Apoptosis Lecture Notes PDF

Programmed cell Death Apoptosis Dr. Manikandan Kathirvel M.Sc., Ph.D., (NET) Assistant Professor, Department of Life Sciences, Kristu Jayanti College (Autonomous), (Reaccredited with "A" Grade by NAAC) Affiliated to Bengaluru North University, K. Narayanapura, Kothanur (PO) Bengaluru...

Programmed cell Death Apoptosis Dr. Manikandan Kathirvel M.Sc., Ph.D., (NET) Assistant Professor, Department of Life Sciences, Kristu Jayanti College (Autonomous), (Reaccredited with "A" Grade by NAAC) Affiliated to Bengaluru North University, K. Narayanapura, Kothanur (PO) Bengaluru 560077 Programmed cell death (PCD) Programmed cell death (PCD) is a regulated process by which cells undergo a self-initiated death as part of normal development, maintenance, or defense in multicellular organisms. The main types of programmed cell death include apoptosis, necrosis, autophagy, pyroptosis, and ferroptosis. 1. Apoptosis Apoptosis is the most well-known form of programmed cell death, often referred to as "cellular suicide." It is highly regulated and non-inflammatory. The main mechanisms involve: Intrinsic Pathway (Mitochondrial Pathway): Triggered by internal stress signals (e.g., DNA damage, oxidative stress), this pathway involves the release of cytochrome c from mitochondria. Cytochrome c activates Apaf-1 (apoptotic protease-activating factor 1), forming the apoptosome complex, which in turn activates caspase-9. This initiates a cascade that activates caspase-3 and -7, which then dismantle the cell. Extrinsic Pathway (Death Receptor Pathway): Triggered by external signals (e.g., Fas ligand binding to the Fas receptor, TNF binding to its receptor). These interactions activate caspase-8, which then activates downstream effector caspases (like caspase-3) to execute cell death. Execution Phase: Activated effector caspases (caspase-3, -6, -7) cleave specific cellular proteins, leading to membrane blebbing, chromatin condensation, and the formation of apoptotic bodies. The cell is then phagocytosed by neighboring cells or immune cells, avoiding inflammation. 2. Necroptosis Necroptosis is a regulated form of necrosis, triggered by death receptors but distinct from apoptosis. It occurs when caspase-8 is inhibited, leading to activation of receptor-interacting protein kinases (RIPK1 and RIPK3). These kinases phosphorylate MLKL (mixed lineage kinase domain-like pseudokinase), causing it to oligomerize and translocate to the plasma membrane, where it disrupts membrane integrity and induces cell rupture. Unlike apoptosis, necroptosis is inflammatory. 3. Autophagy-dependent Cell Death Autophagy is a process where cells degrade damaged organelles or proteins via autophagosomes, which fuse with lysosomes. In some cases, excessive or dysregulated autophagy leads to cell death. This process is mediated by ATG (autophagy-related) proteins and involves the engulfment of cell components, but it lacks the characteristic features of apoptosis and necrosis. 4. Pyroptosis Pyroptosis is a pro-inflammatory form of cell death often triggered by infections. It involves the activation of caspase- 1 or caspase-11/4/5 in response to inflammasome assembly. These caspases cleave gasdermin D (GSDMD), which forms pores in the cell membrane, causing cell swelling and lysis, and releasing pro-inflammatory cytokines (e.g., IL- 1β and IL-18). 5. Ferroptosis Ferroptosis is iron-dependent and is triggered by lipid peroxidation. It is regulated by the accumulation of lipid hydroperoxides due to depletion of glutathione or inactivation of glutathione peroxidase 4 (GPX4). Unlike other forms, ferroptosis is characterized by iron overload and oxidative damage to cell membranes rather than caspase activation. Each type of PCD plays essential roles in development, immunity, and homeostasis, and dysregulation of these processes is implicated in various diseases, including cancer, neurodegeneration, and autoimmune disorders. Cell senescence Cellular senescence is the response of cells that results in stable cell cycle arrest and different phenotypic variations such as metabolic changes, chromatin remodeling, altered gene expression, and induction of autophagy. Cellular senescence is defined as the irreversible exit from the cell cycle. Cellular senescence is a phenomenon characterized by the cessation of cell division. Cellular senescence is a permanent cell cycle arrest that can be induced by several stressors. Conditions such as mitochondrial dysfunction, Telomere shortening (telomere alteration), oxidative stress, DNA damage, mitochondrial dysfunction and epigenetic factors play a crucial role in the induction of cellular senescence. Senescence plays an important role in Aging and age-related diseases such as tissue degeneration, various diseases including cancer.  