DogTag - a genetically encoded proximity labelling strategy to capture problematic protein-protein interactions

DogTag - 一种基因编码的邻近标记策略，用于捕获有问题的蛋白质-蛋白质相互作用

• 批准号: BB/R008787/1
• 负责人: Piers Hemsley
• 金额: $ 32.27万
• 依托单位: University of Dundee
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2018
• 资助国家: 英国
• 起止时间: 2018 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FR008787%2F1
• 关键词: DogTag; genetically; encoded; proximity; labelling

DogTag - how to find a protein's partners in living organismsProteins are the key functional molecules in the cell, forming structural building blocks and performing the many specialized functions a cell requires to survive. Making sure that individual proteins perform the right task at the right time in the right place is largely achieved by regulating which other proteins it interacts with.Defining these interactions is one of the core problems of modern molecular biology. All methods used so far suffer from a number of disadvantages:1. Disruptive -the cells of the organism must be broken apart before the interactions can be assessed. This leads to many interactions falling apart and being missed. This is particularly true for weak or transient interactions such as those occurring between enzymes and their substrates or interactions with proteins found in the cell membrane where detergents have to be used to break the membrane. Enzyme-substrate and membrane protein interactions are some of the most important for understanding disease and developing drugs making this knowledge an important priority in biology. 2. Out of context - many methods use a different organism (heterologous system) to test interactions as it is easier and faster (e.g. yeast is used as a surrogate system for investigating interactions between human or plant proteins). In a heterologous system many components many be missing, this means that many large protein complexes composed of multiple interactions cannot be assessed. 3. Non-physiological - many systems use cell culture as a substitute for whole organisms. While necessary in these instances they ignore the context of the tissue, organism or physiological conditions and therefore miss the real interaction patterns of proteins in a particular situation.4. Outside input needed - Many systems used to look at protein interactions require some form of outside input to reveal the interactions. This means that whole organisms or tissues cannot be used in these circumstances.We have designed a strategy to get around many of these problems, this therefore represents a milestone in protein-protein interaction analysis and opens the door for more physiologically relevant analyses to be performed and reveal more relevant data to researchers.By taking a protein of interest and linking it to a bacterial enzyme capable of adding a small tag onto other proteins we can define the protein environment surrounding our protein of interest. Even more importantly the enzyme and tag are both proteins themselves so can be encoded as DNA and placed into the genome of the study organism. This means that, for the first time, it is possible to identify protein partners in the proteins native cellular environment in a tissue of interest (plant seed, animal liver, etc.) in a whole organism (whole plant, animal, etc.) under physiologically relevant conditions or stresses (low oxygen, drought, etc.) without any outside input.The aim of this work is to take our proof of principle data and demonstrate that this system can be used to discover novel protein interactions from organisms such as fungi, plants and animals and therefore fuel novel primary research in hitherto inaccessible study areas.

DogTag-如何在生物蛋白中找到蛋白质的伴侣是细胞中的关键功能分子，形成结构构建块并执行细胞生存所需的许多专业功能。通过调节其与哪些其他蛋白质相互作用，确保单个蛋白在正确的时间在正确的时间执行正确的任务。定义这些相互作用是现代分子生物学的核心问题之一。到目前为止使用的所有方法都遭受许多缺点：1。破坏性 - 在评估相互作用之前，必须分解生物体的细胞。这导致许多互动崩溃并被错过。对于弱或瞬态相互作用（例如酶及其底物之间发生的相互作用）尤其如此，或与在细胞膜中发现的蛋白质相互作用，必须使用洗涤剂来破坏膜。酶 - 基层和膜蛋白相互作用对于理解疾病和开发药物最重要的相互作用是生物学中的重要优先事项。 2。出于上下文 - 许多方法使用不同的生物体（异源系统）来测试相互作用，因为它更容易，更快（例如，酵母被用作研究人或植物蛋白之间相互作用的替代系统）。在异源系统中，许多组件都缺少许多组件，这意味着无法评估许多由多种相互作用组成的大型蛋白质复合物。 3。非生理学 - 许多系统使用细胞培养作为整个生物的替代品。尽管在这些情况下需要忽略组织，生物或生理条件的背景，因此错过了特定情况下蛋白质的真实相互作用模式。4。所需的外部输入 - 许多用于查看蛋白质相互作用的系统需要某种形式的外部输入以揭示相互作用。这意味着在这种情况下无法使用整个生物体或组织。我们设计了一种解决这些问题的策略，因此，这代表了蛋白质 - 蛋白质相互作用分析的一个里程碑，并为更多的生理相关分析打开了门，并为研究人员添加了一种蛋白质，我们可以将其添加到其他蛋白质中，并将其添加到其他环境中。围绕着我们感兴趣的蛋白质。更重要的是，酶和TAG都是蛋白质本身，因此可以用作DNA并放入研究生物的基因组中。这意味着，这是第一次可以在整个生物体（植物种子，动物肝等）中识别蛋白质的本地细胞环境中的蛋白质伴侣（整个生物，整个植物，动物等）在生理上相关的条件或压力（低氧气，干旱，干旱等）中（无需进行这种习惯），可以证明这一范围的刺激。例如真菌，动植物，因此为迄今难以访问的研究领域提供了新的主要研究。