ApoReview-Introduction to Apoptosis:Page 6 of26 are genetically encoded and are considered to be usually in place in a nucleated cell ready to be activated by a death-inducing stimulus [shizaki,199]. Apoptosis can be triggered by various stimuli from outside or inside the celle.g.by ligation of cell surface receptors,by DNA damage as a cause of defects in DNA repair mechanisms,treatment with cytotoxic drugs or irradiation,by a lack of survival signals,contradictory cell cycle signalling or by developmental death signals.Death signals of such diverse origin nevertheless appear to eventually activate a common cell death machinery leading to the characteristic features of apoptotic cell death. Much of the understanding of cell death has come from genetic studies in the nematode C.elegans by which several genes have been identified that function in the apoptotic killing and elimination of 131 of the initially 109 somatic cells that are generated druing hermaphrodite development [Hengartner. 1999].The proximal cause of apoptosis in C.elegans is the activation of the cysteine protease ced-3. which is mediated by its oligomerization at the activator protein ced-4.Activity of the ced-3/ced-4 complex is regulated by the apoptosis inhibitor ced-9 and the apoptosis inducer egl-1 (Fig.3). Subsequent studies in mammals and in the fly,D.melanogaster,have identidied counterparts for these C.elegans genes,demonstrating that the core components of the cell death machinery are conserved through evolution [Richardson,2002]Accordingly,ced-3 is the single C.elegans member of a family of cysteine proteases,the caspases,whereas ced-4 corresponds to the mammalian apoptotic protease activating factor 1,Apaf-1,which is the core of a caspase-activating signalling complex,the apoptosome.Egl-1 and ced-9 are members of the Bcl-2 family of pro-or antiapoptotic proteins. respectively,which play an important role in the mediation and regulation of apoptotic signalling pathways.All of those central components will be discussed within the following paragraphs. ced-s ced-3 ced-4 →Cell Death (anti-a ototic Bcl-2 members) (ca Fig.3 ans as a model s ystem contains basic components of the cell death machinery.Apoptosis tsalsogan alogous ma malian pro 8H3 teins.ced ne only worm caspase,and ced o ma (according to
ApoReview - Introduction to Apoptosis: Page 6 of 26 are genetically encoded and are considered to be usually in place in a nucleated cell ready to be activated by a death-inducing stimulus [Ishizaki, 1995; Weil, 1996]. Apoptosis can be triggered by various stimuli from outside or inside the cell, e.g. by ligation of cell surface receptors, by DNA damage as a cause of defects in DNA repair mechanisms, treatment with cytotoxic drugs or irradiation, by a lack of survival signals, contradictory cell cycle signalling or by developmental death signals. Death signals of such diverse origin nevertheless appear to eventually activate a common cell death machinery leading to the characteristic features of apoptotic cell death. Much of the understanding of cell death has come from genetic studies in the nematode C. elegans by which several genes have been identified that function in the apoptotic killing and elimination of 131 of the initially 1090 somatic cells that are generated druing hermaphrodite development [Hengartner, 1999]. The proximal cause of apoptosis in C. elegans is the activation of the cysteine protease ced-3, which is mediated by its oligomerization at the activator protein ced-4. Activity of the ced-3/ced-4 complex is regulated by the apoptosis inhibitor ced-9 and the apoptosis inducer egl-1 (Fig. 3). Subsequent studies in mammals and in the fly, D. melanogaster, have identidied counterparts for these C. elegans genes, demonstrating that the core components of the cell death machinery are conserved through evolution [Richardson, 2002]. Accordingly, ced-3 is the single C. elegans member of a family of cysteine proteases, the caspases, whereas ced-4 corresponds to the mammalian apoptotic protease activating factor 1, Apaf-1, which is the core of a caspase-activating signalling complex, the apoptosome. Egl-1 and ced-9 are members of the Bcl-2 family of pro- or antiapoptotic proteins, respectively, which play an important role in the mediation and regulation of apoptotic signalling pathways. All of those central components will be discussed within the following paragraphs. egl-1 ced-9 ced-3 ced-4 Cell Death (pro-apoptotic Bcl-2 members) (anti-apoptotic Bcl-2 members) (caspases, Apaf-1) Fig. 3 C. elegans as a model system contains basic components of the cell death machinery. Apoptosis regulation in C.elegans relies on a simple basic network of factors for which corresponding analogous components also can be found in higher organisms as given within brackets. Thus egl-1 is the worm representative for mammalian proapoptotic BH3-only proteins, ced-9 belongs to the antiapoptotic Bcl-2 family, ced-3 is the only worm caspase, and ced-4 is homologous to mammalian Apaf-1 (according to [Cecconi, 1999])
