apoptosis. normal development e.g. immune system when does apoptosis occur?
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APOPTOSIS
APOPTOSIS
Normal development e.g. immune system
WHEN DOES APOPTOSIS OCCUR?
Disease states e.g. Alzheimer’s disease
WHEN DOES APOPTOSIS OCCUR?
Amyloid plaques in the brain
NECROSIS APOPTOSIS
TYPES OF CELL DEATH
MorphologicalNECROSIS
• loss of membrane integrity
• swelling of cytoplasm & mitochondria
• total cell lysis• no vesicle
formation• disintegration of
organelles
APOPTOSIS • Membrane blebbing, no loss of
integrity• Aggregation of chromatin at
nuclear membrane• Shrinking of cytoplasm &
nuclear condensation• Fragmentation into smaller
bodies• Formation of apoptotic bodies• Mitochondria become leaky
TYPES OF CELL DEATH
Biochemical
NECROSIS• Loss of regulation of
ion homeostasis• No energy
requirement• Random digestion
of DNA• Postlytic DNA
fragmentaion• Tightly regulated
process
APOPTOSIS • Energy dependent• Non random DNA
fragmentation• Prelytic DNA fragmentation• Activation of caspase cascade• Release of various factors into
cytoplasm
physiologicalNECROSIS
• Affects groups of cells
• Evoked by non physiological disturbances
• Phagocytosis by adjacent cells
• Significant inflammatory response
APOPTOSIS • Affects individual cells• Induced by physiological
stimuli• Phagocytosis by macrophages • No inflammatory response
Caspases – key executioners of apoptosis
(cysteinyl aspartate specific proteases)
Highly conserved proteases14 homolgues inactive zymogens
Caspases divided into
1) Initiator caspases: Caspases 2, 8,9,10 or
2) Effector caspases: caspase 3,6,
Properties of proteases
Irreversible -
Autocatalytic: triggered by cofactor
binding or inhibitor removal
Proteases can regulate their own
activation
Have protease, will have inhibitor
specificity
Caspase structure
3 domains 1) highly variable
NH2 domain2) large subunit (~20kD)3) small subunit
( ~10kD)
Highly specific absolute requirement for cleavage after aspartic acid
recognition of at least 4 amino acids NH2 terminals to the cleavage site
Caspase structure
2 key features: variable N domain regulates activation
all domains derived from proenzyme at cleavage specific sites
Basic apoptotic machinery
DNA fragmentation,
chromatin condensation,
membrane blebbing,
cell shrinkage &
disassembly into apoptotic bodies
engulfment. 30-60 min
effectors responsible for cellular changes associated with apoptosis. Caspases inactivate proteins that protect cells from apoptosis
How do caspases disassemble a cell? It slices, it dices
Selective cleavage of specific proteins
eg bcl-2, or CAD/ICAD
e.g. nuclear lamina
eg. Gelsolin
What triggers apoptosis?
• Loss of contact with surroundings
• Irreparable internal damage
• Conflicting signals for cell division
• Specific ‘death ligands’
How are caspases activated?
More than one way to skin a cat
activation of downstream, effector caspases
Proteolytic cleavage
How are caspases activated?
More than one way to skin a cat
Induced proximity
aggregation of multiple procaspase-8 molecules into close proximity somehow results in cross-activation
How are caspases activated?
More than one way to skin a cat
Holoenzyme formation
Activation of caspase-9 is mediated by means of conformational change, not proteolysis
The roads to ruin
The nematode C.elegans
The roads to ruin Mammals
External signalsdriven by death receptors (DR) e.g. CD95 (or Fas/Apo)
Each CD95L trimer binds to 3 CD95 leading to DD clustering.
FADD ( Fas associated death domain/ Mort 1) binds via its own DD
Caspase –8 oligomerisation drives activation through self cleavage
Caspase –8 then activates downstream effector caspases like caspase –9 (CED-9 homolog)
Apoptosis initiation
Internal signals
BCL-2
apoptosis
DNA damage
Death
receptors
Growth
factor
withdrawal
TRIGGER
P53
Bcl-2 family
Death domain
factor
Cytochrome c
oncogenes
REGULATOR
Apaf-1
Caspases
EXECUTIONER