Apoptosis Flashcards
Describe the roles of apoptosis
Harmful cells (e.g. cells with viral infection, DNA damage).
- Developmentally defective cells (e.g. B lymphocytes expressing antibodies against self antigens).- lack of self tolerance
- Excess / unnecessary cells:- important in morphogenesis
(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).- involution- returns to its dormant state
- Exploitation - Chemotherapeutic killing of cells.
What technique can be used to detect dying, apoptotic cells
The TUNEL technique
Define necrosis
Necrosis - unregulated cell death associated with trauma, cellular disruption and an inflammatory response
Define apoptosis
Apoptosis (programmed cell death) - regulated cell death; controlled disassembly of cellular contents without disruption; no inflammatory response
What is important to remember about the different types of cell death
There is a spectrum- and some types have characteristics of both necrosis and apoptosis.
Outline the process of necrosis
Plasma membrane becomes permeable
Cell swelling and rupture of cellular membranes
Release of proteases leading to autodigestion and dissolution of the cell- everything becomes dysregulates as the cell contents are no longer contained within the plasma membrane.
Localised inflammation
Basically the cell explodes.
Describe what happens upon trauma to the cell in necrosis
Trauma to the intestinal epithelium:
Cells and organelles swell
Chromatin condenses
Membrane compromised- fluid rushes in
Dissolution of cellular structures (proteases) Cell lysis (fluid rushing in) Invasion from phagocytic cells and inflammation Proliferation of neighbouring cells to restore the integrity of the epithelium.
Describe the latent phase of apoptosis
Latent phase – death pathways are activated, but cells
appear morphologically the same
Changes are at a molecular level- the cell’s programming is getting ready to apoptose.
Describe the excitation phase of apoptosis
Loss of microvilli and intercellular junctions (so impermeability of the cell is compromised)
Cell shrinkage (unlike the swelling of necrosis).
Loss of plasma membrane asymmetry
(phosphatidylserine lipid appears in outer leaflet)
Chromatin and nuclear condensation
DNA fragmentation
Formation of membrane blebs
Fragmentation into membrane-enclosed apoptotic
bodies
What is the charge of phosphatidylserine
Negative
What is a key feature of apoptosis that distinguishes it from necrosis
Plasma membrane remains intact – no inflammation
Outline the process of apoptosis
Faulty mitosis/excessive DNA damage- usually localised to one cell:
Latent phase
Microvilli contract
intercellular junctions break (impermeability compromised)
Chromatin begins to condense
Cell shrinks- neighbouring cells close around to restore impermeability
Chromatin condenses around nuclear periphery
cell blebs violently
chromatin condensation continunes
cell fragments into membrane-enclosed apoptotic bodies
apoptotic cell bodies phagoyctosed by neighbouring cells and roving macrophages
Describe the DNA modifications that occur during apoptosis
DNA ladders – fragmentation
(agarose gel)- DNA initially intact- too big to fit into channels- but as it condenses- we see fragmentations on the DNA ladder
“TUNEL” assay
DNA fragmentation leads to more “ends” which are labelled by adding an extra fluorescently-tagged base
Describe apoptosis like-PCD
Apoptosis-like PCD - some, but not all, features of apoptosis. Display of phagocytic recognition molecules before plasma membrane lysis
Describe necrosis like-PCD
Necrosis-like PCD - Variable features of apoptosis before cell lysis; “Aborted apoptosis”
What does the fact that there are other types of cell death (other than necrosis and apoptosis) suggest
o The fact that these other forms exist suggests a GRADED response of cell death.
Summarise the mechanisms of apoptotic cell death
o Caspase cascade – the executioners.
o Death response – death receptors and mitochondria- sequential activation of proteins to drive intracellular response
o Bcl-2 family -regulators
o Stopping the death programme.
