Apaf-1, the key element of apoptotic mitochondrial pathway, normally exists in an auto-inhibited forminside the cytosol. WRD-domain of Apaf-1 has a critical role in the preservation of auto-inhibited form;however the underlying mechanism is unclear. It seems the salt bridges between WRD and NOD domainsare involved in maintaining the inactive conformation of Apaf-1. At the present study, we have investi-gated the effect of E546-R907 salt bridge on the maintenance of auto-inhibited form of human Apaf-1.E546 is mutated to glutamine (Q) and arginine (R). Over-expression of wild type Apaf-1 and its E546Qand E546R variants in HEK293T cells does not induce apoptosis unlike – HL-60 cancer cell line. In vitroapoptosome formation assay showed that all variants are cytochrome c and dATP dependent to formapoptosome and activate endogenous procaspase-9 in Apaf-1-knockout MEF cell line. These results sug-gest that E546 is not a critical residue for preservation of auto-inhibited Apaf-1. Furthermore, the behaviorof Apaf-1 variants for in vitro apoptosome formation in HEK293T cell is similar to exogenous wild typeApaf-1. Wild type and its variants can form apoptosome in HEK293T cell with different procaspase-3processing pattern in the presence and absence of exogenous cytochrome c and dATP.
