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

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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.