Proteases 6 Flashcards
What are the three main pathways to apoptosis?
The intrinsic (mitochondrial) pathway
Extrinsic (death receptor) pathway, Cytotoxic T lymphocytes
All of which converge at the level of the caspases
What is the intrinsic pathway to apoptosis?
Certain conditions lead to the activation via phosphorylation of p53 which is typically in the cytosol and inactivated by being associated with the MDM2 regulator
Activation leads to a longer half life via less proteasomal degradation of p53
P53 can act as a transcriptional activator of certain nuclear pro-apoptotic genes through translocating to the outer membrane of the mitochondria
Resulting the release of mitochondrial cytochrome c into the cytosol
This complexes with other cytosolic factors to lead to the formation of a cytosolic apoptosome
This is then activated leading to recruitment of a pro-caspase-9
This oligomerizes leading to auto activation this then activates downstream effector caspases likeprocaspase3
What is the model apoptosome formation?
The cytosolic Apaf-1 protein is associated with dATP
If present in the cytosol mitochondrial cytochrome C will bind to Apaf-1 allowing to hydrolyse the dATP to dADP
If there is sufficient dATP the dADP will be exchanged
Seven apaf-1/cytochrome c/dATP units combine to form the active apoptosome
What are the functional domains on apaf-1?
An N terminal caspase recruitment domain (CARD) which recruite the procaspase-9
A middle nucleotide-binding and oligomerization domain (NOD)
A C-terminal regulatory domain which normally keeps apaf-1 in an autoinhibitory state but binds cytochrome c when present to become active
What does caspase 3 do?
cleaves cellular substrates (breakdown of cytoskeleton, activation of endonucleases which in turn fragment nuclear DNA) which brings about de facto apoptosis
What is the extrinsic death pathway?
Interaction of a death receptor ligand (external stimuli) with a death receptor in the cellular plasma domain
Adapter proteins link death receptors to caspases resulting in autoactivation of the caspases to produce a death-inducing signalling complex which goes on to activate the effector caspase 3
What are death receptors?
Transmembrane proteins with several extracellular cysteine rich domains, trans membrane segment and a death domain of approx. 80 amino acids
What is CD95 and how does it function?
A constitutively expressed death receptor triggered by CD95L released by activated T cells and triggers apoptosis when it crosslinks multiple CD95 receptors causing an open conformation allowing the FADD adaptor protein to align its death domain with that of the receptor
The DED domain of FADD then dimerises with procaspase 8 or 10 inducing a conformational protein allowing the caspases to be active and cleave themselves to from the death inducing signalling complex
What is the cytotoxic T route to apoptosis?
Can bring about caspase activation without the need for mitochondria or death receptors by using their granules which contain granzymes and perforins
If a cell is designated to be attacked then the perforins are used to punch holes in the cells and the granzymes are used to activate caspases
What occurs downstream of the effector caspases?
The cell shrinks as the cytoskeleton is broken down due to cleavage of lamin and actin filaments
The nuclear structural proteins and chromatin (DNA) breakdown slowly causing the shrink and horseshoe shape of the nucleus
The condensing of the cell is a shape that is easily recognizable by macrophages
Membrane blebs appear along with apoptotic bodies and the translocation of phosphatydyl serine to the outer membrane
