Optimization of BRET saturation assays for robust and sensitive cytosolic protein-protein interaction studies

Abstract

Bioluminescence resonance energy transfer (BRET) saturation is a method of studying protein-protein interaction (PPI) upon quantification of the dependence of the BRET signal on the acceptor/donor (A:D) expression ratio. In this study, using the very bright Nluc/YFP BRET pair acquired respectively with microplate reader and automated confocal microscopy, we significantly improved BRET saturation assay by extending A:D expression detection range and normalizing A:D expression with a new BRET-free probe. We next found that upon using variable instead of fixed amount of donor molecules co-expressed with increasing acceptor concentrations, BRET saturation assay robustness can be further improved when studying cytosolic protein, although the relative amounts of dimers (BRETmax) and the relative dimer affinity (BRET50) remain similar. Altogether, we show that our method can be applied to many PPI networks, involving the NF-κB pathway, high-affinity nanobody, rabies virus-host interactions, mTOR complex and JAK/STAT signaling. Altogether our approach paves the way for robust PPI validation and characterization in living cells.

Figure 1

A sensitive method for PPI interaction studies based on BRET saturation assays. (A,B) HEK-293T cells were transfected with 0.025 to 25 ng of YFP-Nluc plasmid per well. Nluc bioluminescence was quantified using various microplate readers (A), and YFP fluorescence was quantified using microplate readers or an automated confocal microscope (B). (C) YFP and Nluc signals were sequentially quantified for 13 sets of plasmids transfected in HEK-293T cells at 1:1 A:D ratios. (D) Bioluminescence spectra properties of reference proteins. Results are the average of three independent experiments. (E) Sequential measurement of the fluorescence of the acceptor (with an automated microscope) and the bioluminescence emitted from both A:D upon BRET (with a plate reader) in order to calculate the net BRET and the A:D expression ratio using the BRET-free Nluc-block-YFP control for BRET saturation assays. (F) Broad range transfection method for BRET saturation assay over 11 ratios (243:1 to 1:243, see Supplementary Fig. S2). (G) Diagram of NF-κB protein domains and sequence homologies. (H,I) BRET saturation assay in HEK-293T cells transfected with the donor (Nluc-p50) and acceptor (YFP-tagged) plasmids using fixed (H) or variable (I) donor. The net BRET was plotted with the YFP/Nluc expression ratios and fitted using a non-linear regression curve to determine both BRETmax and BRET50. The results represent four independent biological replicates, each involving three technical replicates. BRETmax values (J,K) were used to define interacting, non-interactive pairs (grey labeled) and extrapolate a 3 σ-threshold [(J) 0.055 and (K) 0.023]. BRET50 values were reported only for significant interacting pairs (L,M). # not displayed.

Figure 2

Variable donor BRET saturation assay applied to study protein complex with distinctive affinities. (A) Known interaction parameters of YFP and anti-YFP nanobody or SOD1 dimers. (B) Variable donor BRET saturation assay of donor SOD-Nluc or anti-YFP-Nluc with acceptor YFP-SOD1. The net BRET was plotted with the YFP/Nluc expression ratios to determine both BRETmax and BRET50. (C) Rabies virus M_Tha and M_Th4M mutant proteins interact differentially with host proteins p105, TPL2, RelAp43, and ABIN2. (D–G) Variable donor BRET saturation assay of donor Nluc-p105 (D), -TPL2 (E), -RelAp43 (F) or -ABIN2 (G) and acceptor (YFP-M_Tha or M_Th4M) as in Fig. 1 and both BRETmax and BRET50 were plotted. A 3σ-threshold (0.06) was defined, based on the M-p105 interactions considered as negative controls. # not displayed. Unpaired, parametric two-tailed t test were performed with GraphPad (Prism), *p < 0.05.

Figure 3

Monitoring the drug-dependent FRB-FKBP interaction with variable donor BRET saturation assays. (A) Simplified dynamics of the interaction of FKBP with the FRB domain of mTOR mediated by the rapamycin. (B) HEK-293T cells were incubated in presence of rapamycin for 1 h between fluorescence and bioluminescence acquisition. (C) Variable donor BRET saturation assay of FRB-Nluc/YFP-FKBP in the presence or absence of rapamycin. A 3 σ-threshold (0.07) was defined based on SOD-FRB interactions considered a negative control. (D) Variable donor BRET saturation assay of FRB-Nluc/YFP-FKBP in cells treated with various concentrations of rapamycin for 1 h. (E) BRETmax and BRET50 values were plotted according to the different concentrations of rapamycin.

Figure 4

Study of IFN-dependent JAK/STAT pathway and time-lapse monitoring of STAT2/IRF9 interaction. (A) JAK/STAT signaling network. (B) Variable donor BRET saturation assay of Nluc- and YFP-tagged protein pairs from the JAK/STAT pathway as in Fig. 1. Based on the acceptor protein YFP-SOD1 negative control, a 3σ threshold (0.02) was defined. (C) Immediately after the initial bioluminescence acquisition (t = 0), IFNβ was added to the cells at 500 U/mL, and the cells were monitored every 10 min for 1 h. (D) Variable donor BRET saturation assay of Nluc-STAT2/YFP-IRF9 after IFNβ stimulation. (E) BRET50 and BRETmax were represented according to the time of acquisition.