AID2: A POWERFUL TOOL FOR TURNING OFF PROTEINS ON-DEMAND
In the complicated
universe of cellular biology, researchers tend to wonder what occurs when a
particular protein is lost. But what if the protein is necessary for the
survival of the cell? To delete it permanently would destroy the cell, so it
cannot be studied. Conditional protein depletion to the rescue—a sneaky trick
that enables scientists to degrade a protein temporarily and see what occurs in
real time. One of the best systems designed for this end is the Auxin-Inducible
Degron (AID) system, and its mighty counterpart, AID2.
The AID system borrows
from plants, which employ a hormone known as auxin (more precisely,
indole-3-acetic acid or IAA) to control the breakdown of specific proteins. In
this process, auxin binds to a protein known as TIR1, which is incorporated
into an E3 ubiquitin ligase complex (SCF–TIR1). This complex labels AUX/IAA
proteins for breakdown by the cell's protein-waste disposal system—the
proteasome. Scientists used this pathway in yeast and mammalian cells by
expressing a version of the plant TIR1 protein (from Oryza sativa, or rice),
termed OsTIR1. If a protein of interest is tagged with a small tag called
mini-AID (mAID), it becomes subject to degradation in the presence of auxin.
This system allowed
scientists to rapidly eliminate specific proteins and observe immediate
effects, without waiting for long-term genetic changes or compensatory pathways
to kick in. However, despite its utility, the original AID system had a few
limitations. One major issue was leaky degradation—proteins could still be
partially degraded even without adding auxin, which was a serious problem when
studying essential proteins. Another issue was the need for high auxin
concentrations (typically 100–500 µM) to induce degradation, which could be
toxic to cells. These drawbacks also made it difficult to generate stable cell
lines, especially when targeting essential proteins.
To overcome these
limitations, researchers created a better version called AID2. The AID2 system
overcomes all the significant limitations of the initial method. It employs a
mutated form of the TIR1 protein named OsTIR1(F74G), which significantly
minimizes background degradation. It also substitutes natural auxin with a
synthetic compound named 5-phenyl-indole-3-acetic acid (5-Ph-IAA), which
functions more efficiently and specifically. This synergy enables degradation
to be achieved at extremely low ligand concentrations (generally below 1 µM),
reducing cellular stress.
What gives AID2 real potency is
its accuracy. It provides fine control over when and where a
protein gets broken. There is no breakdown in the absence of 5-Ph-IAA,
which makes it particularly suitable for researching genes that
are required for the cell. Also, the system has been found to function in
yeast, mammalian cells, and even mice, so it can be used in a variety of
experimental systems.
In simple terms, first,
the protein you wish to deplete is tagged with the mAID
sequence. Next, you employ a cell line that expresses
OsTIR1(F74G). When you're ready to deplete the protein, you add 5-Ph-IAA.
This causes the SCF–OsTIR1 (F74G) complex to identify and
ubiquitinate the mAID-tagged protein, which is subsequently degraded quickly by
the proteasome. The whole process is rapid, typically in a matter of hours, and
reversible, so the protein can re-emerge once the ligand
is taken away.
Creating a cell line that
uses AID2 involves three main steps. First, a parental cell line is generated
to express OsTIR1(F74G), often using stable integration methods. Next, the gene
of interest is tagged with mAID at its endogenous locus using CRISPR-Cas9 and a
donor plasmid. Finally, when 5-Ph-IAA is added, the tagged protein is quickly
degraded, allowing researchers to study its immediate cellular function.
To demonstrate the
effectiveness of gene tagging with mAID, researchers successfully tagged the N-terminus
of MCM3 and the C-terminus of MCM4, two essential DNA replication
proteins in HCT116 cells. Genotyping results confirmed both mono-allelic
and bi-allelic insertion of mAID tags. Bi-allelic clones produced only
the a-2 PCR products using specific primers, while mono-allelic clones
generated both a-1 and a-2 bands. The bi-allelic clones proved to be fully
functional, since MCM3 and MCM4 are essential, the viability of these clones
confirmed that the tagged proteins retained their activity before degradation.
Further validation was
done via western blotting. In bi-allelic clones, MCM3 and MCM4 fused with mAID
were observed as higher molecular weight bands compared to the untagged
versions in the parental cells. Importantly, upon treatment with 5-Ph-IAA,
these fusion proteins were specifically degraded. Anti-mAID antibodies detected
only the tagged versions of MCM3 and MCM4, confirming the specificity and
efficiency of the system.
Saito Y, Kanemaki MT. Targeted Protein Depletion Using the Auxin‐Inducible Degron 2 (AID2) System. Current Protocols. 2021 Aug;1(8):e219.
IMAGE CREDIT
Current Protocols, https://images.app.goo.gl/1TJ1UtQkTYGXsYSZ9
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