Don’t blame lab mice for shortfalls in their ability to mimic human immune systems — blame their upbringing.
Mice with more experience fighting pathogens have immune system reactions more like humans’, conclude two studies published online April 20. “Dirty” mice bought from pet stores or caught in the wild have more humanlike immune systems than clean lab mice do, researchers report in Nature. And in Cell Host & Microbe, scientists find that infecting lab mice with a series of viruses and parasites alters their immune responses to be similar to those of dirty mice and humans. In recent years, scientists have debated whether mice are adequate stand-ins for humans. Some say mice are poor substitutes, and that money should instead be spent on bolstering human studies (SN: 3/23/13, p. 10). Others look at the same data and conclude that mice do a pretty good job of representing humans (SN: 9/20/14, p. 14). Plus, many important studies could not be done with humans, so mice are a necessity.
But even mouse fans recognize there is room for improvement. “All science is an approximation of the real situation,” says immunologist Andrew Macpherson of the University Hospital of Bern, Switzerland, who relies on mice models. “I don’t think anybody doubts that the models don’t always accurately recapitulate what is happening in humans.” The new papers show where mice fall short and suggest ways to improve their ability to mimic people, he says.
Lab mice’s immune system responses “really do look different” from that of humans’, says immunologist David Masopust, coauthor of both studies. Masopust, of the University of Minnesota in Minneapolis, and colleagues wondered whether those dissimilarities are due to irreconcilable differences in the genetic makeup of mice and humans or if the environment plays a role.
His group counted immune cells in blood from adult lab mice, adult humans and human umbilical cords. Of special interest were “memory CD8+ T cells,” which cull body cells that are infected with viruses or bacteria or that have become cancerous. Lab mice and human infants have few of these memory cells, while adult humans have a plethora. That indicates that lab mice have inexperienced immune systems, much like human babies.
The finding, “is one of those things that once you know it, it’s incredibly obvious,” says E. John Wherry, an immunologist at the University of Pennsylvania. “Mice are like humans raised in a bubble.” Masopust agrees. “They live a preposterously hygienic existence.” Even mice with severe immune deficiencies can thrive in immaculately clean labs.
Ultraclean lab mice can’t emulate the sort of history most human immune systems experience, says Tiffany Reese, a viral immunologist at the University of Texas Southwestern Medical Center in Dallas. Adults carry an average of eight to 12 chronic viruses, such as Epstein-Barr virus (the cause of mononucleosis). Worm parasites infect about 2 billion people worldwide. And by adulthood, people have usually fought off multiple colds, flus and other infections.
Masopust’s team found that the memory T cell profiles of wild and pet-store mice more closely resembled that of adult humans than lab mice’s did. Housing lab mice next to pet-store mice for a month caused their immune system to change, making the lab mice resemble the dirty mice, the researchers reported in Nature. In discrepancies between studies of lab mice and humans, “the mouse may not be at fault,” Masopust says. “It’s the way that they are cared for.”
An experienced immune system not only looks different, it also works differently from an inexperienced one, Reese and colleagues report in Cell Host & Microbe. Reese infected lab mice with two types of herpesviruses, gave them the flu and inoculated them with an intestinal parasite. She then compared how uninfected mice reacted to a yellow fever vaccine with how chronically infected mice reacted. Uninfected mice made more antibodies against the vaccine. The result might help explain why some vaccines that look promising in animal studies don’t pan out in human trials.
Controlled infections may increase understanding of how pathogens interact with each other, with friendly microbes that live in the body and with the host’s immune system, says Reese’s coauthor Herbert Virgin, a viral immunologist at Washington University School of Medicine in St. Louis.
Researchers have a bias that mice are not humans, says Virgin, “But I think that’s too simplistic a view. We shouldn’t be asking whether the mouse is a perfect model for humans, but whether we can make the mouse emulate more closely the basic nature of human physiology.”
Imagine that you’re walking through a secluded forest somewhere in Southeast Asia. You’re far from modern conveniences, like electricity. The trees overhead block out much of the light. It’s quiet, except for the sounds of ruffling leaves or the calls of birds. Suddenly, you’re struck by the scent of popcorn.
