Scientists have known for some time that the biological clock controls many functions related to human health, from cellular energy levels and responses to environmental stress, to immune function and nutrient metabolism. Each of these processes creates feedback that, in turn, also influences the clock. But can these clock-regulated processes and the resulting feedback that affects their timing share a common trigger?
Texas A&M University biologists have determined the answer is yes, thanks to a fundamental and conserved mechanism that contributes to how the circadian clock regulates rhythms in protein production which they recently discovered in their ongoing study of the model fungus Neurospora crassa, commonly thought of as a bread mold. Their findings are reported this week in the Proceedings of the National Academy of Sciences.
“We discovered in this work that the clock controls translation initiation by regulating the activity of a key initiation factor that is conserved from fungi to humans,” said Dr. Deborah Bell-Pedersen, Dr. Terry Thomas University Professor in the Texas A&M Department of Biology and corresponding author on the paper. “This regulation leads to rhythms in protein abundance in cells. Understanding how the clock controls protein abundance rhythms and knowing what proteins cycle is important because more than half of drugs that people take to treat various diseases target a protein that cycles in abundance. Thus, the time of administration of the drug is critical to its efficacy and toxicity.”
Bell-Pedersen’s laboratory within the Center for Biological Clocks Research has shown that the clock generates rhythms in the activity of a conserved protein synthesis factor known as eukaryotic initiation factor 2 (eIF2). This rhythm results from the clock controlling the activity of a specific eIF2 component, eIF2α, in order to ensure that eIF2 maintains high activity at night and low activity during the day to correspond with peak cellular energy levels.
“Protein synthesis is the most energy-demanding process that occurs in cells,” Bell-Pedersen said. “We showed that having increased eIF2α activity — and thus increased protein synthesis activity — at night when the fungus’s cellular energy levels are high and environmental stress is low provides a significant growth advantage to this fungus. In other words, clock control of protein synthesis through control of eIF2α activity is critical to the overall fitness and health of this organism.”
Bell-Pedersen’s group likewise determined that the daily rhythm in the activity of eIF2α is governed by the rhythms in the daily activity of other proteins important for protein synthesis called aminoacyl-tRNA synthetases. All proteins are composed of amino acids, but before the amino acids can be assembled into proteins, they have to be put onto tRNA by tRNA synthetases, which “charge” the tRNA to create aminoacyl-tRNAs that enable the amino acids to be assembled into proteins at the assembly sites. The team’s experiments further revealed that the clock regulates eIF2α activity by also controlling tRNA synthetase levels, which then creates rhythms in the levels of charged versus uncharged tRNAs.
“Levels of tRNA synthetases are higher at night than in the day,” Bell-Pedersen said. “When tRNA synthetase activity is lower, uncharged tRNA levels increase. More uncharged tRNA during the day reduces the activity of eIF2α and reduces protein synthesis.”
The COVID-19 connection
Bell-Pedersen notes that tRNA synthetases also can have many functions beyond their role in charging tRNAs, including roles in nutrient signaling and in cytokine production — a process currently under intense scrutiny as a leading cause of death associated with COVID-19. Cytokines are small proteins released by immune cells to coordinate the body’s response to a pathogen by triggering local inflammation that grants those cells quicker access to the infection site. When the cytokine network goes out of control as it does in many COVID-19 patients, the resulting cytokine storm causes serious damage, including multi-organ failure.
While several labs have shown that the circadian clock controls rhythms in the production of cytokines, Bell-Pedersen says COVID-19 has demonstrated just how critical it is to be able to control cytokine levels. She notes that tRNA synthetases also induce immune cells to release cytokines, contributing to anti-viral immunity. Because they are secreted by cells to boost immunity to infection, the tRNA synthetases effectively function like sensors to warn of impending danger.
“Our work showing that tRNA synthetases are clock-controlled provides a much deeper understanding of their regulation, which in turn will help us better understand their role in immune function and cytokine release,” Bell-Pedersen said. “Blocking the release of tRNA synthetases from cells with inhibitors holds great promise for alleviating cytokine storms. Also, giving these drugs at the right time of day when the levels of tRNA synthetases are high under control of the clock would be expected to have significant additional benefits to patient outcomes.”
Mutations and mis-regulation
Given the broad role of tRNA synthetases in biological activities, Bell-Pedersen says it is not surprising that mutations or changes in expression of tRNA synthetases are linked to several human diseases, including immune system dysfunction, cancer and neurological disorders. She hopes her lab’s work will serve as a basis to look for a possible missing link between protein synthesis and other cellular processes known to be controlled by the clock in humans, including nutrient metabolism and immune system function.
Bell-Pedersen notes that mRNA (messenger RNA) translation is also mis-regulated in cancer, where the stakes are high for the millions of patients affected.
“Showing that the clock controls the activity of a translation factor that is essential for most mRNA translation provides new ideas to inhibit it in cancer cells without affecting normal cells,” Bell-Pedersen said. “Knowing how the clock works to control protein production and what is regulated at this level is essential for understanding how to better treat disease, from cancer to coronaviruses. Our discovery is exciting because it reveals a new mechanism to control clock functions and provides a basis to look for possible missing links between protein synthesis and other cellular processes known to be controlled by the clock in humans. It provides opportunities to use this information for the development of novel therapeutics to combat human disease associated with a defective clock and/or the tRNA synthetases that the clock controls.”
Next steps and goals
After determining that about 30 percent of rhythmic protein synthesis is controlled the rhythmic activity of eIF2α in fungi, the Bell-Pedersen lab currently is working to find out how this specificity is achieved and why some mRNAs are selected for rhythmic translation, while others are not. Bell-Pedersen says their next major goal is to determine how the clock controls rhythms in tRNA synthetase levels and what happens to the organism when this regulation is broken.
“Because these pathways are conserved in humans, our expectation is that determining how tRNA synthetases are regulated in fungi will help us understand the same in humans,” Bell-Pedersen said. “This information will likely lead to new ideas for treating both clock and tRNA synthetase disorders, including immune dysfunction.”
Bell-Pedersen said most of the study’s work was done by Texas A&M biology graduate student Shanta Karki, postdoctoral scientist Kathrina Castillo and senior biology major Olivia Kerr within her lab. Texas A&M biologist Dr. Matthew Sachs, who studies protein synthesis mechanisms in fungi, also contributed intellectually to the study. Others who helped with experiments included research scientist Teresa Lamb, Texas A&M biology graduate student Zhaolan Ding and postdoctoral scientist Cheng Wu.
The team’s research was funded by Bell-Pedersen’s National Institutes of Health Maximizing Investigators’ Research Award (MIRA Grant No. R35GM126966). Their PNAS paper, “Circadian clock control of eIF2α phosphorylation is necessary for rhythmic translation initiation,” can be viewed online along with related figures and captions.
For additional information on Bell-Pedersen and her research, visit https://www.bio.tamu.edu/faculty-page-deb-bell-pedersen/.
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