Biochemical Pathways in Apoptosis

Caspases are a family of proteases that once activated execute apoptosis, a cellular suicide pathway. Activated caspases have a unique property to cleave and activate themselves. Once the first caspase is activated, it generates a chain reaction resulting in robust caspase activity and rapid death. The central question in apoptosis is to understand how the first caspase is activated. To address this question, we present an assay that recapitulates de novo caspase activation in vitro. We describe how this assay was used to purify three proteins that were sufficient to reconstitute caspase activation in vitro: Apaf-1, cytochrome c, and caspase-9. The mechanism of caspase activation in vivo, however, is complicated by these proteins subcelluar localization. In the living cell, Apaf-1 and caspase-9 are cytoplasmic whereas cytochrome c is mitochondria, however, during apoptosis, cytochrome is released to the cytoplasm. In vitro, cytochrome c induces the formation of a stable Apaf-1/caspase-9 complex and caspase-9 autoactivation suggesting that cytochrome c release from the mitochondria to the cytosol is the rate-limiting event that leads to caspase activation. This conclusion shifted the focus of our studies upstream from what initiates caspase activation to what triggers cytochrome c release. The second part of the dissertation uses a biochemical approach to identify how cytochrome c release is regulated after exposure to ultraviolet light. Ultraviolet light irradiation of HeLa cells triggers an apoptotic response mediated by mitochondria. Biochemical analysis of such a response revealed that the initial step leading to cytochrome c release is the complete disappearance of the mRNA of Mcl-1, an anti-apoptotic member of the Bcl-2 family. This event leads to the elimination of Mcl-1 protein from cells due to the short half-life of its protein. The block or delay of Mcl-1 disappearance by either proteasome inhibitors or Mcl-1 over-expression prevents subsequent steps of this apoptotic pathway including the translocation of Bax and Bcl-xL from cytosol to mitochondria and dephosphorylation of BimEL on mitochondria. These sequential events lead to the oligomerization of Bax and Bak on the mitochondria, cytochrome c release and caspase activation.