Small-molecule hydrophobic tagging-induced degradation of HaloTag fusion proteins

Abstract

The ability to regulate any protein of interest in living systems with small molecules remains a challenge. We hypothesized that appending a hydrophobic moiety to the surface of a protein would mimic the partially denatured state of the protein, thus engaging the cellular quality control machinery to induce its proteasomal degradation. We designed and synthesized bifunctional small molecules to bind a bacterial dehalogenase (the HaloTag protein) and present a hydrophobic group on its surface. Hydrophobic tagging of the HaloTag protein with an adamantyl moiety induced the degradation of cytosolic, isoprenylated and transmembrane HaloTag fusion proteins in cell culture. We demonstrated the in vivo utility of hydrophobic tagging by degrading proteins expressed in zebrafish embryos and by inhibiting Hras1(G12V)-driven tumor progression in mice. Therefore, hydrophobic tagging of HaloTag fusion proteins affords small-molecule control over any protein of interest, making it an ideal system for validating potential drug targets in disease models.

Figure 1. Hydrophobic tagging strategy using the HaloTag fusion protein system

(a) Chemical structures of the representative HaloTag Ligands: HyT5, HyT12, HyT13, HyT16, HyT21 and HyT22. (b) HEK 293T cells expressing HA-HaloTag-luciferase were treated with indicated compounds at 1 μM for 24 hours, at which point luciferase assays were performed.

Figure 2. HyT13 leads to degradation of HaloTag fusion proteins

(a) Flp-In 293 cells expressing HA-EGFP-HaloTag were treated with indicated concentrations of HyT13 for 24 hours. The lysates were probed with anti-HA and anti-β-actin antibodies. (b) The same cell line as in (a) was treated for the indicated times with 1 μM HyT13. The rightmost sample was treated with HyT13 for 24 hours, after which HyT13-free media was provided for 24 hours. (c) The same cell line as in (a) was pretreated with proteasome inhibitors MG132 (10 μM) and YU101 (10 μM) for 1 hour prior to addition of 1 μM HyT13. The lysates were prepared from cells 6 hours after HyT13 addition. (d) HeLa cells stably expressing EGFP-HaloTag were treated with vehicle or 1 μM HyT13 for 24 hours, whereupon the intracellular GFP fluorescence was quantified by flow cytometry. MFI = mean fluorescence intensity. (e) HEK 293T cells stably expressing indicated transmembrane HA-HaloTag fusion proteins were treated with 1 μM HyT13 for 24 hours. Shown are representative images from at least three experiments; bands were quantified and mean degradation ± SEM is shown. (f) One-cell stage zebrafish embryos were injected with 100 ng of HA-HaloTag-Smad5 cRNA, grown to 256-cell stage and then treated with 10 μM HyT13 for 24 hours. Shown are representative images from at least three experiments; bands were quantified and mean degradation ± SEM is shown. Full gels are available in Supplementary Results.

Figure 3. Functional validation of HaloTag degradation by HyT13

(a) NIH-3T3 cells were retrovirally infected with a construct expressing either HA-HaloTag-HRas(G12V) or HA-HaloTag(D106A)-HRas(G12V). The cells were then treated with vehicle or 1 μM HyT13 for 24 hours. The lysates were prepared for immunoblotting and the blots were probed with anti-HA and anti-β-actin antibodies. Full gels are available in Supplementary Results. (b) One hundred thousand NIH-3T3 cells infected with HA-HaloTag-HRas(G12V) or HA-HaloTag(D106A)-HRas(G12V) were plated in 10% FBS containing medium onto 10-cm plates. The next day, the medium was replaced with 1% FBS containing medium, along with vehicle or 1 μM HyT13. The media was refreshed every 2 days, and the plates were pictured on day 6. Bar, 5 mm. (c) Quantification of foci as described in (b). The number of foci/cm² was counted from three separate plates, with error bars representing SEM. (d) One hundred thousand HA-HaloTag-HRasG12V-expressing NIH-3T3 cells were injected into the flank of nude mice on day 0. The mice were administered IP injections of vehicle or HyT13 daily from day 0. Tumor size was measured daily, and the tumor volume was calculated. Each treatment group employed 7 mice. Error bars represent SEM.

Figure 4. Schematic of HyT13 mediated degradation of HaloTag fusion proteins

A fusion protein composed of a protein of interest and the HaloTag protein is degraded upon HyT13 treatment by the proteasome.