Tardigrades protect proteins on the brink of death

With their chubby legs and button noses, tardigrades certainly don’t look like they hold the secrets to life and death in their spindly claws, but they do. These charismatic animals — often called water bears or moss piglets — have evolved unique adaptations to thrive in environments where few others can survive.

While they typically live in watery locales from Himalayan snowpacks to moss growing in a parking lot, tardigrades can survive dehydration, freezing, temperatures above the boiling point of water, and even the vacuum and radiation of space (1,2). To do this, tardigrades remove water from their bodies and slow their metabolism to just 0.01 percent of its normal rate. In doing so, they transition into their “tun” form, a state of suspended animation in which they can live for decades. Exposure to just a little bit of water restores tardigrades from tuns to their normal, turgid selves.

“We always hear the adage, ‘Life is water,’ and we know that all metabolism requires water,” said Thomas Boothby, a tardigradologist at the University of Wyoming. “How can you have life without metabolism? It's almost a philosophical question.”

By toeing the line between life and death, tardigrades offer scientists the opportunity to understand what it means to be alive — and perhaps, how to keep living things alive. With the advent of new genetic tools and a growing community of tardigrade researchers, scientists are investigating how tardigrades survive stressful conditions with the hope of translating those lessons into more stable drug formulations. With their ability to protect against dangerous radiation levels, tardigrade biology may even aid humans during long-term space missions to the Moon and Mars.

Finding order in disorder

As the sun climbs high in the sky, the water surrounding tardigrades living in a puddle of pond scum slowly starts to evaporate. As the environment dries, tardigrades’ cells begin to lose water, and the concentration of the molecules inside them grows higher and higher.

“Desiccation isn't an all or none stress,” Boothby explained. “It's a continuum of stresses.”

As cellular structures squeeze together, proteins become more likely to aggregate with one another. When the cell no longer has enough water in it to form hydrogen bonds with proteins, the proteins will unfold and lose their function. Somehow, tardigrades found a way to prevent the loss of protein function due to desiccation, and scientists had no idea how they did this until just a few years ago.

“Five, six years ago, a postdoc from biology here at UNC walked into my office and said he had found the genes that allowed tardigrades to survive desiccation,” said Gary Pielak, a protein chemist at the University of North Carolina (UNC) at Chapel Hill. “That guy was Thomas Boothby.”

Working together, Boothby, Pielak, and their colleagues reported that as conditions dry out, tardigrades express unique proteins called tardigrade intrinsically disordered proteins (TDPs) (3). While tardigrades don’t need TDPs to live their normal aquatic lives, as their watery home dries up, they can’t live without them. When the researchers expressed TDPs in bacteria and yeast, the proteins increased those organisms’ desiccation tolerance by almost 100-fold.

Since this discovery, tardigrade researchers have identified a number of different TDPs involved in protecting tardigrades during desiccation (4). But how exactly TDPs and other aspects of tardigrade biology protect the animal remains an open question. 

For the most part, these studies relied on expressing tardigrade proteins in model systems like yeast and bacteria because many sophisticated genetic manipulation techniques weren’t possible in tardigrades — until now.

Kazuharu Arakawa, a tardigradologist at Keio University, and his team recently developed a system called TardiVec that allows scientists to study tardigrade genes within the animals themselves (5). The first thing they did was express green fluorescent protein (GFP) under control of the promoter for the actin protein, a gene with a high expression pattern.

“We were really excited,” said Arakawa. “To see your favorite organism shine bright is something really, really extra.”

Eager to better understand how tardigrades protect themselves during desiccation, Arakawa and his team tagged proteins known to be involved in desiccation protection with GFP. To their surprise, these TDPs were expressed only in certain cell types — not every cell.

For example, the researchers saw that tardigrades only expressed the TDP cytoplasmic abundant heat soluble protein (CAHS) in epidermal cells. Another desiccation protective protein, secretory abundant heat-soluble protein (SAHS) was almost exclusively expressed in storage cells, which are free floating cells in the tardigrade thought to store energy.

“In humans, if the cells get damaged, our cells go into apoptosis,” said Arakawa. “It doesn't work like that in tardigrades, so every single cell in tardigrades must be protected upon dehydration.” Why tardigrades express some desiccation protective proteins only in certain cells is puzzling.

“It complicates the situation. What we thought we knew was only a part of the story,” said Arakawa.

Moving forward, Arakawa plans to use the TardiVec system to identify the mechanisms driving desiccation tolerance by studying species of tardigrades with different desiccation tolerance abilities. For example, Ramazzottius verieornatus can transition to a tun state in 15 to 30 minutes, Hypsibius exemplaris must first spend about 24 hours in a semi-dry environment, and the species Thulinius ruffoi cannot desiccate at all.

“By introducing the tardigrade specific genes that we found in Ramazzottius into Thulinius or the natural state of Hypsibius without preconditioning, we might be able to engineer those tardigrades to be desiccation tolerant,” explained Arakawa.

