Biochemical Aspects of Cell Death

Biochemical Aspects of Cell Death

Introduction to Cell Death

• Cell death is a fundamental and conserved process crucial for all aspects of biological life.
• Roles of Cell Death:
  • Embryonic development and morphogenesis.
  • Maintaining organismal homeostasis (e.g., normal cell turnover, tissue size control).
  • Eliminating damaged, infected, or abnormal cells (e.g., DNA damage, viral infection, cancer cells).
  • Induced in response to physical damage and infection.
• Classification of Cell Death:
  • Accidental Cell Death (Non-regulated Cell Death, Non-RCD): Typically refers to necrosis, resulting from unexpected cell injury. It lacks tight regulation by intracellular signaling pathways.
  • Regulated Cell Death (RCD): Tightly controlled by evolutionarily conserved signaling molecules and pathways.
    • Apoptosis: The first and most characterized form of RCD, often immunologically silent.
    • Non-Apoptotic RCDs: More recently characterized forms, often lytic and associated with inflammatory responses, including:
      • Autophagic cell death
      • Ferroptosis
      • Necroptosis
      • Pyroptosis
      • PANoptosis
      • Others: Parthanatos, Lysosome-dependent cell death, Autophagy-dependent cell death, Alkaliptosis, Oxeiptosis, Cuproptosis.

Comparison of Apoptosis and Necrosis

Why Apoptosis is Preferred Over Necrosis

• Apoptosis:
  • Apoptotic bodies are engulfed safely by phagocytes.
  • No spillage of cell contents.
  • No inflammatory response.
• Necrosis:
  • Cell and nuclear swelling.
  • Rupture and spillage of cell contents.
  • Significant inflammatory response.

Apoptosis: The Programmed Cell Death

• Etymology: From ancient Greek, meaning "falling of petals from a flower" or "leaves from a tree in autumn." First used by Kerr et al. in { ext{1972}}. Is the most studied RCD.
• Definition: A carefully coordinated collapse of a cell, involving protein degradation, DNA fragmentation, and engulfment of cell corpses by neighboring cells.
• Key Characteristics:
  • Affects single cells scattered in a population of healthy cells.
  • Represents a physiological process to eliminate affected/abnormal cells.
  • Primarily associated with development and homeostasis.
  • Mechanism: Gene-determined single-cell death.
• Importance of Apoptosis:
  • Embryonic Development and Morphogenesis:
    • Eliminates excess cells, sculpting tissues (e.g., resorption of tadpole tail, formation of fingers and toes in fetus, proper neuron connections in the brain).
    • Selection: Eliminates non-functional cells.
  • Adult Life:
    • Normal cell turnover and tissue homeostasis (e.g., sloughing off uterine lining, removal of senescent red blood cells).
    • Induction and maintenance of immune tolerance.
    • Development of the nervous system.
    • Endocrine-dependent tissue atrophy (e.g., mammary gland involution after lactation, prostate gland atrophy).
    • Elimination of activated, damaged, and abnormal cells (e.g., virus-infected cells, immune system cells, cells with DNA damage, cancer cells).
• Consequences of Dysregulation:
  • Too little apoptosis:
    • Can lead to cancer (dysfunctional cells accumulate).
    • Autoimmune diseases (e.g., T cells survive and attack own tissue).
    • Hyperplasia (precancerous lesions).
  • Too much apoptosis:
    • Stroke damage (due to lack of blood flow, stressed cells).
    • Alzheimer's disease (abnormal protein accumulation).
    • Neurodegeneration (Parkinson's, Huntington's).

Phases of Apoptotic Process

1. Induction Phase: Triggered by death signals (e.g., Fas ligand, TNF-{ ext{α}}) or absence of survival signals (growth factors). Reversible at this stage if survival signals return.
2. Cell Cycle Arrest & Capacitation: Cell halts division and molecularly prepares for apoptosis. Mitochondria and signaling pathways are primed.
3. Irreversible Commitment (Pre-apoptosis): Point of no return; the death program continues even if survival factors return. Caspase cascade is initiated.
4. Effector Phase: Executioner caspases are activated, cleaving structural and regulatory proteins. DNA fragmentation and early morphological changes appear.
5. Degradation Phase: Full execution: nucleolysis, chromatinolysis, proteolysis, cytolysis. Cell fragments into apoptotic bodies, cleared by phagocytes without inflammation.

Biochemical Alterations in Apoptotic Phases

1. Breakdown of Energy Metabolism:
   • Decrease in energy-rich nucleosides and ATP production.
   • Depletion in NADH dehydrogenase by DNA repair enzymes.
   • Decrease in glucose concentration.
2. Alterations in Ion Fluxes:
   • { ext{Ca}^{2+}}: Functions as a second messenger at the induction phase, a Bcl-2 cofactor at the effector phase, and a protease/nuclease activator at the degradation phase.
3. Cell Membrane Alterations:
   • Phosphatidylserine translocation to the outer leaflet, serving as an "eat me" signal.
4. Disruption of Anabolic and Catabolic Reactions.

