Team:Slovenia/Protease signaling/Light dependent mediator
nbsp;Light-dependent input signal and proteases - in progress TL
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 cell permeation. In comparison, induction by light as developed by 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 the PhyB/PIF6 system
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
signalling.
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
nbsp;Results
LOVpep and ePDZb
For initial testing and characterization of the system, we fused LOVpep and ePDZb
As luciferase activity was highest with split luciferase on the N-terminus of the ePDZ domain and a 1:3 ratio of the LOVpep:ePDZ constructs, all subsequent experiments were performed with this conditions. 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 real time, by adding luciferin to the medium and measuring luciferase activity upon induction by light ( 2 C). The system exhibited a delayed, but successful induction the first time, but the second induction was much weaker. These results indicate that the LOVpep/ePDZ system in this setup could not be induced more than once, so we decided to test an additional system.
(A) Schematic representation of the light-inducible interaction between proteins containing the ePDZ and LOVpep domains. (B) Light inducible reporter with split luciferase at the N-terminus of the ePDZ domain (nLuc:ePDZb) responded to light more efficiently than ePDZ:nLuc. Corresponding schematic representations of different arrangements of ePDZ fused to the N-terminal segment of split firefly luciferase (nLuc) are shown above the graph. After induction with blue light at 460nm the cells transfected with LOVpep/ePDZ reporter system were lysed and luciferase activity was determined with the dual luciferase assay. (C) The LOVpep/ePDZ system responded to light stimulation only once. Following the addition of luciferin to the medium the cells were induced or left in the dark for indicated periods.
CRY2PHR-CIBN
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.
(A) Response to light depended on the amount of the CIBN:cLuc plasmid. After induction the cells transfected with the CIBN:cLuc and CRY2PHR:nLuc encoding plasmids were lysed and luciferase activity was determined with the dual luciferase assay. (B) CRY2PHR light reporter was induced repeatedly. Following the addition of luciferin to the medium, cells transfected with the CIBN:cLuc and CRY2PHR:nLuc encoding plasmids (ratio 1:3) were induced with blue light at 460nm and left in the dark for indicated periods.
As luciferase activity was highest with a 1:3 ratio of CRY2PHR:CIBN constructs ( 4 A), 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 showed a maximum activity after 2 minutes of induction and dropped to background 10 minutes after the stimulus was removed. The system could be induced repeatedly and reach high levels of activation at each stimulation ( 4 B).
Light inducible proteases
To implement light as one of the input signals for protease-based signalling pathways 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) ( 5 A). Activity and orthogonality were tested by cleavage of a cyclic luciferase reporter . Cleavage of this reporter results in luciferase reconstitution and thus an increase in luminescence. Both the TEV and the PPV split proteases, coupled to the CRY2PHR/CIBN system, showed high catalytic activity and exquisite mutual orthogonality upon blue light illumination ( 5 B and C).
(A) Schematic representation of the CRY2PHR/CIBN light-inducible system with split protease. In the dark, the CRY2PHR and CIBN proteins do not interact (left), while illumination with blue light results in heterodimerization and reconstitution of split protease (right), which in turn cleaves the cyclic luciferase reporter, resulting in increased luciferase activity. HEK293T cells were transfected with 1:3 ratio of plasmids coding for CRY2PHR:CIBN with split TEV (B) or PPV (C) protease. 24 hours after transfection, cells were illuminated with blue light at 460nm for indicated periods of time. The cells were lysed and bioluminescence was measured with dual luciferase assay. Upon illumination with blue light, the protease cleaves only the cyclic reporter with the correct cleavage site (red bars), while the cyclic reporter with the mismatched cleavage site remains uncleaved (white bars).
(A) In the dark, the CRY2PHR and CIBN proteins do not interact (top), while illumination with blue light results in heterodimerization and reconstitution of split protease (bottom), which in turn cleaves the target protein. Activity of (B) TEV, (C) PPV and (D) TEV(E) proteases were analyzed by western blot. 24 hours after transfection, cells were illuminated with blue light at 460nm for indicated periods of time. Cells were immediately lysed and the cleaved products were analysed by Western blot using anti-AU1 antibodies. 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 the AU1 tag at the N-terminus. Uncleaved luciferase appears as a single band on a western blot, partially cleaved luciferase would result in two bands (uncleaved at 65 kDa and cleaved at 55 kDa), while complete cleavage would result in only the smaller band. Our results indicate that substrates carrying specific protease cleavage sites were cleaved after blue light illumination of cells harboring plasmids for expression of CRY2PHR:nTEV and CIBN:cTEV proteins ( 6 ).
Both methods confirmed successful, fast and dose-dependent responses. This is the first time TEVpE and PPVp were prepared as split proteins and shown to function in an inducible system.
References
