细胞凋亡导论（参考文献）Introduction to Apoptosis.pdf_大学文库

ApoReview-Introduction to Apoptosis:Page 2 of26 Introduction to Apoptosis 1. The development of the term apoptosis Already since the mid-nineteenth century,many observations have indicated that cell death plays a considerable role during physiological processes of multicellular organisms,particularly during embryogenesis and metamorphosis [Gluecksmann,1951;Lockshin,2001].The term programmed cell death was introduced in 1964,proposing that cell death during development is not of accidential nature but follows a sequence of controlled steps leading to locally and temporally defined self- destruction [Lockshin,1964]. Eventually,the term apoptosis had been coined in order to describe the morphological processes leading self-destruction and was first introduced na publication by and Currie [Kerr,1972].Apoprosis is of greek origin,having the meaning"falling off or dropping of in analogy to leaves falling off trees or petals dropping off flowers.This analogy emphasizes that the death of living matter is an integral and necessary part of the life cycle of organisms.The apoptotic mode of cell death is an active and defined process which plays an important role in the development of multicellular organisms and in the regulation and maintenance of the cell populations in tissues upon physiological and pathological conditions.It should be stressed that apoptosis is a well-defined and possibly the most frequent form of programmed cell death,but that other,non-apoptotic types of cell death also might be of biological significance [Leist,2001] 2- The significance of apoptosis The development and maintenance of multicellular biological systems depends on a sophisticated interplay between the cells forming the organism,it sometimes even seems to involve an altruistic behaviour of individual cells in favour of the organism as a whole.During development many cells are produced in excess which eventually undergo programmed cell death and thereby contribute to sculpturing many organs and tissues [Meier,2000]

ApoReview - Introduction to Apoptosis: Page 2 of 26 Introduction to Apoptosis 1. The development of the term apoptosis Already since the mid-nineteenth century, many observations have indicated that cell death plays a considerable role during physiological processes of multicellular organisms, particularly during embryogenesis and metamorphosis [Gluecksmann, 1951; Lockshin, 2001]. The term programmed cell death was introduced in 1964, proposing that cell death during development is not of accidential nature but follows a sequence of controlled steps leading to locally and temporally defined selfdestruction [Lockshin, 1964]. Eventually, the term apoptosis had been coined in order to describe the morphological processes leading to controlled cellular self-destruction and was first introduced in a publication by Kerr, Wyllie and Currie [Kerr, 1972]. Apoptosis is of greek origin, having the meaning "falling off or dropping off", in analogy to leaves falling off trees or petals dropping off flowers. This analogy emphasizes that the death of living matter is an integral and necessary part of the life cycle of organisms. The apoptotic mode of cell death is an active and defined process which plays an important role in the development of multicellular organisms and in the regulation and maintenance of the cell populations in tissues upon physiological and pathological conditions. It should be stressed that apoptosis is a well-defined and possibly the most frequent form of programmed cell death, but that other, non-apoptotic types of cell death also might be of biological significance [Leist, 2001]. 2. The significance of apoptosis The development and maintenance of multicellular biological systems depends on a sophisticated interplay between the cells forming the organism, it sometimes even seems to involve an altruistic behaviour of individual cells in favour of the organism as a whole. During development many cells are produced in excess which eventually undergo programmed cell death and thereby contribute to sculpturing many organs and tissues [Meier, 2000]

