Controlling cell death and proliferation with encodable visible light responsive proteins

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Interactions between biomacromolecules play a crucial role in all cellular processes. They are usually weak, i.e. non-covalent and temporal and hence inherently difficult to address chemically. In many cases such as cell cycle control, it would be extremely important to find ways to target these interactions, which could open the way to control cellular processes such as cell death and cell proliferation. We have recently shown that we are able to induce cell death in cancer cells treated with biophotonic nanoswitches, short peptides that interact specifically with protein surfaces. In detail, the interactions between the cell cycle regulators p53/hdm-2, Bcl-xL/bak and Bcl-xL/bid depend on alpha-helices from one partner that bind into groves on the surface of the other. Peptides were synthesised with azobenzene-linkers that enable the light-controlled generation of a stable alpha-helical structure, which then interacts with the binding partner. Unfortunately, UV light is required for the conformational change and for the generation of the alpha-helical, active structure of these peptides and UV light can have damaging effects on cells. Furthermore, UV light cannot penetrate deeply into tissue. It would be advantageous to switch to longer, visible wavelengths of light, which are not damaging and penetrate deeper. An extremely promising approach is to combine such peptides with photo-sensitive domains from blue light receptors. These receptors are used by plants, fungi and bacteria to regulate physiological processes upon a stimulus with blue light. The light-sensitive parts in these proteins are LOV domains, which bind a molecule of a coenzyme called flavine mononucleotide, which in turn acts as the light-harvesting chromophore. Upon illumination a covalent photoadduct between the cofactor and the apoprotein is formed which induces a conformational change in the LOV domain: a C-terminal alpha-helix that is bound to a beta-sheet of the core of the protein becomes flexible and therefore accessible for binding to other partners. We will develop genetically encoded photo-activatable proteins, in which residues of p53 and bid/bak recognition alpha-helices are introduced into the C-terminal helix of LOV domains. In the dark these helices will be tightly bound to the core of the LOV protein and cannot be accessed for binding to other proteins. A blue light pulse will then set the alpha-helix free and allow interaction/binding to hdm-2 and p53 to influence the targeted pathways in live cells. The absorption characteristics of LOV domains allow for wavelengths up to 500 nm, the use of FMN analogues will extend this range up to 550 nm. Light of this range has no detrimental effects on cells and is currently used in dentistry to harden polymer fillings. Thus we will be able to regulate cell cycles processes in a time-dependant manner in exactly defined spatial areas with minimal cell damage. The life-time of the active state can be regulated by the period of illumination, the choice of the LOV domain used and the type of light-sensitive pigment present. The photo-switchable proteins developed will be delivered to cells using our established approach, in which the protein will be tagged with a peptide that enables cellular uptake. Additionally, we will use transient expression of our constructs for short term investigations of the cell cycle and using viral vectors we will engineer cellular systems that allow for stable expression of our proteins for long term cell cycle tracking. The approach described here to intervene in biological process in a targeted fashion is generic in that photoactivatable proteins will be generally applicable to all biomacromolecular interactions based on alpha-helices. It will establish a novel research approach for the reversible modulation of the way in which proteins interact in real time and within live cells with enormous potential for the study of biological processes and for therapy.