6. Apoptosis Flashcards
Outline reasons for programmed cell death (apoptosis)
Programmed Cell Death
We have apoptosis to deal with:
- Harmful cells – e.g. viral infection/DNA damaged.
- Developmentally defective cells – e.g. self-antigen B-cells.
- Excess cells – e.g. brain to eliminate excess neurons / sculpting of hands during embryonic development (get rid of webbing).
- Obsolete cells – e.g. mammary epithelium at the end of lactation.
- Exploitation – e.g. chemotherapeutic killing of cells.
Apoptosis
1. Latent phase – death pathways are activated but cell stays morphologically the same.
2. Execution phase:
- Loss of microvilli and inter-cellular junctions.
- Cell shrinkage.
- Loss of plasma membrane asymmetry: important for lipid composition
- Chromatin and nuclear condensation.
- DNA fragmentation.
- Membrane bleb formations.
- Fragmentation into membrane-enclosed apoptotic bodies.
- So, no inflammation.
DNA modification occurs during apoptosis leading to:
- Fragmentation of DNA ladders – seen in agarose gel.
- Formation of more “ends” – labelled with an extra fluorescently-tagged base.
Necrosis
Necrosis – unregulated cell death associated with trauma, cellular disruption and an inflammatory response.
Apoptosis – regulated cell death, controlled disassembly of cellular contents without an inflammatory response
Necrosis
- Plasma membrane becomes permeable.
- Cell swelling and rupture of cellular membranes
- Release of proteases leading to auto-digestion and dissolution of cell
- Localised inflammation.
Attracts phagocytic cells to remove inflammation
Neighbouring cells over- proliferate
Apoptosis: explain the difference between necrosis and apoptosis
Other Types of Cell Death and Mechanisms
- Apoptosis-like PCD (Programmed Cell Death) – 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”.
The fact that these other forms exist suggests a GRADED response of cell death.
Mechanisms of Cell Death:
- Caspase cascade – the executioners.
- Death response – death receptors and mitochondria.
- Bcl-2 family.
- Stopping the death programme.
Regualated
Plasma membrane intact
Requires ATP
No inflammation
No trauma
No release ofproteases
Not regulated
Plasma membrane not intact
No ATP
Inflammation
Trauma
Releas of proteasees - autodigestion
Outline caspases
Caspases
- Caspases = Cysteine-dependent ASPartate-directed proteASES.
- A cysteine residue in the active site is required for their activity.
- They cut proteins after their aspartate residue.
- They are inactive and need to be activated by a proteolysis cascade (activated by being clipped)
Classes of caspases:
- Initiator caspases – 2, 9, 8, 10 – TRIGGER APOPTOSIS.
- 2 subunits – p20 and P10.
- They have an extra targeting subunit:
- 2,9 CARD ( p20 and P10) – Caspase Recruitment Domain.
-
8,10 DED ( p20 and P10) – Death Effector Domain
These direct them to a location.
- Form homotypic protein-protein interaction (caspase 8 and caspase 8)
- Effector caspases – 3, 6, 7 – CARRY OUT APOPTOSIS PROCESS.
- They start of as a single chain polypeptide with TWO subunits (large and small)
- The subunits are released by proteolytic cleavage during maturation
- p20 and P10 domains
Recall the maturation process of cascades
Caspases – Maturation:
Procaspases are single-chain polypeptides.
- To activate, one pair of procaspases undergoes proteolytic cleavage and dimerization to form LARGE and SMALL subunits.
- Initiator caspases also cleave the targeting subunits (DED and CARD).
- After cleavage, there is a folding of the 2 large and 2 small chains to form an active L2S2 HETEROTETRAMER.
Recall how the proteolytic caspase cascades execute the apoptotic response.
- Bid links the extrinsic (receptor) and intrinsic (mitochondrial) pathways together.
- Caspase 8 from the extrinsic pathway can cleave Bid à enhances activation of the intrinsic pathway.
- Major difference – intrinsic mitochondrial pathway requires energy (ATP).
- Apoptosis always uses intrinsic to some degree so will always use ATP.
- Remember that necrosis uses MUCH LESS ATP.
Summarise how apoptosis may be mediated through death receptors
Caspase Activation – Extrinsic Pathway:
- All cells have death receptors which consist of:
- Extracellular cysteine domains.
- Transmembrane domain.
- Cytoplasmic tail – death domain.
- These receptors are only activated when they encounter secreted or transmembrane trimeric ligands (e.g. TNF-‐alpha or Fas) -‐ these are called death ligands
- TWO Adaptor proteins can interact with these receptors:
- FADD.
- FLIP.
FADD – positive regulator – promotes cell death.
- FADD = DED + DD.
FLIP – negative regulator – inhibits the death pathway and allows regulation.
- FLIP = DED + DED.
Extrinsic Pathway:
- 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 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
Procaspase 8 oligomerisation to cleavage and activation.
- Initiator procaspases bind to FADD (DED -> DED).
- DED regions bind to DED regions.
- This brings procaspases into close contact to allow cleavage.
- Active initiator caspase 8 tetramers release.
