Lecture 15 - Apoptosis Flashcards
Describe the caspase cascade
Initiator caspases dimerise
Dimerised capsases are activated
Initiator caspases cleave executioner caspases
Executioner caspases cleave cellular substrates
Cell undergoes apoptosis, fragmentation and apoptotic bodies are cleared by phagocytosis
What are caspases?
Three classes of caspases:
Activators/Initiators
Executioners/effectors
Inflammatory (unrelated to apoptosis)
Caspases are activated by dimerisation (initiators) OR proteolysis (effectors): one caspase can activate another; an excellent example of positive feedback.
Once executioner caspases are activated, apoptosis is virtually guaranteed. The only way to stop it is to digest the caspases, and this happens at the end of apoptosis.
Describe DNA fragmentation during apoptosis
In healthy cells the endonuclease CAD is bound to its inhibitor iCAD
Apoptosis induces iCAD cleavage
CAD cleaves DNA between nucleosomes
CAD - caspase activated DNAase, normally inactive and bound to iCAD
iCAD is cleaved by executioner caspase, leaving active CAD, which can then go on to cleave the
DNA between the nucleosomes.
Describe how caspases amplify the apoptotic signal
Caspases cleave nuclear (e.g. lamins) and cytoskeletal (e.g. actin) proteins.
Caspases cleave and activate gelsolin, inducing actin severing.
Result: nuclear fragmentation, disruption of the cytoskeleton, membrane blebbing and cell fragmentation.
What are the two apoptosis pathways?
Extrinsic apoptosis: cell receives explicit signal from the extracellular environment to initiate apoptosis (e.g., death ligand expressed on another cell)
Intrinsic apoptosis: this normally involves the release of cytochrome-c (induced by stress, damage or developmental cues)
Describe the role of death receptors
Death receptors are cell surface receptors that initiate apoptosis following ligand binding
Receptors (part of the TNF receptor family) include:
Fas (CD95/APO-1), TNF-R1 (tumour necrosis factor receptor 1)
Ligands:
FasL (CD95L), TNF-a, TRAIL (TNF-related apoptosis-inducing ligand)
Extrinsic apoptosis: Death ligand interacts with death receptor causing the cell with the death receptor to undergo apoptosis. Death receptor has death domain (DD) that allows recruitment of adaptor proteins
Adaptor proteins have DD and DED domains - DED allows recruitment of initiator procaspase (DED binds to DED) allowing activation of procaspase, in turn activating executioner caspases which then cause apoptosis
When receptor activated it undergoes conformational change, allowing the receptor death domain to interact with the appropriate adaptor protein, which also has a death domain
FasL binds to the Fas death receptor
Fas interacts with an adaptor protein FADD (Fas-associated death domain)
FADD complexes with procaspase-8 – a DED-caspase:
DED (death effector domain) domains interact with each other
Forms Death Inducing Signal Complex = DISC
Trimeric Fas ligand on the surface of killer lymphocyte interacting with trimeric Fas receptors on the surface of target cells. This leads to clustering of several ligand-bound receptor trimers. This clustering activates death domains on receptors, which interact with similar domains on adaptor proteins. Each FADD adaptor protein then recruits an initiator caspase via a death effector domain present on both FADD and the caspase, which forms a DISC. Within the DISC two adjacent initiator caspases interact and cleave one another to form an activated protease dimer. This then cleaves itself in the region linking the protease to the effector domain. This stabilises and releases the active caspase dimer into the cytosol, where it activates executioner caspases, which act on multiple substrates in the cell, forming an apoptotic target cell.
Describe the role of Fas in influenza defence
Virus peptide presented by MHC class I, interacts with TCR. Cytotoxic T cell has Fas ligand, which binds to Fas receptor on target cell causing apoptosis, preventing more copies of the virus infected cell being made and limiting spread of the virus
Describe how HIV infection results in loss of CD4+-T-cells
All CD4+-T-cells express Fas, but HIV-infected cells also express high levels of FasL
Induces non-infected cells to commit suicide.
Results in failure of immune response.
The infected cells survive as high FasL levels prevent
interaction with their own Fas.
Describe intrinsic apoptosis
Still caspase mediated process
In this case caspases have CARD domain rather than DED domain
There is an intracellular apoptotic stimulus, causing the release of cytochrome c from the mitochondria. This interacts with a protein called Apaf1. Binding the cytochrome c causes the Apaf1 to unfold slightly, exposing a domain that interacts with the same domain in another activated Apaf1 - these aggregate and form apoptosome. Each Apaf1 protein has a caspase recruitment domain (CARD) which associate with each other, clustering at the centre of the apoptosome. Caspases are recruited into the apoptosome and activated, in turn cleaving and activating executioner caspases
APAF = Apoptotic protease activating factor-1
Caspase recruitment domains (CARDs) associate with each other
Describe how Bcl-2 Proteins Regulate the Intrinsic Pathway of Apoptosis
The Bcl-2 family of proteins contains both pro- and anti-apoptosis members (activators, suppressors, derepressors)
Healthy cells maintain a balance of pro- and anti-apoptosis proteins
BH3-only proteins are thought to promote apoptosis by inhibiting anti-apoptosis Bcl-2 family proteins
In the absence of an apoptotic stimulus, the anti-apoptotic Bcl2 proteins bind to and inhibit the Bcl2 family proteins on the mitochondria outer membrane and prevent cytochrome c from being released
If there is an apoptotic stimulus, BH3-only proteins are activated and bind to the anti-apoptotic Bcl2 proteins, inhibiting them. This means Bcl2 can no longer inhibit the effector Bcl2 on the mitochondrial membrane, so cytochrome c is released
