apoptosis Flashcards
what are the morphological features of apoptosis?
- cell shrinkage
- plasma membrane blebbing
- chromatin condensation
- shrinking of nucleus
- chromatin degradation
- loss of mitochondrial membrane potential
- cell breaks into apoptotic bodies
- translocation of phosphatidylserine to outer lipid bilayer
- secretion of pro-inflammatory cytokines
how is phosphatidylserine involved in apoptosis?
after translocation to the outer lipid bilayer, it binds to receptors on phagocytic cells like macrophages and dendritic cells that engulf the cell fragments
which protein kinase mediates membrane blebbing?
ROCK-I
what is the activation of ROCK1 dependent on?
1- binding to a small GTP binding protein (Rho)
2- binding to arachidonic acid
what is the role of caspase 3 in activation of ROCK1?
cleaves ROCK1 so it is no longer dependent on GTP-Rho or arachidonic acid - becomes cons. active
outline the extrinsic pathway
death ligand binds death receptor, recruiting adaptor proteins and cleavage of initiator caspase, which activates executioner caspase
outline the intrinsic pathway
DNA damage/stress leads to mitochondrial damage, and release of pro-apoptotic proteins from the mitochondrial intermembrane space. this activates initiator caspase -> executioner caspase
what families of proteins constitute the apoptotic machinery in mammalian cells?
1- Bcl-2 protein family 2- caspases 3- death receptor 4- inhibitors of apoptosis proteins 5- mitochondria
what types of Bcl-2 proteins are there?
a - bcl-2 anti-apoptotic proteins
b - bcl-2 pro-apoptotic proteins
c- BH3 only pro-apoptotic proteins
how was Bcl-2 discovered?
discovered in b-cell lymphoma was a gene frequently translocated to the immunoglobulin locus in lymphomas, resulting in ectopic expression of these cancers
overexpression found to block apoptosis
what is the general structure of pro-survival proteins?
9 alpha helices which are separated into 4 separate domains, allow the protein to be anchored to membrane
what is the general structure of BH3-only (pro-apoptotic) proteins?
amphipathic helix roughly comprised of 9-16 amino acids
what is the structure of Bcl-XL?
hydrophobic a-helices in anti-parallel organisation
what is meant by the term: apoptotic switch/
the way in which pro-apoptotic proteins must bind to and inhibit the anti-apoptotic proteins before apoptosis can be induced
which two membrane proteins regulate the mitochondrial membrane permeability?
VDAC: voltage-dependent anion channel
adenosine nucleotide transporter
both of these form a complex that acts as a conductance channel
what is the significance of the mitochondrial intermembrane space in apoptosis?
the site where most pro-apoptotic mediators are present (cytochrome c)
what is the mPTP?
mitochondrial permeability transition pore
how is the mPTP activated?
pro-apoptotic initiator proteins bind BCl-xl and Bcl-2. activating pro-apoptotic functions. there is a sudden increase in permeability which causes the mPTP to be constitutively active, small molecules enter and increase osmolarity -> membrane bursts
how is Bax assembled in apoptotic mitochondria?
forms oligomers that assemble into arcs, rings and pores in mt membrane
what are caspases?
a family of cysteine, aspartate-specific proteases that cleave after aspartate residues in a DxxD motif
which caspases are initiators?
caspases 1,9,8, and 10
which caspases are executioners?
caspase 3, 6 and 7
what is the function of the tail in initiator caspases?
mediates protein-protein interactions
what is the difference between the initiator and executioner caspases?
the executioner caspases lack the pro-domain which is synthesised as a pro-enzyme
what is the unifying feature amongst all caspases?
all synthesised as inactive zymogens containing a pro-domain followed by large and small subunits - activated by cleavage of DXXD motifs at interfaces between subunits
what is meant by ‘the proximity-induced dimerisation model’?
initiator caspases must bring two pro-caspases into close proximity. there is trimerisation of receptor, bringing the death domain and fas-associated death domain together in close proximity. death effector domains dimerise, eventually forming the DISC
what is the ‘DISC’?
death induced signalling complex - site where pro-caspases are assembled
what is apaf-1?
apoptotic protease activating factor-1
what are the components of the apoptosome?
Apaf-1, caspase-9, cytochrome c, ATP
outline formation of the apoptosome
1-cytochrome c binds specific domain of apaf1
2- ATP-ADP exchange -
3- dimerisation of pc-9 catalytic domains - bringing pro-caspase into proximity for cleavage to active caspase 9
how are executioner caspases activated?
primary cleave at DXXD motif by initiator caspase, ssecondary cleavage
what is the consensus cleavage site for caspase 3 (Rock1)?
