BCH3042 Lecture Notes - Lecture 15: Lysis, Caspase 3, Western Blot
25 May 2018
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Lecture 15 – Apoptotic Signalling Pathways, Methods and Models I
Caspases Help Dictate the Cell Death Pathway
• Caspases accelerate apoptosis
• But caspase activity is dispensable for cell death within the intrinsic pathway
of apoptosis (shift is to necrosis)
• Mitochondrial outer membrane permeabilisation (MOMP) is the key
checkpoint (the point of no return)
o Holes in mitochondria – proteins go out and mitochondria is non-
functional → makes no ATP → dies
• Intrinsic (mitochondrial) pathway
• Extrinsic (death receptor) pathway
o Both lead to caspase activation
• Cytochrome c drives apoptosis
Apoptosis and Mitochondrial Connection
• Reagents used didn’t include sucrose – no
osmotic balance → water goes in and
mitochondria membrane englarges and
bursts → releases proteins from
mitochondria into intermembrane space
• Cleabage by western blotting
Development of an in Vitro Assay
• HeLa cell lysate (S-100)
• Monitor caspase 3 cleavage
o Caspase 3 (aka CPP32): cleaved into large (p20) and small (p10)
subunits
▪ Cleavage by western blotting
• Monitor proteolysis of caspase substrates (& end products)
o PARP
o SREBP
o DNA Cleavage
• Use assay to isolate factor that activates caspase 3
• Addition of ATP causes cleavage
• Apoptosis activating factors
o Apaf1 – in FT fraction (PC-FT)
o Apaf 2 – in bound fraction (PC-B) = cytochrome c
Cytochrome c has Dual Functions
• In cytosol of apoptotic cells
• Required for respiratory chain
• Encoded by nuclear gene and
imported into intermembrane space,
folds and binds heme
• Heme bound form is active in
apoptosis – cyt c needs to be
released from mitochondria to
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