Apoptosis Introduction (SLIDE 1) Organisms develop from a single cell, and in doing so an anatomy has to be created. This process not only involves the creation of new cells, but also the removal of cells that are not needed. Fully formed organisms also need to remove old and dysfunctional cells, allowing the replacement of damaged cells. In addition, virally infected cells commit suicide in order to prevent the spread of infection. Apoptosis is the principal mechanism by which cells are physiologically eliminated in animals. During apoptotic death, cells are neatly carved up by caspases and packaged into apoptotic bodies as a mechanism to avoid immune activation. Once cell death has been completed, the remains of the dead cell need to be removed. Neighboring cells and macrophages phagocytize the dead cells before they are able to release their cytosolic contents. There are two main cell signaling pathways that had been identified in the control of apoptosis: the intrinsic pathway (mitochondrial pathway), or core pathway, which involves the mitochondria, and the extrinsic pathway (death receptor pathway), which involves cell surface receptors. Apoptotic pathways (SLIDE 2) (1) The intrinsic pathway is activated by internal surveillance mechanisms or signals sent (or not sent) by other cells. (1a) Signals that induce this pathway include DNA damage, exposure to chemicals that interfere with a variety of cellular pathways, excessive activation of factors that promote cell-cycle progression, and receipt of certain pro-apoptotic stimuli from the surrounding medium. (1b) Withdrawal of nutrients or of nurturing signals from the environment also activates the intrinsic pathway. Survival signals include interleukin-2 and interleukin-3, which are essential for survival of thymocytes; nerve growth factor, which required for survival of many neurons, and extracellular matrix, which is required for survival of epithelial cells. Signals that activate the intrinsic pathway converge on mitochondria, which release key factors that drive the apoptotic response. Mitochondria are the integrators of cell s life and death decisions. (2) Signals from other cells are the primary triggers of the extrinsic pathway. Direct contact with the target cell activates specific receptors that initiate this pathway. Activation of the extrinsic pathway is one strategy that cytotoxic T lymphocytes use to kill cells that are recognized as foreign, or as harboring foreign pathogens. This pathway is also widely used to control cell populations in the immune system. Programmed cell death (apoptosis and autophagy) versus accidental cell death (necrosis) There are many observable morphological and biochemical differences between necrosis and apoptosis. (1) Necrosis occurs when cells are exposed to extreme variance from physiological conditions (e.g., hypothermia, hypoxia) which may result in damage to the plasma membrane. Under physiological conditions direct damage to the plasma membrane is evoked by agents like complement and lytic viruses. Necrosis begins with an impairment of the cell s ability to maintain homeostasis, leading to an influx of water and extracellular ions. Intracellular organelles, most notably the mitochondria, and the entire cell swell and rupture (cell lysis). Due to the ultimate breakdown of the plasma membrane, the cytoplasmic contents including lysosomal enzymes are released into the extracellular fluid. Therefore, in vivo, necrotic cell death is often associated with extensive tissue damage resulting in an intense inflammatory response. Recently, necrosis, once thought of as simply a passive, unorganized way to die, has emerged as an alternate form of programmed cell death whose activation might have important biological consequences, including the induction of an inflammatory response (!). (2) Apoptosis, in contrast, is a mode of cell death that occurs under normal physiological conditions and the cell is an active participant in its own demise ( cellular suicide ). It is most often found during normal cell turnover and tissue homeostasis, embryogenesis, induction and maintenance of immune APOPTOSIS oldal 1
tolerance, development of the nervous system and endocrine-dependent tissue atrophy. Cells undergoing apoptosis show characteristic morphological and biochemical features. These features include chromatin aggregation, nuclear and cytoplasmic condensation, partition of cytoplasm and nucleus into membrane bound-vesicles (apoptotic bodies) which contain ribosomes, morphologically intact mitochondria and nuclear material. These apoptotic bodies are rapidly recognized and phagocytized by either macrophages or adjacent epithelial cells. Due to this efficient mechanism for the removal of apoptotic cells in vivo no inflammatory response is elicited. Activities that cause cells to undergo apoptosis are said to be pro-apoptotic; activities that protect cells from apoptosis are said to be anti-apoptotic. (3) Autophagy resulting in the total destruction of the cell