You’d probably think you were going nuts. But you might have just picked up the trail of a bearcat, also called a binturong, a rare carnivore that sports a bushy, prehensile tail and is most closely related to the civet. And the animals’ popcorn scent is so pronounced that many zookeepers name their bearcats after the buttery movie snack.
The source of the scent, though, has been a mystery. Now Lydia Greene of Duke University and colleagues have found that the chemical that gives popcorn its delightful scent, 2-acetyl-1-pyrroline (2-AP), is also found in bearcat pee. They report their finding in the June issue of The Science of Nature.
As anyone who has ever owned a dog will know, urine is a key way that carnivores mark their territory. But the chemicals inside urine can also help an animal advertise who they are and if they’re ready to mate, as well as provide ways for a group of animals to maintain hierarchies or bonds between individuals.
Bearcats, like other carnivores, leave these chemicals wherever they go — but they’re a bit more aggressive about it than your pet poodle. A binturong pees in a squat, which soaks its feet and tail in smelly urine that is left behind with every step. Greene’s team investigated the composition of the animal’s pee to see how chemicals might influence how females maintain their social dominance over males. Of the 29 chemicals they detected in the urine of 13 female and 20 male bearcats living in captivity at the Carolina Tiger Rescue in Pittsboro, N.C., only one could be found in the pee of every animal: 2-AP.
The chemical, the researchers write, “likely explains the binturong’s signature scent,” and “may serve as a species identifier.”
Just where the chemical is coming from, though, remains unknown. The researchers checked the animals’ food and didn’t find any traces of 2-AP. One possibility is that bacteria in the bearcats’ gut or elsewhere are responsible. Bacillus cereus, commonly found in the intestines of vertebrates, is known to synthesize 2-AP.
But the chemical probably isn’t getting into bearcats in the same way as popcorn. That’s because 2-AP isn’t found in popcorn until the kernels have undergone the high heat necessary to pop the corn — an unlikely activity for jungle-dwelling mammals.
Rachel Dutton’s research is cheesy, by design. The microbiologist at the University of California, San Diego uses cheese rinds to study how microbes form communities.
Dutton, who has a long-standing interest in how bacteria and other microbes interact, got the inspiration for her studies several years ago while visiting the Marine Biological Laboratory in Woods Hole, Mass. In the salt marshes there, multiple species of bacteria, archaea and other microbes were growing in thick, many-layered mats. They would have been perfect for studying microbes in groups. Except for one thing: Many organisms that thrive in those mats won’t grow in captivity. Dutton needed a microbe community that she could pick apart, manipulate and reconstruct in the lab. The solution came with a round of cheese from France. Sliced open, its rind reminded Dutton of the microbial mats on the coastal salt marshes. “I took a piece of cheese into the lab and put it under the microscope,” she says. “Everything I was looking for in a microbial community was present.” Dutton and colleagues did genetic analyses of 137 cheeses from 10 countries and identified 24 genera of bacteria and fungi that are common in cheeses and will grow in the lab, the team reported in 2014 in the journal Cell.
By sampling a Vermont cheese as it aged over 63 days, the group also discovered that rind communities don’t form instantly. At the beginning, community members included Proteobacteria and Leuconostoc bacteria, plus candida yeast commonly found in raw milk. Within a week, Staphylococcus had overwhelmed the Proteobacteria. As the cheese ripened, Brevibacterium and Brachybacterium plus Penicillium and Scopulariopsis fungi became prominent inhabitants of the rind. That pattern held whenever those organisms congregated in a cheese in the lab.
Cheesemakers from Vermont taught Dutton how to ferment cheese curds and create her own lab version of a dry-aged cheddar. Although cheeses can have complex combinations of microbes — stinky cheeses have the most diverse mixes — Dutton’s lab crafts a more simplified rind using three types of fungi and four bacteria. The researchers grow the microbes in pairwise combinations to learn how they interact. Studying cheeses and other fermented foods could teach scientists how microbial communities evolved in different places and lead to the creation of new, tastier and safer foods, Dutton and Benjamin Wolfe of Tufts University wrote last year in Cell.
