Targeting “Proteolethargy” to Restore Protein Mobility May Represent Therapeutic Approach for Chronic Diseases

Chronic diseases such as type 2 diabetes and inflammatory disorders are a major cause of disease burden and deaths around the world, but their treatment has proven difficult because there may not be one cause, such as a single gene mutation, to target therapeutically. Research by Whitehead Institute scientists has now found that many chronic diseases exhibit reduced protein mobility as a common denominator that could be driving their dysfunction.

Headed by Richard Young, PhD, the team’s in vitro studies indicated that the movement of potentially half of all proteins that are active in cells is slowed when the cells are in a chronic disease state, and this reduces protein function. The findings suggest that decreased protein mobility—the team call this mobility defect proteolethargy—may play a key role in decreased cellular function in chronic disease. The studies linked proteolethargy to a dysregulated redox environment that impacts on cysteines on the proteins’ surfaces. The teams also proposes a new therapeutic strategy for treating chronic diseases.

“I’m excited about what this work could mean for patients,” said Alessandra Dall’Agnese, PhD, a co-author on the researchers’ published paper in Cell. “My hope is that this will lead to a new class of drugs that restore protein mobility, which could help people with many different diseases that all have this mechanism as a common denominator.”

Young, Dall’Agnese and colleagues, including graduate students Shannon Moreno and Ming Zheng, and research scientist Tong Ihn Lee, PhD, described their findings in a paper titled “Proteolethargy is a pathogenic mechanism in chronic disease.” In their report the team noted, “There is limited information on the mobility of a range of proteins with diverse functions in human cells and even less information on the effects of pathogenic stimuli on protein mobility. This paucity of knowledge may explain why proteolethargy has apparently not been described as a pathogenic mechanism in chronic diseases…The model described here for proteolethargy in disease has implications for the development of therapeutics for certain chronic diseases.”

Diseases associated with pathogenic or chronic signaling are a leading cause of morbidity and mortality, the authors stated. And in contrast with diseases where there is an evident causal link between gene mutation and disease and for which the cellular pathways impacted are defined, in chronic syndromes causal gene mutations are uncommon, the investigators pointed out, and there may be dysregulation of diverse cellular processes, such as gene regulation, ribosome biosynthesis, and metabolic activity. “For prevalent syndromes such as diabetes and inflammatory disorders, the pathology typically involves a continuous and/or high-level stimulus but not necessarily a known mutation in a specific gene…Thus, how to define hypotheses that will inform therapeutic development on the basis of such a breadth of cellular dysfunction has long vexed clinicians and research scientists.”

The newly reported study focused on protein mobility in the context of chronic diseases. The scientists explained that the billions of protein molecules produced in cells must leave their site of synthesis and arrive at their cellular destinations, where they carry out their specialized functions. To do this the proteins have to move through an environment that is densely packed with biomolecules. “Recently, pathogenic signaling in certain chronic diseases was reported to cause reduced movement of receptor molecules into functional protein assemblies,” the investigators noted.

The team had first suspected that cells affected in chronic disease might have a protein mobility problem after observing changes in the behavior of the insulin receptor, a signaling protein that reacts to the presence of insulin and causes cells to take in sugar from blood. In people with diabetes, cells become less responsive to insulin—a state called insulin resistance—causing too much sugar to remain in the blood. In prior research published on insulin receptors, Young and colleagues reported that insulin receptor mobility might be relevant to diabetes.

The findings from this and other prior work led the team to consider the possibility that dysregulated signaling might be the cause of a more protein mobility defect in cells, and that reduced protein mobility might itself represent be a pathogenic mechanism that is shared across diseases.

To test this hypothesis, for their newly reported studies the team looked at proteins involved in a broad range of cellular functions. Their experiments considered MED1, a protein involved in gene expression; HP1α, a protein involved in gene silencing; FIB1, a protein involved in production of ribosomes; and SRSF2, a protein involved in splicing of messenger RNA. The team applied single-molecule tracking and other methods to measure how each of those proteins moves in healthy cells and in cells in disease states. “Single-particle tracking (SPT) and fluorescence recovery after photobleaching (FRAP) allow for the measurement of the kinetics of protein mobility in living cells…” they commented.

The results showed that all but one of the proteins they investigated showed reduced mobility (about 20–35%) in the disease cells. The team next set out to determine what was causing the proteins to slow down. They suspected that the defect had to do with an increase in cells of the level of reactive oxygen species (ROS), molecules that are highly prone to interfering with other molecules and their chemical reactions. Many types of chronic disease-associated triggers, such as higher sugar or fat levels, certain toxins, and inflammatory signals, lead to an increase in ROS, also known as an increase in oxidative stress.

The researchers measured the mobility of the proteins again, in cells that had high levels of ROS and were not otherwise in a disease state, and saw comparable protein mobility defects, suggesting that oxidative stress might be to blame. The collective results of experiments, they suggested “… are consistent with a model in which diverse pathogenic stimuli known to induce oxidative stress cause suppressed protein mobility in multiple disease-relevant cell types.”

