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PLos ONE 13(5), e0196946. β-Arrestin1 and 2 differentially regulate PACAP-induced PAC1 receptor signaling and trafficking. 2018

Shintani, Y., Hayata-Takano, A., Moriguchi, K., Nakazawa, T., Ago, Y., Kasai, A., Seiriki, K., Shintani, N. and Hashimoto, H.

Notes: The authors demonstrate that β-arrestin1 and β-arrestin2 differ in there interaction with PAC1R by using the Promega NanoBiT® technology. (5082)

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Anal. Biochem. 555, 67–72. Antibody-free detection of cellular neddylation dynamics of Cullin1. 2018

Schwinn, M.K., Hoang, T., Yang, X., Zhao, X., Ma, J., Li, P., Wood, K.V., Mallender, W.D., Bembenek, M.E. and Yan, Z.H.

Notes: The post-transcriptional modification of neddylation helps regulate protein activity, stability and localization. NanoLuc® Binary Technology (NanoBiT) was used to monitor covalent neddylation of Cul1 in a cellular context. SmBiT-Nedd8 and Cul1-LgBiT were expressed in HEK293 cells and luminescence was monitored as a response to a Nedd8-Cul1 covalent protein modification. Further, neddylation was monitored in the presence of both inhibitors and activators, and a concentration- and time-dependent response in luminescence was observed. (5076)

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FEBS J. 285, 46–71. Dynamic control of RSK complexes by phosphoswitch-based regulation. 2018

Gógl, G., Biri-Kovács, B., Póti, Á.L., Vadászi, H., Szeder, B., Bodor, A., Schlosser, G., Ács, A., Turiák, L., Buday, L., Alexa, A., Nyitray, L. and Reményi, A.

Notes: The NanoBiT® PPI MCS Starter System was used to build assays to study the interaction dynamics of RSK1 with ERK2 and MAGl-1 in live cells upon EGF treatment. Various structure-based mutations were studied by transiently transfecting cells with constructions using FuGENE® HD Reagent and assayed with Nano-Glo® Live Cell Reagent. The authors were able to determine which mutations in RSK1 affected MAGl-1 dissociation and determined that it was dependent on the presence of the PDZ-binding motif. (4969)

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Biochem. Pharmacol. 148, 298–307. Molecular dissection of the human A3 adenosine receptor coupling with β-arrestin2. 2018

Storme, J., Cannaert, A., Van Craenenbroeck, K. and Stove, C.P.

Notes: The NanoBiT Protein-Protein Interaction System was utilized to investigate the interaction of human A3 adenosine receptor, a G protein-coupled receptor, and β-arrestin2. Specifically, the importance of specific C-terminal phosphorylation sites for A3AR and β-arrestin2 coupling was determined using protein truncations. To determine the optimal fusion tags for this assay, multiple constructs were tested in the presence of an A3AR selective agonist. Unlike previous studies in rat models, C-terminal human A3AR truncations displayed no major impact on β-arrestin2 coupling. (5077)

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Cancer Immunol. Res. 6(8), 921–929. PD-L1 Binds to B7-1 Only In Cis on the Same Cell Surface. 2018

Chaudhri, A., Xiao, Y., Klee, A.N., Wang, X., Zhu, B. and Freeman, G.J.

Notes: NanoBiT Protein-Protein Interaction System was used to determine an interaction between Programmed death ligand 1 (PD-L1) and B7-1. PD-L1 is an encouraging target for cancer immunotherapy due to its involvement in mediating tumor immune evasion. Previous studies identified an interaction between PD-L1 and B7-1, however here the NanoBiT system was used to show binding between PD-L1 and B7-1 was specific when both are co-expressed on the same cell surface. Interestingly, similar co-expression of PD-L1 and B7-1 was found on tumor-infiltrating myeloid cells. Further, targeted PD-L1 mutants showed a significant decrease in B7-1 binding. (5072)

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J. Biol. Chem. 293(23), 8750–8760. Quantitative interaction analysis permits molecular insights into functional NOX4 NADPH oxidase heterodimer assembly. 2018

O'Neill, S., Mathis, M., Kovačič, L., Zhang, S., Reinhardt, J., Scholz, D., Schopfer, U., Bouhelal, R. and Knaus, U.G.

Notes: The assembly of the NOX4- p22phox integral membrane complex, involved in electron transfer and reactive oxygen species generation, was investigated using the NanoBiT Protein-Protein Interaction System. A total of 31 Small BiT/Large BiT NOX4 or p22phox fusions were tested, and the most active pairs were further analyzed for catalytic activity. Residues involved in complex formation and catalysis were determined using mutational analysis and tested for luciferase activity. Together, the authors identified an approach for determining membrane protein assembly that can be used in the future as a drug discovery tool. (5074)

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Sci. Rep. 8(1). TDP-43 self-interaction is modulated by redox-active compounds Auranofin, Chelerythrine and Riluzole. 2018

Oberstadt, M., Stieler, J., Simpong, D.L., Römuß, U., Urban, N., Schaefer, M., Arendt, T. and Holzer, M.

