Luciferase-YFP fusion tag with enhanced emission for single-cell luminescence imaging

Taking advantage of the phenomenon of bioluminescence resonance energy transfer (BRET), we developed a bioluminescent probe composed of EYFP and Renilla reniformis luciferase (RLuc)--BRET-based autoilluminated fluorescent protein on EYFP (BAF-Y)--for near-real-time single-cell imaging. We show that BAF-Y exhibits enhanced RLuc luminescence intensity and appropriate subcellular distribution when it was fused to targeting-signal peptides or histone H2AX, thus allowing high spatial and temporal resolution microscopy of living cells.     

Analysis of in vitro SUMOylation using bioluminescence resonance energy transfer (BRET)

We demonstrated in vitro small ubiquitin-like modifier (SUMO)-mediated modification (SUMOylation) of RanGTPase activating protein-1 (RanGAP1) by using bioluminescence resonance energy transfer (BRET) for studying protein interactions. Renilla luciferase (Rluc) was fused to SUMO, and RanGAP1, the binding partner of SUMO, was fused to enhanced yellow fluorescence protein (EYFP). Upon binding of SUMO and RanGAP1, BRET was observed between EYFP (donor) and Rluc (acceptor) in the presence of E1 (Aos1/Uba2) and E2 (Ubc9) enzymes, whereas mutation (K524A) of RanGAP1 at its SUMO binding site prevented significant energy transfer. Comparing BRET and fluorescence resonance energy transfer (FRET) efficiencies using this in vitro model system, we observed that BRET efficiency was 3-fold higher than FRET efficiency, due to the lower background signal intensity of EYFP in the BRET system. Consequently, BRET system is expected to be useful for in vitro analysis of SUMOylation as well as studying other protein interactions.     

Levels of neuropeptides nocistatin, nociceptin/orphanin FQ and their precursor protein in a rat neuropathic pain model

Neuropeptides nociceptin/orphanin FQ (N/OFQ) and nocistatin (NST) are related to pain modulation. The amounts of these peptides and their precursor protein, prepronociceptin (ppN/OFQ) in the brain, spinal cord and serum samples of rats with partial sciatic nerve ligation (PSNL) were compared with those in na?ve rats using radioimmunoassay (RIA). There was a significant rise in the levels of ppN/OFQ, N/OFQ and NST in the brains of PSNL rats. Their spinal cords showed significantly increased ppN/OFQ and NST levels but no change in N/OFQ levels. The PSNL rats also had increased serum NST (statistically significant) and N/OFQ (statistically insignificant) with decreased ppN/OFQ suggesting important roles of these peptides in neuropathic pain mechanism.     

Detecting Protein–Protein Interactions in Living Cells: Development of a Bioluminescence Resonance Energy Transfer Assay to Evaluate the PSD-95/NMDA Receptor Interaction

The PDZ domain mediated interaction between the NMDA receptor and its intracellular scaffolding protein, PSD-95, is a potential target for treatment of ischemic brain diseases. We have recently developed a number of peptide analogues with improved affinity for the PDZ domains of PSD-95 compared to the endogenous C-terminal peptide of the NMDA receptor, as evaluated by a cell-free protein–protein interaction assay. However, it is important to address both membrane permeability and effect in living cells. Therefore a bioluminescence resonance energy transfer (BRET) assay was established, where the C-terminal of the NMDA receptor and PDZ2 of PSD-95 were fused to green fluorescent protein (GFP) and Renilla luciferase (Rluc) and expressed in COS7 cells. A robust and specific BRET signal was obtained by expression of the appropriate partner proteins and subsequently, the assay was used to evaluate a Tat-conjugated peptide for its ability to disrupt the PSD-95/NMDA receptor interaction in living cells.     

Detection of heteromerization of more than two proteins by sequential BRET-FRET

Identification of higher-order oligomers in the plasma membrane is essential to decode the properties of molecular networks controlling intercellular communication. We combined bioluminescence resonance energy transfer (BRET) and fluorescence resonance energy transfer (FRET) in a technique called sequential BRET-FRET (SRET) that permits identification of heteromers formed by three different proteins. In SRET, the oxidation of a Renilla luciferase (Rluc) substrate by an Rluc fusion protein triggers acceptor excitation of a second fusion protein by BRET and subsequent FRET to a third fusion protein. We describe two variations of SRET that use different Rluc substrates with appropriately paired acceptor fluorescent proteins. Using SRET, we identified complexes of cannabinoid CB(1), dopamine D(2) and adenosine A(2A) receptors in living cells. SRET is an invaluable technique to identify heteromeric complexes of more than two neurotransmitter receptors, which will allow us to better understand how signals are integrated at the molecular level.     