is another type of programmed cell death; yet, no conclusive evidence exists for such a process. Autophagy is process involving the degradation of a cell's own components through the lysosomal machinery. A variety of autophagic processes exist. The most well-known mechanism involves the formation of a membrane around a targeted region of the cell, separating the contents from the rest of the cytoplasm. The resultant vesicle then fuses with a lysosome and subsequently degrades the contents. Classes of cells that undergo programmed cell death At least five classes of cells undergo programmed cell death: (1) developmentally defective cells; (2) excess cells; (3) cells that serve no function; (4) cells whose cell cycle is perturbed; and (5) virus-infected cells. (I.) The mitochondrial (intrinsic) pathway (SLIDE 3) for the control of apoptosis involves the mitochondria of the cell. This organelle is instrumental in normal cellular metabolism as the major site of ATP production. However, alongside this important role, mitochondria have a role as sensors of the health of the cells. In other words, mitochondria have large parts to play in the decision of whether a cell lives or dies. If cells sense insult from which they cannot recover, mitochondria trigger apoptosis. In response to DNA damage, topoisomerase inhibition, cytoplasmic stress and many other stimuli, the mitochondria become permeabilized and release many proteins into the cytoplasm. The permeabilization involves proteins from the Bcl-2 family. Caspases (SLIDE 4) The central group of player in apoptosis is the caspases, which mediate the cell shrinkage, DNA fragmentation and other changes; however, these molecules are also involved in other events in cell, besides apoptosis. Caspases are cystein proteases, which means that they contain cystein residue in their active site. There are 14 known mammalian caspases, 11 of which have been found in humans. The activation of caspases is itself by proteolytic cleavage; one caspase can cleave and so activate another caspase, leading to activation cascades of caspases. Therefore, inactive caspases are in non-cleaved form, referred to as the pro-caspases. The first cleavage event is an autocatalytic one, where the caspase cleaves and activates itself. This leads to another way of classifying caspases, either as initiator caspases or effector caspases. The initiator caspases can be at the start of a cascade, whereas the effector caspases are down-stream components of the pathway. Permeabilization of the mitochondria by Bcl-2 proteins allows for a release of many proteins, including cytochrome c (Cyt C). The release of cytochrome c has two effects. First, it can act as a signaling molecule in the apoptosis pathway, and secondly, it can no longer function in the electron transport chain and so ATP production is compromised. (SLIDE 4) The released Cyt C binds the Apaf-1 factor and procaspase 9 resulting in the activation of caspase 9, which in turn activate caspase 3. Bcl-2 proteins (SLIDE 5) can be grouped into three subfamilies. (1) Bcl-2 protectors (e.g. Bcl-2 and Bcl- X L ) protect cells against apoptosis. (2) Bcl-2 killers (e.g. Bax and Bak) are pro-apoptotic proteins that actively kill cells. (3) Bcl-2 regulators (e.g. Bad, Bid) promote cell killing by either interfering with the protectors or activating the killers. These proteins primarily regulate the release of death-promoting factors from mitochondria when cells receive signals that activate the intrinsic pathway. Bcl-2 family APOPTOSIS oldal 2
members are defined by the presence of one to four short blocks of conserved protein sequence called BH domains (Bcl-2 homology domains). The role of cytochrome c as a redox protein in mitochondria was established many years ago and it could have been claimed that it is a very well-characterized protein. However, its role in apoptosis seems very disparate from its redox role. This illustrates that a wide range of proteins can be involved in signaling, even ones where their roles are thought to be known. This multifunctional role of proteins is likely to become more common in future, as new roles for old proteins are discovered. Nucleases in apoptosis During apoptosis, the chromosomal DNA is destroyed. The many nucleases involved in cleaving the cellular DNS during and after apoptotic cell death fall into two classes. (1) Cell autonomous nucleases degrade the DNA from within the dying cell. The best known is the caspaseactivated DNase (CAD). CAD is normally present in complex with ICAD (inhibitor of CAD). ICAD is a chaperone that must be present for CAD to fold into an active conformation as it is being translated on the ribosome. However, ICAD also inhibits the nuclease activity of CAD. This dual function of ICAD guarantees that only inactive CAD can be synthesized in healthy cells. During apoptosis, caspase 3 cleaves ICAD and releases active CAD nuclease. Cell autonomous nucleases are dispensable for apoptosis and for life of the organism. (2) Waste