Cancer 10: apoptosis Flashcards
Why is programmed cell death (PCD) required
- Harmful cells (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: brain to eliminate excess neurons; liver regeneration; sculpting of digits and organs). - Obsolete cells (e.g. mammary epithelium at the end of lactation).
- Exploitation - Chemotherapeutic killing of cells.
How is apoptosis important in development of digits
Apoptosis is important in development, for example in the formation of the digits. The induction of apoptosis between digits allows the formation of hands and feet. If this process is dysfunctional, digits won’t separate.
Differentiate necrosis and apoptosis
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 occurs in necrosis
Plasma membrane becomes permeable
Cell swelling and rupture of cellular membranes
Release of proteases leading to autodigestion and dissolution of the cell
Localised inflammation
What happens to the chromatin and organelles duing necrosis
Cells and organelles swell. Chromatin condenses.
Membrane compromisd to fluid rushes in
What are the two phases in apoptosis
Latent phase
Execution phase
What occurs in the latent phase of apopotiss
death pathways are activated, but cells
appear morphologically the same
What happens during execution phase of apoptosis
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
T/F a common feature of apoptosis and necrosis is that the plasma membrane becomes permeable in both
F
Plasma membrane remains intact in apoptosis– no inflammation
T/f chromatin condenses in both apoptosis and necrosis
T
State the sequence of apopotis
Microvilli contract/intercellular junctions break chromatin condenses
Cell shrinks- epithelium of neighbouring cells closes around it
Chromatin condeses around the nuclear periphery
Cell blebs violently…. chromatin condensation continues
Cell fragments into membrane-enclosed apoptotic bodies
Apoptotic bodies phagocytosed by neighburing cells and roving amcrophages
How can the DNA modification in apoptosis be shown
DNA ladders on agarose gel…. due to fragmentation
TUNEL assay shows more flourescently-tagged bases due to more ‘ends’ due to the fragmentation
Differentiate necrosis and necrosis-like PCD
Unregulated cell death associated with cellular disruption and an inflammatory response
Variable features of apoptosis before cell lysis; “Aborted apoptosis”
Differentiate apoptosis and apoptosis-like PCD
Apoptosis (programmed cell death, PCD) - Regulated cell death; controlled disassembly of cellular contents; no inflammatory response
some, but not all, features of apoptosis. Display of phagocytic recognition molecules before plasma membrane lysis
T/F there is clear distinction between apoptosis and necrosis of cells, no in between
F… there can be features of both
What are the components of the mechanisms of apoptotic cell death
- Executioners (caspases)
- Initiating the death programme
- Death recetpros
- Mitochondria - Bcl-2 family
- Stopping the death programme
What are caspases
Cysteine-dependent aspartate-directed proteases
What do caspases do, and how are they activated
Executioners of apoptosis
Activated by proteolysis
Cascade of activation
Categorise caspases
Initator caspases (2,9,10,8)
Effector caspases (3,6,7)
Outline the specific domains seen in caspases
CARD (caspase recruitment domain)… in caspase 2,9
DED (death effector domain) in caspace 10 and 8
What do the specific domains within caspases bind
homotypic
protein-protein interactions
(cf. adaptors)
T/F effector caspases also have specific domains
F
Differentiate caspases to other cascade proteins
They are activated by cleavage, not phosphorylation
Where are the specific domains found within the initator caspases
On the N terminal
Outline protease maturation
Synthesised as pro-caspases (zymogens)
For INITIATOR caspases, the specific domain (DED/CARD) is cleaved. For the effecor casade, there is a smaller pro-domain which is cleaved
There is also then cleave at the sulphide double bond between the LS/SS regions
After the cleavage, you get folding of 2 large and 2 small chains to form an active L2S2 heterotetramer
Characterise caspace cascades
amplification
divergent responses
regulation
What is the function of initiator and effector caspases
Initiator caspases – trigger apoptosis by cleaving and activating..
Effector caspases – carry out the apoptotic programme
What 2 tasks can effector caspases do
Cleave and inactivate proteins or complexes (e.g. nuclear lamins leading to nuclear breakdown)
Activate enzymes
such as protein kinases or nucleases (e.g. Caspase-Activated DNase, CAD is a nuclease) by direct cleavage, or cleavage of inhibitory molecules
This can happen with monomers, or with multiprotein complexes (in the case of nuclear lamins break down)
…..
…….
…….
…….
Outline the mechanisms of caspase activation
Death by design – Receptor-mediated (extrinsic) pathways
Death by default – Mitochondrial (intrinsic) death pathway
T/F all cells contain death receptors
T
Outline structure of the death receptors
Extracellular cysteine-rich domain
Single transcellular domain
Cytoplasmic tail (with a death domain)
Where is the death domain found in the death receptor
In cytoplasmic tail
Outline what follows the activation of death receptor
For example, FAS-Ligand might bind to FAS.