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 tissue Cell death by injury inflammatory reaction reaction -Mechanical damage DNA fragmentation is Ladder-like DNA fragmentation -Exposure to toxic chemicals random, or smeared In vivo, individual cells appear Cell death by suicide In vivo, whole areas of affected -Internal signals the tissue are affected -External signals Mechanism of Apoptosis If cells are no longer needed, they commit suicide by activating an intra- cellular death program. This process is therefore called programmed cell death, although it is more commonly called apoptosis (from a Greek word meaning “falling off,” as leaves from a tree). Apoptosis Is Mediated by an Intracellular Proteolytic Cascade  A cell that undergoes apoptosis dies neatly, without damaging its neighbours. The cell shrinks and condenses. The cytoskeleton collapses, the nuclear envelope disassembles, and the nuclear DNA breaks up into fragments.  The intracellular machinery responsible for apoptosis seems to be similar in all animal cells.  This machinery depends on a family of proteases that have a cysteine at their active site and cleave their target proteins at specific aspartic acids. They are therefore called caspases.  Caspases are synthesized in the cell as inactive precursors, or procaspases, which are usually activated by cleavage at aspartic acids by other caspases.  Once activated, caspases cleave, and thereby activate, other procaspases, resulting in an amplifying proteolytic cascade. Some of the activated caspases then cleave other key proteins in the cell. Mechanism of Intrinsic Pathway (Mitochondrial Pathway): The intrinsic pathway of apoptosis, also known as the mitochondrial pathway, is triggered by internal cellular stress signals such as DNA damage, oxidative stress, hypoxia, or deprivation of growth factors. This pathway is tightly regulated by the Bcl-2 family of proteins and culminates in the activation of caspases, the main executors of apoptosis. 1. Cellular Stress Signal Detection  When a cell encounters stress, such as DNA damage or oxidative stress, it activates pro-apoptotic signals. Key molecules like p53 (a tumor suppressor protein) sense these signals and can act as transcription factors to promote the expression of genes involved in apoptosis.  p53, for instance, can upregulate pro-apoptotic proteins like Bax and PUMA in response to DNA damage. 2. Role of the Bcl-2 Family Proteins  The Bcl-2 family of proteins tightly regulates the intrinsic pathway, and it includes both pro-apoptotic and anti-apoptotic members: o Pro-apoptotic proteins: Bax, Bak, and BH3-only proteins (e.g., Bid, Bad, PUMA, and Noxa) promote cell death. o Anti-apoptotic proteins: Bcl-2, Bcl-XL, and Mcl-1 inhibit apoptosis by binding to and neutralizing pro-apoptotic proteins.  Under normal conditions, anti-apoptotic proteins like Bcl-2 keep the cell alive by inhibiting the activity of pro-apoptotic proteins Bax and Bak. However, in response to cellular stress, BH3-only proteins are activated, binding to and inhibiting anti-apoptotic proteins, therby allowing Bax and Bak to initiate apoptosis. 3. Mitochondrial Outer Membrane Permeabilization (MOMP)  Once activated, Bax and Bak undergo conformational changes and oligomerize (aggregation) to form pores in the outer mitochondrial membrane. This process, known as mitochondrial outer membrane permeabilization (MOMP), is a critical and irreversible step in the intrinsic pathway.  MOMP results in the release of pro-apoptotic factors from the mitochondrial intermembrane space into the cytosol. 