Controlling cell death and proliferation with encodable visible light responsive proteins

生物大分子之间的相互作用在所有细胞过程中起着至关重要的作用。它们通常是弱的，即非共价和暂时的，因此天生难以用化学方法解决。在许多情况下，如细胞周期控制，找到针对这些相互作用的方法将是极其重要的，这可能为控制细胞死亡和细胞增殖等细胞过程开辟道路。我们最近已经证明，我们能够通过生物光子纳米开关诱导癌细胞死亡，生物光子纳米开关是一种与蛋白质表面特异相互作用的短肽。更详细地说，细胞周期调控因子P53/HDM-2、Bcl-XL/BAK和Bcl-XL/BID之间的相互作用依赖于一个伙伴的α-螺旋结合到另一个伙伴表面的凹槽中。用偶氮苯连接物合成肽，这些偶氮苯连接物能够在光控制下产生稳定的α-螺旋结构，然后该结构与结合伙伴相互作用。不幸的是，这些多肽的构象变化和α-螺旋活性结构的产生需要紫外光，而紫外光可以对细胞产生破坏性影响。此外，紫外线不能深入组织。改用更长的可见光波长将是有利的，因为这种光不会造成损害，而且穿透更深。一种非常有希望的方法是将这种多肽与蓝光受体的光敏感区结合起来。这些受体被植物、真菌和细菌用来在蓝光刺激下调节生理过程。这些蛋白质中对光敏感的部分是LOV结构域，它结合了一种名为黄素单核苷酸的辅酶分子，该辅酶分子反过来又充当捕光发色团。在光照下，辅因子和脱辅蛋白之间形成一个共价光加合物，导致LOV结构域的构象变化：与蛋白质核心的β-折叠结合的C-末端α-螺旋变得灵活，因此可以与其他伙伴结合。我们将开发基因编码的光激活蛋白，其中P53残基和Bid/Bak识别α-螺旋被引入LOV结构域的C-末端螺旋中。在黑暗中，这些螺旋将紧密结合在LOV蛋白的核心上，无法与其他蛋白结合。然后，蓝色光脉冲将使α-螺旋自由，并允许与HDM-2和P53相互作用/结合，以影响活细胞中的靶向通路。LOV结构域的吸收特性允许波长达到500 nm，FMN类似物的使用将把这一范围扩大到550 nm。这一范围的光线对细胞没有有害影响，目前用于牙科硬化聚合物填充物。因此，我们将能够在精确定义的空间区域以时间依赖的方式调节细胞周期过程，并将细胞损伤降至最低。活性状态的寿命可以通过光照时间、所使用的LOV结构域的选择和存在的光敏颜料的类型来调节。开发的光可切换蛋白质将使用我们建立的方法传递到细胞，在这种方法中，蛋白质将被标记上一种能够使细胞摄取的多肽。此外，我们将使用我们构建的结构的瞬时表达来短期研究细胞周期，并使用病毒载体来设计细胞系统，使我们的蛋白质能够稳定表达，用于长期细胞周期跟踪。这里描述的以有针对性的方式干预生物过程的方法是通用的，因为光激活蛋白将通常适用于所有基于α-螺旋的生物大分子相互作用。它将为蛋白质实时和活细胞内相互作用方式的可逆调节建立一种新的研究方法，在生物过程研究和治疗方面具有巨大的潜力。

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.