Tunable and reversible drug control of protein production via a self-excising degron

Abstract

An effective method for direct chemical control over the production of specific proteins would be widely useful. We describe small molecule-assisted shutoff (SMASh), a technique in which proteins are fused to a degron that removes itself in the absence of drug, resulting in the production of an untagged protein. Clinically tested HCV protease inhibitors can then block degron removal, inducing rapid degradation of subsequently synthesized copies of the protein. SMASh allows reversible and dose-dependent shutoff of various proteins in multiple mammalian cell types and in yeast. We also used SMASh to confer drug responsiveness onto an RNA virus for which no licensed inhibitors exist. As SMASh does not require the permanent fusion of a large domain, it should be useful when control over protein production with minimal structural modification is desired. Furthermore, as SMASh involves only a single genetic modification and does not rely on modulating protein-protein interactions, it should be easy to generalize to multiple biological contexts.

Figure 1

Small Molecule-Assisted Shutoff (SMASh) concept and development. (a) SMASh concept. Top, a target protein is fused to the SMASh tag via a HCV NS3 protease recognition site. After protein folding, the SMASh tag is removed by its internal protease activity, and is degraded due to internal degron activity. Bottom, addition of protease inhibitor induces the rapid degradation of subsequently synthesized copies of the tagged protein, effectively shutting off further protein production. (b) Amino acid sequence of the SMASh tag. Sequence derived from NS3 protease (orange), NS3 helicase (gray), and NS4A (red) are shown. Secondary structures in the context of the original HCV polyprotein are underlined. The NS4A/4B protease substrate (green), has an arrow indicating site of cleavage. Dotted line indicates putative degron region. (c) Top, organization of fusions of PSD95 with NS3 protease (NS3pro) or NS3pro-NS4A, with predicted protein fragment sizes indicated. Bottom, in the absence of protease inhibitor asunaprevir (ASV), PSD95 was detectable in HEK293 lysates 24 h post-transfection, for both constructs. With asunaprevir, the PSD95-NS3pro fusion was expressed at full-length size, but the PSD95-NS3pro-NS4A failed to exhibit expression. GAPDH served as a loading control. (d) A specific element within NS3pro-NS4A is necessary for degron activity. Transfected HeLa cells expressed either YFP-NS3pro-NS4A, or a variant in which the putative degron (dotted line in b) was mutated to a GGS-repeat linker of the same length (GGS), for 24 h with or without ASV. The GGS mutation restores expression in the ASV condition. β-actin served as a loading control.

Figure 2

Proteins can be regulated by SMASh tags at either terminus. (a) SMASh can regulate YFP when fused to either terminus. SMASh-YFP or YFP-SMASh were expressed in HEK293 cells in the absence or presence of ASV for 24 h. Immunoblotting revealed shutoff of YFP expression by ASV for both constructs. DMSO was used as vehicle control. β -actin served as a loading control. (b) Fluorescence microscopy confirmed shutoff of YFP expression by ASV for both constructs. Scale bar, 50 μm.

Figure 3

Protein regulation by SMASh-tagging is dose-dependent and reversible. (a) To test dose-dependent regulation of protein expression by SMASh, HEK293 cells transfected with YFP-SMASh were cultured for 24 h without or with ASV (15 pM to 15 μM) and YFP was detected by immunoblot. GAPDH served as a loading control. (b) Quantification of YFP levels by immunoblot. Background-subtracted YFP signal was normalized to background-subtracted GAPDH signal, and then plotted as a percent of the signal in the untreated condition (n = 3, error bars represent standard deviations). (c) Restoration of YFP expression following drug washout, assayed by immunoblot. HeLa cells transfected with YFP-SMASh were grown 12 h in the presence of 2 μM ASV, then washed and exchanged into fresh media. Parallel wells were lysed at indicated times afterwards. β -actin served as a loading control. (d) Restoration of YFP expression following drug washout, assayed by fluorescence microscopy. HeLa cells co-transfected with untagged RFP and YFP-SMASh were grown 12 h in the presence of 2 μM ASV, washed, exchanged into fresh media, and imaged at indicated times afterwards. Similarly transfected HeLa cells grown 12 h in DMSO are shown at left for comparison. Representative images are shown. Scale bar, 20 μm.