What are caspases
Cysteine-dependent aspartate-directed proteases (so their cleavage site is between cysteine and aspartame)
Executioners of apoptosis
Activated by proteolysis (secreted as dimers or auto-folded- need to be cleaved by another protease to activate their
Cascade of activation- need both an initiator and effector capsase
State the initiator caspases
2,9,10 and 8
State the effector caspases
3,6 and 7
Describe the structure of initiator caspases
§ 2 subunits – p20 and P10 (P20 is larger)
§ They have an extra targeting subunit:
· CARD – Caspase Recruitment Domain ( 2 and 9)
· DED – Death Effector Domain ( 10 and 8)
o These direct them to a location.
Describe the structure of effector caspases
P20 and P10
What are the key differences between initiator caspases and effector caspases
Initiator caspases: contain N-terminal CARD (CAspase Recruitment Domain) or DED (Death Effector Domain) for homotypic protein-protein interactions (i.e caspase 8 can only interact with caspase 8)
Effector caspases do not contain protein-protein interaction domains.
o Initiator caspases – 2, 9, 8, 10 – TRIGGER APOPTOSIS.
o Effector caspases – 3, 6, 7 – CARRY OUT APOPTOSIS PROCESS.
What structures do caspases recognise
o A cysteine residue in the active site is required for their activity.
o They cut proteins after their aspartate residue.
Describe the synthesis and maturation of caspases
Synthesises as procaspases (zymogens)- inactive
These contain pro-domains at their N-terminal (DED or CARD if initiator) which undergo proteolytic cleavage (either by themselves or another caspase)
They then dimerise with another caspases (e.g caspase 3 and 8) to form a hetero-tetramer
Cleavage of the small chain
Cleavage of the inactive procaspase precursor is followed by folding of 2 large and 2 small chains to form an active L2S2 heterotetramer (this synchronises the two caspases for subsequent steps)
Outline the caspase cascade
Initiator caspases (8 and 9) – trigger apoptosis by cleaving and activating..
Activate caspase 3 and 7:
Caspase 3 can then activate 6,2, and 1
6 can activate 10- which has a feedback loop on 8
…Effector caspases – carry out the apoptotic programme
Describe the importance of the caspase cascade
amplification
divergent responses (different initiator caspases can activate different effector caspases)
regulation
Describe the role of effector caspases in inactivating proteins
Cleave and inactivate proteins or complexes (e.g. nuclear lamins leading to nuclear breakdown)
Can cleave and inactivate monomeric substrates or multi protein complexes (nuclear lamins)
These lead to loss of function of the substrates
Describe the roles of effector caspases in activating proteins
Activate enzymes
(incl. protein kinases; nucleases, e.g. Caspase-Activated DNase, CAD) by direct cleavage, or
cleavage of inhibitory
molecules
Again can be monomeric substrates or multi protein complexes
This leads to a gain of function of the substrates
Outline the two mechanisms for caspase activation
Death by design – Receptor-mediated (extrinsic) pathways- senses something extracellularly
Death by default – Mitochondrial (intrinsic) death pathway
What are death receptors
Secreted or transmembrane ligands (trimeric)- so need 3 to join for activation Include: TNFR1 Fas DR3 DR4 DR5 DR6 NGFR
Adapter proteins can bind to the cytoplasmic tails of death receptors- to bring them to the procaspases
Describe the structure of death receptors
All cells have death receptors which consist of:
o Extracellular cysteine domains (N-terminal -cysteine rich)
o Transmembrane domain.
o Cytoplasmic tail – death domain (C-terminal)- important in transmitting the signal into the cell
Describe the adapter proteins involved in receptor-mediated apoptosis
§ FADD – positive regulator – promotes cell death.
o FADD = DED + DD.
§ FLIP – negative regulator – inhibits the death pathway and allows regulation.
o FLIP = DED + DED.
Describe DD and DED domains
DD and DED domains bind to similar domains on other proteins- i.e DED on FADD will bind to DED on procaspases
Describe signalling through the extrinsic pathway
- Fas receptor is upregulated when the cell needs to apoptose (i.e on infected cells)
- Fas ligand on cytotoxic T-cells binds to the Fas receptor and Fas receptor trimerises.
