Apoptosis and Senescence Flashcards
Give an overview of apoptosis?
AKA programmed cell death
Physiological cell death, in orderly and controlled manner that appears to follow a defined programme
Contrast with necrosis – death following injury, infection or trauma
Apoptosis does not provoke immune response – cell’s contents not liberated
What are some markers for apoptosis?
Cell morphology
DNA ladders - resulting from nuclease digestion/fragmentation of chromatin, therefore generating discrete nucleosomal fragments
Tunel assay - labelling of termini of DNA fragments with fluorescent groups by the enzyme terminal transferase
Adds nucleotides to the 3’ in a non template method
Binding of Annexin V (labelled with a fluorescent dye, FITC) to cells - caused by membrane changes and assayed in flow cytometer
Annexin V recognises phospholipids that are flipped out of the membrane
Activation of caspase enzymes - assayed by Western blotting
What are the stages of apoptosis?
- Decision - survival factors, TNF, Fas, Bcl-2 family, p53/DNA damage
- Execution - caspases (for targeting/digestion), nucleases, surface modifications
- Death - phagocytosis
What are the types of apoptsis?
Extracellular (extrinsic pathway) - signalling from death receptors on the cell surface
Intracellular (intrinsic pathway) - dsDNA breaks, p53, UV radiation, hypoxia
What are caspases?
Cys-catalysed aspartate targeting proteases
They induce apoptosis (except ICE - interleukin converting enzyme)
All have similar substrate specificities and amino acid sequences
Expressed as pro-enzymes (require activation)
Contain 3 domains - N-terminal (regulation of activation), large domain and small domain
They are very specific - recognising and degrading very few proteins = highly effective
How are caspases activated?
Activated by proteolytic cleavage between caspase recognition site between large/small domains
The N-terminal domain has to be cleaved off
The large/small domain has to be digested and moved in position to create a different structure
This leads to a caspase cascade
Describe the categories of caspases?
Initiator caspases - activate a cascade resulting in activation of executioner caspases
Caspase 8 and 10 - activated by death receptors via FADD (extracellular signals)
Caspase 9 - activated by APAF-1, cytochrome C and ATP via CARD (caspase recruitment domain) - activated by p53 levels
Executioner Caspases - activated by initiator caspases
Caspase 7 and 3 induce cell killing
Caspase inhibitors
Inhibitor of apoptosis (IAPs) - binds to the catalytic site of caspases
C-FLIP (FLICE inhibitory protein) inhibitor of initiator caspase 8
Over expression is common in tumours resistant to death inducing ligands
They prevent initiator caspases from activating executioner caspases
What is the caspases cascade?
One molecule of active initiator caspase
Leads to many molecules of executioner caspases
Leads to the cleavage of cytosolic proteins and nuclear lamina proteins
Give an overview of the extrinsic pathway of apoptosis?
Referred to as the death receptor pathway
Induced by extracellular signals - upon ligand binding, shown on a killer lymphocyte, to specific receptors (e.g. Fas, TNFR1)
The death receptors trimerise and recruit FADD adaptor protein and caspases - forming a DISC complex
The DISC complex (death initiation signalling) is formed and caspase 8 activated
The executioner caspases are activated which cleaves caspase 3 into its active site - which brings about apoptosis
Describe signalling though FasL and TRAIL?
Trimerised receptor recruits FADD from the cytoplasm
Binds through death domain cluster
FADD acts as an adaptor protein
FADD also has a death effector domain (DED)
This binds molecules with a DED domain e.g. Caspase 8 and 10
Binding of caspase 8 causes activation and initiation of apoptosis
TRAIL = TNF-related apoptosis-inducing ligand FADD = Fas-associated protein with death domain
Describe signalling via death receptors?
Death receptors are membrane bound receptors which signal death by binding of cytotoxic ligands
Mechanisms to prevent external cell death:
Soluble decoy receptors, membrane bound decoy receptors and intracellular signalling activators and inhibitors
Death receptor activation:
Ligand binding -> receptor trimerisation (homotrimer) -> intracellular death domain cluster signals pro-apoptotic signal
Examples - Death receptor-4 and -5 (TRAIL), Fas (Fas ligand or FasL) and TNFR-1 (TNFa)
Give an overview of the intrinsic pathway of apoptosis?
Referred to as the mitochondrial pathway
Activated following e.g. DNA damage, oxidative stress = release of Cytochrome c from the inner membrane of the mitochondria, when pores are formed in the membrane
Cytochrome C binds to APAF-1 (apoptosis protease-activating factor-1) - requiring ATP hydrolysis, which exposes the CARD domain
It can then form the apoptosome
Procaspase 9 is recruited and activated as it is cleaved to caspase 9, which allow the activation of executioner caspases - which brings about apoptosis = cell death
Cytochrome C is regulated by the BCL2 family of pro and apoptotic proteins
Describe the BCL-2 family?
Bcl-2 is anti-apoptotic
Found in B Cell Lymphomas
25-26kDa membrane protein - associated with membranes, mitochondria
Homologues of Bcl-2 e.g. Bax, pro-apoptotic, forms heterodimers with Bcl-2 and inactivates Bcl-2
Some viruses contain Bcl-2 homologues e.g. adenovirus E1B-19kDa protein
What are the classes of BCL2 family proteins?
- Anti-apoptotic Bcl2 protein (e.g. Bcl2, Bcl-XL) - contains BH1, 2, 3, 4
- Pro-apoptotic BH123 protein (e.g. Bax, Bak) - contains BH1, 2, 3
- Pro-apoptotic BH3-only protein (e.g. Bad, Bim, Bid, Puma, Noxa) - contains BH3
Expression of genes encoding Bax and PUMA activated by p53
PUMA = p53 upregulated modulator of apoptosis
How is BH123, BCL2 and BH3 involved in the mechanism of the intrinsic apoptotic pathway?
BH123 proteins are present in the outer membrane of the mitochondria
When they receive an apoptotic stimulus they form a pore allowing cytochrome C to be released
Bcl2 protein - can inactivate the aggregation of BL123 proteins and not allow the pore to form - when it is present at high levels
BH3 only proteins - they can block the activity of Bcl2 protein, through an apoptotic stimulus and allowing the formation of pores in the membrane
