Journal
CELL REPORTS
Volume 9, Issue 5, Pages 1946-1958Publisher
CELL PRESS
DOI: 10.1016/j.celrep.2014.10.058
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Funding
- NIH [R01-CA61449, R03-MH089489-01]
- Cancer Center Support Grant [CA014195]
- Salk Innovation Grant
- Sanofi-sponsored research grant
- Leona M. and Harry B. Helmsley Charitable Trust grant [2012-PG-MED002]
- Susan G. Komen for the Cure [SAC110036]
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Protein-protein interactions (PPIs) play central roles in orchestrating biological processes. While some PPIs are stable, many important ones are transient and hard to detect with conventional approaches. We developed ReBiL, a recombinase enhanced bimolecular luciferase complementation platform, to enable detection of weak PPIs in living cells. ReBiL readily identified challenging transient interactions between an E3 ubiquitin ligase and an E2 ubiquitin-conjugating enzyme. ReBiL's ability to rapidly interrogate PPIs in diverse conditions revealed that some stapled alpha-helical peptides, a class of PPI antagonists, induce target-independent cytosolic leakage and cytotoxicity that is antagonized by serum. These results explain the requirement for serum-free conditions to detect stapled peptide activity, and define a required parameter to evaluate for peptide antagonist approaches. ReBiL's ability to expedite PPI analysis, assess target specificity and cell permeability, and reveal off-target effects of PPI modifiers should facilitate the development of effective, cell-permeable PPI therapeutics and the elaboration of diverse biological mechanisms.
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