Apoptosis Flashcards
what does apoptosis deal with?
o Harmful cells – e.g. DNA damaged.
o Developmentally defective cells – e.g. self-antigen B-cells.
o Excess cells – e.g. sculpting of hands during embryonic development (get rid of webbing).
o Obsolete cells – e.g. mammary epithelium at the end of lactation.
o Exploitation – e.g. chemotherapeutic killing of cells.
what is necrosis?
- unregulated cell death
- associated with trauma
- cellular disruption
- an inflammatory response.
what is apoptosis?
- regulated cell death
- controlled disassembly of cellular contents
- without an inflammatory response
what are the cellular changes in necrosis?
- Plasma membrane becomes permeable.
- Cell swelling and rupture.
- Release of proteases leading to auto-digestion.
- Localised inflammation.
what are the two phases of apoptosis?
- Latent phase= death pathways are activated but cell stays morphologically the same.
- Execution phase= morphological changes occur
what are the morphological changes that occur in the execution phase?
- Loss of microvilli and inter-cellular junctions.
- Cell shrinkage.
- Loss of plasma membrane asymmetry.
- Chromatin and nuclear condensation.
- DNA fragmentation.
- Membrane bleb formations.
- Fragmentation into membrane-enclosed apoptotic bodies (therefore no inflammation as material is not released)
what can be used to see the DNA modification that occurs in apoptosis?
TUNEL assay shows how DNA modification leads to fragmentation of DNA ladders (in agarose gel)
and the formation of more “ends” labelled with an extra fluorescently-tagged base showing that apoptosis is happening
what are the other 2 methods of cell death?
- Apoptosis-like PCD: some, but not all, features of apoptosis.
Display of phagocytic recognition molecules before plasma membrane lyses. - Necrosis-like PCD: variable features of apoptosis before cell lyses; “Aborted apoptosis”.
what does it suggest that there are different versions of cell death?
cell death is a graded response
what are the components of the mechanism of cell death?
o Caspase cascade – the executioners.
o Death response initiation – death receptors and mitochondria.
o Bcl-2 family – regulators
what do Caspases stand for?
Cysteine-dependent ASPartate-directed proteASES
why is Caspase cysteine-dependent and asparate directed?
what activates the caspase?
A cysteine residue in the active site is required for their activity.
They cut proteins after their aspartate residue.
Activated by a proteolysis cascade (cleavage)
what are the two classes of caspases?
- initiators (trigger apoptosis)
- effectors (carry out apoptosis)
what are the initiator caspases?
2, 9, 8, 10
what are the characteristic subunits of initiator caspases?
p20 and p10
what are the components of INITIATOR Caspases?
which caspases carry the particular subunit?
• N-terminal CARD – Caspase Recruitment Domain.
e.g. 9 involved in the apoptosome, 2
• DED – Death Effector Domain.
e.g 8 involved in receptor mediated (extrinsic) , 10
these produced homotypic protein-protein interactions
these direct them to a location hence “targeting”
what are the effector caspases?
3, 6, 7
- these don’t contain the protein-protein interactions that the initiator caspases have
- these carry out apoptosis
what do caspases mature from?
procaspases (zymogen) which are single-chain polypeptides
they are either activated by themselves or by other caspases
They are activated into a light subunit and a heavy subunit which tetramerise into L2S2 active tetramer.
how does a procaspase become activated into an active enzyme?
needs to be proteolytically cleaved to form large and small subunits (LS and SS)
how is an initiator caspase cleaved?
the targeting subunits (DED, CARD) are cleaved aswell as the large and small subunit
how is an active caspase formed after cleavage?
2 large (heavy subunit) and 2 small chains (light subunit) form an active L2S2 heterotetramer
what does caspase maturation lead to?
caspase cascade
what is the main function of the caspase cascade?
amplification, divergent responses and regulation allowing the effector caspases to carry out their apoptotic function
in which 2 methods do effector caspases carry out apoptotic function?
o Cleave and inactivate proteins/complexes
– e.g. nuclear lamins are targeted leading to nuclear breakdown.
o Activating enzymes by direct cleavage or cleavage of inhibitors
– e.g. nucleases (CAD), protein kinases,
what are the 2 mechanisms of caspase activation?
- Receptor-mediated (extrinsic) pathways–> Fas receptor
2. Mitochondrial (intrinsic) pathways–> cytochrome C release
in receptor mediated caspase activation, what must the cells have on their outer membrane?
death receptors made of:
- Extracellular cysteine domains.
- Transmembrane domain.
- Cytoplasmic tail= death domain.
what interacts with the DD on death receptors on cells?
adaptor proteins so they can recruit signalling proteins e.g. FADD
what are the adaptor proteins in the extrinsic pathway?
