Apoptosis Flashcards
5 reasons for apoptosis
- Harmful cells (e.g. cells with viral infection, DNA damage).
- Developmentally defective cells (e.g. B lymphocytes expressing antibodies against self-antigens).
- Excess/unnecessary cells (embryonic development: brain to eliminate excess neurons; liver regeneration; sculpting of digits and organs).
- Obsolete cells (e.g. mammary epithelium at the end of lactation).
- Exploitation - Chemotherapeutic killing of cells
Definition of necrosis
UNREGULATED CELL DEATH ASSOCIATED WITH TRAUMA, CELLULAR DISRUPTION AND AN INFLAMMATORY RESPONSE
Definition of apoptosis
REGULATED CELL DEATH; CONTROLLED DISASSEMBLY OF CELLULAR CONTENTS WITHOUT DISRUPTION; NO INFLAMMATORY RESPONSE
Execution of apoptosis: 9
- Loss of microvilli and intercellular junctions
- Cell shrinkage
- Loss of plasma membrane asymmetry and the composition changes (phosphatidylserine lipid appears in outer leaflet)
- Chromatin and nuclear condensation
- Epithelium cannot afford a gap in their layer and so close around the cell that’s apoptosing (look at pic)
- DNA fragmentation
- Formation of membrane blebs
- Fragmentation into membrane-enclosed apoptotic bodies
PLASMA MEMBRANE REMAINS INTACT - NO INFLAMMATION - The apoptotic bodies are then phagocytosed by surrounding cells e.g. macrophages
What is APOPTOSIS-LIKE PCD
some, but not all, features of apoptosis e.g. Display of phagocytic recognition molecules before plasma membrane lysis
What is NECROSIS-LIKE PCD-
Variable features of apoptosis before cell lysis; ‘aborted apoptosis’
If you extract DNA from a cell and induce apoptosis, you will see X of the DNA
fragmentation
4 phases of apoptosis?
- Caspases
- Initiating the death programme:
Death receptors (extrinsic)- external stimuli cause cell death
Mitochondria (intrinsic) - The Bcl-2 family (regulators of the whole cell death ting)
- Stopping the death programme
What are Caspases
‘Cysteine-dependent aspartate-directed proteases’
- Executioners of apoptosis
- Activated by proteolysis
- Cascade of activation
Which/what are the initiator caspases
- First ones to be triggered (e.g. caspase 2, 9, 10 and 8)
What special domain do caspase 2 and 9 contain
CARD - CAspase Recruitment Domain
What is the CAspase Recruitment Domain Card domain
this domain localises the caspase at particular sites within the cell and is homotypic
What special domain do caspase 8 and 10 contain
Death Effector Domain (DED)
What is the Death Effector Domain (DED)
this domain mediates homotypic binding protein-protein interactions
What are the effector caspases
Caspases 3, 6 and 7
What are the 2 types of caspase
Initiator (2, 9, 8, 10)
Effector (3, 6, 7)
What do effector caspases contain
p20 and p10
Where do you find the the Death Effector Domain (DED)
caspase 8 and 10
Where do you find the CAspase Recruitment Domain Card domain
caspase 2 and 9
What are caspases originally synthesised as
Procaspases (zymogens)
How many proteolytic cleavages liberate a caspase from a procaspase
2
What type of interactions do DED and CARD domains undergo
Homotypic
What do initiator caspases do
Initiate the caspase cascade and liberate effector caspase
What do effector caspases do (2)
- They cleave and inactivate proteins or complexes (e.g. nuclear lamins leading to nuclear breakdown)
- They also activate enzymes (including protein kinases, nucleases e.g. caspase activated DNase (CAD)) by direct cleavage, or cleavage of inhibitory molecules
2 mechanisms of caspase activation?
Receptor-mediated (extrinsic) pathways
Mitochondrial (intrinsic) pathway
What receptors activate caspases
Death receptors
Where do you find death receptors
Transmembrane with an intracellular cytoplasmic tail
What domain do you find on death receptor tails
Death domain
What do death receptors do
Activate caspase
When activated, what do death receptors attract
Adaptor proteins
Example death receptors?
