Bioluminescence Resonance Energy Transfer Research Articles

KCTD5 Forms Hetero-Oligomeric Complexes with Various Members of the KCTD Protein Family.

Potassium Channel Tetramerization Domain 5 (KCTD5) regulates diverse aspects of physiology, ranging from neuronal signaling to colorectal cancer. A key feature of KCTD5 is its self-assembly into multi-subunit oligomers that seemingly enables participation in an array of protein-protein interactions. KCTD5 has recently been reported to form hetero-oligomeric complexes with two similar KCTDs (KCTD2 and KCTD17). However, it is not known if KCTD5 forms hetero-oligomeric complexes with the remaining KCTD protein family which contains over two dozen members. Here, we demonstrate that KCTD5 interacts with various KCTD proteins when assayed through co-immunoprecipitation in lysed cells. We reinforced this dataset by examining KCTD5 interactions in a live-cell bioluminescence resonance energy transfer (BRET)-based approach. Finally, we developed an IP-luminescence approach to map regions on KCTD5 required for interaction with a selection of KCTD that have established roles in neuronal signaling. We report that different regions on KCTD5 are responsible for uniquely contributing to interactions with other KCTD proteins. While our results help unravel additional interaction partners for KCTD5, they also reveal additional complexities in KCTDs' biology. Moreover, our findings also suggest that KCTD hetero-oligomeric interactions may occur throughout the KCTD family.

Ticagrelor inverse agonist activity at the P2Y12 receptor is non-reversible versus its endogenous agonist adenosine 5´-diphosphate.

Ticagrelor is labelled as a reversible, direct-acting platelet P2Y12 receptor (P2Y12 R) antagonist that is indicated clinically for the prevention of thrombotic events in patients with acute coronary syndrome (ACS). As with many antiplatelet drugs, ticagrelor therapy increases bleeding risk in patients, which may require platelet transfusion in emergency situations. The aim of this study was to further examine the reversibility of ticagrelor at the P2Y12 R. Studies were performed in human platelets, with P2Y12 R-stimulated GTPase activity and platelet aggregation assessed. Cell-based bioluminescence resonance energy transfer (BRET) assays were undertaken to assess G protein-subunit activation downstream of P2Y12 R activation. Initial studies revealed that a range of P2Y12 R ligands, including ticagrelor, displayed inverse agonist activity at P2Y12 R. Only ticagrelor was resistant to washout and, in human platelet and cell-based assays, washing failed to reverse ticagrelor-dependent inhibition of ADP-stimulated P2Y12 R function. The P2Y12 R agonist 2MeSADP, which was also resistant to washout, was able to effectively compete with ticagrelor. In silico docking revealed that ticagrelor and 2MeSADP penetrated more deeply into the orthosteric binding pocket of the P2Y12 R than other P2Y12 R ligands. Ticagrelor binding to P2Y12 R is prolonged and more akin to that of an irreversible antagonist, especially versus the endogenous P2Y12 R agonist ADP. This study highlights the potential clinical need for novel ticagrelor reversal strategies in patients with spontaneous major bleeding, and for bleeding associated with urgent invasive procedures.

Protein kinase B (Akt) blockade inhibits LH/hCG-mediated 17,20-lyase, but not 17α-hydroxylase activity of Cyp17a1 in mouse Leydig cell steroidogenesis

Androgens are produced by adrenal and gonadal cells thanks to the action of specific enzymes. We investigated the role of protein kinase B (Akt) in the modulation of Δ4 steroidogenic enzymes' activity, in the mouse Leydig tumor cell line mLTC1. Cells were treated for 0–24 h with the 3 × 50% effective concentration of human luteinizing hormone (LH) and choriogonadotropin (hCG), in the presence and in the absence of the specific Akt inhibitor 3CAI. Cell signaling analysis was performed by bioluminescence resonance energy transfer (BRET) and Western blotting, while the expression of key target genes was investigated by real-time PCR. The synthesis of progesterone, 17α-hydroxy (OH)-progesterone and testosterone was measured by immunoassay. Control experiments for cell viability and caspase 3 activation were performed as well. We found that both hormones activated cAMP and downstream effectors, such as extracellularly-regulated kinase 1/2 (Erk1/2) and cAMP response element-binding protein (Creb), as well as Akt, and the transcription of Stard1, Hsd3b1, Cyp17a1 and Hsd17b3 genes, boosting the Δ4 steroidogenic pathway. Interestingly, Akt blockade decreased selectively Cyp17a1 expression levels, inhibiting its 17,20-lyase, but not the 17-hydroxylase activity. This effect is consistent with lower Cyp17a1 affinity to 17α-OH-progesterone than progesterone. As a result, cell treatment with 3CAI resulted in 17α-OH-progesterone accumulation at 16–24 h and decreased testosterone levels after 24 h. In conclusion, in the mouse Leydig cell line mLTC1, we found substantial Akt dependence of the 17,20-lyase activity and testosterone synthesis. Our results indicate that different intracellular pathways modulate selectively the dual activity of Cyp17a1.

