10. Apoptosis Flashcards
Why do we need programmed cell death?
Harmful cells that may be damaged (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 e.g. brain to eliminate excess neurons; liver regeneration; sculpting of digits and organs
Obsolete organs (e.g. mammary epithelium at the end of lactation must be reduced in size as mother is no longer breast feeding)
Exploitation - chemotherapeutic killing of cells
What is the difference between apoptosis and necrosis?
Necrosis - unregulated cell death associated with trauma, cellular disruption and an INFLAMMATORY RESPONSE
Apoptosis (Programmed Cell Death) - regulated cell death; controlled disassembly of cellular contents without disruption - NO INFLAMMATORY RESPONSE
What happens in necrosis?
The plasma membrane becomes permeable
There is cell swelling and rupture of cellular membranes
Proteases are released leading to autodigestion and dissolution of the cell
Localised inflammation
What happens in apoptosis (what are the two phases)?
Latent Phase - death pathways are activated by a signal, but cells appear morphologically the same
Execution Phase
Loss of microvilli and intercellular junctions
Cell shrinkage
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
How is DNA modified during apoptosis?
Fragmentation of DNA ladders (seen in agarose gel) where the DNA is degraded
Formation of more ‘ends’, which are labelled by adding an extra fluorescently-tagged base in a TUNEL assay
What are caspases?
Caspase - Cysteine-dependent aspartate-directed proteases
They have a cysteine residue in their active site that is required for their activity
They cut proteins just after their aspartate residue
They are activated by proteolysis
They take part in a cascade of activation
What are the two types of caspases?
Effector Caspases (3, 6 and 7) Initiator Caspases (2, 8, 9 and 10)
Outline the structure of effector caspases
Effector Caspases (3, 6 and 7)
They start of as a single chain polypeptide with TWO subunits (large and small)
The subunits are released by proteolytic cleavage during maturation
Outline the structure of initiator caspases
These also have the same two subunits that are found in effector caspases
They also have an extra targeting subunit (protein-protein interacting domain)
The domains/subunits are found at the end terminals of the caspases
The targeting subunit directs them to a particular location
Targeting subunits:
CARD - Caspase Recruitment Domain
DED - Death Effector Domain
Outline the process of caspase maturation
Caspases are synthesised inside the cell as an inactive component (procaspases)
Procaspases (zymogens) are single chain polypeptides
To become activated, the procaspases must undergo proteolytic cleavage to form large and small subunits
The procaspase has protodomains which contain the DED domain
NOTE: initiator caspases must also be cleaved to release the targeting subunit
These cleavages are done by the caspases themselves
After the cleavage, you get folding of 2 large and 2 small chains to form an active L2S2 heterotetramer
This whole process is called caspase maturation
What are the main purposes of the caspase cascades?
Amplification
Divergent responses
Regulation
How do the caspases react once apoptosis is triggered?
Once apoptosis is triggered, the initiator caspases cleave and activate the effector caspases
What are the two ways that caspases carry out the apoptotic programme
Cleaving and inactivating various proteins and complexes (e.g. nuclear lamins leading nuclear breakdown)
Activating enzymes by direct cleavage, or cleavage of inhibitor molecules (e.g. protein kinases, nucleases such as Caspase-activated Dnase (CAD))
What are the two mechanisms of caspase activation
Death by design - receptor-mediated (extrinsic) pathways
Death by default - mitochondrial (intrinsic) death pathway
What are the two mechanisms of caspase activation?
Death by design - receptor-mediated (extrinsic) pathways
Death by default - mitochondrial (intrinsic) death pathway
Where are death receptors found and what are they made of?
All cells have death receptors on their surface Death receptors consist of: Extracellular cysteine-rich domain Single transcellular domain Cytoplasmic tail (with a death domain)
When are death receptors activated?
These receptors are only activated when they encounter secreted or transmembrane trimeric ligands (e.g. TNF-alpha or Fas) - these are called death ligands
What is found extracellularly and intracellularly of the death receptors?