Apoptotic Regulators

I. Caspases

• Definition: Cysteine aspartate proteases, highly conserved throughout evolution.
• Characteristics:
  • Have an active site cysteine and cleave target proteins after aspartate residues.
  • Synthesized as inactive precursors (pro-caspases).
  • Activation occurs by proteolytic cleavage (intra- or inter-molecular).
  • Inhibiting caspase activity slows or prevents apoptosis.
• Subgroups of Mammalian Caspases ({ ext{14}} identified in humans):
  • Signaling or Initiator Caspases: Caspases { ext{2, 8, 9, 10}}. Cleave and activate effector caspases, leading to amplification of the death signal.
  • Effector or Executioner Caspases: Caspases { ext{3, 6, 7}}. Cleave many cellular targets, leading to apoptotic hallmarks.
  • Inflammatory Caspases: Caspases { ext{1, 4, 5}}. Involved in pyroptosis and inflammation.
  • Other Caspases: Caspases { ext{11, 12, 13, 14}}.
  • Central Role: Caspase { ext{3}} ( ext{CPP32}) plays a central role in the cascade of apoptotic events.
• Mechanisms of Caspase Activation:
  1. Proteolytic Cleavage (e.g., pro-caspase 3): Specific regions of pro-caspase are cut, and subunits assemble to form active caspase.
  2. Induced Proximity (e.g., pro-caspase 8): Death receptors bring pro-caspases together, leading to mutual activation.
  3. Oligomerization (e.g., caspase 9): Cytochrome c (released from mitochondria), Apaf-1, and ATP form the apoptosome complex, which activates caspase 9.
• Caspase Targets and Effects:
  • Caspases selectively cleave a restricted set of target proteins, typically at one or a few positions after an aspartate residue.
  • Caspase-activated DNAse (CAD): Pre-exists in living cells with an inhibitory subunit. Caspase 3 activates CAD, which cuts genomic DNA between nucleosomes, generating fragments.
  • Cleavage of Nuclear Lamins: Leads to nuclear shrinking and budding.
  • Cleavage of Cytoskeletal Proteins (e.g., actin gelsolin): Causes loss of cell shape.
  • Cleavage of PAK-2: Contributes to cell budding.
  • Regulators of DNA repair (e.g., poly ADP-ribose polymerase, DNA-dependent protein kinase).
  • RNA splicing proteins.

II. Bcl-2 Family

• Bcl-2 family members are critical regulators of apoptosis, primarily by controlling mitochondrial outer membrane permeabilization.
• Three Functional Groups:
  1. Anti-apoptotic Proteins (e.g., Bcl-2, Bcl-xL):
     • Characterized by { ext{4}} conserved Bcl-2 homology (BH) domains ({ ext{BH1-BH4}}).
     • Possess a C-terminal hydrophobic tail localizing them to the mitochondrial outer surface or ER.
     • Prevent the release of pro-apoptotic factors.
  2. Pro-apoptotic Proteins (e.g., Bax, Bak):
     • Contain a hydrophobic tail and all but the most N-terminal { ext{BH4}} domain.
     • Promote the release of pro-apoptotic factors.
  3. BH3-Only Pro-apoptotic Proteins (e.g., Bid, Bik):
     • Contain only the { ext{BH3}} domain.
     • Act as initiators by interacting with and activating Bax/Bak.
• Mechanisms of Action:
  • Heterodimerization: Block each other's activity by neutralizing bound pro- and anti-apoptotic proteins (e.g., neutralization of Bax).
  • Mitochondrial Regulation: Regulate the release of pro-apoptotic factors, especially cytochrome c, from the mitochondrial intermembrane compartment into the cytosol.
  • Calcium Efflux Inhibition: Inhibit { ext{Ca}^{2+}} efflux from the ER.
  • ROS Formation Inhibition: Inhibit reactive oxygen species (ROS) formation.

III. Mitochondria: Powerhouse and Arsenal for Apoptosis

• Mitochondria are crucial for activating apoptosis and also involved in necrosis.
• Apoptotic Triggers from Mitochondria:
  1. Disruption of Electron Transport: Decreases ATP production.
  2. Decrease in Mitochondrial Membrane Potential.
  3. Release of Proapoptotic Proteins:
     • Cytochrome c: A key factor that, once released into the cytosol, binds to APAF1 and pro-caspase 9 to form the apoptosome, activating caspase 9.
     • AIF (Apoptosis Inducing Factor)
     • Smac/DIABLO
     • Procaspases (e.g., procaspase-2, 3, 9)
  4. Free Radicals (Reactive Oxygen Species, ROS).
• Role of Bcl-2 Family in Mitochondrial Permeabilization:
  • Pro-apoptotic Bcl-2 family members (Bax, Bak) cause cytochrome c release by forming pores in the outer mitochondrial membrane.
  • Anti-apoptotic members prevent this release.

