Apoptosis Pathways PDF

Apoptosis  Session Learning Outcomes (SLOs): SLO# 1 : Identify the 3 types of cell death and clearly articulate their individual characteristics.  Control of Cell Number and Cell Size Three fundamental processes largely determine organ and body size: Cell growth, cell divis...

Apoptosis  Session Learning Outcomes (SLOs): SLO# 1 : Identify the 3 types of cell death and clearly articulate their individual characteristics.  Control of Cell Number and Cell Size Three fundamental processes largely determine organ and body size: Cell growth, cell division, and cell death. Each of these processes, in turn, depends on programs intrinsic to the individual cell and is regulated by signals from other cells in the body.  Apoptosis helps regulate animal cell numbers ▪ The cells of a multicellular organism are members of a highly organized community. ▪ The number of cells in the multicellular organism is tightly regulated not simply by controlling the rate of cell division, but also by controlling the rate of cell death. ▪ If cells are no longer needed, they can commit suicide by activating an intracellular death program—a process called programmed cell death. ▪ In animals, by far the most common form of programmed cell death is called apoptosis.  Cell Death There are two mechanisms for ensuring other cells in the body are removed, when appropriate: – Apoptosis - suicide - programmed cell death – Necrosis - killing - decay and destruction  Types of cell death ▪ Necrosis: non-apoptotic accidental cell death. Necrotic cell death culminates in cell lysis and provokes inflammation. ▪ Apoptosis: apoptotic cells are packaged into apoptotic bodies that are then engulfed by adjacent cells without an inflammatory response. ▪ Autophagy: Autophagic cell death is characterized by the appearance of cytoplasmic vesicles engulfing bulk cytoplasm and organelles. The contents of the vesicles are digested by the lysosomal system of the same cell after the fusion of the autophagic vesicles with lysosomes. Different forms of cell death  Apoptosis Apoptosis is a process seen in multicellular organisms, by which specific cells are killed and removed for the benefit of the organism. Apoptosis is essential part of life for every multicellular organism from worms to humans. Apoptosis plays a major role from embryonic development to senescence. Apoptosis or programmed cell death, is carefully coordinated collapse of cell, protein degradation , DNA fragmentation followed by rapid engulfment of corpses by neighboring cells.  Apoptosis vs. necrosis Necrotic cell Apoptotic cells Cell death by injury Cell death by suicide (Internal & -Physical Trauma external signals): -Complement-mediated lysis - Development -Exposure to toxic chemicals - Tissue Homeostasis -Lytic viral infection - Cell-Mediated Immunity - Hormone-Mediated Atrophy  Apoptosis Pathway of cell death induced by a tightly regulated suicide program. Controlled by specific genes. Fragmentation of DNA. Fragmentation of nucleus. Blebs form and apoptotic bodies are released. Apoptotic bodies are phagocytized. No neutrophils. Apoptosis Necrosis  Death by Injury vs. Death by Suicide (Necrosis vs. Apoptosis) Necrosis Apoptosis Cellular swelling Cellular condensation Membranes are broken Membranes remain intact ATP is depleted Requires ATP Cell lyses, eliciting an Cell is phagocytosed, no inflammatory reaction tissue reaction DNA fragmentation is Ladder-like DNA random, or smeared fragmentation In vivo, whole areas of In vivo, individual cells the tissue are affected appear affected NECROSIS Vs APOPTOSIS  Consequences of Cell Death Apoptosis Removal of damaged or unnecessary cells Necrosis Loss of fonctionnel tissue Impaired organ function, transient or permanent  Apoptosis Apoptosis results in a quick and clean cell death, without damaging its neighbours, or eliciting an immune response. Every cell is equipped with the ‘cell death pathway’. Apoptosis is an intracellular proteolytic pathway. The DNA is broken into small 200 bp units. 18_20_Apoptosis_.jpg  Apoptosis cells are biochemically recognizable During apoptosis cells die in a regulated, well programmed fashion, following a sequence of morphological changes: 1. Chromatin condensation 2. Cleavage of DNA at internucleosome site. 3. Change in the plasma membrane. 4. Fragmentation of nucleus. 5. Controlled by specific genes 6. Relocation of cytochrome c from the intermembrane space of the mitochondria to the cytosol. 