Bioluminescence Resonance Energy Transfer Signal Research Articles

Bioluminescence Resonance Energy Transfer (BRET)-based Assay for Measuring Interactions of CRAF with 14-3-3 Proteins in Live Cells.

CRAF is a primary effector of RAS GTPases and plays a critical role in the tumorigenesis of several KRAS-driven cancers. In addition, CRAF is a hotspot for germline mutations, which are shown to cause the developmental RASopathy, Noonan syndrome. All RAF kinases contain multiple phosphorylation-dependent binding sites for 14-3-3 regulatory proteins. The differential binding of 14-3-3 to these sites plays essential roles in the formation of active RAF dimers at the plasma membrane under signaling conditions and in maintaining RAF autoinhibition under quiescent conditions. Understanding how these interactions are regulated and how they can be modulated is critical for identifying new therapeutic approaches that target RAF function. Here, I describe a bioluminescence resonance energy transfer (BRET)-based assay for measuring the interactions of CRAF with 14-3-3 proteins in live cells. Specifically, this assay measures the interactions of CRAF fused to a Nano luciferase donor and 14-3-3 fused to a Halo tag acceptor, where the interaction of RAF and 14-3-3 results in donor-to-acceptor energy transfer and the generation of the BRET signal. The protocol further shows that this signal can be disrupted by mutations shown to prevent 14-3-3 binding to each of its high-affinity RAF docking sites. This protocol describes the procedures for seeding, transfecting, and replating the cells, along with detailed instructions for reading BRET emissions, performing data analysis, and confirming protein expression levels. In addition, example assay results, along with optimization and troubleshooting steps, are provided.

Development of a membrane-based Gi-CASE biosensor assay for profiling compounds at cannabinoid receptors.

Introduction: The cannabinoid receptor (CBR) subtypes 1 (CB1R) and 2 (CB2R) are key components of the endocannabinoid system (ECS), playing a central role in the control of peripheral pain, inflammation and the immune response, with further roles in the endocrine regulation of food intake and energy balance. So far, few medicines targeting these receptors have reached the clinic, suggesting that a better understanding of the receptor signalling properties of existing tool compounds and clinical candidates may open the door to the development of more effective and safer treatments. Both CB1R and CB2R are Gαi protein-coupled receptors but detecting Gαi protein signalling activity reliably and reproducibly is challenging. This is due to the inherent variability in live cell-based assays and restrictions around the use of radioactive [35S]-GTPγS, a favoured technology for developing higher-throughput membrane-based Gαi protein activity assays. Methods: Here, we describe the development of a membrane-based Gαi signalling system, produced from membrane preparations of HEK293TR cells, stably overexpressing CB1R or CB2R, and components of the Gαi-CASE biosensor. This BRET-based system allows direct detection of Gαi signalling in both cells and membranes by monitoring bioluminescence resonance energy transfer (BRET) between the α and the βγ subunits. Cells and membranes were subject to increasing concentrations of reference cannabinoid compounds, with 10μM furimazine added to generate RET signals, which were detected on a PHERAstar FSX plate reader, then processed using MARS software and analysed in GraphPad PRISM 9.2. Results: In membranes expressing the Gi-CASE biosensor, the cannabinoid ligands profiled were found to show agonist and inverse agonist activity. Agonist activity elicited a decrease in the BRET signal, indicative of receptor activation and G protein dissociation. Inverse agonist activity caused an increase in BRET signal, indicative of receptor inactivation, and the accumulation of inactive G protein. Our membrane-based Gi-CASE NanoBRET system successfully characterised the potency (pEC50) and efficacy (Emax) of CBR agonists and inverse agonists in a 384-well screening format. Values obtained were in-line with whole-cell Gi-CASE assays and consistent with literature values obtained in the GTPγS screening format. Discussion: This novel, membrane-based Gαi protein activation assay is applicable to other Gαi-coupled GPCRs, including orphan receptors, allowing real-time higher-throughput measurements of receptor activation.

Ratiometric Detection of Zn2+ Using DNAzyme-Based Bioluminescence Resonance Energy Transfer Sensors.

