Apoptosis Lecture Notes

Apoptosis

Learning Outcomes

  • Understand the importance of apoptosis in animal development and function.
  • Explain the intrinsic and extrinsic pathways that trigger apoptosis.
  • Know the three key classes of Bcl2 family proteins and their roles.
  • Understand how apoptosis dysregulation can occur in cancer, using Bcl2 in follicular lymphoma as an example, and how it can be treated.

Types of Cell Death

  • Not all cell deaths are the same.
  • Apoptotic cells have an intact plasma membrane, but the chromatin is distorted and concentrated at the margin of the nucleus.
  • Necrotic cells appear to have exploded.

Apoptosis (Programmed Cell Death)

  • Apoptosis, or programmed cell death (PCD), is a normal process that eliminates unwanted cells.
  • It is critical in embryonic development (e.g., removing skin between digits) and removing damaged cells.
  • An intracellular proteolytic cascade is activated, leading to:
    • Cell shrinkage and condensation.
    • Dissolution of the nuclear envelope.
    • Condensation and fragmentation of chromatin.
    • Collapse of the cytoskeleton.
    • Engulfment and destruction of the cell by phagocytosis (e.g., by a macrophage).
  • No leakage of cell contents occurs, avoiding an inflammatory reaction.

Cell Survival Depends on Extracellular Signals

  • Cells require survival factors to avoid apoptosis.
  • Neurotrophin trophic factors provide survival signals for neurons.
  • Experiment:
    • Wild-type mice express NGF (nerve growth factor) and neurotrophin-3 (NT-3).
    • Knockout mice without NGF or its TrkA receptor lack nociceptive neurons.
    • Mice without NT-3 or its TrkC receptor lack proprioceptive neurons.

Apoptosis is Mediated by a Caspase Cascade

  • An apoptotic signal triggers the assembly of an adaptor-protein complex, which recruits further monomers that dimerise, activating each other.
  • Initiator caspases (8 and 9) cleave and activate executor caspases (3, 6, and 7), which coordinate the apoptosis program.
  • Executor caspases cleave multiple targets, including:
    • Nuclear lamin (by caspase 6).
    • Phospholipid transfer proteins, resulting in the exposure of phosphatidylserine (“eat me” signal).
  • Caspases have a cysteine at their active site and cleave at aspartic acid sites.
  • They exist as inactive soluble monomers in the cytosol.

Apoptosis Acts via the Intrinsic or Extrinsic Pathways

  • Two main activation pathways activate initiator caspases:
    • Extrinsic pathway: signaled from outside the cell by death receptors (e.g., TNF family) activated by Fas ligands.
    • Intrinsic (mitochondrial) pathway: signaled from mitochondria inside the cell.
  • The extrinsic pathway may need to recruit the intrinsic pathway to overcome caspase inhibitors.

The Extrinsic Pathway

  • Tumor necrosis factor (TNF) interacts with cells through TNF receptors (Fas death receptors), which are homotrimeric and bind trimeric Fas ligands.
  • Binding clusters the receptors, exposing death domains that bind and cluster FADD (Fas-associated death domain).
  • Clustered FADD recruits inactive caspase-8, which oligomerizes, forming a death-inducing signaling complex (DISC).
  • Activated caspase-8 cleaves itself to form mature caspase-8 dimers, which activate downstream executioner caspases.

The Intrinsic or Mitochondrial Pathway

  • Activated from within the cell (e.g., developmental signals, DNA damage).
  • Depends on the release of cytochrome c from the mitochondrial intermembrane space into the cytosol.
  • Cytochrome c binds to Apaf1 (apoptotic protease activating factor 1), causing it to bind deoxy-ATP and oligomerize into a heptamer.
  • The heptamer recruits inactive caspase-9 monomers, forming an apoptosome.
  • Caspase-9 is activated by dimerization within the apoptosome and activates downstream executioner caspases.

Bcl2 Proteins are Critical Regulators of Apoptosis

  • The BH3 domain is shared by all Bcl2 family members and mediates direct interactions between pro- and anti-apoptotic members.
  • Three structural and functional classes of mammalian Bcl2 family proteins regulate Mitochondrial Outer Membrane Permeabilisation (MOMP):
    1. Anti-apoptotic (e.g., Bcl2) inhibit apoptosis by preventing MOMP.
    2. Pro-apoptotic effectors (e.g., Bax, Bak) directly induce MOMP.
    3. BH3-only proteins promote apoptosis by regulating the other two classes (e.g., Bim, Puma, Noxa).

How pro- and anti-apoptotic Bcl2 family proteins act

  • Anti-apoptotic proteins Bcl2 and BclxL prevent MOMP by binding to the BH3 domains of Bak and Bax.
  • Pro-apoptotic effectors Bak and Bax trigger MOMP by aggregating into oligomers in the outer mitochondrial membrane, allowing cytochrome c to escape.

Summary of the extrinsic and intrinsic pathways

  • Caspase-8 is the key link between the extrinsic and intrinsic pathways.
  • If apoptosis fails, the necroptosis pathway can be activated, triggered by RIPK1 kinase.
  • This leads to oligomer formation in the plasma membrane, destabilizing it and inducing cell swelling and rupture.

