BTBBCL6 dimers as building blocks for reversible drug-induced protein oligomerization

by Selleckchem | Oct 29 2023

Here, we characterize the BTB domain of the transcription factor BCL6 (BTBBCL6) as a small-molecule-controlled, reversible oligomerization switch, which oligomerizes upon BI-3802 treatment and de-oligomerizes upon addition of BI-3812. We show that the magnitude of oligomerization can be controlled in vitro by BI-3802 concentration and exposure time. In cellular models, exposure to BI-3802/BI-3812 can drive multiple cycles of foci formation consisting of BTBBCL6 fused to EGFP, which are not degraded due to the lack of a degron. We generated an epidermal growth factor receptor (EGFR)-BTBBCL6 fusion. Treatment with BI-3802, as an ON switch, induced EGFR-BTBBCL6 phosphorylation and activation of downstream effectors, which could in part be reversed by the addition of BI-3812, as an OFF switch. Finally, BI-3802-induced oligomerization of the EGFR-BTBBCL6 fusion enhanced proliferation of an EGF-dependent cell line in absence of EGF. These results demonstrate the successful application of small-molecule-induced, reversible oligomerization as a switch for synthetic biology.

Comments:

In this study, the researchers focused on characterizing the BTB domain of the transcription factor BCL6 (referred to as BTBBCL6). They discovered that BTBBCL6 acts as a small-molecule-controlled, reversible oligomerization switch. When treated with a compound called BI-3802, the BTBBCL6 protein forms oligomers (multiple units bound together) and can be reversed back to its individual units by adding BI-3812.

The researchers found that the extent of oligomerization can be regulated in laboratory experiments by adjusting the concentration of BI-3802 and the duration of exposure. They also examined the effects of BI-3802 and BI-3812 in cellular models.

In the cellular models, the researchers introduced a fusion protein called BTBBCL6-EGFP, where the BTBBCL6 domain was fused to a green fluorescent protein (EGFP). Importantly, this fusion protein lacked a degron, a signal for degradation, which allowed the foci (distinct fluorescent spots) formed by the fusion protein to persist.

When the cellular models were exposed to BI-3802, multiple cycles of foci formation were observed, indicating the formation of oligomers of BTBBCL6-EGFP. The addition of BI-3812 reversed this process and led to the dispersion of the foci.

To further explore the potential applications of this reversible oligomerization switch, the researchers created a fusion protein consisting of the epidermal growth factor receptor (EGFR) and BTBBCL6 (referred to as EGFR-BTBBCL6). When treated with BI-3802, the EGFR-BTBBCL6 fusion protein underwent phosphorylation (addition of phosphate groups) and activated downstream signaling pathways. Some of these effects were partially reversed by the addition of BI-3812.

Additionally, the researchers found that the BI-3802-induced oligomerization of the EGFR-BTBBCL6 fusion protein enhanced the proliferation of an EGF-dependent cell line even in the absence of EGF stimulation.

Overall, these findings demonstrate the successful application of small-molecule-induced, reversible oligomerization as a switch in synthetic biology. The ability to control the oligomerization state of proteins using small molecules opens up possibilities for designing novel molecular switches and regulatory systems in various biological contexts.