While fluorescent sensors have been developed for monitoring metal ions in health and diseases, they are limited by the requirement of an excitation light source that can lead to photobleaching and a high autofluorescence background. To address these issues, bioluminescence resonance energy transfer (BRET)-based protein or small molecule sensors have been developed; however, most of them are not highly selective nor generalizable to different metal ions. Taking advantage of the high selectivity and generalizability of DNAzymes, we report herein DNAzyme-based ratiometric sensors for Zn2+ based on BRET. The 8-17 DNAzyme was labeled with luciferase and Cy3. The proximity between luciferase and Cy3 permiQed BRET when coelenterazine, the substrate for luciferase, was introduced. Adding samples containing Zn2+ resulted in a cleavage of the substrate strand, causing dehybridization of the DNAzyme construct, thus increasing the distance between Cy3 and luciferase and changing the BRET signals. Using these sensors, we detected Zn2+ in serum samples and achieved Zn2+ detection with a smartphone camera. Moreover, since the BRET pair is not the component that determines the selectivity of the sensors, this sensing platform has the potential to be adapted for the detection of other metal ions with other metal-dependent DNAzymes.

Effects of 5G-modulated 3.5 GHz radiofrequency field exposures on HSF1, RAS, ERK, and PML activation in live fibroblasts and keratinocytes cells

The potential health risks of exposure to radiofrequency electromagnetic fields from mobile communications technologies have raised societal concerns. Guidelines have been set to protect the population (e.g. non-specific heating above 1 °C under exposure to radiofrequency fields), but questions remain regarding the potential biological effects of non-thermal exposures. With the advent of the fifth generation (5G) of mobile communication, assessing whether exposure to this new signal induces a cellular stress response is one of the mandatory steps on the roadmap for a safe deployment and health risk evaluation. Using the BRET (Bioluminescence Resonance Energy-Transfer) technique, we assessed whether continuous or intermittent (5 min ON/ 10 min OFF) exposure of live human keratinocytes and fibroblasts cells to 5G 3.5 GHz signals at specific absorption rate (SAR) up to 4 W/kg for 24 h impact basal or chemically-induced activity of Heat Shock Factor (HSF), RAt Sarcoma virus (RAS) and Extracellular signal-Regulated Kinases (ERK) kinases, and Promyelocytic Leukemia Protein (PML), that are all molecular pathways involved in environmental cell-stress responses. The main results are (i), a decrease of the HSF1 basal BRET signal when fibroblasts cells were exposed at the lower SARs tested (0.25 and 1 W/kg), but not at the highest one (4 W/kg), and (ii) a slight decrease of As2O3 maximal efficacy to trigger PML SUMOylation when fibroblasts cells, but not keratinocytes, were continuously exposed to the 5G RF-EMF signal. Nevertheless, given the inconsistency of these effects in terms of impacted cell type, effective SAR, exposure mode, and molecular cell stress response, we concluded that our study show no conclusive evidence that molecular effects can arise when skin cells are exposed to the 5G RF-EMF alone or with a chemical stressor.

A Reaction-Based Ratiometric Bioluminescent Platform for Point-of-Care and Quantitative Detection Using a Smartphone.

Fluorescent probes have emerged as powerful tools for the detection of different analytes by virtue of structural tenability. However, the requirement of an excitation source largely hinders their applicability in point-of-care detection, as well as causing autofluorescence interference in complex samples. Herein, based on bioluminescence resonance energy transfer (BRET), we developed a reaction-based ratiometric bioluminescent platform, which allows the excitation-free detection of analytes. The platform has a modular design consisting of a NanoLuc-HaloTag fusion as an energy donor, to which a synthetic fluorescent probe is bioorthogonally labeled as recognition moiety and energy acceptor. Once activated by the target, the fluorescent probe can be excited by NanoLuc to generate a remarkable BRET signal, resulting in obvious color changes of luminescence, which can be easily recorded and quantitatively analyzed by a smartphone. As a proof of concept, a fluorescent probe for HOCl was synthesized to construct the bioluminescent system. Results demonstrated the system showed a constant blue/red emission ratio which is independent to the signal intensity, allowing the quantification of HOCl concentration with high sensitivity (limit of detection (LOD) = 13 nM) and accuracy. Given the universality, this reaction-based bioluminescent platform holds great potential for point-of-care and quantitative detection of reactive species.

Universal Design of Luciferase Fusion Proteins for Epigenetic Modifications Detection Based on Bioluminescence Resonance Energy Transfer.

