Optimization of Experimental Variables Influencing Apoptosome Biosensor in HEK293T Cells

Abstract

The apoptotic protease-activating factor 1 (Apaf-1) split luciferase biosensor has been used as a biological tool for the detection of early stage of apoptosis. The effect of doxorubicin in a cell-based assay and the addition of cytochrome c and ATP in a cell-free system have been used to test the functionality of the reporter for the detection of apoptosome formation. Here, our data established a drug- and cytochrome c/ATP-independent way of apoptosis induction relying on the expression of the biosensor itself to induce formation of apoptosome. Overexpression of Apaf-1 constructs led to increased split luciferase activity and caspase-3 activity in the absence of any drug treatment. Caspase-3 activity was significantly inhibited when caspase-9DN was co-overexpressed, while the activity of the Apaf1 biosensor was significantly increased. Our results show that the Apaf-1 biosensor does not detect etoposide-induced apoptosis.

Apoptosome Formation through Disruption of the K192-D616 Salt Bridge in the Apaf-1 Closed Form

“…A consequence of this variation is that subtle effects are very likely difficult or impossible to detect. Nonetheless, our data on the split luciferase assay for apoptosome formation, together with our previous reports, 25,27 demonstrate the utility of using the assay to quickly test the effects of mutations on different steps in Apaf-1 activation (ATP binding, caspase-9 binding, as well changes in intramolecular interactions) without having to perform resource-and time-demanding protein purification and gel filtration experiments.…”

“…28−30 This cell death is dependent on apoptosome formation as mutating K160 in the NBD to arginine (which prevents ATP binding and apoptosome formation) prevents cell death. 28 Here, we overexpressed split luciferase Apaf-1 22,24,25 in cells to test the effect of various Apaf-1 mutants on apoptosome formation and caspase activity. First, we assessed the increase in luciferase activity and caspase activity induced by expression of split luciferase wild-type (WT) Apaf-1.…”

“…These data further validate the split luciferase complementation assay as a tool for studying apoptosome formation in living cells. 25,27 Disrupting Ionic Interactions between K192 and D616 with K192E. Apaf-1 structure data 13 suggests that a salt bridge between K192 and D616 holds Apaf-1 in an inactive conformation.…”

A Genetically Encoded Bioluminescence Intracellular Nanosensor for Androgen Receptor Activation Monitoring in 3D Cell Models

“…A pivotal work, describing the exploitation of split reporters in protein-fragment complementation assays (PCA) for targeting PPI, was reported by Johnsson and Varshavsky with a split ubiquitin [ 6 ]. Since then, several reporter proteins have been used in PCA, such as the Green Fluorescent Protein (GFP) and its variants [ 7 ], B-galactosidase [ 8 ], and different luciferases [ 9 , 10 , 11 ]. One of the main limitations of split reporters is that they rely on nonspecific reassembly of the fragments in the absence of an interaction between the target proteins and on irreversible protein complementation.…”

Identification of NLRP3PYD Homo-Oligomerization Inhibitors with Anti-Inflammatory Activity

“…This bioluminescent assay presents the critical benefit of providing a robust signal with low background noise [34][35][36]. The optimization of both construct for drug screening is required for cell-based assays [37]. We previously utilized the split-luciferase complementary assay to detail Apaf-1-Apaf-1 interactions during apoptosome formation in vitro and in vivo [38][39][40].…”