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. 2014 Dec 11;9(5):1946-1958.
doi: 10.1016/j.celrep.2014.10.058. Epub 2014 Nov 20.

A Versatile Platform to Analyze Low-Affinity and Transient Protein-Protein Interactions in Living Cells in Real Time

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Free PMC article

A Versatile Platform to Analyze Low-Affinity and Transient Protein-Protein Interactions in Living Cells in Real Time

Yao-Cheng Li et al. Cell Rep. .
Free PMC article

Abstract

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 α-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.

Figures

Figure 1
Figure 1. Description of the ReBiL platform system and expected results generated by PPIs and antagonists
(A) Cartoon depicts BiLC strategy to detect PPIs and their disruption by antagonists. (B) The expression of a ReBiL cassette was controlled by both the TetR-KRAB in the absence of doxycycline (top panel) and rtTA2S-M2 in the presence of doxycycline (bottom panel). The numbers in open circles indicate each key component in the ReBiL platform system (see Table S1).
Figure 2
Figure 2. Detection of low-affinity Ube2t and FANCL interaction with ReBiL
(A) ReBiL detected Ube2t and FANCL interaction with significantly higher signal-to-noise ratio. Luminescent signals in the transient transfection, ReBiL and randomly integrated reporter cells were compared at 35 °C. Data shown are mean ± standard error of mean (SEM) from 3 independent experiments. (B) Western blot analysis of BiLC fusion proteins. The nLuc-HA-Ube2t, cLuc-FLAG-FANCL_WT and cLuc-FLAG-FANCL_C307A were detected by anti-HA and anti-FLAG antibodies respectively. Actin was a loading control. (C) Western blot analysis of the levels of nLuc-Ube2t and endogenous Ube2t by anti-Ube2t antibody (Cell Signaling D2L7H). Actin was a loading control. (D) CellTiter-Glo assay indicated there was no growth difference between FANCL_WT and FNACL_C307A cells. Data shown are mean ± SEM from 3 independent experiments.
Figure 3
Figure 3. ReBiL cells faithfully report PPIs and activity of p53-Mdm2 antagonist using real-time BiLC analyses
(A) Nutlin-3a prevents newly synthesized p53-Mdm2 but not p53-Mdm4 interactions. The Saos-2 p53-Mdm2 and p53-Mdm4 ReBiL cells in 384-well plates (8,500 cells per well) were treated with 500 ng/ml doxycycline, 100 μM D-luciferin and 10 μM Nutlin-3a or DMSO at Time = 0. Luminescence was read every 30 min for 24 hr at 37 °C. Data shown are a representative experiment from more than 3 independent experiments. (B) Western blot analysis of BiLC fusion proteins showed that Nutlin-3a does not affect the expression amounts of nLuc-HA-p53 and cLuc-FLAG-Mdm2 (detected by anti-HA and anti-FLAG antibodies, respectively). Actin served as a loading control. (C) Pre-induced Saos-2 p53-Mdm2 and p53-Mdm4 ReBiL cells were re-seeded into a 384-well plate (5,000 cells per well) together with Nutlin-3a and D-luciferin at Time = 0. The p53-Mdm2 and p53-Mdm4 BiLC signals decayed over time in a biphasic fashion. The first steep decline in BiLC signal is likely due to the temperature changes of the ReBiL cells when moving from the bench (~ 24 °C) to the pre-warmed luminometer at 37 °C. The second slow decay phase of BiLC results from doxycycline withdrawal and the consequent reduction in transcription of the BiLC fusion genes. Luminescence was read every 10 min for 10 hr at 37 °C. Data shown are a representative experiment from more than 3 independent experiments. (D) Western blot analysis showed that Nutlin-3a did not promote nLuc-HA-p53 and cLuc-FLAG-Mdm2 degradation. Actin was a loading control.
Figure 4
Figure 4. Analysis of the ability of SAH peptides to disrupt p53-Mdm2 and p53-Mdm4 complexes in living cells, and antagonism by serum
The Saos-2 p53-Mdm2 and p53-Mdm4 ReBiL cells in 96 well plates (20,000 cells per well) were pre-induced by doxycycline (500 ng/ml for 24 hr). At Time = 0, cells were washed with DMEM and treated with new media containing different PPI antagonists with or without 10% FBS. Luminescent signals were read every 5 minutes for 6 hr by Tecan-M200 at 37 °C. The ReBiL cells were treated with (A) Nutlin-3a and 10% FBS, (B) ATSP-7041 and 10% FBS, (C) Nutlin-3a no FBS, and (D) ATSP-7041 no FBS. Data shown are a representative experiment from more than 3 independent experiments.
Figure 5
Figure 5. Viability assay of Saos-2 ReBiL cells reveals p53-activating SAH peptides possess p53-independent cytotoxicity in the absence of serum
The cell viability was measured at 6 hr after Saos-2 ReBiL cells were treated with indicated SAH peptides without and with 10% FBS by CellTiter Glo assay. (A) Saos-2 p53-Mdm2 ReBiL cells (B) Saos-2 p53-Mdm4 ReBiL cells and (C) Saos-2 BRCA1-BARD1 ReBiL cells. Data shown are a representative experiment from more than 3 independent experiments and normalized to the luminescent reading of DMSO (set to 100%). (D) Saos-2 cells were treated with indicated SAH peptides in 384-well plate (2,000 cells per well) without and with 10% FBS for 24 hr. Cell viability was detected by CellTiter Glo assay. Data shown are mean ± standard deviation from two independent experiments and normalized to the luminescent reading of DMSO (100%).
Figure 6
Figure 6. The BiLC lysate assay reveals that serum does not prevent SAH peptides from disrupting p53-Mdm2 or p53-Mdm4 complexes
(A) The cellular lysates obtained from p53-Mdm2 and p53-Mdm4 ReBiL cells were co-incubated with the indicated PPI antagonists in the absence of FBS in 384-well plates at RT for 10 minutes. Steady-Glo was added, and luminescence was detected at 26 °C. (B) The BiLC lysate assays were identical to (A) except for the inclusion of 10% FBS. Data shown are a representative experiment from more than three independent experiments and normalized to the luminescent reading of DMSO (set to 100%).
Figure 7
Figure 7. Stapled peptides induce membrane leakage by a p53-independent mechanism that is antagonized by serum
(A) Saos-2 cells were treated with the indicated PPI antagonists at 25 μM and 10 μM for 6 hr. Accumulation of cytoplasmic lactate dehydrogenase (LDH) in the growth medium was used as a metric of cell membrane damage. LDH was detected by the CytoTox 96 Non-Radioactive Cytotoxicity Assay Kit (Promega). The Lysed sample represents the maximum LDH leakage in this experiment and its reading was set to 100%. DMSO treatment served as the vehicle control and its value was set to 0%. (B) Normal human fibroblasts (WS1 cells) were treated exactly as in (A). Data are shown as mean ± SEM from two independent experiments.

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