Team:Slovenia/Protease signaling/Split proteases

Orthogonality Split proteases Achievements Introduction Results Light-dependent mediator

 Split orthogonal proteases

• Four new active split site-specific proteases were designed in addition to the previously published TEVp.
• The new split proteases were shown to rapidly respond to regulated by induced chemical dimerization.
• We demonstrated higher cleavage activity of the TEVp homologues against their respective substrates in comparison to the already existing split TEVp.

 

The split protein system based on the inducible dimerization is an attractive method to regulate the protease activity. Wehr et al. Wehr2006 described a split TEVp expressed as two functionally inactive fragments; the N-terminal (1 – 118 aa) and C-terminal (119 – 242 aa) protease fragments (referred to as cTEVp and nTEVp) (1). When the two fragments were coexpressed as fusion constructs with adjacent dimerization partners, the split TEVp was able to reconstitute and regain its catalytic activity, demonstrating that the activity of split TEVp could be controlled through the ligand induced protein – protein interactions.

Reconstituted split-TEVp

Model of nTEVp (residues 1 – 118 in blue) and cTEVp (residues 119 – 242 in orange) reconstituted in the active form (from PDB 1LVB).

Our team hypothesized that the same inducible dimerization approach could also be used with TEVp homologues. We converted all of the tested orthogonal potyviral proteases to split proteases by splitting them at positions corresponding to the position of the previously described split TEV protease. We selected three different types of dimerization domains to induce the activity of the split proteases. The first pair of dimerization domains was the rapamycin responsive FKBP/FRB system Banaszynski, which induces dimerization upon ligand binding. The second pair of dimerization domains was the CRY2PHR/CIBN system, which induces dimerization upon irradiation with blue light. Finally, our third system for dimerization was designed to respond to a Ca²⁺ influx based on the calmodulin-M13 interaction , that we used to detect mechanosensing.

Further explanation ...

Rapamycin and its mechanism of action.

(A) Chemical structure of rapamycin. Binding sites for FRB and FKBP are shown. (B) Schematic presentation of FKBP and FRB binding to rapamycinBanaszynski

Interactions among different proteins play a key role among all living organisms. Chemically induced dimerization (CID) is one of such interactions, which allows two different protein domains to dimerize after the addition of a small molecule. The most widely used CID to date is the FKBP/FRB system which heterodimerizes upon rapamycin addition Inobe2016.

Rapamycin is a 31-membered macrolide antifungal antibiotic that was first isolated from the Streptomyces hygroscopicus and binds with high affinity to the 12-kDa FK506 binding protein (FKBP) as well as to a 100-aminoacid domain (E2015 to Q2114) of the mammalian target of rapamycin (mTOR) protein known as the FKBP-rapamycin binding domain (FRB) (4.10.1) Banaszynski. Besides FKBP/FRB there are also other CID system where small molecules like gibberellin Murase and coumermycin Farrar2000 are used for induced dimerization.

 Results

We tested the full set of four orthogonal proteases with the rapamycin inducible system by measuring their activity with the cycLuc reporter. Increasing luciferase activity was detected correlating with the amount of the transfected protease fragments in stimulated cells (4.10.2.). Luciferase in unstimulated cells remained inactive even at the highest amount of transfected protease fragments, proving low leakage and high inducibility of the split protease system in response to rapamycin.

Activitiy of split proteases based on rapamycin inducible system.

HEK293T cells were trasnfected with indicated cycLuc reporters and rapamycin inducible split proteases. Whole proteases were used as positive control. An increase in luciferase activiy was detected in cells induced with rapamycin.

Kinetics of split PPVp induction with rapamycin.

HEK293T cells were trasnfected with cycLuc_PPVs and rapamycin induclible split PPVp or whole PPVp. Cells were induced with rapamycin, luciferase activity was measured in cells lysed at indicated time points.

Additionally, we tested the kinetics of rapamycin induction with PPVp and its corresponding cycLuc reporter. We showed that luciferase activity starts increasing just a few minutes after the addition of rapamycin, resulting in activity comparable to activity of the reporter in the presence of constitutively active whole protease within one hour after the induction (4.10.3.).

After demonstrating that the new proteases are active as split enzymes with rapamycin-induced complementation, we adapted the same split system to other inputs. To connect the split protease-based signaling to mechanosensing, we prepared Ca²⁺ inducible proteases based on calmodulin and M13 interaction. The first results look promising; however we have to confirm them in the repeated experiments. As an additional type of input we prepared light inducible split proteases.

 References