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

• 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. (C) 2015 Elsevier B.V. All rights reserved.

Ämnesord

Medical and Health Sciences  (hsv)

Medical Biotechnology  (hsv)

Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)  (hsv)

Medicin och hälsovetenskap  (hsv)

Medicinsk bioteknologi  (hsv)

Medicinsk bioteknologi (med inriktning mot cellbiologi (inklusive stamcellsbiologi), molekylärbiologi, mikrobiologi, biokemi eller biofarmaci)  (hsv)

Nyckelord

Apaf-1; Apoptosome; Caspase-9