Molecular mystery solved – Harvard scientists have discovered a previously unknown way for cells to break down proteins

This mechanism breaks down short-lived proteins that support brain and immune function

Short-lived proteins control gene expression in cells and carry out critical roles ranging from aiding brain communication to boosting the body’s immune response. These proteins originate in the nucleus, and are rapidly degraded after serving their purpose.

For decades, the mechanism behind the breakdown of these essential proteins and their removal from cells has remained a mystery to researchers, until now.

In an interdepartmental collaboration, researchers from Harvard Medical School have identified a protein called medullinulin that plays a key role in the degradation of several short-lived nuclear proteins. The study shows that meduline does this by directly grabbing proteins and dragging them into the cellular waste disposal system, called the proteasome, where they are destroyed.

The results were recently published in the journal Sciences.

“These specifically short-lived proteins have been known for more than 40 years, but no one has determined how they actually degrade,” said co-lead author Shen Guo, a research fellow in neurobiology at HMS.

Because the proteins broken down by this process modulate genes with important functions related to the brain, immune system, and development, scientists may eventually be able to target the process as a way to control protein levels to alter these functions and correct any malfunctions.

“The mechanism we found is very simple and very elegant,” added co-lead author Christopher Nardoni, a PhD candidate in genetics at HMS. “It is a fundamental scientific discovery, but there are many implications for the future.”

Molecular puzzle

It is well established that cells can break down proteins by binding them to 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 another ubiquitin-independent mechanism of proteolysis.

“There was scattered evidence in the scientific literature that the proteasome could somehow directly degrade unmarked proteins, but no one understood how that could happen,” Nardoni said.

One group of proteins that appears to be degraded by an alternative mechanism are stimuli-induced transcription factors: proteins that are rapidly synthesized in response to cellular stimuli that travel to the cell nucleus to turn on genes, after which they are rapidly destroyed.

“What initially blew me away is that these proteins are very unstable and have a very short half-life – once they’re produced, they do their job, and very quickly after that they degrade,” Joe said.

These transcription factors support a range of important biological processes in the body, but even after decades of research, “the mechanism of their turnover has been largely unknown,” said Michael Greenberg, the Nathan March Posey Professor of Neurobiology at HMS Blavatnik Institute and Harvard University. . Co-author of this paper with Stephen Eledge, Gregor Mendel Professor of Genetics and Medicine at HMS and Brigham and Women’s Hospital.

From a handful to hundreds

To investigate this mechanism, the team started with two familiar transcription factors: Fos, which has been studied extensively in Greenberg’s lab for its role in learning and memory, and EGR1, which is involved in cell division and survival. Using cutting-edge proteomics and genetic analyzes developed in Elledge’s lab, the researchers focused on meduline as a protein that helps break down transcription factors. Follow-up experiments revealed that in addition to Fos and EGR1, meduline may also be involved in degrading hundreds of other transcription factors in the nucleus.

Gu and Nardone remember being shocked and skeptical about their findings. To confirm their findings, they decided they needed to know exactly how meduline targets and degrades many different proteins.

“Once we identified all of these proteins, there were many tantalizing questions about how the medullinolenic mechanism actually works,” Nardoni said.

With the help of a machine learning Using a tool called AlphaFold that predicts protein structures, as well as the results of a series of lab experiments, the team was able to elucidate the details of the mechanism. They demonstrated that meduline has a ‘capture domain’ – a region of the protein that grabs other proteins and feeds them directly to the proteasome, where they are broken down. This catch domain consists of two separate, linked regions Amino acids (think gloves on a string) that capture a relatively unstructured region of the protein, allowing medullin to capture many different types of proteins.

Of note are proteins such as Fos that are responsible for turning on genes that trigger neurons in the brain to connect and rewire themselves in response to stimuli. Other proteins such as IRF4 activate genes that support the immune system by ensuring that cells are able to form functional B and T cells.

“The most exciting aspect of this study is that we now understand a new general mechanism independent of ubiquitylation that degrades proteins,” Elledge said.

Tantalizing translation potential

In the short term, the researchers want to dig deeper into the mechanism they discovered. They plan to conduct structural studies to better understand the fine details of how proteins are captured and degraded. They are also making mice that lack linulin, to understand the protein’s role in different cells and stages of development.

The scientists say their findings have tantalizing translation potential. It may provide a pathway that researchers can harness to control levels of transcription factors, thereby modulating gene expression and, in turn, related processes in the body.

“Protein degradation is a critical process, and its deregulation underlies many disorders and diseases,” Greenberg said, including some neuropsychiatric conditions, as well as some types of cancer.

For example, when cells contain too many or too few transcription factors such as Fos, learning and memory problems may arise. In multiple myeloma, cancer cells become addicted to the immune protein IRF4, so its presence can fuel disease. Researchers are particularly interested in identifying diseases that may be good candidates for developing therapies that work through the mandulin proteasome pathway.

“One area we are actively exploring is how to adjust the specificity of the mechanism so that it can specifically analyze proteins of interest,” said Gu.

Reference: “The Medulin Proteasome Pathway Captures Proteins for Ubiquitin-Independent Degradation” by Shen Guo, Christopher Nardoni, Nolan Kamitaki, Uyo Mao, Stephen J. Eledge, and Michael E. Greenberg, Aug. 25, 2023, Available here. Sciences.
doi: 10.1126/science.adh5021

Funding was provided by a National Mah Jongg League Fellowship from the Damon Runyon Foundation for Cancer Research, a National Science Foundation Graduate Research Fellowship, and National Institutes of Health (T32 HG002295; R01 NS115965; AG11085).