Summary: Researchers identified a critical protein called midnolin that degrades short-lived nuclear proteins, solving a long-standing biological mystery. These proteins play essential roles in gene expression, affecting brain development, learning, and immune responses.
Midnolin ushers these proteins into the cell’s waste disposal system—the proteasome—for destruction. This groundbreaking discovery has promising implications for therapies targeting neurological disorders and cancers.
Midnolin directly targets short-lived proteins for degradation, facilitating crucial cellular processes such as gene modulation and immune response.
This discovery resolves decades-long questions about a ubiquitin-independent mechanism for protein degradation.
Manipulating the midnolin-proteasome pathway may lead to new therapies for a range of diseases including neurological disorders and certain cancers.
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.
Despite their importance, the process by which these proteins get broken down and removed from cells once they are no longer needed has eluded scientists for decades — until now.
In a cross-departmental collaboration, researchers from Harvard Medical School identified a protein called midnolin that plays a key role in degrading many short-lived nuclear proteins.
The study shows that midnolin does so by directly grabbing the proteins and pulling them into the cellular waste-disposal system, called the proteasome, where they are destroyed.
The findings are published Aug. 24 in Science.
“These particular short-lived proteins have been known for over 40 years, but no one had established how they are actually degraded,” said co-lead author Xin Gu, a research fellow in neurobiology at HMS.
Because the proteins broken down by this 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.
“The mechanism we found is very simple and quite elegant,” added co-lead author Christopher Nardone, a PhD candidate in genetics at HMS. “It is a basic science discovery, but there are many implications for the future.”
A molecular mystery
It is well established that cells can break down proteins by tagging them with a small molecule called ubiquitin. The tag tells the proteasome that the proteins are no longer needed, and it destroys them. Much of the pioneering research on this process was done by the late Fred Goldberg at HMS.
However, sometimes the proteasome breaks down proteins without the help of ubiquitin tags, leading researchers to suspect that there was 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: Proteins rapidly made in response to cellular stimuli that travel to the nucleus of a cell to turn on genes, after which they are rapidly destroyed.
“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,” Gu said.
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,” said 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.
From a handful to hundreds
To investigate this mechanism, the team began with 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.
Using sophisticated protein and genetic analyses developed in the Elledge lab, the researchers homed in on midnolin as a protein that helps break down both transcription factors.
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.
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, plus results from a series of lab experiments, the team was able to flesh out the details of the mechanism. They established that midnolin has a “Catch domain” — a region of the protein that grabs other proteins and feeds them directly into the proteasome, where they are broken down.
This Catch domain is composed of two separate regions linked by amino acids (think mittens on a string) that grab a relatively unstructured region of a protein, thus allowing midnolin to capture many different types of proteins.
Of note are proteins like Fos that are responsible for turning on genes that prompt neurons in the brain to wire and rewire themselves in response to stimuli. Other proteins like IRF4 activate genes that support the immune system by ensuring that cells can make functional B and T cells.
“The most exciting aspect of this study is that we now understand a new general, ubiquitination-independent mechanism that degrades proteins,” Elledge said.
Tantalizing translational potential
In the short term, the researchers want to delve deeper into the mechanism they discovered. They are planning structural studies to better understand the fine-scale details of how midnolin captures and degrades proteins. They are also making mice that lack midnolin to understand the protein’s role in different cells and stages of development.
The scientists say their finding has tantalizing translational potential. It may offer a pathway that researchers can harness to control levels of transcription factors, thus modulating gene expression, and in turn, 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. In multiple myeloma, cancer cells become addicted to the immune protein IRF4, so its presence can fuel the disease.
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.
Authorship, funding, disclosures
Additional authors on the paper include Nolan Kamitaki of HMS and Aoyue Mao of HMS, Brigham and Women’s, and Harvard University.
Funding: Funding was provided by a National Mah Jongg League Fellowship from the Damon Runyon Cancer Research Foundation, a National Science Foundation Graduate Research Fellowship, and the National Institutes of Health (T32 HG002295; R01 NS115965; AG11085).
