site-directed mutagenesis in Apaf-1 gene for determining the mechanism of action using split luciferase

Apoptosis (programmed cell death) is considered a vital component of various processes including normal cell turnover, proper development and functioning of the immune system, hormone-dependent atrophy, embryonic development and chemical-induced cell death and is initiated through intrinsic and extrinsic pathways. Inintrinsic pathway, different stimuli cause cytochrome c to be released from mitochondria, which is then bindsand activates Apaf-1 as well as procaspase-9, forming an apoptosome. Binding of cytochrome c, induces someconformational changes in Apaf-1 and put it in an appropriate stereochemical structure to switch theautoinhibited state of Apaf-1 into active form. In this study we have tried to make better understanding of the molecular mechanism in which Apaf-1 molecules change into active state. For this, a point mutation was introduced into cloned Apaf-1 gene by quick chang PCRin order to disrupt a critical salt bridge, which connects two different domains in Apaf-1. Based on X-raycrystallographic data, this disruption is necessary for the formation of apoptosome. Thereafter, the mutant gene transfected into Human Embryonic Kidney 293 cells (HEK293) and formation of apoptosome complex was assayed through split luciferase. By inducing apoptosis in transfected cells, no apoptosome formation and no caspase activity was observed, which inferred that the Apaf-1 molecules were locked in their autoinhibited state.

Keywords:

Apaf-1 , Apoptosis , Split luciferase , apoptosome , cytochrome c