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Controlling cell death and proliferation with encodable visible light responsive proteins

用可编码的可见光响应蛋白控制细胞死亡和增殖

基本信息

批准号：
BB/I021396/1
负责人：
Rudolf Allemann
金额：
$ 56.24万
依托单位：
CARDIFF UNIVERSITY
依托单位国家：
英国
项目类别：
Research Grant
财政年份：
2012
资助国家：
英国
起止时间：
2012 至无数据
项目状态：
已结题

来源：
https://gtr.ukri.org/projects?ref=BB%2FI021396%2F1
关键词：
Controlling cell death proliferation encodable

项目摘要

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.

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，即非共价的和暂时的，因此固有地难以化学处理。在许多情况下，如细胞周期控制，找到靶向这些相互作用的方法将是非常重要的，这可以打开控制细胞过程，如细胞死亡和细胞增殖的方法。我们最近已经证明，我们能够在用生物光子纳米开关处理的癌细胞中诱导细胞死亡，生物光子纳米开关是与蛋白质表面特异性相互作用的短肽。详细地说，细胞周期调节剂p53/hdm-2、Bcl-xL/巴克和Bcl-xL/bid之间的相互作用依赖于来自一个伴侣的α-螺旋结合到另一个伴侣表面上的格罗夫斯中。肽与偶氮苯-接头一起合成，所述偶氮苯-接头使得能够在光控制下产生稳定的α-螺旋结构，所述α-螺旋结构然后与结合配偶体相互作用。不幸的是，这些肽的构象变化和α-螺旋活性结构的产生需要UV光，并且UV光可能对细胞具有破坏作用。此外，紫外线不能深入穿透组织。最好改用波长更长的可见光，因为它不会造成损害，而且穿透更深。一种非常有前途的方法是将此类肽与蓝光受体的光敏结构域结合起来，联合收割机。这些受体被植物、真菌和细菌用来调节蓝光刺激下的生理过程。这些蛋白质中的光敏部分是LOV结构域，它结合一种称为黄素单核苷酸的辅酶分子，而黄素单核苷酸反过来又充当捕光发色团。在光照下，辅因子和脱辅基蛋白之间形成共价光加合物，其诱导LOV结构域中的构象变化：与蛋白质核心的β-折叠结合的C-末端α-螺旋变得柔性，因此可与其他配偶体结合。我们将开发基因编码的光活化蛋白，其中p53和bid/巴克识别α-螺旋的残基被引入LOV结构域的C-末端螺旋。在黑暗中，这些螺旋将紧密结合到LOV蛋白的核心，并且不能与其他蛋白结合。蓝光脉冲将释放α-螺旋，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高达500 nm，使用FMN类似物将该范围扩展至550 nm。该范围的光对细胞没有有害影响，目前用于牙科中硬化聚合物填充物。因此，我们将能够以时间依赖性的方式在精确定义的空间区域内以最小的细胞损伤调节细胞周期过程。活性状态的寿命可以通过照射时间、所用LOV域的选择和存在的光敏色素的类型来调节。开发的光可转换蛋白质将使用我们既定的方法递送到细胞，其中蛋白质将用能够使细胞吸收的肽进行标记。此外，我们将使用我们的构建体的瞬时表达进行细胞周期的短期研究，并使用病毒载体，我们将设计细胞系统，使我们的蛋白质稳定表达，用于长期细胞周期跟踪。这里描述的以靶向方式干预生物过程的方法是通用的，因为光活化蛋白质通常适用于基于α-螺旋的所有生物大分子相互作用。它将为蛋白质在真实的时间和活细胞内相互作用的可逆调节方式建立一种新的研究方法，具有研究生物过程和治疗的巨大潜力。