New discovery about nature’s ‘high-end machines’ could have big implications for biology

Working with tiny bacteria, Michigan State University researchers led by Lee Kroos have made a discovery that could have big implications for biology.

Researchers have revealed a new way nature can inhibit or deactivate important proteins called intramembrane proteases -; pronounced “ace of tea pro” -; whose team reported on April 26 in the newspaper eLife.

Although the Spartans made this discovery using a model organism, a microbe known as Bacillus subtilis, this type of protein is highly conserved, as evolutionary biologists say, “it’s everywhere.”

These types of proteases are found in organisms that span all kingdoms of life, from single-celled bacteria to humans. In fact, the first intramembrane protease was discovered in humans in 1997 and perhaps the best known member of this family, named gamma-secretase, is implicated in Alzheimer’s disease.

“Our paper shows the first example of regulation of an intramembrane protease with natural inhibitory proteins,” said Kroos, a professor in the Department of Biochemistry and Molecular Biology and the Department of Microbiology and Molecular Genetics at the College of Natural Sciences. “It gives us some ideas of how we could use and emulate that.

“Will this tell us how to modulate gamma-secretase? No, Kroos said. “But it might give people some ideas about what decorations they could put on inhibitors to try as therapeutics.”

Using this information to design drugs to treat Alzheimer’s disease will take years, Kroos said, but the results could have more immediate impacts in the fight against particularly nasty and stubborn bacterial pathogens. This includes Bacillus anthracis, the bacteria that causes anthrax infections, and other bacteria that cause tetanus, botulism, and food poisoning.

“Many, many bacteria have intramembrane proteases that are quite closely related to the one we studied,” said Kroos, who has been a fellow of the American Association for the Advancement of Science since 2014, thanks in part to his work to advance our understanding of biology with bacteria. “If we find them, we might find a way to make the bacteria less resistant to stress and easier to treat with antibiotics.”

A better understanding of these proteases could also help develop applications in other fields, including agriculture and environmental protection, eLife noted in a summary featuring the Spartans’ work. Beyond that, it helps paint a fuller picture of how life works.

“This work should have a broad impact on our understanding of the regulation of this class of proteins in the tree of life,” wrote Petra Anne Levin, editor-in-chief of eLife and professor of biology at the University of Washington. in St. Louis.

Scissors, Spores and Corvettes

A protease is an enzyme, a type of protein machine, that nature uses to chop up other proteins. It is a fundamental biological process that cells use to achieve various goals. An intramembrane protease is an enzyme which has its active site -; where the enzyme does the cutting -; buried inside a cell membrane.

“Sometimes you’ll hear them call ‘scissors in the membrane,'” Kroos said. “These intramembrane proteases do really important things in cells.”

Lee Kroos, researcher, Michigan State University

The protease studied by the Spartans, for example, is part of the biological system that B. subtilis uses to make spores when food is scarce. Spores are basically dormant cells covered in protein armor that can withstand harsh conditions and then reactivate once things improve (other bacteria, including B. anthracis, also form spores, this which is one of the reasons why these pathogens are so persistent).

Because intramembrane proteases do their job within the confines of a cell membrane, it has been difficult for researchers to pinpoint exactly how they work. Adding to the complexity of Project Spartan, the researchers believed their protease might work in a sophisticated way that had never been documented before.

“When you look at other related organisms, you see this system has evolved a lot,” Kroos said. “B. subtilis is like the Corvette. It has high-end machinery.”

Understanding this high-end machinery required extensive genetic and biochemical testing, which was conducted by Sandra Olenic, a PhD student in Kroos’ lab. Olenic earned his Ph.D. after completing this project and is now a postdoctoral researcher at Tufts University.

As Olenic designed and conducted experiments, she and Kroos realized that their results would not provide all the answers they were looking for. They turned to one of Kroos’ longtime collaborators, Michael Feig, a professor of biochemistry and molecular biology at MSU, to provide computer modeling and help complete the large puzzle.

Lim Heo, a postdoctoral research associate in Feig’s lab, had expertise in a computational technique that can predict protein structures. The technique has recently received more attention thanks to Google and other big names in artificial intelligence developing software packages that make it more accessible to the scientific community.

Before such tools became available, however, Heo and Feig still had the know-how to help Kroos and Olenic begin to assemble a model explaining how intramembrane protease works.

“I think it’s a really cool story and a great collaboration that took a lot of hard work and perseverance,” Kroos said. “Sandra has shown incredible perseverance and dedication. It is also a credit to Lim and Michael to have done such a good job with computer modelling.”

Complete the puzzle

Taken together, the team’s findings imply that this B. subtilis intramembrane protease is kept inactive -; that is to say not to cut its target protein or its substrate -; with the help of two other proteins. One of these inhibitory proteins works like a clamp, keeping the second protein lodged in the active site of the scissor enzyme.

The researchers hypothesize that the bacterium can then activate the protease by releasing the clamp, letting the blocking protein escape and letting the target protein in.

“It was like putting together a 5,000 piece jigsaw puzzle without knowing what it looks like,” Kroos said. While the puzzle isn’t completely solved, the team has enough data and results to be sure they have a reasonable model of how things look and work. But the researchers don’t stop there.

Their next step is already underway, which is to test the model’s predictions -; such as when the clamp is attached to both protease and inhibitor protein -; and see how well these correspond to reality. Two undergraduate researchers from Kroos’ lab are leading these experiments.

Another key step is to determine the structures of the proteins involved using analytical techniques such as X-ray crystallography and cryogenic electron microscopy. Kroos has been working on this part for about a decade with collaborators at MSU, but the proteins are in no rush to reveal their secrets.

Kroos suspects he might retire before the puzzle is fully completed, but he doesn’t seem to mind. On the one hand, he confessed that he might try to sneak in and help out if his colleagues let him. And he seems mostly excited to see just how much of a puzzle he and his team can solve with the few years he has left.

When Kroos started at MSU in 1988, science didn’t know about intramembrane proteases. But soon after their discovery in 1997, Kroos decided he needed to change his research focus to include these proteins.

“It’s not an easy thing to do,” Kroos said, because everyone -; funding bodies included -; believes you can make the change. But he found support and willing collaborators at MSU.

“Our department is really strong in membrane proteins,” he said. “I thought it would be a good chance to bloom where I was planted.”

It is safe to say that the hypothesis has been confirmed.

Michigan State University has been advancing the common good with uncommon will for more than 165 years. One of the world’s leading research universities, MSU pushes the boundaries of discovery to create a better, safer, and healthier world for all while providing life-changing opportunities to a diverse and inclusive academic community through more of 200 study programs in 17 degrees. colleges.


University of Michigan

Journal reference:

Olenic, S., et al. (2022) Inhibitory proteins block substrate access by occupying the active site cleft of the intramembrane protease from Bacillus subtilis SpoIVFB. eLife.

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