Team:NUDT CHINA/Poster

Poster: NUDT_CHINA

Predator Pro: a modularized toolbox for signal-controlled Targeted Protein Degradation

Poster presented by Team NUDT_CHINA 2020

Tianyi Zhang, Zhenyu Zhou, Changtai Xiao, Lunhao Ju, Xinlin Liu, Yongjiang Li, Huiying Liu, Qingyi Liu, Linjie Li, Ruoxi Wang, Yuxin Liu, Yingqian Ye, Junzi Gu, Haoyu Zhang, Yulan Chen, Zhiliang Pan, Hanxiao Feng, Guangyi Lin, Yanchen Gou, Yuxuan Wang, Weiqian Zhou.

Abstract

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 FRB-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.

Introduction

A key aspect of synthetic biology is the selection and reengineering of multiple biological elements who can control designer cells over time and space. These intelligent cells could fulfil various biomedical needs.

Over the past decades, tremendous synthetic biological circuits have been designed and the basis of them is to manipulate protein abundance either spatially or temporally in alternative splicing, translational and post translational levels.

As is known that protein synthesis and protein degradation control the overall cellular protein homeostasis. While regulating protein expression has been put into much efforts in synthetic biology, only a few tools have been developed for targeted protein degradation.

There are three targeted protein degradation methods that has been applied based on ubiquitin proteasome system (UPS), namely degron, PROTACs and Trim-Away. However, a degron could only degrade tagged protein. PROTACs, although controllable by small molecules, cannot be genetically-encoded. And it is expensive and complicated for Trim-Away to microinject or electroporate to introduce antibodies into cells.

In iGEM 2018’s Predator System, we used genetically-encoded, nanobody-fused Fc domain to recruit Trim21 and thus degrade the target to avoid microinjection or electroporation. In iGEM 2019, we achieved glucose-sensing protein degradation with PREDATOR system. Although these approaches provided a controllable protein degradation method, it was still worth noticing that the transcriptionally-controlled feature has given them obvious defects. Therefore, a robust control of the target protein degradation cannot be achieved, which leads us to a question: could we manipulate protein degradation in a more direct, accurate and controllable way?

Project Goals

This year, we oriented our efforts towards a highly modularized, genetically-encoded target protein degradation directly controlled by exogenous signals to manipulate untagged protein abundance.

Proposed Implementation

To sum up, PREDATOR Pro is tunable, reversible and highly modularized protein degradation tool with promising applications.

A: First of all, PREDATOR Pro would be helpful for Symbiology researchers to manipulate untagged endogenous protein abundance in a freely selectable temporal and dose-dependent combinations.

B: For scientists working on gene function researches, PREDATOR Pro can also provide solution on developing cellular or animal model to knockdown the expression of specific gene under given stimulus, especially useful for genes that are lethal. With light controlled or thermal controlled PREDATOR Pro system, highly precise tissue specific knockdown can be achieved.

C: It's possible that our project might be used for therapeutic purposes. In this case, PREDATOR Pro can be used to control the target protein abundance within physical level, thus maintaining the original function of such protein. The delivery and expression of PREDATOR Pro system would raise safety concerns especially regarding the specificity of the delivery. Also, huge amount of optimization and trial experiments should have been conducted before the application is pushed this far.

Design

Upon investigation on literatures, we had a deeper understanding of the mechanism of Trim21. It is a multi-domain protein consisting of an N-terminal RING domain with E3 ligase activity, a B-box domain, a coiled-coil dimerization domain and C-terminal PRYSPRY domain. Its PRYSPRY domain has a high affinity with constant Fc domain of the antibody.

Once the antibody-antigen complex enters the cytosol, Trim21 binds the Fc domain of the antibodies and form a Trim21-antibody-antigen trimer. Thereafter, the RING domain dimerizes, and ubiquitin proteasome system is recruited to mediate proteasomal degradation of the antigen. Since it has demonstrated in previous research and our PREDATOR system that the complete structure of antibody is not necessarily needed for Trim21, we hypothesized that the PRYSPRY domain of the Trim21 can also be replaced with other protein domains that can dimerize with other proteins under given signal input.