Nocistatin and nociceptin given centrally induce opioid-mediated gastric mucosal protection

Nociceptin (N/OFQ) and nocistatin (NST) are two endogenous neuropeptides derived from the same precursor protein, preproN/OFQ. The aim of the present work was to study the effect of NST on the ethanol-induced mucosal damage compared with that of N/OFQ following intracerebroventricular (i.c.v.) administration in the rat and to analyze the mechanism of the gastroprotective action. It was found that both NST and N/OFQ reduced the mucosal lesions in the same dose range (0.2–1 nmol i.c.v.), but in higher doses (2–5 nmol i.c.v.) the gastroprotective effect of both peptides was highly diminished. The gastroprotective effect of N/OFQ (1 nmol), but not that of NST (1 nmol), was reduced by the selective nociceptin receptor antagonist J-113397 (69 nmol i.c.v.). Similarly, decrease of the gastroprotective effect was observed after the combination of NST (1 nmol) with N/OFQ (0.6 or 1 nmol). However, addition of the gastroprotective effects was observed, when lower dose (0.2 nmol) of NST was given prior to N/OFQ (0.6 nmol). The gastroprotective effect of both N/OFQ and NST was antagonized by naloxone (27 nmol), β-funaltrexamine (20 nmol), naltrindole (5 nmol) and norbinaltorphimine (14 nmol), the μ-, δ- and κ-opioid receptor antagonists, respectively, given i.c.v. The mucosal protection was significantly decreased after bilateral cervical vagotomy. The present findings suggest that NST similar to N/OFQ, may also induce gastric mucosal protective action initiated centrally in a vagal-dependent mechanism. Opioid component is likely to be involved in the gastroprotective effect of both NST and N/OFQ.     

Demonstration of a homogeneous noncompetitive immunoassay based on bioluminescence resonance energy transfer

We describe a noncompetitive homogeneous bioluminescent immunoassay based on the antigen-dependent reassociation of antibody variable domains (open sandwich bioluminescent immunoassay, OS-BLIA). The reassociation of two chimeric proteins, an antibody heavy-chain fragment (V(H))-Renilla luciferase (Rluc) and an antibody light-chain fragment (V(L))-enhanced yellow fluorescent protein (EYFP), was monitored by a bioluminescence resonance energy transfer (BRET) between the two. Upon simple mixing of the reagents with the sample, an antigen-dependent increase in BRET was observed with a measurable concentration range of 0.1 to approximately 10 microg/ml antigen hen egg lysozyme. Compared with our comparable assays based on fluorescence resonance energy transfer (FRET), a 10-fold improvement in the sensitivity was attained, probably due to a reduction in reagent concentration.     

Assembly and signaling of CRLR and RAMP1 complexes assessed by BRET

Biochemical and functional evidence suggest that the calcitonin receptor-like receptor (CRLR) interacts with receptor activity-modifying protein-1 (RAMP1) to generate a calcitonin gene-related peptide (CGRP) receptor. Using bioluminescence resonance energy transfer (BRET), we investigated the oligomeric assembly of the CRLR-RAMP1 signaling complex in living cells. As for their wild-type counterparts, fusion proteins linking CRLR and RAMP1 to the energy donor Renilla luciferase (Rluc) and energy acceptor green fluorescent protein (GFP) reach the cell surface only upon coexpression of CRLR and RAMP1. Radioligand binding and cAMP production assays also confirmed that the fusion proteins retained normal functional properties. BRET titration experiments revealed that CRLR and RAMP1 associate selectively to form heterodimers. This association was preserved for a mutated RAMP1 that cannot reach the cell surface, even in the presence of CRLR, indicating that the deficient targeting resulted from the altered conformation of the complex rather than a lack of heterodimerization. BRET analysis also showed that, in addition to associate with one another, both CRLR and RAMP1 can form homodimers. The homodimerization of the coreceptor was further confirmed by the ability of RAMP1 to prevent cell surface targeting of a truncated RAMP1 that normally exhibits receptor-independent plasma membrane delivery. Although the role of such dimerization remains unknown, BRET experiments clearly demonstrated that CRLR can engage signaling partners, such as G proteins and beta-arrestin, following CGRP stimulation, only in the presence of RAMP1. In addition to shed new light on the CRLR-RAMP1 signaling complex, the BRET assays developed herein offer new biosensors for probing CGRP receptor activity.     

