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

狗标签-如何在活着的有机体中找到蛋白质的伴侣蛋白质是细胞中的关键功能分子，形成结构构件，并执行细胞生存所需的许多专门功能。要确保单个蛋白质在正确的时间、正确的地点执行正确的任务，很大程度上是通过调节它与哪些其他蛋白质相互作用来实现的。确定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有一些缺点：1.破坏性--在评估相互作用之前，有机体的细胞必须被分解。这导致许多互动失败，并被错过。对于酶及其底物之间的弱相互作用或瞬时相互作用尤其如此，或者与细胞膜中发现的蛋白质的相互作用，其中必须使用洗涤剂来打破细胞膜。酶-底物和膜蛋白的相互作用对于理解疾病和开发药物是最重要的，使这一知识成为生物学中的一个重要优先事项。2.脱离上下文-许多方法使用不同的有机体(异源系统)来测试相互作用，因为它更容易和更快(例如，酵母被用作研究人类或植物蛋白质之间相互作用的替代系统)。在一个异源系统中，许多成分可能会丢失，这意味着许多由多种相互作用组成的大型蛋白质复合体无法被评估。3.非生理性--许多系统使用细胞培养作为整个生物体的替代品。尽管在这些情况下是必要的，但它们忽略了组织、有机体或生理条件的背景，因此错过了特定情况下蛋白质的真实相互作用模式。需要外部输入--许多用于观察蛋白质相互作用的系统需要某种形式的外部输入来揭示相互作用。这意味着整个生物体或组织不能在这种情况下使用。我们设计了一种策略来绕过其中的许多问题，因此这代表了蛋白质相互作用分析的一个里程碑，并为进行更多生理相关分析打开了大门，并向研究人员揭示了更多相关数据。通过提取感兴趣的蛋白质并将其与能够在其他蛋白质上添加小标签的细菌酶相关联，我们可以定义感兴趣的蛋白质周围的蛋白质环境。更重要的是，酶和标签本身都是蛋白质，因此可以被编码为DNA，并放入研究有机体的基因组中。这意味着，第一次有可能在感兴趣的组织(植物种子、动物肝脏等)中确定蛋白质天然细胞环境中的蛋白质伙伴。在整个有机体(整个植物、动物等)中在生理相关的条件或压力下(低氧、干旱等)这项工作的目的是获得我们的基本数据的证据，并证明该系统可以用于从真菌、植物和动物等生物中发现新的蛋白质相互作用，从而推动迄今无法进入的研究领域的新的初级研究。

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.