Figure 4

SMASh functions on a variety of proteins. (a) SMASh functions on multimerizing protein, CaMKIIα. TimeSTAMP2-tagged CaMKIIα (TS2-CaMKIIα) or SMASh-CaMKIIα were expressed in HEK293 cells for 24 h in the absence or presence of ASV. The TimeSTAMP2 contain a cis-cleaving NS3 protease domain but lacks NS4A, verifying that drug inhibition of protein expression is specific to SMASh. Immunoblotting revealed shutoff of CaMKIIα expression by ASV when it was tagged with SMASh but not when it was tagged with TimeSTAMP2. GAPDH served as a loading control. The asterisk indicates a cross-reactive protein also detected in untransfected cells. The expected locations of the uncleaved higher molecular weight protein and the cleaved protein are indicated with arrows. (b) GluRIIA-CFP fused to TimeSTAMP2 with an orange fluorescent protein readout (GluRIIA-CFP-TS2:OFP) or GluRIIA-CFP-SMASh were expressed in HEK293 cells for 24 h in the absence or presence of ciluprevir (CLV). Immunoblotting revealed shutoff of GluRIIA expression by CLV. The non-degron-containing TS2:OFP tag verified that drug inhibition of protein expression is specific to SMASh. Cross-reactive bands at 80 kDa (asterisk) served as a lysate loading control. (c) CYP21A2 was either fused to TimeSTAMP2 or SMASh and tested with the same method as in (a). CYP21A2 level was detected by immunoblotting. β -actin served as a loading control.

Figure 5

SMASh functions in budding yeast. (a) YFP-SMASh under strong GPD promoter is integrated into the yeast chromosomal LUE locus. Recombinated yeast were cultured in SD media in the absence or presence of ASV for 24 h. Immunoblotting revealed shutoff of YFP expression by ASV. DMSO was used as vehicle control. GAPDH served as a loading control. (b) Fluorescence images of yeast cultures in (a) show that chromosomally-expressed YFP signal is controlled in a drug-dependent manner. Imaging was done in SD media. Scale bar, 10 μm. (c) An HA tag and the SMASh tag were inserted at the C-terminus of the endogenous SEC14 coding sequence, and serial dilutions of cells were plated and incubated for 48 h at 30 °C and 23 °C in the absence or presence of ASV (3 μM). (d) A SMASh tag was inserted at the C-terminus of the endogenous YSH1 coding sequence, and serial dilutions of cells were plated and incubated for 48 h at 30 °C and 37 °C in the absence or presence of ASV (10 μM).

Figure 6

Generation of a drug-controllable “SMAShable” measles vaccine virus. (a) Concept of controlling MeV replication with P-SMASh. In the absence of the drug, essentially unmodified phosphoprotein (P, blue) is released and can successfully form replication complexes with nucleocapsid (N, orange) and large (L) proteins. (b) Genome organization of MeV-EGFP-P-SMASh. Scale bar is 1 kilobase. (c) Regulation of MeV-EGFP-P-SMASh by drug. Vero cells infected with MeV-EGFP or MeV-EGFP-P-SMASh at multiplicity of infection (MOI) of 1 were grown for 72 h in the absence or presence of ASV. Drug inhibited syncytium formation and GFP expression in MeV-EGFP-P-SMASh-infected but not MeV-EGFP-infected cells. Scale bar, 50 μm. (d) Quantification of fluorescence from Vero cells infected with MeV-EGFP-P-SMASh at MOI 1 and 0.1 in the absence or presence of 3 μM ASV (n = 3, error bars are standard deviation). (e) Drug inhibited P expression in MeV-EGFP-P-SMASh-infected but not MeV-EGFP-infected cells, as assayed by immunoblotting. GAPDH served as a loading control.