Luciferase Complementation Assay Research Articles

The cotton 70-kDa heat shock protein GhHSP70-26 plays a positive role in the drought stress response

The 70-kDa heat shock protein (HSP70) is a molecular chaperone that plays an important role in the response of plants to abiotic stress, but the function and molecular mechanism of HSP70 in the cotton drought stress response are unclear. In this study, the HSP70-encoding gene GhHSP70-26, which belongs to the cytoplasmic HSP70 subgroup, was cloned from upland cotton (Gossypium hirsutum L.). The transcript level of GhHSP70-26 in cotton leaves was higher than that in cotton stems and roots. GhHSP70-26 was found to respond at the transcriptional level to polyethylene glycol (PEG) and abscisic acid (ABA) treatment and to cold, salt, and heat stress, and its transcript level was positively correlated with the drought resistance of different cotton varieties. Heterologous overexpression of the GhHSP70-26 gene improved the drought resistance of transgenic tobacco compared with that of wild-type (WT) tobacco, and the transgenic tobacco plants were characterized by low leaf wilting, high survival, low leaf water loss, increased root length and high chlorophyll content. Moreover, the transgenic tobacco plants overexpressing GhHSP70-26 had higher proline contents, higher superoxide dismutase (SOD) and peroxidase (POD) enzyme activities, and lower malondialdehyde (MDA) and reactive oxygen species (ROS) contents than the WT tobacco plants. The transcript levels of the stress response-related genes NtLEA, NtERD10D, NtPOD, NtSOD, and NtNCED3-1 in transgenic tobacco overexpressing GhHSP70-26 were significantly higher than those in WT tobacco. In contrast, under drought stress, unlike the results obtained with the pTRV2 empty vector (EV)-transformed and WT cotton plants, the use of virus-induced gene silencing (VIGS) technology to silence the GhHSP70-26 gene in cotton resulted in plants with severely wilted leaves, an increased water loss rate, a higher MDA content, and higher relative electrical conductivity (REC). Furthermore, yeast one-hybrid assays showed that GhbZIP43 and GhHSF8 bind to ABA-responsive elements (ABREs) and heat shock elements (HSEs) in the GhHSP70-26 promoter, respectively. Moreover, yeast two-hybrid, bimolecular fluorescence complementation and luciferase complementation assays showed that the GhHSP70-26 protein interacts with the ascorbate peroxidase (APX) GhAPX2 protein. These results indicate that by reducing the degrees of cell membrane damage and cellular damage caused by ROS stress, the GhHSP70-26 protein plays a positive role in the response of plants to drought stress.

Apoptosome Formation through Disruption of the K192-D616 Salt Bridge in the Apaf-1 Closed Form

The molecular mechanism of apoptosome activation through conformational changes of Apaf-1 auto-inhibited form remains largely enigmatic. The crystal structure of Apaf-1 suggests that some ionic bonds, including the bond between K192 and D616, are critical for the preservation of the inactive “closed” form of Apaf-1. Here, a split luciferase complementation assay was used to monitor the effect of disrupting this ionic bond on apoptosome activation and caspase-3 activity in cells. The K192E mutation, predicted to disrupt the ionic interaction with D616, increased apoptosome formation and caspase activity, suggesting that this mutation favors the “open”/active form of Apaf-1. However, mutation of D616 to alanine or lysine had different effects. While both mutants favored apoptosome formation such as K192E, D616K cannot activate caspases and D616A activates caspases poorly, and not as well as wild-type Apaf-1. Thus, our data show that the ionic bond between K192 and D616 is critical for maintaining the closed form of Apaf-1 and that disrupting the interaction enhances apoptosome formation. However, our data also reveal that after apoptosome formation, D616 and K192 play a previously unsuspected role in caspase activation. The molecular explanation for this observation is yet to be elucidated.