By understanding how tardigrades survive the harsh stresses of dehydration, Arakawa and his team hope to apply these lessons to preserving biological materials for human health, whether that’s stabilizing vaccines and biologics to preserving blood donations for longer periods of time.

Need blood? Add TDPs

While multiple research teams have used TDPs to protect model proteins from degrading due to dehydration, Boothby wanted to know if that protection could extend to lifesaving biologics.

In a recent preprint, he and his team tested whether tardigrade CAHS proteins could stabilize human blood clotting factor VIII (FVIII) in a dry state under a variety of temperature stresses (6). FVIII is an important component in the blood clotting pathway, and clinicians use it to stop bleeding in trauma patients and in people with the genetic blood clotting disorder hemophilia. Currently, manufacturers add polyethylene glycol to FVIII to make it stable at room temperature, but this level of stabilization is still not good enough for people living in certain hot locations.

“Room temperature, what that really means, is up to about 30°C, but of course, there are many places in the world where ambient temperatures are well above 30°C,” said Boothby. Doctors in Texas reported that “people have trouble storing these pharmaceuticals in their homes because their house gets hotter than 30°C during the summer,” he added.

The team developed variants of the CAHS D protein with different biophysical properties and found that certain variants protected FVIII better under specific conditions. For example, one variant called CAHS D 2X Linker forms a gel more readily than the standard CAHS D protein. When Boothby and his team mixed FVIII with 2X Linker, they could dehydrate and rehydrate FVIII for six consecutive cycles without damaging its blood clotting function. This variant also kept FVIII stable for 10 weeks in a dry state.

“This is really important because in the field, you don't often have really precisely controlled humidity, so you can get partial rehydration or more severe desiccation,” Boothby explained. “Being able to tolerate these fluctuations in hydration is important.”

When they mixed FVIII with a different variant called CAHS D Linker Region that cannot form a gel at all, they found that this variant stabilized FVIII in a dry state at temperatures as high as 95°C for two days, which is almost double the highest temperature ever recorded on Earth — 56.7 °C in Death Valley in 1913.

“We're really interested in this question of the biophysical state of these proteins,” said Boothby. “A non-gelling variant, how does it protect a protein versus the gelling variant? That really helps us with future engineering [and] applied goals where we want to modify the protein to stabilize a certain type of pharmaceutical or biologic.”

For example, if they want to stabilize a particular protein-based vaccine that is prone to aggregation in a dry state, “then we have a pretty good idea of what our top candidates from our variants would be,” he added. “We want to dig into the mechanisms by which these proteins work, so then we can make them work for us and do the things we want them to do in formulations.”

Protecting human cells

For Roger Chang, a computational biologist at Albert Einstein College of Medicine, tardigrade proteins don’t have to stop at preserving biologics. They may one day inspire new drugs for human diseases too. Chang has always been interested in organisms that live on the extremes of survivability. He started his research career working on the bacterial species Deinococcus radiodurans, the most radiation resistant organism ever known.

“It's a bacterium, not a complex animal system, which is one reason why studying tardigrades became interesting because it's evolutionarily certainly much closer to humans,” said Chang. He and his team wondered if disordered proteins from tardigrades and other extremotolerant organisms could protect human cells from stress, in particular, chemical stress from a chemotherapy drug (7).

Chang and his team screened approximately 300 proteins or protein fragments with disordered regions by expressing them in human cells. They included proteins from extremophile organisms including tardigrades, nematodes, and the Chinese giant salamander, but they also considered both full length and truncated forms of human proteins. 

“If we want to develop a therapeutic, it might be the case that if you were able to extract something like this from the human proteome, you would have less concerns about immunogenic effects perhaps because it's more human and not so foreign,” said Chang.

The researchers then treated the cells with the common chemotherapy drug camptothecin, which kills cells by triggering apoptosis. They found that even though most of the proteins had no evolutionary relationship to one another, they all protected against the chemical stress to a similar extent.

What surprised the team the most was that their top protective proteins were not tardigrade ones, but fragments of the human proteins APOE4 and APOE2. (APOE4 is best known for its role in Alzheimer’s disease.) They realized that these APOE protein fragments contained a series of alpha-helical repeats, which disordered proteins from extremophile organisms also have.

“If you pull out just that repeat region, that is the part that resembles the extremotolerant species’ intrinsically disordered proteins. That is the variant of APOE that is the most protective,” said Chang.

The researchers noticed that the protective APOE fragments formed tiny clusters in the cytoplasm, so they wondered if they and potentially their other disordered protein hits could protect cells from apoptosis by sequestering proteins involved in apoptosis within condensates, membraneless structures that set specific cellular components apart from the rest of the cell. While they saw that many of their most protective hits formed condensates, for the most part, they did not form condensates around key components of the apoptosis pathway. Only one synthetic disordered protein did.