management nucleases clean up the debris after cells die. They either function within lysosomes of cells that have phagocytosed apoptotic cell fragments or are secreted and function in the extracellular space. DNase II, one of the most important waste management nuclease, is essential for life. Mouse embryos that lack this enzyme become overwhelmed with undegraded DNA and die. Regulation of apoptosis by Bcl-2 proteins (SLIDE 6) (A) In the absence of an apoptotic stimulus, anti-apoptotic Bcl-2 proteins bind to and inhibit the BH123 proteins on the mitochondrial outer membrane (and in the cytosol). (B) In the presence of an apoptotic stimulus, BH-3 only proteins are activated and bind to the anti-apoptotic Bcl-2 proteins so that they can no longer inhibit the BH123 proteins, which now become activated and aggregate in the outer mitochondrial membrane and promote the release of intermembrane mitochondrial proteins into the cytosol. Some activated BH3-only proteins may stimulate mitochondrial release more directly by binding to and activating the BH123 proteins. Roles of IAPs and anti-iaps in apoptosis (SLIDE 7) (A) In the absence of an apoptotic stimulus, IAPs prevent accidental apoptosis caused by the spontaneous activation of procaspases. The IAPs are located in the cytosol and bind to and inhibit any spontaneously caspases. Some IAPs are also ubiquitin ligases that mark the caspases for degradation in proteasomes. (B) When an apoptotic stimulus activates the intrinsic pathway, among the proteins released from the mitochondrial intermembrane space are anti-iap proteins, which bind to and block inhibitory activity of the IAPs. At the same time, the released cytochrome C triggers the assembly of apoptosomes, which can now activate a caspase cascade, leading to apoptosis. Caspase inhibitors and activators (SLIDE 8) Because most healthy cells express initiator procaspases with the potential to oligomerize by mistake and kill the cell, it is important to have mechanisms that dampen this noise in the pro-apoptotic pathway. The inhibitor of apoptosis proteins (IAP) inactivate caspases in two ways. First, they bind the caspase and invade the active site, thereby blocking its access to substrates. Secondly, several IAPs are also ubiquitin ligases, which tag caspases for destruction by proteasomes. If IAP proteins inactivate caspases, then how is the apoptotic response ever initiated? Cells also express an antidote for the IAPs. These protein, known as second mitochondrial activator of caspases (smac or DIABLO), is normally sequestered in mitochondria. It is released when the intrinsic pathway of apoptosis is initiated. The ER stress (SLIDE 9) In resting conditions, the pro-apoptotic Bax and Bak (Bax/Bak) are kept inactive by interaction with BCL2 both on the mitochondrial and endoplasmic reticulum (ER) APOPTOSIS oldal 3
membranes, whereas Bim (BH3) is inhibited by binding to cytoskeletal dynein. Severe ER stress leads to activation of c-jun N-terminal kinase (JNK) and induction of C/EBP homologous protein (CHOP; initiation phase). Both JNK and CHOP eliminate the anti-apoptotic effect of BCL2; CHOP blocks expression of BCL2, whereas JNK phosphorylates it. JNK also phosphorylates Bim, which leads to its release from the cytoskeleton and to its activation (commitment phase). Collectively, these changes allow activation of Bax and Bak, transmission of the signal from the ER to the mitochondria and execution of death (execution phase). Caspases are activated possibly on the ER membrane itself, as well as in the apoptosome, after transmission of the death signal to mitochondria and the release of cytochrome c. Blue labels show inactive molecules, whereas red labels indicate active molecules, with the rounded shapes representing the pro-apoptotic molecules and rectangles representing the anti-apoptotic molecules. ATF6, activating transcription factor 6; IRE1, inositol-requiring enzyme 1; PERK, pancreatic ER kinase (PKR)-like ER kinase; TRAF2, TNF-receptor-associated factor 2; UP, uniporter. (II.) The death receptor (extrinsic) pathway (SLIDE 10) is activated by the binding or FAS or TRAIL ligands to their death receptors (DR4 or DR5), stimulating receptor aggregation. This aggregation stimulates recruitment of FADD and caspase (casp) 8 activation. Casp 8 activation leads to casp 3 cleavage, which initiates multiple proapoptotic processes, including CAD stimulation of DNA cleavage. Granzymes are serine proteases that are released by cytoplasmic granules within cytotoxic T cells and natural killer cells. Their purpose is to induce apoptosis within virus-infected cells, thus destroying them. Granzymes cleave caspases (especially caspase-3), which in turn activates caspaseactivated DNase. This enzyme degrades DNA, thus inducing apoptotic cascades. Death Receptor Signaling Death receptors are cell surface receptors that transmit apoptotic signals initiated by specific ligands such as Fas ligand, TNF and TRAIL. They play an important role in apoptosis and can activate