APOPTOSIS. Normal development e.g. immune system WHEN DOES APOPTOSIS OCCUR?

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