IV. ROS and Oxidants

• Types: Superoxide radical ({ ext{O}2^{ullet-}}), hydrogen peroxide ({ ext{H}2 ext{O}_2}), organic peroxides.
• Sources: Electron Transport Chain (mitochondria, ER), fatty acid oxidation.
• Mechanism: Through lipid peroxidation and protein-nucleic acid alterations, ROS provoke cell death.
• Antioxidant Systems: Nonenzymatic ({ ext{glutathione, thioredoxin}}) and enzymatic ({ ext{catalase, superoxide dismutase}}) antioxidants mitigate ROS damage.
• Signaling Role: ROS also act as signaling molecules for apoptosis.

Pathways of Apoptosis

I. Intrinsic Apoptosis (Mitochondrial Pathway)

• Inducing Perturbations:
  • DNA damage (often p53-mediated).
  • ER stress (accumulation of misfolded proteins, unfolded protein response activation).
  • Replication stress, microtubular alterations, mitotic defects.
  • Golgi apparatus- and lysosome-associated stress (caspase-2 and some death receptors).
  • Cytosolic ROS.
• Key Event: Mitochondrial Outer Membrane Permeabilization (MOMP).
  • Controlled by pro-apoptotic and anti-apoptotic members of the BCL2 family.
  • BAX and BAK form pores across the outer mitochondrial membrane in response to apoptotic stimuli.
  • Anti-apoptotic BCL2 family members antagonize MOMP by binding to pro-apoptotic members.
• Apoptosome Formation and Caspase Activation:
  • MOMP promotes the cytosolic release of apoptogenic factors (cytochrome c, SMAC).
  • Cytosolic cytochrome c binds to APAF1 and pro-caspase { ext{9}} ( ext{CASP9}) to form the apoptosome.
  • The apoptosome activates CASP9.
  • Activated CASP9 activates the downstream effector caspases, CASP3 and CASP7.
• Nuclear Mechanisms (p53):
  • The p53 gene is located on chromosome { ext{17}}. p53 levels are very low in normal cells.
  • Function: p53 acts as a guardian of the genome. It stops the cell cycle at G1 to allow DNA repair. If DNA damage is extensive and repair fails, p53 induces apoptosis.
  • Activation: Upon injury, p53 is phosphorylated and activated, leading to increased p53 levels within minutes of DNA damage.
  • Consequences of p53 Dysfunction: Inactivated by mutation in over half of human cancers, leading to tumor formation.
  • p53 is the checkpoint decision between apoptosis and survival, initiated by genotoxic agents (oxidative stress, genome modification).

II. Extrinsic Apoptosis (Death Receptor Pathway)

• Initiated through the engagement of plasma membrane receptors:
  1. Death Receptors (ligand-dependent):
     • Examples: Fas (CD95), TNF receptor (TNFR1), TRAIL receptors (TR1-4), DR2.
     • Mechanism: Ligand binding causes a conformational change that recruits adaptor proteins, leading to the conversion of pro-CASP8 to its active form, CASP8.
     • Physiological Roles: Peripheral deletion of activated T cells, killing of virus-infected cells or cancer cells by cytotoxic T cells, killing of inflammatory cells.
  2. Dependence Receptors:
     • Mechanism: Activated when their ligand level drops below a threshold, leading to CASP9 activation, which then activates effector CASP3.
• Execution Pathways (Cell-type Dependent):
  1. Type I Cells (e.g., thymocytes, mature lymphocytes):
     • CASP8-dependent proteolytic activation of CASP3 and CASP7 is sufficient to execute cell death.
     • Direct pathway: CASP8 {
       ightarrow} CASP3/7 {
       ightarrow} apoptosis.
  2. Type II Cells (e.g., hepatocytes, pancreatic ext{β}-cells, some cancer cells):
     • Activation of CASP3 and CASP7 by CASP8 is restrained.
     • Indirect pathway (involves mitochondria): CASP8 proteolytically cleaves BID to form truncated BID (tBID).
     • tBID translocates to mitochondria, initiating cytochrome c release.
     • Cytochrome c binds APAF1 to form the apoptosome, activating CASP9.
     • CASP9 then activates CASP3 and CASP7.
• Mitochondrial Bypass: Apoptosis induced by death receptors can sometimes bypass the mitochondrial pathway (cytochrome c release is a result, not a cause, of caspase activation), making it insensitive to Bcl-2 protection.

Non-Apoptotic Regulated Cell Death Mechanisms

I. Autophagic Cell Death

• Involves the formation of autophagosomes that encapsulate cellular material for degradation by lysosomes, effectively consuming the cell from within.

II. Ferroptosis

• Mechanism: An iron-dependent form of RCD driven by the buildup of toxic lipid peroxides, leading to oxidative damage.
• Cytological Changes: Decreased mitochondrial cristae and damaged mitochondrial membranes.
• Consequences: Loss of selective permeability of the plasma membrane due to intense membrane lipid peroxidation and oxidative stress.
• Metabolic Aspects: Involves amino acid, lipid, NADPH, and microelement metabolism.
• Triggers: Cysteine depletion, glutathione peroxidase ({ ext{GPX4}}) inactivation, and iron overload.

III. Necroptosis

• Mechanism: A form of lytic RCD induced by specific death receptors (Fas, TNFR1) or pathogen recognition receptors (PRRs) when caspase activation is inhibited.
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