This then causes adaptor proteins to bind to the receptor
What are the 2 types of adapter proteins associated with death receptors
FADD- ACTIVATE- contain DED and DD.
FLIP- INHIBITION. Contain DEATH EFFECTOR DOMAIN (DED and DED)
(remember that these specific domain regions are HOMOTYPIC)
How does signalling occur through death receptors (e.g. Fas/Fas-liand)
- Receptor (Fas) trimerisation by ligand (Fas-L on lymphocyte)
- Recruitment of adapter protein (FADD) through its DD to DD of Fas
- Recruitment and oligomerisation of procaspase 8 through its DED to FADD DED –> Death-Inducing Signalling Complex (DISC)
What is oligomerisation
process that links monomeric compounds (e.g. amino acids, nucleotides or monosaccharides) to form dimers, trimers, tetramers, or longer chain molecules (oligomers)
How is an initiator procaspase (e.g. initiator procaspase 8) activated
Procaspase 8 binds to the trimerised receptor tails/FADD adaptor protein (which is associated with the receptor tails by DD domains)
The inititiator procaspase which contains DED associates with the DED of the FADD. This forms DISC
The initiator procaspase is in close proximity to the other 2 initator procaspases (as one is assoaited with each receptor cytoplasmic tail), and TRANSCLEAVAGE occurs because of this
Outline the two ways that the initator procaspase can be activated when they bind to the DED domain of the FADD adapter protein (which is linked to the death receptor)
- activated by conformational change on oligomerisation (I.e activated as its DED domain associates with the DED domain of the FADD adapter protein)
- Some initiator procaspases have intrinsic low catalytic activity – oligomerisation allows transcleavage
In transcleavage the initaitr procaspases can cleave each other too due to close proximity
How many procaspases must be bound to the receptor to form an active tetramer
At least 2
What is the structure of a procaspase
Prodomain + LS (long strand)+ SS (short strand)
Note the prodomains have specific domains in initiator caspases (CARD and DED), but this is not case for effector caspases
What occurs following cleavage of the pro-caspase at the receptor
Active initiator caspase 8 tetramer released from receptor
What inhibits activation of caspase 8 (the initiator caspase) at the death receptor
How
FLIP
FLIP contains 2 DED domains, so can bind to the FADD/receptor tails
Incorporates into receptor-procaspase complexes and interferes with transcleavage
What is the effect of activated caspase 8
activates downstream effector caspases
Outline how mitochonria regulate apoptosis
If loss of mitochondrial membrane potential (ΔΨ)
Cytochrome C is released
Other apoptosis inducing factors are released
The apoptosome complex is formed
What can cause loss of mitochondrial membrane potential (ΔΨ)
Cellular stresses e.g. lack of or overstimulation by growth factors, DNA damage (p53), ROS
What is the apoptosome composed of
Apaf1, caspase 9
Outline the composition of the apoptosome (wheel of death)
7 X Apaf-1
apoptotic activating factor- 1
What is the structure of the Apaf-1
There is a CARD domain (CAspase Recruitment Domain)
and many WD-40 repeats (protein protein-interactions)
How is the apoptosome activated, and what is its effect
Cyt C (from mitochondrial damage) binds to WD40 domain USING ATP
This allows procaspase 9 to bind to the CARD domain
Oligomerisation brings multiple procaspase 9s close together, resulting in cleavage, activation and release as active caspase 9 tetramer, which initiates a caspase cascade leading to apoptosis
T/f only certain Apaf-1 within the heptameric apoptosome can bind procaspase 9
F: each Apaf-1 in the heptameric apoptosome can potentially bind a procaspase 9
How does the apoptosome/ wheel of death influence whether apoptosis or necrosis occurs
ATP!
The apoptosome requires ATP….
