Chapter 18: Molecular Mechanisms and Functions of Cell Death

Overview of Cell Death mechanisms

• The development and maintenance of multicellular organisms depend not only on cell growth and cell division but also on cell death.
• In animal development, orchestrated patterns of cell growth, division, and death determine the size and shape of limbs and organs.
• Cell Turnover: A process in adult animals where cells die at the same rate as they are produced to maintain tissue size.
• Normal cell death occurs in:   - Animals (development and adult maintenance).   - Plants (development and senescence of flowers and leaves).   - Unicellular organisms (yeasts and bacteria).
• Most normal cell deaths are suicides where molecular events destroy cells from within.
• Apoptosis: A molecularly distinct form of cell suicide occurring in animal cells.   - Etymology: Derived from the Greek word meaning ‘falling off,’ like leaves from a tree.   - Thought to occur in all animals and is the most common way for cells to die.   - Morphological and biochemical changes package the cell to be eaten and digested quickly by nearby cells.

Morphological and Biochemical Characteristics of Apoptosis

• Morphological Changes:   - The cell shrinks and condenses.   - The cytoskeleton collapses.   - The nuclear envelope disassembles.   - The nuclear chromatin condenses and fragments into pieces.   - Blebs: Large protrusions that form on the apoptotic cell surface.   - Apoptotic Bodies: Membrane-enclosed fragments produced if the cell is large.
• Clearing Process:   - The cell surface and fragments are chemically altered.   - Neighboring cells, often macrophages in vertebrates, rapidly engulf the fragments before they spill their contents.   - This neat process prevents a damaging inflammatory response.   - Because of rapid digestion, few dead cells are typically visible even in tissues with high death rates.
• Contrast with Cell Necrosis:   - Definition: A passive form of death where cells lyse in response to acute tissue insult (trauma or blocked blood supply).   - Mechanism: Cells swell and burst, spilling contents and eliciting an inflammatory response.   - Active Necrosis: Named forms like necroptosis or pyroptosis involve active cell participation depending on inducing stress and molecular mechanisms.

Biological Functions of Apoptosis

• Scale of Death:   - In a healthy adult human, at least 1 imes 10^6 cells die via apoptosis every second.
• Sculpting During Development:   - Hand and foot development: Appendages start as spade-like structures; digits separate as cells between them die (interdigital cell death).   - Metamorphosis: In tadpoles, tail cells die by apoptosis as the tail is no longer needed in the adult frog.
• Quality Control:   - Eliminates abnormal, misplaced, nonfunctional, or dangerous cells.   - Adaptive Immune System: Eliminates developing T and B lymphocytes that fail to produce antigen-specific receptors or produce self-reactive receptors.   - Post-infection: Eliminates most lymphocytes activated by infection after they have performed their function.
• Regulation of Tissue Size:   - Example (Rat Liver): If part of the liver is removed, proliferation increases. If treated with phenobarbital (stimulating growth) and then stopped, apoptosis increases until the liver returns to its normal size (usually within a week).
• Response to Damage:   - Cells detect damage in organelles and kill themselves if the damage is great.   - DNA Damage: Apoptosis eliminates cells with cancerous mutations if the DNA cannot be repaired.

The Caspase Proteolytic Cascade

• Definition: Apoptosis is triggered by a family of specialized intracellular proteases called caspases.
• Chemical Nature:   - Cysteine at their active site (‘c’).   - Cleave target proteins at specific aspartic acids (‘asp’).
• Procaspases: Inactive precursors present in the cytosol of nearly all cells.
• Initiator Caspases:   - Primary mammals: Caspase-8 and Caspase-9.   - Made as inactive soluble monomers.   - Activated by dimerization triggered by adaptor-protein complexes.   - Activated monomers cross-cleave their partners to form mature activated initiator caspase dimers.
• Executioner Caspases:   - Primary vertebrates: Caspase-3, Caspase-6, and Caspase-7.   - Exist as inactive soluble dimers.   - Activated by cleavage mediated by initiator caspases.   - Each initiator can activate many executioners, creating an amplifying cascade.   - The process is destructive and irreversible.

Target Proteins of Executioner Caspases

• Nuclear Lamins: Cleaved by Caspase-6, causing the irreversible breakdown of the nuclear lamina.
• DNA Fragmentation:   - Caspase-3 cleaves an inhibitor protein (iCAD) that normally holds a DNA-degrading endonuclease (CAD) inactive.   - Active CAD cuts chromosomal DNA between nucleosomes.   - This produces a ‘ladder pattern’ on gel electrophoresis ($180–200$ base pair increments).
• Cytoskeletal Changes:   - Cleave actin regulators, causing actin polymerization in the cell cortex responsible for surface blebbing.   - Cleave cell–cell adhesion proteins, helping the cell round up and detach.
• Eat Me Signals:   - Cleave phospholipid transfer proteins, resulting in the exposure of phosphatidylserine on the cell surface.

The Extrinsic Pathway of Apoptosis

• Trigger: Extracellular signal proteins binding to cell-surface death receptors.
• Death Receptors:   - Transmembrane proteins with an extracellular ligand-binding domain, a single transmembrane domain, and an intracellular death domain.   - Homotrimers belonging to the tumor necrosis factor (TNF) receptor family (8 members total, including Fas and TNF receptor).
• Process (Fas Example):   - Trimeric Fas ligands on a killer (cytotoxic) lymphocyte bind to trimeric Fas receptors on a target cell.   - Clustering of receptors exposes death domains on the tails.   - Adaptor protein FADD (Fas-associated death domain) binds the receptor death domain and exposes a death effector domain (DED).   - FADD recruits monomeric Caspase-8 via DED-DED interactions.   - Formation of the DISC (death-inducing signaling complex).   - Caspase-8 oligomerizes, dimerizes, and activates to cleave downstream executioners.
• Inhibitors:   - FLIP: A protein resembling Caspase-8 but lacking protease activity (missing the key cysteine).   - It dimerizes with Caspase-8 in the DISC, setting an inhibitory threshold.