Global hypomethylation and promoter hypermethylation of tumor-suppressor genes are the hallmarks of cancer. We previously reported a global DNA methylation level sensing system based on dual-color bioluminescence resonance energy transfer (BRET) using methyl-CpG binding domain (MBD)-fused firefly luciferase (Fluc) and unmethyl-CpG binding domain (CXXC)-fused Oplophorus luciferase (Oluc). Moreover, BRET-based hydroxymethylation and hemi-methylation level sensing systems have been developed using hydroxymethyl-CpG and hemi-methyl-CpG binding domain-fused Fluc. These studies suggest that target epigenetic modifications can be simultaneously quantified using target-modification-binding protein-fused luciferases. In this study, we focused on the SnoopTag (SnT)/SnoopCatcher (SnC) protein ligation system to establish a universal design for fusion protein construction for any combination. SnT spontaneously forms an isopeptide bond with SnC; therefore, any kind of fusion protein would be constructed by the SnT/SnC system. To establish the proof of concept, MBD-SnT, CXXC-SnT, and SnC-Oluc were prepared and ligated MBD-SnT or CXXC-SnT to SnC-Oluc. The ligation products of MBD-SnT-SnC-Oluc and CXXC-SnT-SnC-Oluc showed luciferase activity and specific binding activity to methyl-CpG and unmethyl-CpG, respectively. The BRET signal using MBD-SnT-SnC-Oluc and CXXC-SnT-SnC-Oluc increased the amount of methyl-CpG and unmethyl-CpG in genomic DNA, respectively. There was a significant negative correlation between the BRET signals; therefore, the global DNA methylation level was quantified using the BRET signals (R2 = 0.99, and R.S.D. <3.5%). These results indicate that the SnT/SnC protein ligation system can be utilized to construct target modification-binding protein-fused luciferases in any combination that detects target modifications in genomic DNA based on BRET.

Modular and genetically encodable RNA-based bioluminescence resonance energy transfer (BRET) sensors for cellular target detection.

Genetically encodable bioluminescent sensors have been broadly used for highly sensitive, low-background, low-phototoxicity bioanalysis and imaging in living biological systems. To achieve real-time quantitative measurement and high-throughput screening, powerful bioluminescence resonance energy transfer (BRET) sensors have been designed, especially based on interactions between a luciferase/luciferin donor and a fluorescent protein acceptor. While fluorescent protein-based BRET sensors have been widely applied in cellular and in vivo studies, more modular and easy-to-engineer RNA-based BRET sensors have never been reported. In this project, we report, for the first time, genetically encoded fluorogenic RNA-based BRET sensors for cellular imaging and quantification of different target analytes and RNA-protein interactions. Fluorogenic RNAs are single-stranded RNA aptamers that can bind specific small-molecule fluorophores and activate the fluorescence signals. By coupling a NanoLuc luciferase donor with different types of fluorogenic RNA acceptor, without reliance on external light, sensitive BRET signals are generated in response to target metabolite, signaling molecule, or RNA levels inside living cells. We envision that this novel genetically encoded RNA-based BRET system can be widely used in bioanalysis and nanomedicine.

Differential effects of glucose-dependent insulinotropic polypeptide receptor/glucagon-like peptide-1 receptor heteromerization on cell signaling when expressed in HEK-293 cells.

The incretin hormones: glucose‐dependent insulinotropic polypeptide (GIP) and glucagon‐like peptide‐1 (GLP‐1) are important regulators of many aspects of metabolism including insulin secretion. Their receptors (GIPR and GLP‐1R) are closely related members of the secretin class of G‐protein‐coupled receptors. As both receptors are expressed on pancreatic β‐cells there is at least the hypothetical possibility that they may form heteromers. In the present study, we investigated GIPR/GLP‐1R heteromerization and the impact of GIPR on GLP‐1R‐mediated signaling and vice versa in HEK‐293 cells. Real‐time fluorescence resonance energy transfer (FRET) and bioluminescence resonance energy transfer (BRET) saturation experiments confirm that GLP‐1R and GIPR form heteromers. Stimulation with 1 μM GLP‐1 caused an increase in both FRET and BRET ratio, whereas stimulation with 1 μM GIP caused a decrease. The only other ligand tested to cause a significant change in BRET signal was the GLP‐1 metabolite, GLP‐1 (9–36). GIPR expression had no significant effect on mini‐Gs recruitment to GLP‐1R but significantly inhibited GLP‐1 stimulated mini‐Gq and arrestin recruitment. In contrast, the presence of GLP‐1R improved GIP stimulated mini‐Gs and mini‐Gq recruitment to GIPR. These data support the hypothesis that GIPR and GLP‐1R form heteromers with differential consequences on cell signaling.

