Team:ESBS-Strasbourg/proteolux/scientific/proteoluxpro

• Proteolux P
• Proteolux Plus ®
• Proteolux Pro ®

As shown previously the Proteolux system offers a light-inducible specific mechanism to degrade tagged protein. This system may be applied to any protein expressed in the cytoplasm of the host cell. For the original system a homologue recombination step is necessary in order to fuse the PIF-DAS tag to the target protein in the host cell or organism. This step requires additional time in order to prepare the experiment and might further change the normal protein physiology, which should be observed, due to the creation of a fusion protein. It is known, that the function of a fusion protein can be different of the wild-type protein due to different stability of the mRNA after the homologue recombination leading to a different translation rate or the expressed protein does not fold correctly and forms aggregates in the cytoplasm. These drawbacks of the original system are solved in Proteolux’s P (Plus and Pro) system.

The Proteolux Plus and Pro system allows the observation of the native target protein functions without long experiment preparation and interference with the cell metabolism. The developed strategy is to use a specifically engineered intrabody against the target protein. The binding of the intrabody to the protein can lead to the direct inhibition of the protein function. In the case, that the protein is not directly inhibited and to prevent re-establishment of the protein function the intrabody-protein complex is targeted to the proteasome. The binding of the intrabody to the target protein has to be regulated to avoid constant degradation of the target protein. Therefore, Proteolux offers the Plus and Pro system of different complexity, according to the experimenter demand.

Intrabodies are antibody derived proteins that bind to an intracellular protein within the cell. The normal antibody (figure X) has to be modified due to the reducing environment within the cytoplasm that prevents disulfide bridge formation. Therefore, single-chain antibodies, which are fusion proteins of the variable region of the light chain (VL) and the heavy chain (VH) connected by a short linker, are often expressed.

Light-controlled regulation of the intrabody activity.

The idea is to have an inactive intrabody which can be assembled on demand into its active form. Therefore, the structure of a general IgG antibody has to be regarded in greater detail (figure 1) [Garrett RH and Grisham CM, Biochemistry 2nd edition].

Figure 1: Overview over the structure of an IgG antibody. The active IgG antibody is composed of two heavy (H) chains (outer long chains) and the small light (L) chains (inner short chains). The constant domains are shown in green (C) and the variable domains in violet (V). The heavy chains consist of 3 constant domains (CH1, CH2 and CH3) and a variable domain VH. The light chains are composed of a constant (CL) and a variable (VL) domain. The Fab region which is composed of the variable domain and the CH1 domain is connected via a flexible hinge region to the Fc part of the antibody (CH2 and CH3 region). The chains are intra- and interconnected by disulfide bridges (yellow lines). There are two disulfide bridges that interconnect the two heavy chains and each variable chain is connected to one heavy chain via one disulfide bridge. The active antibody is only formed when the light chain is connected to the heavy chain in order to form the antigen binding site, which is situated between the variable domains.

The formation of the antigen binding site is dependent on the correct binding of the VL to the VH domain, which is enhanced by a strong affinity between the constant regions and the disulfide bridge between the light and the heavy chain. In the Proteolux Pro system, this permanent disulfide bridge is replaced by a light inducible interaction between two proteins. In order to avoid interference with the PhyB-PIF interaction, other interaction partners were searched which are active under light conditions that do not activate phytrochrome B (figure 2).

Figure 2: Absorption spectrum of phytrochome B containing the PCB chromophore. The absorption maxima of the two red light forms are visible as two distinct peaks at 658 nm for the red light form and at 720 nm for the far-red light form. The absorbance is lowest in the blue/green light part of the spectrum between 450nm and 550nm. Therefore, it is save to work under blue/green light conditions to avoid the activation of phytrochrome B.

The absorbance spectrum of phytrochrome B reveals that it does not absorb under light conditions between 450 nm and 550 nm which correlates to blue/green light. Therefore, the blue light (475 nm) regulated interaction between the Arabidopsis thaliana photoreceptor Zeitlupe (ZTL, Gene ID: 835842) and Gigantea (GI, Gene ID: 838883) is used to replace the disulfide bridge function. Zeitlupe contains a light sensing PAS-like LOV domain at its N-terminal end and a C-terminal Kelch repeat domain, which is involved in protein-protein interaction [Lariguet P and Dunand C, 2005; http://www.uniprot.org/uniprot/Q94BT6]. The GI protein has a more than 4 fold affinity to ZTL under blue light conditions [Kim WY, 2007].
In order to prevent the strong affinity between the constant domains of the light and the heavy chain, only the low affinity variable domains are used in the construction.
The ZTL sequence is fused together with the normal PIF-DAS-tag to the C-terminus of the VL domain and the GI sequence followed by a GST sequence to the C-terminus of the VH domain. The GST dimerizes and connects therefore two VH domains. This allows the formation of bivalent intrabodies with higher protein binding capacity.
The expression of such a modified intrabody allows the formation of the active intrabody only under blue light conditions. The protein expressing phenotype can be analyzed under far-red light conditions. When the protein should be degraded, the intrabody formation is triggered under blue light (475nm) resulting in the formation of an active antibody binding site and fixation of the target protein by the intrabody. To ensure, that the protein is not active anymore the intrabody-protein complex can be tethered to the protease using red light conditions. This procedure is summarized in figure 3.

Proteolux Pro offers the first light-controlled intrabody activity which can be used to analyze any protein in its native conformation without interference with the cell metabolism.

Figure 3: Light controlled intrabody formation and specific target protein degradation. A: Structure of an IgG antibody. Shown are the two long heavy (H) chains and the small light (L) chains. The constant domains are shown in green (C) and the variable domains in violet (V). The heavy chains consist of 3 constant domains (CH1, CH2 and CH3) and a variable domain VH. The light chains are composed of a constant (CL) and a variable (VL) domain. The chains are intra- and interconnected by disulfide bridges (yellow lines). B1.1 and B1.2: Fusion constructs for the light and heavy chains. B1.1: The blue light photoreceptor Zeitlupe (ZTL), the Phytochrome interacting factor (PIF) and the C-terminal degradation tag DAS are fused to the C-terminus of the light chain. B1.2: The gigantea (GI) sequence is fused in the hinge region of the antibody. The two conserved cysteine residues for the disulfide bridge are maintained, the two constant domains (CH2 and CH3) of the heavy chain are deleted. C: Blue light dependent photo conversion of ZTL. The absorption of blue light (475 nm) by the PAS-like LOV domain of ZTL leads to a conformational change allowing GI binding (D). D: Blue light induced formation of the active intrabody. The binding of GI to ZTL under blue light brings the light and heavy chain together and restores the natural disulfide bridge between the two. The two variable domains can now form an active antigen binding site that binds to the protein of interest. E: Red light induced protein degradation. PIF binds to PhyB under red light conditions bringing the DAS tag in proximity to the ClpXP protease leading to degradation of the protein of interest bound to the intrabody.