There’s one drawback to the cheesy research: The lab has a ripe odor, Dutton says. “We look like a normal microbiology lab, but we don’t smell like one.”
Years ago, when I visited the Daintree Rainforest in northeastern Australia, I couldn’t help but notice the signs — several of them — warning of crocodiles. Australia is home to two species of the ferocious reptiles, freshwater and saltwater — the latter of which can be found in the Daintree River. And the signs are no joke. Croc attacks aren’t common, but a few do happen every year, and some result in deaths.
Crocodiles aren’t terrifying just because they have huge teeth and a deadly bite, though. It’s that an attack appears to come from nowhere. The animals lurk just beneath the water, with only their eyes keeping a lookout for something tasty — like one of us. Now, new research shows that, while a croc may not see as well as you or I, its eyesight is quite good and well adapted for lying in wait at the water’s surface.
Nicolas Nagloo and colleagues from the University of Western Australia in Crawley took a detailed look at eyes from three young saltwater and two young freshwater crocodiles. “Both Australian species possess a bright yellow iris, a slit pupil and a relatively large lens,” the team notes May 4 in the Journal of Experimental Biology. Such features, which were known before this study, are helpful for seeing in dim light. (The animals, though, don’t have great vision underwater.) Crocs are also equipped with a “mobile slit retina” that helps the animals control how much light reaches the eye during daylight. Dissections and examinations of the cells of the eyes revealed that both species have three types of single cones, a type of double cone and one type of rod. This means that the animals can see colors well. But the freshwater crocs appear to be a bit more sensitive to red than their saltwater counterparts (known as “salties” in Australia); that may help the freshies see in streams and rivers where there is more red light. Both species also have a horizontal streak of high spatial acuity, which allows the reptiles to scan back and forth for prey without ever moving their heads. That the two species have eyes that are so similar is somewhat surprising given that they are separated by some 12 million years of evolution, live in different habitats and prefer different prey, with the freshwater crocs preferring smaller animals and more fish. But both species have adopted a similar hunting style in which the animals hide just beneath the water and scan the flat environment for a suitable meal. Their eyes, this study shows, are specialized to aid in such attacks.
Hair standing on end during a thunderstorm is a bad sign — it means lightning is on the way. On exoplanet HAT-P-11b, though, static hair might be the least of your worries. A barrage of lightning striking 530 times as often per square kilometer as storms in the United States could be the cause of a surge of radio waves detected from the planet several years ago, Gabriella Hodosán of the University of St. Andrews in Scotland and colleagues suggest online April 23 in Monthly Notices of the Royal Astronomical Society.
In 2009, astronomers recorded radio waves coming from the HAT-P-11 system that ceased when the planet slipped behind its star, suggesting the planet was the source of the signal. A second look in 2010 found no radio waves.
Scientists have detected lightning on planets closer to home, including on Venus and Jupiter, but not on a planet orbiting another star. HAT-P-11b is too close to its star for astronomers to see visible flashes of light. But an infrared telescope might pick up a stockpile of hydrogen cyanide created by the electrical discharge.
In 2013, nearly three times as many U.S. high school students smoked cigarettes as vaped electronic cigarettes. By 2015, the trend reversed. Nearly twice as many vaped as smoked, a new study finds. For middle school students, the preference for vaping over smoking cigarettes was even stronger (2.3-fold).
Federal researchers analyzed data from the National Youth Tobacco Survey and found that some 2.39 million U.S. high school kids vaped in 2015. Fewer high schoolers, about 1.37 million, smoked cigarettes, the researchers report in the April 15 Morbidity and Mortality Weekly Report. Since 2011, teen use of tobacco products has fallen for all categories but two: e-cigarettes and hookahs (although even hookah use has dropped since 2014). In fact, the federal researchers conclude, the steep growth in vaping seems to be keeping overall teen tobacco use stable. The vast majority of U.S. states have banned sales of vaping supplies to minors, according to the National Conference of State Legislatures. Yet “minors do not face any significant barrier in purchasing liquid nicotine over the Internet” to vape in e-cigs, a second new study shows.