A BRET Ca2+ sensor enables high-throughput screening in the presence of background fluorescence.

The intrinsic fluorescence of samples confounds the use of fluorescence-based sensors. This is of particular concern in high-throughput screening (HTS) applications using large chemical libraries containing intrinsically fluorescent compounds. To overcome this problem, we developed a bioluminescence resonance energy transfer (BRET) Ca2+ sensor, CalfluxCTN. We demonstrated that it reliably reported changes in intracellular Ca2+ concentrations evoked by an agonist and an antagonist of the human muscarinic acetylcholine receptor M1 (hM1R) even in the presence of the fluorescent compound fluorescein, which interfered with a standard fluorescent HTS sensor (Fluo-8). In an HTS using a chemical library containing fluorescent compounds, CalfluxCTN accurately identified agonists and antagonists that were missed or miscategorized using Fluo-8. Moreover, we showed that a luciferase substrate that becomes activated only when inside cells generated long-lasting BRET signals in HTS, enabling results to be reliably compared among replicate samples for hours. Thus, the use of a self-luminescent sensor instead of a fluorescent sensor could facilitate the complete screening of chemical libraries in a high-throughput context and enable analysis of autofluorescent samples in many different applications.

BRET-Based Self-Cleaving Biosensors for SARS-CoV-2 3CLpro Inhibitor Discovery.

ABSTRACTThe 3C-like protease (3CLpro) of SARS-CoV-2 is an attractive drug target for developing antivirals against SARS-CoV-2. A few small molecule inhibitors of 3CLpro are in clinical trials for COVID-19 treatments, and more inhibitors are under development. One limiting factor for 3CLpro inhibitors development is that the cellular activities of such inhibitors should be evaluated in Biosafety Level 3 (BSL-3) laboratories. Here, we design DNA-coded biosensors that can be used in BSL-2 laboratories to set up cell-based assays for 3CLpro inhibitor discovery. The biosensors were constructed by linking a green fluorescent protein (GFP2) to the N-terminus and a Renilla luciferase (RLuc8) to the C-terminus of SARS-CoV-2 3CLpro, with the linkers derived from the cleavage sequences of 3CLpro. After overexpression of the biosensors in human embryonic kidney (HEK) 293T cells, 3CLpro can be released from GFP2 and RLuc by self-cleavage, resulting in a decrease of the bioluminescence resonance energy transfer (BRET) signal. Using one of these biosensors, pBRET-10, we evaluated the cellular activities of several 3CLpro inhibitors. These inhibitors restored the BRET signal by blocking the proteolysis of pBRET-10, and their relative activities measured using pBRET-10 were consistent with their previously reported anti-SARS-CoV-2 activities. We conclude that the biosensor pBRET-10 is a useful tool for SARS-CoV-2 3CLpro inhibitor discovery.IMPORTANCE The virus proteases 3CLpro are validated drug targets for developing antivirals to treat coronavirus diseases, such as COVID-19. However, the development of 3CLpro inhibitors relies heavily on BSL-3 laboratories. Here, we report a series of BRET-based self-cleaving biosensors that can be used to set up cell-based assays to evaluate the cell permeability and cellular activity of SARS-CoV-2 3CLpro inhibitors in BSL-2 laboratories. The cell-based assay is suitable for high-throughput screening for 3CLpro inhibitors because of the simplicity and good reproducibility of our biosensors. The design strategy can also be used to design biosensors for other viral proteases for which the activation processes involve the self-cleavage of polyproteins.

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

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.

Quantification of Global DNA Hydroxymethylation Level Using UHRF2 SRA-Luciferase Based on Bioluminescence Resonance Energy Transfer.

