The Legend of PREDATOR Pro
Exactitude temporal control of protein abundance is critical for the robustness and dynamics of synthetic circuits. While multiple approaches have been developed to manipulate the protein synthesis, few tools have been demonstrated to precisely control untagged protein degradation. Here, we present Predator Pro, a modularized and signal-controllable method for target protein degradation, on the basis of the Predator system we demonstrated in iGEM 2018-19. By rationally reengineer the Trim21 protein, we demonstrated that the interaction between Trim21 and antibody Fc domain can be replaced with other constitutive or inducible protein dimerization pairs. We demonstrated that constitutive DocS-Coh2 interaction or rapamycin-induced FRP-FKBP interaction enabled constitutive or drug-controlled degradation of untagged EGFP protein. As an effective expansion of the current synthetic biological tools for protein abundance control, this system may provide a modularized and convenient platform for controlled protein degradation, which might be applied in fundamental researches and clinical applications.
In the past 20 years, Synthetic Biology, regarded as one of the most hyped research topics this century, has revolutionarily reshaped biotechnology and biomedicine. As the basis of complicated synthetic regulatory networks, dramatic achievements have been made in the past two decades to achieve the spatial and temporal manipulation of protein abundance1. In mammalian cells, wide variety of tools have been developed to precisely control the protein synthesis on genomic, transcriptional and translational level2-6, resulting in designer cells carrying increasingly complicated synthetic circuits to address multiple biomedical needs.
With these tremendous achievements, however, it is still worth noticing that as the other end of the leverage controlling cellular protein homeostasis, only a few synthetic biological tools have been reported to precisely control the protein degradation process. In mammalian cells, ubiquitin-proteasome system is responsible for about 80% of the endogenous protein degradation. Target proteins are tagged with ubiquitin chain by ubiquitin ligase, which triggers 26S proteasome mediated protein degradation7,8. Utilizing such mechanism, multiple targeted protein degradation methods have been developed. For tagged proteins like exogenous expressed ones, degrons can be fused to manipulate their degradation, thus altering the dynamics of these proteins9. For untagged proteins, i.e. endogenous proteins, methods such as PROTAC10 and Trim-away11 have also been developed to attach ubiquitin to target proteins to trigger their degradation. PROTAC utilizes a set of target-specifically designed small molecules, which interacts with both target protein and endogenous ubiquitin ligase to trigger the ubiquitylation and degradation of the target protein. While such elegant tool provides new approach to control the protein degradation, its small-molecule-dependent nature somehow lowered the compatibility with other synthetic biological tools we already have, the high throughput customization of these small molecules also remains problematic and costly.
Trim-away was initially reported in 2017 as an acute and rapid endogenous protein degradation method11. Trim-away utilizes Tripartite Motif containing-21 (Trim21), an E3 ubiquitin ligase that specifically recognizes the Fc domain of antibodies, to trigger the degradation of antibody tagged target protein11-13 (Figure 1a). Antibodies can be either microinjected or electroporated into the cells to initiate the Trim-away process. To further adapt such tool into synthetic biology toolbox and avoid the inconvenience of antibody microinjection or electroporation, in iGEM 2018 and iGEM 2019, our team developed and upgraded PREDATOR system (and 2019). The previous PREDATOR system shares a similar mindset with the Trim-away system, they both utilize Trim21 to trigger the targeted degradation of specific endogenous proteins. Instead of microinjecting or electroporating the antibodies into the cells, we hereby used genetically encoded, Antibody Fc domain fused nanobodies or single-chain variable fragments (scFv) to trigger the interaction of the target protein and Trim21 (see NUDT_CHINA on iGEM 2018). Seeing the limited availability of the nanobodies and ScFvs, we then demonstrated that the targeting of the PREDATOR system can also be achieved with other protein-protein interactions such as ligand-receptor interaction (see NUDT_CHINA on iGEM 2019). We also showed that such system can be linked to classical synthetic transcriptional circuits to achieve signal controlled degradation of target protein (see NUDT_CHINA on iGEM 2019) (Figure 1b). However, we also noticed that the current PREDATOR still need to be transcriptionally controlled to achieve signal-responsiveness, which is both slow and indirect. A new system that can be directly controlled by endogenous signal is hence designed in iGEM 2020.
Figure 1. Schematic representation of two systems and their characteristics. (A) The molecule structure of Trim21 which is vital in the Trim-Away pathway. (B) The molecule structure of improved Trim21 which is used in Predator System.
