Researchers have found a way to control protein levels inside different tissues of a whole, living animal for the first time. The method lets scientists dial protein levels up or down with great precision during the animal's entire life, a technological advance which can help them study the molecular underpinnings of ageing and disease.
Scientists at the Centre for Genomic Regulation in Barcelona and the University of Cambridge successfully tested the technique by controlling how much protein was present in the intestines and neurons of the nematode worm Caenorhabditis elegans. Their findings are described today in the journal Nature Communications.
The study paves the way for designing completely new experiments that were impossible to carry out with current techniques, like understanding how much of a protein is needed to maintain good health, or tracing how small perturbations to a protein in one tissue can ripple across the whole body.
"No protein acts alone. Our new approach lets us study how multiple proteins in different tissues cooperate to control how the body functions and ages," says Dr. Nicholas Stroustrup, researcher at the Centre for Genomic Regulation and senior author of the paper.
The new method will have particularly important implications for studying whole-body, systemic processes like ageing, which are shaped by constant interactions between different organs. If a protein affects lifespan in different ways across different body parts, traditional methods can't always separate those effects using typical on/off experiments with genes.
This lack of precise, lifelong, tissue-by-tissue control has made it difficult to understand how different parts of our body drive ageing, how they talk to each other and how subtle molecular changes ripple through the entire organism over time. According to the authors of the study, what's also been missing until now is finesse and calibration.
"To unpick nuance in biology, sometimes you need half the concentration of a protein here and a quarter there, but all we've had up till now are techniques focused on wiping a protein out. We wanted to be able to control proteins like you turn the volume up or down on a TV, and now we can now ask all sorts of new questions," explains Dr. Stroustrup.
The technique is an adaptation of existing technology which originates from plant biology. Plants use a hormone called auxin to control growth. Researchers working with yeast created a popular lab tool known as the auxin-inducible degron system, also known as the AID system.
It works by tagging a protein with a tag, also known as a degron. An enzyme called TIR1 recognises the degron and destroys the protein, but only when auxin is present. Remove the plant hormone and the protein comes back. Since its discovery, the AID system has become a widely used tool in cells and model organisms for rapid, reversible protein control.
Now, by engineering different versions of the TIR1 enzyme and degrons and testing them across more than one hundred thousand nematodes, the researchers have created a newer, more flexible version which they call a "dual-channel" AID system.
Rather than switching proteins on or off, the technique allows scientists to control how much of a protein remains, where in the body it is controlled and when the change happens. All this while the animal continues to live normally: eating, moving and growing as the system quietly adjusts protein levels inside the tissues of its body.
The new technique works by attaching to the end of the target protein a degron tag and then genetically engineering worms to produce a TIR1 enzyme in specific tissues only. When the worms are fed auxin-containing food, the plant hormone activates TIR1, which recognises the degron tag and tells the cell to remove just the right amount of that protein.
The important innovation was combining two different TIR1 enzymes, each triggered by a different auxin compound. By placing them in different tissues, they could independently control the same protein in the worm's intestine and in its neurons or even control two different proteins at the same time.
The researchers also overcame another hurdle, which is that AID systems often fail to work in reproductive tissues. The team traced this to a biological process in the germline and adapted their new system to get around it, creating a tool that works across the whole body, including reproductive cells.
"Getting this to work was quite an engineering challenge. We had to test different combinations of synthetic switches to find the perfect pair that didn't interfere with one another. Now that we've cracked it, we can control two separate proteins simultaneously with incredible precision. It's a powerful tool that we hope will open up new possibilities for biologists everywhere," concludes Dr. Jeremy Vicencio, postdoctoral researcher at the Centre for Genomic Regulation and coauthor of the study.
