Molecular tools for optogenetic control allow for spatial and temporal regulation of cell behavior. In particular, light controlled protein degradation is a valuable mechanism of regulation because it can be highly modular, used in tandem with other control mechanisms, and maintain functionality throughout growth phases. Here, we engineered LOVtag, a protein tag that can be appended to a protein of interest for inducible degradation in Escherichia coli using blue light. We demonstrate the modularity of LOVtag by using it to tag a range of proteins, including the LacI repressor, CRISPRa activator, and the AcrB efflux pump. Additionally, we demonstrate the utility of pairing the LOVtag with existing optogenetic tools to enhance performance by developing a combined EL222 and LOVtag system. Finally, we use the LOVtag in a metabolic engineering application to demonstrate post-translational control of metabolism. Together, our results highlight the modularity and functionality of the LOVtag system, and introduce a powerful new tool for bacterial optogenetics.

/pdf/light-inducible-protein-degradation-in-e-coli-with-lovtag-8xcofg2m.pdf

Light inducible protein degradation in E. coli with LOVtag

iTAG an optimized IMiD-induced degron for targeted protein degradation in human and murine cells

Auxin-inducible degron (AID) technology is powerful for chemogenetic control of proteolysis. However, generation of human cell lines to deplete endogenous proteins with AID remains challenging. Typically, homozygous degron-tagging efficiency is low and overexpression of an auxin receptor requires additional engineering steps. Here, we establish a one-step genome editing procedure with high-efficiency homozygous tagging and auxin receptor expression. We demonstrate its application in 5 human cell lines, including embryonic stem (ES) cells. The method allowed isolation of AID single-cell clones in 10 days for 11 target proteins with >80% average homozygous degron-tagging efficiency in A431 cells, and >50% efficiency for 5 targets in H9 ES cells. The tagged endogenous proteins were inducibly degraded in all cell lines, including ES cells and ES-cell derived neurons, with robust expected functional readouts. This method facilitates the application of AID for studying endogenous protein functions in human cells, especially in stem cells.

/pdf/one-step-generation-of-auxin-inducible-degron-cells-with-2hizx2sr.pdf

One-step generation of auxin-inducible degron cells with high-efficiency homozygous tagging

https://academic.oup.com/toxsci/advance-article-pdf/doi/10.1093/toxsci/kfad049/50453174/kfad049.pdf

Use of the dTAG system in vivo to degrade CDK2 and CDK5 in adult mice and explore potential safety liabilities

Conditional degron tags (CDTs) are a powerful tool for target validation that combines the kinetics and reversible action of pharmacological agents with the generalizability of genetic manipulation. However, successful design of a CDT fusion protein often requires a prolonged, ad hoc cycle of construct design, failure, and re-design. To address this limitation, we report here a system to rapidly compare the activity of five unique CDTs: AID/AID2, IKZF3d, dTAG, HaloTag, and SMASh. We demonstrate the utility of this system against 16 unique protein targets. We find that expression and degradation are highly dependent on the specific CDT, the construct design, and the target. None of the CDTs leads to efficient expression and/or degradation across all targets; however, our systematic approach enables the identification of at least one optimal CDT fusion for each target. To enable the adoption of CDT strategies more broadly, we have made these reagents, and a detailed protocol, available as a community resource. 

/pdf/systematic-profiling-of-conditional-degron-tag-technologies-3t86o8nr.pdf

Systematic profiling of conditional degron tag technologies for target validation studies


 Translocations involving Fibroblast Growth Factor Receptor 2 (FGFR2) are found in up to 45% of Intrahepatic Cholangiocarcinoma (ICC). Treatment of patients bearing FGFR2 fusions with FGFR kinase inhibitors induces tumor regressions, though response rate and durability of response appear limited due to the emergence of resistance or to sub-optimal dosing which is limited by drug adverse effects. Thus, therapeutics that drive activity specifically towards FGFR2 are needed to enhance drug efficacy and reduce adverse effects. We first asked whether the extracellular domain (ECD) of FGFR2-BICC1 fusion is functional and important for receptor dimerization and signaling using NanoBiT system and western blotting. FGFR ECD consists of 3 Ig-fold domains D1, D2, and D3. Using focus transformation and viability assays, we found that deletions in D1, D2, D3, and D2-3 ECD FGFR2-BICC1 decreased growth and transformation of FGFR2 fusion driven cells. Next, we sought to investigate if antibodies against the ECD could perturb the growth of FGFR2 fusion transformed cells. We identified six FGFR2 specific antibodies that bind to epitopes in the ECD and confirmed the ligand blocking activity of these bivalent antibodies. In the absence of ligand, however, most of the bivalent antibodies were relatively inactive. As standard bivalent antibodies engage two receptors at a time, they are at risk for agonism or the maintenance of tonic signaling. Thus, to enhance efficacy and prevent the potential agonist or tonic activity of bivalent antibodies, all possible biparatopic antibodies (15), recognizing two distinct epitopes on the FGFR2 ECD, were engineered from the six parental antibodies. The biparatopic antibodies exhibited enhanced efficacies in FGFR2-fusion driven NIH3T3/BaF3 and ICC13-7 cancer cell lines and significantly improved binding affinities (~10-fold) as analyzed by Octet, MSD-SET, and SPR. Two biparatopic antibodies (BpAb-1, BpAb-2) showed the highest antiproliferative activity and induced apoptosis in FGFR2 fusion driven cells. BpAb-1, BpAb-2 were superior to their parental antibodies in reducing FGFR2-fusion driven growth, transformation and downstream signaling (pFGFR2, pFRS) in vitro and in reducing tumor growth in xenograft models. Importantly, BpAb-1 and BpAb-2 significantly induced FGFR2 internalization and lysosomal-mediated degradation as compared to their parental antibodies. Moreover, BpAb-1 and BpAb-2 had anti-growth activities against ICC patient-derived oncogenic FGFR2 ECD in-frame deletions, and FGFR kinase resistance mutations, including the gatekeeper mutation V565F. BpAb-1 and BpAb-2 were also active in FGFR2 WT amplified cells SNU-16. These results suggest that these FGFR2 biparatopic antibodies are a novel treatment option for ICC patients with tumors harboring FGFR2 fusions.
 Citation Format: Saireudee Chaturantabut, Sydney Oliver, John Kim, Dennie Frederick, Foxy Robinson, Alessandro Sinopoli, Diego J. Rodriguez, Liang Chang, Nabeel Bardeesy, William Sellers. Novel FGFR2 biparatopic antibodies for the treatment of cholangiocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2647.

Sydney Oliver

Papers

Systematic profiling of conditional degron tag technologies for target validation studies

Abstract 2647: Novel FGFR2 biparatopic antibodies for the treatment of cholangiocarcinoma