Notes: Determining target engagement of potential therapeutics and their target protein is commonly assessed through Thermal Shift Assays (TSA). Cell-based TSA (CETSA) now provide a more physiologically relevant information on target engagement however, these assays require use of antibodies which selectively identify the target protein and commonly have problems with reproducibility. The NanoLuc^® luciferase thermal shift assay (NaLTSA) uses NanoLuc^® luciferase activity to measure the remaining soluble fraction of the target protein making it a simplified procedure with higher reproducibility. (5055)

Notes: The presence of methyl-cytosine binding domain (MBD) containing proteins and ability to remodel methylated chromatin was investigate in sponges. Specifically, the sponge MBD2 and GATAD2A protein interaction was monitored in cells using the NanoBRET™ Protein-Protein Interaction System. Multiple coiled-coil fusion constructs were tested to determine optimal signal intensity. Interestingly, while DNA methylation was observed in sponges, this interaction was much lower affinity than in vertebrate organisms. (5057)

Notes: Modified antisense oligonucleotides (ASOs) show increased delivery and stability in cells, however these modifications have off-target effects. Localization of ASOs to cytoplasmic ribonucleoprotein (RNP) granules is observed to be mediated by RNA binding proteins, FUS and PSF. These interactions are further investigated using the NanoBRET™ Protein-Protein Interaction System. NanoLuc^® tagged protein is produced using an in vitro transcription and translation system, purified, and bound to an acceptor oligonucleotide (AlexaFluor594-ASO). Various backbone and 2′ ASO modifications were screened for interaction with FUS truncations to determine the interaction domain. (5061)

Notes: PRDM14 is dysregulated in a variety of cancers, including breast and pancreatic cancer, and overexpression leads to stem-cell-like phenotypes associated with aggressive tumors. Here, PRDM14 interacting proteins are identified using the HaloTag^® Mammalian Pull-down System. The interactions of PRDM14 and glucoseregulated protein 78 (GRP78) and heat shock protein 90-a (HSP90a) were validated in vivo with the NanoBRET™ assay. (5053)

Notes: Vascular endothelial growth factor (VEGF) receptor interactions are observed using the NanoBRET™ Protein-Protein Interaction System. A novel method of labeling VEGF at a single N-terminal cysteine (TMR) to maintain full activity is presented. NanoLuc^®-VEGFR2 and VEGF-TMR are used in conjunction to monitor an interaction and internalization into intracellular endosomes. The effect of receptor tyrosine kinase inhibitors such as Cediranib and vandetanib on internalization in living cells is assessed. (5060)

Notes: Adenomatous polyposis coli protein (APC) is shown to directly interact with HIV-1 Gag protein to stimulate Gag multimerization and spread of viral particles. Direct measurements of the Gag-Gag protein interaction were measured using the NanoBRET™ system. HeLa cells were co-transfected with Gag-HaloTag^® and Gag-NanoLuc^® expression vectors and BRET signal was monitored after 24 hours. ADC knockdown displayed a substantial decrease in viral production and Gag-Gag interaction. Together, the authors show ADC regulates Gag localization to the PM and viral packaging. (5058)

Notes: G protein coupled receptor (GPCR) oligomerization and protein interaction has been commonly investigated using bioluminescence resonance energy transfer (BRET). The need for exogenous expression of fusion proteins has been a short coming of the BRET assay. Here CRISPR/Cas9 mediated homology directed repair was used to generate protein-NanoLuc^® fusions under endogenous expression. The GPCR- β-arrestin2 interaction serves as a model of this system, where interaction and internalization are monitored under conditions where the donor luciferase endogenously expressed. (5059)

Notes: Oligomerization of the glucose transporter (GLUT1) within this plasma membrane was assessed using the NanoBRET™ system. When expressed at high levels, GLUT1 has been shown to form tetrameric complexes with higher transport efficiency. Theoretical NanoBRET™ efficiency was determined for a variety of fluorescent protein acceptors and experiments were performed with mCherry as the NanoBRET™ acceptor. GLUT1 was shown to form a range of higher order complexes in live cells. Flow cytometry and immunoblotting were used in parallel to estimate GLUT1 density in cells. (5054)

Notes: Agonist antibodies targeting T cells and other immune cells to simulate immune activation have shown to be promising for cancer therapeutics. Here, the NanoBRET™ Protein-Protein Interaction Assay was used to measure hexamerization of anti-OX40 antibodies on the cell surface. Specific anti-OX40 antibody mutations were analyzed for increased antibody multimerization and engagement. Mutations promoting IgG hexamerization showed enhanced agonistic activity. (5056)

Notes: This paper introduces NanoBRET technology, which provides an improved alternative to conventional BRET protein interaction assays. NanoBRET assays combine the extremely bright NanoLuc luciferase with a means for tagging intracellular proteins with a long-wavelength fluorophore (HaloTag). The greater light intensity and improved spectral resolution of the NanoBRET assay results in increased detection sensitivity and dynamic range over current BRET technologies. Performance of the assay is demonstrated using several model systems, and the ability to image BRET in individual cells is illustrated. The  authors also demonstrate the application of NanoBRET in a novel assay developed for analyzing the interactions of bromodomain proteins with chromatin in living cells. (4575)