Researchers from Harvard Medical School (HMS) have discovered how a protein called midnolin plays a key role in degrading many short-lived nuclear proteins. Their study showed that midnolin acts by directly grabbing these proteins and pulling them into the cellular waste-disposal system, called the proteasome, where they are destroyed. And while cells typically tag proteins destined for proteasomal destruction with the small molecule ubiquitin, the newly reported study found that midnolin-proteasome mechanism bypasses this canonical ubiquitination system.

Because the proteins broken down by the newly identified process modulate genes with important functions related to the brain, the immune system, and development, scientists may eventually be able to target the process as a way of controlling protein levels to alter these functions and correct any dysfunction. “These particular short-lived proteins have been known for over 40 years, but no one had established how they are actually degraded,” said Xin Gu, PhD, a research fellow in neurobiology at HMS. “The mechanism we found is very simple and quite elegant,” commented Christopher Nardone, a PhD candidate in genetics at HMS. “It is a basic science discovery, but there are many implications for the future.”

Co-lead authors Gu, Nardone, and colleagues reported on their findings in Science, in a paper titled, “The midnolin-proteasome pathway catches proteins for ubiquitination-independent degradation,” in which they concluded, “Our study suggests that the midnolin-proteasome pathway may represent a general mechanism by which the proteasome bypasses the canonical ubiquitination system to achieve selective degradation of nuclear proteins, many of which are crucial for transcription.”

Short-lived proteins control gene expression in cells to carry out a number of vital tasks, from helping the brain form connections, to helping the body mount an immune defense. These proteins are made in the nucleus and are quickly destroyed once they’ve done their job. Yet despite the importance of these proteins, the process by which they get broken down and removed from cells once they are no longer needed has eluded scientists for decades.

It is well established that cells can break down proteins by tagging them with ubiquitin. The tag tells the proteasome that the proteins are no longer needed, and it destroys them. However, sometimes the proteasome breaks down proteins without the help of ubiquitin tags, pointing to the existence of another, ubiquitin-independent mechanism of protein degradation. “There has been sporadic evidence in the literature that somehow the proteasome can directly degrade unmarked proteins, but no one understood how that can happen,” Nardone said.

One group of proteins that seemed to be degraded by an alternative mechanism are stimuli-induced transcription factors. These proteins are made in response to cellular stimuli and travel to the nucleus of a cell to turn on genes, after which they are rapidly destroyed. “In mammals, the transcriptional response to growth factor, neuronal, and immune stimuli is mediated by a group of genes called immediate-early genes (IEGs),” the authors noted. “The IEG mRNAs accumulate within minutes after the initial stimulus and, once translated, their proteins are rapidly degraded to allow for a transient burst of protein expression.” Gu added, “What struck me in the beginning is that these proteins are extremely unstable and they have a very short half-life—once they are produced, they carry out their function, and they are quickly degraded afterwards.”

These transcription factors support a range of important biological processes in the body, yet even after decades of research, “the mechanism of their turnover was largely unknown,” noted Michael Greenberg, the Nathan Marsh Pusey Professor of Neurobiology in the Blavatnik Institute at HMS and a co-senior author on the paper with Stephen Elledge, the Gregor Mendel Professor of Genetics and of Medicine at HMS and Brigham and Women’s Hospital. The authors continued, “Although the mechanisms that regulate IEG transcription are well-characterized, how IEG proteins are swiftly targeted for destruction has remained mysterious for many years … it is unclear how IEG proteins are degraded.”

To identify the mechanism by which these proteins are destroyed once they’ve completed their job, the team focused on two familiar transcription factors, Fos, studied extensively by the Greenberg lab for its role in learning and memory, and EGR1, which is involved in cell division and survival. “We hypothesized that there exists a cellular pathway dedicated to the rapid destruction of c-Fos and other IEG proteins,” they wrote.

The team carried out genome-wide CRISPR-Cas9 screens to search for genes that regulated the stability of the IEG proteins in human cell lines engineered to express a reporter. The top hit from the screens was the MIDN gene, which, in mammals, encodes the largely uncharacterized midnolin protein. The team validated their screening results by confirming that knocking out MIDN in human cell lines increased the stability of both EGR1 and FosB. Interestingly, they found, overexpression of midnolin decreased levels of the cFos, FosB, and EGR.

