Trigger of our project design
Achieving signal-controlled protein degradation has been preliminarily tried in our previous iGEM projects. In the project of NUDT_CHINA iGEM 2019, we coupled endogenous CHREBP glucose sensing pathway to a CHREBP activating hybrid promoter controlling a PREDATOR element to target glucagon receptor in hepatocytes. This system could degrade glucagon receptors in a glycemic dependent way, subsequently arresting hyperglycemia. Also, other systems including Doxycycline -inducible, or blue light-inducible systems have also been developed based on our PREDATOR V1 or V2 systems (unpublished).
Although these approaches provided a controllable protein degradation method, it was still worth noticing that the transcriptionally-controlled feature has given them obvious defects. First, given that PREDATORs are regulated by transcriptional factors, it will take hours until the PREDATORs are expressed after acute stimulation. Second, other known or unknown factors may also crosstalk with the transcriptional factors and interfere prior to the translation of the PREDATORs. Therefore, a robust control of the target protein degradation cannot be achieved, which leads us to a question: could we manipulate protein degradation in a more direct, accurate, acute way?
Upon investigation on literatures, we had a deeper understanding of the mechanism of Trim21. As a functional protein to mediate proteasomal degradation, it is a multi-domain protein consisting of an N-terminal RING domain with E3 ligase activity, a B-box domain, a coiled-coil dimerization domain and C-terminal PRYSPRY domain. Its PRYSPRY domain has a high affinity with constant Fc domain of the antibody2. Once the antibody-antigen complex enters the cytosol, Trim21 binds the Fc domain of the antibodies and form a Trim21-antibody-antigen trimer. Thereafter, the RING domain dimerizes, and ubiquitin proteasome system is recruited to mediate proteasomal degradation of the antigen3(Figure 2). Since we’ve already demonstrated in our previous PREDATOR system that the complete structure of antibody is not necessarily needed for Trim21, we hypothesized that the PRYSPRY domain of the Trim21 can also be replaced with other protein domains that can dimerize with other proteins under given signal input.
Figure 2. Schematic representation of Trim21 and its mechanism
Preliminary trial--Replaceability of the Trim21-antibody interface
To validate our hypothesis, we first tested whether the Trim21-antibody interface can be uncoupled from the PREDATOR system. For such matter, we reengineered the GFP PREDATOR system we reported in iGEM 2018 by replacing the Trim21 PRYSPRY domain and its interacting partner IgG-Fc domain into the DocS and Coh2, another set of protein that constitutively dimerizes, respectively. The new system was termed as GFP PREDATOR Pro (GFP PrePro)(Figure 3). Upon GFP expression, it would be first recognized by the target module- GFP nanobody. Then, the specific interaction of DocS and Coh2 would induce the formation of a ternary (Trim21-DocS, coh2-GFPnanobody, GFP) complex with high affinity, thus triggering the ubiquitination and degradation of GFP4.The fluorescent imaging and western blotting results showed significant decrease of GFP protein abundance in GFP PrePro expressing groups comparing to the control group.(click here to view more in the Result)
Nonetheless, we consulted Professor Zanxian Xia, an expert on protein ubiquitylation and degradation, on our project and presented our preliminary data in CCiC meetup(click here to view more in the Human Practice)We were suggested to try some other approaches to normalize the irrelevant factors that may affect the GFP abundance. Hence, we changed the GFP reporter system to a Dual Luciferase Reporter systemin which the firefly luciferase (Fluc) fused with GFP was linked with a renilla luciferase (Rluc) reporter via P2A peptide. Herein, Fluc serves as the reporter of GFP abundance and Rluc serves as the internal control. With this reporter, we repeated the preliminary experiment, results illustrated a noticeable decrease of the Fluc to Rluc ratio in contrast to the control(click here to view more in the Result), indicating that GFP PrePro has impressive efficiency in degrading the target protein.
Figure 3. Schematic representation of GFP Predator Pro
Towards signal responsiveness
In an attempt to accomplish a goal that the PREDATOR Pro system could be assembled and initiated by external signals, such as small molecule chemicals , we substituted the interface with a set of widely used and fully characterized heterodimerizing components: the FK506 binding protein (FKBP) domain and the T2089L mutant of FKBP-rapamycin binding domain (FRB)5.In this design, the presence of rapamycin would induce the heterodimerization of FRB and FKBP and the formation of a ternary complex, which, subsequently, trigger the proteasomal degradation of target protein.
To test such design, we constructed a Rapamycin-induced PREDATOR Pro plasmid (termed RiPrePro1.0) targeted for GFP, comprised of two components——FRB-Trim21-HA and FKBP-GFP nanobody (Figure 4).Results showed a mild dip of Fluc/Rluc under rapamycin induction, which implied a low degradation efficiency of RiPrePro1.0.
To promote the degradation efficiency, we turned to the model group for help. After performing Sensitivity Analysis of our system, (click here to view more in the Result), they discovered that a parameter regarding the protein interaction intensity of FKBP and FRB was pivotal to the degradation efficiency. Therefore, how to enhance the interaction intensity of FKBP-FRB turns out to be a challenge for us.
The first solution that came to us was to elevate the concentration of rapamycin. With the increment of rapamycin concentration, the degradation efficiency of RiPrePro did increase impressively (click here to view more in the Model). However, it has been well established that exorbitant amount of rapamycin would sigificantly reduce the cellular protein synthesis. Consequently, we proposed another solution: multiplying the copies of FKBP for a higher intensity of protein interaction. Hereby, we designed a Rapamycin-inducible PREDATOR Pro2.0 plasmid (RiPrePro2.0) composing GFPnano-FKBP*2 and HA-Trim21-FRB.
Having transfected HEK-293T cells with RiPrePro2.0, we were surprised to find out that in comparison to the control group and RiPrePro1.0 transfected groups, the Fluc/Rluc of RiPrePro-2 transfected groups has plunged substantially under equivalent rapamycin stiumlation(click here to view more in the Result), which verified our concept that a multiplied copy of FKBP could boost the degradation efficiency.
Figure 4. Schematic representation of RiPrePro1.0
Above all, our results demonstrated rapamycin inducible target protein degradation with the PREDATOR Pro system, which further supported our preliminary hypothesis.