The Intrinsic (Mitochondrial) Pathway of Apoptosis

• Trigger: Internal stimuli, developmental signals, or injury like DNA damage.
• Role of Mitochondria:   - Depends on the release of proteins from the mitochondrial intermembrane space.   - Cytochrome c: A water-soluble component of the electron-transport chain released into the cytosol.
• Mechanism:   - Cytochrome c binds to Apaf1 (apoptotic protease activating factor 1).   - Apaf1 binds deoxy-ATP (dATP) and oligomerizes into a wheel-like heptamer.   - The heptamer recruits inactive Caspase-9 monomers via CARD (caspase recruitment domain).   - Formation of the Apoptosome.   - Caspase-9 is activated by dimerization and activates executioners.

Bcl2 Family Proteins: Controllers of MOMP

• MOMP: Mitochondrial outer membrane permeabilization.
• Functional Classes:   - Anti-apoptotic: e.g., Bcl2, BclxL. Inhibit apoptosis by preventing MOMP. Share four BH domains (BH1–4).   - Pro-apoptotic Effectors: e.g., Bak and Bax. Directly induce MOMP by aggregating into oligomers to form membrane openings. Structurally similar to Bcl2 but lack BH4.   - Pro-apoptotic BH3-only proteins: e.g., Bad, Bim, Bid, Puma, Noxa. Promotes apoptosis by sharing sequence homology only in the BH3 domain.
• Regulation Process:   - Stimuli activate BH3-only proteins.   - Some BH3-only (Bad) inhibit anti-apoptotic proteins (Bcl2) by binding their BH3-binding grooves, allowing Bak/Bax to oligomerize.   - Others (Bim, Bid) directly bind and activate Bak (bound to mitochondria) or Bax (relocated from cytosol).
• Specific Examples:   - p53: Accumulates in response to DNA damage, activating transcription of Puma and Noxa.   - Bid: Serves as a link between extrinsic and intrinsic pathways; cleaved by Caspase-8 and translocates to mitochondria.

Cytosolic Caspase Inhibition and Anti-IAPs

• IAPs (Inhibitors of Apoptosis):   - First identified in insect baculoviruses to prevent infected host cells from killing themselves.   - XIAP: X-linked IAP in humans. Binds and inhibits Caspase-9, -3, and -7.   - Contains a ubiquitin-ligase domain to mark caspases for proteasome destruction.
• Anti-IAPs:   - Smac and Omi are released from the mitochondrial intermembrane space during MOMP.   - They bind to XIAP to neutralize its anti-caspase activity, allowing apoptosis to proceed.

Extracellular Survival Factors

• Function: Promote cell survival by inhibiting apoptosis, ensuring cells live only where needed.
• Nerve Cell Competition: Cells are produced in excess and compete for limited survival factors from target tissues. Those with insufficient signals die by apoptosis.
• Mechanisms of Action:   - Stimulating transcription of genes for anti-apoptotic proteins (Bcl2, BclxL).   - Activating the protein kinase Akt: Akt phosphorylates and inactivates the BH3-only protein Bad.   - Akt also phosphorylates transcription factors that would otherwise stimulate the synthesis of Bim.
• Unoccupied Receptor Strategy: In some neurons, receptors stimulate apoptosis when empty and stop when a survival factor binds.

Phagocytosis and "Eat Me" Signals

• Phosphatidylserine (PS):   - In healthy cells, PS is confined to the inner leaflet of the plasma membrane by ATP11C (a phospholipid flippase using ATP hydrolysis).   - In apoptotic cells, PS accumulates on the outer surface (the ‘eat me’ signal).
• Caspase-dependent Exposure Mechanism:   - Executioner caspases cleave and inactivate the ATP11C flippase.   - Executioner caspases cleave and activate Xkr8 (a phospholipid scramblase) which transfers phospholipids nonspecifically between leaflets.
• Engulfment:   - PS is recognized by soluble ‘bridging’ proteins that interact with phagocytic receptors on macrophages.   - To be eaten, cells must also remove or inactivate ‘don’t eat me’ signals that normally block phagocytosis of healthy cells.

Apoptosis and Human Disease

• Excessive Apoptosis:   - Heart attacks and strokes: Caused by acute blood supply interruption. Initial death is necrotic, but reperfusion can trigger apoptosis in surviving oxygen-deprived tissue.
• Insufficient Apoptosis:   - Autoimmune disorders: Inactivation of Fas or its ligand prevents lymphocyte elimination.   - Lymphoma: Chromosome translocation causing excessive synthesis of Bcl2 (B-cell lymphoma-2) prolongs survival and increases resistance to drugs.   - p53 Mutations: Mutated in about $50\%$ of human cancers. Lack of p53 prevents apoptosis in response to DNA damage, allowing mutation accumulation.
• Therapeutic Approaches:   - BH3-mimetic drugs: Small chemicals designed to bind the BH3-binding groove of anti-apoptotic proteins.   - Venetoclax: A drug designed to bind human Bcl2 tightly. It is in clinical use for chronic lymphocytic leukemia.

Summary of Death Pathways

• Extrinsic: Ligand → Death Receptor → FADD → DISC → Caspase-8 dimerize → Executioners.
• Intrinsic: Stimulus → MOMP → Cytochrome c release → Apaf1 + dATP → Apoptosome → Caspase-9 dimerize → Executioners.
• Key Regulators: Bcl2 family (Anti-apoptotic, Effectors, BH3-only), IAPs (XIAP), and Anti-IAPs (Smac, Omi).