Abstract
Technologies that convert transient protein-protein interactions (PPIs) into stable expression of a reporter gene are useful for genetic selections, high-throughput screening, and multiplexing with omics technologies. We previously reported SPARK (Kim et al., 2017), a transcription factor that is activated by the coincidence of blue light and a PPI. Here, we report an improved, second-generation SPARK2 that incorporates a luciferase moiety to control the light-sensitive LOV domain. SPARK2 can be temporally gated by either external light or addition of a small-molecule luciferin, which causes luciferase to open LOV via proximity-dependent BRET. Furthermore, the nested 'AND' gate design of SPARK2-in which both protease recruitment to the membrane-anchored transcription factor and LOV domain opening are regulated by the PPI of interest-yields a lower-background system and improved PPI specificity. We apply SPARK2 to high-throughput screening for GPCR agonists and for the detection of trans-cellular contacts, all with versatile transcriptional readout.
Highlights
Because protein-protein interactions (PPIs) are central to every biological signaling pathway, there has been keen interest in developing ever-more specific, sensitive, and versatile reporters for detecting them in living cells
These results demonstrate that NanoLuc can regulate the LOV domain of SPARK2 in place of external blue light, enabling users to toggle between luciferin-control and light-control of SPARK2 for PPI detection in living cells
We confirmed that in the absence of 9cr, there was no significant increase in the Citrine/Flag intensity with either blue light or furimazine (Figure 5—figure supplement 1D–E). These results provide a proof-of-concept demonstration that SPARK2 can be combined with intercellular NanoLuc-ob2AR BRET to enable luciferin-gated, bioluminescence-mediated transcriptional readout of cell-cell contacts
Summary
Because protein-protein interactions (PPIs) are central to every biological signaling pathway, there has been keen interest in developing ever-more specific, sensitive, and versatile reporters for detecting them in living cells. Existing tools to detect PPIs fall into two broad categories: realtime reporters such as those based on FRET (Truong and Ikura, 2001) or protein complementation assays (Kerppola, 2006), and transcriptional reporters such as yeast two-hybrid (Miller and Stagljar, 2004) and TANGO (Barnea et al, 2008). We previously reported ‘SPARK’, for ‘Specific Protein Association tool giving transcriptional Readout with rapid Kinetics’, a transcriptional PPI reporter that improves upon yeast two-hybrid and TANGO designs by incorporating light-gating. Instead of integrating PPI events over many hours or days, as yeast two-hybrid and TANGO do, SPARK integrates PPI events over just a 5 min user-selected time window determined by exogenous blue light delivery
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
