Notes: These authors developed a sensitive receptor-binding assay for detection of interactions between the peptide hormone Insulin-like peptide-3 (INSL3) and the relaxin family peptide receptor RXFP2.  Recombinant INSL3 was tagged with NanoLuc® Luciferase by chemical modification of INSL3 to include an active disufide bond, and engineering of a 6× His-Cys-NanoLuc with an exposed N-terminal cysteine. The NanoLuc®-conjugated INSL3 was used to monitor the receptor-binding of a variety of ligands. (4438)

Notes: To understand the mechanism of Panicle blast 1 (Pb1) gene-mediated resistance to rice blast, a rice fungal disease, researchers investigated Pb1 interacted with a transcription factor involved in resistance, WRKY45 that is regulated by the ubiquitin system. To study how these proteins interacted, inner rice leaf sheaths were bombarded with gold particles coated with 0.5 µg of effector plasmid, 0.3 µg of NanoLuc® luciferase reporter and 0.1 µg of reference Renilla luciferase. After incubating overnight at 28°C, samples were ground in liquid nitrogen and reporter activities assayed using the Dual-Glo® Luciferase Reporter Assay System and Nano-Glo® Luciferase Assay System. The Renilla luciferase gene was also split into an N-terminal construct and C-terminal construct, expressed in rice protoplasts and assayed for reconstituted Renilla luciferase activity. Expression was normalized to firefly luciferase. (4510)

Notes: In vivo imaging using bioluminescent reporters is a powerful tool for real-time detection of viral load and spread in an animal over time. However, construction of influenza reporter viruses is complicated because the small viral genome does not tolerate large insertions and all the viral genes are critical in vivo, making it impossible to replace any gene with a reporter. These authors describe construction of a replication-competent influenza reporter virus containing the small (19kDa), bright NanoLuc® luciferase gene. NanoLuc® luciferase activity was then used to monitor viral infection in real time in an animal model.  Bioluminescent imaging of the reporter virus allowed serial observations of viral load and dissemination in infected animals, even following clearance of a sublethal challenge. (4435)

Notes: These authors demonstrated use of NanoLuc® Luciferase bioluminescence for imaging applications in mice. They showed that bioluminescence could be imaged in superficial and deep tissues, and were able to monitor changes in bioluminescence over time to quantify tumor growth. Secreted NanoLuc® Luciferase was also detectable in small volumes of serum. The paper also details use of both NanoLuc® and firefly luciferase reporters in a dual assay to quantify two steps in TGFβ signaling both in intact cells and in living mice. (4439)

Notes: These authors analyzed the performance of the following reporter enzymes used to measure biological pathway modulation by small molecules: firefly luciferase, Renilla reniformis luciferase, β-lactamase, mutated forms of Renilla luciferase emitting either blue- or green-shifted luminescence, a red-light emitting form of Luciola cruciata firefly luciferase, a mutated form of Gaussia princeps luciferase, and NanoLuc® luciferase. They screened a collection of more than 42,000 PubChem compounds using purified enzyme preparations to determine hit rates and then examined structure:activity relationships. The study evaluated hit rates and inhibitor overlap between reporters. Based on these results, the authors suggest strategies to improve the construction and interpretation of assays using these reporter enzymes. (4437)

Notes: These authors set out to determine how TET2 and TET3 proteins are involved in epigenetic regulation. To characterize proteins that interact with TET, the authors expressed full-length TET1, TET2 and TET3 as HaloTag® fusion proteins and performed protein pull-downs. They identified novel interactions between all three TET proteins and O-GlcNAc transferase (OGT), which catalyzes the addition of N-acetylglucosamine (GlcNAc) to numerous transcription factors, regulatory proteins and histones to activate or inhibit the target protein or recruit additional proteins. In this paper, they focused on TET2 and TET3, which showed the strongest interaction with OGT. They mapped TET2, TET3 and OGT binding throughout  the genome by expressing these proteins as HaloTag® fusion proteins in HEK293T cells, crosslinking the proteins and DNA, then capturing the fusion proteins and associated DNA fragments and performing high-throughput sequencing to show that TET2/3 and OGT colocalize at active gene promoters and were tightly clustered near transcription start sites.

For expression of HaloTag® fusion proteins and controls, HEK-293 cells were plated at 12 ×10⁶ cells in a 150mm dish and grown to 70–80% confluency before transfection with 30µg of plasmid using the FuGENE® HD Transfection Reagent.

To assess whether TET2/3-OGT activity affects the interaction of SET1/COMPASS with chromatin, the authors used bioluminescence resonance energy transfer (BRET). They created a fusion protein consisting of the H3K4 methyltransferase SETD1A and NanoLuc® luciferase as the energy donor and a fluorescently labeled histone H3.3-HaloTag® fusion protein as the energy acceptor.  These BRET experiments confirmed that TET2/3-OGT activity is necessary for SET1/COMPASS complex function and showed that TET and OGT activities promote binding of SETD1A, a component of the SET1/COMPASS complex, to chromatin. This binding increases H3K4me3 levels. Thus, the authors’ data support a TET2/3-OGT-mediated mechanism for regulating the SET1/COMPASS complex and thus H3K4me3. (4262)

Notes: These authors describe the engineering of an enzyme and substrate to create a novel highly efficient bioluminescence system. The paper introduces NanoLuc™ Luciferase, a small luciferase subunit (19kDa) from the deep-sea shrimp Oplophorus gracilirostris, which provides ∼2.5 millionfold improved luminescence activity in mammalian cells by merging optimization of protein structure with development of a novel imidazopyrazinone substrate (furimazine). The glow-type luminescence (signal half-life >2 hours) produced by the new luciferase has a specific activity ∼150-fold greater than either firefly or Renilla luciferases similarly configured for glow-type assays. In mammalian cells, NanoLuc™ Luciferase shows no evidence of post-translational modifications or subcellular partitioning. The enzyme exhibits high physical stability, retaining activity with incubation up to 55°C or in culture medium for >15 hours at 37°C.
The authors discuss utility of NanoLuc™ Luciferase as a genetic reporter configured for high sensitivity or for response dynamics through addition of a degradation sequence to reduce intracellular accumulation. Data shows the effect of adding a signal sequence to allow export of NanoLuc™ Luciferase to the culture medium, allowing measurement of enzyme activity without cell lysis. (4295)