- Trimerised DD domains recruit adaptor proteins such as FADD (via the DD domains on FADD)
- FADD binding causes recruitment and oligomerisation (links monomers to form dimers/trimers/etc.) of procaspase 8 through DED à FADD DED.
- Procaspase 8 + FADD à DISC (Death-inducing-signalling-complex).
- DISC cross-activates other procaspase 8 molecules.
- Active caspase 8 is released to cleave effector caspases.
Describe the importance of oligomerisation of procaspase 8 in the extrinsic pathway
Some initiator procaspases have intrinsic low catalytic activity – oligomerisation allows transcleavage
others are activated by conformational change on oligomerisation
Need at least 2 procaspases to form an active tetramer
DD cleaved- 2 of these join with internal domains to form active tetramer
Describe FLIP
Death Receptor activation of caspase 8 is inhibited by FLIP
c-FLIPs -only DED domains
c-FLIPl- DED domains and P20, P10
FLIP - caspase homology in DED domain, but no proteolytic activity therefore competes with procaspase -so binds to the DED domain of FADD- but prevents oligomerisation
Describe how FLIP inhibits procaspase 8 inactivation
Competes for binding to receptor tails / FADD via DED domains
Incorporates into receptor-procaspase complexes and interferes with transcleavage
Hence no formation of active tetramer- no activation of effector caspases.
What are the consequences of caspase 8 activation
Active caspase 8 can then go on to activate the other effector caspases that then carry out the apoptotic process.
Acticates caspase 3 and 7
Outline the mitochondrial regulation of apoptosis (intrinsic pathway)
Cellular stresses e.g. lack of or overstimulation by growth factors, DNA damage (p53), ROS
This leads to a loss of mitochondrial membrane potential
So you get release of cytochrome c and other intracellular mitochondrial factors- including apoptosis-promoting factors (Apaf1, caspase 9)
Formation of the apoptosome complex (Apaf1, caspase 9)
What does the apoptosome consist of
§ The Apoptosome – “The Wheel of Death” – consists of:
o APAF-1 – Apoptotic Factor 1.
o Cytochrome C.
o ATP.
o Procaspase 9.
Describe the formation of the apoptosome
§ APAF1 is composed of CARD, ATPase and WD-40 repeats.
§ When cytochrome C binds to WD-40 repeats on APAF-1, it forms a heptamer (the apoptosome) which requires ATP.
§ The CARD domain then binds to CARD on procaspase 9 (so seven procaspases can bind to one apoptosome).
§ Proximity of procaspases then allows cross-cleaving.
§ Activated caspase 9 is then released to trigger apoptosis.
Describe the activation of Caspase 9 at the apoptosome
Caspase-9: attach to any of the Apaf-1 CARD domains at the centre of the ring in a dimer with caspase-3
- oligomerisation brings multiple procaspase 9s close together- can dimerise or trimerise
- cleavage, activation and release as active caspase 9 tetramer initiates a caspase cascade leading to apoptosis
activated caspase 9s can then go on to activate effector caspases, such as caspase 3
Describe how apoptosis requires energy
The apoptosome requires ATP
Energy levels in the cell may determine whether
death is by necrosis or apoptosis- not enough ATP will mean that the cell dies by necrosis.
Which pro-apoptotic protein links the intrinsic pathway to the extrinsic pathway
Bid
Caspase 8 cleaves Bid which enhances release of mitochondrial proteins (CytC), thus engaging the intrinsic pathway
§ Major difference – intrinsic mitochondrial pathway requires energy (ATP).
o Apoptosis always uses intrinsic to some degree so will always use ATP.
§ Remember that necrosis uses MUCH LESS ATP
Which family of proteins act as regulators of the intrinsic pathway
The Bcl-2 family
The large family of Bcl-2 family proteins can be pro-apoptotic or anti-apoptotic. Their only common feature is the presence of so-called BH3 protein-protein interaction domains that mediate binding between different members of the family.
