome c APAF-1 CARD association between CRADD/RAIDD and PIDD is mediated by their shared death domains (DD), while Pro-caspase-9 CARD APAF-1 NUCLEUS CARD domains mediate the interaction between CRADD/RAIDD and Pro-caspase-2. Formation of the PIDDOSOME INTERACTING DOMAINS PIDD DD PIDDosome leads to the cleavage of Pro-caspase-2. CRADD/RAIDD DD/CARD Pro-caspase-2 CARD Autocatalytic cleavage of the initiator pro-caspases occurs at aspartic acid residues located after the pro- INFLAMMASOME INTERACTING DOMAINS domain, and in between the large and the small subunits. Upon cleavage, mature caspases form a NALP3, NALP1 or IPAF Pyrin DOMAIN KEY proteolytically active heterotetramer consisting of two small and two large subunits. Once activated, Death Domain (DD) ASC Pyrin/CARD Death Effector Domain (DED) initiator caspases cleave downstream effector caspases that promote the ordered disassembly of the cell Caspase Recruitment Pro-caspase-1 CARD Domain (CARD) by targeting a number of critical cellular proteins including structural proteins, DNA repair proteins, and Pyrin Domain proteins involved in signal transduction pathways. NOTE: This poster conveys a general overview and should be considered neither comprehensive nor definitive. The details of this information are understood to be subject to interpretation. © R&D Systems 2016 Learn more | rndsystems.com/apoptosis Global [email protected] [email protected] North America TEL 800 343 7475 Europe | Middle East | Africa TEL +44 (0)1235 529449 China [email protected] TEL +86 (21) 52380373 Rest of World bio-techne.com/find-us/distributors TEL +1 612 379 2956 Rnlu Wall Poster RnDSy-lu-2945 Caspase Activation & Apoptosis Extrinsic & Intrinsic Pathways of Caspase Activation CASPASE CLEAVAGE & ACTIVATION Caspases are a family of aspartate-specific, cysteine proteases that serve as the primary mediators of Pro-Domain Large Subunit (p20) Small Subunit (p10) apoptosis. Mammalian caspases can be subdivided into three functional groups, apoptotic initiator TRAIL Pro-Domain α chain β chain caspases (Caspase-2, -8, -9, -10), apoptotic effector caspases (Caspase-3, -6, -7), and caspases involved Asp-x Asp-x Proteolytic cleavage in inflammatory cytokine processing (Caspase-1, -4, -5, 11, and -12L/12S). All caspases are synthesized α chain as inactive zymogens containing a variable length pro-domain, followed by a large (20 kDa) and a small Fas Ligand TRAIL R1 Pro-Domain β chain TRAIL R2 Heterotetramer Formation (10 kDa) subunit. FADD FADD α chain α chain Pro-caspase-8, -10 Active caspase Pro-caspase-8, -10 TWEAK β chain Apoptotic caspases are activated upon the receipt of either an extrinsic or an intrinsic death signal. The Fas/CD95 extrinsic pathway (green arrows) is initiated by ligand binding to cell surface death receptors (TNF RI, Fas/ MAMMALIAN CASPASE DOMAINS & CLEAVAGE SITES FADD FADD TNF-α APOPTOTIC CASPASES CD95, DR3, TRAIL R1/DR4, TRAIL R2/DR5) followed by receptor oligomerization and cleavage of Pro- Extrinsic Pathway DR3 (or another TWEAK R) INITIATOR CASPASES 152 316 331 Pro-caspase-8, -10 Pro-caspase-8, -10 1 435 caspase-8 and -10. Activation of Caspase-8 and Caspase-10 results in the cleavage of BID and Caspase-2 CARD TRADD FLIP TRADD downstream effector caspases. The intrinsic pathway of caspase activation (purple arrows) is initiated by TNF RI ARC 216 374 385 FADD TRAF-2 1 479 Caspase-8, -10 Caspase-8 DED DED RIP1 events such as DNA damage, growth factor withdrawal, or loss of contact with the extracellular matrix. TRADD BID Pro-caspase-8, -10 DD RIP1 TRADD DED 138 315 331 tBID 1 416 These events lead to changes in the integrity of the mitochondrial membrane that result in the release of TRAF-2 Caspase-9 CARD Pro-caspase-8, -10 BAK Cytochrome c pro-apoptotic proteins including Cytochrome c, Smac/Diablo, HTRA2/Omi, Apoptosis-Inducing Factor (AIF), Death Domain (DD) 219 415 SMAC/Diablo 1 521 Death Effector Domain (DED) Caspase-9 IAPs Caspase-10 DED DED and Endonuclease G. tBID HTRA2/Omi IAPs tBID tBID tBID 14-3-3 Bad Bad BAK BAK Bcl-2 Bax 28 175 P P EFFECTOR CASPASES 1 277 P Caspase-3 Mitochondrial membrane permeability is regulated by the Bcl-2 family of proteins.