Trim-Away degrades proteins by directly ubiquitinating them, but this does not require lysine residues or free N-termini
Protein depletion is a powerful strategy to study protein function and treat disease. Unlike CRISPR and RNAi approaches that alter a cells genetic information or how it is translated, protein depletion methods act directly at the protein level. Trim-Away is a depletion technology developed by Dean Clift, Melina Schuh and Leo James at the LMB that allows widely-available, off-the-shelf antibodies to be used to degrade targets. Leo James’ group in the LMB’s PNAC Division has now identified surprising protein modifications that underly Trim-Away protein degradation technology, which may explain its broad applicability to deplete almost any cellular protein.
Trim-Away exploits the natural function of the intracellular antibody receptor TRIM21, which targets antibody-bound pathogens for degradation when they enter the cell. To deplete a protein using Trim-Away, researchers can simply choose an antibody that binds to their protein of interest and deliver this antibody to cells by giving them a mild electric shock. Once inside cells the antibody binds its protein target, forming a protein-antibody complex that is recognised by TRIM21 and destroyed within minutes. However, how TRIM21 triggers the degradation of an antibody-bound protein has not been shown until now.
What is ubiquitination?
Ubiquitin is a small protein that can be attached to other proteins through a process called ubiquitination, where it acts as a signal to recruit the cell’s protein degradation machinery. Ubiquitin is normally attached to protein lysine residues, but serine, threonine, cysteine and N-terminal ubiquitination can also occur. TRIM21 is a ubiquitin ligase enzyme, meaning that it can catalyse the attachment of ubiquitin molecules to proteins.
Previous work from Leo’s group showed that TRIM21 can ubiquitinate itself specifically at its N-terminus. This type of modification was thought to be important for TRIM21 function and also the function of other proteins within the TRIM protein family. To directly test if TRIM21 N-terminal ubiquitination is required for TRIM21 function, Leo Kiss in collaboration with Jonas Weidenhausen in Irmgard Sinning’s lab at the University of Heidelberg developed a method to modify the TRIM21 protein N-terminus with an acetyl group to prevent its self-ubiquitination. Indeed, Leo found that this N-terminally acetylated TRIM21 was no longer degraded in cells because it could not ubiquitinate itself. Surprisingly, however, in infection and Trim-Away experiments carried out together with Tyler Rhinesmith, he found that the N-terminally acetylated TRIM21 was still able to efficiently degrade antibody-bound cellular proteins and invading pathogens.
Reconstituting Trim-Away in vitro revealed that, in addition to ubiquitinating itself, TRIM21 can also directly ubiquitinate antibody-bound proteins. Furthermore, proteins targeted by Trim-Away inside living cells were found to be ubiquitinated prior to their degradation. The finding that Trim-Away degrades proteins by directly ubiquitinating them led the researchers to ask which residues ubiquitin is attached to. Surprisingly, they found that if they removed all lysine residues from a protein and blocked its N-terminus by acetylation the protein was still efficiently degraded by Trim-Away.
The authors speculate that TRIM21 may have evolved a promiscuous ubiquitination mechanism to remain one step ahead of rapidly-evolving pathogens that can easily mutate residues normally prone to ubiquitination. This mode of action may also explain why Trim-Away technology can be used to degrade a wide-range of cellular proteins.
This work was funded by UKRI MRC, Boehringer Ingelheim Fonds and Wellcome Trust.
Trim-Away ubiquitinates and degrades lysine-less and N-terminally acetylated substrates. Kiss, L., Rhinesmith, T., Luptak, J., Dickson, CF., Weidenhausen, J., Smyly, S., Yang, JC., Maslen, SL., Sinning, I., Neuhaus, D., Clift, D., James, LC. Nature Communications
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