a caspase cascade within seconds of ligand binding. Induction of apoptosis via this mechanism is therefore very rapid. Although there are differences in the signaling pathways activated by the different death receptors it is possible to outline a general apoptotic signaling pathway. Binding of the death inducing ligand to its receptor can lead to lipid raft fusion which results in clustering of the death receptors. The large scale receptor clustering is important because it helps amplify the apoptotic signaling. In the absence of receptor clustering some cells, such as lymphocytes, are still able to trigger apoptosis but in most cases amplification of the signaling pathway is needed to activate the full apoptotic response. Following ligand binding a conformational change in the intracellular domains of the receptors reveals the presence of a "death domain" which allows the recruitment of various apoptotic proteins to the receptor. This protein complex is often called the DISC (Death Inducing Signaling Complex). The final step in this process is the recruitment of one of the caspases, typically caspase 8, to the DISC. This results in activation of caspase 8 and the initiation of apoptosis. Signaling through the Fas receptor is slightly simpler than through the TNF receptor. The adapter protein FADD can be recruited directly to the death domain on the Fas receptor, without requiring the prior recruitment of TRADD. In addition the Fas receptor is generally though to only activate apoptosis and does not play an important role in other aspects of cell signaling like the TNF receptor. The response of the extrinsic pathway can be enhanced by the interaction with the intrinsic pathway (see details in SLIDE 11). TNF receptor signaling TNF is produced by T-cells and activated macrophages in response to infection. By activating its receptor, TNFR1, TNF can have several effects. In some cells it leads to activation of NF-kB and AP-1 which leads to the induction of a wide range of genes. In some cells, however, TNF can also induce apoptosis, although receptor ligation is rarely enough on its own to initiate apoptosis as is the case with Fas ligand binding. Binding of TNF alpha to TNFR1 results in APOPTOSIS oldal 4
receptor trimerisation and clustering of intracellular death domains. This allows binding of an intracellular adapter molecule called TRADD (TNFR-associated death domain) via interactions between death domains. TRADD has the ability to recruit a number of different proteins to the activated receptor. Recruitment of TRAF2 (TNF-associated factor 2) can lead to activation of e.g. the NF-kB pathway. TRADD can also associate with FADD, which leads to the induction of apoptosis via the recruitment and cleavage of pro-caspase 8. No survival signals The PI 3 kinase/akt pathway (SLIDE 12) An extracellular survival signal (e.g. IGF, insulin-like growth factor) activates a receptor tyrosine kinase (RTK), which recruits and activates PI 3-kinase. Pi 3 kinase produces PIP3 (from PIP2), which serves as a docking site for two serine/threonine kinases, PDK1 (phoshoinositol-dependent kinase) and Akt. The Akt is phosphorylated by a third kinase (mtor) on a serine, which alters the conformation of the Akt so that it can be phosphorylated on a threonine by PDK1, which activates Act. The activated Akt now dissociates from the plasma membrane and phosphorylates various target proteins, including the BAD protein. When unphosphorylated, BAD holds one or more apoptosis-inhibitory factors in an inactive state. Once phosphorylated, BAD releases the inhibitory proteins, which now can block apoptosis and thereby promote cell survival. BAD binds to a ubiquitous cytoplasmic protein called 14-3-3, which keeps BAD out of function. There are two other ways that extracellular signals can inhibit apoptosis, see SLIDE 13. The role of p53 in apoptosis (SLIDE 14, 15) In many cell types, p53-mediated growth inhibition is dependent on induction of p21, which is an inhibitor of cyclin-dependent kinases that are required for cell cycle progression. Failure of mutant p53 proteins to transactivate p21 may lead to uncontrolled proliferation. (1) In the cytosolic p53 apoptotic pathway, nuclear p53 induces Puma expression, which in turn releases cytosolic p53 held inactive in the cytoplasm through binding to Bcl-X L. Then, cytosolic p53 induces Bax oligomerization and mitochondrial translocation. Accumulation of p53 in the cytosol as a consequence of normal intracellular transport or stable monoubiquitination is the major source for mitochondrial p53. (2) Mitochondrial apoptotic pathway In the mitochondria, p53 induces Bax and Bak oligomerization, antagonizes the Bcl-2 and Bcl-X L antiapoptotic effect, and forms a complex with cyclophilin D in the mitochondrial inner membrane. These changes result in marked disruption of mitochondrial membranes and subsequent release of both soluble and insoluble apoptogenic factors. MPT, mitochondrial permeability transition; U, ubiquitin. APOPTOSIS oldal 5