In our iGEM 2020 project, we reanalyzed the previous PREDATOR working model, and modularized the PREDATOR system into two modules (a.k.a. Targeting module and Functional module). These two modules were connected by a protein-protein interaction-based interface. We then hypothesized that the previous [antibody Fc domain – Trim21 PRYSPRY domain] interface can be replaced with other protein dimerization pairs. Our results showed that replacing the interface in GFP targeting PREDATOR into constitutively dimerizing DocS-Coh2 pair didn’t impair the PREDATOR mediated degradation of the GFP protein, which proved the basic concept of our design. Furthermore, we demonstrated that the interface in GFP PREDATOR can also be replaced into Rapamycin mediated FRB-FKBP interaction pairs allowing the direct control of GFP degradation of GFP by Rapamycin (Figure 2).
Figure 2. Schematic representation of Predator Pro systems.
In the meantime, our HP members also established wide connection to academic professionals and iGEMers around China. The feedbacks from these experts and iGEMers provided tremendously important suggestions for the development and execution of our project.
In general, with modularized mindset and brand-new function, we hereby proudly announce PREDATOR Pro system, a not-yet-being reported toolbox in mammalian cells to achieve targeted protein degradation directly controlled by exogenous signals. By changing the targeting modules and the protein dimerization pair forming the interface, we believe that PREDATOR Pro system can be applied to degrade wide rage of proteins under the control of a variety of signals including small molecule, light, etc. for different purposes.
1 Xie, M. & Fussenegger, M. Mammalian designer cells: Engineering principles and biomedical applications. Biotechnol J 10, 1005-1018, doi:10.1002/biot.201400642 (2015).
2 Gaj, T., Gersbach, C. A. & Barbas, C. F., 3rd. ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. Trends Biotechnol 31, 397-405, doi:10.1016/j.tibtech.2013.04.004 (2013).
3 Bai, P. et al. A fully human transgene switch to regulate therapeutic protein production by cooling sensation. Nature Medicine 25, 1266-1273, doi:10.1038/s41591-019-0501-8 (2019).
4 Xue, S. et al. A Synthetic-Biology-Inspired Therapeutic Strategy for Targeting and Treating Hepatogenous Diabetes. Molecular Therapy the Journal of the American Society of Gene Therapy 25, 443, doi:10.1016/j.ymthe.2016.11.008 (2017).
5 Wang, H., Xie, M., Hamri, G. C.-E., Ye, H. & Fussenegger, M. Treatment of chronic pain by designer cells controlled by spearmint aromatherapy. Nat Biomed Eng 2, 114-123, doi:10.1038/s41551-018-0192-3(2018).
6 Shao, J. et al. Smartphone-controlled optogenetically engineered cells enable semiautomatic glucose homeostasis in diabetic mice. Science Translational Medicine 9, eaal2298, doi:10.1126/scitranslmed.aal2298 (2017).
7 Kleiger, G. & Mayor, T. Perilous journey: a tour of the ubiquitin-proteasome system. Trends Cell Biol 24, 352-359, doi:10.1016/j.tcb.2013.12.003 (2014).
8 Chassin, H. et al. A modular degron library for synthetic circuits in mammalian cells. Biotechnol J 10, 2013, doi:10.1038/s41467-019-09974-5 (2019).
9 Natsume, T. & Kanemaki, M. T. Conditional Degrons for Controlling Protein Expression at the Protein Level. Annu Rev Genet 51,83-102, doi:10.1146/annurev-genet-120116-024656 (2017).
10 Toure, M. & Crews, C. M. Small-Molecule PROTACS: New Approaches to Protein Degradation. Angew Chem Int Ed Engl 55, 1966-1973, doi:10.1002/anie.201507978 (2016).
11 Clift, D. et al. A Method for the Acute and Rapid Degradation of Endogenous Proteins. Cell 171, 1692-1706.e1618, doi:10.1016/j.cell.2017.10.033 (2017).
12 James, L. C., Keeble, A. H., Khan, Z., Rhodes, D. A. & Trowsdale, J. Structural basis for PRYSPRY-mediated tripartite motif (TRIM) protein function. Proceedings of the National Academy of Sciences 104, 6200, doi:10.1073/pnas.0609174104 (2007).
13 Zeng, J. et al. (bioRxiv, 2020).