ApoReview-Introduction to Apoptosis:Page 7 of 26 42 Caspases are central initiators and executioners of apoptosis The caspases,cysteine proteases homologous to C.elegans ced-3,are of central importance in the apoptotic signalling network which are activated in most cases of apoptotic cell death [Bratton,2000]. Actually,strictly defined,cell death only can be classified to follow a classical apoptotic mode if execution of cell death is dependent on caspase activity [Leist2001]. The term caspases is derived from ysteine-dependent aspartate-specific proteases their catalytical activity depends on a critical cysteine-residue within a highly conserved active-site pentapeptide QACRG,and the caspases specifically cleave their substrates after Asp residues.So far,7 different caspases have been identified in Drosophla,and 14 different members of the caspase-family have been described in mammals,with caspase-11 and caspase-12 only identified in the mouse [Denault, 202:Richardson,2002]According to a unified nomenclature,the caspases are referred to in the order of their publication:caspase-1 is ICE(Interleukin-1B-Converting Enzyme).the first mammalian caspase described to be a homologue of Ced-3 [Creagh,2001;Miura,1993].Caspase-1 as well as caspases-.,-11,and-12 appear to be mainly involved in the proteolytic maturation of pro inflammatory cytokines such as pro-IL-1B and pro-L-1and their contribution to the execution of apoptosis remains questionable [Denault,2002].Indeed,mice deficient for caspase-1 or caspase-11 develop normally and cells from those knockout mice remain sensitive to various death stimuli [L 1995;Wang.1998].In contrast,gene knockout experiments targeting caspase-3 and caspase-9 resulted in perinatal mortality as a result of severe defects in brain development [Kuida.99:Kuida.199] whereas caspase-deficient embryos died after day 12 [Varfolomeev,1]This and the observation that cell lines derived from those knockout experiments are resistant to distinct apoptosis stimuli underlines the importance of caspases as proapoptotic mediators.Indeed,caspase-3.caspase-9, caspase-and additionally caspases-27,and-10have been recognized to play an important rol in the apoptotic signalling machinery [Earnshaw,1999]. In the cell,caspases are synthesized as inactive zymogens,the so called procaspases,which at their N- terminus carry a prodomain followed by a large and a small subunit which sometimes are separated by a linker peptide.Upon maturation,the procaspases are processed between the large and small subunit,resulting in a small and a large subunit.The prodomain is also frequently but not necessarily removed during the activation process.A heterotetramer consisting of each two small and two large subunits then forms an active caspase.The proapoptotic caspases can be divided into the group of initiator caspases including procaspases-2.-9and-10,and into the group of executioner caspases including procaspases-3.-6.and-7.Whereas the executioner caspases possess only short prodomains,the initiator caspases possess long prodomains,containing death effector domains(DED) in the case of procaspases-8 and-10 or caspase recruitment domains (CARD)as in the case of procaspase-9 and procaspase-2
ApoReview - Introduction to Apoptosis: Page 7 of 26 4.2 Caspases are central initiators and executioners of apoptosis The caspases, cysteine proteases homologous to C. elegans ced-3, are of central importance in the apoptotic signalling network which are activated in most cases of apoptotic cell death [Bratton, 2000]. Actually, strictly defined, cell death only can be classified to follow a classical apoptotic mode if execution of cell death is dependent on caspase activity [Leist, 2001]. The term caspases is derived from cysteine-dependent aspartate-specific proteases: their catalytical activity depends on a critical cysteine-residue within a highly conserved active-site pentapeptide QACRG, and the caspases specifically cleave