Apoptosis Flashcards
Why do we need programmed cell death?
To remove- Harmful cells: Developmentally defective cells Excess/unnecessary cells: Embryonic development Obsolete organs Exploitation
Give an example of developmentally defective cells?
B lymphocytes expressing antibodies against self-antigens
Give example of excess/unneccesary cells from embryonic development?
Brain to eliminate excess neurones
Liver regeneration
Sculpting of digits and organs
What is necrosis?
Unregulated cell death associated with trauma, cellular disruption and an inflammatory response
What is apoptosis?
Regulated cell death, controlled disassembly of cellular contents without disruption- no inflammatory response
What happens in cells during necrosis?
Plasma membrane becomes permeable
There is cell swelling and rupture of cellular membranes
Proteases are released leading to auto digestion and dissolution of the cell
Localised inflammation
What happens in latent phase apoptosis?
Death pathways are activated but cells appear morphologically the same
What happens in the execution phase of apoptosis?
Loss of microvilli and intercellular junctions
Cell shrinkage
Loss of plasma membrane asymmetry
Chromatin and nuclear condensation
DNA fragmentation
Formation of membrane blebs
Fragmentation into membrane-enclosed apoptotic bodies
Why is there no inflammation in apoptosis?
Plasma membrane remains intact
What happens to apoptotic bodies once the cells have been broken down?
They are taken up by macrophages
DNA modification occurs during apoptosis, what does this lead to?
Fragmentation of DNA ladders
Formation of more ‘ends’ which are labelled by adding an extra fluorescently-tagged base in a TUNEL assay
What is apoptosis like programmed cell death?
Has some but not all features of apoptosis and display of phagocytic recognition molecules before plasma membrane lysis
What is necrosis like programmed cell death?
Displays variable features of apoptosis before cell lysis- like an aborted apoptosis that ends up being necrosis
What is important to remember about cell death?
It is a graded response and cells often die of something that is between necrosis and apoptosis
What does caspase mean?
Cysteine-dependent aspartate-directed proteases
What is required for caspase activity?
Cysteine residue in active site
Where do caspases cut proteins?
Just after their aspartate residue
What activates caspases?
Proteolysis
What are caspases known as?
Executioners
Which caspases are the effector caspases?
3, 6 and 7
How do effector caspases start off and what happens to them?
They start off as single chain polypeptide with two subunits (large and small) and these subunits are released by proteolytic cleavage during maturation
Which caspases are the initiator caspases?
2, 8, 9 and 10
How do initiator caspases exist?
They also have the same two subunits that are found in effector caspases and an extra targeting subunit
What are the two types of targeting subunit and what do they do?
They direct caspases to a particular location and
CARD- Caspase recruitment domain
DED- Death effector domain
What are procaspases?
Single chain polypeptides (zymogens)
How do procaspases become activated?
They must undergo proteolytic cleavage to form large and small subunits
What carries out the proteolytic cleavage that activates caspases?
Caspases
What happens after the proteolytic cleavage of procaspases?
You get folding of 2 large and 2 small chains to form an active L2S2 heterotetramer
What is the main purpose of caspase cascades?
Amplification
Divergent responses
Regulation
What happens once apoptosis is triggered in terms of caspases?
Initiator caspases cleave and activate effector caspases
Which caspases execute the apoptotic programme?
Effector
In what two ways do effector caspases carry out apoptotic programme?
Cleaving and inactivating various proteins and complexes (e.g. nuclear laming leading nuclear breakdown)
Activating enzymes by direct cleavage or cleavage of inhibitor molecules (protein kinases, nucleases such as caspase activated DNase
What are the mechanisms of caspase activation?
Death by design- receptor-mediated (extrinsic pathways)
Death by default- Mitochondrial (intrinsic) death pathway
Which cells have death receptors on their surface?
All cells
What does a death receptor consist of?
Extracellular cysteine-rich domain Single transcellular domain Cytoplasmic tail (with a death domain)
When are death receptors only activated?
When they encounter secreted or transmembrane trimeric ligands (e.g TNF alpha or Fas)- death ligands
Which two adapter proteins are very important in this pathway and how?
FADD- positive regulator (required for pathway to become activated) promotes cell death
FLIP- Negative regulator (inhibits death pathway and allows regulation)
What is the structure of FADD and FLIP?
Different:
FADD- DED and DD
FLIP- DED and DED
What is Fas/
A death receptor that is unregulated if apoptosis is required e.g. a cell is infected by a virus
What happens when the fas ligand binds to the fas receptor on the surface of cytotoxic T lymphocytes?
The Fas receptors then undergo trimerisation which brings the three cytoplasmic D domains together
What do trimerised death domains do?
Recruit positive adaptor protein FADD by its own DD
What happens when FADD binds to the trimerised death domain?
Recruitment and oligomerisation of procaspase 8 through its own DED to the FADD DED
What is oligomerisation?
Chemical process that links monomeric compounds (e.g. amino acids, nucleotides or monosaccharides) to form dimers, trimers, tetramers or longer chain molecules
What happens when procaspase 8 binds to FADD?
A death inducing signalling complex (DISC) forms
What does DISC formation result in?
Cross-activation of procaspase 8 whereby they cleave each other within the complex due to close proximity
What then happens to the active caspase 8?
It is released and it cleaves effector caspases to execute the death programme
How do initiator procaspases bind to FADD?
Via their DED domains to the DED domains of FADD which brings the three initiator procaspase 8s into close contact which allows cleavage and this releases the active initiator initiator caspase 8 tetramer
How many procaspases do you at least need to form an active tetramer?
2
What inhibits death receptor activation of procaspase 8?
FLIP
How does FLIP antagonise procaspase 8?
