Smc - a Mitochondrial Protein That Promotes C-Dependant Caspase Activation by Eliminating Iap Inhibition

Introduction to biochemical methodologies

Paper1- Smac, a Mitochondrial protein that promotes c-Dependant Caspase Activation by eliminating IAP inhibition

Describe what triggered the investigations of this paper.

• Scientists discovered that certain types of cells are not responsive to microinjected cytochrome c. They performed a test on healthy neurons and observed that they do not respond to microinjected cytochrome c unless they have been subjected to NGF (nerve growth factor) withdrawal for a certain period of time which gave these neurons the status “competent to die”. This suggests additional caspase-activating protein factors that are potentially regulated by the Bcl-2 family proteins that also regulate cytochrome-C

Briefly discuss the proposed mechanism of action of Smac and the evidence provided for this mechanism in the paper.

• They found that Smac stimulates caspase activation but they have 2 theories as to how this occurs:

1. Eliminating an inhibitor(s) in the S-100

2. Or directly acting on Apaf-1, cytochrome C and procaspase-9 to enhance the activation efficiency of caspases.

• Smac was coupled with nickel andwas added to S-100, this was then centrifuged and tested for caspase-3 activation. Result: activation was observed to be higher in the S-100 treated with Smac and nickel compared to that of the S-100 treated with just nickel.
• Adding free Smac to extracts with uncoupled nickel were responsive whereas extracts which had already been exposed to Smac did not increase in caspase-3 activity
• They tested whether the inhibitors that came out from the aforementioned process was IAPs, using Western Blot Analysis.
• Adding Smac to the extract that does not have inhibitors anymore, didn’t increase caspase-3 activity, which therefore suggests that Smac does not activate caspase-3 but rather inhibits the IAPs, therefore allowing caspase-3 to act.