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Title
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Role of the salt bridge between glutamate 546 and arginine 907 inpreservation of autoinhibited form of Apaf-1
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Type
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JournalPaper
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Keywords
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Apaf-1, Apoptosome, Caspase-9
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Abstract
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Apaf-1, the key element of apoptotic mitochondrial pathway, normally exists in an auto-inhibited forminside 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 domainsare involved in maintaining the inactive conformation of Apaf-1. At the present study, we have investi-gated 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 E546Qand E546R variants in HEK293T cells does not induce apoptosis unlike – HL-60 cancer cell line. In vitroapoptosome formation assay showed that all variants are cytochrome c and dATP dependent to formapoptosome and activate endogenous procaspase-9 in Apaf-1-knockout MEF cell line. These results sug-gest that E546 is not a critical residue for preservation of auto-inhibited Apaf-1. Furthermore, the behaviorof Apaf-1 variants for in vitro apoptosome formation in HEK293T cell is similar to exogenous wild typeApaf-1. Wild type and its variants can form apoptosome in HEK293T cell with different procaspase-3processing pattern in the presence and absence of exogenous cytochrome c and dATP.
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Researchers
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Sussan Kaboudanian Ardestani (Not In First Six Researchers), Artur Cieslar-Pobuda (Not In First Six Researchers), Mayur Vilas Jain (Fifth Researcher), Mehrdad Rafat (Not In First Six Researchers), Marek Jain Los (Third Researcher), Saman Hosseinkhani (Second Researcher), Jamshid Davoodi (Fourth Researcher), Raheleh Shakeri (First Researcher)
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