Last summer, Dmitriy Nikitin of the University of California, Irvine and his colleagues recruited three teens to buy e-liquids from 120 different U.S. online vaping-supply vendors. Only four stores did not sell e-liquids to the 16- and 17-year-olds. “I was really blown away,” Nikitin says. His team published its findings online March 19 in Nicotine & Tobacco Research.
Some of the stores packaged their e-liquids with child-friendly bonuses: toy frogs, stickers, little green army men or candy such as SweeTarts. On May 5, the U.S. Food and Drug Administration announced sweeping new regulations for e-cigs, hookahs and other tobacco products.
Solar outbursts may have supplied early Earth with the right stuff for life.
Based on telescope observations of young sunlike stars, researchers estimate that “super” solar flares bombarded Earth with energetic particles at least once a day around 4 billion years ago. Collisions between the particles and molecules in Earth’s atmosphere produced nitrous oxide, a planet-warming greenhouse gas, and hydrogen cyanide, a crucial component for building DNA, the researchers propose May 23 in Nature Geoscience.
Those chemical products warmed and fostered emerging life, says study coauthor Vladimir Airapetian, an astrophysicist at NASA’s Goddard Space Flight Center in Greenbelt, Md. “Our sun, worshipped by ancient civilizations, wasn’t just a source of warmth, it also produced ingredients for life,” he says. Life’s earliest traces date back to around 4.1 billion years ago (SN Online: 10/19/15). At the time, the sun was about 25 to 30 percent dimmer than it is today — too faint to keep Earth’s temperatures above freezing without the help of additional greenhouse gases (SN: 5/4/13, p. 30). Simulating the early sun using star data collected by NASA’s Kepler space telescope as a guide, Airapetian and colleagues found that while dim, the sun was probably wilder in its youth. Solar flares, also called coronal mass ejections, probably erupted more often and with more ferocity, producing storms 1,000 times as powerful as the most intense flares on record.
Those storms made a big impact on Earth, the researchers propose. The storms temporarily squeezed the magnetosphere, the protective magnetic bubble surrounding Earth, to one-sixth its normal height. That squashing allowed more solar particles to rain into the atmosphere. The dive-bombing particles ionized and broke apart nitrogen molecules in the air. Those molecules reassembled into new ones such as hydrogen cyanide, which can produce DNA bases and amino acids.
Another product, nitrous oxide, is a greenhouse gas nearly 300 times as potent as carbon dioxide. The additional nitrous oxide could have kept Earth from freezing during the sun’s dim days, the researchers propose.
The solar flares would have impacted more than just Earth. Similar effects would also have occurred on Mars, Airapetian adds, potentially improving the Red Planet’s habitability.
The interactions create the right molecules but in the wrong place, says Ramses Ramirez, an astrobiologist at Cornell University, who wrote an accompanying perspective piece in the same issue of Nature Geoscience. The flare-formed molecules would have originated in the upper atmosphere, not near the surface where most greenhouse warming takes place and where life would have taken root. While the idea is plausible, some other mechanism is needed to “get the molecules down so the critters can utilize them,” he says.
When scientist George Yancopoulos speaks about his experience with the Science Talent Search, he uses words like “life-changing.” Named a finalist in the competition decades ago, he credits it with helping him launch a career in medical research. Now, Yancopoulos, chief scientific officer at Regeneron Pharmaceuticals Inc., of Tarrytown, N.Y., and his fellow STS alum Leonard Schleifer, Regeneron CEO and president, want to give back to the competition.
On May 26, Regeneron and Society for Science & the Public, which created the STS program in 1942 and publishes Science News, announced that the biotechnology company will take over as the third lead sponsor of the Science Talent Search. The competition was sponsored by Westinghouse for more than five decades; in 1998, Intel became the lead sponsor. “We are honored to be the new sponsors of the Science Talent Search, a national treasure that highlights the critical role science plays in advancing society,” Yancopoulos said in a press release. “For me, participating in the Science Talent Search was a life-changing experience that inspired my future scientific career.” The sponsorship will include $100 million in support over 10 years, increasing the value of the scholarships and other awards offered to winners of the competition to $3.1 million annually. Regeneron will also dedicate $30 million of the total to growing the Society’s efforts in outreach and equity, designed to encourage more young people to engage in original research as part of their explorations of science.
