A surprising tag

Ubiquitin can mark drug-like molecules in cells

Ubiquitin determines the fate of many proteins in human cells: It decides whether they remain active or are broken down and which functions they perform. Acting as molecular tagging machines, ubiquitin ligases are indispensable in this process. They attach the small protein ubiquitin to the target proteins to be controlled. Researchers at the Max Planck Institute (MPI) for Multidisciplinary Sciences in Göttingen (Germany) have now discovered that the human ubiquitin ligase HUWE1 can attach ubiquitin to more than just cellular proteins. It can tag externally added drug-like molecules, with effects on the cellular ubiquitin system. These findings could open up new avenues in drug discovery and biotechnology.

In a nutshell:

• Surprising tag: Ubiquitin can be attached to drug-like molecules added to living cells. Cellular ubiquitination had previously only been observed for proteins, sugars, and other biomolecules.
• Widespread principle: In addition to HUWE1, other ubiquitinating enzymes can mark synthetic molecules with ubiquitin in cells.
• Effect on the ubiquitin system: This discovery could provide new molecular tools for externally interfering with the ubiquitin system and thus influencing disease processes.

Small but powerful: Ubiquitin controls the lifespan and distribution of proteins in the cell, but it can also determine their shape, function, or interactions with other cellular components. Ubiquitin ligases are key to this process because they reliably recognize the relevant proteins among tens of thousands of molecules and confer the correct instructions. Disruptions of this precise tagging routine can result in faulty cellular processes and diseases such as cancer. Conversely, drugs can be designed to modulate ubiquitin ligases, directing them to tag specific disease-promoting proteins with ubiquitin for degradation However, this principle is currently only applicable to a few of the over 600 human ubiquitin ligases.

The ubiquitin ligase HUWE1 is an interesting, yet unexploited target for therapeutic strategies with important roles in tumors and neurodevelopmental disorders. Although drug-like compounds have been used to inhibit HUWE1 in scientific research, their mechanism of action remained unclear, hindering their advancement into potential therapeutic agents.

Challenging discovery process

A team led by Sonja Lorenz at the MPI for Multidisciplinary Sciences has now unraveled the unexpected mechanism by which these compounds act. In an interdisciplinary approach, the researchers combined protein biochemistry, cell biology, and click chemistry. Other key methods for the project’s success were tailor-made molecular syntheses from medicinal chemists led by Matthias Gehringer at the University of Tübingen, as well as the mass spectrometric measurements by Henning Urlaub’s research group and molecular dynamics simulations by colleagues from Helmut Grubmüller’s department at the MPI, Lorenz emphasizes.

“We found that the drug-like compounds marketed as HUWE1 inhibitors indeed interact with this enzyme – but they do not inhibit it. Rather, HUWE1 recognizes the compounds themselves as target molecules and tags them with ubiquitin,” reports Lorenz. “When the drug-like compounds are supplied in excess over a natural target protein in the test tube, they consume the ubiquitin. This competition between the drug-like compounds and the target protein is what had previously been misinterpreted as HUWE1 inhibition,” adds Pavel Pohl, one of the lead authors of the study now published in the journal Nature Communications.

The team also succeeded in demonstrating, for the first time, that the synthetic compounds are tagged with ubiquitin in living cells. However, the situation here is much more complex than in the test tube. “In the cell, HUWE1 promotes the ubiquitination of the compounds, but it does not drive it exclusively,” explains Barbara Orth, another lead author of the study. This implies that cellular ubiquitination enzymes other than HUWE1 can also tag drug-like molecules with ubiquitin. This finding is of fundamental importance because ubiquitin signals had previously only been observed on proteins, sugars, and other biomolecules in cells, but not on synthetic compounds.

“The novel substrate spectrum will be particularly interesting for therapeutic and biotechnological applications in the ubiquitin field. Our discovery offers tangible strategies for developing new molecular tools to modulate the cellular ubiquitin system and, in turn, influence disease processes,” explains Lorenz. (cr/sl)