Lecture 20 - Apoptosis Flashcards
What is apoptosis often referred to as?
Programmed cell death or cell suicide
How is apoptosis cell death?
Via an active cellular response to extracellular or intracellular signals
What was studied to look at apoptosis as a developmental process?
- Salamander tail
- Removal of interdigital cells during digit formation
- Development of visual system and other neural circuits - neurons that fail to connect undergo apoptosis
- Regulation of tissue size by cell competition
- C. elegans development - the ced genes
What did Sidney Brenner develop?
C. elegans as a model system - early 60s
What did John Sulston’s lineae map reveal?
That many cells undergo programmed cell death - early 70s
What did Robert Horvitz screen for?
Genes that when mutated allow these cells to survive - ced genes - early 80s
How is apoptosis different than necrosis?
Apoptosis involves the activation of a cell death pathway while necrosis is a passive process resulting from damage to the cell
What are some features of apoptotic cells?
- Cytoskeleton collapse
- Nuclear envelope breaks down
- Chromatin condenses and breaks into fragments
- Cell surface blebbing
- Apoptotic bodies
- Apoptotic cells signal to phagocytic cells to be phagocytosed
What are some features of necrotic cells?
Swell, burst, and their cellular contents illicit an inflammatory response
How were apoptotic cells identified?
By incubation in Acridine Orange
- TUNEL labeling (label DNA ends with Terminal deoxynucleotidyl transferase): apoptotic cells have fragmented chromosomes
What apoptosis assays were done?
1) Plasma membrane loses activity
- Uptake of cell impermeable DNA dyes - Acridine orange
2) Chromatin breakage
- Ladder of DNA seen by gel electrophoresis
- TUNEL labeling
3) Membrane flipping
- Labeled Annexin5 - Phosphatidylserine flips to outside of membrane (acts as a signal for macrophages)
4) Mitochondrial activity is lost
- Positively charged fluorescent dyes that accumulate in active mitochondria - rhodamine 123
5) Pro-caspase cleavage
- Antibodies against cleaved caspases
What are caspases?
Proteases that mediate apoptosis made as pro-caspases
What keeps caspases inactive?
Pro-domain
What may mediate procaspase cleavage?
Another caspase
What are the classes of caspases?
1) Initiator (activator) caspases - Cleave other caspases
2) Effector (executioner) caspases - Cleave specific cellular targets to cause apoptosis
What does initiator caspase activation lead to?
Rapid amplification of a caspase cascade
What does caspase activation lead to?
Rapid and irreversible initiation of apoptosis
Activation of caspases: Intrinsic pathway
- Cellular response to various stresses, DNA damage, and depletion of survival factors
- BH-domain family of proteins regulate release of proteins from mitochondrial intermembrane space to activate procaspases
- Inhibitor caspases activated by apoptosome
Activation of caspases: Extrinsic pathway
- Apoptosis induced via a signal from specialized cells - e.g., cytotoxic T-lymphocytes
- Cell surface “death receptors” - e.g., Fas, TNF, TRAIL respond to TNF-related ligands
- Adaptor proteins interact with receptors and activate procaspases
How is the intrinsic pathway of apoptosis regulated by the BH-Domain family of domains?
- Proapoptotic Bax and other BH1,2,3 proteins from mitochondrial channels that promote the release of cytochrome c and other proteins from the intermembrane space of mitochondria
- Cytochrome c promotes the formation of apoptosome that in turn stimulates activator caspases
What induces the intrinsic pathway of apoptosis?
- Various stresses acting through p53
- External signals
What are the BH-domain proteins?
- Anti-apoptotic Bcl2 protein
- Pro-apoptotic BH123 protein
- Pro-apoptotic BH3-only protein
What do all Bcl2-related proteins (BH-domain proteins) have?
1 or more Bcl2 homology domains (BH1-4)
What do the anti-apoptotic Bcl2 proteins do?
Inhibit BH123 pore formation
What do the pro-apoptotic BH123 proteins do?
- Form mitochondrial pore
- Release cytochrome c
What do pro-apoptotic BH3-only proteins do?
Inhibit Bcl2
What does cytochrome c do after it’s released from the mitochondria?
It binids Apf1 to promote procaspase 9 binding and apoptosome formation
How is procaspase 9 activated?
By cleavage
What does activated procaspase 9 do?
Cleaves executioner caspases
What are apoptosis inhibitors?
Inhibitors of apoptosis (IAPs) and anti-inhibitors of apoptosis (anti-IAPs)
What were IAPs first identified as?
Viral proteins that prevent host cell apoptosis
What do IAPs do?
Block caspase-9 activity
- Prevent spontaneous activation of caspases by biding cleaved caspases
- Bind activated caspases and prevent their activity
What do anti-IAPs do?
Promote caspase-9 activity
- Compete with caspases for IAP binding –> promote caspase activation
- Expression is induced in response to apoptotic stimuli (anti-IAP genes are p53 targets)
What are anti-IAPs released with in vertebrates?
Cytochrome c from the mitochondria
How is the intrinsic pathway regulated by survival factors?
Anti-apoptotic
- Many growth factors/mitogens are also survival factors
- Receptor tyrosine kinases activate PI3 kinase and Ras –> Akt phosphorylates and inhibits Bad (BH3); MAPK phosphorylates and inhibits Hid (anti-IAP)
How does p53 regulate the intrinsic pathway?
Pro-apoptotic
- Inhibits Bcl2 expression
- Induces BH123 expression
- Induces BH3-only expression
- Induces the expression of anti-IAPs
What leads to p53 induction?
DNA damage and other cellular stresses (e.g., anoxia)
How does p53 induce cell cycle arrest?
By activating p21 expression (and other targets)
How does p52 induce apoptosis?
By activating the expression of BH123, BH3-only, Fas receptor, anti-IAPs. repressing Bcl2 expression (and by other mechanisms)
How is cytotoxic T-cell-mediated apoptosis carried out?
Virus-infected cells display virus peptides on their cell surface
- Cells of the immune system, cytotoxic T-cells, bind to MHC proteins and peptides via cadherin-mediated adhesion
*If peptide is self –> no recognition
*If peptide is foreign –> recognition
What is FasL?
A ligand of the Tumour Necrosis Factor (TNF) family of proteins expression of most or all cells - integral membrane protein ligands that function as homotrimers
What does the activation of Fas receptor lead to?
Caspase 8 (initiator caspase) activation
How does the Fas-mediated extrinsic apoptotic pathway get carried out?
1) Activation of the Fas receptor gene expression by p53
2) Recruitment of FADD
3) FADD interacts with procaspases
4) Complex of receptors, FADD, and procaspases makeup the death induced signalling complex (DISC), which is the functional equivalent of apoptosome
5) Procaspases are activated by their close proximity to each other within the DISC and cleave each other so they can be released