Death domains are found at the c-terminals of the cytoplasmic tails of death receptors so these are found intracellularly
On the extracellular side of the death receptor there are cysteine rich domains which are the ligands that can be recognised by signalling molecules that are released when apoptosis needs to occur
Why are adaptor proteins needed by death receptors?
When the death receptors cluster together as a trimer, they need adaptors to come to their tails and recruit signalling molecules
What are the two regulator proteins that modulate the action of death receptors? What effect do they have on apoptosis?
FADD - POSITIVE regulator (required for the death pathway to become activated) and promotes cell death
FLIP - negative regulator (inhibits the death pathway and allows it to be regulated)
What are the domains that make up the adaptor proteins FADD and FLIP?
FADD = DED + DD FLIP = DED + DED
Where:
DED = Death Effector Domain
DD = Death Domain
What is upregulated when apoptosis is required?
Fas - this is a death receptor and is upregulated if apoptosis is required e.g. if a cell is infected by a virus
How is apoptosis induced by the Fas receptor?
The Fas ligand (produced by immune cells) binds to the Fas receptor on the surface of cytotoxic T lymphocytes
The Fas receptors then undergo trimerisation, which brings the three cytoplasmic DD domains together
The trimerised death domains recruit the positive adapter protein FADD by its own DD
The binding of FADD causes recruitment and oligomerisation of procaspase 8 (which is an initiator caspase) through its DED to the FADD DED
NOTE: oligomerisation = a chemical process that links monomeric compounds (e.g. amino acids, nucleotides or monosaccharides) to form dimers, trimers or longer chain molecules (oligomers)
The binding of procaspase 8 to FADD forms a Death-Inducing Signalling Complex (DISC)
DISC formation results in cross-activation of procaspase 8, whereby they cleave each other within the complex (due to close proximity)
The active caspase 8 is then released, and it cleaves effector caspases to execute the death programme
What is the role of caspase 8
It activates downstream effector caspases which go on to carry out the apoptotic programme
How are mitochondria able to regulate apoptosis?
This is the intrinsic pathway whereby cellular stresses (e.g. lack of/overstimulation by growth factors, DNA damage etc.)
The intrinsic pathway involves no external stimulus that comes into the cell but is triggered due to changes within the cell itself (e.g. damage in the cell, changes in metabolism)
These all cause a loss of mitochondrial membrane potential (which acts like an initiator of apoptosis)
This results in the activation of the mitochondrial apoptotic program and there will be a release of cytochrome C and other apopotosis-inducing factors
These stimulate the formation of an apoptosome complex - this is a large molecule that is able to carry out apoptosis
What does the apoptosome consist of?
APAF-1 (apoptotic activating factor 1)
Cytochrome C
ATP
Procaspase 9
What is the mechanism by which the apoptosome can cause apoptosis?
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 each other to produce caspase 9
The activated caspase 9 is then released, which is able to trigger the caspase cascade, which leads to apoptosis
This includes cleaving caspase 3, 6 and 7
What links the receptor-mediated and mitochondrial death pathways?
Bid - When one pathway is triggered, it can trigger the other pathway through the activation of Bid
How does Bid work to interlink the two death pathways?
Caspase 8 from the receptor-mediated pathway can cleave Bid, which enhances release of mitochondrial proteins, thus engaging the intrinsic pathway
Bid promotes the release of cytochrome C from the mitochondrion, which triggers the mitochondrial death pathway
What is the difference between the two pathways of apoptosis?
The difference between the two mechanisms is that the mitochondrial pathway requires ATP
How can you determine if a cell is undergoing apoptosis or necrosis?
Apoptosis is an ACTIVE process unlike necrosis, which requires energy so the energy levels of a cell may determine whether death is by necrosis (less ATP) or apoptosis (more ATP)
What is the function of Bcl-2 proteins?
These are intrinsic modulators of apoptosis
Whats is the structure of the Bcl-2 proteins?