Phase I: Preliminary trial--Replaceability of the Trim21-antibody interface

Replaceability verification

To test the replaceability of the interface, we replaced the natural PRYSPRY-Fc pair in the GFP targeting PREDATOR system (Fig A, left panel) with DocS-Coh2, a previously reported constitutive dimerization pair (Fig A, right panel). The new GFP-targeting system was termed as GFP Predator Pro (GFP PrePro).

To validate the function of GFP PrePro, HEK-293T cells were co-transfected with GFP expressing reporter plasmids and GFP PrePro expressing plasmids as the experimental group (GFP PrePro group). Florescent imaging showed that comparing to the control group, GFP fluorescence in GFP PrePro group was ~60% lower (Figure B). Western blotting analysis also showed ~60% lower GFP abundance in GFP PrePro group (Figure C).. These results suggested that the original PRYSPRY/IgG-Fc interface in the PREDATOR system could be replaced with other protein dimerization pairs while maintaining the protein targeting and degradation activity.

Phase I: Preliminary trial--Replaceability of the Trim21-antibody interface

New reporter system

We used a dual luciferase reporter (pEFR), in which firefly luciferase (Fluc) was fused with GFP, to obtain more accurate GFP abundance changes (Figure A). Fluc activity was used as an indicator of GFP abundance, and the activity of a constitutively expressed Renilla luciferase (Rluc) reporter was used to normalize the noise caused by irrelevant factors such as transfection efficiency, cellular protein synthesis and cell growth. Results also showed significant lower (~50% lower) Fluc/Rluc ratio in GFP PrePro group comparing to the Ctr group (Figure B), indicating that GFP PrePro has impressive efficiency in degrading the target protein.

Phase II: Towards signal responsiveness

Increase of Rapamycin Concentration

We observed fluorescence image detecting GFP and GFP-coupled firefly luciferase showing the degradation of GFP in RiPrePro-1 transfected groups with different concentrations of Rapamycin. As expected, the degradation effect could be significantly improved with the increasing concentration of rapamycin. However, it has been well established that exorbitant amount of rapamycin would significantly reduce the cellular protein synthesis. Therefore, we come to another solution to multiplying FKBP copies for a higher intensity.

Phase II: Towards signal responsiveness

Two FKBP domains

To further push the PREDATOR Pro system towards direct responsiveness on exogenous signals, we then constructed Rapamycin Induced PREDATOR Pro (RiPrePro) system by replacing the DocS-Coh2 dimerization pair in the GFP PrePro into well-characterized, rapamycin inducible FRB-FKBP dimerization pair (RiPrePro1.0 or RiPrePro-1, Fig A). RiPrePro system with two FKBP copies fused with GFP nanobody was also constructed (RiPrePro2.0 or RiPrePro-2, Fig 3A) to improve the system performance. Also, dual luciferase reporter pEFR was used to normalize the difference on protein synthesis caused by rapamycin.

Similarly, for RiPrePro1.0/2.0 group, HEK-293T cells were co-transfected with pEFR and plasmid expressing RiPrePro1.0/2.0. In Ctr group, HEK-293T cells were co-transfected with pEFR and empty vector. Dual luciferase assay showed that both RiPrePro1.0 and RiPrePro2.0 groups showed significantly lower Fluc/Rluc ratio comparing to the Ctr group at 24, 48 and 72 hours post 2 ng/μL rapamycin induction. The normalized GFP abondance in RiPrePro2.0 group was significantly lower than the RiPrePro1.0 group in most time points (Fig B), indicating an improved degradation efficiency under increased FKBP copies. Further analysis on RiPrePro2.0 further revealed that the GFP degradation activity of RiPrePro2.0 was dose dependent to the rapamycin concentration (Fig C). Similarly, western blotting analysis showed ~40% lower GFP protein level in RiPrePro2.0 group comparing to the Ctr group (Fig D), which is in alignment with the dual luciferase assay results. Moreover, 30%-60% reduction on Fluc/Rluc ratio in the RiPrePro2.0 group comparing to the Ctr group could be observed in different host cell lines we tested 48 h post 2 ng/μL rapamycin induction, suggesting good robustness of such RiPrePro system (Fig E). In general, these results showed that our PREDATOR Pro system can be successfully engineered to degrade the target protein under the control of exogenous signals with decent performance and satisfying robustness.

