Team:NUDT CHINA/Design
DESIGN
Overview of our project design
In our project we intend to achieve the light-mediated control of cell cycle in mammalian cells by degrading cyclins in a light dependent manner.
To achieve this, we made some major modification on the PREDATOR system, a programmable protein degradation tool we’ve been working on for years, as shown in Figure 1: a) The core module that governs the signal responsiveness was changed into light inducible protein dimerization/disassociation pairs; b) The targeting module that controls the specificity was changed into cyclin-specific binding proteins.
Figure 1. Schematic representation of Predator Pro and Blue Light Predator.
With such modularized mindset, we were able to design, optimize and experimentally validate these two major changes in parallel.
Achieving Blue Light-Inducible Protein Degradation
2.1 Testing Protein Dimerizatioin Pairs Affinity
To achieve blue-light stimulation of the cells, we built a illumination device with blue light LEDs and a Raspberry Pi controller. With a simple python script we can precisely control the illumination time and strength in the incubator. By taking researches in the blue light responsive proteins, we have found few protein dimerization pairs. To achieve light responsiveness, we in tend to replaced the original core module of PREDATOR system into either Cry2/CIB1, Lov2/Zdk or PixD/PixE. CRY2 is a blue light-absorbing photosensor binding CIB1 in its photoexcited state 1, thereby dragging the catalytic domain of Trim21 to the target protein, while Lov2/Zdk 2and PixD/PixE 3,work in an opposite manner, in which blue light illumination disassociates protein dimers or oligomers into monomers, thereby stopping the targeted protein degradation.
To figure out the best frequency and intensity of the blue light and testify how these protein dimerization/disassociation pairs work with our own device, we constructed a tetR based transcriptional activation system and reporter gene SEAP. By fusing one member of the pairs to tetR and the other one with VP64, their dimerization/disassociation in blue light can be reported by the expression of SEAP reporter down stream of tetO.
The result showed that under 3mW/cm2 power, dark/blue 2/58s cycle of illumination, Cry2/CIB1 works optimally in initiating SEAP transcription.
Figure 2. Schematic representation of the tetR system.
2.2 Construction and Optimization of Light-inducible PREDATOR Pro (LiPrePro) system
With the Cry2/CIB1 chosen as our photoswitch, we then started to construct light inducible PREDATOR system. To simplify the testing and speed things up, we used GFP as the target protein to prove the light responsiveness of the light inducible PREDATOR system. As we’ve shown the Cry2/CIB1 was the optimal pair in our hardware setup, we then focused on testing the Cry2/CIB1 based PREDATOR system. Upon GFP expression, we were able the quantify the effect of light inducible protein degradation by fluorescent imaging. Unfortunately, preliminary results showed that these constructs were not capable of degrading GFP in HEK-293 cells under blue light illumination(See Results page for more information).
To understand why our preliminary design fails, we then consulted Mirta Viviani, a phD student focused on mammalian cell synthetic biology in Westlake University. Upon showing great interests in our project, she pointed out that the length of linker between Cry2 and GFPnano, or the linker between CIB1 and Trim21 could be important for the proteins to fold correctly. Building on her suggestions, we changed these linkers from 1×GS linker into 3× or even 5×GS linkers. Results showed significant improve of GFP degradation in HEK-293 cells transfected with these new constructs.
Figure 3. Schematic representation of the Blue-Light on switch1 and off switch2,3.
Targeting Cyclin and Control Cell Proliferation
3.1 ScFv based Cyclin E targeting
On the other hand, we tried multiple approaches to achieve cyclin targeting. As intracellular antibodies and ScFvs are the most obvious approaches for specific recognition of target protein, we first focused on finding the published ScFvs targeting specific cyclin protein. To simplify the experiments, we kept the original DocS/Coh2 core module of the PREDATOR system. With this constitutively binding core module, we expect to see significant reduction of cell proliferation in Cyclin PREDATOR transfected cells. The ScFv of Cyclin E was published by Strube et al. in 2002. Authors reported two potential ScFvs specifically targeting Cyclin E and provided their DNA sequences. While specific binding were demonstrated, their paper also showed that the full-length of ScFv might already be sufficient to trigger the dysfunction of cyclins. Therefore, inspired by the precision immunotherapy of a split T cell-engaging antibody4,we designed another set of constructs that split the ScFv into VH and VK fragments, and fused these fragments on either N terminus of C terminus of Predator proteins. In this case, the dimerization of the core module domains would also trigger the reconstitution of ScFv, therefore reducing the potential effect of full-length ScFv on Cyclins.
