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

2015; Elsevier BV; Volume: 81; Linguagem: Inglês

10.1016/j.ijbiomac.2015.08.027

1879-0003

Raheleh Shakeri, Saman Hosseinkhani, Marek Łoś, Jamshid Davoodi, Mayur Vilas Jain, Artur Cieślar‐Pobuda, Mehrdad Rafat, Sussan Kaboudanian Ardestani,

ATP Synthase and ATPases Research

Apaf-1, the key element of apoptotic mitochondrial pathway normally rests in an autoinhibited form inside the cytosol.Typically, cellular stress-triggered cytochrome c release from the mitochondria and its binding to Apaf-1 is required to transform the "closed" (inactive) molecule to open form.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 domain are involved in maintaining the inactive configuration 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 full length human Apaf-1.E546residue is mutated to glutamine (Q) and arginin (R) and the effects of such aa exchange on apoptosome formation and procaspase-9 activation in the presence or absence of exogenous cytochrome c and dATP is evaluated.Overexpression of wild type, E546Q and E546R Apaf-1 in HEK293T cells does not induce apoptosis unlike in HL-60 cancer cell line.In vitro apoptosome formation assay showed that all mutants 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, exogenous (overexpressed) Apaf-1 can form an in vitro apoptosome in the absence of exogenous cytochrome c and dATP.Exogenous Apaf-1 in the presence of exogenous cytochrome c and dATP processes procaspase-3 in two sites.