Identification of novel potential interaction partners of UDP-galactose (SLC35A2), UDP-N-acetylglucosamine (SLC35A3) and an orphan (SLC35A4) nucleotide sugar transporters

Nucleotide sugar transporters (NSTs) are ER and Golgi-resident members of the solute carrier 35 (SLC35) family which supply substrates for glycosylation by exchanging lumenal nucleotide monophosphates for cytosolic nucleotide sugars. Defective NSTs have been associated with congenital disorders of glycosylation (CDG), however, molecular basis of many types of CDG remains poorly characterized. To better understand the biology of NSTs, we identified potential interaction partners of UDP-galactose transporter (SLC35A2), UDP-N-acetylglucosamine transporter (SLC35A3) and an orphan nucleotide sugar transporter SLC35A4 of to date unassigned specificity. For this purpose, each of the SLC35A2-A4 proteins was used as a bait in four independent pull-down experiments and the identity of the immunoprecipitated material was discovered using MS techniques. From the candidate list obtained, we selected a few for which the interaction was confirmed in vitro using the NanoBiT system, a split luciferase-based luminescent technique. NSTs have been shown to interact with two ATPases (ATP2A2, ATP2C1), Golgi pH regulator B (GPR89B) and calcium channel (TMCO1), which may reflect the regulation of glycosylation by ion homeostasis, and with basigin (BSG). Our findings provide a starting point for the NST interaction network discovery in order to better understand how glycosylation is regulated and linked to other cellular processes. SignificanceDespite the facts that nucleotide sugar transporters are a key component of the protein glycosylation machinery, and deficiencies in their activity underlie serious metabolic diseases, biology, function and regulation of these essential proteins remain enigmatic. In this study we have advanced the field by identifying sets of new potential interaction partners for UDP-galactose transporter (SLC35A2), UDP-N-acetylglucosamine transporter (SLC35A3) and an orphan transporter SLC35A4 of yet undefined role. Several of these new interactions were additionally confirmed in vitro using the NanoBiT system, a split luciferase complementation assay. This work is also significant in that it addresses the overall challenge of discovering membrane protein interaction partners by a detailed comparison of 4 different co-immunoprecipitation strategies and by custom sample preparation and data processing workflows.

Abstract 1947: Brought to light: the homotypic interaction pattern of Bcl-2 effector transmembrane domains in a bimolecular luciferase complementation assay

Abstract The intrinsic apoptosis signaling pathway comprises the intimate interaction network of the Bcl-2 protein family, in which both pro- and antiapoptotic members pushing cell fate to either survival or death. As a critical event, oligomerization of pore-forming effector proteins BAX, BAK and BOK drive mitochondrial outer membrane permeabilization (MOMP), which results in Cytochrome C release and ultimately apoptotic cell death. Thus far, investigations focus on protein-protein interactions mediated by the BH3-domain and hydrophobic groove. These interactions are targeted by BH3 mimetics that represent highly efficient anti-cancer drugs. In contrast, the C-terminal α-9 helices, known as transmembrane domains (TMD), tend to be neglected as a potential secondary interaction site with other Bcl-2 proteins. However, TMDs strongly affect subcellular localization and there is increasing evidence that TMDs crucially influence Bcl-2 protein interactions and therefore apoptosis signaling. Although TMDs are widely abundant in pro- and anti-apoptotic Bcl-2 proteins, their contribution to the complex Bcl-2 interaction network remains largely in the dark. Aiming to shed more light on the interactions of TMD among effector Bcl-2 proteins, we generated a bimolecular complementation assay in which effector TMD peptides are coupled to a bimolecular split-luciferase system. In addition, vectors encode for simultaneous expression of fluorophores to allow control of transgene expression. Human cell lines were co-transfected with combinations of TMD expression constructs yielding luminescence-based interaction data with a fluorescence-based normalization. Interestingly, we found an almost exclusively homotypic TMD interaction among the TMDs of BAX, BAK and BOK. Moreover, confocal microscopy with modified fluorophore-coupled TMD peptides revealed that TMD sequences were sufficient to locate BAX TMD and BAK TMD peptides effectively to mitochondria, while BOK TMD peptides were mainly localized to the endoplasmic reticulum. Strikingly, exchanging TMD sequences in full-length effector proteins profoundly modulated cell death induction. These results strongly suggest a central functional relevance of the α-9-helices in promoting homotypic interactions of effectors BAX, BAK and BOK during oligomerization and pore formation. In contrast, putative heterotypic interaction among effector proteins is not reflected by the newly generated TMD interaction assay. Thus, effector TMDs are a vital factor for specific protein localization and cell death induction displaying specific functionality in each effector protein. Our findings underline the importance of further investigating the TMD region as a potential oncogenic feature influencing interaction of Bcl-2 proteins and therefore as a potential target for therapeutic intervention. Citation Format: Tobias B. Beigl, Sandra Weller, Benjamin Schäfer, Kathrin Böpple, Lara-Sophie Rieder, Hans-Georg Kopp, Markus Rehm, Walter E. Aulitzky, Frank Essmann. Brought to light: the homotypic interaction pattern of Bcl-2 effector transmembrane domains in a bimolecular luciferase complementation assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1947.