7. Lost of adhesion and shrinkage of the cell 8. Membrane blebbing. 9. Apoptotic bodies clearance via phagocytosis  DNA DAMAGE p53 A caspase-activated deoxyribonuclease (CAD) and its inhibitor (ICAD) Cleavage of chromosomal DNA into a characteristic ladder of fragments Lauber et al. (2004) Fig 6 The three steps of apoptotic cell removal. Steps in apoptosis: 1- The decision to activate the pathway; 2- The actual "suicide" of the cell; 3- Engulfment of the cell remains by specialized immune cells called phagocytes; 4- Degradation of engulfed cell. The actual steps in cell death require: 1- condensing of the cell nucleus and breaking it into pieces 2- Condensing and fragmenting of cytoplasm into membrane bound apoptotic bodies; 3- Breaking chromosomes into fragments containing multiple number of nucleosomes (a nucleosome ladder)  Regulators of apoptosis Inducers of apoptosis Physiological Damage related inducers Therapy-associated death activator 1- Heat Shock agents 1-TNF family (TNF- , 2- Viral infection 1- Chemotherapeutic TNF- β), Fas ligand 3- Bacterial toxins 2- Drugs FasL) 4- Oncogenes 3- Gamma radiation 2- TGF-beta 5- Tumor supressors (p53) 4- UV radiation 3- Neurotransmitters 6- Cytolytic T cells 4- Growth factor Toxins 7- Oxidants increased withdrawal 1- Some levels of oxidants 5- Loss of matrix chemotherapeutic within the cell attachment drugs 8- Free radicals Sustained rise in 2- Ethanol 9- Nutrient deprivation 6- Calcium 3- Beta-amyloid 10- UV radiation (X-ray peptide 6-accumulation of or UV light) improperly folded 11- Gamma radiation proteins, 7- Glucocorticoids  Session Learning Outcomes (SLOs): SLO# 1 : Describe the purpose of apoptosis and identify the physiological and pathological context of cell death.  Apoptosis: the necessity for cell death in multicellular organisms Apoptosis plays important roles in many biological processes Physiological conditions – An intrinsic and integral component of physiology, just like proliferation and differentiation. – Embryonic development: e.g. in Caenorhabditis elegans (worm), 131 out of a total of 1090 somatic cells are programmed to undergo apoptosis at predefined stages. – Cellular homeostasis: e.g. lymphocytes Pathological conditions – Down-regulation of apoptosis: e.g. cancer, autoimmune disorders, persistent viral infections... – Up-regulation of apoptosis: e.g. many forms of degenerative disorders such as Alzheimer’s disease, ischemic injury from stroke (heart disease), post-menopausal osteoporosis...  Physiologic Apoptosis - Development - Metamorphosis - Regulation of cell number in tissues (homeostasis and tumorigenesis) - Development of B and T cells via negative selection (Immune systems maturation) - Immune defense (cytotoxic T cell activity)  Biological Roles for Apoptosis ❑ Apoptosis is needed for proper development Examples: 1- normal physiology / development ▪ In the developing vertebrate nervous system, for example, more than half of the nerve cells produced normally die soon after they are formed. ▪ In a healthy adult human, billions of cells die in the bone marrow and intestine every hour. ❑ Apoptosis is needed for proper development Embryonic morphogenesis Examples: 1- The resorption of the tadpole tail The cells in the tadpole tail are induced to undergo apoptosis stimulated by the increases in thyroid hormone that occurs during metamorphosis Apoptosis during the metamorphosis of a tadpole into a frog. ❑ Apoptosis is needed for proper development Examples: 2- The formation of the fingers and toes of the fetus Mouse paws and Human hands and feet are sculpted by apoptosis during embryonic development: they start out as spade like structures, and the individual fingers and toes separate because the cells between them die. Sculpting the digits in the developing mouse paw by apoptosis  Developmentally-regulated apoptosis Types of cells that Apoptosis in the embryonic mouse paw sculpts the undergo apoptosis digits. Apoptosis occurs between 12.5 to 14.5 days in embryogenesis Regulation of cell number in tissues (homeostasis and tumorigenesis): In adult tissues, cell death usually exactly balances cell division, unless the tissue is growing or shrinking. Example: If part of the liver is removed in an adult rat, liver cells proliferate to make up for the loss. Conversely, if a rat is treated with the drug phenobarbital, which stimulates liver cell division, the liver enlarges. However, when the phenobarbital