Rapid and tunable control of protein stability in Caenorhabditis elegans using a small molecule

Abstract

Destabilizing domains are conditionally unstable protein domains that can be fused to a protein of interest resulting in degradation of the fusion protein in the absence of stabilizing ligand. These engineered protein domains enable rapid, reversible and dose-dependent control of protein expression levels in cultured cells and in vivo. To broaden the scope of this technology, we have engineered new destabilizing domains that perform well at temperatures of 20-25°C. This raises the possibility that our technology could be adapted for use at any temperature. We further show that these new destabilizing domains can be used to regulate protein concentrations in C. elegans. These data reinforce that DD can function in virtually any organism and temperature.

Figure 1. Schematic showing that the stability of a protein of interest (POI) fused to a destabilizing domain (DD) can be controlled using a high-affinity, stabilizing ligand.

Figure 2. Temperature dependence of existing DDs.

Destabilizing domains derived from either FKBP or ecDHFR were fused to the N-terminus of YFP (F-Y and D-Y) or the C-terminus of YFP (Y-F and Y-D). The indicated fusion proteins were stably expressed in NIH 3T3 cells for 24 h treated with vehicle (–) or stabilizing ligand (+, 2 µM Shield-1 for FKBP-derived DD and 10 µM trimethoprim for ecDHFR-derived DD) at 25°C or 37°C. The expression levels of the fusion proteins were then measured by flow cytometry. (FKBP mutations in F-Y: L106P, Y-F: E31G/R71G/K105E, DHFR mutations in D-Y: R12Y/G67S/Y100I, Y-D: R12H/N18T/A19V/G67S).

Figure 3. Ligand-dependent stability of new DDs screened at 25°C.

NIH 3T3 cells were stably transduced with the indicated ecDHFR mutants fused to YFP and treated with vehicle (–) or 10 µM trimethoprim (+) for 24 hours at 25°C. (37°C DDs are the ones engineered at 37°C; same mutants as Y-D and D-Y in Figure 2).

Figure 4. Trimethoprim rapidly stabilizes DDs expressed in C. elegans in a dose-dependent manner.

(A) Representative images of transgenic worms expressing eft-3pro:YFP-DD clone #7 from a single copy MosSci insertion grown in the absence or presence of 1 mM trimethoprim (TMP) for 24 hours at 20°C. (B) Wild type (N2) and YFP-DD transgenic worms were placed on plates containing 0.008, 0.04, 0.2, or 1 mM trimethoprim or control plates (DMSO vehicle only) as synchronized L1 larvae and imaged after 24 hours at 20°C. Graph shows average fold-induction of YFP expression relative to a non-transgenic control at increasing doses of trimethoprim (n>20 worms per dose). Error bars are ± SEM. (C) Wild-type (N2) or YFP-DD transgenic worms were placed on plates containing either 1 mM trimethoprim or DMSO vehicle control and imaged after 2, 6, 12, 24, and 48 h at 20°C. The graph shows the YFP expression of trimethoprim treated worms normalized to age-matched DMSO treated worms at each time point (n>20 worms per dose). Error bars are ± SEM. (D) YFP-DD transgenic worms were placed on plates containing 1 mM trimethoprim or DMSO vehicle control as L1s and grown for 24 hours, at which point one group was maintained on trimethoprim (“+TMP”), one was moved from trimethoprim to control plates (“washout”), and one was maintained on DMSO control plates. The graph shows background subtracted YFP expression of washout worms relative to +TMP worms at each timepoint (n>20 worms per condition). Error bars are ± SEM.