Team:NUDT CHINA/Description
DESCRIPTION
Optogenetic tools provide essential approaches to control designer cell function. Our project intends to optimize Predator Pro, a modularized protein degradation system we’ve been working on in the past few years, to achieve light-mediated degradation of endogenous cyclin protein, the key regulator protein family in cell cycle, thereby manipulating cell cycle in mammalian cells. Integrating with a raspberry pie-based blue light illumination device, our system may provide novel tools for cell cycle synchronization and spatiotemporal control of cell division. Our work may provide novel tools for synthetic biology community and researchers in other fields, it will also provide new hints for treating diseases caused by cell abnormal division.
Cell cycle is the most basic biological process controlling the proliferation and environmental response of living cells. Loss of robustness in cell cycle control is related to multiple diseases including cancer, fibrosis, cardiovascular diseases, diabetes, and neurodegenerative diseases1,2. Therefore, tools for artificial control of cell cycle would provide us opportunity to study or manipulate cell behavior in different conditions. As suggested by medical professionals during our HP interview, developing novel cell-cycle regulation tools would not only provide new tool for fundamental research, but also shed light in the treatment of multiple diseases.
The molecular mechanisms underlying cell cycle regulation has been well-established in the past decades. Among the large family of proteins regulating cell cycle (Figure 1), cyclin/ CDKs (cyclin-dependent kinase) play essential roles as the key driver of cell cycle3. Cyclins(Cyc), as the core of the precise regulation of the cell cycle, are binding partners and activators of CDKs, which are required for substrate specification and localization of the CDK–cyclin complex2. The protein levels of cyclins and cdks are strictly regulated during cell cycle. Generally in mammalian cells, Cdk4 and Cdk6 together with D-type cyclins promote the transition from G0, a resting phase in which cells are in a quiescent state, into the cell cycle by initiating phosphorylation of the Rb family proteins Rb, p107, and p130 and thereby releasing the transcription of early E2F target genes (including CycE and CycA) from their repression by Rb proteins2. Subsequently, Cdk2 controls entry into and progression through S phase in complex with CycE and CycA by completing the phosphorylation of Rb proteins, leading to further activation of E2F target genes and culminating in the initiation of S phase4-6. Cdk1, in conjunction with CycA and CycB, then controls the entry and progression through M phase7,8. Exit from mitosis requires degradation of mitotic A- and B-type cyclins by the APC/C9.
Figure 1. Regulation of the cell cycle and cyclin-dependent kinase inhibitors in development.1
With the regulation of cyclin levels been broadly considered as the main approach of endogenous cell cycle control, interfering the synthesis or degradation of cyclins via synthetic biological approaches provides new opportunities to integrate exogenous signals into the control of cell cycle. Luckily, in our projects throughout iGEM 2018-2020, we have developed a novel tool, the PREDATOR system, to control the degradation of specific target proteins. The Predator system consists of a functional module, which functions as a E3 ubiquitin ligase; a targeting module, which binds target protein; and a core module, which is an artificially designed interface that regulates the interaction of functional module and targeting module in a signal-responsive way, which subsequently render the degradation process controllable (Figure 2a).
By changing the targeting module into cyclin-specific binding domains, e.g. Scfvs, nanobodies or binding domains from endogenous binding partners, we hypothesized that we could interfere the cell cycle by directly degrading cyclins (Figure 3). Our results also showed that with either cyclin D or cyclin E targeting modules, the modified PREDATOR system was capable of degrading cyclin D or cyclin E in HEK-293T cells, therefore reducing the proliferation of these cells (Figure 2b).
Figure 2. Schematic representation of previous system and the changes of target module. (A) Schematic representation of Predator Pro systems in 2020. (B) The characteristics of new target modules.
To make things more interesting, with the suggestions from experts working in mammalian cell synthetic biology, we then designed a blue light-controlled PREDATOR system to allow light inducible targeted protein degradation. To achieve this, we tested multiple blue-light inducible protein dimerization/dissociation pairs, including Cry2/CIB1, aslov2/zdk1 and PixD/PixE, in HEK-293T cells to test the fitness of these protein pairs to our home-made illumination hardware. The best-performing pair was then used to replace the original core module in the PREDATOR pro system (Figure 3). Using GFP as the target, we demonstrated that the modified PREDATOR system with Cry2/CIB1 based core module allowed a robust blue light induced degradation of GFP in HEK-293T cells.
Figure 3. Schematic representation of previous system and the changes of core module which can be controlled by blue light.
By combining these attempts, we then present “CycleBlue” (Figure 4), a light inducible controller of cyclin degradation, therefore achieving light-based control of cell cycle in mammalian cells. As both our wet lab and modelling results showed significant degradation of cyclin and reduction of cell proliferation under blue light illumination. We believe that our tool can possibly be applied for either research or medical purposes by controlling cell cycle synchronization and spatiotemporal control of cell division.
In the meantime, our HP members also established wide connection to academic professionals and iGEMers around China. In addition, our Hp group conducted a popular science lecture, delivered an online questionnaire, gave students who are in middle school a simple science popularization of our project. The feedbacks from those experts, iGEMers, and even students provided tremendously important suggestions for the development and execution of our project.
Figure 4. Schematic representation of “CycleBlue”.
- Sarsour, E. H. , Kumar, M. G. , Chaudhuri, L. , Kalen, A. L. , & Goswami, P. C. . (2009). Redox control of the cell cycle in health and disease. Antioxidants & Redox Signaling, 11(12), 2985.
- Harashima, H. , Dissmeyer, N. , & Schnittger, A. . (2013). Cell cycle control across the eukaryotic kingdom. Trends in Cell Biology, 23(7), 345-356.
- 李志琴, & 章静波. (2002). 细胞周期及其调控——兼论2001年诺贝尔生理学或医学奖. 生理科学进展, 33(2), 3.
- Sherr, C.J. and Roberts, J.M. (1999) CDK inhibitors: positive and negative regulators of G1-phase progression. Genes Dev. 13, 1501– 2112
- Dyson, N. (1998) The regulation of E2F by pRB-family proteins. Genes Dev. 12, 2245–2262
- Weinberg, R.A. (1995) The retinoblastoma protein and cell cycle control. Cell. 81, 323–330
- Furuno, N. et al. (1999) Human cyclin A is required for mitosis until mid prophase. J. Cell Biol. 147, 295–306
- Riabowol, K. et al. (1989) The cdc2 kinase is a nuclear protein that is essential for mitosis in mammalian cells. Cell. 57, 393–401
- Peters, J.M. (2006) The anaphase promoting complex/cyclosome: a machine designed to destroy. Nat. Rev. Mol. Cell Biol. 7, 644–656