The time-dependent characteristic of GFP PrePro system in silico and in vitro

In order to determine the experimental time of degradation of GFP PrePro and GFP, we simulated the function according to the experimental data, and drew the corresponding function curve. According to the above figure, we can see that the degradation rate of GFP is basically stable within 48 hours. In other words, the reaction between p18 and GFP was basically completed. Therefore, we think that the experimental group can take 48 hours as the node of experimental data collection, so as to obtain stable data and follow-up experimental observation results.

Interview to Prof. Xia & CCiC meetup

After the preliminary trial experiment of replacing PRYSPRY/Fc domain with DocS-Coh2 protein pairs, we consulted Professor Zanxian Xia and presented our data in CCiC meetup. We were suggested to change the GFP reporter into a Dual Luciferase Reporter system to normalize irrelevant factors affecting GFP abundance.

With this new reporter, the abovementioned experiments were repeated and the results clearly showed that GFP PrePro could degrade the target protein significantly.

Using our model to improve the degradation effect

Increase of Rapamycin concentration

To improve the degradation effect of our RiPrePro system, we approached our modeling group to figure out the most important factors affecting the PREDATOR Pro system. By performing Sensitivity Analysis (Figure A) among all parameters in our model, we noticed that the degradation efficiency was highly sensitive to the parameter reflecting the interaction strength of the protein dimerization pair(Figure B and C). In the case of RiPrePro system, such result implied that the degradation effect can be improved by increasing the concentration of rapamycin or increasing the binding strength of the interface part.

With the increment of rapamycin concentration, the degradation efficiency of RiPrePro did increase impressively. However, it has been well established that exorbitant amount of rapamycin would significantly reduce the cellular protein synthesis. Therefore, we come to another solution to multiply FKBP copies for higher binding strength.

Literature research to determine protein dimerization pairs regulated by exogenous signals

As a set of widely used and fully characterized heterodimerizing components, the dimerization of FK506 binding protein (FKBP) domain and the T2089L mutant of FKBP-rapamycin binding domain (FRB) could be initiated by external rapamycin signals. Therefore, we substituted the interface with FRB-FKBP protein interaction pairs.

Using our model to improve the degradation effect

Two FKBP domains

To promote the degradation efficiency, we turned to the model group for help. After performing Sensitivity Analysis of our system, they discovered that a parameter regarding the protein interaction intensity of FKBP and FRB was pivotal to the degradation efficiency. Therefore, we designed a Rapamycin-inducible PREDATOR Pro2.0 plasmid (RiPrePro2.0) composing GFPnano-FKBP*2 and HA-Trim21-FRB. It was observed that under 2 ng/μL rapamycin induction, the fluorescence intensity of HEK-293T cells co-transfected with pEGFP and RiPrePro2.0 plasmid was significantly lower than the control group transfected with pEGFP and empty vector.

Integrated HP

As a foundational advance project, our work in iGEM 2020 attempted to provide a novel toolbox to control the homeostasis of specific target proteins in mammalian cells. Hence, our lab work was mainly directed to serve synthetic biologists with new toys for synthetic circuit design, as well as scientists in other fields with new approach to develop cell models, etc. With these proposed applications in mind, our human practice work mainly focused on gathering ideas and suggestions from scientists and experts and collecting information on how our project could be shaped to meet their demands. Their supportive feedbacks helped us to reshape the project design and inspired us on the possible future application of our project as well.

Here we demonstrate how our integrated human practice interacts with our project design and the wet-lab experiments.

