By transplanting a plant-specific degradation pathway controlled by a phytohormone, auxin, into non-plant cells, researchers at the Japanese National Institutes of Genetics (NIG) have developed a rapid protein degradation process – the auxin-inducible degron (AID) technology.
In drug discovery, high hopes ride on degradation-based methods as a strategy to target proteins that are not easily subdued by classical inhibitors.
And in basic research, too, degradation can complement current knockout and knockdown methods to gain insights into protein function.
While genetic perturbation is a powerful approach to the analysis of protein function in vivo, genes essential for cell viability cannot be perturbed constitutively, and therefore, conditional inactivation or depletion of encoding proteins is useful for the functional analysis.
The AID technology works by attaching a small protein tag, known as a degron, fused to proteins of interest. This induces a rapid proteasomal degradation of the tagged protein subsequent to the addition of the plant hormone auxin to the culture medium.
AID technology allows for conditional depletion that is very rapid, typically with a half-life of 10-20 min. Examining the phenotype that is triggered by the depletion of the protein enables researchers to determine the function of the protein.
This technology, however, suffered from two major drawbacks -- one, a leaky degradation process where there is a weak degradation of the protein in the absence of auxin, and secondly, a requirement for high amounts of auxin. These negative features precluded the use of this technology for generating conditional gene knockout mice models.
"It can be difficult to precisely control the expression level of a protein of interest in living cells and apply this method to animal models," Masato Kanemaki told BioWorld Science. "The ability to knock out genes in mice is a critical step in biological research. An approach may work well in cultured cells, but it must work in a whole model system."
Kanemaki is a professor at the Molecular Cell Engineering Laboratory at NIG, Japan where his group focuses on the various applications of the AID technology.
In a paper published in the November 12, 2020, online issue of Nature Communications, senior author Kanemaki and his group described an improved version of AID technology-AID version 2. AID2 shows no detectable leaky degradation, and requires a significantly lower ligand concentration, and achieves even quicker degradation than the conventional AID.
AID2: An improved version
To establish the AID2 system, the researchers employed a "bump-and-hole" strategy to create an empty space in a mutant version of the TIR1 plant protein that recognizes and induces the degradation of degron-fused proteins. Additionally, they used an auxin analog, 5-Ph-IAA (5-phenyl-indole acetic acid) that can bind directly to the TIR1 mutant and initiate the degradation process, instead of using the actual auxin hormone.
Since the approach is very efficient, minimum quantities of the auxin analogue are needed. The researchers found that depletion could be induced at a concentration about 670 times lower than in the original system.
The AID2 system also allowed sharper and quicker control of functional protein in human cells than the original AID system. Further, using the AID2 technology, they were able to generate degron mutants that were difficult to establish using the AID system. This underscores the increased utility of the improved system. The AID2 system is also seen to control protein expression in xenograft tumors in mice and can prove to be an immensely useful tool in cancer drug discovery research.
Kanemaki and his group are open to collaborating with industry partners to develop this technology for application in drug discovery. They themselves plan to focus on basic science applications.
"With the AID2 system, it is possible to rapidly deplete a protein of interest in both cultured cells and mice-this greatly improves the application potential of this technology", Kanemaki said. "Next, we plan to apply the AID2 system to other model organisms and study different aspects of developmental and chromosomal biology. Considering the original AID system has already been applied to different eukaryotic organisms including yeast, Drosophila, C. elegans and zebrafish, we predict that AID2 system can also be applied to these organisms." (Yesbolatova, A. et al. Nat Commun 2020, 11(1): 5701).