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.
Institute of Biochemistry and Biophysics, Department of Biochemistry, University of Tehran, Tehran, Iran.
Raheleh
R
Shakeri
Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran. Electronic address: saman_h@modares.ac.ir.
Saman
S
Hosseinkhani
Department of Clinical & Experimental Medicine (IKE), Division of Cell Biology, Integrative Regenerative Med. Center (IGEN), Linköping University, Linköping, Sweden; Department of Pathology, Pomeranian Medical University, Szczecin, Poland.
Marek J
MJ
Los
Institute of Biochemistry and Biophysics, Department of Biochemistry, University of Tehran, Tehran, Iran.
Jamshid
J
Davoodi
Department of Clinical & Experimental Medicine (IKE), Division of Cell Biology, Integrative Regenerative Med. Center (IGEN), Linköping University, Linköping, Sweden.
Mayur V
MV
Jain
Department of Clinical & Experimental Medicine (IKE), Division of Cell Biology, Integrative Regenerative Med. Center (IGEN), Linköping University, Linköping, Sweden; Institute of Automatic Control, Silesian University of Technology, Gliwice, Poland.
Artur
A
Cieślar-Pobuda
Department of Biomedical Engineering, Linköping University, Linköping, Sweden.
Mehrdad
M
Rafat
Institute of Biochemistry and Biophysics, Department of Biochemistry, University of Tehran, Tehran, Iran. Electronic address: ardestany@ibb.ut.ac.ir.
Sussan Kaboudanian
SK
Ardestani
D000818:Animals; D053478:Apoptosomes / Q000378:metabolism; D053477:Apoptotic Protease-Activating Factor 1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001120:Arginine / Q000737:chemistry* / Q000378:metabolism; D053453:Caspase 9 / Q000378:metabolism; D003062:Codon; D045304:Cytochromes c / Q000378:metabolism; D003838:Deoxyadenine Nucleotides; D015870:Gene Expression; D055786:Gene Knockout Techniques; D018698:Glutamic Acid / Q000737:chemistry* / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D008958:Models, Molecular; D009154:Mutation; D011487:Protein Conformation
D053478:Apoptosomes; D053477:Apoptotic Protease-Activating Factor 1; D003062:Codon; D003838:Deoxyadenine Nucleotides; D018698:Glutamic Acid; D045304:Cytochromes c; D001120:Arginine; D053453:Caspase 9; C026600:2'-deoxyadenosine triphosphate
Apaf-1; Apoptosome; Caspase-9