Pharmacological profiles of 5‐HT 2A Receptor (5‐HT 2A R):5‐HT 2C R Interactions In Vitro

Aims The serotonin 5-HT2A receptor (5-HT2AR) and 5-HT2CR have garnered intense interest from drug discovery programs due to their role in metabolic (e.g., obesity) and neuropsychiatric disorders (e.g., addiction, depression, psychosis). Functional and physical interactions between the 5-HT2AR and 5-HT2CR occur in living cells (Felsing et al., PLoS One 13(8):e0203137, 2018) and rat brain (Price et al., ACS Chem Neurosci 10:3241, 2019). Intriguingly, when the two receptors are in complex, the 5-HT2CR is functionally dominant, i.e., silences signaling from the 5-HT2AR in vitro (Moutkine et al., J Biol Chem 292:6352, 2017). In the present study, we have extended these observations to uncover additional cellular signaling output associated with the 5-HT2AR:5-HT2CR complex and explore disruption of the receptor interfaces important for their interaction and signaling. Methods . A split luciferase complementation assay (LCA) was employed to assess the direct coupling of 5-HT2AR and 5-HT2CR in live HEK293 cells. The cDNA for the human 5-HT2AR and 5-HT2CR fused with complementary N-terminal (NLuc) and C-terminal (CLuc) inactive fragments, respectively, were transfected into HEK293 cells using template mammalian expression vectors and standard cloning techniques. Association of the receptor constructs in vitro within ~50 nm reconstitutes luciferase activity and light is released in the presence of D-luciferin. The expression of the activity-regulated immediate early gene early growth response 1 (EGR1) was used to trace cellular responsivity to 5-HT in cells. A truncated version of the 5-HT2CR transmembrane helix IV (TM4) domain conjugated to a polyethylene glycol chain (PEG-TM4 5-HT2CR) was synthesized as a tool to disrupt formation of the 5-HT2AR:5-HT2CR complex. Results . Serotonin (5-HT) readily induced robust expression of EGR1 mRNA in cells solely expressing 5-HT2AR, but not 5-HT2CR; 5-HT-evoked EGR1 expression was blunted in cells co-expressing the 5-HT2CR plus 5-HT2AR. In the LCA, vehicle (0.1% DMSO) treated cells exhibit robust complementation (i.e., increased luminescence) between the 5-HT2AR and 5-HT2CR. The PEG-TM4 5-HT2CR peptide significantly decreased complementation (p<0.05) in a concentration-dependent manner indicating an inhibition of association between the 5-HT2AR and 5-HT2CR. Conclusions Blunted 5-HT-evoked EGR1 expression in cells expressing both receptors supports the “master regulator” role for the 5-HT2CR when in complex with the 5-HT2AR. Secondly, the formation of the 5-HT2AR:5-HT2CR complex in vitro is dependent on an interaction interface located at the 5-HT2CR TM4. Ongoing studies are testing the hypothesis that the PEG-TM4 5-HT2CR peptide restores a critical signaling output of 5-HT2AR function, which will indicate the importance of allosteric regulation by the heteromer complex. Future studies with our novel TM domain peptides will help elucidate determinants of receptor complex formation and signaling and the physiological relevance of the 5-HT2AR:5-HT2CR complex in vivo.

β-arrestin2 recruitment at the β2 adrenergic receptor: A luciferase complementation assay adapted for undergraduate training in pharmacology.