The final part of the puzzle was why some, but not all, proteins slow down in the presence of ROS. SRSF2 was the only one of the proteins studied that was unaffected in the experiments, and it had one clear difference from the others. The SRSF2 surface does not contain any cysteines, an amino acid building block of many proteins. Cysteines are especially susceptible to interference from ROS because it will cause them to bond to other cysteines. When this bonding occurs between two protein molecules, it slows them down because the two proteins cannot move through the cell as quickly as either protein alone.

About half of the proteins in our cells contain surface cysteines, so this single protein mobility defect can impact many different cellular pathways. “We estimate that 50% of human proteins contain at least one surface-exposed cysteine, so there is potential for half of the proteome to be directly susceptible to proteolethargy in high ROS environments,” the authors wrote.

This makes sense when one considers the diversity of dysfunctions that appear in cells of people with chronic diseases, which may be associated with dysfunctions in cell signaling, metabolic processes, gene expression and gene silencing. All of these processes rely on the efficient functioning of proteins, including proteins studied by the researchers.

How do proteins moving more slowly through a cell lead to widespread and significant cellular dysfunction? Dall’Agnese likens every cell to a tiny city, with proteins as the workers who keep everything running. Proteins have to commute in dense traffic in the cell, traveling from where they are created to where they work. The faster their commute, the more work they get done.

Normally, most proteins zip around the cell bumping into other molecules until they locate the molecule they work with or act on. The slower a protein moves, the fewer other molecules it will reach, and so the less likely it will be able to do its job. Young and colleagues found that such protein slow-downs lead to measurable reductions in the functional output of the proteins. When many proteins fail to get their jobs done in time, cells begin to experience a variety of problems—as they are known to do in chronic diseases.

Young and colleagues performed several experiments to confirm that decreased protein mobility does in fact decrease a protein’s function. Their results indicated, for example, that when an insulin receptor experiences decreased mobility, it acts less efficiently on IRS1, a molecule to which it usually adds a phosphate group.

Discovering that decreased protein mobility in the presence of oxidative stress could be driving many of the symptoms of chronic disease provides opportunities to develop therapies to rescue protein mobility. In the course of their experiments, the researchers treated cells with an antioxidant drug—something that reduces ROS—called N-acetyl cysteine (NAC) and saw that this partially restored protein mobility. “These results are consistent with the possibility that elevated levels of ROS cause a decrease in the mobility of certain proteins and suggest that the change in protein behavior is caused by an alteration in the oxidative environment,” they commented.

In their paper the team further suggested, “Redox homeostasis is regulated by many pathways and proteins, which counteract transient increases in ROS that occur normally in diverse cellular processes so it is possible that therapeutic targeting of these natural pathways will prove beneficial for treating or preventing proteolethargy. The rescue of protein mobility with NAC treatment, as described here, is a proof of principle for this concept.”

The researchers are pursuing a variety of follow ups to this work, including the search for drugs that safely and efficiently reduce ROS and restore protein mobility. They developed an assay that can be used to screen drugs to see if they restore protein mobility by comparing each drug’s effect on a simple biomarker with surface cysteines to one without. They are also looking into other diseases that may involve protein mobility, and are exploring the role of reduced protein mobility in aging.

“Restoring protein mobility might be considered among the therapeutic hypotheses for these chronic diseases,” they wrote. “Protein mobility biosensors, such as the one developed for this study, may prove to be valuable for high-throughput screening for drugs that restore normal protein mobility under pathogenic signaling conditions.”

Added Young, who is  also a professor of biology at the Massachusetts Institute of Technology, “The complex biology of chronic diseases has made it challenging to come up with effective therapeutic hypotheses. The discovery that diverse disease-associated stimuli all induce a common feature, proteolethargy, and that this feature could contribute to much of the dysregulation that we see in chronic disease, is something that I hope will be a real game changer for developing drugs that work across the spectrum of chronic diseases.”

Lee stated, “This work was a collaborative, interdisciplinary effort that brought together biologists, physicists, chemists, computer scientists, and physician-scientists. Combining that expertise is a strength of the Young lab. Studying the problem from different viewpoints really helped us think about how this mechanism might work and how it could change our understanding of the pathology of chronic disease.”

Zheng in addition noted, “I’m excited that we were able to transfer physics-based insight and methodology, which are commonly used to understand the single-molecule processes like gene transcription in normal cells, to a disease context and show that they can be used to uncover unexpected mechanisms of disease.” “This work shows how the random walk of proteins in cells is linked to disease pathology,” Moreno concurred: “In school, we’re taught to consider changes in protein structure or DNA sequences when looking for causes of disease, but we’ve demonstrated that those are not the only contributing factors. If you only consider a static picture of a protein or a cell, you miss out on discovering these changes that only appear when molecules are in motion.”