5-Methylcytosine (5mC) plays an important role in the regulation of gene expression. Ten-eleven translocation (TET) continuously oxidizes 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). High levels of 5hmC are found in the brain and embryonic stem cells, while global hydroxymethylation levels are reduced in several cancer cells. Moreover, alterations in hydroxymethylation levels occur in neurological diseases, such as Alzheimer's disease and Parkinson's disease. In this study, a convenient sensing method for the determination of global hydroxymethylation levels was developed. A bioluminescence resonance energy transfer (BRET) assay for global methylation level determination has been previously reported. In the assay, BOBO-3 DNA intercalating dye is excited by the bioluminescence of methyl-CpG-binding domain-fused firefly luciferase (MBD-Fluc); that is, the BRET signal depends on the content of methylated CpG on genomic DNA. To develop a hydroxymethylation level sensing method, SET- and RING-associated (SRA) domain of ubiquitin-like with PHD and RING finger domains 2 (UHRF2)-fused Fluc (UHRF2 SRA-Fluc) was prepared. UHRF2 SRA is known to bind to both hydroxymethylated and methylated CpG sites; thus, MBD was utilized to mask the methylated CpG on genomic DNA. We demonstrated that the BRET signal between UHRF2 SRA-Fluc and BOBO-3 depends on the global hydroxymethylation level in the presence of MBD (R2 = 0.99, and relative standard deviation < 2.3%). The limit of detection for hydroxymethylated genomic DNA was 0.75 ng μL-1. In this assay, the global hydroxymethylation level was quantified within 40 min in a single tube, indicating that the assay would be utilized not only for clinical diagnostics but also for the elucidation of 5hmC functions.

Development of a Human Estrogen Receptor Dimerization Assay for the Estrogenic Endocrine-Disrupting Chemicals Using Bioluminescence Resonance Energy Transfer.

Endocrine-disrupting chemicals (EDCs) are found in food and various other substances, including pesticides and plastics. EDCs are easily absorbed into the body and have the ability to mimic or block hormone function. The radioligand binding assay based on the estrogen receptors binding affinity is widely used to detect estrogenic EDCs but is limited to radioactive substances and requires specific conditions. As an alternative, we developed a human cell-based dimerization assay for detecting EDC-mediated ER-alpha (ERα) dimerization using bioluminescence resonance energy transfer (BRET). The resultant novel BRET-based on the ERα dimerization assay was used to identify the binding affinity of 17β-estradiol (E2), 17α-estradiol, corticosterone, diethylhexyl phthalate, bisphenol A, and 4-nonylphenol with ERα by measuring the corresponding BRET signals. Consequently, the BRET signals from five chemicals except corticosterone showed a dose-dependent sigmoidal curve for ERα, and these chemicals were suggested as positive chemicals for ERα. In contrast, corticosterone, which induced a BRET signal comparable to that of the vehicle control, was suggested as a negative chemical for ERα. Therefore, these results were consistent with the results of the existing binding assay for ERα and suggested that a novel BRET system can provide information about EDCs-mediated dimerization to ERα.

Identification of the contact region responsible for the formation of the homomeric CYP1A2•CYP1A2 complex.

Previous studies showed that cytochrome P450 1A2 (CYP1A2) forms a homomeric complex that influences its metabolic characteristics. Specifically, CYP1A2 activity exhibits a sigmoidal response as a function of NADPH-cytochrome P450 reductase (POR) concentration and is consistent with an inhibitory CYP1A2•CYP1A2 complex that is disrupted by increasing [POR] (Reed et al. (2012) Biochem. J. 446, 489-497). The goal of this study was to identify the CYP1A2 contact regions involved in homomeric complex formation. Examination of X-ray structure of CYP1A2 implicated the proximal face in homomeric complex formation. Consequently, the involvement of residues L91-K106 (P1 region) located on the proximal face of CYP1A2 was investigated. This region was replaced with the homologous region of CYP2B4 (T81-S96) and the protein was expressed in HEK293T/17 cells. Complex formation and its disruption was observed using bioluminescence resonance energy transfer (BRET). The P1-CYP1A2 (CYP1A2 with the modified P1 region) exhibited a decreased BRET signal as compared with wild-type CYP1A2 (WT-CYP1A2). On further examination, P1-CYP1A2 was much less effective at disrupting the CYP1A2•CYP1A2 homomeric complex, when compared with WT-CYP1A2, thereby demonstrating impaired binding of P1-CYP1A2 to WT-CYP1A2 protein. In contrast, the P1 substitution did not affect its ability to form a heteromeric complex with CYP2B4. P1-CYP1A2 also showed decreased activity as compared with WT-CYP1A2, which was consistent with a decrease in the ability of P1-CYP1A2 to associate with WT-POR, again implicating the P1 region in POR binding. These results indicate that the contact region responsible for the CYP1A2•CYP1A2 homomeric complex resides in the proximal region of the protein.

