apoptosis Flashcards
why do we need programmed cell death *
remove harmful cells eg cells with a viral infection/dna damage
developmentally defective cells eg B lymphoscyetes expressing Ab against self-ag
excess/unnecessary cells - embryonic development ie in brain to eliminate excess neurons, liver regeneration, sculpting digits (apoptosis of the membrane between the digits) and organogenesis
obsolete cells - eg mammary epithelium is apoptosed at end of lactation to return the breast to the normal size
exploitation - used in chemo - induce apoptosis to reduce number of tumour cells
what is the TUNEL technique
labels apoptopic cells
what is necrosis *
unregulated cell death associated with trauma cellular disruption and an inflammatory response
what is apoptosis *
programmed cell death
regulated cell death, controlled disassembly of cellular contents without diruption - no inflammatory response
it is stimulated and timely
describe pathway of necrosis *
the plasma membrane becomes permeable
water enters = cells swell = rupture of cell membranes = everything is deregulated because it is not contained in the plasma membrane
chromatin condenses
dissolution of cellular structure - degregation of organelles
lysis
release of proteases leading to autodigestion and dissolution of the cell
phagocytes clean the debris
causes localised inflamm because proteins are at sites that they shouldnt be
neighbouring cells proliferate and fill the gap
what is the pathway of apoptosis *
can be localised - ie only 1 cell effected
latent phase
- death pathways are activated (molecular changes) but cells appear morphologically the same
execution phase
- loss of microvilli and intracellular junctions (comprimise the permeability of a cell)
- cell shrinkage
- loss of plasma mem asymmetry (normally membrane at top of cell is differnet from that at bottom etc ie phosphatidylserine lipid appears in outer leaflet of the lipid bilayer, normally only on inside)
- chromatin and nuclear condensation
- DNA fragmentation
- surrounding epi close around the apoptotic cell - restores permeability
- formation of membrane blebs
- fragmentation into membrane enclosed apoptopic bodies
- apoptotic boduies are phagocytosed by neighbouring cells and and reving macrophages
plasma membrane remains intact - therefore no inflammation because there is no release of contents out of the cell
SEM of apoptotic bodies *
dark areas are the apoptotic bodies - being taken up by macrophages
describe DNA modification in apoptosis, and how it can be seen *
DNA ladders - fragmentation - separate the DNA based on size in agarose gel
at 0 mins the DNA is intact - bulky and too large to go into the gel - the is DNA condensation but no freagmentation in the nucleus
at 45mins - intact DNA disappears and can see ladder of fragmented DNA
this is increased at 70 mins - blebs of DNA
using TUNEL assay - the flurescence labels fragments of DNA - cells that have flurescened nuclei are apoptotic - those that dont are not
what are the 4 main types of cell death and explain them *
necrosis - unregulated cell eath associated with cellular disruption ad an inflammatory response
apoptosis - programmed cell death (PCD) - regulated, controlled disassembly of cellular contents, no inflammation
apoptosis-like PCD - some but not all features of apoptosis, display phagocytic recognition molecules before plasma membrane lysis
necrosis-like PCD - variable features of apoptosis before cell lysis - called aborted apoptosis
is there a graded response of cell death *
yes - apoptosis at one end and necrosis at other
many differnet types in middle
apoptosis requires ATP, necrosis doesnt
what are the mechanisms of apoptotic cell death *
the executioners - caspases
initiating the death program via death receptors/mt - sequential actions of proteins leading to steps in cell
the Bcl-3 family
stopping the death program
what are caspases *
Cysteine-dependant ASPartate-directed proteASES
they are proteases with specific cysteine and aspartate recognition sites
they are the excutioners of apoptosis