In the context of pharmacology teaching, hands‐on activities constitute an essential complement to theoretical lectures. Frequently, these activities consist in exposing fresh animal tissues or even living animals to selected drugs and qualitatively or quantitatively evaluating functional responses. However, technological advancements in pharmacological research and the growing concerns for animal experimentation support the need for innovative and flexible in vitro assays adapted for teaching purposes. We herein report the implementation of a luciferase complementation assay (LCA) enabling to dynamically monitor β‐arrestin2 recruitment at the β2 adrenergic receptor in the framework of pharmacological training at the faculty of Pharmacy and Biomedical Sciences. The assay allowed students to quantitatively characterize the competitive antagonism of propranolol, and to calculate pEC50, pKB, and pA2 values after a guided data analysis session. Moreover, the newly implemented workshop delivered highly reproducible results and were generally appreciated by students. As such, we report that the luciferase complementation‐based assay proved to be a straightforward, robust, and cost‐effective alternative to experiments performed on animal tissues, constituting a useful and flexible tool to enhance and update current hands‐on training in the context of pharmacological teaching.

High-Throughput Screening for Drugs That Inhibit Papain-Like Protease in SARS-CoV-2.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in late 2019 has triggered an ongoing global pandemic whereby infection may result in a lethal severe pneumonia-like disease designated as coronavirus disease 2019 (COVID-19). To date, millions of confirmed cases and hundreds of thousands of deaths have been reported worldwide, and there are currently no medical countermeasures available to prevent or treat the disease. The purported development of a vaccine could require at least 1–4 years, while the typical timeline from hit finding to drug registration of an antiviral is >10 years. Thus, repositioning of known drugs can significantly accelerate the development and deployment of therapies for COVID-19. To identify therapeutics that can be repurposed as SARS-CoV-2 antivirals, we developed and initiated a high-throughput cell-based screen that incorporates the essential viral papain-like protease (PLpro) and its peptide cleavage site into a luciferase complementation assay to evaluate the efficacy of known drugs encompassing approximately 15,000 clinical-stage or US Food and Drug Administration (FDA)-approved small molecules. Confirmed inhibitors were also tested to determine their cytotoxic properties. Here, we report the identification of four clinically relevant drugs that exhibit selective inhibition of the SARS-CoV-2 viral PLpro.

Effects of Linker Flexibility and Conformational Changes of IP3 Receptor on Split Luciferase Complementation Assay.

Background: IP3-induced Ca2+ release, mediated by IP3R, is one of the most momentous cellular signaling mechanisms that regulate in a wide variety of essential cellular functions. Involvement of disrupted IP3 signaling pathways in numerous pathophysiology conditions is implicated to find the best methods for its measurement. Hence, several different biosensors have developed to monitor temporal changes of IP3 by using the IP3-binding domain of IP3 receptors.Objectives: Based on a previous study, we developed and characterized a series of bioluminescent biosensors using the human type-II IP3 receptor ligand binding domain (residues 1–604), named LAIRE (luminescent analyzer for IP3 receptor element) to study the effect of flexible and rigid linkers on the luminescence intensity of split luciferase. The effect of a mutation in IP3 binding residues and suppressor domain in the IP3 binding domain on luciferase complementary assay is also investigated.Materials and Methods: In the present study, first IP3-binding domain (residues 1-604) of IP3-receptor type 2 (LAIRE) was fused between complementary non-functional fragments of firefly luciferase and then the rigid linker sequence (LLRAIEAQQHLL), selected by ProDA database, introduced between Nluc and ligand binding domain and compared with that of the flexible linker ((GGGGS)2) in LAIRE chimera. The IP3-insensitive mutant of the biosensor was constructed using the Stratagene QuikChange® procedure. In order to the analysis of the dynamical movements of selected structures in the large-scale, coarse-graining method of molecular dynamics simulation (1µs) was applied.Results: As expected, the flexible linker brings two inactive fragments of luciferase together relative to the rigid linker and leads to complementation of luciferase activity, which is detected using luciferin. However, this conformational flexibility in linker increases background to noise ratio and attenuates fold induction.Conclusions: It seems that the ligand binding properties of IP3 binding core make it more suitable for the design of biosensor than the ligand binding domain.

Demonstration of Heterologous Complexes formed by Golgi-Resident Type III Membrane Proteins using Split Luciferase Complementation Assay.