their substrates after Asp residues. So far, 7 different caspases have been identified in Drosophila, and 14 different members of the caspase-family have been described in mammals, with caspase-11 and caspase–12 only identified in the mouse [Denault, 2002; Richardson, 2002]. According to a unified nomenclature, the caspases are referred to in the order of their publication: caspase-1 is ICE (Interleukin-1ß-Converting Enzyme), the first mammalian caspase described to be a homologue of Ced-3 [Creagh, 2001; Miura, 1993]. Caspase-1 as well as caspases-4, -5, -11, and –12 appear to be mainly involved in the proteolytic maturation of proinflammatory cytokines such as pro-IL-1ß and pro-IL-18 and their contribution to the execution of apoptosis remains questionable [Denault, 2002]. Indeed, mice deficient for caspase-1 or caspase-11 develop normally and cells from those knockout mice remain sensitive to various death stimuli [Li, 1995; Wang, 1998]. In contrast, gene knockout experiments targeting caspase-3 and caspase-9 resulted in perinatal mortality as a result of severe defects in brain development [Kuida, 1998; Kuida, 1996], whereas caspase-8 deficient embryos died after day 12 [Varfolomeev, 1998]. This and the observation that cell lines derived from those knockout experiments are resistant to distinct apoptosis stimuli underlines the importance of caspases as proapoptotic mediators. Indeed, caspase-3, caspase-9, caspase-8, and additionally caspases-2, -6, -7, and –10 have been recognized to play an important role in the apoptotic signalling machinery [Earnshaw, 1999]. In the cell, caspases are synthesized as inactive zymogens, the so called procaspases, which at their Nterminus carry a prodomain followed by a large and a small subunit which sometimes are separated by a linker peptide. Upon maturation, the procaspases are proteolytically processed between the large and small subunit, resulting in a small and a large subunit. The prodomain is also frequently but not necessarily removed during the activation process. A heterotetramer consisting of each two small and two large subunits then forms an active caspase. The proapoptotic caspases can be divided into the group of initiator caspases including procaspases-2, -8, -9 and –10, and into the group of executioner caspases including procaspases-3, -6, and –7. Whereas the executioner caspases possess only short prodomains, the initiator caspases possess long prodomains, containing death effector domains (DED) in the case of procaspases-8 and –10 or caspase recruitment domains (CARD) as in the case of procaspase-9 and procaspase-2
ApoReview-Introduction to Apoptosis:Page 8 of26 Via their prodomains,the initiator caspases are recruited to and activated at death inducing signalling complexes either in response to the ligation of cell surface death receptors (extrinsic apoptosis pathways)or in response to signals originating from inside the cell(intrinsic apoptosis pathways). In extrinsic apoptosis pathways(Fig.4).e.g procaspase-is recruited by its DEDs to the death inducing signalling complex(DISC),a membrane receptor complex formed following to the ligation of a member of the tumor necrosis factor receptor (TNFR)family [Sartorius,2001].When bound to the DISC.several procaspase-8 molecules are in close proximity to each other and therefore are assumed to activate each other by autoproteolysis [Denault,2002]. Procaspaso-8 aa6.7一→APOPTOSIS active Caspase-8 Fig.4 Roceptomctetedoca5aseactaronmptcheDssgepn3sgeoae%ag9at69escbe possessing DDs,the adaptors additionally contain death effector domains (DED)which recruit which isa het bunits he initiatior caspase-8 cleaves and ereby a cror caspases for t si
ApoReview - Introduction to Apoptosis: Page 8 of 26 Via their prodomains, the initiator caspases are recruited to and activated at death inducing signalling complexes either in response to the ligation of cell surface death receptors (extrinsic apoptosis pathways) or in response to signals originating from inside the cell (intrinsic apoptosis pathways). In extrinsic apoptosis pathways (Fig. 4), e.g. procaspase-8 is recruited by its DEDs to the death inducing signalling complex (DISC), a membrane receptor complex formed following to the ligation of a member of the tumor necrosis factor receptor (TNFR) family [Sartorius, 2001]. When bound to the DISC, several procaspase-8 molecules are in close proximity to each other and therefore are assumed to activate each other by autoproteolysis [Denault, 