Team:Slovenia/Protease signaling/Light dependent mediator
nbsp;Light-depended mediator
Achivements
We designed and successfully tested three light inducible split proteases: CIBN/CRY2PHR light inducible split TEV protease, CIBN/CRY2PHR light inducible split PPVp and CIBN/CRY2PHR light inducible split TEVpE.
Introduction
In the recent years, light has been extensively explored as a trigger signal for activation of different biological processes. Small molecules and other chemical signals lack spatial resolution and their temporal resolution is limited by the time required for the cell permeation. In comparison, induction by light as developed by the optogenetics offers many advantages. It is fast as well as inexpensive and allows for excellent spatial, temporal and dose-dependent control.
There is a plethora of various light inducible systems available; however, not many are applicable to our purpose. Red light induced systems like Phy/PIF require an
additional phytochrome
Initially we decided to test the LOVpep/ePDZ system. This system has been used previously at iGEM, by
EPF_Lausanne 2009, Rutgers 2011 and Rutgers 2012
and in mammalian cells by Freiburg_2014. AsLOV2 is a small photosensory domain from Avena sativa
phototropin 1 with a C-terminal Jα helix. The Jα helix is caged in darkness but unfolds upon blue light (< 500 nm) photoexcitation, which is crucial for phototropin
signaling.
A photosensor has been prepared by engineering the AsLOV2 domain to contain a peptide epitope SSADTWV on the C-terminus of the Jα helix (LOVpep), binding an engineered
Erbin PDZ domain (ePDZ) upon blue light stimulation
Results
For initial testing and characterization of the system, we fused the LOVpep and ePDZb
We tested two orientations of ePDZ (ePDZ fused to split luciferase to N or C terminus) and ratios of the constructs ePDZ vs. cLuc:LOVpep were tested (2).
(A) Schematic representation of the light-inducible interaction between proteins containing ePDZ and LOVpep domains. (B) Light inducible reporter with split luciferase at the N-terminus of ePDZ domain (nLuc:ePDZb) responded to light more efficiently than ePDZ:nLuc. Schematic representation of different arrangements of ePDZ to split firefly luciferase (nLuc). After induction the cells transfected with LOVpep/ePDZ reporter system were lysed and bioluminescence was measured with dual luciferase assay. (D) LOVpep/ePDZ reporter reacted to light only once. Following the addition of luciferin to the medium the cells were induced and left in the dark for indicated periods.
As the measured signal was the highest with split luciferase on the N-terminus of the ePDZ domain and a 1:3 ratio of LOVpep:ePDZ, all subsequent experiments were performed with this ratio. An important feature for real life applications is the ability of the system to be stimulated multiple times. Therefore, repeated association and dissociation was tested in the real time, by adding luciferin to the medium and measuring bioluminescence upon induction by light (2C). The system showed a delayed, but successful induction the first time, but the second induction was much weaker. The results indicated that the LOVpep/ePDZ system in this setup could not be induced more than once, so we decided to test another system.
As it has previously been shown on the example of split Cre recombinase
CRY2 is a cryptochrome, originating from Arabidopsis thaliana and is a blue light–absorbing photosensor that binds a helix-loop-helix DNA-binding protein CIB1 in
its photoexcited state. In our system, we used the conserved N-terminal photolyase homology region of CRY2 (CRY2PHR; aa 1-498) that mediates light-responsiveness and
the truncated version of the CIB1 protein (CIBN; aa 1-170) without the helix-loop-helix region, which mediates DNA binding
We adapted this system for the reconstitution of split luciferase to create a blue-light sensor, which enables easy characterization for further experiments. The N- and C-terminal split fragments of the firefly luciferase were fused to the C-terminus of the CRY2PHR and the CIBN proteins, since this topology has previously been shown to work with the Cre recombinase (3).
(A) Response to light depended on concentration of CIBN:cLuc. After induction the cells transfected with CIBN:cLuc and CRY2PHR:nLuc were lysed and bioluminescence was measured with dual luciferase assay. (B) CRY2PHR light reporter was induced repeatedly. Following the addition of luciferin to the medium of the cells transfected with CIBN:cLuc and CRY2PHR:nLuc (ratio 1:3) were induced and left in the dark for indicated periods.
As the measured signal was the highest with a 1:3 ratio of CRY2PHR/CIBN (4A), all subsequent experiments were performed with this ratio. Next we tested if this system could be induced repeatedly in real time. The CRY2PHR/CIBN system shows maximum activity after 2 minutes of induction, returns to background in 10 minutes after the stimulus is removed, and can be induced repeatedly (4B).
(A)Schematic representation of CRY2PHR/CINB light-inducible system with split protease linked to cyclic luciferase reporter. Cells were transfected with 1:3 ratio of plasmids coding for CRY2PHR:CIBN with split TEVp (B) or split PPVp protease (C). The system was tested with the corresponding cyclic reporter (red bars), while orthogonality was tested with mismatched cyclic reporter (white bars). After indicated periods of induction the cells were lysed and bioluminescence was measured with dual luciferase assay.
To implement light as one of the input signals for protease-based signaling pathway or logic functions, we fused the CRY2PHR and the CIBN domains to the N- and C-terminal split domains of 3 different proteases (TEVp, TEVpE and PPVp) (5A) and tested their activity and orthogonality with bioluminescence assays (5B) and western blotting (5C). The bioluminescence assay was based on the split firefly luciferase with cleavage site for either TEVp, TEVpE or PPVp. Cleavage of this reporter results in the luciferase activity reconstitution and thus an increase in the luminescence.
(A) Schematic representation of CRY2PHR/CIBN light-inducible system with split protease and substrate with protease target sequence. Activity of (B) TEV, (C) PPV and (D) TEV:E proteases were analyzed by Western blot analysis. 24 hours after transfection of cells with plasmids expressing split proteases and protease substrate, the formation of active protease was induced by light. After the indicated time periods cells were immediately lysed and formation of cleaved products were analysed by Western blot using primary antibodies against AU1 tag. Excluding the non-specific upper band, uncleaved samples show only one band, while the cleaved ones show two bands.
The reporter used for the western blot analysis was the luciferase reporter with the appropriate cleavage substrate inserted at a permissible site and an AU1 tag at the N-terminal. Uncleaved luciferase appears as a single band on a western blot, while partial cleavage of luciferase results in two bands (uncleaved at 65 kDa and cleaved at 55 kDa) and complete cleavage results in only the smaller band (6). We showed that substrates carrying specific target peptide for proteases were cleaved after induction of split protease reporters.
Both methods showed a successful, fast and dose-dependent response. This is the first time split TEV protease has been shown to work as a light inducible system. Also this is the first time TEVpE and PPVp were prepared as split proteins and shown to function in an inducible system.