Heterodimerization of Chemoreceptors TAS1R3 and mGlu2 in Human Blood Leukocytes.

The expression of canonical chemosensory receptors of the tongue, such as the heteromeric sweet taste (TAS1R2/TAS1R3) and umami taste (TAS1R1/TAS1R3) receptors, has been demonstrated in many extra-oral cells and tissues. Gene expression studies have revealed transcripts for all TAS1 and metabotropic glutamate (mGlu) receptors in different types of immune cells, where they are involved, for example, in the chemotaxis of human neutrophils and the protection of T cells from activation-induced cell death. Like other class-C G protein-coupling receptors (GPCRs), TAS1Rs and mGlu receptors form heteromers within their families. Since mGlu receptors and TAS1R1/TAS1R3 share the same ligand, monosodium glutamate (MSG), we hypothesized their hitherto unknown heteromerization across receptor families in leukocytes. Here we show, by means of immunocytochemistry and co-IP/Western analysis, that across class-C GPCR families, mGlu2 and TAS1R3 co-localize and heterodimerize in blood leukocytes. Expressing the recombinant receptors in HEK-293 cells, we validated their heterodimerization by bioluminescence resonance energy transfer. We demonstrate MSG-induced, mGlu2/TAS1R3 heteromer-dependent gain-of-function and pertussis toxin-sensitive signaling in luminescence assays. Notably, we show that mGlu2/TAS1R3 is necessary and sufficient for MSG-induced facilitation of N-formyl-methionyl-leucyl-phenylalanine-stimulated IL-8 secretion in neutrophils, using receptor-specific antagonists. In summary, our results demonstrate mGlu2/TAS1R3 heterodimerization in leukocytes, suggesting cellular function-tailored chemoreceptor combinations to modulate cellular immune responses.

Pharmacological Inhibition of PTEN Rescues Dopaminergic Neurons by Attenuating Apoptotic and Neuroinflammatory Signaling Events.

Parkinson's disease (PD) is a progressive neurodegenerative disorder characterized by the selective degeneration of dopaminergic neurons in the substantia nigra pars compacta resulting in an irreversible and a debilitating motor dysfunction. Though both genetic and idiopathic factors are implicated in the disease etiology, idiopathic PD comprise the majority of clinical cases and is caused by exposure to environmental toxicants and oxidative stress. Fyn kinase activation has been identified as an early molecular signaling event that primes neuroinflammatory and neurodegenerative events associated with dopaminergic cell death. However, the upstream regulator of Fyn activation remains unidentified. We investigated whether the lipid and tyrosine phosphatase PTEN (Phosphatase and Tensin homolog deleted on chromosome 10) could be the upstream regulator of Fyn activation in PD models as PTEN has been previously reported to contribute to Parkinsonian pathology. Our findings, using bioluminescence resonance energy transfer (BRET) and immunoblotting, indicate for the first time that PTEN is a critical early stress sensor in response to oxidative stress and neurotoxicants in in vitro models of PD. Pharmacological attenuation of PTEN activity rescues dopaminergic neurons from neurotoxicant-induced cytotoxicity by modulating Fyn kinase activation. Our findings also identify PTEN's novel roles in contributing to mitochondrial dysfunction which contribute to neurodegenerative processes. Interestingly, we found that PTEN positively regulates interleukin-1β (IL-1β) and the transcription of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). Taken together, we have identified PTEN as a disease course altering pharmacological target that may be further validated for the development of novel therapeutic strategies targeting PD.