treatment is stopped, apoptosis in the liver greatly increases until the organ has returned to its original size, usually within a week or so. Thus, the liver is kept at a constant size through regulation of both the cell death rate and the cell birth rate. ❑ Apoptosis is needed for proper development Examples: 3- Neural development: The formation of the proper connections between neurons in the brain. Apoptosis is also important in the development of the nervous system Mechanisms of immunological tolerance to self antigens  Apoptosis in Pathologic Conditions DNA damage due to radiation, and chemotherapy. Accumulation of misfolded proteins leads to ER stress which ends with apoptosis. Cell death in viral infections that induce apoptosis such as HIV and Adenovirus or by the host immune response such as hepatitis. Organ atrophy after duct obstruction.  Apoptosis in Pathologic Conditions The disturbance is implicated in numerous pathological conditions ranging from degenerative disorders to autoimmunity to cancer Excess apoptosis – Neurodegenerative diseases Deficient apoptosis – Cancer – Autoimmunity – Infectious diseases  Session Learning Outcomes (SLOs): SLO# 3: Describe the molecular events leading to apoptosis in general.  The molecular basis of apoptosis in mammals The machinery that is responsible for apoptosis seems to be similar in all animal cells. The two major players in apoptosis are: Caspases Adaptor proteins TNF & TNFR family Bcl-2 family Caspases  Caspases ▪ Cysteine-dependent aspartate- specific proteases ▪ Have a cysteine at the active site ▪ Cleave target just after aspartic acid residues ▪ Substrate specificity is determined by the 4 residues upstream of the cleavage site  Caspases ▪ The members of caspase family of proteases are proteolytic enzymes activated by both intrinsic and extrinsic pathways ▪ Exist in cytosol as inactive precursors called procaspases. ▪ Procaspases are typically activated by proteolytic cleavage in response to signals that induce apoptosis. ▪ The activated caspases cleave, and thereby activate, other members of the procaspase family, resulting in an amplifying proteolytic cascade ▪ Also cleave key proteins in the cell, causing the characteristic morphology and biochemistry of apoptosis.  Caspases ▪ Procaspases: Contain N terminal pro-domain followed by region that forms a 2 subunit catalytic effector domain Downstream, or effector or executioner caspases with short prodomains Upstream, or initiator caspases with long prodomains prodomain Asp Second cleavage Asp First cleavage Prodomain: for protein-protein interactions; allows it to bind upstream regulators and effector proteins; examples include: DED: death effector domain (e.g. caspase 8) CARD:caspase activation and recruitment domain (e.g. caspase 9)  Caspases Procaspases may possess low but significant activity, e.g. procaspase-8 has ~1-2% of the activity of the mature caspase-8. Caspases are fully activated by a first proteolytic cleavage between the large and small subunits and a second cleavage to remove the prodomain. Active caspases : heterotetramers composed of two large and two small subunits with two active sites per molecule  Apoptosis depends on an intracellular proteolytic cascade that is mediated by caspases 1. Each caspase is made as an inactive proenzyme, a procaspase. 2. Procaspases are typically activated by proteolytic cleavage by another member of the same protease family in response to signals that induce apoptosis. 3. Two cleaved fragments from each of two procaspase molecules associate to form an active caspase. 4. The activated caspases cleave, and thereby activate, other members of the procaspase family, resulting in an amplifying proteolytic cascade 5. The activated caspases also cleave key proteins in the cell, causing the characteristic morphology and biochemistry of apoptosis. 6. This cleavage causes the irreversible breakdown of the nuclear lamina. 7. The cell dismantles itself quickly and cleanly, and its corpse is rapidly taken up and digested by another cell. Apoptosis is mediated by an intracellular proteolytic cascade  Caspases are specialized proteases that mediate apoptosis Procaspase activation during apoptosis  Caspases Two types: - Related to caspase 1 (Caspases 1, 4, 5, 13, and 14); role in cytokine processing during inflammation - Involved in apoptosis (Caspases 2, 3, 6, 7, 8, 9 and 10) Initiators : activate downstream effector caspases to initate activation cascades Effectors: cleave target proteins resulting in morphological and biochemical markers of apoptosis  Effector Caspases Effector caspases (such as caspase-3, -6 and -7 in mammals) function to breakdown cell structures through cleavage of specific substrates. Caspases target: 1. FAK (focal adhesion kinase): inactivation of FAK disrupt cell adhesion, leading to detachment of the apoptotic cell from its neighbors. 