Elledge is a founder of TScan Therapeutics, Maze Therapeutics, ImmuneID, and Mirimus, serves on the scientific advisory boards of Homology Medicines, ImmuneID, Maze Therapeutics, X-Chem, and TScan Therapeutics, and is an advisor for MPM Capital.
About this genetics research news
Author: Dennis Nealon Source: Harvard Contact: Dennis Nealon – Harvard Image: The image is credited to Neuroscience News
The midnolin-proteasome pathway catches proteins for ubiquitination-independent degradation
In mammals, the transcriptional response to growth factor, neuronal, and immune stimuli is mediated by a group of genes called immediate-early genes (IEGs), which encode transcription factors of the Fos, EGR, and NR4A families.
IEG proteins are activated stereotypically in virtually all mammalian cells but promote the transcription of late-response genes (LRGs) that are cell-type specific and crucial for the appropriate response to the initial stimulus. The physiological importance of IEGs is underscored by the fact that misregulation of their expression can lead to cancer, immune deficiencies, and neurological disorders.
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. Although the mechanisms that regulate IEG transcription are well characterized, how IEG proteins are swiftly targeted for destruction has remained mysterious for many years.
Eukaryotic cells rely on a macromolecular protease called the proteasome that canonically degrades proteins marked with ubiquitin. It has been suggested that the Fos family is targeted to the proteasome by both ubiquitination-dependent and -independent mechanisms, but the molecular events that orchestrate these processes have remained elusive. We hypothesized that there exists a cellular pathway dedicated to the rapid destruction of c-Fos and other IEG proteins. By harnessing the power of forward genetic screens, we sought to identify the machinery that controls the degradation of these proteins.
We performed genome-wide CRISPR-Cas9 screens to search for genes that regulate the stability of IEG proteins. We found that midnolin, a largely uncharacterized protein in mammals, promoted the proteasomal destruction of IEG proteins from structurally distinct families including c-Fos, FosB, EGR1, and NR4A1. These results prompted us to search for additional midnolin targets.
We used the global protein stability (GPS) assay with a human open reading frame library (ORFeome) to assess changes in protein stability for ~12,000 human proteins simultaneously. In addition to IEG proteins, midnolin promoted the degradation of IRF4, NeuroD1, PAX8, GATA1, and many other cell-type–specific transcriptional regulators in the nucleus, where midnolin itself resides.
Diverse stimuli that activate IEGs also induced midnolin, and midnolin overexpression was sufficient to cause the destruction of its targets by a mechanism that does not require ubiquitination. Multiple lines of evidence support this ubiquitination-independent mechanism of protein degradation.
Midnolin still bound to and promoted the degradation of many targets that had been mutated to lack lysine residues. Moreover, inhibition of the proteasome, but not E1 ubiquitin–activating enzymes, abrogated midnolin function.
Additionally, midnolin does not contain RING or HECT domains that are characteristic of E3 ubiquitin ligases or ubiquitin-binding domains found in proteasomal processivity factors such as Rad23.
Instead, midnolin engaged substrates using its “Catch” domain, which was necessary and sufficient to interact with unstructured regions within substrates that have the potential to form a β strand upon binding midnolin. These unstructured regions with the propensity to form a β strand were also necessary and sufficient to bind the Catch domain, thus functioning as a midnolin degron.
In addition, midnolin stably associated with the proteasome through a C-terminal α helix and promoted the degradation of Catch-bound targets using its N-terminal ubiquitin-like domain.
Thus, midnolin contains three conserved structural domains that function in concert to directly target a large set of nuclear proteins to the proteasome for ubiquitination-independent degradation.
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. Within substrates, midnolin recognizes relatively degenerate amphipathic regions with the potential to form β strands, so the midnolin degron may be a common structural component of numerous proteins. How the midnolin-proteasome pathway is regulated by various cues in diverse cell types to control transcriptional programs will be an important subject of future exploration.