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.

Gα Protein Signaling Bias at Serotonin 1A Receptor.

Serotonin 1A receptor (5-HT1AR) is a clinically relevant target because of its involvement in several central and peripheral functions, including sleep, temperature homeostasis, processing of emotions, and response to stress. As a G protein coupled receptor (GPCR) activating numerous Gα i/o/z family members, 5-HT1AR can potentially modulate multiple intracellular signaling pathways in response to different therapeutics. Here, we applied a cell-based bioluminescence resonance energy transfer assay to quantify how ten structurally diverse 5-HT1AR agonists exert biased signaling by differentially stimulating Gα i/o/z family members. Our concentration-response analysis of the activation of each Gα i/o/z protein revealed unique potency and efficacy profiles of selected agonists when compared with the reference 5-hydroxytryptamine, serotonin. Overall, our analysis of signaling bias identified groups of ligands sharing comparable G protein activation selectivity and also drugs with unique selectivity profiles. We observed, for example, a strong bias of F-15599 toward the activation of Gα i3 that was unique among the agonists tested: we found a biased factor of +2.19 when comparing the activation of Gα i3 versus Gα i2 by F-15599, while it was -0.29 for 8-hydroxy-2-(di-n-propylamino) tetralin. Similarly, vortioxetine showed a biased factor of +1.06 for Gα z versus Gα oA, while it was -1.38 for vilazodone. Considering that alternative signaling pathways are regulated downstream of each Gα protein, our data suggest that the unique pharmacological properties of the tested agonists could result in multiple unrelated cellular outcomes. Further investigation is needed to reveal how this type of ligand bias could affect cellular responses and to illuminate the molecular mechanisms underlying therapeutic profile and side effects of each drug. SIGNIFICANCE STATEMENT: Serotonin 1a receptor (5-HT1AR) activates several members of the Gi/o/z protein family. Here, we examined ten structurally diverse and clinically relevant agonists acting on 5-HT1AR and identified distinctive bias patterns among G proteins. Considering the diversity of their intracellular effectors and signaling properties, this data reveal novel mechanisms underlying both therapeutic and undesirable effects.

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.

Analysis of the complex between MBD2 and the histone deacetylase core of NuRD reveals key interactions critical for gene silencing.

The nucleosome remodeling and deacetylase (NuRD) complex modifies nucleosome positioning and chromatin compaction to regulate gene expression. The methyl-CpG-binding domain proteins 2 and 3 (MBD2 and MBD3) play a critical role in complex formation; however, the molecular details of how they interact with other NuRD components have yet to be fully elucidated. We previously showed that an intrinsically disordered region (IDR) of MBD2 is necessary and sufficient to bind to the histone deacetylase core of NuRD. Building on that work, we have measured the inherent structural propensity of the MBD2-IDR using solvent and site-specific paramagnetic relaxation enhancement measurements. We then used the AlphaFold2 machine learning software to generate a model of the complex between MBD2 and the histone deacetylase core of NuRD. This model is remarkably consistent with our previous studies, including the current paramagnetic relaxation enhancement data. The latter suggests that the free MBD2-IDR samples conformations similar to the bound structure. We tested this model of the complex extensively by mutating key contact residues and measuring binding using an intracellular bioluminescent resonance energy transfer assay. Furthermore, we identified protein contacts that, when mutated, disrupted gene silencing by NuRD in a cell model of fetal hemoglobin regulation. Hence, this work provides insights into the formation of NuRD and highlights critical binding pockets that may be targeted to block gene silencing for therapy. Importantly, we show that AlphaFold2 can generate a credible model of a large complex that involves an IDR that folds upon binding.