How survival factors inhibit apoptosis

  • Survival factors can stimulate transcription of anti-apoptotic Bcl2 family proteins (e.g., Bcl2 or BclxL).
  • They can activate RTKs and Akt/PKB, which phosphorylates and inactivates the pro-apoptotic BH3-only protein Bad.
  • Unphosphorylated Bad promotes apoptosis by binding to and inhibiting anti-apoptotic Bcl2 proteins.
  • Phosphorylated Bad dissociates, freeing Bcl2 to suppress apoptosis.
  • Akt can also suppress apoptosis by phosphorylating and inactivating transcription regulatory proteins that stimulate the transcription of genes encoding proteins that promote apoptosis, such as the BH3-only protein Bim.

Summary of pathway integration regulating MOMP

  • Integration of multiple signaling pathways regulate MOMP and apoptosis:
    1. Growth factor inhibition of apoptosis.
    2. Absence of growth/survival factor promotes apoptosis.
    3. DNA damage or UV stimulates apoptosis.

Apoptosis is triggered by a variety of pathways

  • Apoptosis can be activated by direct signaling or activation of DNA damage pathways.
  • It can also occur if critical survival factors are withdrawn.

Excess or insufficient apoptosis can cause disease

  • Heart attack and stroke can lead to cell death by necrosis, followed by apoptosis in the affected area.
  • Mutations in the Fas receptor or ligand prevent normal lymphocyte death, leading to autoimmunity and lymphomas.
  • Decreased apoptosis rates contribute to many cancers.

Dysregulated apoptosis and cancer

  • Resistance to apoptosis is a hallmark of cancer.
  • Mechanisms of evasion include:
    1. Disrupted balance of pro- and anti-apoptotic proteins.
    2. Reduced caspase function.
    3. Impaired death receptor signaling.

Tumour cells are frequently resistant to Fas-induced apoptosis

  • Mechanisms include:
    • Decreased receptor expression.
    • Expression of soluble Fas.
    • Increased expression of c-FLIP.
    • Decreased expression of caspase-8.
    • Increased expression of Bcl-2 and/or decreased Bax/Bad/Bak.
    • Defects in signaling and gene expression.

Dysregulated apoptosis and haematopoietic cancers

  • A disrupted balance of pro- and anti-apoptotic proteins plays a role in leukemias and lymphomas.
  • In chronic lymphocytic leukemia (CLL), malignant cells have an anti-apoptotic phenotype.
  • The BCL2 gene was identified due to the t(14; 18) chromosome translocation, present in $>90\%$ of follicular B cell lymphomas, resulting in Bcl2 overexpression.
  • Mcl1 is another commonly expressed pro-survival protein in hematological malignancies.

Therapy: Venetoclax inhibits Bcl2

  • Drugs have been developed to block the anti-apoptotic proteins Bcl2 and BclXL by binding to the BH3-binding groove, stimulating the intrinsic pathway.
  • Venetoclax binds to Bcl2 and is used in chronic lymphocytic leukemia.

Bcl2 and Mcl1 inhibitors for haematological cancers

  • BAX/BAK are essential to drive MOMP and are required for the action of venetoclax.
  • MCL1 inhibition could be effective for multiple myeloma (MM) since MCL1 is the predominant survival protein.
  • Some subtypes of multiple myeloma are also highly susceptible to BCL2 inhibition.
  • The degree of AML dependence on BCL2 or MCL1 varies.
  • Overall, both BCL2 and MCL1 appear to play prominent prosurvival roles in most AML cases.

Cancer therapeutic approaches targeting apoptosis

  • Examples:
    • Targeting death receptor ligands (DRL)
    • Targeting growth factor receptors
    • BH3 mimetics
    • Targeting ER stress
    • Inhibiting MDM2
    • Targeting IAPs
    • Caseinolytic protease P (ClpP)

Key points

  • Apoptosis eliminates unwanted cells without triggering inflammation. Extracellular survival factors inhibit apoptosis, and DNA damage can trigger it.
  • Apoptosis relies on a proteolytic cascade mediated by initiator (caspase 8 & 9) and executor caspases (3, 6 & 7), activated by extrinsic or intrinsic pathways.
  • Extrinsic pathway: Fas ligands bind Fas death receptors, clustering FADD proteins and caspase-8 to form the DISC.
  • Intrinsic pathway: Cytochrome c is released into the cytosol, binding Apaf1, which oligomerizes and recruits caspase-9 to form the apoptosome.
  • Bcl2 family proteins regulate MOMP: anti-apoptotic (Bcl2, Bclxl) prevent MOMP; pro-apoptotic (Bax, Bak) induce MOMP; BH3-only proteins are regulators (e.g., Bim, Puma, Noxa).
  • Cancer is often caused by disrupted balance, reduced caspase function, or impaired death receptor signaling, common in leukemias and lymphomas (e.g., Bcl2 translocation in follicular lymphoma).
  • Venetoclax blocks Bcl2, treating chronic lymphocytic leukemia.