Short-lived proteins control gene expression in cells and perform many important tasks, from helping the brain form connections to aiding the body's immune defenses. These proteins are produced in the nucleus and are quickly destroyed once they have done their job. Despite their importance, these proteins have been unknown for decades by the process by which they are degraded and removed from cells when they are no longer needed.

In a new study, researchers from Harvard Medical School have identified a protein called midnolin that plays a key role in degrading many short-lived nuclear proteins. Their research shows that midnolin works by grabbing these proteins directly and pulling them into the cell's waste disposal system, the proteasome, where it destroys them. The relevant research results were published in Science on August 25, 2023, with the title of the paper "The midnolin-proteasome pathway catches proteins for ubiquitination-independent degradation".

"These particular short-lived proteins have been known for more than 40 years, but no one was sure exactly how they were degraded," said co-first author Xin Gu, a neurobiology researcher at Harvard Medical School.

Because short-lived proteins that undergo degradation during this process regulate genes with important functions related to the brain, immune system, and development, scientists may eventually be able to target this process as a way to control protein levels to alter these functions and correct any dysfunctional approach.

Co-first author Christopher Nardone, a Ph.D. student in genetics at Harvard Medical School, added, "The mechanism we discovered is remarkably simple and elegant. It's a fundamental science discovery, but it has many implications for the future."

Molecular mystery

Cells are known to degrade them by using a small marker protein called ubiquitin. This tag tells the proteasome that the proteins are no longer needed, thus destroying them. The late Fred Goldberg did much of the groundbreaking research on this process at Harvard Medical School. However, sometimes the proteasome degrades proteins without the help of the ubiquitin tag, leading scientists to speculate on the existence of another ubiquitin-independent protein degradation mechanism.

"There's anecdotal evidence in the literature that somehow the proteasome can directly degrade proteins that aren't tagged with ubiquitin, but no one understands how that happens," Nardone said.

A group of proteins that appear to be degraded by another mechanism are stimulus-induced transcription factors: proteins that are produced rapidly in response to a cellular stimulus, travel to the nucleus to turn on genes, and are quickly destroyed.

"What struck me at first was that these proteins are extremely unstable, and they have a short half-life, once produced, they perform their function and are then degraded very quickly," Gu said.

"These transcription factors support a host of important biological processes in the body, but even after decades of research, their turnover mechanisms remain largely unknown," said co-corresponding author Michael Greenberg, professor of neurobiology at the Blavatnik Institute at Harvard Medical School. Another co-corresponding author of the paper is Stephen Elledge, professor of genetics and medicine at Harvard Medical School and Brigham and Women's Hospital.

From few to hundreds

To investigate this mechanism, the authors started with two familiar transcription factors: Fos, whose role Greenberg's lab has extensively studied in learning and memory, and EGR1, which is involved in cell division and survival.

Using complex protein and gene analysis methods developed in Elledge's lab, the authors focused on midnolin, a protein that helps degrade the two transcription factors. Subsequent experiments found that, in addition to Fos and EGR1, midnolin may also be involved in degrading hundreds of other transcription factors in the nucleus.

Gu and Nardone recall being shocked and skeptical by their findings. To confirm their discovery, they decided to find out how midnolin targets and degrades so many different proteins.

"Once we've identified all of these proteins, there are still a lot of puzzling questions about how exactly this midnolin degradation mechanism works," Nardone said.

Using a machine-learning tool called AlphaFold, which predicts protein structure, and the results of a series of laboratory experiments, the authors were able to flesh out the details of the mechanism. They found that midnolin has a "catch domain", a region of the protein that catches other proteins and sends them directly to the proteasome for subsequent degradation. This "capture domain" consists of two separate regions linked together by amino acids that grab onto a relatively unstructured region of the protein, allowing midnolin to capture a variety of different types of proteins.

Notably, proteins like Fos are responsible for switching on genes that prompt neurons in the brain to wire and rewire in response to stimuli. Other proteins, such as IRF4, activate genes that support the immune system by ensuring that cells can make functional B and T cells.

"What's most exciting about this new study is that we now understand a new general mechanism for degrading proteins that doesn't depend on ubiquitination," Elledge said.

Attractive conversion potential

In the short term, the authors hope to delve deeper into the mechanisms they discovered. They are planning structural studies to better understand the details of how midnolin traps and degrades proteins. They are also creating midnolin-deficient mice to understand the protein's role in different cells and developmental stages.

According to the authors, their findings have tantalizing translational potential. It may provide a way for scientists to control the levels of transcription factors that regulate gene expression and, in turn, related processes in the body.

"Protein degradation is a critical process, and dysregulation of protein degradation underlies many diseases, including certain neurological and psychiatric disorders, as well as some cancers.” Greenberg said.

For example, learning and memory problems can arise when cells have too much or too little of a transcription factor like Fos. In multiple myeloma, cancer cells become addicted to the immune protein IRF4, so its presence fuels the disease. The authors were particularly interested in identifying which diseases might be treated by developing therapies based on the midnolin-proteasome pathway.

"One area we're actively exploring is how to tune the specificity of this mechanism so that it specifically degrades the protein of interest," Gu said.