Regeneron, founded in 1988, developed the cholesterol-fighting drug Praulent that went on sale last year and Eylea, a treatment for the vision disease wet macular degeneration, among other products. It also has a $1.7 billion deal to develop new immunotherapies for cancer with Sanofi, the French pharmaceutical firm.
To Maya Ajmera, CEO and president of Society for Science & the Public and publisher of Science News, the expansion of the competition and related outreach efforts is particularly exciting. “Through the dedication of Regeneron not only to continue but to advance the Science Talent Search, we will be expanding the program’s reach like never before,” she said.
The influenza virus is a quick-change artist. In a few decades, its genome can evolve as much as animal genomes can over millions of years. That means that the viral proteins, including those that alert our bodies to an infection, constantly reinvent themselves, threatening our immune systems and frustrating vaccine developers.
For Jesse Bloom, a biologist studying how evolution affects proteins, that relentless change is an opportunity. Thanks to data collected during past flu seasons, Bloom knows the exact genetic makeup of some ancestors of today’s influenza viruses. His lab group at the Fred Hutchinson Cancer Research Center in Seattle uses that information to figure out how the viruses made their immunity-dodging transformations.
Bloom and others are part of a growing group of scientists who practice “evolutionary biochemistry.” They seek to explain life’s tremendous diversity and determine exactly how that diversity emerged. Rather than focusing on how plants or animals adapted to different environments, however, these researchers consider diversity on a much smaller scale: Their work aims to explain how the small set of proteins that powered primitive life-forms evolved into the millions of specialized proteins that drive biological processes today.
Exploiting the genetic records, Bloom can assemble virus proteins that existed in bygone times, then reconstruct how they evolved, one amino acid at a time. Other researchers are analyzing modern species to resurrect the ancestral forms of biological molecules that have evolved over millions of years.
With a historical protein in hand, researchers can test how swapping out a single amino acid — as evolution might have done — changes how the protein flexes or folds and connects (or doesn’t) with other molecules. By trying out alternate versions of a protein’s history through stepwise amino acid changes, scientists can learn how a protein’s physical form has both enabled and constrained its evolution.
Ultimately, this work might answer some long-standing questions: To what extent does evolution depend on chance events? Can evolution reach the same point by traveling different paths? How does biological complexity evolve? Such experiments are also helping researchers who study modern proteins sort out how the order of amino acids relates to biological function. That ordered series of amino acids is spelled out by the gene that holds the blueprint for a protein. Once the proper amino acids are strung together, they origami-fold into tiny structures with nooks and protrusions that determine what the protein does inside a cell. A protein’s folded shape lets it grab on to specific bits of DNA or hasten certain chemical reactions. Mutations in a gene can shift the resulting protein’s shape or alter subtle aspects of its behavior so that, over time, a protein’s function can change. But the possibilities are not endless. New proteins that fall apart, fail to fold or don’t perform as needed don’t survive the tests of natural selection.
“The physical determinants of folding, stability, solubility, function and specificity are absolutely essential aspects of the evolutionary process,” says University of Chicago biologist Joe Thornton. “That has not been widely appreciated or explicitly addressed until pretty recently.” Now, Thornton says, it’s clear that to understand molecular evolution, it’s important to study proteins as functioning, physical objects.
As they reconstruct proteins’ pasts, researchers are finding that genetic mutations sometimes remodel a molecule just enough to give a chance to other mutations that would have failed earlier. That creates opportunities for new features and functions to evolve — an idea that biologists have considered for decades but have only just begun to explore in the lab.
Bloom and colleagues, for instance, used an influenza virus protein called nucleoprotein to examine how interactions among mutations have affected the overall evolution of the virus. Understanding the combined effects of several mutations could allow researchers to anticipate the short-term effects of new genetic variation. That knowledge could help improve forecasts of which viral strains are likely to circulate in upcoming flu seasons, important information for designing effective vaccines.