Notes: Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by formation of TDP-43 protein aggregates. The NanoBiT Protein-Protein Interaction System was used to measure self-interaction of TDP-43. Multiple tag locations were tested, and fusion of the Large BiT and Small BiT tags on the N-terminus resulted in the highest signal. Drug screening for potential ALS therapeutics was performed by monitoring for the disruption of the TDP-43 aggregates and loss of luciferase activity using the NanoBiT system. Drug screening resulted in the identification of Auranofin as a potential therapeutic, which showed redistribution of insoluble aggregates to PBS soluble protein. (5073)

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ACS Chemical Biology 12(4), 1028-1037. Highly Potent Cell-Permeable and Impermeable NanoLuc Luciferase Inhibitors. 2017

Walker, J.R., Hall, M.P., Zimprich, C.A., Robers, M.B., Duellman, S.J., Machleidt, T., Rodriguez, J., and Zhou, W.

Notes: The development of NanoLuc Luciferase has led to a need for selective NanoLuc inhibitors to allow for bioluminescent suppression and multiplexing compatibility with existing assays. A lead compound with an IC50 of 600 nM against NanoLuc was further derivatized to create a family of potent and cell permeable inhibitors. These compounds were additionally developed to generate a second class of cell impermeable inhibitors. These inhibitors were tested for selectivity against Firefly luciferase and the NanoBiT system with no cross-reactivity.  (5106)

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Anal. Chem. 89, 9527–36. Activity-based detection of consumption of synthetic cannabinoids in authentic urine samples using a stable cannabinoid reporter system. 2017

Cannaert, A., Franz, F., Auwärter, V. and Stove, C.P.

Notes: The NanoBiT® system was used to develop CB1 and CB2 receptor activation bioassays based on β-arrestin recruitment, and stable cell lines were generated for these assays. The cell lines were used to screen for synthetic cannabinoids in urine samples and assayed using the Nano-Glo® Live Cell Reagent. (4966)

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J. Med. Chem. 60, 4869–81. Design and discovery of N-(2-Methyl-5'-morpholino-6'-((tetrahydro-2H-pyran-4-yl)oxy)-[3,3'-bipyridin]-5-yl)-3-(trifluoromethyl)benzamide (RAF709): A potent, selective, and efficacious RAF inhibitor targeting RAS mutant cancers. 2017

Nishiguchi, G.A., Rico, A., Tanner, H., Aversa, R.J., Taft, B.R., Subramanian, S., Setti, L., Burger, M.T., Wan, L., Tamez, V., Smith, A., Lou, Y., Barsanti, P.A., Appleton, B.A., Mamo, M., Tandeske, L., Dix, I., Tellew, J.E., Huang, S., Mathews Griner, L.A., Cooke, V.G., Van Abbema, A., Merritt, H., Ma, S., Gampa, K., Feng, F., Yuan, J., Wang, Y., Haling, J.R., Vaziri, S., Hekmat-Nejad, M., Jansen, J.M., Polyakov, V., Zang, R., Sethuraman, V., Amiri, P., Singh, M., Lees, E., Shao, W., Stuart, D.D., Dillon, M.P. and Ramurthy, S.

Notes: The major goal of this study was to identify selective RAF inhibitors that would suppress the RAF-MEK-ERKK pathway. A NanoBiT® cRAF:bRAF interaction assay was used to study the potency of identified compounds to stabilize this dimer using dose-response assays in transfected HCT116 cells (4967)

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Anal. Biochem. 522, 10-17. Development of a bimolecular luminescence complementation assay for RGS: G protein interactions in cells. 2017

Bodle, C.R., Hayes, M.P., O'Brien, J.B. and Roman, D.L.

Notes: NanoBit™ technology was used to characterize interactions between regulators of G protein signaling proteins (RGS proteins) and target G proteins in this study. (4898)

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eLife 6, e26857. Estrogen receptor coregulator binding modulators (ERXs) effectively target estrogen receptor positive human breast cancers. 2017

Raj, G.V., Sareddy, G.R., Ma, S., Lee, T.K., Viswanadhapalli, S., Li, R., Liu, X., Murakami, S., Chen, C.C., Lee, W.R., Mann, M., Krishnan, S.R., Manandhar, B., Gonugunta, V.K., Strand, D., Tekmal, R.R., Ahn, J.M. and Vadlamudi, R,K.

