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

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Development of a New DHFR-Based Destabilizing Domain with Enhanced Basal Turnover and Applicability in Mammalian Systems
ACS Chemical Biology ( IF 3.5 ) Pub Date : 2022-09-19 , DOI: 10.1021/acschembio.2c00518
Emi Nakahara ₁ , Vishruth Mullapudi ₂ , Gracen E Collier ₁ , Lukasz A Joachimiak _{2,

3} , John D Hulleman _{1,

4}

Affiliation

Destabilizing domains (DDs) are an attractive strategy allowing for positive post-transcriptional small molecule-regulatable control of a fusion protein’s abundance. However, in many instances, the currently available DDs suffer from higher-than-desirable basal levels of the fusion protein. Accordingly, we redesigned the E. coli dihydrofolate reductase (ecDHFR) DD by introducing a library of ∼1200 random ecDHFR mutants fused to YFP into CHO cells. Following successive rounds of fluorescence-activated cell sorting, we identified six new ecDHFR DD clones with significantly enhanced proteasomal turnover in the absence of a stabilizing ligand, trimethoprim (TMP). One of these clones, designated as “C12”, contained four unique missense mutations (W74R/T113S/E120D/Q146L) and demonstrated a significant 2.9-fold reduction in basal levels compared to the conventional ecDHFR DD (i.e., R12Y/G67S/Y100I). This domain was similarly responsive to TMP with respect to dose response and maximal stabilization, indicating an overall enhanced dynamic range. Interestingly, both computational and wet-lab experiments identified the W74R and T113S mutations of C12 as the main contributors toward its basal destabilization. However, the combination of all the C12 mutations was required to maintain both its enhanced degradation and TMP stabilization. We further demonstrate the utility of C12 by fusing it to IκBα and Nrf2, two stress-responsive proteins that have previously been challenging to regulate. In both instances, C12 significantly enhanced the basal turnover of these proteins and improved the dynamic range of regulation post stabilizer addition. These advantageous features of the C12 ecDHFR DD variant highlight its potential for replacing the conventional N-terminal ecDHFR DD and improving the use of DDs overall, not only as a chemical biology tool but for gene therapy avenues as well.

中文翻译：

开发一种新的基于 DHFR 的去稳定域，具有增强的基础周转和在哺乳动物系统中的适用性

去稳定结构域 (DD) 是一种有吸引力的策略，可以对融合蛋白的丰度进行积极的转录后小分子调节控制。然而，在许多情况下，目前可用的 DD 的融合蛋白基础水平高于所需水平。因此，我们通过将约 1200 个与 YFP 融合的随机 ecDHFR 突变体库引入 CHO 细胞，重新设计了大肠杆菌二氢叶酸还原酶 (ecDHFR) DD。经过连续几轮荧光激活细胞分选后，我们鉴定了 6 个新的 ecDHFR DD 克隆，在没有稳定配体甲氧苄啶 (TMP) 的情况下，蛋白酶体周转率显着增强。其中一个克隆被命名为“C12”，包含四个独特的错义突变（W74R/T113S/E120D/Q146L），与传统的 ecDHFR DD（即R12Y/G67S/Y100I）相比，基础水平显着降低 2.9 倍。）。该结构域在剂量响应和最大稳定性方面对 TMP 具有类似的响应，表明总体动态范围增强。有趣的是，计算和湿实验室实验均发现 C12 的 W74R 和 T113S 突变是导致其基础不稳定的主要因素。然而，需要所有 C12 突变的组合来维持其增强的降解和 TMP 的稳定性。我们通过将 C12 与 IκBα 和 Nrf2（这两种以前难以调节的应激反应蛋白）融合，进一步证明了 C12 的实用性。在这两种情况下，C12 都显着增强了这些蛋白质的基础周转率，并改善了添加稳定剂后调节的动态范围。 C12 ecDHFR DD 变体的这些有利特征突出了其替代传统 N 端 ecDHFR DD 并改善 DD 整体使用的潜力，不仅作为化学生物学工具，而且还用于基因治疗途径。

更新日期：2022-09-19

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