How cellular processes round and unload damaged proteins

In a new paper with findings that senior author Eric Strieter of the University of Massachusetts Amherst calls “incredibly surprising,” he and his chemistry lab team report that they have discovered how an enzyme known as UCH37 regulates the management system of waste of a cell.

Strieter says, “It took eight years to figure this out, and I’m very proud of this work. We had to develop many new methods and tools to understand what this enzyme is doing.”

As he explains, a very large protease called a proteasome is responsible for the degradation of the vast majority of proteins in a cell; it can be composed of as many as 40 proteins. It has been known for more than 20 years that UCH37 is one of the regulatory enzymes that binds to the proteasome, he adds, “but nobody understood what it was doing.”

It turns out that the crux of the whole process, he adds, is how complicated the changes can be in a small protein called ubiquitin. “In addition to modifying other proteins, ubiquitin modifies itself to produce a wide range of chains. Some of these chains can have extensive branching. We found that UCH37 removes branchpoints from the chains, enabling the degradation process.”

Writing this week in Molecular cell, he and lead author and Ph.D. candidate Kirandeep Deol, who led and conducted the experiments, with co-authors Sean Crowe, Jiale Du, Heather Bisbee, and Robert Guenette, discuss how they answered the question. The work was supported by the NIH National Institute of General Medical Sciences.

This advance could eventually lead to a new cancer treatment, Strieter says, because cancer cells need the proteasome to grow and proliferate. “Many cancer cells are essentially dependent on proteasome function,” he points out. “Its cells produce proteins at such a high rate that mistakes are made and, if they are not eliminated, the cells cannot function. Since UCH37 helps eliminate proteins, it could be a useful therapeutic target to add to inhibitors of the proteasomes that have already been successful in the clinic “.

To begin their years-long process, says Strieter, “we had to find a way to generate a wide variety of ubiquitin chains that would represent potential diversity in a cell. Using that new ubiquitin chain library allowed us to interrogate the activity of UCH37 in a controlled environment. That series of experiments gave us the first clue that this enzyme was doing something unique. “

Another new method they have developed uses mass spectrometry to characterize the architecture of ubiquitin chains in complex mixtures. “This allowed us to see that the activity we discovered with our substrate library was also present in a more heterogeneous mix,” says Strieter. Finally, the chemists used the CRISPR gene editing tool to remove UCH37 from cells to measure the impact of UCH37 on proteasome-mediated degradation in vitro and in cells.

This technique led to another surprise. “Instead of acting as intended and opposing the degradation process, it turned out that UCH37 was removing branchpoints from ubiquitin chains to help break down proteins,” says Strieter. “One would think that by removing the signal for degradation the degradation would be compromised,” he adds, “but it didn’t work that way.”

In future experiments, Strieter and colleagues hope to further explore the degradation process and learn in more detail how UCH37 manages to regulate cellular function.