2. Lamins: important component of the nuclear envelope, cleavage of lamins leads to disassembly of the nuclear lamina 3. Proteins required for cell structure: actin, intermediate filaments, etc…cleavage of these proteins lead to changes in cell shape and the surface blebbing 4. Endonuclease Caspase Activated Dnase CAD: responsible for chromosome fragmentation. CAD cuts DNA into small fragments. CAD normally binds to an inhibitor protein. Caspases cleaves the inhibitor protein to activate CAD 5. Enzymes involved in DNA repair  Regulation of Caspases The proteolytic cascade is not only destructive and self-amplifying but also irreversible. Thus, it is important that the decision to die is tightly controlled. Which are the cytoplasmic proteins that impact on mitochondria and control apoptosis ? The Bcl-2 family of proteins, which may be pro- or anti-apoptotic  Bcl-2 family of proteins A family of apoptosis proteins has been discovered in mammalian cells. The first member of the Bcl-2 family was identified during a study of B cell lymphoma. The oncogenic version is formed through a reciprocal chromosomal translocation in which parts of the chromosome 14 and chromosome 18 are exchanged. The translocated bcl-2 gene is now under the control of an active immunoglobulin promoter that drives high levels of constitutive expression.  Bcl-2 family of proteins Bcl-2 localises to the outer membrane of the mitochondria. The major role of Bcl-2 family of proteins is to control the mitochondrial outer membrane permeability (MOMP). MOMP controls the release into the cytoplasm of proteins contained in the mitochondrial intermembrane space, including cytochrome c. How do proteins get released from mitochondria? BcL-2 family proteins regulate the release of apoptogenic cytochrome c by forming channels in mitochondria membrane  The Bcl-2 family The Bcl-2 family of proteins may be pro- or anti-apoptotic The three classes of Bcl2 proteins The three classes of Bcl2 proteins  Groups of Bcl-2 proteins There are at least 24 Bcl-2-related proteins: 6 are anti- apoptotic and 18 are pro-apoptotic. Anti-apoptotic proteins (Bcl-2, Bcl-XL; Bcl-w; Mcl-1 and A1/Bfl-1) Display the BH1, BH2, BH3 and BH4 domains, and a transmembrane domain.  Caspase Pathways The two best understood signaling pathways that activate a caspase cascade in mammalian cells are: The intrinsic pathway and the extrinsic pathway Each pathway uses its own initiator procaspases and activation complex SLO# 4: Explain the difference between the intrinsic and extrinsic pathways of apoptosis and where they converge. SLO# 5: Describe the molecules that control the apoptosis intrinsic pathway. SLO# 6: Explain how the balance between pro- and anti-apoptotic members of the Bcl2 family regulates the intrinsic apoptotic pathway.  Caspase Pathways I. Intrinsic pathway – mitochondria mediated; caspase 9. II. Extrinsic pathway – involves death receptors (TNF receptor, Fas); caspase 8 Converge to active executioner caspases 3 and 7  Intrinsic apoptosis Intrinsic apoptosis is mitochondria-dependent and is induced by DNA damage, heat, radiation, nutrient deprivation, viral infection, and increased intracellular calcium concentration Internal damage to the cell (e.g., from reactive oxygen species) causes Bcl-2 to activate a related protein, which punches holes in the outer mitochondrial membrane, causing cytochrome c to leak out. The role of BH123 pro-apoptotic Bcl2 proteins in the release of mitochondrial intermembrane proteins in the intrinsic pathway of apoptosis Two of the most important death-promoting family members are proteins called Bcl-2 associated X protein (Bax) and Bcl-2 associated killer (Bak). Bax and Bak proteins are themselves activated by other death-promoting members of the Bcl2 family, which are produced or activated by various insults to the cell, such as DNA damage. Other members of the Bcl2 family, including Bcl2 itself, act to inhibit, rather than promote, procaspase activation and apoptosis. The role of BH123 pro-apoptotic Bcl2 