Team:NUDT CHINA/Human Practices/Backup
Backup
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Literature
Background：
Based on our iGEM 2018’s project, this time, we aim at creating a system which can directly regulate the protein degradation system. To start with, we read a great amount of literatures about targeted protein degradation and how this process can be controlled.
What we learnt:
1. Signal controlled protein degradation has been introduced into Synthetic biology in recent years. Typical approaches include inducible degrons and PROTACs. Inducible degrons can specifically degrade tagged protein under control of small molecule or light signal, however, it was difficult to tag the endogenous protein with inducible degrons.
2. PROTACs has the advantage that it can controlled by small molecule and degrade target proteins efficiently. Reports also demonstrated light induced PROTACs recently. However, its high molecular weight leads to poor water solubility, oral absorption, and membrane permeability. The high cost of synthesizing and screening of the PROTAC molecules also limited its application.
3. The Trim21 protein we engineered in the last two years interacts with the IgG-Fc domain with its PRYSPRY domain. It seemed that the PRYSPRY domain can be replaced by other interactions to trigger the targeting of Trim21.
Representative References
[1]. Ding, W.X. and X.M. Yin, Sorting, recognition and activation of the misfolded protein degradation pathways through macroautophagy and the proteasome. Autophagy, 2008. 4(2): p. 141-50.
[2]. Bennett, E.J., et al., Global impairment of the ubiquitin-proteasome system by nuclear or cytoplasmic protein aggregates precedes inclusion body formation. Mol Cell, 2005. 17(3): p. 351-65.
[3]. Goldberg, A.L., %J Nature, Protein degradation and protection against misfolded or damaged proteins. 2003. 426(6968): p. 895-9.
[4]. Chan, N.C., et al., Broad activation of the ubiquitin-proteasome system by Parkin is critical for mitophagy. 2011. 20(9): p. 1726-1737.
[5]. Jiaqing, H., et al., Cancer-testis antigen HCA587/MAGE-C2 interacts with BS69 and promotes its degradation in the ubiquitin-proteasome pathway. 2014. 449(4): p. 386-391.
[6]. Hochstrasser, M., Ubiquitin, proteasomes, and the regulation of intracellular protein degradation. Curr Opin Cell Biol, 1995. 7(2): p. 215-23.
[7]. Ottis, P. and C.M.J.A.C.B. Crews, Proteolysis-Targeting Chimeras: Induced Protein Degradation as a Therapeutic Strategy. 2017. 12(4): p. 892-898.
[8]. Zheng, J., et al., Proteolysis targeting peptide (PROTAP) strategy for protein ubiquitination and degradation. 2016. 470(4): p. 936-940.
[9]. Zou, Y., D. Ma, and Y. Wang, The PROTAC technology in drug development. Cell Biochem. Funct., 2019.
[10]. Zhou, P., et al., Harnessing the ubiquitination machinery to target the degradation of specific cellular proteins. Mol. Cell, 2000. 6(3): p. 751-6.
[11]. Sakamoto, K.M., et al., Protacs: chimeric molecules that target proteins to the Skp1-Cullin-F box complex for ubiquitination and degradation. 2001. 98(15): p. 8554-8559.
[12]. Sakamoto, K.M., et al., Development of Protacs to Target Cancer-promoting Proteins for Ubiquitination and Degradation. 2003. 2(12): p. 1350-1358.
[13]. Gu, S., et al., PROTACs: An Emerging Targeting Technique for Protein Degradation in Drug Discovery. 2018. 40(4): p. 1700247.
[14]. Marina, V., et al., Intracellular antibody receptor TRIM21 prevents fatal viral infection. 2013. 110(30): p. 12397-12401.
[15]. Keeble, A.H., et al., TRIM21 is an IgG receptor that is structurally, thermodynamically, and kinetically conserved. 2008. 105(16): p. 6045-6050.
Interview with Prof. Xia
Brief Introduction of Prof. Zanxian Xia
The interview process:
We approached Dr. Xia after we obtained the first batch of data proving that the DocS-Coh2 can be used as the protein interface to initiate the protein degradation process. During the interview, we began with a brief presentation of our project design and preliminary data, we also presented our future plan on pushing our project towards signal responsiveness. Prof. Xia was very excited about our project and believed that it has a good application prospect. But he also pointed out some flaws we missed in the designing of our experiments: he suggested that efforts should be made to avoid the affection of transfection efficiency on the GFP fluorescence. He also offered some generous help on how we could prove that our system did work as the way we expected.
Figure 1. Interview with Dr. Zanxian Xia
What we learnt:
1. We should normalize the influence of the transcription efficiency and cellular status on experiment degradation.
2. We can try using protein synthesis inhibitors or proteosome inhibitors to determine whether our system actually works.
The seventh Conference of China iGEMers community
Introduction:
We participated in the CCiC online meetup on August 28 to 31, 2019, during which more than 60 teams all over China presented and exchanged their ideas. At the same time, our instructors and some experts and scholars in synthetic biology also gave us lectures in related fields, which made us deeply feel the charm of synthetic biology.
Figure 1
During the meetup, we obtained important feedbacks from both judges and other iGEM teams. For example, the differences on cell activities of the experimental group and control group may result in different transcription efficiency of GFP, which weakens the data’s integrity on proving the degradation ability of GFP PrePro.
Figure 2
Figure 3
Also, in the poster session, we are delighted to find that we have many similar ideas with Team SCU_WestChina in the synthetic biology education related public engagement. We hence established further connections on with them.
What we learnt:
HP: In the HP part, we should consider how to achieve the interaction between HP and wet-lab experiments, further researches related to practical application direction of targeted protein degradation tools are still needed.
Experiment: In the experimental work, we should focus on how to push our project towards signal responsiveness. Also, we should consider the affection of the signal we use on the transcriptional and translational programs of the chassis.
Model: We may try to use model to understand the key factors in our project.
Feedback
In order to make the experimental design more perfect and reliable, we carefully consider the valuable Suggestions put forward by Dr. Xia and some members from CCiC. We hence developed a new reporter on which GFP was fused with firefly luciferase so dual luciferase assay can be used to normalize the irrelevant factors.
Literature2
What we learnt:
1. 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.
Interview with Dr. Mingqi Xie
Brief Introduction of Dr.Mingqi Xie
The interview process
We approached Dr. 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.
Figure 1:interview with Dr. Mingqi Xie
What we learnt:
1. We learnt that the interface module of Predator Pro system can be substituted with better protein dimerization system, aiming to be more effective and compatible in human body.
2. We were suggested to start looking for some therapeutic targets for our predator system.
3. 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.
Feedback
1. Based on Dr.Xie’s suggestions and our own research, A blue-light-inducible PREDATOR Pro plasmid has been constructed as the constitution of the core module, which is more compatible to human body. Relevant experiments are within our plan.
2. We made a more reasonable plan to explore the effect of Predator Pro system in degrading TDP-43, which may make it more realistic to cure the ALS patience in the future.
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