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

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• 作者：Raheleh Shakeri^a ; Saman Hosseinkhani^b ; ^{saman_h@modares.ac.ir" class="auth_mail" title="E-mail the corresponding author} ; Marek J. Los^c ; ^d ; Jamshid Davoodi^a ; Mayur V. Jain^c ; Artur Cieślar-Pobuda^c ; ^e ; Mehrdad Rafat^f ; Sussan Kaboudanian Ardestani^a ; ^{ardestany@ibb.ut.ac.ir" class="auth_mail" title="E-mail the corresponding author}
• 关键词：Apaf-1 ; apoptotic protease activating factor-1 ; CARD ; caspase recruitment domain ; NOD ; nucleotide-binding and oligomerization domain ; WRD ; WD40-repeat domain ; NBD ; nucleotide-binding domain ; HD1 ; helical domain 1 ; WHD ; winged-helix domain ; HD2 ; helical domain 2 ; Apaf-1-knockout MEF ; Apaf-1-knockout murine embryonic fibroblasts ; HEK293T ; human embryonic kidney 293T ; HL-60 ; human myeloid leukemia ; Mw ; molecular weight
• 刊名：International Journal of Biological Macromolecules
• 出版年：2015
• 出版时间：November 2015
• 年：2015
• 卷：81
• 期：Complete
• 页码：370-374
• 全文大小：979 K

文摘

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.