Role of the Proximal Region of CYP1A2 in the Homomeric P450 Complex Formation

Previous studies have established the formation of the CYP1A2-CYP1A2 homomeric complex and its effect on metabolic function. Preliminary evidence has shown that CYP1A2 activity exhibits a sigmoidal response in the CYP1A2/POR binary system as a function of POR. As the CYP1A2‑CYP1A2 homomeric complex is disrupted, CYP1A2 activity is increased, which is consistent with cooperativity. The goal of this study was to identify the regions of CYP1A2 involved in the homomeric complex formation. Structural evidence suggested that the proximal face of CYP1A2 may play a role in the homomeric complex formation. Consequently, a part of the proximal face (residues L91-K106: P1 region) was examined. Residues in this region of CYP1A2 were replaced with the homologous region of CYP2B4 (T81‑S96) using molecular cloning and was expressed in HEK-293T cells. Bioluminescence resonance energy transfer (BRET) was used to measure complex formation and disruption. The chimeric CYP1A2 (with the mutated P1 region) showed a decreased BRET signal when co‑transfected with wild-type CYP1A2, thereby indicating a decrease in complex formation. This was further verified by competition studies, which showed that the CYP1A2-P1 chimera was less effective at disrupting the CYP1A2 homomeric complexes as compared to wild‑type CYP1A2. This effectively demonstrated the impaired binding of the CYP1A2-P1 chimera to wild-type CYP1A2. Lower activity was observed as well with the CYP1A2‑P1 chimera as compared to wild‑type CYP1A2. As POR associates with CYP1A2 on the proximal face, the decreased activity of the chimera indicated that the ability of the chimera to interact with POR was affected. The chimera exhibited a decreased BRET signal when co‑transfected with wild‑type CYP1A2. Competition studies demonstrated that the chimera was less effective than wild‑type CYP1A2 at disrupting the CYP1A2‑POR heteromeric complex. Therefore, the binding of the chimera to POR was impaired, which was consistent with the decreased activity of the chimera. Therefore, these results not only establish the P1 region as a contact region in the CYP1A2‑CYP1A2 homomeric complex formation but also that modification of P1 region also affects formation of the CYP1A2‑POR heteromeric complex.

Physical Interactions between Heme Oxygenase‐1 and the Cytochromes P450 Lead to Complex Changes in Enzyme Activity

The cytochromes P450 (P450s) catalyze the oxygenation of endogenous and exogenous organic substrates, including most prescribed drugs. Heme oxygenase-1 (HO-1) is an inducible enzyme responsible for the rate-limiting step of heme catabolism. HO-1 and the P450s are membrane-bound proteins expressed on the cytosolic face of the endoplasmic reticulum where they must interact with NADPH-cytochrome P450 reductase (POR) for activity. Because POR levels are often limiting, HO-1 and the P450s must compete for POR binding in order to function. However, simple competition for POR is often complicated by homomeric and heteromeric P450•P450 interactions. We hypothesized that HO-1 might participate with the P450s in their competition for POR and that this competition might lead to changes in P450 and/or HO-1 activity. We were also interested in determining whether HO-1 could physically interact with P450s and measuring what functional effects any P450•HO-1 complex might have. Using bioluminescence resonance energy transfer (BRET), we were able to establish that HO-1 formed physical complexes with CYP1A1, CYP1A2, and CYP2D6 in transiently transfected HEK293T cells. The stability of these complexes was determined by measuring the BRET signal generated by GFP- and Renilla luciferase-tagged P450 and HO-1 in the presence and absence of untagged POR. The presence of POR did not have a significant effect on the interactions of HO-1 with CYP1A2 and CYP2D6 but caused an increase in the BRET signal generated by the HO-1/CYP1A1 pair. Further BRET studies of these three P450s showed that the presence of HO-1 inhibited the formation of POR•P450 complexes, while, conversely, the presence of untagged P450 caused marginal disruption of POR•HO-1 complex formation. The differences in the response of the HO-1•CYP1A1 complex and the HO-1•CYP1A2 complex to POR indicated the possibility of different functional consequences of these P450/HO-1 interactions. Activity measurements of purified enzymes in lipid reconstituted systems confirmed that this was the case. CYP1A1 and HO-1 had large inhibitory effects on each other over a large range of POR concentrations. HO-1 activity in the presence of CYP1A1 approached that of HO-1 alone only in the presence of a large excess of POR. The parallel experiments with HO-1 and CYP1A2 yielded results that differed both qualitatively and quantitatively from those seen with CYP1A1. Like CYP1A1, CYP1A2 caused inhibition of HO-1 activity, though the effect of CYP1A2 was much milder. The effect of HO-1 on CYP1A2 was also smaller in magnitude than was observed with CYP1A1, but CYP1A2 activity increased in the presence of HO-1 and subsaturating POR. In the presence of HO-1 and excess POR, CYP1A2 activity was slightly inhibited. Kinetic analysis of these results showed that they were inconsistent with a simple model of competition between HO-1 and P450 for POR.