A Semisynthetic Bioluminescence Sensor for Ratiometric Imaging of Metal Ions In Vivo Using DNAzymes Conjugated to An Engineered Nano‐Luciferase

AbstractDNA‐based probes have gained significant attention as versatile tools for biochemical analysis, benefiting from their programmability and biocompatibility. However, most existing DNA‐based probes rely on fluorescence as the signal output, which can be problematic due to issues like autofluorescence and scattering when applied in complex biological materials such as living cells or tissues. Herein, we report the development of bioluminescent nucleic acid (bioLUNA) sensors that offer laser excitation‐independent and ratiometric imaging of the target in vivo. The system is based on computational modelling and mutagenesis investigations of a genetic fusion between circular permutated Nano‐luciferase (NLuc) and HaloTag, enabling the conjugation of the protein with a DNAzyme. In the presence of Zn2+, the DNAzyme sensor releases the fluorophore‐labelled strand, leading to a reduction in bioluminescent resonance energy transfer (BRET) between the luciferase and fluorophore. Consequently, this process induces ratiometric changes in the bioluminescent signal. We demonstrated that this bioLUNA sensor enabled imaging of both exogenous Zn2+ in vivo and endogenous Zn2+ efflux in normal epithelial prostate and prostate tumors. This work expands the DNAzyme sensors to using bioluminescence and thus has enriched the toolbox of nucleic acid sensors for a broad range of biomedical applications.

A Semisynthetic Bioluminescence Sensor for Ratiometric Imaging of Metal Ions In Vivo Using DNAzymes Conjugated to An Engineered Nano-Luciferase.

DNA-based probes have gained significant attention as versatile tools for biochemical analysis, benefiting from their programmability and biocompatibility. However, most existing DNA-based probes rely on fluorescence as the signal output, which can be problematic due to issues like autofluorescence and scattering when applied in complex biological materials such as living cells or tissues. Herein, we report the development of bioluminescent nucleic acid (bioLUNA) sensors that offer laser excitation-independent and ratiometric imaging of the target in vivo. The system is based on computational modelling and mutagenesis investigations of a genetic fusion between circular permutated Nano-luciferase (NLuc) and HaloTag, enabling the conjugation of the protein with a DNAzyme. In the presence of Zn2+ , the DNAzyme sensor releases the fluorophore-labelled strand, leading to a reduction in bioluminescent resonance energy transfer (BRET) between the luciferase and fluorophore. Consequently, this process induces ratiometric changes in the bioluminescent signal. We demonstrated that this bioLUNA sensor enabled imaging of both exogenous Zn2+ in vivo and endogenous Zn2+ efflux in normal epithelial prostate and prostate tumors. This work expands the DNAzyme sensors to using bioluminescence and thus has enriched the toolbox of nucleic acid sensors for a broad range of biomedical applications.

REGA-SIGN: Development of a Novel Set of NanoBRET-Based G Protein Biosensors.

Despite G protein-coupled receptors (GPCRs) being important theapeutic targets, the signaling properties of many GPCRs remain poorly characterized. GPCR activation primarily initiates heterotrimeric G protein signaling. To detect ligand-induced G protein activation, Bioluminescence Resonance Energy Transfer (BRET)-based biosensors were previously developed. Here, we designed a novel set of Nanoluciferase (NLuc) BRET-based biosensors (REGA-SIGN) that covers all Gα protein families (i.e., Gαi/o, GαSs/L, Gα12/13 and Gαq/15). REGA-SIGN uses NLuc as a bioluminescent donor and LSS-mKATE2, a red-shifted fluorophore, as an acceptor. Due to the enhanced spectral separation between donor and acceptor emission and the availability of a stable substrate for NLuc, this donor-acceptor pair enables sensitive kinetic assessment of G protein activity. After optimization, the NLuc integration sites into the Gα subunit largely corresponded with previously reported integration sites, except for GαSs/L for which we describe an alternative NLuc insertion site. G protein rescue experiments validated the biological activity of these Gα donor proteins. Direct comparison between EGFP and LSS-mKATE2 as acceptor fluorophores revealed improved sensitivity for nearly all G protein subtypes when using the latter one. Hence, REGA-SIGN can be used as a panel of kinetic G protein biosensors with high sensitivity.

Chemoproteomics Yields a Selective Molecular Host for Acetyl-CoA.