There are THREE main groups of Bcl-2 proteins, all of which contain a mixture of Bcl 2 homology (BH) domains ranging from BH 1-4. The BH3 domain is present in all the groups
Some of the proteins contain other domains including a transmembrane domain
What is the importance of BH3
BH3 is a dimerisation motif (for protein-protein interaction) that allows proteins in the Bcl-2 family to associate and dimerise with each other
This is why it is found in all of the Bcl-2 protein groups
What are the two categories of the Bcl proteins?
Anti-apoptotic proteins - localised to the mitochondrial membrane and INHIBIT apoptosis. E.g. BCl-2 and Bcl-xL
Pro-apoptotic proteins - move between the cytosol and the mitochondrial membrane and they PROMOTE apoptosis. E.g. Bid, Bad, Bax and Bak
How do growth factors inhibit apoptosis?
Growth factors may activate TWO growth factor pathways associated with anti-apoptotic effects
Ligand binding causes dimerisation and cross-phosphorylation of the tyrosine kinase receptors
Phosphorylation of the tyrosine kinase receptor initiates signal transduction pathways as well as creating docking sites for adapter proteins (e.g. Grb2), which can bind and mediate the protein-protein interactions within the pathways (e.g. activating Ras, which leads to activation of the MAPK/ERK cascade)
Another phosphorylation site on the tyrosine kinase receptors triggers the PI3-Kinase pathway, which is involved in cell survival and has anti-apoptotic effects
What is Phosphatidylinositol 3-kinase?
PI3-K is a lipid kinase involved in growth control and cell survival – even though it is a kinase it doesn’t phosphorylate an amino acid but instead phosphorylates a lipid and changes the lipid composition of the membrane
What is the structure of PI3-K?
It has three main subunits:
Targeting subunit
Adapter subunit
Catalytic subunit
How is PI3-K recruited and how does it have anti-apoptotic effects?
Growth factors first dimerise and bring in an adaptor protein that recruits PI3-K
This phosphorylates PIP2 to PIP3, which is then recognised by the adapter subunit 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 (part of the Bcl-2 family)
How are the Bcl-2 family proteins involved in reducing the effect of pro-apoptotic proteins?
Other pro-apoptotic proteins (such as Bax and Bak) are held in their inactive heterodimers (by their BH3 domains) to the anti-apoptotic Bcl-2 proteins
As the pro-apoptotic proteins are held in the inactive heterodimers, they cannot have their effect so cell survival and proliferation are promoted
How does an absence of the PI3-kinase pathway cause apoptosis?
When growth factors are ABSENT or the cell is under stress, the PI3-kinase pathway is not activated, so PIP3 is NOT generate and, hence, PKB is NOT recruited to the cell membrane and activated
This means that Bad can NOT be phosphorylated and held in an inactive heterodimer (with PKB)
So the Bad is dephosphorylated and released from the heterodimer
Bad can then go to the mitochondrial membrane, where it can bind through its BH3 domain to the BH3 domains of the anti-apoptotic Bcl-2 family members thus DISPLACING the pro-apoptotic Bcl-2 family members
Once the pro-apoptotic Bcl-2 family members (e.g. Bax and Bak) are released from inhibition by the anti-apoptotic Bcl-2 family members, they form a pore in the mitochondrial membrane, which allows cytochrome C to escape into the cytosol and induce apoptosis
How is the intrinsic activity of apoptosis regulated?
PTEN is a lipid phosphatase that counteracts the production of PKB, therefore reducing the regulation of cell survival and promoting apoptosis
IAPs (Inhibitor of Apoptosis Proteins) bind to procaspases and prevent activation
IAPs also bind to active caspases and inhibit their activity
What are the inhibitor molecules of the different apoptotic pathways?
Bcl-2, Bcl-xL = intrinsic pathway
FLIP, IAPs = extrinsic pathway
Growth factor pathways via PI3-kinase and PKB/Akt
What are the different proto-oncogenes and tumour suppressors associated with apoptosis?
Bcl-2 (oncogene - because over-expression of Bcl-2 will promote cancer)
PKB/Akt (oncogene - because over-expression of PKB/Akt will promote cancer)
PTEN (tumour suppressor - because inactivation of this gene promotes cancer)
How can DNA cleavage be stimulated in cancer therapy?
Dexamethasone
