Identification of hydrophobic tags for the degradation of stabilized proteins

Abstract

New HyTs are a knockout: we previously reported that labeling HaloTag proteins with low molecular weight hydrophobic tags (HyTs) leads to targeted degradation of HaloTag fusion proteins. In this report, we employed a chemical approach to extend this hydrophobic tagging methodology to highly stabilized proteins by synthesizing and evaluating a library of HyTs, which led to the identification of HyT36.

Figure 1

Hydrophobic tagging-induced protein degradation. a) Fusion proteins composed of a protein-of-interest and HaloTag can be specifically targeted for proteasomal degradation with low molecular weight hydrophobic tags (HyTs). b) Chemical structures of HyT13 (1) and the more active HyT36 (2).

Figure 2

HyT13 does not significantly degrade the stabilized HaloTag7. a) and b) HEK 293 cells expressing GFP-HaloTag2 or GFP-HaloTag7 were treated with vehicle or HyT13 (1) (10 μM) for 24 hr followed by analysis of fusion protein levels by flow cytometry or immunoblot with specific antibodies as indicated. Images are representative of at least three independent experiments; band intensities were quantified and data presented as mean % degradation ± s.d.

Figure 3

HyT36 degrades HaloTag2 fusion proteins to a greater extent than HyT13. a) and b) HEK 293 cells expressing GFP-HaloTag2 were treated with vehicle, HyT13 (1) or HyT36 (2) for 24 hr followed by analysis of fusion protein levels by immunoblot or flow cytometry. c) HEK 293T cells expressing Fz4-HaloTag2 were treated with vehicle, HyT13 (1) or HyT36 (2) for 24 hr followed by detection of this fusion protein by immunoblot. Images are representative of at least three independent experiments; band intensities were quantified and data presented as mean degredation ± s.d.

Figure 4

Effect of HyT36 on HaloTag7 fusion proteins. a) and b) HEK 293 cells expressing GFP-HaloTag7 were treated with vehicle, HyT13 (1) (10 μM) or HyT36 (2) (10 μM) for 24 hr followed by analysis of fusion protein levels by immunoblot or flow cytometry. c) Melting curves for HaloTag7 in the presence of vehicle (closed circles, solid line) or HyT13 (1) (10 μM, open circles, dashed line). d) Melting curves for HaloTag7 in the presence of vehicle (closed circles, solid line) or HyT36 (2) (10 μM, open circles, dashed line) or HyT36 (2) (40 μM, closed triangles, dotted line). Images are representative of at least three independent experiments; band intensities were quantified and data presented as mean % degredation ± s.d. Errors bars on thermal shift plots represent the mean ± s.d for each temperature.