4. Release of Cytochrome c and Other Pro-apoptotic Factors  Cytochrome c: One of the key factors released into the cytosol, cytochrome c binds to apoptotic protease-activating factor-1 (Apaf-1), adaptor protein, leading to the formation of a complex known as the apoptosome.  Other factors: Additional pro-apoptotic molecules, such as Smac/DIABLO and HtrA2/Omi, are also released from mitochondria. These factors inhibit inhibitors of apoptosis proteins (IAPs), which normally suppress caspase activation, further ensuring the progression of apoptosis. 5. Apoptosome Formation and Activation of Caspase-9  In the cytosol, cytochrome c binds to Apaf-1 in the presence of ATP/dATP. This complex undergoes a conformational change and assembles into the apoptosome, a large, heptameric complex.  The apoptosome then recruits and activates pro-caspase-9, converting it into its active form, caspase-9. 6. Caspase Cascade Activation  Activated caspase-9 initiates a cascade by cleaving and activating executioner caspases (specifically caspase-3 and caspase-7).  Caspase-3 and caspase-7 are responsible for the proteolytic cleavage of various cellular substrates, leading to the characteristic morphological changes of apoptosis, such as chromatin condensation, nuclear fragmentation, cell shrinkage, and membrane blebbing. 7. Execution Phase of Apoptosis  Executioner caspases degrade structural and regulatory proteins, leading to the dismantling of the cell. Specific proteins targeted include: o PARP (poly ADP-ribose polymerase), involved in DNA repair, which is cleaved to prevent unnecessary repair processes. o Lamins, which stabilize the nuclear envelope, are also degraded, leading to nuclear fragmentation.  The cell breaks apart into membrane-bound fragments known as apoptotic bodies, which are then engulfed and removed by neighboring phagocytic cells, avoiding inflammatory responses.  APOPTOSIS: Morphology organelle reduction membrane blebbing & changes cell mitochondrial leakage shrinkage nuclear chromatin fragmentation condensation Hacker., 2000  APOPTOSIS: Morphological events cell shrinkage organelle reduction mitochondrial leakage chromatin condensation nuclear fragmentation membrane blebbing & changes Summary of Key Steps 1. Stress signal detection activates pro-apoptotic proteins. 2. Bcl-2 family regulation allows mitochondrial pore formation. 3. MOMP leads to cytochrome c and other factor release. 4. Apoptosome formation activates caspase-9. 5. Caspase cascade leads to cellular dismantling. 6. Apoptotic bodies are formed and cleared. This intrinsic pathway is crucial for normal development, tissue homeostasis, and the elimination of damaged or infected cells. Dysregulation can lead to various diseases, including cancer, where the apoptosis pathway is often suppressed to promote cell survival.  Pathways of Cellular Apoptosis Hotchkiss R et al. N Engl J Med 2009;361:1570-1583 Extrinsic Pathway (also known as the Death Receptor Pathway):  The extrinsic pathway is triggered by extracellular signals that bind to specific cell surface receptors (e.g., Fas ligand binding to the Fas receptor, TNF binding to its receptor).  Killer lymphocytes, for example, can induce apoptosis by producing a protein called Fas ligand, which binds to the death receptor protein Fas on the surface of the target cell.  The clustered Fas proteins then recruit intracellular adaptor proteins (FADD) that bind to it.  These interactions, aggregate procaspase-8 molecules, which cleave and activate one another.  The activate caspase-8 molecules, which then activates downstream effector caspases (like caspase-3) to to induce apoptosis/execute cell death.  Thus this pathway is regulated by death receptors in the TNF (tumor necrosis factor) receptor superfamily, which include receptors like Fas (CD95) and TNF receptor-1 (TNFR1). Activation of these receptors initiates a cascade of events that ultimately lead to apoptosis. The extrinsic pathway of apoptosis is initiated by extracellular signals that bind to death receptors on the cell surface, leading to programmed cell death. Here are some key examples of extracellular signals involved in this pathway: 1.Tumor Necrosis Factor-alpha (TNF-α) TNF-α is a cytokine produced by immune cells, such as macrophages, and it binds to TNF receptors (mainly TNFR1). This interaction activates the extrinsic pathway, particularly during inflammation and immune responses, often to eliminate infected or cancerous cells. 