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

● Spatial and temporal manipulation of protein abundance is the basis of designing and constructing complicated synthetic regulatory networks (Figure 1)

Figure 1. The mindset of mammalian cell synthetic biology

● Wide variety of synthetic biological tools have been developed to precisely control the protein synthesis on genomic, transcriptional, and translational level.

● Limited tools have been demonstrated to control protein degradation in synthetic biology (Figure 2).

Figure 2. Schematic representation of TPD methods in SynBio (A)Degron (B)PROTACs (C)Trim-Away

● Genetically encoded tool for signal-responsive degradation of untagged proteins is still needed.

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: PREDATOR Pro would be helpful for SynBio 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 in a tissue specific manner under given stimulus.

C: It's possible that our project might be used for therapeutic purposes. PREDATOR Pro can be used to control the target protein abundance within physical level, thus maintaining the original function of such protein.

Design

Figure 3. the design of our PREDATOR Pro system.

Based on knowledge regarding the structure of Trim21 and the working model of Trim21-mediated degradation, we 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 (see Figure 3).

Our Hypothesis:

The interface of the Predator system can be replaced with other well-characterized protein-protein interactions (Replaceability) .

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

Replaceability verification

Figure 4. Characterization of GFP PREDATOR Pro system based on the DocS-Coh2 interaction.

● The constitutive dimerization of DocS and Coh2 would function as the interface to bring the truncated Trim21 into proximity with GFP protein and trigger the degradation of GFP (Fig 4A).

● ~55% decrease of green fluorescence was observed in pEGFP plasmid and GFP PrePro plasmid co-transfected groups 48 hours after transfection (Fig 4B).

● Western blotting analysis showed that GFP was significantly degraded to ~40% of the control group (Fig 4C), which confirmed that PREDATOR Pro could be used to degrade target protein with high efficiency.

● Significant lower GFP fluorescence could be observed in GFP PrePro expressing group in every time point from 12 hours to 144 hours post transfection. Of note, such trend was also well aligned to the prediction of our mathematical model (Fig 4D and 4E).

Conclusion:

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-Fc interface

New reporter system

Figure 5. Dual luciferase assay showing the GFP degradation mediated by GFP PrePro system.

● A dual luciferase reporter (pEFR) was used to obtain more accurate GFP abundance changes (Figure 5A), in which firefly luciferase (Fluc) was fused with GFP.

● Results showed significant lower (~50% lower) Fluc/Rluc ratio in GFP PrePro group comparing to the Ctr group (Figure 5B).

Conclusion:

GFP PrePro has impressive efficiency in degrading the target protein

Phase II: Towards signal responsiveness

RiPrePro1.0 with FRB-FKBP dimerization pair

Figure 6. Rapamycin-induced PREDATOR Pro system based on the FRB-FKBP interaction module.

● Version 1.0 of Rapamycin-induced PREDATOR Pro system (RiPrePro1.0 or RiPrePro-1) was constructed by replacing DocS-Coh2 with FRB-FKBP.

● Slight decrease (~20%) of GFP fluorescence in RiPrePro1.0 expressing group under 1 ng/μL rapamycin induction.

How to improve?

● With the input provided by the mathematical model, the parameter reflecting the interaction strength of the protein dimerization pair:

> increasing the concentration of rapamycin

> increasing the binding strength of the interface part of the protein.

Increase Rapamycin concentration:

Figure 7. Fluorescence Heatmap in cells treated with increasing concentrations of Rapamycin.

● The degradation effect could be significantly improved with the increasing concentration of rapamycin.

● Increasing rapamycin would significantly reduce the cellular protein synthesis.

Phase II: Towards signal responsiveness

Two FKBP domains

With the implications obtained from the model, we changed the GFPnano-FKBP unit into GFPnano-FKBP-FKBP(Fig 8A).

Figure 8. Rapamycin-induced PREDATOR Pro system based on the FRB-FKBP-FKBP interaction module.

● Dual luciferase assay showed that the normalized GFP abundance in RiPrePro2.0 group was significantly lower than the RiPrePro1.0 group in most time points (Fig 8B), 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 8C).

● Western blotting analysis(Fig 8D) is in alignment with the dual luciferase assay results.

● Reduction on Fluc/Rluc ratio in the RiPrePro2.0 group could be observed in different host cell lines , suggesting good robustness of such RiPrePro system (Fig 8E).

Conclusion:

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

Figure 9. Schematic representation showing the experimental workflow (left panel) and quantified GFP fluorescence heatmap 48 h post transfection of corresponding amount of plasmid (right panel).

● To find the optimal GFP PrePro transfection dosage, fluorescent intensity was transformed into heatmap for better visualization. Through the difference of color shade, a GFP PrePro dose dependent GFP degradation could be clearly observed.

● Furthermore, we provided the data to Model hoping that they could obtain the optimal concentration ratio of GFP and GFP PrePro plasmids. With their advice, the plasmid ratio was set as 1:1 in further experiment.

Interview Prof. Xia & CCiC meetup

Figure 10. Interview Prof.Xia.

● Interview Professor Zanxian Xia with our preliminary trial experiment data, We were suggested to:

> Change the GFP reporter into a Dual Luciferase Reporter system to normalize irrelevant factors affecting GFP abundance.

Figure 11. CCiC meet-up.

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

Figure 12. the sensitivity analysis of parameters in the model.

● We approached our modele group to figure out the most important factors affecting the PREDATOR Pro system. By performing Sensitivity Analysis (Figure 12A), we noticed 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.

