In Vivo Analysis of Protein–Protein Interactions with Bioluminescence Resonance Energy Transfer (BRET): Progress and Prospects

Abstract

Proteins are the elementary machinery of life, and their functions are carried out mostly by molecular interactions. Among those interactions, protein–protein interactions (PPIs) are the most important as they participate in or mediate all essential biological processes. However, many common methods for PPI investigations are slightly unreliable and suffer from various limitations, especially in the studies of dynamic PPIs. To solve this problem, a method called Bioluminescence Resonance Energy Transfer (BRET) was developed about seventeen years ago. Since then, BRET has evolved into a whole class of methods that can be used to survey virtually any kinds of PPIs. Compared to many traditional methods, BRET is highly sensitive, reliable, easy to perform, and relatively inexpensive. However, most importantly, it can be done in vivo and allows the real-time monitoring of dynamic PPIs with the easily detectable light signal, which is extremely valuable for the PPI functional research. This review will take a comprehensive look at this powerful technique, including its principles, comparisons with other methods, experimental approaches, classifications, applications, early developments, recent progress, and prospects.

Human Metapneumovirus M2-2 Protein Acts as a Negative Regulator of Alpha Interferon Production by Plasmacytoid Dendritic Cells

“…After the addition of furimazine, a substrate for Nluc from the Nano-Glo luciferase assay system (Promega), luminescence and fluorescence signals were immediately detected using an Infinite F500 microplate reader (Tecan). The BRET ratio is defined as the light signal emitted by YFP (530 to 570 nm) relative to the light signal emitted by Nluc (370 to 450 nm) (45,46). The actual BRET ratio was calculated by subtracting a background BRET ratio, which was obtained for cells expressing Nluc-IRF7 alone, from the directly measured BRET ratio of each sample.…”

Divide et Impera: Identification of Small-Molecule Inhibitors of HCMV Replication Interfering with Dimerization of DNA Polymerase Processivity Factor UL44

“…This is interesting and consistent with the evidence that the F121A variant binds to DNA with a 10-fold greater affinity than the L86A/L87A variant and the hypothesis that it still presents weak DNA binding upon dimerization (16). Regardless of the effects of DNA binding on the HCMV holoenzyme conformation, which are currently being investigated in our laboratory, our data show that UL44 interactions with UL54 and with itself occur with similar affinity, as implied by the very similar B 50 values (Figure 3 and Table I; (56)). Therefore, given the fact that previous studies have identified peptides and SMs able to successfully block HCMV replication by interfering with the interaction between UL54 and UL44 (50, 51, 65), these results prompted us to perform a virtual screening aimed at identifying SMs inhibiting UL44 dimerization (Figure 4).…”

“…BRET experiments were performed essentially as described previously using a reader compatible with BRET measurements (VICTOR X2 Multilabel Plate Reader, Perkin Elmer) (55). BRET saturation curves were generated using the GraphPad Prism software (Graphpad Software Inc.) by plotting each individual BRET ratio value to the YFPnet/RLuc signal, and interpolating such values using the one-site binding hyperbola function of GraphPad Prism (Graphpad Software Inc.) to calculate BRETmax (Bmax) and BRET 50 (B 50 ) values, indicative of maximum energy transfer and relative affinity of each BRET pair tested (56). For further details please refer to the Appendix section.…”

Exploring Protein–Protein Interaction in the Study of Hormone-Dependent Cancers

“…Results of those described above indicated that evaluation of intracellular PPIs could offer significant advantages for understanding hormonal signaling in hormone-dependent cancers. Many methodologies have been proposed for the evaluation of PPI [ 23 ]. PPI analysis by in vivo imaging using xenograft or transgenic mice is possible [ 23 , 24 ].…”

“…Many methodologies have been proposed for the evaluation of PPI [ 23 ]. PPI analysis by in vivo imaging using xenograft or transgenic mice is possible [ 23 , 24 ]. Although gene recombination and antibody engineering technologies are employed for investigation of PPI, it is mostly examined using in vitro cell culture models.…”