The orphanin FQ/nociceptin gene: structure, tissue distribution of expression and functional implications obtained from knockout mice

Like other neuropeptides, orphanin FQ/nociceptin (OFQ/N) is encoded by a larger precursor protein. The cDNA for the OFQ/N precursor has been cloned from human, rat, mouse and bovine tissue demonstrating that this peptidergic system serves important functions that have been conserved during evolution. The structural organization of the precursor protein is similar to opioid peptide precursors, supporting the view of a common origin for the opioid systems and the OFQ/N system. In addition to OFQ/N, the precursor may encode two other biologically active peptides. Anatomic studies have revealed high levels of expression of the OFQ/N messenger RNA in brain structures involved in sensory, emotional and cognitive processing. In particular, high levels of OFQ/N mRNA were detected in the limbic system, underlining the stress attenuating activities that have been described as an important function of OFQ/N. Recently, mutant mice have been generated that lack the precursor protein of OFQ/N to further define the physiological functions of the OFQ/N system. The OFQ/N-deficient mice are characterized by an increased sensitivity to stressful stimuli and a lack of habituation to chronic and repeated stress. This review will summarize recent findings on the molecular biology of the OFQ/N precursor and relate it to possible physiological functions of this newly discovered neuropeptide system.     

Identification of NIPSNAP1 as a nocistatin-interacting protein involving pain transmission

4-Nitrophenylphosphatase domain and non-neuronal SNAP25-like protein homolog 1 (NIPSNAP1) is a molecule of physiologically unknown function, although it is predominantly expressed in the brain, spinal cord, liver, and kidney. We identified NIPSNAP1 as a protein that interacts with the neuropeptide nocistatin (NST) from synaptosomal membranes of mouse spinal cord using high-performance affinity latex beads. NST, which is produced from the same precursor protein as an opioid-like neuropeptide nociceptin/orphanin FQ (N/OFQ), has opposite effects on pain transmission evoked by N/OFQ. The calculated full-length pre-protein of NIPSNAP1 was 33 kDa, whereas the N-terminal truncated form of NIPSNAP1 (29 kDa) was ubiquitously expressed in the neuronal tissues, especially in synaptic membrane and mitochondria of brain. The 29-kDa NIPSNAP1 was distributed on the cell surface, and NST interacted with the 29-kDa but not the 33-kDa NIPSNAP1. Although intrathecal injection of N/OFQ induced tactile allodynia in both wild-type and NIPSNAP1-deficient mice, the inhibition of N/OFQ-evoked tactile allodynia by NST seen in wild-type mice was completely lacking in the deficient mice. These results suggest that NIPSNAP1 is an interacting molecule of NST and plays a crucial role in pain transmission.     

Sequential bioluminescence resonance energy transfer-fluorescence resonance energy transfer-based ratiometric protease assays with fusion proteins of firefly luciferase and red fluorescent protein

We report here the preparation of ratiometric luminescent probes that contain two well-separated emission peaks produced by a sequential bioluminescence resonance energy transfer (BRET)–fluorescence resonance energy transfer (FRET) process. The probes are single soluble fusion proteins consisting of a thermostable firefly luciferase variant that catalyze yellow-green (560 nm maximum) bioluminescence and a red fluorescent protein covalently labeled with a near-infrared fluorescent dye. The two proteins are connected by a decapeptide containing a protease recognition site specific for factor Xa, thrombin, or caspase 3. The rates of protease cleavage of the fusion protein substrates were monitored by recording emission spectra and plotting the change in peak ratios over time. Detection limits of 0.41 nM for caspase 3, 1.0 nM for thrombin, and 58 nM for factor Xa were realized with a scanning fluorometer. Our results demonstrate for the first time that an efficient sequential BRET–FRET energy transfer process based on firefly luciferase bioluminescence can be employed to assay physiologically important protease activities.     