Notes: The authors are studying synthetic peptides (peptidomimetics) and their ability to modulate estrogen receptor signaling. The authors developed NanoBiT® PPI assays to study estrogen receptor and androgen receptor dimerization. Using these assays, they were able to demonstrate compound ERX-11 blocks estrogen receptor signaling and can specifically disrupt ER dimerization, but does not affect AR dimerization. The authors also developed a NanoBiT® assay to study the interaction of ER with the coregulator PELP1 and found this interaction could also be decreased with exposure to ERX-11. (4968)

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J. Virol. 91(23), e01455–17. Genome-Wide Mutagenesis of Dengue Virus Reveals Plasticity of the NS1 Protein and Enables Generation of Infectious Tagged Reporter Viruses. 2017

Eyre, N.S., Johnson, S.M., Eltahla A.A., Aloi, M., Aloia, A.L., McDevitt, C.A., Bull, R.A. and Beard, M.R.

Notes: An insertional mutagenesis screen of the Dengue virus genome was used to identify regions tolerating small 15 amino acid insertions. The NS1 protein, which is essential for viral genome replication, was shown to be highly tolerant to insertions. NS1 was tagged with both a minimal fusion tag (Small BiT) and NanoLuc Luciferase. Tagged protein variants were assessed for viral infectivity, and used to investigate the protein localization and levels of NS1. (5075)

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6. The prohibitin-repressive interaction with E2F1 is rapidly inhibited by androgen signalling in prostate cancer cells. 2017

Koushyar, S., Economides, G., Zaat, S., Jiang, W., Bevan, C.L. and Dart, D.A.

Notes: To study PHB-mediated gene repression and cell cycle arrest we generated promoter-reporter fusions using the pGL4 Luciferase Vectors. The PHB-E2F1 interaction was investigated using the CheckMate™ Mammalian two-hybrid system. The NanoBiT™ PPI interaction system was used for additional analysis of the PHB-E2F1 interaction. cDNA was synthesized using the Reverse Transcription System. (4900)

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Cell Biochem. Funct. 34(7), 497-504.
SOD1 dimerization monitoring using a novel split NanoLuc, NanoBit.

Oh-Hashi K., Hirata Y., and Kiuchi K.

Notes: These authors used a NanoBiT® complementation assay to investigate interactions between proteins implicated in development of amyotrophic lateral sclerosis (ALS). Mutations in superoxide dismutase 1 (SOD1) and in TAR‐binding protein 43 kDa (TDP43) have been associated with ALS. The authors observed formation of SOD1 homodimers in neuronal cells transfected with SOD1 tagged with NanoBiT complementation partners, and found that SOD1 mutants associated with ALS were unable to form homodimers.  Next, possible interactions between SOD1 and TDP43 were assessed. When NanoBiT-tagged constructs of TBP43 and SOD were transfected into Neuro 2A cells, only NanoBiT-tagged SOD generated a luciferase signal. TBP43 did not self-associate to form homodimers, and SOD1 and TBP43 did not interact with each other to form heterodimers. The authors state that NanoBiT is a useful and sensitive tool for analyzing protein interactions under physiological conditions, and may provide a convenient way to monitor the modulation of SOD1 conformation by candidate treatments. (4765)

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Anal. Chem. 88, 11476–85. Detection and activity profiling of synthetic cannabinoids and their metabolites with a newly developed bioassay. 2016

Cannaert, A., Storme, J., Franz, F., Auwärter, V. and Stove, C.P.

Notes: The authors used NanoLuc® Binary Technology (NanoBiT®) to build β-arrestin recruitment assays for two G-protein coupled receptors (CB1 and CB2) to create live-cell cannabinoid reporter assays. The assays were used to compare activity of synthetic cannabinoids and their metabolites and also to screen urine for CB receptor activity using the Nano-Glo® Live Cell Assay System. (4965)

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ACS Chemical Biology 11, 400–408. NanoLuc Complementation Reporter Optimized for Accurate Measurement of Protein Interactions in Cells. 2016

Dixon, A.S., Schwinn, M.K., Hall, M.P., Zimmerman, K., Otto, P., Lubben, T.H., Butler, B.L., Binkowski, B.F., Machleidt, T., Kirkland, T.A., Wood, M.G., Eggers, C.T., Encell, L.P., Wood, K.V.

Notes: These authors designed a complementation reporter for detecting protein interactions under physiological conditions in mammalian cells. The engineered reporter (NanoBiT) is based on NanoLuc luciferase and is composed of two small subunits (18kDa and 1.3kDa), which associate weakly. The formation of a luminescent signal is dependent on the interaction characteristics of the proteins onto which the subunits are attached. The paper demonstrates utility of the NanoBiT assay for detecting protein interactions in live cells using  FRB/FKBP as an example, shows that response dynamics are rapid and reversible, and also illustrates use of the system to measure the pharmacology of kinase inhibitors that induce interaction between BRAF and CRAF. (4585)

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