Team:NUDT CHINA/Proof Of Concept
Proof Of Concept
The ultimate goal of our project was to control cell cycle of mammalian cells using light signal based on the targeted protein degradation system PREDATOR Pro we developed previously. As our proposed applications were mainly related to lab uses and therapeutic applications, the control of target cell proliferation would be the major consequence of cell cycle control under these scenarios. Therefore, as a proof of concept, we hereby demonstrate how our system could be used to control the growth of HEK-293 cells.
Light-mediated control of mammalian cell cycle
In our project, we made two major modification on the PREDATOR system. On the one hand, the core module that governs the signal responsiveness was changed into light inducible protein dimerization/disassociation pairs, on the other hand, the targeting module that controls the specificity was changed to specifically target cyclin with high affinity. During our lab work, we independently optimized the light-mediated control interface and the targeting module (See details in the Designand Resultspage).
With the optimal protein-protein pairs, optimization of linkers and Cyclin-targeting module, we then build a blue light inducible Predator system to mediate the regulation and intervention of the cell cycle as a proof of our concept. Here we mainly show the data directly related to ultimate constructs under optimized parameter and plasmid design. Other supporting data and how our modeling results and HP feedbacks affect the development of our project are located in the Result page.(Click the link here to see our Results page)
Through rounds of iteration, Cry2/CIB1was chosen as the optoswitch. protein pair that dimerize under blue light in our next step. Then, we have made many attempts to further optimize the light inducible PREDATOR (LiPrePro) system, such as changing the length of linker between Cry2 and GFPnano and the linker between CIB1 and truncated Trim21 from 1x GS linker into 3x or even 5x GS linkers. With reported sequence and preliminary trials, we designed multiple targeting modules. The αCHelix region of CDK4 and Rb were proved to be an optimal targeting module.
Integrating with a raspberry pie-based blue light illumination device, light-controlled dimerization of protein pairs may occur and subsequently ternary degradation complex formed, leading to the degradation of Cyclin. Herein, two CycleBlue systems namely CycleBlue-1 and CycleBlue-2 were build, using CDK4 αCHelix or Rb αCHelix as targeting module, respectively. In detail, Cry2/CIB1was chosen as the core module of such system: Cry2 was fused with the truncated Trim21 protein with a 5xGS linker, while CIB1 was linked to tandem repeats of either CDK4 αCHelix or Rb αCHelix with a a 5xGS linker (Fig.1A).
Figure 1. CycleBlue: Blue-light controllable cell growth. (A)Schematic representation showing the design of CycleBlue system. (B) Representative Western blotting determining the Cyclin abundance in HEK-293T cells 48 h post transfected with CycleBlue-1 plasmid and CycleBlue-2 plasmid under blue light illumination condition. (C) CCK8 cell proliferation assay on CycleBlue-1/ CycleBlue-2 transfected HEK-293T cells under dark or blue light illuminated conditions. For (B-C, E-F), cells were illuminated with 3 mW/cm2 of 405nm blue light, the illumination was programed as the repeat of [2 s ON /58 s OFF] cycle for indicated hours. Data represent mean ± s.e.m. (n=3 biological replicates); ***P ＜ 0.001; two-tailed unpaired Student’s t-test.
To characterize our CycleBlue system, HEK-293 cells were transfected with either CycleBlue-1 or CycleBlue-2 expressing plasmids and illuminated with blue light 24h post transfection. Western Blotting and CCK8 assay were performed to analyze Cyclin levels and cell proliferation, respectively. Intriguingly, under blue light illumination, western blotting showed significantly reduced cyclin D1 level in cells transfected with CycleBlue-1 comparing to the control group, while the reduction on cyclin D1 level in cells transfected with CycleBlue-2 was not observed (Fig. 1B). Interestingly, CCK8 assay(Fig. 1C) showed significant reduction of cell proliferation under blue light illumination in both CycleBlue-1 and CycleBlue-2 transfected cells. (Fig.1C, right panel, p ＜ 0.001), while the effect of CycleBlue-1 and CycleBlue-2 transfection in dark condition was insignificant (Fig.1C, left panel, p ＜ 0.001).
Furthermore, the results of model group fits well with the experimental results under either light or dark conditions. As is shown in figure 2, simulation showed similar trend as the wet lab results, in which cell growth would be significantly inhibited in blue light illuminated, CycleBlue transfected cells, while the effect of CycleBlue under dark condition should be insignificant.(Click here to know more about model)
Figure 2. Comparison diagram of experimental data and model results.
In general, these results showed that our CycleBlue system can successfully degrade Cyclin D1 and regulate cell proliferation under the control of blue light with decent performance. This proof of concept also showed the potential of CycleBlue system to be further developed into a novel synthetic biology toolbox that can be used in our purposed implementation.