Chemoproteomic profiling is a powerful approach to define the selectivity of small molecules and endogenous metabolites with the human proteome. In addition to mechanistic studies, proteome specificity profiling also has the potential to identify new scaffolds for biomolecular sensing. Here, we report a chemoproteomics-inspired strategy for selective sensing of acetyl-CoA. First, we use chemoproteomic capture experiments to validate the N-terminal acetyltransferase NAA50 as a protein capable of differentiating acetyl-CoA and CoA. A Nanoluc-NAA50 fusion protein retains this specificity and can be used to generate a bioluminescence resonance energy transfer (BRET) signal in the presence of a CoA-linked fluorophore. This enables the development of a ligand displacement assay in which CoA metabolites are detected via their ability to bind the Nanoluc-NAA50 protein "host" and compete binding of the CoA-linked fluorophore "guest". We demonstrate that the specificity of ligand displacement reflects the molecular recognition of the NAA50 host, while the window of dynamic sensing can be controlled by tuning the binding affinity of the CoA-linked fluorophore guest. Finally, we show that the method's specificity for acetyl-CoA can be harnessed for gain-of-signal optical detection of enzyme activity and quantification of acetyl-CoA from cellular samples. Overall, our studies demonstrate the potential of harnessing insights from chemoproteomics for molecular sensing and provide a foundation for future applications in target engagement and selective metabolite detection.

Functional characterization of CYP1 enzymes: Complex formation, membrane localization and function

CYP1A1, CYP1A2, and CYP1B1 have a high degree of sequence similarity, similar substrate selectivities and induction characteristics. However, experiments suggest that there are significant differences in their quaternary structures and function. The goal of this study was to characterize the CYP1 proteins regarding their ability to form protein-protein complexes, lipid microdomain localization, and ultimately function. This was accomplished by examining (1) substrate metabolism of the CYP1s as a function of NADPH-cytochrome P450 reductase (POR) concentration, and (2) quaternary structure, using bioluminescence resonance energy transfer (BRET). Both CYP1As were able to form BRET-detectable homomeric complexes, which was not observed with CYP1B1. When activities were measured as a function of [POR], CYP1A1 and CYP1B1 showed a hyperbolic response, consistent with mass-action binding; however, CYP1A2 produced a sigmoidal response, suggesting that the homomeric complex affected its function. Differences were observed in their ability to form heteromeric complexes. Whereas CYP1B1 and CYP1A1 formed a complex, neither the CYP1A1/CYP1A2 nor the CYP1B1/CYP1A2 pair formed BRET-detectable complexes. These proteins also differed in their lipid microdomain localization, with CYP1A2 and CYP1B1 residing in ordered membranes, and CYP1A1 in the disordered lipid regions. Taken together, despite their sequence similarities, there are substantial differences in quaternary structures and microdomain localization that can influence enzymatic activities. As these proteins exist in the endoplasmic reticulum with other ER-resident proteins, the P450s need to be considered as part of multi-enzyme systems rather than simply monomeric proteins interacting with their redox partners.

Characterisation of tyrosine kinase inhibitor-receptor interactions at VEGFR2 using sunitinib-red and nanoBRET

Vascular endothelial growth factor (VEGF) is an important mediator of angiogenesis, proliferation and migration of vascular endothelial cells. It is well known that cardiovascular safety liability for a wide range of small molecule tyrosine kinase inhibitors (TKIs) can result from interference with the VEGFR2 signalling system. In this study we have developed a ligand-binding assay using a fluorescent analogue of sunitinib (sunitinib-red) and full length VEGFR2 tagged on its C-terminus with the bioluminescent protein nanoluciferase to monitor ligand-binding to VEGFR2 using bioluminescence resonance energy transfer (BRET). This NanoBRET assay is a proximity-based assay (requiring the fluorescent and bioluminescent components to be within 10 nm of each other) that can monitor the binding of ligands to the kinase domain of VEGFR2. Sunitinib-red was not membrane permeable but was able to monitor the binding affinity and kinetics of a range of TKIs in cell lysates. Kinetic studies showed that sunitinib-red bound rapidly to VEGFR2 at 25 °C and that cediranib had slower binding kinetics with an average residence time of 111 min. Comparison between the log Ki values for inhibition of binding of sunitinib-red and log IC50 values for attenuation of VEGF165a-stimulated NFAT responses showed very similar values for compounds that inhibited sunitinib-red binding. However, two compounds that failed to inhibit sunitinib-red binding (dasatinib and entospletinib) were still able to attenuate VEGFR2-mediated NFAT signalling through inhibition of downstream signalling events. These results suggest that these compounds may still exhibit cardiovascular liabilities as a result of interference with downstream VEGFR2 signalling.