Death receptor activation of procaspase 8 is inhibited by FLIP (negative regulator).
FLIP incorporates into the trimer but it has NO PROTEOLYTIC ACTIVITY and so cannot cleave the other procaspases.
- It can still bind to the DED regions on FADD though*
Active caspase 8 can then go on to activate the other effector caspases that then carry out the apoptotic process.
Summarise how apoptosis may be mediated through the mitochondria
Caspase Activation – Intrinsic (Mitochondrial) Pathway:
- Cellular stresses – e.g. lack of growth factor.
- Loss of mitochondrial membrane potential.
- Release of cytochrome C (and other apoptosis-inducing factors).
- Stimulation of formation of “apoptosome complex”.
Caspase Activation – Intrinsic (Mitochondrial) Pathway:
The Apoptosome – “The Wheel of Death” – consists of:
- APAF-1 – Apoptotic Factor 1.
- Cytochrome C.
- ATP.
- Procaspase 9.
APAF1 is composed of CARD, ATPase and WD-40 repeats.
At one end, APAF-‐1 contains a number of repeats that are involved in protein-‐ protein interactions
- 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 in some initiator caspases (e.g. caspase 9)
- When cytochrome C binds to the WD-‐40 repeats on APAF-‐1, it forms a
- heptamer (the apoptosome)
- This process also requires ATP
- 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 can cross-‐cleave and activate eachother to produce caspase 9
- The activated caspase 9 is then released, which is able to trigger the caspase cascade, which leads to apoptosis
- Activated caspase 9 is then released to trigger apoptosis.
Recall the proteolytic caspase cascades
Main purposes of the caspase cascades:
- Amplification
- Divergent responses
- Regulation
Once apoptosis is triggered, the initiator caspases cleave and activate the effector caspases
Effectors and Initiators:
Effector caspases carry out the apoptotic programme in two ways:
- Cleave and inactivate proteins/complexes – e.g. nuclear lamins -> nuclear breakdown.
- Activating enzymes by direct cleavage or cleavage of inhibitors – e.g. nucleases (CAD).
Mechanisms of Caspase Activation
- Death by design -‐ receptor-‐mediated (extrinsic) pathways
- Death by default -‐ mitochondrial (intrinsic) death pathway
Recall how Bcl-2 family proteins may modulate apoptosis.
Bcl-2
- These are the intrinsic modulators of apoptosis.
- There are 3 main groups (all containing BH3 domains).
- Some proteins contain TM domains.
- BH3 is required to dimerise with each other.
There are 2 main categories for the Bcl-2 proteins:
Anti-apoptotic – Bcl-2, Bcl-xL.
- Localised in mitochondrial membrane.
Pro-apoptotic - Bid, Bad, Bax, Bak.
- Move between cytosol and mitochondrial membrane.
Apoptosis by Bcl-2 via BH3 Heterodimerisation
“A” picture:
- Pro-apoptotic proteins (e.g. Bax, Bak) are held INACTIVE in their heterodimers (by the BH3 domain) to Bcl-2 and Bcl-xL on the mitochondrial membrane.
“B” picture:
- GF is absent à no PI3-K pathway à PIP3 not generated à PKB not recruited à Bad NOT phosphorylated and so is released from heterodimer with 14-3-3.
- Bad goes to mitochondrial membrane and binds to Bcl-2 and Bcl-xL displacing the pro-apoptotic Bcl-2 family proteins.
- Bcl-2 family proteins (Bax, Bak) then form a pore in the mitochondrial membrane to allow cytochrome C to escape to induce apoptosis.
PTEN and PI3-K
- PTEN (lipid phosphatase) counteracts PI3-K signalling and production of PKB and so PROMOTES APOPTOSIS.
- IAPs (Inhibitor of Apoptosis Proteins) bind to:
- Procaspases and prevent activation.
- Caspases and inhibit activity.
Anti-Apoptotic Pathways
- Bcl-2, Bcl-xL – via the intrinsic pathway.
- FLIP, IAPs – via the extrinsic pathway.
- Growth factor pathways via PI3-K and PKB/Akt.
Proto-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.
Explain the PI3 kinase signalling pathway
PI3’-Kinase Signalling Pathway
- GFs may activate TWO growth factor pathways associated with anti-apoptotic effects.
- Ligand binds -> dimerisation -> 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).
- Another phosphorylation site on tyrosine kinase receptors triggers the PI3-kinase pathway à cell survival and anti-apoptotic effects.
- PI3-K = Phosphatidylinositol 3-kinase = a lipid (not protein) kinase.
- Three main subunits:
- Targeting subunit.
- Adapter subunit.
- Catalytic subunit.
- PI3-K phosphorylates PIP2àPIP3 which then binds PKB/Akt (protein kinase B) adapter subunit.
- PKB has anti-apoptotic effects by:
- Phosphorylating and inactivating Bad (a Bcl-2).
- Phosphorylating and inactivating caspase 9.
- Inactivating FOXO (promote expression of apoptosis-promoting genes- ubiquitly expressed and always are in the nucleus and get inactivated by nucler exlusion) transcription factors.
- Other – stimulates ribosome production.
