Development of a Novel Cellular Assay For Assessing Protein:Protein Interactions (PPIs) Between Extrinsic Apoptosis Proteins


Year:

Session type:

Luke Humphreys1,Catherine Higgins1,Joanna Majkut1,Timothy Harrison1,Daniel B. Longley1
1Cell Death and Drug Resistance Group, Centre For Cancer Research and Cell Biology, Queens University, Belfast

Abstract

Background

Evasion of apoptosis is well recognised as one of the hallmarks of cancer. Extrinsic apoptosis occurs when death receptors (such as CD95/Fas, TRAIL-R1 and TRAIL-R2) bind their cognate ligands expressed by immune effector cells, stimulating the formation of the Death Inducing Signalling Complex (DISC). This complex contains FLIP, FADD and procaspase 8, which interact with each other through homotypic DED interactions. FLIP is an endogenous inhibitor of caspase-8 that, if recruited to the DISC to a sufficient level, will prevent full processing of procaspase-8, allowing cells to evade death. FLIP is frequently overexpressed in a number of cancers, including Non-Small Cell Lung Cancer, Prostate and Colorectal cancer.

Method

To enable monitoring of FLIP’s PPIs, we have developed a novel assay using Promega’s NanoBiT™ technology. This is an ATP-independent, luminescence-based assay that uses NanoLuciferase. In this assay, the NanoLuciferase is split into large and small subdomains (Lg-BiT and Sm-BiT) and then fused to the proteins or protein domains of interest. The NanoLuciferase activity is only reconstituted when the Lg-BiT and Sm-BiT are brought together by interactions between the regions to which they have been fused.

Results

A NanoLuc PPI assay for FLIP has been established and verified. Site-directed mutagenesis was performed to alter key residues to demonstrate that NanoLuciferase activity was dependent on previously characterised FLIP protein-protein interaction domains. Novel FLIP PPI inhibitors have been shown to have activity in this assay that is consistent with their activity in an alternative low throughput “DISC IP” assay. Confounding factors such as fusion protein stability and loss of cell viability have been investigated.

Conclusion

The NanoLuc FLIP PPI assay provides a novel cellular method for monitoring FLIP’s ability to bind to its key protein partners and is now being used as a medium throughput screen to assess the activity of small molecule FLIP PPI inhibitors.