The goal of this protocol is to explore the applicability of the most recent variant of split luciferase complementation for demonstrating heterologous complexes formed by nucleotide sugar transporters (NSTs). These ER- and Golgi-resident multitransmembrane proteins carry the cytoplasmically synthesized nucleotide sugars across organelle membranes to supply enzymes that mediate glycosylation with their substrates. NSTs exist as dimers and/or higher oligomers. Heterologous interactions betweendifferent NSTs have also been reported. To verify whether the technique is suitable for studying the phenomenon of NST heteromerization, we tested it against a combination of the two Golgi-resident NSTs that have been previously shown to associate by several other means. Theluciferase complementation assay appears to be particularly suitable for studying interactions between Golgi-resident membrane proteins, as it does not require high expression levels, which often trigger protein mislocalization and increase the risk of false positives.

Demonstration of Heterologous Complexes formed by Golgi-Resident Type III Membrane Proteins using Split Luciferase Complementation Assay

Demonstration of Heterologous Complexes formed by Golgi-Resident Type III Membrane Proteins using Split Luciferase Complementation Assay

SnRK1 Phosphorylates and Destabilizes WRKY3 to Enhance Barley Immunity to Powdery Mildew

Plants recognize pathogens and activate immune responses, which usually involve massive transcriptional reprogramming. The evolutionarily conserved kinase, Sucrose non-fermenting-related kinase 1 (SnRK1), functions as a metabolic regulator that is essential for plant growth and stress responses. Here, we identify barley SnRK1 and a WRKY3 transcription factor by screening a cDNA library. SnRK1 interacts with WRKY3 in yeast, as confirmed by pull-down and luciferase complementation assays. Förster resonance energy transfer combined with noninvasive fluorescence lifetime imaging analysis indicates that the interaction occurs in the barley nucleus. Transient expression and virus-induced gene silencing analyses indicate that WRKY3 acts as a repressor of disease resistance to the Bgh fungus. Barley plants overexpressing WRKY3 have enhanced fungal microcolony formation and sporulation. Phosphorylation assays show that SnRK1 phosphorylates WRKY3 mainly at Ser83 and Ser112 to destabilize the repressor, and WRKY3 non-phosphorylation-null mutants at these two sites are more stable than the wild-type protein. SnRK1-overexpressing barley plants display enhanced disease resistance to Bgh. Transient expression of SnRK1 reduces fungal haustorium formation in barley cells, which probably requires SnRK1 nuclear localization and kinase activity. Together, these findings suggest that SnRK1 is directly involved in plant immunity through phosphorylation and destabilization of the WRKY3 repressor, revealing a new regulatory mechanism of immune derepression in plants.

MYB44 competitively inhibits the formation of the MYB340-bHLH2-NAC56 complex to regulate anthocyanin biosynthesis in purple-fleshed sweet potato

BackgroundAnthocyanins, which have important biological functions and have a beneficial effect on human health, notably account for pigmentation in purple-fleshed sweet potato tuberous roots. Individual regulatory factors of anthocyanin biosynthesis have been identified; however, the regulatory network of anthocyanin biosynthesis in purple-fleshed sweet potato is unclear.ResultsWe functionally determined that IbMYB340 cotransformed with IbbHLH2 in tobacco and strawberry receptacles induced anthocyanin accumulation, and the addition of IbNAC56a or IbNAC56b caused increased pigmentation. Furthermore, we confirmed the interaction of IbMYB340 with IbbHLH2 and IbNAC56a or IbNAC56b via yeast two-hybrid and firefly luciferase complementation assays; these proteins could form a MYB340-bHLH2-NAC56a or MYB340-bHLH2-NAC56b transcriptional complex to regulate anthocyanin biosynthesis by binding to the IbANS promoter rather than the IbUFGT promoter. Furthermore, it was found by a transient expression system in tobacco leaves that IbMYB44 could decrease anthocyanin accumulation. Moreover, the interaction of IbMYB44 with IbMYB340 and IbNAC56a or IbNAC56b was verified. This result suggested that IbMYB44 acts as a repressor of anthocyanin in sweet potato.ConclusionsThe repressor IbMYB44 affected anthocyanin biosynthesis by competitively inhibiting the IbMYB340-IbbHLH2-IbNAC56a or IbMYB340-IbbHLH2-IbNAC56b regulatory complex formation. Overall, the present study proposed a novel regulatory network whereby several vital TFs play key roles in regulating anthocyanin biosynthesis, and it provides strong insight into the potential mechanism underlying anthocyanin biosynthesis in sweet potato tuberous roots with purple color.