Fusion of Aequorea victoria GFP and aequorin provides their Ca(2+)-induced interaction that results in red shift of GFP absorption and efficient bioluminescence energy transfer

The bioluminescence emitted by Aequorea victoria jellyfish is greenish while its single bioluminescent photoprotein aequorin emits blue light. This phenomenon may be explained by a bioluminescence resonance energy transfer (BRET) from aequorin chromophore to green fluorescent protein (GFP) co-localized with it. However, a slight overlapping of the aequorin bioluminescence spectrum with the GFP absorption spectrum and the absence of marked interaction between these proteins in vitro pose a question on the mechanism providing the efficient BRET in A. victoria. Here we report the in vitro study of BRET between homologous Ca(2+)-activated photoproteins, aequorin or obelin (Obelia longissima), as bioluminescence energy donors, and GFP, as an acceptor. The fusions containing donor and acceptor proteins linked by a 19 aa peptide were purified after expressing their genes in Escherichia coli cells. It was shown that the GFP-aequorin fusion has a significantly greater BRET efficiency, compared to the GFP-obelin fusion. Two main factors responsible for the difference in BRET efficiency of these fusions were revealed. First, it is the presence of Ca(2+)-induced interaction between the donor and acceptor in the aequorin-containing fusion and the absence of the interaction in the obelin-containing fusion. Second, it is a red shift of GFP absorption toward better overlapping with aequorin bioluminescence induced by the interaction of aequorin with GFP. Since the connection of the two proteins in vitro mimics their proximity in vivo, Ca(2+)-induced interaction between aequorin and GFP may occur in A. victoria jellyfish providing efficient BRET in this organism.     

Chemically modified firefly luciferase is an efficient source of near-infrared light

Bioluminescence and bioluminescence resonance energy transfer (BRET) are two naturally occurring light emission phenomena that have been adapted to a wide variety of important research applications including in vivo imaging and enzyme assays. The luciferase enzyme from the North American firefly, which produces yellow-green light, is a key component of many of these applications. Recognizing the heightened interest in the potential of near-infrared (nIR) light to improve these technologies, we have demonstrated that spectral emissions with maxima of 705 and 783 nm can be efficiently produced by a firefly luciferase variant covalently labeled with nIR fluorescent dyes. In one case, an outstanding BRET ratio of 34.0 was achieved emphasizing the importance of selective labeling with fluorescent dyes and the efficiency provided by the intramolecular BRET process. Additionally, we constructed a biotinylated fusion protein that similarly produced nIR light. This novel material was immobilized on solid supports containing streptavidin, demonstrating, in principle, that it may be used for receptor-based imaging. Also, the matrix-bound labeled fusion protein was used to measure factor Xa activity at physiological concentrations in blood. We believe this to be the first report of bright nIR light sources produced by chemical modification of a beetle luciferase.     

Leukotriene C4 synthase homo-oligomers detected in living cells by bioluminescence resonance energy transfer

Leukotrienes (LTs) are biologically active compounds derived from arachidonic acid which have important pathophysiological roles in asthma and inflammation. The cysteinyl leukotriene LTC(4) and its metabolites LTD(4) and LTE(4) stimulate bronchoconstriction, airway mucous formation and generalized edema formation. LTC(4) is formed by addition of glutathione to LTA(4), catalyzed by the integral membrane protein, LTC(4) synthase (LTCS). We now report the use of bioluminescence resonance energy transfer (BRET) to demonstrate that LTCS forms homo-oligomers in living cells. Fusion proteins of LTCS and Renilla luciferase (Rluc) and a variant of green fluorescent protein (GFP), respectively, were prepared. High BRET signals were recorded in transiently transfected human embryonic kidney (HEK 293) cells co-expressing Rluc/LTCS and GFP/LTCS. Homo-oligomer formation in living cells was verified by co-transfection of a plasmid expressing non-chimeric LTCS. This resulted in dose-dependent attenuation of the BRET signal. Additional evidence for oligomer formation was obtained in cell-free assays using glutathione S-transferase (GST) pull-down assay. To map interaction domains for oligomerization, GFP/LTCS fusion proteins were prepared with truncated variants of LTCS. The results obtained identified a C-terminal domain (amino acids 114-150) sufficient for oligomerization of LTCS. Another, centrally located, interaction domain appeared to exist between amino acids 57-88. The functional significance of LTCS homo-oligomer formation is currently being investigated.     