need to be tightly regulated - they are present in inactive form(auto-folded on itself or as dimers) and are activated by proteolysis
describe the structure of initiator caspases, what are the 4 initiator caspases *
they are caspase 2, 9, 10 and 8
they have homogous domains
2 and 9 have CARD - CAspase Recruitment Domain - this domain places the caspases at a specific subsite in a cell
all have p20 and p10 domains
caspase 10 and 8 have DED domains - Death Effector Domains
10 and 8 undergo homotypic protein-protein interactions ie 8 binds to 8
describe the structure of effector caspases, what are the effector caspases *
they are caspase 3 6 7
they have p20 and p10 domains
describe caspase maturation *
caspases are synthesised as procaspases/zymogens - have prodomain and LS and SS domains
prodomain is cleaved and procaspase is activated by dimerisation
SS domain is also cleaved - so only LS domains are involved in dimerisation (ie a light and heavy subunit are left)
the folding of 2 large adn 2 small chains forms an active L2S2 heterotetramer
eg heterodimerisation of capsase 8 and 3 - therefore they are close together so can synchronise the following steps
summarise the caspase kinases *
there is amplification, divergent responses (depending on caspases involved) and regulation
caspase 8 and 9 are the initiater caspases nd tigger apoptosis by cleaving and activating 3 and 7
3 and 7 are the effector caspases that carry out the apoptotic program
caspase 3 actiaves 6 2 and 1
caspase 10 feedsback tp 8
describe the 4 actions of the effector caspases *
cleave and inactivate proteins or complexes
- monomeric substrates
- multiprotein complexes eg nuclear lamins - lead to nuclear breakdown
activate enzymes - including protein kinases, nucleases eg Caspase-Activated Dnase (CAD)
- direct cleavage
- cleavage of inhibitory molecules
what does CAD do *
clip DNA in nuclear fragmentation
what are the2 mechanisms of caspase activation *
death by design - receptor mediated (extrinsic) pathways - sense something on outside of cell
death by default - mt (intrinsic) death pathway - no stim outside of cell
describe death receptors in extrinsic pathway *
the ligands are found in trimeric form (3 molecules forming 1 unit of recognition)
all have single transmembrane domain
have extracellular domains of varying composition - all cysteine rich
intracellular have different cytoplasmic tails - all have Death Domain (DD) to hook specific kinases
the receptors recruit adaptor proteins that connect to other proteins
descrieb the adaptor proteins in receptor-mediated apoptosis *
they are FADD or FLIP
contain conserved domains
FADD - DED (death effector domain) and DD (death domain)
FLIP - DED and DED
FADD - activates apoptosis
FLIP - inactivates it
DD and DED are modular domains and bind to similar domains on other proteins
describe the signalling from the death receptors *
Fas death receptor trimerisation occurs as it binds to death ligand Fas-L on lymphocyte (trimeric)
this causes recruitment of adaptor protein FADD
the DD on the receptor (Fas) binds to DD on FADD
the DED on FADD binds to DED on procaspase 8 - this is recruitment and oligomerisation of procaspase 8 - this mores the Death-Inducing Signalling Complex (DISC)
3 procaspase 8s are recurited to the trimeric receptor
this causes oligomerisation which allows transcleavage and activation of caspase 8 - active initiator caspase 8 teteramer is released from the receptor
[some initiator procaspases have low intrinsic activity - the oligomerisation allows transcleavage, others are activated by a conformational change on oligomerisation; either way need at least 2 procaspases to form an active tetramer because one has to activate the other]
describe the inhibition of death receptor activation of caspase 8 *
happens by FLIP
short and long FLIP - short doesnt have p20 and p10 domains, long does
both FLIP has DED - but no proteolytic activity - therefore competes with procaspase for DED of FADD
FLIP is incorperated into the receptor-procaspase complexes and interfers with transcleavage