Heterodimerization of apelin and opioid receptor-like 1 receptors mediates apelin-13-induced G protein biased signaling

The apelin receptor (APJ) and the opioid-related nociceptin receptor 1 (ORL1) are family A G protein-coupled receptors that participate in a variety of physiological processes. The distribution and function of APJ and ORL1 in the nervous system and peripheral tissues are similar; however, the detailed mechanism of how these two receptors modulate signaling and physiological effects remains unclear. Here, we examined whether APJ and ORL1 form dimers, and investigated signal transduction pathways. The endogenous co-expression of APJ and ORL1 in SH-SY5Y cells was confirmed by western blotting and RT-PCR. Bioluminescence and fluorescence resonance energy transfer assays, as well as a proximity ligation assay and co-immunoprecipitation experiments, demonstrated that APJ and ORL1 heterodimerize in HEK293 cells. We found that the APJ-ORL1 heterodimer is selectively activated by apelin-13, which causes the dimer to couple to Gαi proteins and reduce the recruitment of GRKs and β-arrestins to the dimer. We showed that the APJ-ORL1 dimer exhibits biased signaling, in which G protein-dependent signaling pathways override β-arrestin-dependent signaling pathways. Our results demonstrate that the structural interface of the APJ-ORL1 dimer switches from transmembrane domain TM1/TM2 in the inactive state to TM5 in the active state. We used mutational analysis and BRET assays to identify key residues in TM5 (APJ L2185.55, APJ I2245.61, and ORL1 L2295.52) responsible for the receptor–receptor interaction. These results provide important information on the APJ-ORL1 heterodimer and may assist the design of new drugs targeting biased signaling pathways for treatment of pain and cardiovascular and metabolic diseases.

Mu-opioid receptor selective superagonists produce prolonged respiratory depression

Synthetic opioids are increasingly challenging to combat the opioid epidemic and act primarily at opioid receptors, chiefly the G protein-coupled receptor (GPCR) μ-opioid receptor (MOR), which signals through G protein-dependent and β-arrestin pathways. Using a bioluminescence resonance energy transfer (BRET) system, we investigate GPCR-signaling profiles by synthetic nitazenes, which are known to cause overdose and death due to respiratory depression. We show that isotonitazene and its metabolite, N-desethyl isotonitazene, are very potent MOR-selective superagonists, surpassing both DAMGO G protein and β-arrestin recruitment activity, which are properties distinct from other conventional opioids. Both isotonitazene and N-desethyl isotonitazene show high potency in mouse analgesia tail-flick assays, but N-desethyl isotonitazene shows longer-lasting respiratory depression compared to fentanyl. Overall, our results suggest that potent MOR-selective superagonists may be a pharmacological property predictive of prolonged respiratory depression resulting in fatal consequences and should be examined for future opioid analgesics.

Detecting GPCR Signals With Optical Biosensors of Gα-GTP in Cell Lines and Primary Cell Cultures.

G protein-coupled receptors (GPCRs) are the largest class of transmembrane receptors and mediate a wide variety of physiological processes. GPCRs respond to a plethora of extracellular ligands and initiate signaling pathways inside cells via heterotrimeric G proteins (Gαβγ). Because of the critical role GPCRs play in regulating biological processes and as pharmacological targets, the availability of tools to measure their signaling activity are of high interest. Live-cell biosensors that detect the activity of G proteins in response to GPCR stimulation have emerged as a powerful approach to investigate GPCR/G protein signaling. Here, we detail methods to monitor G protein activity through direct measurement of GTP-bound Gα subunits using optical biosensors based on bioluminescence resonance energy transfer (BRET). More specifically, this article describes the use of two types of complementary biosensors. The first protocol explains how to use a multicomponent BRET biosensor that relies on expression of exogenous G proteins in cell lines. This protocol yields robust responses that are compatible with endpoint measurements of dose-dependent ligand effects or with kinetic measurements of subsecond resolution. The second protocol describes the implementation of unimolecular biosensors that detect the activation of endogenous G proteins in cell lines expressing exogenous GPCRs or in primary cells upon stimulation of endogenous GPCRs. Overall, using the biosensors as described in this article will help users characterize the mechanisms of action of many pharmacological agents and natural ligands that modulate GPCR and G protein signaling with high precision. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Using bimolecular BRET biosensors to monitor Gα-GTP formation of tagged Gα in live cells Alternate Protocol 1: Measuring GPCR dose-dependent Gα-GTP responses in endpoint format Basic Protocol 2: Using unimolecular BRET biosensors to study endogenous G protein activity Alternate Protocol 2: Using unimolecular BRET biosensors to study endogenous G protein activity in mouse cortical neurons.