Using our model to improve the degradation effect

Two FKBP domains

Figure 13. Representative fluorescence images of HEK-293T cells carrying pEFR and RiPrePro-2 plasmids in comparison to the Ctr group 48 h post 2 ng/μL rapamycin induction.

● The model group discovered that a parameter regarding the protein interaction intensity of FKBP and FRB was pivotal to the degradation efficiency after performing Sensitivity Analysis. Therefore, we designed RiPrePro2.0 composing GFPnano-FKBP*2 and HA-Trim21-FRB.

● 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.

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.

Aim:

> Serve synthetic biologists with new toys for synthetic circuit design

> Serve scientists in other fields with new approach to develop cell models, etc.

Our human practice work mainly focused on:

> Gathering ideas and suggestions to meet their demands.

> Supportive feedbacks helped us to reshape the project design and inspired us on the possible future application of our project.

Model

Figure 14. Framework of the mathematical model

To understand the performance of our design comprehensively and offer guidance on wet-lab work, a mathematical model composed of two modules (protein interaction module and a protein degradation module) was constructed based on fundamental biochemical principles.

Figure 15. the side-by-side comparison of model prediction and experimental data

● The simulation showed that the model predictions matched well with the corresponding wet-lab data.

Figure 16. the influence of plasmid dosage and ratio on GFP degradation rate

● Model results showed that 1:1 is the optimal plasmid ratio and 0.25 mg dosage for both plasmids should be prioritized.

Figure 17. the sensitivity analysis of parameters in the model

● The Sensitivity Analysis of all parameters in the model calculate the time-dependent sensitivities (derivatives) of GFP with respect to each parameter.

k3f, the rate parameter characterizing the dimerization of FRB and FKBP, manifested the highest sensitivity across the board(Figure 17).

Results indicated that we could optimize the RiPrePro by increasing the value of k3f, 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 Prof. Xie

Figure 18. Interview Prof.Xie

● We approached Dr. Mingqi Xie after we obtained most of our data. Through the interview, we first showed Dr.Xie the design of Predator Pro system, as well as the data we've already obtained.

● Suggestion:

Looking for some therapeutic targets.

Conducting further researches on whether our system could be used to establish new disease cell or animal models.

Future Work

Figure 19. Schematic representation of our future work

● New interface: Apply other signal-sensitive protein dimerization, Using CIBN-CRY2 interaction to achieve a blue-light-inducible PREDATOR Pro plasmid has been constructed and relevant experiments are within our plan.

● New target: TAR DNA-binding protein-43(TDP-43), a hallmark of amyotrophic lateral sclerosis (ALS), to provide a promising solution to the treatment of ALS and other neurodegenerative diseases.

Partnership

Long-term cooperation mechanism with CSU_CHINA

Figure 20. The online and offline meeting with CSU_CHINA

Figure 21. Project collaboration with CSU_CHINA

Figure 22. The joint brochure for education

● Held multiple online and offline meetings for project and idea sharing.

● CSU_CHINA helped us with the visualization of our massive amount of fluorescent images.

● We helped CSU_CHINA to measure the cadmium-uptaking levels of Synechocystis in different growth states.

● We designed our joint brochure for education purposes.

Science Communication

● Spread the basic knowledge and mindsets of synthetic biology and iGEM to our college and Yali High School to deepen the public's understanding of synthetic biology

Figure 23. Spreading the SynBio

● Prepare the brochure about synthetic biology and our projects for students in lower age

Figure 24. The joint brochure for education

● 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

Figure 25. The localized propaganda of our SynBio 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

Figure 26. The truncated Trim21

Our favorite Basic parts are Truncated Trim21 and CMV-Replaceable-1-Fluc-P2A-Rluc. Truncated Trim21 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.

Figure 27. a new reporter system

Another part is a reporter system to quantify the abundance of specific target protein.

Favorite composite part

Figure 28. a composite part for controllable protein degradation

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

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).

Attribution

Experiment: Tianyi Zhang, Hanxiao Feng, Junzi Gu, Yuxin Liu, Changtai Xiao, Yingqian Ye, Zhenyu Zhou, Yuxuan Wang, Huiying Liu, Weiqian Zhou.

Model: Jie Cai, Yanchen Gou, Yongjiang Li, Zhiliang Pan, Haoyu Zhang. Our PI Xinyuan Qiu and Instructor Chuanyang Liu helped us with modeling ideas and methods.

Human Practice: Lunhao Ju, Zuyu Dai, Qingyi Liu, Xinlin Liu, Yuxin Liu, Linjie Li, Yulan Chen

Art Design: Guangyi Lin, Ruoxi Wang, Tianyi Zhang.

Wiki Design: Qinrui Jiang, Guangyi Lin, Ruoxi Wang.

Support

General Support: The project was supported by the Department of Biology and Chemistry of College of liberal art and Science, National University of Defense Technology.

Project Support: PI Lingyun Zhu, Xinyuan Qiu; Instructor Chuanyang Liu, Lu Min, Lvyun Zhu, Xiaomin Wu, Jingyu Kuang, Wenying Li; Advisor Jiaxin Ma.

Fundraising help and advice: PI Lingyun Zhu, advisor Lu Min was in charge of expenses management.

Lab support: Department of Biology and Chemistry of College of Liberal arts and Sciences. Difficult technique support: Sisi Xie, Wenying Li, Xinyuan Qiu.

Human Practice & Wiki support: Our advisor Xinyuan Qiu, Chuanyang Liu provided us with solutions when we face difficulties in the process of wiki production.

Acknowledgement

Dr. Zanxian Xia

Dr. Mingqi Xie