2002]. Ligand (FasL, TNF-α, TRAIL) Death Receptor (Fas, TNFR1, DR5, .) Adaptors (FADD, TRADD) D D D D D D D E D D D D E D D D D E D D E D Procaspase-8 D E D D E D active Caspase-8 Activation of Caspases-3, -6, -7 APOPTOSIS Ligand (FasL, TNF-α, TRAIL) Death Receptor (Fas, TNFR1, DR5, .) Adaptors (FADD, TRADD) D D D D D D D E D D D D E D D D D E D D E D Procaspase-8 D E D D E D active Caspase-8 Activation of Caspases-3, -6, -7 APOPTOSIS Fig. 4 Receptor-mediated caspase activation at the DISC. Upon ligation by its cognate ligand, the trimeric death receptor recruits adaptor molecules via its cytoplasmic death domains (DD). Besides possessing DDs, the adaptors additionally contain death effector domains (DED) which recruit procaspase-8 to the receptor complex which now is called the death-inducing signalling complex (DISC). Procaspase-8 is activated by autoproteolytic cleavage and forms the active caspase-8 which is a heterotetramer of two small and two large subunits. The initiatior caspase-8 cleaves and thereby activates effector caspases for the execution of apoptosis
ApoReview-Introduction to Apoptosis:Page 9 of 26 Intrinsic apoptosis pathways (Fig.5)involve procaspase-9 which is activated downstream of mitochondrial proapoptotic events at the so called apoptosome,a ytosolic death signalling protein complex that is formed upon release of cytochrome c from the mitochondria [Salvesen,2002b].In this case it is the dimerization of procaspase-9 molecules at the Apaf-I scaffold that is responsible for caspase-activation [Denault,2002]Once the initiator caspases have been activated,they can proteolytically activate the effector procaspases-3.6,and-7 which subsequently cleave a specific set of protein substrates,including procaspases themselves,resulting in the mediation and amplification of the death signal and eventually in the execution of cell death with all the morphological and biochemical features usually observed [Earnshaw,1999]. A.Mitochondrial pathway of caspase activation →→ B.Apoptosome formation and activation Apoptosome that allows the fo of a hep enc. vhee like structure.the apoptosome. Procaspase-g 5oset5e的1op9ypouogeeeep9uBu3969eUse西GWNpyd
ApoReview - Introduction to Apoptosis: Page 9 of 26 Intrinsic apoptosis pathways (Fig. 5) involve procaspase-9 which is activated downstream of mitochondrial proapoptotic events at the so called apoptosome, a cytosolic death signalling protein complex that is formed upon release of cytochrome c from the mitochondria [Salvesen, 2002b]. In this case it is the dimerization of procaspase-9 molecules at the Apaf-1 scaffold that is responsible for caspase-9 activation [Denault, 2002]. Once the initiator caspases have been activated, they can proteolytically activate the effector procaspases-3, -6, and -7 which subsequently cleave a specific set of protein substrates, including procaspases themselves, resulting in the mediation and amplification of the death signal and eventually in the execution of cell death with all the morphological and biochemical features usually observed [Earnshaw, 1999]. WD40 CARD WD40 Apaf-1 Cyto C dATP WD40 WD40 CARD Cyto C 7 x Active Caspase-9 dimers WD40 WD40 Cy ot C WD40 WD40 Cy ot C CARD CARD „hub“ plane CARD CARD CARD CARD CARD CARD ProCaspase-9 Mitochondria Apoptotic Stimulus Cyto c release Formation of the Apoptosome Activation of Caspase-9 A. Mitochondrial pathway of caspase activation B. Apoptosome formation and activation Apoptosome WD40 CARD WD40 Apaf-1 Cyto C dATP WD40 WD40 CARD Cyto C 7 x Active Caspase-9 dimers WD40 WD40 Cy ot C WD40 WD40 Cy ot C CARD CARD „hub“ plane CARD CARD CARD CARD CARD CARD ProCaspase-9 Mitochondria Apoptotic Stimulus Cyto c release Formation of the Apoptosome Activation of Caspase-9 A. Mitochondrial pathway of caspase activation B. Apoptosome formation and activation Apoptosome Fig. 5 Mitochondria-mediated caspase activation at the apoptosome. A. Apoptotic stimuli trigger the release of apoptogenic factors from the mitochondrial intermembrane space to the cytosol, such as cytochrome c which induces the formation of the apoptosome and the activation of procaspase- 9. B. By the action of cytochrome c (Cyto C) and dATP the Apaf-1 protein adopts a conformation that allows the formation of a heptameric, wheel-like structure, the apoptosome. Procaspase-9 molecules can bind to the inner “hub” region of the apoptosome and are activated by dimer formation. Active caspase-9 dimers further mediate activation of effector caspases [Acehan, 2002]