proteins in the release of mitochondrial intermembrane proteins in the intrinsic pathway of apoptosis BAX undergoes extensive conformational change stimulated by death signals leads to activation and membrane insertion of BAX The protein changes from a soluble cytoplasmic protein in healthy cells to one that appears to have at least 3 helices inserted in the mitochondrial membrane in apoptotic cells. Conformational changes in BCL-2 family members during apoptosis The role of BH123 pro-apoptotic Bcl2 proteins (Bax and Bak) in the release of mitochondrial intermembrane proteins in the intrinsic pathway of apoptosis Process of Intrinsic apoptosis: 1. Bax forms homo-dimers in the presence of apoptotic signals, opening a channel that translocates cytochrome c from the intermembrane space to the cytoplasm 2. In the cytosol, the released cytochrome c binds to Apaf-1 ("apoptotic protease activating factor-1") Using the energy provided by ATP CARD: caspase activation and recruitment domain 3. These complexes aggregate to form apoptosomes. Cytochrome c promotes the assembly of a large, seven-armed pinwheel-like structure that recruits specific procaspase molecules, forming a protein complex called an apoptosome. Effector caspases Nature 407, 770-776 (12 October 2000) The procaspase molecules become activated within the apoptosome, triggering a caspase cascade that leads to apoptosis Bax and Bak activate procaspases indirectly, by inducing the release of cytochrome c from mitochondria into the cytosol. 4. The apoptosomes bind to and activate caspase-9. 5. Caspase-9 cleaves and, in so doing, activates other caspases (caspase-3 and -7). 6. The activation of these "executioner" caspases creates an expanding cascade of proteolytic activity which leads to digestion of structural proteins in the cytoplasm, degradation of chromosomal DNA, and phagocytosis of the cell.  Intrinsic pathway Effector caspases The intrinsic pathway of apoptosis depends on mitochondria I. Intrinsic -Mitochrondrial pathway Cellular stress (UV, cytotoxic drugs etc.) Alterations in mitochondria membrane potential (MMP) Release of pro-apoptotic molecules (cytochrome c) from intermembrane space out into the cytosol Cytochrome c combines with dATP, Apaf-1 (apoptotic protease activating factor-1), and caspase 9 Formation of a catalytic complex called the apoptosome Apoptosome bounds Caspase 9 Activation of Caspase 9 Activation of effector caspases (caspases 3 and 7) How pro-apoptotic BH3-only and anti-apoptotic Bcl2 proteins regulate the intrinsic pathway of apoptosis The three classes of Bcl2 proteins  Regulation of intrinsic pathway How pro-apoptotic BH3-only and anti-apoptotic Bcl2 proteins regulate the intrinsic pathway of apoptosis 1- Anti-apoptotic molecules (Bcl-2) Synthesis of anti-apoptotic molecules (Bcl-2) promoted by Growth factors which regulate the release of pro-apoptotic factors from mitochondria. Interaction of pro-apoptotic Bax with anti-apoptotic Bcl-xL: Bax-Bcl-xL complexes shuttle from the cytoplasm to the outer mitochondrial membrane OMM, thus preventing accumulation of Bax in the OMM, oligomerization of Bax and permeabilization of the OMM (MOMP), and consequently release of cytochrome c  Activation of anti-apoptotic Bcl2 Increase production of the anti-apoptotic Bcl2 Interaction of anti-apoptotic Bcl-xL with pro-apoptotic Bax Inhibition of Apoptosis  The BH3-only proteins 2- The BH3-only proteins interact with anti- apoptotic proteins (i.e. Bad with Bcl-2 or Bcl-xL) and allow Bax-Bak oligomerization in the mitochondial membrane, followed by cytochrome C release and apoptosis. They act as sensors for cellular stress, cell damage, infection, growth factor deprivation, and any other signal that causes apoptosis.  Regulation of intrinsic pathway When cells are deprived of growth factors or subjected to stress anti-apoptotic molecules (Bcl-2) are lost. Mitochondrial membrane becomes permeable and proteins that activate caspase leak out.  Bcl-2 and related proteins The anti-apoptotic protein, BCL-XL , is inhibited by binding of the pro-apoptotic BH3 only protein (orange) in the groove between BH1 and BH3  Regulation of intrinsic pathway Bad (BH3-only protein) sequesters Bcl-2 (an anti-apoptotic protein) in cells responding to apoptotic stimuli. When the Akt kinase phosphorylates Bad, Bcl-2 is released and its anti-apoptotic potential restored Inhibite apoptosis.  