Lecture 7 (apoptosis) Flashcards
Types of cell death mechanisms
-Apoptosis
-Autophagy
-Pyroptosis
-Necroptoisis
-Necrosis
Key morphological changes that apoptotic cells undergo:
-cell shrinkage
-blebbing
-nuclear fragmentation
-formation of apoptotic bodies
Morphological steps of apoptosis:
- Apoptotic signal
- Cell shrinkage and chromatin condensation
- Nuclear fragmentation and membrane blebbing
- Apoptotic body formation
Triggers of apoptosis
-Radiation
-Cytotoxic agents
-Toxins
-Growth factor/nutrient/oxygen deprivation
Caspases involvement in apoptosis:
Acts as a enzymatic mediator of apoptosis.
-Expressed as larger inactive pro-enzymes (pro-caspases).
Function and structure of caspase
-It is a cysteine protease that cleaves after aspartic acid residues.
-It is composed of a large and small subunit.
Two types of Caspases
- Initiator caspase
- Executioner caspase
Types of initiator caspases and their domains
-Caspase 8, 9, and 10
-Prodomain has DED in procaspase 8 and 10 and CARD in procaspase 9.
-These domains allow for homophilic interactions with adaptor proteins
Types of executioner caspases and how they are activated:
-Important in both intrinsic and extrinsic pathways
-Caspase 3 and 7
-Activation by proteolytic cleavage, mediated by initiator caspases
-Procaspase cleavage leads to tetramer of small and large heterodimers
3 ways executioner caspases kill cells?
-It disassembles the cell structures by degradation of nuclear lamina.
-It inactivates apoptotic inhibitors e.g i-CAD
-It deregulates other protein activity e.g gelsolin to actin depolymerisation
The Intrinsic Pathway of Apoptosis:
-Also known as mitochondrial pathway
-Activated by stresses signals being relayed to the mitochondria. Leading to mitochondrial outer membrane permeabilization (MOMP), which releases apoptotic proteins into the cytoplasm
Pro-apoptotic proteins released in the intrinsic pathway:
- Cytochrome C which associates with other proteins such as APaf-1 to allow for the formation of apoptosome.
- Smac (Diablo) inactivates a group of anti-apoptotic proteins called IAPs (inhibitors of apoptosis)
Cytochrome c fuction:
-Cytochome c + Apaf-1 + procaspase 9 = activation of caspase 9
-Caspase9 leads to activation of
effector caspases
What is APAF-1:
-It is a scaffold protein and stands for apoptosis protease activating factor 1.
-Together with cytochrome c and procaspase 9 it activates caspase 9.
-It also oligomerizes in response to cytochrome c release and forms a large complex known as apoptosome.
Apoptotic proteins:
-Proapoptotic (BH3 only): BIM, BAD, NOXA, PUMA, BID which activate Bax/Bak
-Proapoptotic (BAX subfamily): BAX, BOK and BAK which are essential for MOMP
-Anti-apoptotic: Bcl-2, Bcl-XL, MCL-1, Bcl-w are necessary for cell survival
BH3 proteins:
-Activate BAX/BAK either through direct binding and/or indirectly by binding to their repressors, prosurvival BCL2 protein
How are BH3 proteins regulated and activated and give examples of some:
-BH3 proteins are regulated at the transcriptional and post-translational level
and activated in response to given stimuli:
Examples:
-UV activates Bim
-Genotoxic damage activates Noxa, PUMA.
-Cytokine deprivation activates Bad
-Death receptors activates tBid
What determines cytochrome c release
-The relative levels of pro and anti-apoptotic proteins within each channel determine whether cytochrome c will be released from the mitochondrion.
Inhibitors of apoptosis (IAPs):
-Family of 8 proteins including c-IAP1, c-IAP2, XIAP.
-X-IAP inhibits caspase-3 and -9
-cIAP1 ubiquitinates caspase-3 and -7
Apoptosis in cancer cells:
-Upregulation of anti-apoptotic proteins is common in cancer (e.g. Bcl-2 and Mcl-1).
-Inactivation of pro-apoptotic proteins (e.g. Bax is inactivated in the majority of colon cancers)
Signaling regulation of the intrinsic pathway is controlled by:
-AKT
-P53
AKT role in intrinsic pathway:
AKT is a kinase activated by numerous growth factor receptor signals.
-AKT is anti apoptosis and does this by:
-Inactivating Bad and indirectly downregulating BIM and PUMA expression through the transcription factor, FOXO3a.
-It also prevents cleavage of Bid from extrinsic pathway.
P53 role in intrinsic pathway:
-P53 is proapoptotic and induces apoptosis by inducing expression of BAX, PUMA and NOXA (pro-apoptotic) and inactivating Bcl-2 (anti-apoptotic).
Extrinsic pathway of apoptosis:
-Also known as death receptor pathway
-Induced by external signalling caused by ligand binding to a specific membrane receptor
Two ways to activate death receptor pathway signalling:
- Increase ligand expression
- Increase receptor expression
Ligands and their Death receptor:
-Death receptors/ligands are members of the TNF family of proteins
TRAIL - DR4 and DR5
TNF - TNFR1
FasL - Fas (APO-1/CD95)
Key steps in the extrinsic pathway:
1.Ligand-receptor binding
2. Receptor trimerization
3. Recruitment and formation of Death
Inducing Signalling Complex (DISC)
4. DISC recruits and activates the initiator
caspase. DISC triggers self cleavage, thereby activating, caspases.
5. Active initiator caspases then activates
executioner caspases
What happens when the death receptors timerisation:
It brings the 3 Death Domains (DD) together which allows for recruitment of FADD (Fas associated death domain), which forms the Death Inducing Signalling Complex (DISC).
Active Caspase 8:
-Also known as initiator caspase as it can lead to direct activation of caspase 3 and activation of BID which promotes MOP
What is anoikis?
-Greek for homeless. In biology it means anchorage independent.
-It is a unique mode of apoptosis induced after loss of cell adhesion to ECM (through integrin).
-This results inactivation of pro-survival signaling (PI3K-Akt pathway or NK—κB) and caspase 8 activation.
Directly killing through the extrinsic pathway:
-Done by FasL
-Fas expression can be increased due to p53 activation
FasL:
-Can be membrane bound on nearby cell or truncated-soluble form.
-FasL (ligand) on cytotoxic T cell binds to Fas (receptor) on tumour cell
-Stimulates extrinsic pathway in tumour cell and perforin and granzyme release from cytotoxic T cell.
How does perforin and granzyme B cause apoptosis:
They are proteins that break holes in the cell membrane to release its contents.
How does the body prevent CD8+ cells from attacking any cells containing FasL?
When DNA damage occurs then MHC I will display a new antigen that will show CD8+ to attack it.
How do phagocytic cells clean up destroyed apoptotic cells.
-They recognize phosphatidylserine on the apoptotic cell surface and then engulf them.
-As a result the PCs release anti-inflammatory cytokines.