FADD
FLIPP
what is FADD?
positive regulator – promotes cell death
the adaptor protein involved in receptor mediated (extrinsic) apoptosis
what are the components of FADD?
DED + DD
death effector domain and death domain
what is FLIPP?
negative regulator – inhibits the death pathway and allows regulation.
what are the components of FLIPP?
DED+DED
both are death effector domains
has no efficacy
what is upregulated in the extrinsic pathway when a cell needs to apoptosis?
Fas receptor (will be the death receptor)
what will bind to the death receptor Fas and what does this lead to?
Fas ligand on cytotoxic T-cells binds to the Fas receptor and Fas receptor trimerises.
what is the effect of the Fas receptor trimerising?
the DD domains of the receptor trimerise and recruit adapter proteins, in apoptosis’ case, FADD
what is the effect of FADD binding to the Fas receptor’s DD domain?
causes recruitment and oligomerisation of procaspase 8 through DED
what is produced when the FADD binds to procaspase 8?
DISC (Death-inducing-signalling-complex)
what does DISC do?
allows cross-activation of other procaspase 8 molecules due to all their close proximities
what will the active caspase 8 do?
- the initiator caspase will now cleave effector caspases
- it will activate effector procaspase 3
summary of extrinsic pathway of apoptosis
- Fas receptor is upregulated when the cell needs to apoptose.
- Fas ligand on cytotoxic T-cells binds to the Fas receptor and Fas receptor trimerises.
- Trimerised DD domains recruit adaptor proteins such as FADD.
- FADD binding causes recruitment and oligomerisation (links monomers to form dimers/trimers/etc.) of procaspase 8 through DED –> FADD DED.
- Procaspase 8 + FADD –> DISC (Death-inducing-signalling-complex).
- DISC cross-activates other procaspase 8 molecules.
- Active caspase 8 is released to cleave effector caspases.
how does procaspase 8 bind to the trimeric receptor with FADD?
the DED region of the procaspase binds to the DED region of the FADD (made of DED and DD)
what is the effect of the DED regions binding in procaspase 8 oligomerisation?
brings procaspases into close contact to allow cleavage
eventually cleavage and activation occurs
what is the final product of procaspase 8 oligomerisation?
Active initiator caspase 8 tetramers (released from the receptor)
what effect will FLIP have on the trimeric receptor?
binds to the trimer but has no proteolytic activity, so can’t cleave other procaspases
it can bind to DED regions of FADD
what is the outline of the intrinsic pathway of caspase activation?
- Cellular stresses – e.g. lack of growth factor, DNA damage detected by p53
- Loss of mitochondrial membrane potential.
- Release of cytochrome C (and other apoptosis-inducing factors).
- Stimulation of formation of “apoptosome complex”.
what is the apoptosome and what does it consist of?
o APAF-1 – Apoptotic Factor 1.
o Cytochrome C (bound to Apaf-1)
o ATP
o Procaspase 9s bound to Apaf-1 via the CARD domain
what is APAF-1 made of?
CARD, ATPase and WD-40 repeats.
when is the apoptosome heptamer formed?
when Cytochrome C binds to the WD-40 repeats on APAF-1
this heptamer formation process requires ATP
Where does the procaspase 9 bind in the apoptosome?
the procaspase 9’s CARD domain binds to the CARD domain of APAF-1
[initiator caspases 9 (and 2) made of CARD+p20+10]
as the apoptosome is a heptamer, seven procaspase 9s can bind to it
what is the effect of the proximity of the procaspase 9s on the apoptosome?
Proximity of procaspases then allows cross-cleaving and the activated caspase 9 is then released to trigger apoptosis.
how are the intrinsic and extrinsic pathways of caspase activation linked?
by Bid
how is Bid produced?
Caspase 8 from the extrinsic pathway can cleave Bid which can enhance the activation of the intrinsic pathway by going into the mitochondria to release cytochrome C
what is the major difference between the extrinsic and intrinsic pathway?
the intrinsic pathway requires ATP (in the apoptosome)
- Apoptosis always uses intrinsic to some degree so will always use ATP.
which process , apoptosis or necrosis, required less ATP? what does it mean for cells with high ATP content?
necrosis requires much less ATP
cells with high levels of ATP will die by apoptosis whilst those with low ATP will die by necrosis
what are the intrinsic modulators of apoptosis?