It is evolutionarily related to caspases but has lost its catalytic activity- it has no proteolytic activity so can compete with procaspase 8 to bind to the DED domains of FADD
What does caspase 8 activate?
Downstream effector caspases which carry out the apoptotic programme
What activates the intrinsic pathway of apoptosis?
Cellular stresses (lack of/overstimulation by growth factors, DNA damage etc) cause a loss of mitochondrial membrane potential which results in release of cytochrome c and other apoptosis inducing factors
What does cytochrome c and other apoptosis inducing factors stimulate?
The formation of an apoptosome complex
What does the apoptosome consist of?
APAF-1 (apoptotic activating factor 1)
Cytochrome C
ATP
Procaspase 9
What is within APAF-1?
At one end, it 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 is a caspase recruitment domain (CARD) which is also found in some initiator caspases
What does cytochrome c bind to and what happens when it binds?
The WD-40 repeats on APAF-1- it forms a heptamer (the apoptosome)
This process requires ATP
What do the CARD domains at the centre of the apoptosome interact with?
CARD domains on procaspase-9
How many procaspase-9s can bind to apoptosome?
Seven
What does the close proximity of the procaspase 9s that bind to the CARD domains of the apoptosome mean?
They can cross-cleave and activate each other to produce caspase 9
What happens once activated caspase 9 is formed?
It is released and able to trigger the caspase cascade which leads to apoptosis
What does Bid enable?
Links the receptor-mediated and mitochondrial death pathways- when one is triggered, it can trigger the other pathway
How can caspase 8 from the receptor-mediated pathway trigger the intrinsic pathway?
It can cleave Bid which enhances release of mitochondrial proteins thus engaging the intrinsic pathway
What is a major difference between the two pathways for triggering apoptosis?
Mitochondrial pathway requires ATP
How does Bid trigger mitochondrial death pathway?
It promotes release of cytochrome c from mitochondrion which triggers death pathway
How do the energy levels of a cell affect cell death?
Apoptosis is an active process which requires energy so energy levels of a cell may determine whether death is by necrosis (less ATP) or apoptosis
What are the intrinsic modulators of apoptosis?
Three main groups of Bcl-2 proteins, all of which contain BH3 domain
What is BH3?
Dimerisation motif (for protein-protein interaction) that allows proteins in Bcl-2 family to associate and dimerise with each other
What are the two categories of proteins in the Bcl-2 family?
Anti-apoptotic- Bcl-2 Bcl-xL
Pro-apoptotic- Bid, Bad, Bax and Bak
What are anti-apoptotic proteins?
Proteins are localised to the mitochondrial membrane and inhibit apoptosis
What are pro-apoptotic proteins?
They move between the cytosol and mitochondrial membrane and promote apoptosis
What effect can growth factors have on apoptosis?
They may activate two growth factor pathways associated with anti-apoptotic effect
What happens when growth factor (EGF, insulin) binds to a growth factor receptor (EGFR, insulin R)?
It causes dimerisation and cross-phosphorylation of tyrosine kinase receptors which initiates signal transduction pathways
What else does phosphorylation of growth factor receptors do?
It creates docking sites for adapter proteins (Grb2) which can bind and mediate the protein-protein interactions within the pathways (e.g. activating Ras which leads to activation of MAPK/ERK cascade)
What else does another phosphorylation site on tyrosine kinase receptors trigger?
PI3-Kinase pathway
What is the PI3-Kinase pathway involved in?
Cell survival and has anti-apoptotic effects
What is PI3-K?
Phosphatidylinositol 3-kinase is a lipid kinase involved in growth control and cell survival
What are the three main subunits of PI3-K?
Targeting subunit
Adapter subunit
Catalytic subunit
What does PI3-K do?
It phosphorylates PIP2 to PIP3
What is PIP3 recognised by?
Adapter subunit of PKB/Akt
What happens to PKB after it PIP3 formation
It is recruited to the cell membrane and is activated
What does active PKB do?
Anti-apoptotic effects- it phosphorylates and inactivates Bas (part of the Bcl-2 family)
How are other apoptotic proteins such as Bax and Bak held?
In their inactive heterodimers (by their BH3 domains) to anti-apoptotic Bcl-2/xL proteins- this promotes cell survival and proliferation
What happens when growth factors are absent?
PI3 kinase pathway isn’t activated so PIP3 isn’t formed and hence PKB is not recruited to the cell membrane and activated- this means that Bad can’t be phosphorylated and held in inactive heterodimer so it’s dephosphorylated and released from heterodimer
What can Bad do when it is dephosphorylated and released from heterodimer?
It can go to 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
What happens once pro-apoptotic Bcl-2 family members are released from inhibition by anti-apoptotic Bcl-2 family members?
They form a pore in the mitochondrial membrane which allows cytochrome c to escape into cytosol and induce apoptosis
Give a summary of the effects of PKB/Akt and on cell survival?
It phosphorylates and inactivates Bad
Phosphorylates and inactivates caspase 9
Inactivates FOXO transcription factors (these normally promote expression of apoptosis promoting genes)
What is PTEN and what does it do?
It is a lipid phosphatase that counteracts the production of Pkb therefore reducing regulation of cell survival and promoting apoptosis
What are IAPs?
Inhibitors of apoptosis proteins that bind to procaspases and prevent activation, they also bind to active caspases and inhibit their activity
What anti-apoptotic pathways are there?
Bcl-2, Bcl-xL = intrinsic
FLIP, IAPs = extrinsic
Growth factor pathways via PI3 kinase and PKB/Akt
What porto-oncogenes/tumour suppressors are associated with apoptosis?
Bcl-2 - oncogene because overexpression will promote cancer
PKB/Akt- oncogene
PTEN- tumour supressor
What does apoptosis form the basis of?
Chemotherapeutic killing of tumour cells involving for example DNA cleavage