Analysis of RAS protein interactions in living cells reveals a mechanism for pan-RAS depletion by membrane-targeted RAS binders

HRAS, NRAS, and KRAS4A/KRAS4B comprise the RAS family of small GTPases that regulate signaling pathways controlling cell proliferation, differentiation, and survival. RAS pathway abnormalities cause developmental disorders and cancers. We found that KRAS4B colocalizes on the cell membrane with other RAS isoforms and a subset of prenylated small GTPase family members using a live-cell quantitative split luciferase complementation assay. RAS protein coclustering is mainly mediated by membrane association-facilitated interactions (MAFIs). Using the RAS-RBD (CRAF RAS binding domain) interaction as a model system, we showed that MAFI alone is not sufficient to induce RBD-mediated RAS inhibition. Surprisingly, we discovered that high-affinity membrane-targeted RAS binding proteins inhibit RAS activity and deplete RAS proteins through an autophagosome-lysosome-mediated degradation pathway. Our results provide a mechanism for regulating RAS activity and protein levels, a more detailed understanding of which should lead to therapeutic strategies for inhibiting and depleting oncogenic RAS proteins.

Investigating the Kinetics of β1 and β2 Adrenoceptor Effector Recruitment using Luciferase Complementation Assays

Nanoluc‐based luciferase complementation assays (NanoBiT; Dixon et al, 2016) provide new approaches to measure G protein‐coupled receptor (GPCR) signalling, by assessing the kinetics of receptor subtype recruitment of effector proteins in real‐time. Here, we use NanoBiT systems to compare β adrenoceptor subtype (β1AR/β2AR), recruitment of β‐arrestin2 and an engineered Gαs GTPase domain sensor (mini Gαs; mGαs)( Wan et al, 2018).HEK293T cells stably expressed SNAP‐tagged βAR (β1AR or β2AR) fused to C‐terminal 18 kDa LgBiT luciferase fragment, and the 11 amino acid SmBiT fused to either β‐arrestin2 or mGαs. NanoBiT assays were conducted in cells in 96 well plates in HBSS/0.1% BSA at 37°C. Cells were pre‐incubated with furimazine (5min; 37°C), before the addition of agonist. Luminescence was monitored for 31 min (BMG Pherastar Platereader, 37°C). Concentration response curves were fitted in GraphPad Prism v7, with pharmacology quoted as mean±s.e.m (n≥3)Both βAR subtypes stimulated transient β‐arrestin2 recruitment upon 10μM isoprenaline stimulation (Figure 1a). At 31 min, both receptor subtypes reported partial agonism of salbutamol and salmeterol, with increased potency and efficacy (relative to isoprenaline) at β2AR compared to β1AR (Figure 1c–d). 10μM Isoprenaline also stimulated recruitment of mGas at each subtype, increasing rapidly between 0 and 5 min post agonist addition. Association of β2AR‐mGαs complexes was maintained stably throughout incubation, whilst at β1AR, mGαs recruitment continued to increase over incubation window, reaching 322.0±39.1% by 31 min (%10μM isoprenaline, at 3 min) (Figure 1b).NanoBiT complementation assays report real‐time recruitment of β‐arrestin2 and mGαs protein, at both β1AR and β2AR. Transient β‐arrestin2 recruitment was reported at both receptor subtypes responses distinguished full and partial agonists. Further studies of the continued βAR‐mGαs association will probe whether mGαs sensors remain stably associated with membrane or endosomal pools of adrenoceptors.Support or Funding InformationPhD funded by Biotechnology and Biological Sciences Research Council (BBSRC).NanoBiT luciferase complementation monitors βAR recruitment of signalling effector proteins. Signalling timecourse of (a.) β‐arrestin2 and (b.) mGαs recruitment, at β1AR and β2AR. Concentration response curves of β‐arrestin2 recruitment, at (c.) β1AR and (d.) β2AR. Data points represent mean±s.e.m.; n≥3.Figure 1

