https://pubs.acs.org/doi/10.1021/acschembio.2c00518

Development of a New DHFR-Based Destabilizing Domain with Enhanced Basal Turnover and Applicability in Mammalian Systems

Key figures

• [Figure 3B]: Quantitatively shows C12’s markedly lower basal abundance while preserving TMP-stabilized levels, expanding dynamic range.
• [Figure 5A–B]: Dissects which C12 mutations drive low basal signal versus inducibility, showing the mutations act concertedly.
• [Figure 7A–E]: Demonstrates that C12 improves regulation of a challenging signaling factor (Nrf2ΔNeh2/6) and downstream output (HO-1), not just a reporter.

1) Thesis (one sentence)

To address leaky basal expression from existing ecDHFR destabilizing domains, in mammalian cells, engineering the C12 ecDHFR DD (W74R/T113S/E120D/Q146L) causes lower basal fusion-protein abundance with preserved TMP-induced stabilization by enhancing proteasomal turnover of the ligand-free state, supported by cell-based fluorescence imaging and western blot quantification.

2) Evidence card (three bullets only)

• Strongest result: C12 has the lowest basal abundance among screened variants while reaching TMP-stabilized levels comparable to the conventional ecDHFR DD (increased dynamic range) and shows similar TMP dose responsiveness. (Fig 3A–C).
• Method enabler: An error-prone ecDHFR library (~1200 variants) fused to YFP was lentivirally introduced into CHO cells and enriched by serial FACS for “dark” cells then TMP-responsive clones, enabling discovery of multiple independent DD solutions. (Fig 1; Table 1; screening + FACS/Celigo imaging).
• Critical limitation: C12’s improved behavior does not tolerate substantial truncation: tested truncations lose either basal destabilization or TMP-induced stabilization, so the full-length ecDHFR sequence is required to retain both key regulatory features. (Fig S7).

Optional

Quote bank (2–4 short excerpts)

• Quote 1: “Herein, we sought to improve ecDHFR DD characteristics by generating and screening for new ecDHFR mutants that have improved basal degradation without sacrificing TMP-induced stabilization.” (Introduction, p.2).
• Quote 2: “These results emphasize the difficulty in achieving a substantial shortening to the ecDHFR DD sequence without compromising its most important regulatory features.” (Results, p.9).
• Quote 3: “Overall, our work in developing and verifying the C12 ecDHFR DD marks an important milestone highlighting a second-generation ecDHFR DD system.” (Discussion, p.10).

Key comparisons (1–3 lines)

• Compared to: Conventional N-terminal ecDHFR DD (R12Y/G67S/Y100I).
• Win: Lower basal fusion-protein levels with similar TMP-induced stabilization, improving usable dynamic range across reporters and client proteins (IκBα, Nrf2ΔNeh2/6).
• Tradeoff: Cannot be meaningfully shortened without breaking either destabilization or inducibility, limiting size-driven vector design.

Methods I might copy (protocol hooks)

• Construct design / Models: ecDHFR mutant library fused to a C-terminal YFP-HA reporter; C12 sequence contains missense W74R/T113S/E120D/Q146L plus a synonymous A143A; N-terminal fusions tested with IκBα and Nrf2ΔNeh2/6. Cell lines used include CHO/dhFr- (CRL-9096), HEK-293A, HEK-293T (CRL-3216), and Neuro-2a (N2A, CCL-131).
• Conditions / Instruments: CHO library transduction at MOI ~1 using 1 μg/mL polybrene and 250 μL crude lentivirus (24 h), then puromycin selection (10 μg/mL, 2 weeks); FACSAria sorting twice for YFP-negative (“dark”) cells, then TMP 10 μM for 24 h and single-cell sorting into 96-well plates; transient TMP stabilization typically 10 μM for 24 h (dose series 10 nM–10 μM for 24 h). Basal/transient imaging performed on a Celigo imaging cytometer with YFP captured at 150,000 ms exposure (after switching to Fluorobrite DMEM + 1% FBS).
• Readout / Analysis: Western blot workflow includes RIPA lysis with protease inhibitor + benzonase (15 min, RT), clarified at 14,000 rpm (10 min, 4 °C), 15–20 μg protein on 4–20% Tris-Gly SDS-PAGE, transfer via iBlot 2, imaging on Odyssey CLx with ImageStudio/Image Quant quantification; key antibodies include anti-ecDHFR, anti-IκBα, anti-Nrf2, anti-HO-1, anti-GFP(YFP), and loading controls (GAPDH/β-actin). Computational stability analysis used Rosetta (v3.12) ddG calculations on ecDHFR structure (PDB 6XG5) for apo vs holo (TMP+NADPH) states.

Open questions / Theoretical implications (2–5 bullets)

• Does C12 improve basal turnover primarily by increased polyubiquitination versus engagement of ubiquitin-independent proteasome routes (suggested as possibilities), and can co-IP identify distinct binding partners relative to the conventional DD?
• Can computational design explicitly optimize the stability gap between apo and holo states (rather than absolute destabilization) to systematically generate “ideal” DDs for new ligand/domain pairs?
• How general is the C12 advantage across cell types and pathway-sensitive contexts beyond HEK-293A screening/validation, especially where basal leak is most phenotoxic?