BH3 only protein binding specificity for BCL-2 homologues BIM and PUMA bind to all BCL-2 family members tested; by contrast NOXA only binds to A1 and MCL1. These binding specificities recapitulate the ability of these proteins to activate apoptosis e.g. BIM et al can induce apoptosis alone whereas a combination of NOXA and BAD is required. Inactivation of anti-apoptotic Bcl2 By pro-apoptic BH3 only protein Activation of Apoptosis  IAPs inhibit caspases 3- A proposed model for the roles of the Inhibitors of apoptosis proteins (IAPs) and anti-IAPs in the control of apoptosis in mammalian cells  A model for the function of Bcl-2 family proteins 1. Pro-survival Bcl-2 and Bcl-xL contain all four BH domains. 2. Pro-apoptotic Bax and Bak contain BH1, BH2 and BH3. 3. Pro-apoptotic Bid and PUMA contain only BH3 domain.  To summarize….. BCL-2 family of proteins have opposing apoptotic activities 1st set (e.g. Bcl-2 itself) inhibit apoptosis. 2nd set (e.g. BAX) promotes apoptosis. 3rd set (e.g. the BH3 only proteins) bind and regulate the anti-apoptotic BCL-2 proteins to promote apoptosis. The three major mammalian fractions of the Bcl-2 family. The BH3-only proteins (yellow) are essential initiators of apoptosis that primarily antagonize their pro-survival relatives (blue), whereas either Bax or Bak (red) is required downstream of Bcl-2. Three ways that extracellular survival factors can inhibit apoptosis Summary of the intrinsic apoptotic pathway  Extrinsic (Death receptor initiated) pathway of apoptosis ▪ Death receptors are members of the tumor necrosis factor receptor family and a related protein called Fas (CD95). ▪ These molecules contain a death domain.  Extrinsic pathway of apoptosis The death receptor pathway is activated by external cytokines and is mitochondria-independent The ligands of the death receptors are members of the tumor necrosis factor (TNF) family of proteins, including TNF-alpha, Fas ligand (FasL), TRAIL/Apo2L, Apo3L Binding of ligand to the death receptors results in homotrimerization of the receptors. Death receptors contain a death domain in the cytoplasmic region that is required for apoptosis signaling. Death Receptors Trimerization of the receptor death domains allows binding and activation of Fas-associated death domain protein (FADD) and formation of death-inducing signaling complex (DISC), which recruits and activates procaspase 8 and 10 to caspase 8 and 10. Death receptors  Extrinsic pathway Death receptor-mediated apoptosis Initiated by death receptors: 1- Start with ligand binding, clustering or aggregation of death receptors 2- Cytoplasmic tails of death receptor bind adaptor proteins 3- Adaptor proteins recruit and activate procaspase 8 to yield caspase 8 (active) 4- Caspase 8 triggers downstream effector caspases, such as caspase 3 Fas – Fas ligand system Extrinsic pathway Fas: Prototypical cell death receptor of the TNF CD95L (FasL) only expressed by receptor superfamily activated T cells No intrinsic enzymatic activity Signals via adaptor proteins Fas ligand (FasL): Only expressed by activated T cells (Transmembrane TNF-like protein) The expression of FasL is induced on the T cell when the TCR (T cell receptor) of an antigen specific CTL (cytotoxic T lymphocyte) binds antigen on MHC I FasL binds Fas (present on most cells of body) that is on the presenting cell to induce death of that cell Cytoplasmic tail of Fas binds its adaptor protein FADD Fas-FADD complex binds to and activates Caspase 8 Cleavage of vital cellular substrates e.g. actin, lamin Initiates lethal proteolytic cascade DNAase activation Two strategies by which effector cytotoxic T cells kill their target cells  Cell-surface death receptors activate the extrinsic pathway of apoptosis The extrinsic pathway of apoptosis activated through Fas death receptors Convergence of intrinsic and extrinsic apoptotic pathways Convergence of intrinsic and extrinsic apoptotic pathways 1- Fas receptor signalling pathways employ a cytoplasmic protein motif known as the death domain (DD) in the receptors and certain adaptor proteins (MORT1/FADD, TRADD and RIP). 2- The death domain of Fas binds to MORT1/FADD. 3- MORT1/FADD interacts with caspase-8, a member of the ICE/Ced-3 protease family, through another motif designated the death effector domain (DED). 4- Caspase-8 in turn cleaves Bid, a Bcl-2 family protein which may regulate mitochondrial integrity in a manner which further activates the apoptotic cascade. Convergence of intrinsic and extrinsic apoptotic pathways