Ligand-Activatable BRET9 Probes for Imaging Molecular Events in Living Mammalian Cells.

Bioluminescence resonance energy transfer (BRET) is a commonly used assay system for studying protein-protein interactions. The present protocol introduces a conceptually unique ligand-activatable BRET system (termed BRET9), where a full-length artificial luciferase variant 23 (ALuc23), acting as the energy donor, is sandwiched in between a protein pair of interest, FRB and FKBP, and further linked to a fluorescent protein as the energy acceptor for studying protein-protein interaction. A specific ligand, rapamycin, which initiates intramolecular interactions of FRB and FKBP inside the probe, which develops molecular strain in the sandwiched ALuc23 to complete its folding, thus, the probe system greatly enhances both the overall bioluminescence (BL) spectrum and the BRET signal in the far-red (FR) region. This new BRET system provides a robust ligand-activatable platform that efficiently reports FR-BL signals in mammalian cells.

BRET-based assay to specifically monitor β2AR/GRK2 interaction and β-arrestin2 conformational change upon βAR stimulation.

The β-adrenergic receptors (βARs) are members of G protein-coupled receptor (GPCR) family and have been one of the most important GPCRs for studying receptor endocytosis and signaling pathway. Agonist binding of βARs leads to an activation of G proteins and their canonical effectors. In a parallel way, βAR stimulation triggers the termination of its signals by receptor desensitization. This termination process is initiated by G protein-coupled receptor kinase (GRK)-induced βAR phosphorylation that promotes the recruitment of β-arrestins to phosphorylated βAR. The uncoupled βARs which formed a complex with GRK and β-arrestin subsequently internalize into the cytosol. In addition, GRKs and β-arrestins also act as scaffolding proteins and signal transducers in their own functions to modulate various downstream effectors. Upon translocation to the βAR, β-arrestin is believed to undergo an important conformational change in the structure that is necessary for its signal transduction. The bioluminescence resonance energy transfer (BRET) technique involves the fusion of donor (luciferase) and acceptor (fluorescent) molecules to the interested proteins. Co-expression of these fusion proteins enables direct detection of their interactions in living cells. Here we describe the use of our established BRET technique to track the interaction of βAR with both GRK and β-arrestin. The assay described here allows the measurement of the BRET signal for detecting the interaction of β2AR with GRK2 and the conformational change of β-arrestin2 following βAR stimulation.