Fas, DR3,4,5,6 NGFR, TNFR1
What adaptor proteins do death receptors recruit
FADD and FLIP
What domains does FADD contain, what is it, what recruits it, and what does it do
FADD is an adaptor protein recruited by an activated death receptor
contain the DED domain and the Death Domain (DD)-> FAD activates the apoptotic pathway
What domains does FLIP contain, what is it, what recruits it, and what does it do
FLIP is an adaptor protein recruited by an activated death receptor
proteins have 2 DED domains but have inhibitory activity
Describe signalling through the Fas/Fas-ligand
- Receptor (Fas) trimerisation by ligand (Fas-L on lymphocyte)
- Recruitment of adapter protein (FADD) though its DD to the DD of Fas
- Recruitment and oligomerisation of procaspase 8 through its DED to the DED domain of FADD This is the DEATH INDUCING SIGNALLING COMPLEX (DISC)
Describe activation of initiator procaspase 8
- Initiator procaspases bind, via their DED domains, to the DED domains of FADD which is bound via its DD domain to the DR
- This brings 3 initiator procaspase 8s into close contact, which allows cleavage
- This releases the active initiator caspase 8 tetramer
Describe adaptor protein FLIPs actions (what does it compete with, what does it bind to)
- FLIPs contain DED domains on their end terminals which can interact with DED domains on the FADD
- FLIP has no proteolytic activity so it can compete with procaspase 8 to bind the DED domains on FADD
It can incorporate into receptor-procaspase complexes and interfere with cleavage to inhibit formation of caspase 8
Describe mitochondrial regulation of apoptosis (intrinsic pathway)
- Cellular stress causes loss of mitochondrial membrane potential
- This causes release of cytochrome C and other factors as the mitochondria begin to die and their membrane breaks down
- You get release of other apoptosis-inducing factors
- An apoptosome complex is formed, which includes Apaf1 and caspase 9 -> This is the equivalent of DISC- it gathers proteins to cause degradation
What does the apoptosome consist of
APAF-1 (apoptotic activating factor 1)
Cytochrome C
ATP
Procaspase 9
Where is most of an apoptosomes constituents released from
Mitochondria
Describe the structure of apoptosome (repeats, 2 domains)
- At one end, APAF-1 contains a number of WD-40 repeats that are involved in protein-protein interactions and basically is a scaffolding protein that allows other molecules to bind to it
- There is also an ATPase domain within APAF-1
- At the other end of APAF-1 there is a caspase recruitment domain (CARD), which is also found on some initiator caspases (e.g. caspase 9)
what does CytC bind to to form the apoptosome
WD40 repeats on APAF1
What does APAF1 have on it
WD40 repeats
How many CARD domains on an apoptosome
7
What is the relationship between procaspase X, caspase X, and the apoptosome
X = 10
- The CARD domains at the centre of the apoptosome can interact with the CARD domains on procaspase 9 (so seven procaspase 9s can bind to the apoptosome)
- The close proximity of the procaspase 9s that bind to the CARD domains of the apoptosome means they can cross-cleave and activate eachother to produce caspase 9
What is one of the major factors deciding whether a cell undergoes apoptosis or necrosis
ATP, apoptosis needs it
What links the receptor-mediated and mitochondrial death pathways
Bid
- When one pathway is triggered, Bid can trigger the other pathway
How does Bid trigger the intrinsic pathway
Bid promotes the release of cytochrome C from the mitochondrion, which triggers the mitochondrial death pathway
How does Bid trigger the extrinsic pathway
Caspase 8 from the receptor-mediated pathway can cleave Bid, which enhances release of mitochondrial proteins, thus engaging the intrinsic pathway
1 difference between the extrinsic and intrinsic pathway?
Intrinsic requires ATP for CytC to bind to WD40
What family of proteins does Bid belong to
Bcl-2
What characterises the Bcl-2 family
BH-3 homology domains
What is special about group 3 Bcl-2 proteins
They only have BH3 domains and can dimerise
Which Bcl-2 proteins are anti-apoptotic
Bcl-2
Bcl-XL
Which Bcl-2 proteins are pro-apoptotic
Bid
Bax
Bad
Bak
Describe inactivation of Bad
- Phosphatidylinositol 3-kinase (PI3-K) is a lipid kinase involved in growth control and cell survival
- It phosphorylates PIP2 to PIP3, which is then recognised by the adapter unit of PKB/Akt (protein kinase B)
- PKB is then recruited to the cell membrane and it is activated It has anti-apoptotic effects
- PKB phosphorylates and inactivates Bad (of the Bcl-2 family)
Anti-apoptotic effect for PKB? (4)
- Phosphorylates and inactivates Bad
- Phosphorylates and inactivates caspase 9
- Inactivates FOXO transcription factors (FOXOs promote expression of apoptosis-promoting genes)
- Other, e.g. stimulates ribosome production and protein synthesis
Describe activation of bad
GF absent -> PI3K pathway inactive -> PIP3 ungenerated -> PKB not recruited to membrane and activated -> Bad is dephosphorylated and released -> Bad goes to mitochondrial membrane and binds to anti-apoptotic Bcl-2 members which displaces pro-apoptotic Bcl-2 -> released pro-apoptotic Bcl-2 family members form a pore in mitochondrial membrane allowing CytC to escape and induce apoptosis
What is PTEN and what does it do
- PTEN is a lipid phosphatase that counteracts the production of PKB, therefore reducing the regulation of cell survival and promoting apoptosis
What binds to procaspases and prevents their activation?
IAP Inhibitor of apoptosis proteins
What binds to caspases and inhibits their action?
IAPs Inhibitor of apoptosis proteins
IAPs Inhibitor of apoptosis proteins bind to and inhibit
Procaspases and caspases
What counteracts the production of PKB
PTEN
What mechanisms protect the intrinsic pathway
- Bcl-2, Bcl-xL
What mechanisms protect the extrinsic pathway
- FLIP, IAPs
What mechanisms protect the GF pathways
PI3’-K and PKB
Example of proto-oncogene associated with apoptosis
Bcl-2 and PKB/Akt
Example of TSG associated with apoptosis
PTEN - inactivation of this (which increases chance of apoptosis) leads to CANCER