DETD
what are death receptors?
cell surface receptors that transmit apoptotic signals upon binding specific ligands, activating a caspase cascade within seconds of ligand binding
what gene family do death receptors belong to?
tumour necrosis factor
what are the best characterised death receptors?
CD95 (Fas)
TNFR1
TRAIL
how were IAPs first discovered?
first discovered as virally-encoded proteins used by baculovirus as a strategy to suppress apoptosis of host cells
what do IAPs do?
bind directly to caspases, inhibiting their activity
what is a BIR domain?
required for binding to and inactivating caspases
what is Smac?
second mitochondria derived activator of apoptosis
what is Diablo?
direct IAP binding protein with low pI
how can apoptosis be induced?
via extrinsic or intrinsic pathways
how is apoptosis induced in the extrinsic pathway?
involves signals transduced from the outside of the cell via death receptors such as Fas, TNF-Rs, TRAIL
how is apoptosis induced via the intrinsic pathway?
- signals are induced from within cell
- as a result of exposure to damage inducing agent
how might tumour cells evade apoptosis?
up regulation/overactivation of anti-apoptotic proteins
down regulation or inactivation of pro-apoptotic proteins
which pathways are modified in apoptosis in the cancer cell?
- loss of pro-apoptotic signals (e.g. LKB1)
- increase in pro-survival signals e.g. Bcl-2
- checkpoint defects (Loss of checkpoint 2)
- loss of sensitivity to apoptotic signalling
- mutations of DNA damage sensing systems
- oncogene activation
what is the importance of the mTOR kinase?
only pathway that integrates signals from growth factors and nutrients with levels of O2
what is mTOR?
serine//threonine kinase; present as a complex of mTOR/mTOR2
what processes does mTOR control?
cell growth/proliferation
angiogenesis
cell survival
how is p53 regulated?
p53 and mdm2 form an auto regulated feedback loop. mdm2 is a transcriptional target of p53, when synthesised, it inhibits p53
what is ATM?
a kinase that senses DNA damage - activation of these kinases leads to phosphorylation of p53, triggering tetramerisation
induces expression of a number of TFs
what is the basis for B cell lymphoma?
translocation of antibody H chain enhancer on chromosome 14 to chromosome 18, where bcl-2 is located
what advantage does the Bcl-2 translocation in B cell lymphoma offer?/
constitutive expression offers an advantage for tumour cell survival
what is FLIP?
a decoy adapter protein for caspases - over expression is associated with cancer
how do decoy receptors function to regulate apoptosis?
bind trail ligand, but do not activate pro-caspases. prevents apoptosis from becoming activated
what is the difference between the death receptors and decoy receptors?
death receptors have a transmembrane region which recruits adaptors. decoy receptors may lack CTD or have a truncated CTD
in which ways may the receptor and DISC be defective in cancer?
1- epigenetic silencing
2- receptor gene mutation
3- increased expression of decoy receptors
4- increased c-flip expression
what are the 3 Ras genes?
N–Ras
Ha-Ras
Ki-Ras
what is the most common mutation in K-Ras?
glycine-> valine mutation that suppresses the GTPase activity of the protein
what is p10?
commonly mutated tumour suppressor that converts PIP3 back to PIP2
why are mutations to PI3K common in cancer?
1-PI3K phosphorylates the membrane lipid PIP2
2- PIP3 recruits akt, which is phosphorylated at 2 specific residues
3- phosphorylation of pro-apoptotic BAD
mutation to PI3K prevents stimulation of BAD
in PI3K signalling, which protein phosphorylates Akt?
mTOR
what are the therapeutic strategies for targeting apoptosis?
1- inhibiting p53-mdm2 interaction
2- Bcl-2 family inhibitors
3-XIAP inhibitors
4- activation of the trail receptor
what is the therapeutic advantage of mdm2 inhibitors??
- increased levels of p53
- tumour suppressor
how does the drug Nutlin 2 work?
mimics binding of p53 and mdm2 via specific residues on hydrophobic groove (L26, W23, F19)
what are the effects of Nutlin 2 on cancer cells?
- accumulation and activation of p53
- cell cycle arrest in G1 and G2
- inhibits growth of tumours in xenograft models without causing significant toxicity
- cells with WT p53 have increased sensitivity to Nutlin 2
what characterises inhibitor of apoptosis proteins (IAPs)?
presence of 1 or more BIR domains - which bind or inhibit caspases
what is the domain structure of IAP proteins?
BIR domain - caspase binding domain
CARD domain
RING - e3 ubiquitin ligase catalyses transfer of ubiquitin to substrate bound by RING domain. promotes ubiquitinylation and degradation of IAPs and destruction of caspases
