Role of the salt bridge between glutamate 546 and arginine 907 in preservation of autoinhibited form of Apaf-1

• Published in: International journal of biological macromolecules 2015-11, Vol.81, p.370-374
• Main Authors: Shakeri, Raheleh, Hosseinkhani, Saman, Los, Marek J., Davoodi, Jamshid, Jain, Mayur V., Cieślar-Pobuda, Artur, Rafat, Mehrdad, Ardestani, Sussan Kaboudanian
• Format: Article
• Language: English
• Subjects: Animals; Apaf-1; Apoptosome; Apoptosomes - metabolism; Apoptotic Protease-Activating Factor 1 - chemistry; Apoptotic Protease-Activating Factor 1 - genetics; Apoptotic Protease-Activating Factor 1 - metabolism; Arginine - chemistry; Arginine - metabolism; Caspase 9 - metabolism; Caspase-9; Codon; Cytochromes c - metabolism; Deoxyadenine Nucleotides; Gene Expression; Gene Knockout Techniques; Glutamic Acid - chemistry; Glutamic Acid - metabolism; HEK293 Cells; Humans; Mice; Models, Molecular; Mutation; Protein Conformation
• ISSN: 0141-8130; 1879-0003; 1879-0003
• DOI: 10.1016/j.ijbiomac.2015.08.027

Apaf-1, the key element of apoptotic mitochondrial pathway, normally exists in an auto-inhibited form inside 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 domains are involved in maintaining the inactive conformation of Apaf-1. At the present study, we have investigated 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 E546Q and E546R variants in HEK293T cells does not induce apoptosis unlike – HL-60 cancer cell line. In vitro apoptosome formation assay showed that all variants are cytochrome c and dATP dependent to form apoptosome and activate endogenous procaspase-9 in Apaf-1-knockout MEF cell line. These results suggest that E546 is not a critical residue for preservation of auto-inhibited Apaf-1. Furthermore, the behavior of Apaf-1 variants for in vitro apoptosome formation in HEK293T cell is similar to exogenous wild type Apaf-1. Wild type and its variants can form apoptosome in HEK293T cell with different procaspase-3 processing pattern in the presence and absence of exogenous cytochrome c and dATP.