Bioluminescence resonance energy transfer biosensor for measuring activity of a protease secreted by Pseudomonas fluorescens growing in milk

Bacterial proteases are sporadic contributors to milk spoilage, reducing the quality of ultra-heat treated (UHT) milk and other dairy products. Current methods for measuring bacterial protease activity in milk are insensitive and too slow to be used in routine testing in dairy processing plants. We have designed a novel bioluminescence resonance energy transfer (BRET)-based biosensor to measure the activity of proteases secreted by bacteria in milk. The BRET-based biosensor is highly selective for bacterial protease activity compared with other proteases tested, notably including plasmin, which is abundant in milk. It incorporates a novel peptide linker that is selectively cleaved by P. fluorescens AprX proteases. The peptide linker is flanked by green fluorescent protein (GFP2) at the N-terminus and a variant Renilla luciferase (RLuc2) at the C-terminus. Complete cleavage of the linker by bacterial proteases from Pseudomonas fluorescens strain 65, leads to a 95% decrease in the BRET ratio. We applied an azocasein-based calibration method to the AprX biosensor using standard international enzyme activity units. In a 10-min assay, the detection limit for AprX protease activity in buffer was equivalent to 40 pg/mL (≈0.8 pM, 22 μU/mL) and 100 pg/mL (≈2pM, 54 μU/mL) in 50% (v/v) full fat milk. The EC50 values were 1.1 ± 0.3 ng/mL (87 μU/mL) and 6.8 ± 0.2 ng/mL (540 μU/mL), respectively. The biosensor was approximately 800x more sensitive than the established FITC-Casein method in a 2-h assay, the shortest feasible time for the latter method. The protease biosensor is sensitive and fast enough to be used in production settings. It is suitable for measuring bacterial protease activity in raw and processed milk, to inform efforts to mitigate the effects of heat-stable bacterial proteases and maximise the shelf-life of dairy products.

On-the-spot trace lactose test for flavored and unflavored lactose-free dairy beverages

We previously reported a novel Bioluminescence Resonance Energy Transfer (BRET)-based biosensing construct able to estimate trace levels of lactose (>0.01% (w/v)), directly in lactose-modified white milk, without the need for sample pre-treatment. In that study, the method was performed by highly trained researchers using a BRET-capable microplate reader in a research laboratory. It is desirable for the method to be adapted for on-the-spot use by production staff in a very different setting, the quality control lab of a milk processing factory. In such a setting, there is a growing requirement to produce and quality-control lactose-modified milks that are colored and flavored. Measurement of trace lactose in these matrices presents additional challenges over white milk because colors, flavors and added sugars potentially interfere with existing electrochemical or colorimetric biosensors. In the current work we therefore describe the development and validation of a method that uses the aforementioned BRET-based lactose biosensing construct to measure trace lactose in white, colored and flavored milks using a dedicated user-friendly device. Milk specific calibrations allowed the estimation of lactose at concentrations ranging from ≈ 7% (w/v) “full-lactose” to 0.01% (w/v), which is the most stringent quantified “lactose-free” level currently mandated globally. Limits of detection were 0.004% (w/v) for white milk and 0.003% (w/v) for both strawberry- and coffee-flavored milks. The assay could be performed in less than 5 min. We demonstrated the use of this assay to follow, effectively in real-time, simulated commercial lactase hydrolyses in white, strawberry- and coffee-flavored milks where the milk was aseptically incubated with food-grade lactase enzyme at 4 °C. These experiments highlighted the potential value of such a point-of-need lactose test for monitoring production, expediting release of finished product and accelerating new product development of a range of lactose-modified beverages.