The follow-up experiments revealed that in addition to Fos and EGR1, midnolin may also be involved in breaking down hundreds of other transcription factors in the nucleus. “Through a gain-of-function genetic screen, we identified a large group of potential targets of midnolin strongly enriched for nuclear proteins, especially transcriptional regulators, revealing that midnolin functions broadly to promote the degradation of proteins in the nucleus, where midnolin itself is predominantly localized,” the investigators noted.

Gu and Nardone recall being shocked and skeptical about their results. To confirm their findings, they decided they needed to figure out exactly how midnolin targets and degrades so many different proteins. “Once we identified all these proteins, there were many puzzling questions about how the midnolin mechanism actually works,” Nardone said.

With the aid of a machine learning tool called AlphaFold that predicts protein structures, in combination with data from a series of lab experiments, the team was able to flesh out the details of the mechanism. “To gain insight into how midnolin interacts with the proteasome and its numerous substrates, we used AlphaFold to obtain a predicted structure of midnolin, which revealed three confidently predicted and highly conserved regions with defined structure,” they wrote. The researchers called one of these regions in midnolin the “Catch domain,” as it represents the part of midnolin protein that effectively grabs other proteins and feeds them directly into the proteasome, where they are broken down. The team found that the Catch domain is composed of two separate regions linked by amino acids. And interestingly, they noted, “Deletion of the regions that fold together to form the Catch domain (the N-terminal Catch1 and C-terminal Catch2 subdomains) abolished the interaction of midnolin with its substrates without affecting its ability to bind the proteasome.” The Catch domain effectively grabs a relatively unstructured region of a protein, allowing midnolin to capture many different types of proteins.

Of note are proteins such as Fos, which are responsible for turning on genes that prompt neurons in the brain to wire and rewire themselves 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. Summarizing their findings, the authors noted, “Our results suggest that the midnolin-proteasome pathway may represent a general mechanism by which the proteasome bypasses the traditional ubiquitination system to achieve selective degradation of many nuclear proteins. Elledge added, “The most exciting aspect of this study is that we now understand a new general, ubiquitination-independent mechanism that degrades proteins.”

In the short term, the researchers want to delve deeper into the newly discovered midnolin mechanism. They are planning structural studies to better understand the finer details of how midnolin captures and degrades proteins. They are also creating engineered mice that lack midnolin, to help gain new insights into the protein’s role in different cells and stages of development.

However, they also acknowledged that the mechanism by which midnolin kicks off the destruction of its bound proteins isn’t yet fully recognized. “How midnolin initiates the degradation of bound substrates is not completely understood mechanistically. We do not know if midnolin interacts with its targets before binding to the proteasome or if it associates with the proteasome constitutively and then recruits its substrates, thus defining a new subclass of proteasomes in the nucleus.”

The scientists do suggest that the finding has tantalizing translational potential. It may offer a pathway that could be harnessed to control levels of transcription factors, thus modulating gene expression, and in turn the associated processes in the body. “Protein degradation is a critical process and its deregulation underlies many disorders and diseases,” including certain neurological and psychiatric conditions, as well as some cancers, Greenberg said.

For example, when cells have too much or too little of transcription factors such as Fos, problems with learning and memory may arise. As the authors further suggested, “Given that many IEG proteins are efficiently targeted for degradation by midnolin and that the precise expression of IEGs is critical for learning and memory, it is possible that disrupting or boosting midnolin function could affect the ability of animals to efficiently learn and store information in the brain.” And in multiple myeloma, cancer cells become addicted to the immune protein IRF4, so its presence can fuel the disease. “ … our finding that IRF4 is potently targeted for degradation by midnolin may provide insights into the function of midnolin in the immune system,” they added.

The researchers are especially interested in identifying diseases that may be good candidates for the development of therapies that work through the midnolin-proteasome pathway. “One of the areas we are actively exploring is how to tune the specificity of the mechanism so it can specifically degrade proteins of interest,” Gu said.

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