ApoReview - Introduction to Apoptosis: Page 3 of 26 Fig. 1 Examples of physiological cell death Development and Morphogenesis: - 131 of the 1,090 somatic cells die during C.elegans development - during limb formation separate digits evolve by death of interdigital mesenchymal tissue (a) - ablation of cells no longer needed such as the amphibian tadpole tail during metamorphosis (b) - demise of cells allows sculpturing of hollow structures (c) - formation of reproductive organs (d) (Müllerian duct Æ uterus, deleted in males;Wolffian duct Æ male organs, deleted in females) - massive cell death occurs during early development of the nervous system (> 50 percent of all neurons die) In the human body about 100,000 cells are produced every second by mitosis and a similar number die by apoptosis (Vaux and Korsmeyer, 1999, Cell) ! Homeostasis: - a paradigm for the involvement of apoptosis in homeostasis is the immune system: several millions of B and T cells are generated every day and the majority (> 95 percent) of those die during maturation (death by neglect, negative selection) or by AICD of peripheral immune cells) Deletion of damaged and dangerous cells: - Cells with severely damaged DNA that cannot be repaired appropriately usually are removed by apoptosis - Inappropriate mitogenic signalling that is in conflict with the environmental or cellular status of the cell usually results in cell cycle arrest or apoptosis - Autoreactive cells of the immune system are deleted by apoptosis - Elimination of infected cells (a) (b) (c) (d) Development and Morphogenesis: - 131 of the 1,090 somatic cells die during C.elegans development - during limb formation separate digits evolve by death of interdigital mesenchymal tissue (a) - ablation of cells no longer needed such as the amphibian tadpole tail during metamorphosis (b) - demise of cells allows sculpturing of hollow structures (c) - formation of reproductive organs (d) (Müllerian duct Æ uterus, deleted in males;Wolffian duct Æ male organs, deleted in females) - massive cell death occurs during early development of the nervous system (> 50 percent of all neurons die) In the human body about 100,000 cells are produced every second by mitosis and a similar number die by apoptosis (Vaux and Korsmeyer, 1999, Cell) ! Homeostasis: - a paradigm for the involvement of apoptosis in homeostasis is the immune system: several millions of B and T cells are generated every day and the majority (> 95 percent) of those die during maturation (death by neglect, negative selection) or by AICD of peripheral immune cells) Deletion of damaged and dangerous cells: - Cells with severely damaged DNA that cannot be repaired appropriately usually are removed by apoptosis - Inappropriate mitogenic signalling that is in conflict with the environmental or cellular status of the cell usually results in cell cycle arrest or apoptosis - Autoreactive cells of the immune system are deleted by apoptosis - Elimination of infected cells (a) (b) (c) (d) A particularly instructive example for the implication of programmed cell death in animal development is the formation of free and independent digits by massive cell death in the interdigital mesenchymal tissue [Zuzarte-Luis, 2002]. Other examples are the development of the brain, during which half of the neurons that are initially created will die in later stages when the adult brain is formed [Hutchins, 1998] and the development of the reproductive organs [Meier, 2000]. Also cells of an adult organism constantly undergo physiological cell death which must be balanced with proliferation in order to maintain homeostasis in terms of constant cell numbers. The majority of the developing lymphocytes die either during genetic rearrangement events in the formation of the antigen receptor, during negative selection or in the periphery, thereby tightly controlling the pool of highly efficient and functional but not self-reactive immune cells and at the same time keeping lymphocyte numbers relatively constant [Rathmell, 2002]. Taken together, apoptotic processes are of widespread biological significance, being involved in e.g. development, differentiation, proliferation/homoeostasis, regulation and function of the immune system and in the removal of defect and therefore harmful cells. Thus, dysfunction or dysregulation of the apoptotic program is implicated in a variety of pathological conditions. Defects in apoptosis can result in cancer, autoimmune diseases and spreading of viral infections, while neurodegenerative disorders, AIDS and ischaemic diseases are caused or enhanced by excessive apoptosis [Fadeel, 1999a]

ApoReview-Introduction to Apoptosis:Page 4 of26 Due to its importance in such various biological processes,programmed cell death is a widespread phenomenon,occuring in all kinds of metazoans [Tittel,2000]such as in mammals,insects [Richardson,2002]nematodes [Liu,199and cnidaria [Cikala,199].Moreover,programmed cell death also might play a role in plant biology [Solomon,1999],and apoptosis-like cell death mechanisms even have been observed and used as a model system in yeast [Frohlich.2000. Skulachev,2002].Fascinating insights into the origin and evolution of programmed cell death might possibly be given by the fact,that programmed cell death is also an integral part of the life cycle of other unicellular eukaryotes (such as the kinetoplastid parasite Trypanosoma brucei brucei,the ciliate Tetrahymena thermophila.and the slime mold Dictvostelium discoideum)and that even prokaryotes (such as Bacillus subrilis,Streptomyces and Myxobacteria)sometimes undergo regulated cell death [Ameisen,20021 3. Morphological features of apoptosis Apoptotic cells can be recognized by stereotypical morphological changes:the cell shrinks,shows deformation and looses contact to its neighbouring cells.Its chromatin condenses and marginates at the nuclear membrane,the plasma membrane is blebbing or budding.and finally the cell is fragmented into compact membrane-enclosed structures,called 'apoptotic bodies which contain cytosol,the condensed chromatin,and organelles(Fig.2).The apoptotic bodies are engulfed by macrophages and thus are removed from the tissue without causing an inflammatory response.Those morphological changes are a consequence of characteristic molecular and biochemical events occurring in an apoptotic cell,most notably the activation of proteolytic enzymes which eventually mediate the cleavage of DNA into oligonucleosomal fragments as well as the cleavage of a multitude of specific protein substrates which usually determine the integrity and shape of the cytoplasm or organelles [Saraste,2000].Apoptosis is in contrast to the necrotic mode of cell-death in which ease the cells suffer a major insult,resulting in a loss of membrane integrity.swelling and disrupture of the cells. During necrosis,the cellular contents are released uncontrolled into the cell's environment which results in damage of surrounding cells and a strong inflammatory response in the corresponding tissue [Leist.20011