Comparing nucleoprotein genes from strains of the virus isolated in 1968 and 2007, Bloom’s team mapped out the most likely steps by which the 1968 protein morphed into its newer form. Though nucleoprotein still plays the same role that it did in 1968 — aiding in the assembly of viral RNA — 33 of its 498 amino acids changed over those four decades, and a few changed more than once, the researchers reported in 2013 in eLife.
Bloom’s team built the 1968 nucleoprotein, then tested the effects of introducing each historical mutation. Some of the mutations affected parts of the protein that tip off a person’s immune cells that an invader is present — they probably helped the flu virus avoid detection. But on their own, some of those changes were bad for the virus: The nucleoprotein could no longer stay properly folded long enough to do its job.
During the course of the nucleoprotein’s evolution, some mutations boosted the protein’s stability, giving it a bit of a buffer. When later mutations occurred, allowing the virus to buck immune recognition, these earlier changes probably held the structure stable so the protein could still function.
When a mutation’s effects depend on other mutations, this interplay is called epistasis. These interactions within individual molecules have been important in shaping evolutionary trajectories, says University of Oregon biophysicist Michael Harms, who is studying how diverse functions evolved in a group of proteins called s100s. He calls epistasis “the common feature in all of evolution.”
Codependent interactions don’t occur just between pairs of mutations. They can be significantly more complex. Analyzing data from other labs, Harms has found epistatic interactions involving up to six different mutations. Such interplay means that in many cases, if genes had transformed themselves just a bit differently, evolution would have veered onto a different course.
Green light Scientists call mutations that lay the groundwork for future change “permissive” mutations. Some protein functions came about only after permissive mutations modified an evolving molecule in highly improbable ways.
Thornton uses ancestral protein reconstruction to study how steroid hormones — which control stress responses, growth and sexual developmental in vertebrates — evolved partnerships with their receptors. Receptors are proteins that bind to specific partners to activate responses in the cell. By comparing steroid receptors in different species, Thornton can map the evolutionary relationships between the molecules and infer the likely amino acid sequence of their common ancestor. Then he introduces a DNA molecule that encodes the long-extinct protein into lab-grown cells. Those cells use the genetic instructions to manufacture a tiny piece of the deep past.
Many of Thornton’s studies begin with a 450-million-year-old receptor protein that he and colleagues reconstructed in 2006. The protein gave rise to modern receptor molecules that are activated by different hormones. One receptor, the glucocorticoid receptor, responds to the stress hormone cortisol. The other, the mineralocorticoid receptor, controls levels of salt and other electrolytes in response to the hormone aldosterone. Thornton’s team found that their reconstructed ancestor could be activated by both cortisol and mineralocorticoids.
A receptor that responded only to cortisol appeared 40 million years after the promiscuous receptor, Thornton showed. His team found a set of amino acid changes that converted the general ancestral receptor into the cortisol-specific one. But the mutations that changed the ancient receptor’s preference couldn’t have generated a functional receptor by themselves, experiments showed.
“The function-switching mutations are not tolerated on their own,” Thornton says. They destabilize parts of the receptor. Like the flu virus’s evolving nucleoprotein, the ancestral receptor’s structure had to be buttressed before it could withstand the mutations that would make the receptor choosier.
Two amino acid changes quietly readied the ancient receptor for its transformation, Thornton and colleagues reported in 2009 in Nature. Without them, the path to the function-switching mutation would have been inaccessible. “If we were to wind back the clock and set history rolling again, it’s very unlikely that those permissive mutations would occur,” he says. “We would have ended up with a very different glucocorticoid receptor and a very different endocrine system.”
Thornton and Harms, then a postdoctoral researcher in Thornton’s lab at University of Oregon in Eugene, explored whether evolution could have taken an alternate route to the same end. Harms created and screened thousands of variants of the ancestral protein, searching for alternative mutations that might have set it up for the same functional switch. He found none, the researchers reported in Nature in 2014. Evolution, it seems, had acted on a rare opportunity.
Biophysical analyses of variant receptor proteins showed why so few mutations enabled cortisol-specific binding to evolve. Although certain parts need extra support, the receptor also needs to be able to transition between two forms: an inactive conformation when no cortisol is present, and a gene-activating conformation when the hormone binds. Some mutations stabilize the active form of the receptor too much, locking it into an “always-on” configuration. Mutations also had to be compatible with the ancestral protein on their own, before the function-switching mutations were introduced.
“A mutation has to fulfill all these requirements, and that is not easy to do,” Thornton says. “That seems to be the explanation for why permissive mutations [for this functional switch] are so rare.”
But not every new function is the result of complicated epistatic interactions. In January in eLife, Thornton and Ken Prehoda of the University of Oregon described an ancient protein that gained a completely new function by way of a single amino acid change.
The team studied the origins of an animal protein that helps cells orient themselves in space before dividing. Doing so is vital for positioning new cells in the right places within a growing body. Single-celled life-forms had to get this right before multicellular organisms could evolve. Thornton, Prehoda and colleagues focused on a segment of the protein called GK PID (for GK protein-interaction domain), which orients cells by acting as a scaffold during division. The billion-year-old ancestor of GK PID did nothing of the sort. It was an enzyme predecessor to the modern guanylate kinase, which catalyzes a chemical reaction that cells use to make some of the building blocks of DNA. Amazingly, Thornton says, one mutation was enough to transform the ancestral protein from an enzyme to a working scaffold. That surprising result is an example of why developing general theories about the physical principles shaping evolution requires a grasp of the evolutionary histories of a broader collection of proteins.
“Every time people take [a protein] apart, they see a new feature,” Harms says. Fortunately, he says, thanks to faster computers, better software and a growing number of genomes to reference, research on ancestral protein reconstruction is on the rise.
Roads taken While chance events can shift the landscape of evolution’s possibilities, evolving proteins also have some freedom to explore. They can take more than one path to some functions.
Douglas Theobald, a biochemist at Brandeis University in Waltham, Mass., has seen this in his own investigations of an enzyme that many cells use to produce energy without oxygen. The enzyme, lactate dehydrogenase, evolved from structurally similar enzymes not just once, but at least four times in different groups of organisms. By reconstructing the evolutionary events that transformed a similar enzyme, malate dehydrogenase, into lactate dehydrogenase, Theobald and colleagues found that two groups of single-celled parasites came by the same enzyme in different ways. The researchers reported the findings in eLife in 2014 and in Protein Science in February.
The work demonstrates that different genetic backgrounds may steer evolution along different paths in different organisms but still lead to similar outcomes, Theobald says. “Even if there is a lot of epistasis, there’s still lots of different ways to the same function.”
Biochemist Susan Marqusee of the University of California, Berkeley has also found that there’s more than one way for a protein to do something new.
Marqusee collaborated with Thornton’s team to compare how two bacteria, Escherichia coli and the heat-loving Thermus thermophilus, evolved enzymes that do the same job at very different temperatures. T. thermophilus thrives in hot springs, at temperatures that would cause most proteins to fall apart. Biochemists are eager to borrow from nature’s strategies to engineer heat-tolerant proteins but have struggled to find general principles that account for this property. By reconstructing the common ancestor of the RNA-snipping enzyme known as H1 from E. coli and T. thermophilus, Marqusee’s team found out how the bacterial protein takes the heat.
That 3-billion-year-old common ancestor was less stable than the enzyme that T. thermophilus uses today, the team reported in 2014 in PLOS Biology. As the heat-tolerant protein evolved, its stability steadily increased — not because of any one innovation, but by virtue of distinct biophysical strategies at different points in time.
“The physical chemistry doesn’t really matter as long as in the end, they add up to the right phenotype,” Marqusee says. Because evolution was able to take advantage of different amino acids to boost stability in a variety of ways, the enzyme’s growing resilience to hot environments didn’t depend on the chance occurrence of a particular set of mutations.
