Apoptosome formation upon overexpression of native and truncated Apaf-1 in
cell-free and cell-based systems
#MMPMID29410086
Noori AR
; Hosseini ES
; Nikkhah M
; Hosseinkhani S
Arch Biochem Biophys
2018[Mar]; 642
(?): 46-51
PMID29410086
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Apaf-1 is a cytosolic multi-domain protein in the apoptosis regulatory network.
When cytochrome c releases from mitochondria; it binds to WD-40 repeats of Apaf-1
molecule and induces oligomerization of Apaf-1. Here in, a split luciferase assay
was used to compare apoptosome formation in cell-free and cell-based systems.
This assay uses Apaf-1 tagged with either N-terminal fragment or C-terminal
fragment of P. pyralis luciferase. In cell based-system, the apoptosome formation
is induced inside the cells which express Apaf-1 tagged with complementary
fragments of luciferase while in cell-free system, the apoptosome formation is
induced in extracts of the cells. In cell-free system, cytochrome c dependent
luciferase activity was observed with full length Apaf-1. However, luciferase
activity due to apoptosome formation was much higher in cell based system
compared to cell-free system. The truncated Apaf-1 which lacks WD-40 repeats
(?Apaf-1) interacted with endogenous Apaf-1 in a different fashion compared to
native form as confirmed by different retention time of eluate in gel filtration
and binding to affinity column. The interactions between endogenous Apaf-1 and
?Apaf-1 is stronger than its interaction with native exogenous Apaf-1 as
indicated by dominant negative effect of ?Apaf-1 on caspase-3 processing.
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