ApoReview - Introduction to Apoptosis: Page 4 of 26 Due to its importance in such various biological processes, programmed cell death is a widespread phenomenon, occuring in all kinds of metazoans [Tittel, 2000] such as in mammals, insects [Richardson, 2002], nematodes [Liu, 1999], and cnidaria [Cikala, 1999]. Moreover, programmed cell death also might play a role in plant biology [Solomon, 1999], and apoptosis-like cell death mechanisms even have been observed and used as a model system in yeast [Frohlich, 2000; Skulachev, 2002]. Fascinating insights into the origin and evolution of programmed cell death might possibly be given by the fact, that programmed cell death is also an integral part of the life cycle of other unicellular eukaryotes (such as the kinetoplastid parasite Trypanosoma brucei brucei, the ciliate Tetrahymena thermophila, and the slime mold Dictyostelium discoideum) and that even prokaryotes (such as Bacillus subtilis, Streptomyces and Myxobacteria) sometimes undergo regulated cell death [Ameisen, 2002]. 3. Morphological features of apoptosis Apoptotic cells can be recognized by stereotypical morphological changes: the cell shrinks, shows deformation and looses contact to its neighbouring cells. Its chromatin condenses and marginates at the nuclear membrane, the plasma membrane is blebbing or budding, and finally the cell is fragmented into compact membrane-enclosed structures, called 'apoptotic bodies' which contain cytosol, the condensed chromatin, and organelles (Fig. 2). The apoptotic bodies are engulfed by macrophages and thus are removed from the tissue without causing an inflammatory response. Those morphological changes are a consequence of characteristic molecular and biochemical events occurring in an apoptotic cell, most notably the activation of proteolytic enzymes which eventually mediate the cleavage of DNA into oligonucleosomal fragments as well as the cleavage of a multitude of specific protein substrates which usually determine the integrity and shape of the cytoplasm or organelles [Saraste, 2000]. Apoptosis is in contrast to the necrotic mode of cell-death in which case the cells suffer a major insult, resulting in a loss of membrane integrity, swelling and disrupture of the cells. During necrosis, the cellular contents are released uncontrolled into the cell's environment which results in damage of surrounding cells and a strong inflammatory response in the corresponding tissue [Leist, 2001]

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 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]

ApoReview-Introduction to Apoptosis:Page 10of 26 4.3 Extrinsic apoptosis pathways of type I and type ll Extrinsic apoptosis signalling is mediated by the activation of so called"death receptors"which are cell surface receptors that transmit apoptotic signals after ligation with specific ligands.Death receptors belong to the tumor necrosis factor receptor(TNFR)gene superfamily,including TNFR-1, Fas/CD95,and the TRAIL receptors DR-4 and DR-5 [Ashkenazi,2002].All members of the TNFR family consist of cysteine rich extracellular subdomains which allow them to recognize their ligands with specificity,resulting in the trimerization and activation of the respective death receptor [Naismith,1998].Subsequent signalling is mediated by the cytoplasmic part of the death receptor which contains a conserved sequence termed the death domain (DD).Adapter molecules like FADD or TRADD themselves possess their own DDs by which they are recruited to the DDs of the activated death receptor,thereby forming the so-called death inducing signalling complex(DISC).In addition to its DD.the adaptor FADD also contains a death effector domain(DED)which through homotypic DED-DED interaction sequesters procaspase-8 to the DISC(Fig.).As described above.the loca concentration of several procaspase-8 molecules at the DISC leads to their autocatalytic activation and release of active caspase-8.Active caspase-8 then processes downstream effector caspases which subsequently lave specific substrates resulting incell death.Cells harboring the capacity to induce such direct and mainly caspase-dependent apoptosis pathways were classified to belong to the so called type I cells[Scaffidi,199] In type II cells.the signal coming from the activated receptor does not generate a caspase signalling cascade strong enough for execution of cell death on its own.In this case,the signal needs to be amplified via mitochondria-dependent apoptotic pathways.The link between the caspase signalling cascade and the mitochondria is provided by the Bcl-2 family member Bid.Bid is cleaved by caspase 8 and in its truncated form(tBID)translocates to the mitochondria where it acts in concert with the proapoptotic Bel-2 family members Bax and Bak to induce the release of cytochrome c and other mitochondrial proapoptotic factors into the cytosol [Luo,1998]).Cytosolic cytochrome c is binding to monomeric Apaf-1 which then,in a dATP-dependent conformational change.oligomerizes to assemble the apoptosome,a complex of wheel-like structure with 7-fold symmetry,that triggers the activation of the initiator procaspase-9 [Acehan,20021).Activated caspase-9 subsequently initiates a caspase cascade involving downstream effector caspases such as caspase-3.caspase-7,and caspase-6. ultimately resulting in cell death [Slee,19991

ApoReview - Introduction to Apoptosis: Page 10 of 26 4.3 Extrinsic apoptosis pathways of type I and type II Extrinsic apoptosis signalling is mediated by the activation of so called “death receptors” which are cell surface receptors that transmit apoptotic signals after ligation with specific ligands. Death receptors belong to the tumor necrosis factor receptor (TNFR) gene superfamily, including TNFR-1, Fas/CD95, and the TRAIL receptors DR-4 and DR-5 [Ashkenazi, 2002]. All members of the TNFR family consist of cysteine rich extracellular subdomains which allow them to recognize their ligands with specificity, resulting in the trimerization and activation of the respective death receptor [Naismith, 1998]. Subsequent signalling is mediated by the cytoplasmic part of the death receptor which contains a conserved sequence termed the death domain (DD). Adapter molecules like FADD or TRADD themselves possess their own DDs by which they are recruited to the DDs of the activated death receptor, thereby forming the so-called death inducing signalling complex (DISC). In addition to its DD, the adaptor FADD also contains a death effector domain (DED) which through homotypic DED-DED interaction sequesters procaspase-8 to the DISC (Fig. 4). As described above, the local concentration of several procaspase-8 molecules at the DISC leads to their autocatalytic activation and release of active caspase-8. Active caspase-8 then processes downstream effector caspases which subsequently cleave specific substrates resulting in cell death. Cells harboring the capacity to induce such direct and mainly caspase-dependent apoptosis pathways were classified to belong to the so called type I cells [Scaffidi, 1998]. In type II cells, the signal coming from the activated receptor does not generate a caspase signalling cascade strong enough for execution of cell death on its own. In this case, the signal needs to be amplified via mitochondria-dependent apoptotic pathways. The link between the caspase signalling cascade and the mitochondria is provided by the Bcl-2 family member Bid. Bid is cleaved by caspase- 8 and in its truncated form (tBID) translocates to the mitochondria where it acts in concert with the proapoptotic Bcl-2 family members Bax and Bak to induce the release of cytochrome c and other mitochondrial proapoptotic factors into the cytosol [Luo, 1998]). Cytosolic cytochrome c is binding to monomeric Apaf-1 which then, in a dATP-dependent conformational change, oligomerizes to assemble the apoptosome, a complex of wheel-like structure with 7-fold symmetry, that triggers the activation of the initiator procaspase-9 [Acehan, 2002]). Activated caspase-9 subsequently initiates a caspase cascade involving downstream effector caspases such as caspase-3, caspase-7, and caspase-6, ultimately resulting in cell death [Slee, 1999]