describe the mitochondrial regulation of apoptosis *
cellular stress eg lack/overstim by GF, DNA damage (p53), ROS cause mt to lose membrane potential
this causes mt to release Cyt c and other factors
this triggers the formation of the apoptosome complex
describe the apoptososme *
it is a localising signal - brings in proteins to activate the pathway
main proteins are Apaf-1 (apoptotic activating factor 1), Cytochrome C, ATP and procaspase 9
Apaf-1 has an ATPase, CARD domain, WD-40 repeats (WD at C terminus)
WD-40 repeats are modular structures that mediate protein-protein interactions
Apaf-1 proteins oligomerise as a heptamer - form a wheel; CARD domain is in centre, WD-40 are at edge
WD-40 domain interacts with cyt C that is released form mt
CARD domain binds to procaspase 9 dimer = oligomerisation bringing 9s together = cleavage and release as active caspase 9 tetramer initiates a cascade leading to apoptosis ie caspase 3 activated
this requires ATP and cytochorme C
describe how the intrinsic and extrinsic mechanisms of apoptosis are linked *
caspase 8 that is activated in extrinsic pathway cleaves Bid
Bid enhances the release of mt proteins - ie increases the intrinsic pathway
describe modulation of the mitochondrial pathway *
modulators are Bcl-2 proteins - divided into 3 groups
- gp 1 - Bcl-2 - contains Bcl-2 homologous domains - 4 in transmembrane domain hooked at mt
- gp 2 - Bax - doesnt have Vcl-2 homology 4
- gp 3 - Bid Bik Bad - have differnet combinations of the domains - some are not membrane bound
all have BH3 domain that forms the dimerisation motif
describe the Bcl-2 family proteins *
anti-apoptotic - Bcl-2 and Bcl-xl - hooked by membrane of the mt
pro-apoptotic - Bid, Bad, Bax, Bak = move between cytosol and mt
describe the PI3’-kinase signalling pathway *
PI3’ kinase regulate teh cell cycle and apoptosis
GF stimulation (eg EGF, insulin) can = Ras = Erk = growth
GF stim can also stim Pi3’K = PDK-1 = PKB/Akt = survival and proliferation
the phosphorylated GF receptor recruits PI3k - has p85 nad p110 components - p85 is adaptor protein and recognises tyrosine kinase domains
p85 is hooked to kinase domain p110
together p85 and p110 form - Phosphatidylinositol 3’-kinase (PI3’-K) – a lipid kinase
p110 is close to the membrane that contains a lot of lipid so will phosphorylate the PI4,5 bisphosphate (PIP2) to PI3,4,5 triphosphate (PIP3)
PIP3 provides docking sites for many proteins
via the PH domain - PIP3 recruits and activates protein kinase PKB/Akt that will have an anti-apoptotic effect and transduce mitogenic signals
describe the action of PKB/Akt *
it phosphorylates and inactivates Bad
phosphorylates and inactivates capsase 9 (main regulator of the intrinsic pathway)
inactivates FOXO TF (FOXOs promote the expression of apoptosis promoting genes)
stimulates ribosome production and protein synth - necessary for functioning and proliferating cells
summarise the regulation of apoptosis by Bcl-2 when GF is present *
GF present = PKB/Akt active = phosphorylation of Bad (phospho=Bad) - therefore Bad is kept in the cytoplasm by 14-3-3- inactive
Bcl-2 or Bcl-xl and Bax heterodimerise by BH3 and are inactivated
= cell survival
summarise the regulation of apoptosis by Bcl-2 when GF is not present *
Bad is dephosphorylated - not in cytoplasm - goe sinto mt and displaces Bcl-2/xl from Bax Bcl-2 heterodimer
Bax forms a pore at the mt mem = release of Cyt C = apoptosis
describe PTEN *
it is a lipid phosphtase
converts PIP3 to PIP2
tehrefore PKB/Akt are not produced
describe Inhibitor of Apoptisis Proteins (IAPs) *
regulate the extrinsic process of apoptosis
they bind to procaspases and prevent activation, or bind to activated caspases and inhibit their activity
what are the cytoprotextive/anti-apoptotic pathways *
Bcl-2/xl
FLIP IAPs
PI3’-K
how can cancer cells avoid apoptosis *
apoptosis regulators as oncogenes or tumour suppressors
how can we use apoptosis therapeutically *
onchogenic cells whave DNA damage
in chemp dexamethasone stimulates DNA cleavage
