Mutation of Caspase Cleavage Site in XIAP and its Effect on Interaction
محل انتشار: کنگره بین المللی علوم زیست پزشکی اصفهان
سال انتشار: 1399
نوع سند: مقاله کنفرانسی
زبان: انگلیسی
مشاهده: 228
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شناسه ملی سند علمی:
ICIBS01_176
تاریخ نمایه سازی: 2 آذر 1399
چکیده مقاله:
Apoptosis as a form of programmed cell death includes a proteolytic cascade that dismantles damaged or unwanted cells. At the heart of the apoptosis pathway are caspases with clear role for cleaving their substrates. In general, there are two ways to stop caspases activity in both extrinsic and intrinsic pathways of apoptosis: first, remove it from the cell by proteasome degradation machinery; second, direct inhibition of its enzymatic activity. There is evidence that inhibitor of apoptosis protein (IAP) family members can terminate caspases activity at both ways. XIAP as a famous IAP binds amino-terminal of the small subunit of monomeric caspase-9 in apoptosome complex through its BIR3 and also binds caspase-3 and caspase-7 through its linker-BIR2 resulting in obliterating their enzymatic activity and inhibition of cell apoptosis. In this study, an effect of a mutant form of XIAP on interaction with caspase-9 has been investigated.METHODS: This investigation is based on protein complementation assay approach using split-luciferase to detect XIAP and caspase-9 interaction. In this method, caspase-9 and XIAP proteins have been fused to each amino and carboxy terminal fragments of firefly luciferase and a mutation was introduced on XIAP by site-directed mutagenesis. The co-transfected cells of these constructs have been assessed for luciferase activity with and without treatment using apoptosis-inducing agents. Protein expression level was also confirmed with immunoblotting.RESULT AND CONCLUSION: Split luciferase-activity showed that native form of XIAP interacts with caspase-9, however this interaction is affected by caspase-3 mediated cleavage of XIAP. Mutagenesis of caspase cleavage site on XIAP showed this mutant has inhibitory effect on caspase-3 activation but XIAP/caspase-9 interaction is preserved.
نویسندگان
R Hamidi
Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran
F Ataei
Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran
S Hosseinkhani
Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran