Horses are among the world’s most elite athletes: When galloping, they can consume twice as much oxygen per kilogram as the fittest humans. All that oxygen supercharges horses’ cells’ energy-producing compartments as they crank out ATP, the chemical needed to power their impressive muscles. But making so much cellular fuel so quickly comes with a catch: the manufacture of pernicious byproduct molecules called reactive oxygen species (ROS), which can wreak havoc in cells.
How horses dealt with this biological trade-off and evolved into premier endurance athletes has long intrigued biologists. Researchers report today in Science that they have uncovered a big part of it, identifying a key mutation that lets horses safely produce so much ATP. The trait helped pave the way for horses to go from dog-size critters millions of years ago to the high-endurance athletes we know today.
The study’s detailed molecular work makes it “exceptional,” says José Calbet, an expert on the cellular responses to exercise at the University of Las Palmas de Gran Canaria who wasn’t involved with the study.
The mutation in question occurs in the gene that encodes a protein called KEAP1, which acts as a biochemical bouncer, binding to a different protein called NRF2 to prevent it from entering the cell’s nucleus, where it would otherwise activate stress-response genes that help blunt cell damage.
But ROS can help NRF2 sneak in by causing KEAP1 to release its bind on the protein, allowing it to enter the nucleus and trigger the cell’s stress-response genes.
Johns Hopkins University ophthalmologist and clinician scientist Elia Duh, a senior author of the new study, didn’t set out to study horses. Initially, Duh was interested in the KEAP1-NRF2 system because its role in activating stress-response genes makes it a tempting target for treating inflammation—and aging-related conditions, such as blinding retinal diseases, irritable bowel syndrome, and neurodegeneration.
Duh wondered whether any insights could be gleaned from studying the evolution of these proteins in different animals. So, he teamed up with Gianni Castiglione, an evolutionary biologist and biochemist at Vanderbilt University. Together, they scanned hundreds of vertebrate genomes looking for notable mutations to the gene for KEAP1.
The team’s genomic work revealed birds had almost completely lost the gene, presumably an adaptation to the extreme demands of flight. When they looked in horses, researchers noticed what initially appeared to be a DNA sequence that encoded an unusually short—and therefore presumably nonfunctional—version of the KEAP1 protein. But when Duh’s and Castiglione’s team grew horse cells in culture, it discovered the protein was very much there and working. “Naturally, I was worried I was doing something wrong,” Castiglione says. “Then one day, a light bulb went off.”
As it turns out, the computer algorithm scientists had used to scan the horse genome had made a mistake. The algorithm had spotted a specific kind of mutation in the part of the KEAP1 gene that changed the messenger RNA from CGA—which codes for the amino acid arginine—to UGA, which is what’s known as a “stop codon.”
Normally, the cellular machinery interprets UGA as a sign to stop translating the RNA into a protein. But instead, the horses’ genetic machinery recodes the stop codon into a different amino acid, cysteine, causing it to ignore that order. This phenomenon, known as a stop codon read-through, is common among viruses but rare in multicellular organisms.
“The identification of this evolutionarily significant UGA recoding event represents a potentially seminal finding, offering a model for uncovering other yet-unidentified cases of stop codon read-through,” says Hozumi Motohashi, a biologist at Tohoku University who has studied KEAP1 and NRF2.
That the replacement is a cysteine is particularly notable, Castiglione says. KEAP1 senses cellular stress through its cysteines, which contain sulfur atoms whose reactions with ROS, induce the chemical changes that cause KEAP1 to let go of NRF2. The mutation the researchers had identified adds another place on KEAP1 for ROS to interact, which makes the protein more sensitive to stress—and lets horse cells respond much faster to the cellular stress of intense exercise. “It does make complete sense [that] by introducing another cysteine, another sulfur, you would have heightened sensitivity,” Castiglione says.
What’s more, this tweaking of KEAP1 is a “[key] genetic component to the puzzle of the evolution of horses,” Duh says. “Once they figured out how to run, they could occupy all kinds of ecological niches,” Castiglione adds.
The finding could also point the way toward new kinds of drugs to treat diseases by targeting the specific parts of the KEAP1 protein that help horses hoof it. “By looking at what evolution has figured out, we know this is a viable strategy,” Castiglione says.
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