Bcl-2 family of proteins
what are the components of the Bcl-2?
they can contain BH1, BH2, BH3, BH4 and TM
all of them have BH3 in common
what is the purpose of the BH3 domain in all the Bcl-2 proteins ?
required for dimerisation between different members of the Bcl-2 family
what are the 2 main categories of Bcl-2 proteins? what are their locations?
o Anti-apoptotic: localised in the mitochondria membrane
- Bcl-2, Bcl-xL
o Pro-apoptotic: move between cytosol and mitochondrial membrane
- Bax and Bak
what are the anti-apoptotic modulator proteins?
Bcl-2
Bcl-xL
both have BH1, BH2, BH3 and BH4
what are the pro-apoptotic modulator proteins?
Bid (BH3 only) Bad (BH3 only) --> inhibited by PKB - these bad boys stop the anti-apoptotic proteins binding to the pro-apoptotic proteins below: Bax (BH1,2,3) Bak (BH1,2,3)
what are the two pathways that can be triggered by Growth Factors?
- activate Ras for the ERK cascade (Ras-Raf-MEK-ERK) for growth effects
- activate the PI3-K’ pathway for cell survival and anti-apoptic effects
the different pathways proceed when different adaptor proteins bind to the phosphorylated domains of the GF receptors
what does PI3-K’ stand for?
Phosphatidylinositol 3-kinase = a lipid (not protein) kinase
what are the main subunits of PI3-K’?
o Targeting subunit.
o Adapter subunit.
o Catalytic subunit.
what does PI3-K’ do?
PI3-K phosphorylates PIP2–>PIP3
PIP3 can bind to PKB to activate it
what does phosphorylated PIP3 bind to?
PKB (or Akt) - protein kinase B adapter subunit.
PI3-K is the growth factor control over PKB
This is antagonised by PTEN (often deleted in tumour cells)
what sort of effects does PKB have?
anti-apoptotic effects and therefore induced mitogenic signal by:
o Phosphorylating and inactivating Bad (a pro-apoptotic)
o Phosphorylating and inactivating caspase 9 (apoptosis effector).
o Inactivating FOXO transcription factors (promote expression of apoptosis-promoting genes)
o Other – stimulates ribosome production.
what are the components of PI3-K’?
p85 + p110
what does GF binding promote in terms of cells existence?
cell survival
GF stimulates PKB action (anti-apoptotic) via PI3-K
GF also stimulates the normal Ras-Raf-MEK-ERK pathway for proliferation
(absence of GF–> apoptosis)
how does PKB ensure the inactivation of Bad?
phosphorylates Bad so inactivates it
what happens to the other Bcl proteins when Bad and Bid have been held inactive when inducing cell survival ?
pro-apoptotic proteins Bax and Bak are bound to anti-apoptotic Bcl-2 and Bcl-xl via their BH3 domains [dimerisation domain] on the mitochondrial membrane
what happens when GF is not present?
- no PI3-K pathway
- PIP3 not generated
- PKB not recruited
- Bad not phosphorylated by PKB
- Bad released from heterodimer with 14-3-3.
what is the effect of Bad being released from 14-3-3?
Bad will bind to Bcl-2 and Bcl-xl who can not longer hold onto Bax and Bak.
Bax and Bak escape to form a pore into the mitochondrial membrane to allow the release of cytochrome C into the cytosol for the formation of the apoptosome–> apoptosis
which Bcl proteins does Bcl-2 bind to?
Bax and Bak
pro-apoptotic
which Bcl protein does Bcl-xl bind to?
Bak and Bax
pro-apoptotic
how can PI3’K and PKB’s cell survival signal be counteracted?
by PTEN (lipid phosphatase)
converted PIP3 back to PIP2 (dephosphorylation)
what happens to Bcl-2 proteins in cell survival mode?
pro-apoptotic proteins are held inactive
what do IAPs (Inhibitor of Apoptosis Proteins) bind to?
o Procaspases and prevent activation.
o Caspases and inhibit activity.
what are the anti-apoptotic proteins involved in the intrinsic pathway?
Bcl-2 and Bcl-xl
what are the anti-apoptotic proteins involved in the extrinsic pathway?
FLIP and IAPs
IAPs bind to procaspases and caspases
what pathway promotes cell survival/anti-apoptotic?
Growth factor pathways via PI3-K using PKB/Akt.
what are the oncogenes involved in cell survival of cancer? how can overexpression lead to cancer?
Bcl-2 and Bcl-xL– as overexpression of Bcl-2 results in cancer.
PKB/Akt – as overexpression of PKB leads to increased cell survival and thus cancer.
these proteins all stop apoptosis i.e allow cancer cells to survive
what are the TSGs involved in cell death and dissolution of cancer? how can deactivation lead to cancer?
PTEN – if PTEN is inactivated–> raises the PI3-K pathway and thus allows increased survival of cancer.
Bid and Bad
Bax and Bad
Caspases
these all promote apoptosis