Constitutive and agonist-dependent homo-oligomerization of the thyrotropin-releasing hormone receptor. Detection in living cells using bioluminescence resonance energy transfer

The ability of G-protein-coupled receptors (GPCRs) to interact to form new functional structures, either forming oligomers with themselves or forming associations with other intracellular proteins, has important implications for the regulation of cellular events; however, little is known about how this occurs. Here, we have employed a newly emerging technology, bioluminescence resonance energy transfer (BRET), used to study protein-protein interactions in living cells, to demonstrate that the thyrotropin-releasing hormone receptor (TRHR) forms constitutive homo-oligomers. This formation of TRHR homo-oligomers in the absence of ligand was shown by demonstration of an energy transfer between TRHR molecules fused to either donor, Renilla luciferase (Rluc) or acceptor, enhanced yellow fluorescent protein (EYFP) molecules. This interaction was shown to be specific, since energy transfer was not detected between co-expressed tagged TRHRs and either complementary tagged gonadotropin-releasing hormone (GnRH) or beta(2)-adrenergic receptors. Furthermore, generation of a BRET signal between the TRHRs could only be inhibited by co-expression of the wild-type TRHR and not by other GPCRs. Agonist stimulation led to a time- and dose-dependent increase in the amount of energy transfer. Inhibition of receptor internalization by co-expression of dynamin mutant K44A did not affect the interaction between TRHRs, suggesting that clustering of receptors within clathrin-coated pits is not sufficient for energy transfer to occur. BRET also provided evidence for the agonist-induced oligomerization of another GPCR, the GnRH receptor (GnRHR), and the presence of an agonist-induced interaction of the adaptor protein, beta-arrestin, with TRHR and the absence of an interaction of beta-arrestin with GnRHR. This study supports the usefulness of BRET as a powerful tool for studying GPCR aggregations and receptor/protein interactions in general and presents evidence that the functioning unit of TRHRs exists as homomeric complexes.     

Real-time bioluminescence imaging of a protein secretory pathway in living mammalian cells using Gaussia luciferase

Using photon counting and charge-coupled device (CCD) cameras, we have applied the method of real-time bioluminescence imaging to investigate protein trafficking in mammalian cells. In the living cells of Chinese hamster ovary and PC12D cells, exocytotic secretion of protein and protein targeting on the cell surface were visualized using the secreted Gaussia luciferase (GLase) as a reporter protein in a minute. After incubation of the cells with luciferin (coelenterazine) for 10min, luciferin was imported into the cells and the vesicle transport network in the cells could be shown by luminescence images of GLase activity. Further, we demonstrate that GLase with a heterologous signal peptide sequence is targeted to the cell surface in neuronally differentiated PC12D cells and luminescence signals could be detected in a few seconds.     

Functional complementation of high-efficiency resonance energy transfer: a new tool for the study of protein binding interactions in living cells

Green bioluminescence in Renilla species is generated by a approximately 100% efficient RET (resonance energy transfer) process that is caused by the direct association of a blue-emitting luciferase [Rluc (Renilla luciferase)] and an RGFP (Renilla green fluorescent protein). Despite the high efficiency, such a system has never been evaluated as a potential reporter of protein-protein interactions. To address the question, we compared and analysed in mammalian cells the bioluminescence of Rluc and RGFP co-expressed as free native proteins, or as fused single-chain polypeptides and tethered partners of self-assembling coiled coils. Here, we show that: (i) no spontaneous interactions generating detectable BRET (bioluminescence RET) signals occur between the free native proteins; (ii) high-efficiency BRET similar to that observed in Renilla occurs in both fusion proteins and self-interacting chimaeras, but only if the N-terminal of RGFP is free; (iii) the high-efficiency BRET interaction is associated with a dramatic increase in light output when the luminescent reaction is triggered by low-quantum yield coelenterazine analogues. Here, we propose a new functional complementation assay based on the detection of the high-efficiency BRET signal that is generated when the reporters Rluc and RGFP are brought into close proximity by a pair of interacting proteins to which they are linked. To demonstrate its performance, we implemented the assay to measure the interaction between GPCRs (G-protein-coupled receptors) and beta-arrestins. We show that complementation-induced BRET allows detection of the GPCR-beta-arrestin interaction in a simple luminometric assay with high signal-to-noise ratio, good dynamic range and rapid response.     