2.Fas Ligand (FasL) / CD95 Ligand FasL, also known as CD95L, is a protein expressed on the surface of certain immune cells, like cytotoxic T cells. It binds to the Fas receptor (CD95) on target cells, which triggers the apoptosis pathway to remove cells that may be damaged or potentially harmful. 3.TRAIL (TNF-Related Apoptosis-Inducing Ligand) TRAIL is another cytokine that binds to death receptors DR4 (TRAIL-R1) and DR5 (TRAIL-R2). This ligand-receptor binding can selectively induce apoptosis in cancer cells without affecting most normal cells, making it a potential target for cancer therapies. 4.TNF-β (Lymphotoxin) TNF-β, also known as lymphotoxin, is a cytokine produced by lymphocytes. It also binds to TNFR1 and TNFR2 on cells and is involved in promoting apoptosis in certain immune responses. These signals, upon binding to their respective death receptors, lead to the recruitment and activation of adaptor proteins like FADD (Fas-associated protein with death domain) and the activation of caspase-8, which is the initiator caspase in the extrinsic pathway. This cascade ultimately results in apoptosis. Steps of the Extrinsic (Death Receptor) Apoptosis Pathway 1. Binding of Death Ligands to Death Receptors: o The pathway is initiated when death ligands (such as Fas ligand [FasL] or TNF-α) bind to their corresponding death receptors on the cell membrane (e.g., Fas receptor [FasR] or TNFR1). o Ligand binding causes receptor oligomerization (aggregation of receptor molecules), which allows the recruitment of adapter proteins (FADD). 2. Formation of the Death-Inducing Signaling Complex (DISC): o The death receptors have intracellular domains known as death domains (DDs), which interact with adapter proteins (FADD). o For example, the Fas receptor recruits an adapter protein called FADD (Fas-associated death domain), which also contains a death domain. o FADD recruits pro-caspase-8 (or pro-caspase-10 in some cells) through an effector domain called the death effector domain (DED), forming the Death-Inducing Signaling Complex (DISC). 3. Activation of Initiator Caspases: o Within the DISC, pro-caspase-8 molecules are brought close together, leading to their dimerization and activation. o Active caspase-8 is an initiator caspase, and it triggers downstream events in apoptosis. It can directly activate executioner caspases or initiate a mitochondrial amplification loop. 4. Executioner Caspase Activation and Execution Phase: o Activated caspase-8 cleaves and activates executioner caspases, such as caspase-3 and caspase-7. o Executioner caspases then cleave a variety of cellular substrates, leading to DNA fragmentation, degradation of cytoskeletal proteins, and formation of apoptotic bodies. 5. Cross-talk with the Intrinsic Pathway (Amplification via Bid): o In some cells, the extrinsic pathway can amplify its signal by engaging the intrinsic pathway. Caspase-8 cleaves a protein called Bid (a BH3-only protein), converting it into tBid. o tBid translocates to the mitochondria, where it promotes mitochondrial outer membrane permeabilization (MOMP)-(pores in the outer membrane of mitochondria ) releasing cytochrome c and activating the intrinsic pathway. o This amplification loop is especially important in cells that require both extrinsic and intrinsic signaling for full apoptosis activation. 6. Formation and Clearance of Apoptotic Bodies: o The cell undergoes structural changes, blebbing, and fragmentation into apoptotic bodies, which are then phagocytosed by immune cells or neighboring cells.  Summary  Death Ligands (FasL, TNF-α, TRAIL) binding to Death Receptors (FasR, TNFR1) on the cell membrane.  Receptor Oligomerization and recruitment of adapter proteins (FADD).  Formation of the DISC with caspase-8 activation.  Executioner Caspase Activation and cell dismantling.  Optional cross-talk with intrinsic pathway through Bid cleavage.