Biochemical Analysis of Apoptosome Formation
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Apoptosis is an active cell death program executed by proteases named caspases. One of the major caspase-activating pathways is initiated by mitochondria. Upon various apoptotic stimuli, the mitochondria releases cytochrome c into the cytosol where it binds to apoptotic protease activating factor 1 (Apaf-1). Then, the cytochrome c-bound Apaf-1 forms a heptameric complex named apoptosome. Apoptosome provides a platform to activate downstream caspases. The initiator caspase, procaspase-9 is recruited to apoptosome and gets activated to cleave downstream effector caspases. The series of this activation cascade is tightly regulated at each step. However, the role of cytochrome c and nucleotides during apoptosome formation is not clear. Also, how the apoptosome activity is stimulated by the positive regulator PHAP proteins is yet to be determined. Thus, my thesis work includes studies regarding these questions using biochemical analysis. I reconstituted apoptosome pathway using recombinant proteins in vitro. As a result, I discovered several biochemical steps during apoptosome formation that were previously unknown. And I identified a new mediator that positively regulates apoptosome formation. The new findings are, 1) Recombinant Apaf-1 obtained from insect cell expression system was already associated with dATP. 2) The Apaf-1-bound dATP got hydrolyzed upon cytochrome c binding. 3) The hydrolyzed nucleotide on Apaf-1 needed to be exchanged with dATP/ATP to form an active apoptosome. 4) CAS is a mediator of PHAP proteins. PHAPI and CAS enhanced the nucleotide exchange on Apaf-1 to stimulate apoptosome formation.