Naturally occurring small molecules correct mutant proteins in living cells

Yeast screens explore the therapeutic potential of chemical rescue.

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Anyone who’s worked in a lab knows that sinking feeling of discovering that the temperature of an incubator, carefully set the night before, has crept up high enough to ruin the experiment. While such a mishap usually spells disaster, occasionally, it can lead to an unexpected discovery.

One such revelation was prompted by an uncooperative incubator in the lab of Michael McMurray, a cell biologist at University of Colorado’s Anschutz Medical Campus. McMurray studies the septin family of cytoskeletal proteins, and inside the incubator were plates of yeast with temperature-sensitive septin mutations. The mutant yeast could survive only at mild temperatures, so the incubator was set to a comfortable 27°C.

For this experiment, a chemical called guanidine hydrochloride had been added to some of the plates, to test whether it would stop the mutants from growing at the permissive temperature. When the incubator was found roasting away at more than 30°C, however, all of the yeast should have been dead.

“Amazingly, one of the mutants actually grew,” says McMurray. “The guanidine restored its viability.”

That discovery launched an investigation of how, exactly, guanidine had protected the mutant from normally lethal conditions. In a paper in the September issue of G3: Genes|Genomes|Genetics, Hassell et al. report several mutants that can be rescued by guanidine. They also show that another naturally occurring small molecule can correct an even broader range of mutants.

Guanidine can stand in for a lost arginine

Guanidine’s molecular structure mimics the side chain of the amino acid arginine. Researchers had previously shown that guanidine could restore function to an enzyme that had been mutated to lack an arginine in its active site. But all of this work had been done in vitro. This piqued McMurray’s interest even more. “Arginine is the most commonly mutated amino acid in human disease,” he says. “If guanidine can restore function to arginine mutant proteins, why has no one explored this in living cells?”

McMurray’s team began by testing enzymes in which a single arginine mutation disabled the enzyme enough to cause disease, such as ornithine transcarbamylase (OTC). OTC deficiency is an inherited metabolic disease that leads to a buildup of toxic ammonia in the body. The researchers created yeast with the same OTC arginine mutation that causes the human disease, making the yeast unable to grow without nutritional supplementation. Adding guanidine hydrochloride to the growth media restored some of the lost enzyme function.

“The effect was pretty small,” McMurray says. “It wasn’t a full rescue, but it was something.”

Next, the researchers decided to broaden their investigation. Instead of testing candidate enzymes, they screened hundreds of yeast mutants to see if guanidine restored function to any of them. “We decided to let the cells tell us what would work best,” McMurray says. “That’s when things started to get interesting.”

The screen uncovered 11 new candidates, the most interesting of which was an arginine mutant of actin, another cytoskeletal protein. “It just so happens that arginine is also mutated in human cardiac beta actin, and that mutation causes disease,” McMurray says.

As an ATPase, actin is technically an enzyme, but the arginine mutation is far from the active site, and guanidine isn’t restoring catalytic activity per se. Instead, McMurray says, it’s helping the protein fold into its proper 3D shape. “All proteins have to fold,” McMurray says. Protein folding results from chemical interactions between the side chains of various amino acids. “To rescue the mutant, the guanidine just has to be able to fix what’s missing and restore the folding.”

The idea of rescuing mutants by restoring proper protein folding led them to investigate other chemicals that can influence protein folding. “From a biological perspective, what are other cases in nature in which organisms have to deal with alterations in protein folding?” McMurray says. “Then we thought of sea creatures — sharks and rays.”

Moving beyond guanidine

Because they live in saltwater, sharks maintain high concentrations of urea in their bodies to keep from losing water through osmosis. Urea, however, is toxic to proteins, and causes them to unfold. To counteract the urea, these animals also have high levels of a chemical called trimethylamine oxide (TMAO), which promotes protein folding.

Does the shark’s protein protection trick work in other contexts? To follow up, research assistant Daniel Hassell screened yeast mutants using TMAO. He turned up hundreds of mutants that were rescued by the molecule. The genes and mutant types were all very different from each other, suggesting that TMAO has a more general stabilizing effect rather than specifically replacing a particular amino acid. This broad effect suggests a potential role for the molecule in synthetic biology, as a way to design proteins with an on/off switch system.

For its part, guanidine is already FDA-approved as a treatment for an inherited autoimmune disorder called Lambert-Eaton myasthenic syndrome. McMurray remains curious about whether it has the potential to treat other genetic diseases.

“That would be my ultimate hope, that someone would be inspired by our work to try it in an animal model or the clinic,” McMurray says.

CITATION

Chemical rescue of mutant proteins in living Saccharomyces cerevisiae cells by naturally occurring small molecules
Daniel S Hassell, Marc G Steingeisser, Ashley S Denney, Courtney R Johnson, Michael A McMurray
G3 Genes|Genomes|Genetics 2021; jkab252
https://doi.org/10.1093/g3journal/jkab252