Model

To understand the performance of our design comprehensively and offer guidance on wet-lab work, a mathematical model comprised of two modules was constructed based on fundamental biochemical principles. To be specific, a protein interaction module describing the formation of GFP PrePro or RiPrePro-mediated ternary degradation complex, and a protein degradation module simulating the ubiquitination and degradation of target protein triggered by the Predator Pro.

With the wet-lab data, we adjusted the parameter of the GFP PrePro model. We challenged the model with different input plasmid levels. Simulation showed that the model predictions matched well with the corresponding wet-lab data.

In order to find the optimal ratio and dosage of plasmids, we calculated the second derivative of the degradation efficiency with respect to the ratio. As is shown in figure C, a relatively stationary phase could be reached when the plasmid ratio was greater than 1. Therefore, we chose 1:1 as the optimal plasmid ratio to avoid dosage waste. Also, given that when introducing zere point two five microgram GFP plasmid, the degradation rate was relatively steady and considerable, we believed that zere point two five microgram dosage for both plasmids should be prioritized.

In order to determine which parameter have the most direct and dramatic influence on GFP degradation process, we used the built-in sensitivity analysis program to calculate the time-dependent sensitivities (derivatives) of GFP with respect to each parameter.

By checking the magnitude of the computed sensitivities integrated over time, we could observe that k3f, the rate parameter characterizing the dimerization of FRB and FKBP, manifested the highest sensitivity across the board, which suggested that GFP protein level was most sensitive to k3f (Figure A). In order to further verify the effects of variations in k3f (k) on GFP abundance, the value of k was scanned within a range. Simulation showed that when k was set to zero, the GFP abundance resulted in a significant high level, which corresponded to the situation that the rapamycin was absent. With the growth of k value, GFP level stabled at an obviously lower value, suggesting an ever-increasing degradation efficiency. Such results indicated that we could optimize the RiPrePro by increasing the value of k, or in other words, increasing the dosage of rapamycin or the number of FKBP domains. Considering the toxicity of rapamycin to cells, the future experiment focused on adding up the number of FKBP domains.

Interview to Prof. Xie

We approached Dr. Mingqi Xie after we obtained most of our data. Considering his record as a synthetic biologist, we were hoping that he could provide us some hint on how our project could be implemented in the real world. Through the interview, we first showed Dr.Xie the design of Predator Pro system, as well as the data we’ve already obtained. After knowing about our project, Dr. Xie praised our project for sufficient experiments and adequate data. Although some points still need to be improved, he thought that our project was logical and rigorous, and he had expectation that our work would be a useful tool for Synthetic Biology and Medicine. More importantly, he provided us some more suggestions on how to further improve our system. We were suggested to start looking for some therapeutic targets for our predator system. We were also suggested that further researches should be conducted on whether our system could be used to establish new disease cell or animal models..

Future Work

On the one hand, we plan to apply other well-characterized, signal-sensitive protein dimerization systems, for instance, blue light-inducible CIBN-CRY2 interaction. A blue-light-inducible PREDATOR Pro plasmid has been constructed and relevant experiments are within our plan.

On the other hand, we are seeking a way out for the application of PREDATOR Pro. Through literature investigation, HP group has discovered a protein-TAR DNA-binding protein-43(TDP-43) that could be a candidate for the target of Predator Pro. As a DNA/RNA binding protein that is extensively localized in the cell nucleus, TDP-43 has various important functions in RNA metabolism. However, a pathological behavior of this protein, in which an abnormal self-aggregation and mis-localization in the cytoplasm occur, has been proved to be a hallmark of amyotrophic lateral sclerosis (ALS), a serious neurogenerative disease with a symptom of muscle atrophy, complete paralysis and even death7. Seeing this, we expect to harness PrePro to control the aggregation of TDP-43 in the cell for ALS patients. The construction of PrePro targeted for TDP-43 is in our future work and it is firmly believed to be a promising solution to the treatment of ALS and other neurodegenerative diseases.

