Taking aim at neurodegenerative diseases

A research team led by Harvard Medical School researchers zeroes in on an enzyme called Usp14

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BOSTON, Mass.—Scientists have discovered a small molecule that helps human cells get rid of the misfolded, disfigured proteins implicated in Alzheimer's disease and other neurodegenerative ailments. According to researchers, this finding could also have applications for other drugs as well.

Cells create and discard proteins continuously, a process that relies on a balance between the speed with which new proteins are created and damaged ones destroyed. Protein destruction occurs through a sophisticated system that marks proteins for disposal by tagging them with a small molecule called ubiquitin. Ubiquitin latches onto these proteins, often forming long chains. The cell's protein waste-disposal system, the proteasome, recognizes these ubiquitinated proteins and breaks them down.

If that finely tuned system malfunctions, damaged or misfolded proteins begin to accumulate in the cell and may become toxic. A number of ailments, including Parkinson's and Alzheimer's, have been linked to this buildup of misfolded proteins.

To better understand just what causes this malfunction, a research team led by Harvard Medical School researchers Daniel Finley, a professor of cell biology, and Randall King, associate professor of cell biology, zeroed in on an enzyme called Usp14. They found that, when activated, Usp14 disassembles the ubiquitin chain, slowing down the proteasome's ability to rid the cell of bad proteins. As a result, the cell makes new proteins more quickly than it rids itself of the old ones, leading to a build-up of misfolded proteins.

According to Finley, Usp14 inhibits the proteasome by attacking ubiquitin chains on the substrate protein.

"It was pure speculation when we set out, but happily it held up," he explains.

Finley explains that the Usp14 project grew out of another project on the lab, the work of an MD-PhD student, John Hanna.

"John used yeast as a model organism to study the regulation of the proteasome and he fixed on Ubp6, which is simply the yeast version of Usp14," he says. "Ubp6 was so interesting in John's hands that we tried our luck with the human form of the protein. Although, funny enough, what we then learned about Usp14 was something that we had not yet appreciated about Ubp6."

The researchers also faced technical challenges, the biggest being the ability to find a small-molecule inhibitor that was truly specific for Usp14, because it belongs to a large family of closely related enzymes, having almost 100 members—the deubiquitinating enzymes, or DUBs.

"The worry was that the inhibitors would all hit multiple members of this enzyme family, making them useless," Finley says. "That almost proved to be true; almost every inhibitor we found was 'nonspecific' in this way, but when we were about at the end of our rope we got lucky and got a couple of good ones from an initial pool of about 300 hits.

Another challenge was to find the right proteasome substrates to assay the effect of Usp14 on proteasome function.

"Here again, we probably benefited from some luck as we looked first at the substrates that were simply sitting around in our labs, and they worked just like that," Finley says. "But this challenge is still in front of us in many ways, because we now need to test what substrates respond to IU1 on a broader and maybe global scale."

The researchers wanted to see if they could find a molecule that inhibited Usp14, thus allowing the proteasome to work effectively. To identify such a selective inhibitor, Byung-Hoon Lee, a postdoctoral researcher, developed a special screening assay with assistance from the Institute of Chemistry and Cell Biology-Longwood Screening Facility at HMS. Lee screened 63,000 compounds, looking for molecules that inhibited only Usp14 and could easily infiltrate the cell. The strongest candidate was a small molecule they named IU1.
 Experimenting with both human and mouse cell cultures, Min Jae Lee, also a postdoctoral researcher, and his coworkers found that IU1 inhibited Usp14 and allowed the proteasome to dispose of proteins more quickly. In other words, adding IU1 to cells boosted proteasome activity.

"We think that IU1 has a simple mechanism: IU1 inhibits Usp14, which is a deubiquitinating enzyme," Finley says. "Deubiquitination is a reaction in which ubiquitin is cut away from the substrate protein of the proteasome, in this case, right before the subsrate is itself broken down in another subcomplex of the proteasome. We think that IU1 blocks the active site of Usp14."

Though scientists are still investigating just how IU1 works, it appears that the molecule suppresses Usp14's ability to trim the ubiquitin chain.

In addition to discovering IU1, the research also shed light on an aspect of proteasome function that was not previously understood, King explains. Scientists had thought that the proteasome was not involved in regulating the speed of protein degradation, but other proteins work with ubiquitin to modulate the process.

"Our work suggests that there is another level of control where the rate at which the proteasome can degrade the ubiquinated proteins is also controlled," King says. "It looks like there are multiple control steps along the way in this pathway."

As scientists learn more about the link between misfolded proteins and human disease, interest in proteasome has increased. While much of that focus has been on ways to inhibit proteasome function, there may be an advantage to developing a drug that boosts proteasome activity rather than hindering it, Finley speculates.

"If you take a typical cell growing in culture and kill its Usp14 activity, the cell will continue to thrive," he says. "If you kill its proteasome activity, it would immediately die."

This tool ultimately could have far-reaching implications for the development of drugs to treat not only neurodegenerative diseases, but also other illnesses that have been linked to an accumulation of misfolded proteins, King says.

For example, when a cell suffers oxidative damage—say from a stroke or heart attack—proteins could fold improperly and be marked for degradation by the ubiquitin system. If the proteasome becomes overwhelmed, misfolded proteins could accumulate in the cell, triggering a cascade of problems.

In this latest study, Finley explains that researchers induced  protein oxidation in cells and then treated them with IU1, which resulted in rapid elimination of the oxidized proteins. At the same time, the ability of cells to survive oxidative insult was enhanced.   

Patents are pending for IU1 and the assay used to identify the molecule.
While the discoveries offer plenty of hope, Finley says researchers have a lot to do before this could be a drug, including addressing for example safety and potency issues.

"If no compound from the IU1 series ever reaches the clinic, the idea is still there and maybe something acting in this way—by enhancing proteasome function—could ultimately work," he says.

The next step, Finley adds, is to learn what are the proteasome substrates that will respond to this compound and find the specific reasons why some respond and others don't.

"This should keep us occupied over the foreseeable future," he notes. "And importantly it could give us a better idea of what diseases our strategy is best suited for."

A major concern for the team, as scientists, is to determine how much their findings help in the understanding of how the proteasome works.

"Second, we want to know whether this research has a direct or indirect influence on medical practice sometime down the line," Finley concludes.

The research was funded by the National Institutes of Health, Harvard Technology Development Accelerator Fund, Merck & Co. and Johnson & Johnson.

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