Inactive 5‐HT7 Receptors Associate with Inactive and Active Gs Heterotrimers

G protein‐coupled receptors (GPCRs) interact cooperatively with agonists and heterotrimeric G proteins to form ternary complexes, leading to activation of G proteins. Following exchange of GDP for GTP the complex is thought to rapidly disassemble as nucleotide‐bound G proteins adopt conformations that have low affinity for GPCRs, and heterotrimers dissociate into Gα and Gβγ subunits. Unlike most GPCRs, serotonin 5‐HT7 receptors are known to preassemble with GDP‐bound Gs heterotrimers prior to agonist binding. Here we show that these receptors also interact with GTP‐bound Gs heterotrimers. We used bioluminescence resonance energy transfer (BRET) and luciferase complementation assays to assess the effects of ligands and nucleotides on 5‐HT7‐Gs coupling. We find that stimulation with agonist (5‐HT) in intact cells promotes net dissociation of 5‐HT7‐Gs complexes, whereas inverse agonists (e.g. methiothepin) produce net association. To determine how nucleotide binding by Gs affects interactions with receptors, we replaced endogenous nucleotides with the non‐hydrolyzable analogs GTPγS or GDPβS in permeabilized, apyrase‐treated HEK293 cells. Unexpectedly, ligand‐mediated effects on 5‐HT7‐Gs complexes were largely maintained in the various nucleotide conditions. Agonist‐induced 5‐HT7‐Gs dissociation still occurred when only GDPβS was present, indicating that dissociation does not require Gs activation. Similarly, inverse agonist‐induced 5‐HT7‐Gs association still occurred when only GTPγS was present, suggesting that activated Gs heterotrimers retain affinity for inactive 5‐HT7 receptors. Moreover, we found that inverse agonist‐bound 5‐HT7 sequestered free Gβγ in the presence of GTPγS, suggesting that inactive 5‐HT7 receptors stabilized active Gs heterotrimers. These results reveal unconventional coupling of inactive 5‐HT7 receptors to both inactive and active Gs heterotrimers. This represents a significant departure from the standard allosteric model of GPCR‐G protein coupling, wherein G proteins interact primarily with active GPCRs, and GTP‐bound Gα subunits dissociate from both receptors and Gβγ subunits.Support or Funding InformationSupported by GM130142 (to N.A.L.)

PyWRKY26 and PybHLH3 cotargeted the PyMYB114 promoter to regulate anthocyanin biosynthesis and transport in red-skinned pears

Red pear is favored because of its bright appearance and abundant anthocyanins. Anthocyanin biosynthesis is controlled by transcription factors (TFs) forming regulatory complexes. In red-skinned pears, the WRKY TFs have a significant relationship with anthocyanin biosynthesis, but the molecular mechanism of the WRKY TFs involved in regulating color formation in red-skinned pear is unclear. In this study, the TFs PyWRKY31 and PyWRKY26 were screened as candidate genes for controlling anthocyanin biosynthesis by transcriptome data and bioinformatics analysis. The effect of anthocyanin accumulations after cotransformation of PyWRKY31 or PyWRKY26 with its partners PyMYB10, PyMYB114, and PybHLH3 was verified in tobacco leaves and strawberry receptacles by a transient expression system. RT-qPCR analysis and a dual-luciferase reporter system further confirmed that this cotransformation activated the expression of PyDFR, PyANS, and PyUFGT in anthocyanin biosynthesis and PyGST in anthocyanin transport instead of the PyABC transporter and PyAVP. Furthermore, the cotransformed PyWRKY26 and PybHLH3 could bind to the PyMYB114 promoter, and PyWRKY26 directly activated the transcription of PyMYB114. In addition, the TF PyWRKY26 could interact with PybHLH3, as confirmed by firefly luciferase complementation and yeast two-hybrid (Y2H) assays. These results showed that the interaction of PyWRKY26 and PybHLH3 could cotarget the PyMYB114 promoter, which resulted in anthocyanin accumulation in red-skinned pear. This study further strengthened the understanding of the regulatory mechanism of anthocyanin accumulation and contributed to improving the appearance of red-skinned pears.

Analysis of homologous and heterologous interactions between UDP-galactose transporter and beta-1,4-galactosyltransferase 1 using NanoBiT

Split luciferase complementation assay is one of the approaches enabling identification and analysis of protein-protein interactions in vivo. The NanoBiT technology is the most recent improvement of this strategy. Nucleotide sugar transporters and glycosyltransferases of the Golgi apparatus are the key players in glycosylation. Here we demonstrate the applicability of the NanoBiT system for studying homooligomerization of these proteins. We also report and discuss a novel heterologous interaction between UDP-galactose transporter and beta-1,4-galactosyltransferase 1.

