Deprived of one of its proteins, a cell may betray the protein’s function—but not always. If the cell is deprived of the protein indirectly, at the level of protein-coding DNA or RNA (that is, via CRISPR/Cas9 knockout or RNA interference [RNAi] knockdown), the cell will experience a deficit only after many hours or even days have passed. The cell may have time to develop mechanisms to compensate for the loss of a protein. And so, the cell may effectively hide the protein’s function, particularly if the protein is long-lived.
To assess protein function more directly—and quickly—scientists based at the Medical Research Council (MRC) in the U.K. and the Max Plank Institute (MPI) in Germany developed a new technique. It’s called Trim-Away. Unlike CRISPR/Cas9 and RNAi, it can deplete a targeted protein from any cell type, including nondividing primary cells, which resist DNA- and RNA-targeting techniques. Also, Trim-Away can distinguish between different variants of a protein, opening new paths to disease research or even, eventually, the development of new therapies.
Details about Trim-Away appeared November 16 in the journal Cell, in an article entitled “A Method for the Acute and Rapid Degradation of Endogenous Proteins.” The article emphasizes that because Trim-Away utilizes antibodies, it can be applied to a wide range of target proteins using off-the-shelf reagents.
“Here, we present Trim-Away, a technique to degrade endogenous proteins acutely in mammalian cells without prior modification of the genome or mRNA,” wrote the article’s authors. “Trim-Away harnesses the cellular protein degradation machinery to remove unmodified native proteins within minutes of application.”
Trim-Away works so rapidly, the authors indicated, it minimizes the risk that phenotypes are compensated and that secondary, nonspecific defects accumulate over time.
Central to the new technique is a protein—Trim21—that had been discovered in the lab of Leo James, Ph.D., at the MRC Laboratory of Molecular Biology. Trim21 recognizes antibodies that enter the cell attached to viruses. It binds to these antibodies, tags the antibody–virus complex as “garbage,” and hands it over to the cell's “garbage chute,” the proteasome.
“Basically, Nature's toolbox provided us with all the components we needed,” explained Melina Schuh, Ph.D., one of the Cell article’s corresponding authors and director of biophysical chemistry at the MPI. The trick was to choose the right ones and to combine them into a system that works for our purpose.
Schuh realized that this ability of Trim21 could help her to overcome a problem she had been facing in her research: It had proven exceptionally difficult to deplete specific proteins from egg cells by genome editing or RNAi, as many proteins in these cells are very long-lived. Schuh now wanted to use Trim21 as a molecular tool.
Together with colleagues at the MRC, Schuh introduced antibodies into the egg cells that were directed against a specific cellular protein, instead of being directed against viruses. Trim21 recognized the antibody and delivered the antibody-bound protein to the proteasome for destruction. Within minutes, the protein disappeared from the cell. This redirection of Trim21 to the protein of interest is the central principle of Trim-Away.
A difficulty was that many cell types do not have sufficient amounts of Trim21 to cope with the task of removing all of the antibody-bound protein. The researchers overcame this problem by delivering additional Trim21 protein into the cell together with the antibody. A small “electric shock” made the cell take up the proteins.
“When we first identified Trim21 as an antibody receptor over ten years ago and subsequently showed how efficiently it destroys viral proteins, we realized it could be a powerful tool if retasked against cellular proteins. However, the results are even more remarkable than we could have imagined,” James says. This also holds true for Trim-Away's applicability to long-lived proteins and primary cells, which are cells that are taken directly from a tissue.
Another application is in macrophages, a type of white blood cell: “Macrophages are completely inaccessible to genome editing or RNAi because they are particularly good at recognizing foreign DNA and RNA, which are central components of those techniques”, James noted. “With Trim-Away, it is now possible to deplete proteins from macrophages to study their function in this specific cell type.”
A feature of Trim-Away is it can take advantage of the remarkable specificity of antibodies, which can not only distinguish between different proteins but also between two different variants of the same protein. Such variants play important roles in many diseases.
A prominent example is Huntington's disease, an inheritable neurodegenerative disorder caused by a mutation in one of an individual's two copies of the gene coding for the protein huntingtin. The scientists showed that Trim-Away can be used to remove the disease-causing variant of huntingtin from tissue culture cells while leaving the “normal” variant unscathed.
“Of course, getting this to work in cell culture is something completely different than curing the disease,” Schuh emphasized. “A therapeutic application is still far off. But our work may open up new venues for treating diseases with antibodies in the future.”