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 can be dysregulated in cancer, with the example of Bcl2 in follicular lymphoma and its treatment.
Types of Cell Death
Apoptotic cells have an intact plasma membrane, distorted chromatin, and concentrated nuclei.
Necrotic cells appear to have exploded.
Apoptosis (Programmed Cell Death)
Apoptosis, or programmed cell death (PCD), is a normal process for eliminating unwanted cells.
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, avoiding an inflammatory reaction.
Cell Survival Depends on Extracellular Signals
Cells require survival factors (e.g., growth factors) to avoid apoptosis.
Neurotrophin trophic factors provide survival signals for neurons.
Experiment:
Wild-type mice express NGF (nernerve growth factor) and NT-3 (neurotrophin 3) which generate motor, nociceptive, and proprioceptive neurons.
Knockout mice lacking NGF or TrkA receptor lack nociceptive neurons.
Mice without NT-3 or TrkC receptor lack proprioceptive neurons.
Apoptosis is Mediated by a Caspase Cascade
An apoptotic signal triggers the assembly of an adaptor-protein complex, recruiting monomers that dimerize, activating them through cleavage.
Initiator caspases (8 and 9) cleave and activate executor caspases (3, 6, and 7), coordinating the apoptosis program.
Executor caspases cleave multiple targets in a cascading, non-reversible event.
Nuclear lamin is cleaved by caspase 6.
Phospholipid transfer proteins are cleaved, exposing phosphatidylserine (“eat me” signal).
Caspases have a cysteine at their active site and cleave at specific aspartic acid sites.
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, activated by death receptors (e.g., TNF family) and Fas ligands.
Intrinsic (mitochondrial) pathway: signaled from mitochondria inside the cell.
The extrinsic pathway needs to overcome caspase inhibitors by recruiting the intrinsic pathway in some cell types.
The Extrinsic Pathway
Tumor necrosis factor (TNF) interacts with cells through TNF receptors (Fas death receptors), which are homotrimeric receptors that bind trimeric Fas ligands.
Binding clusters receptors, exposing death domains on receptor tails, which bind and cluster FADD (Fas-associated death domain).
Clustered FADD proteins recruit inactive, monomeric initiator caspase-8, which oligomerize, forming a large death-inducing signaling complex (DISC).
Activated caspase-8 cleaves itself to form mature, active caspase-8 dimers that 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 proteins from the mitochondrial intermembrane space, especially cytochrome c.
Cytochrome c binds to Apaf1 (apoptotic protease activating factor 1), causing it to bind deoxy-ATP and oligomerize into a wheel-like heptamer.
The heptamer recruits inactive initiator 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 interactions between pro-apoptotic and anti-apoptotic family members.
Three classes of mammalian Bcl2 family proteins:
Anti-apoptotic Bcl2 family proteins (e.g., Bcl2) inhibit apoptosis by preventing Mitochondrial Outer Membrane Permeabilization (MOMP).
Pro-apoptotic Bcl2 family effectors (e.g., Bax, Bak) induce MOMP by creating openings in the outer mitochondrial membrane.
BH3-only proteins (e.g., Bim, Puma, Noxa) promote apoptosis by regulating the other two classes and link apoptotic stimuli to the intrinsic pathway.
How Pro- and Anti-Apoptotic Bcl2 Family Proteins Act
Anti-apoptotic proteins Bcl2 and BclxL are located on the outer mitochondrial membrane, preventing MOMP by binding to the BH3 domains of Bak and Bax, preventing their oligomerization.
When an apoptotic stimulus activates the intrinsic pathway, Bak and Bax are activated and trigger MOMP by aggregating into oligomers, allowing cytochrome c to escape.
Summary of the Extrinsic and Intrinsic Pathways
Caspase-8 is the key link between the extrinsic and intrinsic pathways.
When apoptosis fails (e.g., absence of caspase-8 or FADD), the necroptosis pathway can be activated, triggered by RIPK1 kinase.
This activates a chain of proteins that form oligomers in the plasma membrane, destabilizing it and inducing cell swelling and rupture.
How Survival Factors Inhibit Apoptosis
Some survival factors suppress apoptosis by stimulating the transcription of genes encoding anti-apoptotic Bcl2 family proteins (e.g., Bcl2 or BclxL).
Many activate RTKs and the protein kinase Akt/PKB, which phosphorylates and inactivates the pro-apoptotic BH3-only protein Bad.
When not phosphorylated, Bad promotes apoptosis by binding to and inhibiting anti-apoptotic Bcl2 family proteins (e.g., Bcl2).
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
Growth factor inhibition of apoptosis.
Absence of growth/survival factor promotes apoptosis.
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 (e.g., growth factors) are withdrawn from the cell.
Excess or Insufficient Apoptosis Can Cause Disease
Conditions such as heart attack and stroke can lead to initial cell death by necrosis, with subsequent apoptosis in the affected area.
Mutations in the Fas receptor or ligand prevent normal lymphocyte death, leading to an excess in lymph nodes and spleen, triggering autoimmune disorders and lymphomas.
Decreased rates of apoptosis are important factors in many cancers, as normal inhibitory controls on apoptosis are defective in cancer cells.
Dysregulated Apoptosis and Cancer
Resistance to apoptosis is one of the hallmarks of cancer.
Mechanisms by which evasion of apoptosis occurs:
Disrupted balance of pro-apoptotic and anti-apoptotic proteins.
Reduced caspase function.
Impaired death receptor signaling.
Tumour Cells are Frequently Resistant to Fas-Induced Apoptosis
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 which may potentiate any/all of these effects.
Dysregulated Apoptosis and Haematopoietic Cancers
A disrupted balance of pro-apoptotic and anti-apoptotic proteins plays a role in many cancers of the blood and lymphoid tissues (leukemias and lymphomas).
In chronic lymphocytic leukemia (CLL), malignant cells have an anti-apoptotic phenotype with high levels of anti-apoptotic Bcl-2 and low levels of pro-apoptotic proteins such as Bax, so leukemogenesis is due to reduced apoptosis rather than increased proliferation.
The BCL2 (B cell lymphoma 2) gene was first identified because of the t(14; 18) chromosome translocation (present in >90% of follicular B cell lymphomas), resulting in overexpression of Bcl2 and inhibition of apoptosis.
Mcl1 (myeloid leukemia 1) 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 with high affinity to the BH3-binding groove.
These BH3 mimetic drugs stimulate the intrinsic pathway of apoptosis, particularly in cells that are heavily dependent on a particular anti-apoptotic Bcl2 family member for their survival.
Venetoclax binds to Bcl2 and is used effectively 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 and all BH3 mimetics.
In normal plasma cells and in many cases of multiple myeloma (MM), the predominant survival protein expressed is MCL1. MCL1 inhibition could prove effective for this plasma cell malignancy.
Some subtypes of multiple myeloma are also highly susceptible to BCL2 inhibition.
In acute myeloid leukemia (AML), the degree of dependence on BCL2 or on MCL1 varies, with some subtypes being more BCL2 reliant than others.
Overall, both BCL2 and MCL1 appear to play prominent pro-survival roles in most AML cases.
Cancer Therapeutic Approaches Targeting Apoptosis
Examples include DRL, TRAIL, Mapatumumab, Apo2L/TRAIL, TLY012, ABBV-621, GEN1029, conatumumab, ONC201, and MM-201.
Growth factor receptors and dopamine receptors (DRD2 & DRD3) are also targets.
BH3 mimetics such as Venetoclax, S55746, and APG-2575.
Other approaches target MDM2, p53, IAP, and ER stress.