Foggy future Studies of how proteins have evolved in the past are unlikely to spell out how evolution will proceed in the future. “The emerging picture is that the role of chance is so great that long-term predictions of the future evolution of any protein is a very risky enterprise,” Thornton says. But recent research does offer insights into how and why today’s proteins do what they do. One example comes from Thornton’s work on how the DNA-binding sites on steroid receptors have evolved along with their DNA targets. The hormone-activated receptors act as transcription factors, binding to specific sections of DNA to switch on certain genes. In 2014, Thornton’s team reported in Cell that a bulky amino acid in an ancestral protein prevented the protein from binding to the stretch of DNA favored by many of today’s steroid receptors. The ancestral protein awkwardly bumped up against the DNA, unable to make enough contact to really grab on. The receptor gained its new specificity when mutations ended those obstacles and introduced new clashes that blocked its access to the former binding site.
Researchers often can’t tell which differences between two related proteins make them behave differently. But reconstructing evolutionary paths can point them in the right direction.
Using ancestral reconstruction, Theobald and Brandeis colleague Dorothee Kern studied how Abl, a growth-promoting protein linked to chronic myelogenous leukemia, diverged from the related Src protein. The researchers wanted to know why the anticancer drug Gleevec binds to and shuts off Abl without obstructing Src, even though Src has a very similar structure. Theobald, Kern and colleagues identified 15 amino acids in Abl that are crucial for Gleevec binding. The amino acids influence how the protein transitions between two different configurations (that shape-shifting is disrupted in some patients with Gleevec-resistant cancers). The finding, published last year in Science, suggests that researchers may be able to develop better drugs by considering these conformational shifts.
Some proteins, or parts of proteins, might even be inherently more able to evolve than others. Certain parts of the fast-evolving viral protein hemagglutinin are unusually tolerant of change, Bloom and Bargavi Thyagarajan, who was a postdoctoral researcher in Bloom’s lab, reported in 2014 in eLife. Antibodies against hemagglutinin are the immune system’s best defense against influenza, but the protein is adept at escaping detection.
The researchers used a relatively new method called deep mutational scanning to build and test hemagglutinin proteins with nearly every possible amino acid change — about 10,000 in all — in viruses grown in the lab. In a host, changes that disguise hemagglutinin from the immune system would be advantageous. Even though there was no immune system to hide from in the lab, viruses still survived more changes to parts of hemagglutinin that would be recognized by an immune system than they did changes to other parts of the protein. Bloom and his graduate student Michael Doud reported a more detailed view of the protein and the areas that are more and less likely to tolerate mutations online on bioRxiv.org in April. That’s good for the virus, but bad for people. Hemagglutinin seems capable of accumulating change in the very sites that vaccine developers would like to remain the same. But the finding also suggests that flu vaccines designed to target less mutation-tolerant regions of hemagglutinin might be more likely to protect against the flu from season to season. That’s a strategy some labs are already exploring — targeting the less-evolvable stalk of hemagglutinin’s lollipop-shaped structure.
It’s not yet clear why certain parts of the hemagglutinin protein tolerate change so well; Bloom hopes that studying the mutational tolerance of other proteins will help researchers figure that out.
“We’re never going to be able to predict evolution precisely, because it’s a highly stochastic process,” Bloom says. “But I think we can make better forecasts about many of the evolutionary processes that affect us. These are really challenging problems, but I think we are getting to the point where we can use experiments and molecular understanding to help us think about these processes.”
At the end of the last Ice Age, humans undertook an epic American road trip — trekking from a northern land bridge into interior of North America. But details about the route and timing of that trip are hotly debated.
Some researchers think that humans followed a so-called “ice-free corridor” along the eastern Rocky Mountains. Studies have suggested, though, that the corridor froze over and became impassable around 21,000 years ago. Now, a bread crumb trail of fossils showing the movement of ancient bison indicates that the corridor may have reopened a few thousand years later, researchers report in the Proceedings of the National Academy of Sciences the week of June 6.
Analyzing DNA samples from 78 bison fossils unearthed in Canada, a team led by Beth Shapiro of the University of California, Santa Cruz, found that genetically distinct northern and southern populations moved north and south along the corridor by 13,000 years ago. That means humans may have also hiked along the slopes of the Rockies at the same time.
These probably weren’t the first humans to head south. Recent archaeological evidence points to early Americans trekking as far as Chile more than 15,000 years ago. But the corridor could have served as a later route, the team argues.
