Apoptosis Part 1

necrosis

• swelling and bursting of cells

• loss of membrane integrity

• generates inflammatory signals from release of contents

apoptosis - what happens to structure of cell

• Cell shrinkage

• Cytoskeleton collapses

• Loss of nuclear membrane

• Chromatin condenses and DNA is cleaved into fragments

• Membrane blebs which break off into apoptotic bodies

• Cell surface alters to attract phagocytes

autophagy basic overview

• happens when cells lack nutrients 

• digestion of internal structures

• plasma membrane remains intact

• may not lead to cell death 

are all capsases involved in apoptosis 

no

function of caspases

• endopeptidases cleave after an aspartate in the substrate

• regulate apoptosis

caspase activated DNase (CAD)

breaks down DNA as part of apoptosis

bound to an inhibitor

inhibitor of caspase activated DNase

• caspase substrate 

• caspases bind to inhibitor, cleavage

• CAD dimerises and can cut any open DNA

• happens at regular intervals between histones 

iPLA2 and caspase 3

• caspase 3 cleaves iPLA2 to activate it

• phosphatidylcholine → iysophosphatidylcholine

• this signals for apoptosis

Xkr8

phospholipid scramblase that promotes phosphatidylserine exposure on apoptopic cell surface

caspase 3 interaction with Xkr8

• cleavage by caspase 3

• disregulates phospholipids

• phosphatidylserine exposure on cell surface

• acts as receptor, attracting macrophages

are flippases activated or inactivated by executioner caspase mediated cleavage

inactivated

are scramblases activated or inactivated by executioner caspase mediated cleavage

activated

size of pro domain for executioner and initiator caspases

• executioner caspases - small pro domain

• initiator caspases - large pro domain

executioner caspases examples

• 3, 6 and 7

• exist as inactive dimers

active sites of executioner caspases

• 2 potential on each side

• connecting loop from large and small subunit prevents from being exposed

• cleavage by initiator caspase causes rearrangement to expose

caspase cascade

• each initiator caspase can activate many copies of one or more executioner caspases

examples of pro enzyme initiator caspases 

mainly 8 and 9

problem with spontaneous dimerisation of initiator caspases

conc is too small

two types of pro domain in proenzyme initiator caspases

• death effector domain DED

• CAspase recruiter domain CAD

caspase 8, 9 and 1 activation

• dimerisation

• N-terminal pro domain recruiting the monomers to an activating complex 

apoptosome formation and dimerisation of caspase 9

• WD-40 of APAF-1 binds to cytochrome c

• binding of dATP creates heptamer

• CARD domains cluster in centre

• CARD domains interact with caspase-9 CARD domain monomers

• allows dimerisation

APAF-1

• cytosolic monomer that assembles into a heptamer when it binds to cytochrome c

• binds to caspase-9 through the CARD-CARD domains

how is cytochrome c recruited outside of the mitochondria

• MOMP - mitochondria outer membrane permeabilisation

• Bcl-2 protein family controls this

disocvery of Bcl-2

• follicular lymphoma

• translocated oncogene t(14;18) stopped lymphoma cells from dying

• due to over expression of Bcl-2

pro apoptopic Bcl2 family 

• Bak

• Bax

pro apoptopic BH3 only proteins

• Bad

• Bim

• Bid

• Puma

• Noxa

anti apoptopic Bcl2 family proteins

Bcl2, BclXL

advantage of differences in apoptopic priming

• sensitivity to apoptopic signals

• shows how cancer cells respond to chemotherapy

• can influence toxic side effects from chemotherapy and radiotherapy

threshold for BH3 explained

• if there is inc of damage, anti apoptopic Bcl-2 proteins will be inhibited

• if damage is over threshold, BH3 will interact with Bax/Bak, inc permeability of OMM

• apoptosis

what happens if anti apoptopic protein Bcl-2 inc with t(14;18)

• over expression of Bcl-2

• same amount of damage, never exceeds threshold

• cells are resistant to apoptosis

p53

transcription factor usually activated by kinases from DNA damage 

• transcription of PUMA, activates Bax → apoptosis 

which mutation is present in 50% of cancers

p53

how does bad work - phosphorylation and no phosphorylation outcome

No apoptosis:

• phosphorylation through kinase pathway

• allows binding of Bad to chaperone protein 14-3-3

• moves Bad away from mitochondria so no apoptosis

Apoptosis:

• no phosphorylation of bad

• bad binds to the mitochondria

• apoptosis

effect of oncogene and tumour suppressor gene mutations

• shift the trreshold for Bcl-2 regulation of MOMP

• over expression of EGF receptor

• so over expression of Bcl-2

• so not enough Bad to activate Bax for MOMP

• no apoptosis

venetoclax

• ABT-737 bound to hydrophobic groove at very low affinity - inhibited Bcl-2 proteins in tumours and promoted apoptosis

  • mimics BH3- only proteins

  • was modified to venetoclax, inc specificity, less side effects

  • drug reduces excess Bcl-2 to reset the apoptopic threshold

which proteins restrain the extrinsic pathway

FLIP

XIAP

• inhibits caspase 9 (initiator), caspase 3 and 7 (executioners)

which protetins bind to XIAP

• anti-IAP

• prevent it from inhibiting caspases

• promotes caspase activation and apoptosis