Celastrol is a novel selective agonist of cannabinoid receptor 2 with anti-inflammatory and anti-fibrotic activity in a mouse model of systemic sclerosis

BackgroundIncreasing evidence indicated that the cannabinoid receptors were involved in the pathogenesis of organ fibrogenesis. PurposeThe purpose of this study was to discover novel cannabinoid receptor 2 (CB2) agonist and assess the potential of CB2 activation in treating systemic sclerosis. MethodsA gaussia princeps luciferase–based split luciferase complementation assay (SLCA) was developed for detection of the interaction between CB2 and β-arrestin2. A library of 366 natural products was then screened as potential CB2 agonist using SLCA approach. Several GPCR functional assays, including HTRF-based cAMP assay and calcium mobilization were also utilized to evaluated CB2 activation. Bleomycin-induced experimental systemic sclerosis was used to assess the in vivo anti-fibrotic effects. Dermal thickness and collagen content were evaluated via H&E and sirius red staining. ResultsCelastrol was identified as a new agonist of CB2 by using SLCA. Furthermore, celastrol triggers several CB2-mediated downstream signaling pathways, including calcium mobilization, inhibition of cAMP accumulation, and receptor desensitization in a dose-dependent manner, and it has a moderate selectivity on CB1. In addition, celastrol exhibited the anti-inflammatory properties on lipopolysaccharide (LPS) treated murine Raw 264.7 macrophages and primary macrophages. Finally, we found that celastrol exerts anti-fibrotic effects in the bleomycin-induced systemic sclerosis mouse model accompanied by reduced inflammatory conditions. ConclusionTaken together, celastrol is identified a novel selective CB2 agonist using a new developed arrestin-based SLCA, and CB2 activation by celastrol reduces the inflammatory response, and prevents the development of dermal fibrosis in bleomycin-induced systemic sclerosis mouse model.

Split Nano luciferase complementation for probing protein-protein interactions in plant cells.

Deciphering protein-protein interactions (PPIs) is fundamental for understanding signal transduction pathways in plants. The split firefly luciferase (Fluc) complementation (SLC) assay has been widely used for analyzing PPIs. However, concern has risen about the bulky halves of Fluc interfering with the functions of their fusion partners. Nano luciferase (Nluc) is the smallest substitute for Fluc with improved stability and luminescence. Here, we developed a dual-use system enabling the detection of PPIs through the Nluc-based SLC and co-immunoprecipitation assays. This was realized by coexpression of two proteins under investigation in fusion with the HA- or FLAG-tagged Nluc halves, respectively. We validated the robustness of this system by reproducing multiple previously documented PPIs in protoplasts or Agrobacterium-transformed plants. We next applied this system to evaluate the homodimerization of Arabidopsis CERK1, a coreceptor of fungal elicitor chitin, and its heterodimerization with other homologs in the absence or presence of chitin. Moreover, split fragments of Nluc were fused to two cytosolic ends of Arabidopsis calcium channels CNGC2 and CNGC4 to help sense the allosteric change induced by the bacterial elicitor flg22. Collectively, these results demonstrate the usefulness of the Nluc-based SLC assay for probing constitutive or inducible PPIs and protein allostery in plant cells.

Identifying Inhibitors of the HBx-DDB1 Interaction Using a Split Luciferase Assay System.

There is an urgent need for novel therapeutic agents for hepatitis B virus (HBV) infection. Although currently available nucleos(t)ide analogs potently inhibit viral replication, they have no direct effect on the expression of viral proteins transcribed from a viral covalently closed circular DNA (cccDNA). As high viral antigen load may play a role in this chronic and HBV-related carcinogenesis, the goal of HBV treatment is to eradicate viral proteins. HBV regulatory protein X (HBx) binds to the host DNA damage-binding protein 1 (DDB1) protein to degrade structural maintenance of chromosomes 5/6 (Smc5/6), resulting in activation of viral transcription from cccDNA. Here, using a split luciferase complementation assay system, we present a comprehensive compound screening system to identify inhibitors of the HBx-DDB1 interaction. Our protocol enables easy detection of interaction dynamics in real time within living cells. This technique may become a key assay to discover novel therapeutic agents for treatment of HBV infection.