Describe the Bcl-2 family of proteins
Group (1)- Bcl-2 -consist of a transmembrane domain, and four Bcl-2 homologous domains (BH1-4)
Groups 2 (Bax)- transmembrane domains and BH1-3
Group (3):
Bid- only BH3, not bound to mitochondrial membrane
Bik,Bad- TM domain and BH3
BH3 is the dimerisation motif
State the anti-apoptotic Bcl-2 proteins
Anti-apoptotic
Bcl-2 Bcl-xL
(Mitochondrial)
State the pro-apoptotic Bcl-2 family of proteins
Pro-apoptotic
Bid Bad Bax Bak
(move between Cytosol and Mitochondria)
What two signalling pathways can GFs activate to stimulate growth and anti-apoptotic effects
§ GFs (EGF, insulin) may activate TWO growth factor pathways associated with anti-apoptotic effects.
§ Ligand binds à dimerisation à cross-phosphorylation à signal transduction and docking of adapter proteins (e.g. Grb2) to adapt pathway direction (e.g. activating Ras à MAPK/ERK cascade)- leading to growth
§ Another phosphorylation site on tyrosine kinase receptors triggers the PI3-kinase pathway à cell survival and anti-apoptotic effects.
Outline the PI3-kinase pathway
PI3-K– PDK-1 — PKB/Akt — Survival, proliferation
What is PI3-K
Phosphatidylinositol 3’-kinase (PI3’-K) – a lipid kinase (not a protein kinase) involved in growth control and cell survival
Activates protein kinase PKB/Akt which is anti-apoptotic
Consists of 3 subunits:
o Targeting subunit.
o Adapter subunit.
o Catalytic subunit.
Describe the PI3-K signalling pathway
Adapter subunit (P85) of PI3-K binds to phosphorylating tyrosine residue Targeting subunit (P110) of PI3-K recognises PIP2 and phosphorylates it (requiring ATP) to PIP3 PIP3 then sits on PH domain of mitochondrial membrane- where it recruits PKB/Akt- which is anti-apoptotic.
What are the roles of PKB/Akt
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
Induce cell survival by blocking apoptosis.
Describe how the PKB/Akt (PI3’-K, GF pathways) lead to cell-survival
Activation of PKB/Akt will lead to the phosphorylation of Bad- allowing it to be held in an inactive heterodimer with protein 14-3-3.
As Bad is not free it means that the inactive mitochondrial membrane heterodimers of Bcl-2-Bax and Bcl-xL- Bak remain inactive (joined via their BH3 domains)
So as the heterodimers are inactive and phsphobad is inactive we get cell survival.
Describe how the absence of growth factor will lead to apoptosis
Bad dephosphorylated and released
displaces Bcl-2/-xL from -> Bax/Bak (pro-apoptotic)
§ Bcl-2 family proteins (Bax, Bak) then form a pore in the mitochondrial membrane to allow cytochrome C to escape to induce apoptosis.
What are the end results of the PKB/Akt pathways
Cell survival
Protein synthesis
What is the role of PTEN
PTEN (lipid phosphatase) counteracts PI3’-K signalling
Removes a phosphate from PIP3 to form PIP2
· Counteracts the activation of PKB
· Reduces cell survival and promotes apoptosis
What is the role of the inhibitors of apoptosis proteins (IAPs)
Extrinsic pathway
Bind to procaspases and prevent activation
Bind to active caspases and inhibit their activity
Outline the apoptotic/cyto-protective pathways
Bcl-2, Bcl-xL: intrinsic pathway
FLIP, IAPs: extrinsic pathway
growth factor pathways via PI3’-K and PKB/Akt
Summarise the oncogenes/TSGs associated with apoptosis
§ Bcl-2 Oncogene – as overexpression of Bcl-2 results in cancer.
§ PKB/Akt Oncogene – as overexpression of PKB leads to increased cell survival and thus cancer.
§ PTEN TSG – as inactivation raises the PI3-K pathway and thus allows increased survival of cancer.
Summarise the Therapeutic uses of apoptosis
Harmful (oncogenic) cells (e.g. cells with viral infection, DNA damage)
Chemotherapeutic killing of tumour cells, e.g. Dexamethasone stimulates DNA cleavage- therefore stimulating apoptosis.