A comparative analysis of resonance energy transfer methods for Alzheimer related protein-protein interactions in living cells

Fluorescence resonance energy transfer (FRET) and bioluminescence resonance energy transfer (BRET) are extensively used to analyze protein interactions occurring in living cells. Although these two techniques are broadly applied in cellular biology, comparative analysis of their strengths and limitations is lacking. To this end, we analyzed a small network of proteins involved in the amyloidogenic processing of the Alzheimer β-amyloid precursor using FRET based cytometry, BRET, and fluorescence lifetime imaging microscopy (FLIM). Using all three methods, we were able to detect the interactions of the amyloid precursor protein with APBB1, APBB2, and APP itself. And we found an unreported interacting pair, APP-APH1A. In addition, we show that these four interacting pairs exhibit a strong FRET correlation with the acceptor/donor expression ratios. Overall the FRET based cytometry was the most sensitive and reliable approach to screen for new interacting proteins. Therefore, we applied FRET based cytometry to study competitive binding of two proteins, APBB1 and APBB2, with the same APP target.     

B族G蛋白偶联受体PAC1的N端基序HSDCIF对其二聚化和上膜运输的影响

B族G蛋白偶联受体（G protein-coupled receptors, GPCRs）PAC1是垂体腺苷酸环化酶激活多肽（pituitary adenylate cyclase activating polypeptide, PACAP）的特异受体,介导PACAP神经保护等功能,是神经系统疾病药物开发的重要靶点之一. HSDCIF（His-Ser-Asp-Cys-Ile-Phe）为位于PAC1的N端胞外1区（extracellar domain 1, EC1）的一段短肽序列,与特定负责激活PAC1受体的激动域PACAP（1-6）具有极高的同源性.利用基因敲除技术构建出缺陷HSDCIF基序的PAC1突变体（简称D PAC1）|利用基因工程原理和技术构建系列真核表达重组载体,包括融合了增强型黄色荧光蛋白（enhanced yellow fluorescent protein, EYFP）的表达载体D-PAC1-EYFP|用于生物发光能量转移（bioluminescence resonance energy transfer, BRET）检测的D-PAC1-Rluc|以及用于双分子荧光互补（bimolecular fluorescence complementation, BiFC）实验的D-PAC1-EYFP/N和D-PAC1-EYFP/C.免疫荧光检测（immunofluorescence assay）测定D PAC1的表达|荧光共聚焦显微观察D-PAC1的细胞运输,然后通过 Western印迹、BRET与BiFC方法来检测D PAC1的二聚化情况,综合评价HSDCIF基序对PAC1二聚化和在细胞中定位的影响.检测结果显示,缺陷HSDCIF基序的突变体D PAC1不能发生二聚化,也不能正常的进行上膜运输,而是滞留在内质网中,同时外源化学合成的寡肽HSDCIF可以竞争性地抑制正常PAC1的二聚化.     

Oligomerization of adenosine A2A and dopamine D2 receptors in living cells

We investigated whether oligomerization of adenosine A(2A) receptor (A(2A)R) and dopamine D(2) receptor (D(2)R) exists in living cells using modified bioluminescence resonance energy transfer (BRET(2)) technology. Fusion of these receptors to a donor, Renilla luciferase (Rluc), and to an acceptor, modified green fluorescent protein (GFP(2)), did not affect the ligand binding affinity, subcellular distribution, and coimmunoprecipitation of the receptors. BRET was detected not only between Myc-D(2)R-Rluc and A(2A)R-GFP(2) but also between HA-tagged A(2A)R-Rluc and A(2A)R-GFP(2). These results indicate A(2A)R, either homomeric or heteromeric with D(2)R, exists as an oligomer in living cells.