ENERGY LEVELS IN THE CELL DETERMINE WHETHER DEATH IS BY NECROSIS OR APOPTOSIS OR SOMETHING IN BETWEEN
What is the relationship between the extrinsic and the intrnsic pathway of apoptosis
The death receptor binding etc. is all extrinsic
The mitochondrial damage/Cyt C binding to the apoptosome is the intrinsic pathway
Caspase 8 (activated by extrinsic pathway) cleaves Bid, which enhances release of the mitochondrial proteins, engaging the intrinsic pathway
Which 2 factors can cause release of mitochondrial proteins to activate the apoptosome
- Mitochondrial damage
2. Caspase 8 activated through the extrinsic pathway via Bid
Both the extrinsic and intrinsic pathway cause activation of which caspase
Caspase 3, which then leads to proteolysis and cell death
Which family of proteins modulates apoptosis
Bcl-2 family proteins
Classify Bcl-2 family proteins in terms of structure
Group 1-3
All contain dimerisation motif called BH3
Some contain other domains e.g. Transmembrane
Group 1: Bcl-2
Group 2: Bax
Group 3: Bid (activated by caspase 8 to cause mitochondrial damage), Bik and Bad these are BH3 only.
t/f group 1, 2 and 3 all contain TM domain
Yes, group 1 and 2 do, but not necessarily all in group 3
Give examples of antiapoptotic and pro-apoptotic proteins within the Bcl-2 apoptosis modulating family
Anti-apoptotic
Bcl-2 Bcl-xL
Pro-apoptotic
Bid Bad Bax Bak
Where are the anti-apoptotic and the pro-apoptocic Bcl-2 family proteins located in the cell
Anti apoptotic: MITOCHONDRIA
Pro apoptotic: move between Cytosol and Mitochondria, where they cause release of CyC/other apoptotic proteins(
The PI3’-kinase signalling pathway is involved in what
Cell Cycle and Apoptosis Regulation
T/f the PI3’-kinase signalling pathway occurs at the same receptor as the Ras/ERK pathway
T!
Outline how the PI3’-kinase pathway is activated
Upon growth factor binding, PI3’K binds to p85 (an adapter protein bound to the receptor tyrosine kinase)
This then phsophorylates PIP2–>PIP3
This activates PDK1 then PKB/AKT.
AKT is anti-apoptotic
What is PI3’K
Phosphatidylinositol 3’-kinase (PI3’-K) – a lipid kinase (not a protein kinase) involved in growth control and cell survival
PIP3 has phopshorylation on the 3, 4 and 5 position. Whereas PIP2 only has phosphorylation on the 4 and 5 position
It is essentially part of the plasma membrane
How is PI3-k different to other kinases
It phosphorylates a phospholipid not a protein
What does PKB/Akt bind to
The PH domain of these proteins bind to the PIP3
What is the effect of PKB/Akt
Phosphorylates and inactivates Bad
Phosphorylates and inactivates caspase 9
Inactivates FOXO transcription factors (FOXOs promote expression of apoptosis-promoting genes) such as p27Kip and apoptotic protein Fas
ligand
Other, e.g. stimulates ribosome production and protein synthesis
Which transcription factos promote transcription of pro-apoptotic genes
FOXOs
How does the PKB/Akt proteins, from the GF pathways, interact with apoptosis regulation by mitocondria
Normally Bad is is phosphorylated and inactivated by PKB/Akt.
This allows Bax or Bak to be bound to Bcl2, and bcl2-xL (group 1 of the bcl2 family)
However, when GF is absent, Bad will displace Bax and Bak, and Bad will bind to Bcl2 or Bcl-xL instead.
This then releases the Bax or Bak, such that they combine to form a pore, out of which mitochondrial contents (like Cyt C) can flow, and activate apoptosis
How are Bcl2 and Bax linked
Via BH3 domains (common to all Bcl2 proteins)
What type of molecule is PTEN
(Llipid phosphatase)
How does PTEN work to inhibit PI3’-K pathway
It removes the phosphate from PIP3, forming PIP2, which cannot activate the PKB/Akt proteins which induce cell survival and protein syntehsis
Compare PTEN and PI3’-K
PTEN is a lipid phosphatase and PI3’-K is a lipid kinase….
allows for control of cell survival easily
What are the anti-apoptotic pathways for the intrinsic and extrinsic pathway
What other antiapoptotic pathway is there
INTRINSIC: Bcl-2, Bcl-xL (which prevent BAX and Bak from heterodimertising to form the pore allowing escape of Cyt C, inducing the apoptosome, and eventually activation of caspase 9
EXTRINSIC: FLIP and IAPs (inhibitor of Apoptosis Proteins)
Growth factor pathways via PI3’-K and PKB/Akt
What are inhibitors of apoptosis proteins
regulate Programed Cell Death via Extrinsic pathway
Bind to procaspases and prevent activation
Bind to active caspases and inhibit their activity
How can cancer cells avoid apoptosis
Apoptosis regulators as
oncogenes or tumour suppressors?
How can programmed cell death be used therapeutically
Harmful (oncogenic) cells can be virally infected to cause DNA damage
Chemotherapeutic killing of tumour cells, e.g. Dexamethasone stimulates DNA cleavage, which damages DNA and sends these cells down the apoptotic pathway
