To investigate a protein’s function, one of the most important strategies is to remove it from a cell and to study the effects. Currently, researchers have two main techniques: genome editing by CRISPR/Cas, and RNA interference (RNAi). By targeting a cell’s DNA or RNA, respectively, they efficiently shut down the production of a protein. However, these methods affect protein levels only indirectly and are not applicable to every type of cell and protein.
This new technique, published in Cell, utilises a previous discovery at the MRC LMB: the protein Trim21. Trim21 recognises antibodies which 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.
Targeting specific proteins
The researchers co-opted this natural system to target specific cellular proteins, by delivering antibodies directed against the target protein into the cell. Trim21 recognised the antibody and delivered the antibody-bound protein to the proteasome for destruction. Within minutes, the protein disappeared from the cell.
“When we first identified Trim21 as an antibody receptor over ten years ago and showed how efficiently it destroys viral proteins, we realised it could be a powerful tool if re-tasked against cellular proteins. However, the results are even more remarkable than we could have imagined,” said Dr Leo James, an author on the study from the MRC LMB.
A difficulty was that many cell types do not have enough 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’ (electroporation) made the cell take up the proteins.
“We can now take basically any cell from the body and rapidly destroy proteins inside this cell, allowing us to immediately study the effects on cellular processes,” said Dr Dean Clift, an author on the study from the MRC LMB.
Dr Melina Schuh, an author on the study from the MPI for Biophysical Chemistry, said: “With Trim-Away, it is possible for the first time to directly target almost any protein in any type of cell. It is very simple to use and removes proteins within minutes. This is much faster than anything you can achieve with genome editing or RNAi – with these techniques, it typically takes many hours or even days to deplete a protein. This gives the cell time to develop mechanisms to compensate for the loss, which sometimes masks the actual effects. Moreover, genome editing and RNAi are unsuitable for studying long-lived proteins or proteins in primary cells. With Trim-Away, we can now close this gap.”
Trim-Away destroyed disease-causing protein variant
The researchers then took advantage of the remarkable specificity of antibodies to distinguish between two different variants of the same protein. Such variants play important roles in many diseases. A prominent example is Huntington’s disease, the inheritable neurodegenerative disorder caused by a mutation in one of an individual’s two copies of 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,” Dr Schuh emphasised. “A therapeutic application is still far-off. But our work may open up new venues for treating diseases with antibodies in the future.”
Dr Nathan Richardson, MRC head of molecular and cellular medicine, commented: “The ability to directly target and knockdown specific proteins and study the effect within live cells is an important development and will be a key tool to allow scientists to better understand that protein’s role in normal and disease states. Applying this technique in cells taken from clinical samples and non-dividing cells that are currently intractable to existing knockdown techniques is particularly exciting, as are the potential opportunities to develop this technique as a new therapeutic approach.”
This research was funded by the MRC, Wellcome Trust, European Research Council and European Commission.
Image: Trim-Away halves the amount of fluorescent protein in an egg cell within nine minutes (from left: before introduction of antibodies and 10, 30, and 60 minutes after). Credit: Dean Clift / MRC LMB.