Partnership

We focused on various ways for collaborations to occur, both online and offline. Faced with the dilemma of experimental progress stagnation and HP communication obstruction, we reached out to the Team CSU_CHINA to find a team that was able to help us out of the dilemma and at the same times achieve mutual progress. Through the online meet of exchange, we have established a long-term cooperation mechanism with Central South University. Teachers, leaders and members of both teams have set up an online communication group, and we also held the NUDT_CHINA×CSU_CHINA offline meet of exchange on October 8th to deepen the communication between our teams. The two teams carried out all-round cooperation on respective projects this year.

After several times online discussions, on 8 October 2020, we held the NUDT_CHINA×CSU_CHINA offline meet of exchange to deepen the communication between our teams. To our relief, both teams agreed on further and more concrete collaboration.

In experiment exchange, we provided CSU_CHINA with our fluorescent images, after qualifying, they transformed them into heatmap using R language for better visualization (Fig A). As CSU_CHINA has limited access to the cadmium ion detection kits, we lent atomic absorption spectrometer and helped them measure the cadmium-uptaking levels of Synechocystis while in different growth states(Fig B).

Furthermore, we designed our joint brochure for educational propaganda of synthetic biology.

Science Communication

We raised public awareness program to spread the basic knowledge and mindsets of synthetic biology and iGEM, targeting our college and Yali High School. The purpose of our activities is to deepen the public's understanding of synthetic biology, so that the project can be applied better in the future.

In addition, we prepared some brochures about synthetic biology and our projects for students in lower age.

After we finished our project promotion video, we prepared a Chinese version and uploaded it online allowing others to know our team and our project.

Parts

This year, we handed in 16 high-quality, well documented bio-brick parts, including all those we used in our project this year, and several brilliant designed others inspired by our iGEM projects within these two years.

Favorite basic parts

Our favorite Basic parts are Truncated trim 21 and CMV-Replaceable-1-Fluc-P2A-Rluc. Truncated trim 21 is the core of the newly registered Predator Pro system. This truncate protein maintained the E3 ubiquitin ligase activity, and provided an open interface for other protein dimerization pairs to be added.

Another part is CMV-Replaceable-1-Fluc-P2A-Rluc, which is a reporter system to quantify the abundance of specific target protein.

Favorite composite part

Our favorite composite part is Replaceable-1-Replaceable-2-P2A-TRIM21-Replaceable-3. It is a plasmid platform on which different targeting proteins and protein dimerization pairs can be easily installed to achieve the controllable degradation of specific target protein.

Part Collection

Our part collection “Predator” consists of 44 parts of our Predator Pro system and original Predator system we demonstrated in iGEM 2018-19. Using these parts, we can construct a Predator Pro system that can target any endogenous target protein regulated by exogenous signals.

Reference & Acknowledgement

1  Xie, M., Haellman, V. & Fussenegger, M. Synthetic biology—application-oriented cell engineering-2016-Current Opinion in Biotechnology. Current Opinion in Biotechnology 40, 139-148, doi:10.1016/j.copbio.2016.04.005 (2016).

2  Foss, S. et al. TRIM21-From Intracellular Immunity to Therapy. Front Immunol 10, 2049, doi:10.3389/fimmu.2019.02049 (2019).

3  Zeng, J., Santos, A., Mukadam, A. & Osswald, M. Substrate-induced clustering activates Trim-Away of pathogens and proteins. bioRxiv 225359 ,doi: 10.1101/2020.07.28.225359 (2020).

4  Barak, Y. et al. Matching fusion protein systems for affinity analysis of two interacting families of proteins: the cohesin-dockerin interaction. J Mol Recognit 18, 491-501, doi:10.1002/jmr.749 (2005).

5  Gaj, T. et al. ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. Trends Biotechnol 31, 397-405, doi:10.1016/j.tibtech.2013.04.004 (2013).

6  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).

7  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).

8  Wang, H. et al. 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).

9  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).

10  Kleiger, G. et al. Perilous journey: a tour of the ubiquitin-proteasome system. Trends Cell Bio 124, 352-359, doi:10.1016/j.tcb.2013.12.003 (2014).

11  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).
Acknowledgement

Dr. Zanxian Xia

Dr. Mingqi Xie