APOPTOSIS

APOPTOSIS

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

Disease states e.g. Alzheimer’s disease WHEN DOES APOPTOSIS OCCUR? Disease states e.g. Alzheimer’s disease Amyloid plaques in the brain

TYPES OF CELL DEATH NECROSIS APOPTOSIS

Morphological NECROSIS loss of membrane integrity TYPES OF CELL DEATH Morphological NECROSIS 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

Biochemical NECROSIS APOPTOSIS Energy dependent 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

physiological NECROSIS APOPTOSIS Affects individual cells 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 proteases 14 homolgues inactive zymogens Caspases divided into 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 domain 2) 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 Proteolytic cleavage activation of downstream, effector caspases

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 signals driven 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 TRIGGER REGULATOR EXECUTIONER DNA damage Death receptors Growth factor withdrawal P53 Bcl-2 family Death domain factor Cytochrome c oncogenes Apaf-1 Caspases