(Invited) A Protein-Based Artificial Receptor for Insulin Sensing

Insulin is one of the most important hormones that controls the glucose level in blood. Therefore, the investigation of insulin is very important for the diabetic research. There have been many reports analyzing the role of insulin with insulin detecting methods. Most of these methods utilized antibodies or aptamers for insulin recognition. Although these sensors are highly sensitive and specific to insulin attributed to the target recognition ability of antibodies or aptamers, there is a possibility to detect physiologically inactive insulin such as partially digested or mutated insulin because the antibodies or the aptamers recognize only a part of insulin molecule. However, what we need is to measure the concentration of functionally active insulin for accurate investigation in our body. Therefore, it is necessary to develop a novel method based on a new target recognition mechanism for the accurate evaluation of insulin.To meet the demand, we have paid attention to the mechanism of recognition in an insulin receptor, because the recognition by the insulin receptor is the first step and one of the most essential processes for insulin to exhibit its function in nature. The insulin receptor is a dimer protein and each monomer contains the carboxy-terminal α-chain (αCT) segment and the first leucine-rich repeat with the cysteine-rich (L1CR) domain as an insulin recognition site. The insulin molecule is captured by the αCT segment from one monomer and the L1CR domain from the other monomer. However, a part of insulin receptor is buried in a cellular membrane and thus it is difficult to use the receptor in a soluble format. Hence, we have developed a protein-based artificial receptor which is soluble in aqueous environment. In this presentation, we demonstrate the applications of the artificial receptor for biosensing such as online monitoring of insulin secretion from drug-stimulated living cells and for imaging analysis of insulin secretion in a single cellular level.The genes of the αCT segment and the L1CR domain in the primary binding site on the insulin receptor were connected with an appropriate length of peptide linker to be the main component of protein-based artificial insulin receptor. To detect the structural change of the artificial receptor in response to the insulin recognition, the genes of a bioluminescent protein (Nanoluc) and a fluorescent protein (YPet) were further connected. By connecting these proteins, the structural change could be evaluated by a bioluminescence resonance energy transfer (BRET) system. The genetically fused artificial protein termed as NαLY, was expected to work as a biosensing protein probe, exhibiting BRET signal in response to insulin. The gene was successfully expressed utilizing mammalian cells and the expressed protein was purified utilizing His-tag affinity in aqueous solution.The feasibility of insulin detection with NαLY was confirmed by measuring luminescence spectra. The luminescence spectra indicated a clear BRET signal in response to insulin. The emission being attributed to Nluc decreased and the emission from YPet increased. The BRET units were proportional to the concentration of insulin, and thus we concluded that the artificial receptor-based insulin sensing probe has an insulin recognition ability presumably reflecting that of the insulin receptor and works as a biosensing probe for insulin. Then, the artificial-receptor-based biosensing probe was applied for the online monitoring of insulin secretion. When the probe was added in the culture medium, it was proved that a time-course in BRET signal reflected the insulin secreting response of drug-stimulated pancreatic-β-cells (MIN6 cell line). To analyze the secretion of insulin from a single-living cell, insulin sensor cells which express the artificial-receptor-based biosensing probe on a cellular surface were fabricated. The sensor cells were then co-cultured with insulin-secreting cells. Imaging analysis revealed that some of the sensor cells nearby pancreatic β cells showed a spike-shaped BRET signal response when co-cultured MIN6 cells were stimulated by glucose.From these results, it is concluded that the protein-based artificial receptor has a great potential to be a platform to create a novel biosensing probe for insulin sensing.

Twohuman menopausal gonadotrophin (hMG) preparations display different early signaling in vitro.

Commercial hMG drugs are marketed for the treatment of infertility and consist of highly purified hormones acting on receptors expressed in target gonadal cells. Menopur® and Meriofert® are combined preparation of FSH and hCG and are compared in vitro herein. To this purpose, the molecular composition of the two drugs was analyzed by immunoassay. The formation of FSH receptor and LH/hCG receptor (FSHR; LHCGR) heteromer, intracellular Ca2+ and cAMP activation, β-arrestin 2 recruitment and the synthesis of progesterone and estradiol were evaluated in transfected HEK293 and human primary granulosa lutein cells treated by drugs administered within the pg-mg/ml concentration range. Molecular characterization revealed that Meriofert® has a higher FSH:hCG ratio than Menopur® which, in turn, displays the presence of LH molecules. While both drugs induced similar FSHR-LHCGR heteromeric formations and intracellular Ca2+ increase, Meriofert® had a higher potency than Menopur® in inducing a cAMP increase. Moreover, Meriofert® revealed a higher potency than Menopur® in recruiting β-arrestin 2, likely due to different FSH content modulating the tridimensional structure of FSHR-LHCGR-β-arrestin 2 complexes, as evidenced by a decrease in bioluminescence resonance energy transfer signal. This drug-specific activation of intracellular signaling pathways is consistent with the molecular composition of these preparations and impacts downstream progesterone and estradiol production, with Menopur® more potent than Meriofert® in inducing the synthesis of both the steroids. These findings are suggestive of distinct in-vivo activities of these preparations, but require cautious interpretation and further validation from clinical studies.