Mitochondrial Pathway

MCB Movie

Apoptosis mitochondrial pathway

begins with permeabilisation of mitochondrial outer membrane

either permeability transition (PT) pore dependent or independent

Permeability Transition (PT) Pore

Structure formed from matrix, inner membrane, outer membrane proteins

matrix protein cyclophilin D

inner mitochondrial membrane protein adenine nucleotide translocator (ANT)

outer mitochondrial membrane protein voltage dependent anion channel (VDAC)

Opening the PT pore

triggers the dissipation of the proton gradient created by electron transport

uncoupling of oxidative phosphorylation

also causes water to enter the mitochondrial matrix

results in swelling of the intermembranal space

rupturing of the outer membrane

releasing apoptogenic proteins - cytochrome c, apoptosis inducing factor (AIF),

endonuclease G

Apoptosome

Apoptosome formation

Formed by Cytochrome c, apoptosis protease activating factor (APAF-1) and procaspase 9 PNAS - Model of apoptosome formation

complex promotes the activation of caspase 9

activates effector caspases that collectively orchestrate the execution of

apoptosis

AIF and endonuclease G - DNA fragmentation and chromosomal condensation

Other proteins released upon mitochondrial outer membrane permeabilisation

Smac/DIABLO - second mitochondria-derived activator of caspases/direct IAPassociated binding protein with low pI)

Omi/HtrA2 (high temperature requirement A2), which antagonize IAPs thereby promoting
caspase activation

Bcl-2 Family

Apoptosis Bax regulation

PT pore independent mitochondrial membrane permeabilisation is regulated by Bcl-2 family

members anchored to the mitochondria membrane by hydrophobic C-terminal
Anti-apoptotic members

Bcl-2 and Bcl-xL

Bcl-2 proteins protect integrity of mitochondria

prevent cytochrome C release

block caspase 9 activation

Pro-apoptotic members

two categories based on expression of Bcl-2 homology (BH) domains

Multi-domain proteins comprise BH domains 1-3 and include Bax, Bak, and Bok

form channels in outer mitochondrial membrane releasing intermembranal space

apoptogenic proteins

BH3 only proteins consists of Bad, Bik, Bid, Puma, Bim, Bmf and Noxa.

activate multi-domain pro-apoptotic species

disrupt the function of anti-apoptotic Bcl-2 family members

Possible Bcl Mechanisms

Formation of a pore - cytochrome c and other intermembrane proteins escape

Heterodimerization - between pro- and anti-apoptotic family members

Direct regulation of caspases - by adaptor molecules

Interaction with mitochondrial proteins - generate a pore or to modulate mitochondrial

homeostasis

Oligomerization- form a weakly selective ion channel

p53
Induced by pro-apoptotic stimuli activates apoptosis by intrinsic mitochondrial pathway
Normally p53 low levels

by murine double minute-2 (MDM2) or the human homologue (HDM2)

inhibit p53 transcription

promote p53 degradation (by proteosome)

Activation of p53

stabilization of p53 by post-translational modifications

disrupt interaction between p53 and MDM2

p53 drives expression of APAF-1 and pro-apoptotic Bcl-2 family members and

transcriptional independent death pathways

Caspases

Apoptosome formation

cysteine proteases

central regulators of apoptosis

14 different caspases identified

expressed as pro-enzymes

activation by proteolytic processing between domains

three domains NH2 terminal, a large subunit 20 kDa and a small subunit 10 kDa

allows association of large and small subunits

active caspases are a tetramer 2 heterodimers of large and small subunits

Initiator caspases

caspase 8, 9, 10 and 12

closely linked to pro-apoptotic signals

once caspases activated cleave and activate effector

Effector caspases

caspases 3, 6, and 7

cleave cytoskeletal and nuclear proteins

induce apoptosis

FasL and Fas-expressing Cells

Caspase Cleavage

leads to diverse results

depending on substrate and position of cleavage site

loss of biological activity eg lamin network

limited proteolysis by caspases result in a gain of biological activity eg Bcl-2 or Bcl-x,

PAK2, Bid and CAD/ICAD

Caspase Activation Mechanisms

(a) proteolytic cleavage by an upstream caspase activation of downstream effector caspases
induction of non-caspase proteases (granzyme B)
(b) Induced proximity aggregation of procaspase-8 molecules somehow results in crossactivation
(c) holoenzyme formation cytochrome c and ATP dependent oligomerization of Apaf-1
recruitment of procaspase-9 into apoptosome complex Activation of caspase-9 mediated by
conformational change

Apoptosis Inhibitors

directly inhibit caspases or prevent activation

Akt

serine/threonine protein kinase

important anti-apoptotic factor

inhibits pro-apoptotic Bcl-2 family member, Bad

directly inhibits caspase 9

TNF-alpha

Tumour Necrosis Factor -alpha

both anti-apoptotic and pro-apoptotic effect

activate the transcription factor NF-kB

which then induces expression of IAP

an inhibitor of caspases 3, 7,and 9

Neurotrophins

regulate apoptosis through protein kinase cascades

phosphoinositide 3-kinase/Akt

mitogen-activated protein kinase pathways

Final Stages of Apoptosis

The membrane enclosed cell fragments are phagocytosed by macrophages and other
cells.