Figure 4. Schematic representation of the ScFv and split ScFv strategy.
However, both Western Blotting and cell proliferation assay showed unsatisfying degradation of Cyclin E by these constructs. CCK8 assay was also performed to evaluate the effect of these constructs on HEK-293 cell proliferation, which showed only modest but unsatisfying reduction on cell cycle(See Results page for more information).
3.2 αHelix of CDK4/Rb based Cyclin E targeting
In the meantime, during the discussion with Prof. Chun Meng, who specializes on ScFv design, he suggested us to check for other binding candidates as the strongest ScFv were often hidden in patents instead of scientific publications. With his suggestions, we found some protein binding domains from the natural binding partners of cyclins that could be feasible for our targeting module. We noticed that CDK4 and Rb have been widely reported as the endogenous binding partner of Cycliln D1. Research shows the Cdk4 and Rb can bind to cyclinD via a C-terminal helix and that this interaction is a major driver of cell proliferation5,6.
Hence, we then built a set of PREDATOR constructs using a tendon repeat of CDK4 αChelix or Rb αChelix as targeting module. Western Blotting showed significantly reduced Cyclin D1 level in HEK-293 cells transfected with these constructs. Consistently, cell proliferation assay showed significantly reduced cell proliferation upon transfection of these constructs(See Results page for more information).
Figure 5. CDK4/Rb αCHelix mediated degradation of cyclinD1.
Light-mediated control of mammalian cell cycle
With the optimal optoswitch, optimal liner, and optimal targeting module, we then designed CycleBlue to achieve light-mediated control of cell cycle in mammalian cells. The CycleBlue construct consists of a truncated Trim21 domain as the functional module, linked with Cry2 with 3×GS linkers, and either CDK4 or Rb αChelix as the targeting module, linked with CIB1 with 3×GS linkers.
Western Blotting showed significant reduction of Cyclin D1 level in blue light illuminated, CycleBlue transfected cells comparing to control cells exposed to blue light. Significant reduction of cell proliferation was also observed in CycleBlue transfected cells comparing to control cells under light illuminated conditions, while such difference was not observed in cells kept under dark condition. These results showed a proof of concept that we were capable of regulating mammalian cell cycle by blue light signals(See Results page for more information).
Figure 6. Schematic representation of CycleBlue construct.
- Kennedy, M. J. et al.(2010). Rapid blue-light–mediated induction of protein interactions in living cells. Nat. Methods, 7, 973–975.
- Wang, H. et al.(2016) LOVTRAP: an optogenetic system for photoinduced protein dissociation. Nat. Methods， 13, 755–758.
- Dine, E., Gil, A. A., Uribe, G., Brangwynne, C. P. & Toettcher, J. E. (2018) Protein Phase Separation Provides Long-Term Memory of Transient Spatial Stimuli. Cell Syst. 6, 655-663.e5
- Banaszek, A. , Bumm, T. , Nowotny, B. , Geis, M. , & Stuhler, G. . (2019). On-target restoration of a split t cell-engaging antibody for precision immunotherapy. Nat. Commun., 10(1), 5387.
- Céline Bouclier, Simon, M. , Laconde, G. , Pellerano, M. , & Morris, M. C. . (2020). Stapled peptide targeting the cdk4/cyclin d interface combined with abemaciclib inhibits kras mutant lung cancer growth. Theranostics, 10(5).
- Topacio, B. R. , Zatulovskiy, E. , Cristea, S. , Xie, S. , Tambo, C. S. , & Rubin, S. M. , et al. (2019). Cyclin d-cdk4,6 drives cell-cycle progression via the retinoblastoma protein's c-terminal helix. Molecular Cell.