There are definitely a lot of fascinating and impressive evolved adaptations in nature that we can learn from, and I think we're really just scratching the surface. 
– Roger Chang, Albert Einstein College of Medicine

“It's likely that these condensates that are forming are either broadly or specifically interacting with other cellular components and either stabilizing them or simply taking them away from the rest of the cytoplasm and not allowing them to function normally,” said Chang.

He and his team are currently puzzling over the potential connection between the protective APOE protein fragments and Alzheimer’s disease. Most of all, Chang wants to develop a platform to create synthetic disordered proteins that can bind and stabilize any human protein. For example, they could create disordered proteins that bind to proteins such as those sensitive to oxidative stress to prevent the development of disease.

“It's fascinating that nature has come up with solutions for surviving really extreme conditions,” said Chang. “There are definitely a lot of fascinating and impressive evolved adaptations in nature that we can learn from, and I think we're really just scratching the surface.”

To the Moon and Mars

One of the most hostile environments to life is space, but even there, the tough little tardigrade persists. Scientists have launched tardigrades into low Earth orbit, exposing them to both the vacuum and intense radiation of space, and found that they survived the trip (8). When an Israeli lunar lander carrying tardigrades among other cargo crashed into the Moon in 2019, some tardigrades ended up calling space home — although recent research suggests that they likely didn’t survive the impact (9).

While tardigrades may be prolific spacefarers, they did not naturally evolve to be little animal astronauts.

“Obviously, tardigrades do not live in an environment where there is very high radiation,” said Arakawa. “They are not made to tolerate radiation in the first place, but they're made to tolerate desiccation, which is as strong a stress as a high dose of radiation.”

Radiation causes two main stresses in cells: oxidative stress and DNA damage. Tardigrades have to deal with both of these stresses during desiccation as well, so researchers think that tardigrades likely use similar mechanisms to protect themselves from both dehydration and radiation.

Researchers identified one protein in particular called Damage suppressor protein (Dsup) which is unique to tardigrades. When expressed in human cells, it bound to nucleosomes, sections of DNA wrapped around chromatin, and it suppressed X-ray-induced DNA damage by 40 percent (10,11). In a recent preprint, Chang and his colleagues at Weill Cornell Medicine and Harvard University investigated how Dsup protected DNA in human cells from radiation damage (12). The researchers found that Dsup acts on an epigenetic level to lead to a less repressive chromatin environment.

“Even though it comes from tardigrades, it does seem to have a particular response in terms of transcriptional activity in human cells that is quite interesting,” said Chang.

They also confirmed some prior reports that human cells engineered to express Dsup have increased adhesive, proliferative, and antiapoptotic properties. The researchers found that the Hippo and Wnt signaling pathways, which increase cellular proliferation and antiapoptotic effects and also promote radiation resistance in cancer cells when dysregulated, were enriched in Dsup expressing cells (13).

“At this stage, we're probably several steps away from an actual therapeutic coming out of this, but it's a starting point,” Chang said. Additional research on Dsup and other TDPs in human cells may help researchers better understand how cancer cells become radiation resistant and perhaps one day, how to make a drug to increase astronauts’ radiation tolerance.

Long term space missions to the Moon and Mars will expose astronauts to higher radiation levels than those they currently experience on the International Space Station (ISS). In addition to the radiation, astronauts also live in a microgravity environment for months at a time. To better understand the long-term effects of life in space, Boothby teamed up with scientists at NASA Ames Research Center to send tardigrades up to the ISS on a SpaceX Dragon cargo spacecraft.

“If we take a tardigrade that was born here on Earth and we send it up into space, how does it respond to that stress?” asked Boothby. He and his team wanted to learn not only how microgravity and radiation affected tardigrades flown from Earth to space, but also how those stresses affect tardigrades born in space.

Spending two months on the ISS, the tardigrades went through four generations in space with the astronauts preserving tardigrade samples at specific timepoints. Boothby and his team recently received the tardigrade samples back from space, sequenced them, and are now digging into the sequencing data.

“By understanding how tardigrades cope with and adapt to the stresses of spaceflight, we hope that not only can we develop better countermeasures or therapies for protecting humans in space, but also for protecting people who work around radioactive material or are exposed to those situations here on Earth,” said Boothby.

Whether they’re reaching for the stars with their microscopic claws or floating in a water droplet, tardigrades are one of very few organisms that come to the brink of death and can return unscathed. For Arakawa, this is what fascinates him most about tardigrades, and it’s an idea that he thinks is best exemplified by a tardigrade species that lives on moss growing in concrete in Sapporo, Japan.

“They dehydrate every time there's a sunny day and then rehydrate every time there's a rainy day. They constantly go into this desiccated stage, almost every day,” Arakawa explained. “The halt of life is a very common event, which is quite unusual compared to other species.” But, he added, “The transition between matter/no matter is the critical subject to study if we want to really understand what life is.”

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