Helping a Puzzled Clock

Chronobiologists study how a key protein keeps time inside our bodies. Yasemin Saplakoglu ticks off the possible benefits. Illustrated by Bailee DesRocher and Fiorella Ikeue.

Illustration: Bailee DesRocher

Carrie Partch draws a circle on a crinkled piece of paper. “This is our internal clock,” she says, tracing it over and over. “It turns day in and day out.” Keeping her eyes poised on me, she then flicks her pen off into the corner of the page, breaking her perfect circle. “You know what just happened?” she asks with a smile. “I took away CRY.”

You can’t hear the ticking, but inside almost every one of our cells there is a clock—a protein cluster that is attuned to our planet’s rotation. CRY, short for “cryptochrome,” is one of these proteins that keep the body’s clock on a near-24-hour rhythm.

Partch, a mammalian clock researcher at UC Santa Cruz, recently found a key to how cryptochromes help to control the body’s clock. CRY makes sure that our cells know when night falls, she found, by interacting with the other proteins in the cluster through a small pocket. While scientists had long known that cryptochromes were essential to a well-functioning body clock, they had yet to understand how they exerted their control. Now, Partch’s research shows how cryptochromes dock to the cluster, keeping the whole system running on time.

The pocket provides an opportunity for fine-tuning the molecular clock.

Her discovery opens up the possibility of finding a cure for when the clock goes awry—a phenomenon that happens often, usually starting with a step onto an airplane, taking on shift work, or just staying up late one night. The pocket in the protein cluster has a beckoning shape, Partch says, forming a C with her hand: “It’s just screaming for a drug.” She envisions a pill that people could take to prevent jet lag on long-distance trips. Rather than just overriding control of the circadian rhythm by inducing sleep, as melatonin does, such a pill would have a direct and more potent ability to reset the entire clock—and with it, everything it controls, including metabolism and blood pressure.

A different clock is now ticking. The U.S. Patent Office has given Partch one year of exclusive research on the pocket to see whether she can find a small molecule capable of knocking out a confused CRY, resetting the clock to its morning state.

Our ticking cells

Every human carries a solar-powered clock in the suprachiasmatic nucleus, a small region in the hypothalamus of our brain just above where the optic nerves cross. This mammalian master clock plays conductor: By using cues from the sun, it makes sure every cell in the body knows the correct time and is playing together in synchrony. The master clock sends signals to the rest of the body’s cells, telling them to set their own “molecular clocks”—what scientists call the cycling interactions among four main proteins. Almost every cell in the body has a molecular clock. The only cells that can’t tell time are embryonic stem cells and some types of cancer cells.

CRY’s role is to inactivate this protein complex at night to prepare the body for slower times.

The biological clock runs without glimpsing any light, but its synchronization with the sun is what keeps the body on a healthy rhythm. This clock system varies only slightly from the average time cycle of 24 hours, 10 minutes, and 48 seconds among humans. Usually, these slight variations don’t cause noticeable effects on processes controlled by the clock, such as metabolism, immune function, hormone release, body temperature, blood pressure, and of course, sleep. But once the clock begins to sway more than an hour away from a terrestrial day, as happens with certain sleep disorders, the de-synchronization between the world and the internal clock brings about a weariness that feels like constant jet lag.

Top figures are the front and back of an arbitrary model of CRY taken from Carrie Partch’s thesis. Bottom figures are the front and back of the new model of CRY, including the recently discovered pocket. Courtesy of Carrie Partch. Illustration: Carrie Partch

But even an accurate clock is surprisingly easy to break. Flying across time zones confuses the master clock in the suprachiasmatic nucleus, so it cannot give out the signals it usually does based on the sun. Your inner clock might think it’s 3 p.m., but your new environment says it’s 6 p.m., causing the haziness and grumpiness so familiar to frequent travelers. It takes most people a few days, sometimes up to a week, to re-sync to their environment and recover from jet lag.

Upsetting the clock is more than an inconvenience. Mounting evidence raises the possibility that disrupting the body’s clock—a system that controls more than 40% of our body’s entire set of genes—can contribute to diseases such as cancer, cardiovascular disease, obesity, diabetes, depression, anxiety, and mania.

Now, chronobiologists such as Partch hope to find a solution.

Conserved by evolution

Partch joined Nobel Laureate Aziz Sancar’s lab at the University of North Carolina in Chapel Hill as a graduate student in biochemistry and biophysics, right after Sancar’s discovery of the first mammalian cryptochromes. He was working on figuring out how cryptochrome’s ancient ancestor, photolyase, repairs damaged DNA in plant and bacterial cells.

At the time, researchers in the field were fairly certain that humans didn’t have photolyase to repair DNA damage caused by sun exposure. Meanwhile, during the mid- to late 1990s, scientists were racing to figure out the human genome, and labs around the world were publishing everything they knew about human genes.

CRY docks to the clock complex in our cells twice a night, once around midnight and once right before dawn. Illustration and animation: Fiorella Ikeue

One day, Sancar was investigating the genetic sequence of plant and bacterial photolyase genes and found strikingly similar sequences in human genes. At first, thinking it was a mistake, he cloned the human genes and tested whether they repaired DNA. They didn’t, and Sancar realized he was face to face with a group of proteins that had evolved from photolyases called cryptochromes, which were known to exist in plants. No one knew what these mammalian cryptochromes looked like or how they interacted in the body.

Although her desk stood in a distant corner of the lab against a white cinder block wall, Partch was always visible by her bright-red dyed hair that she tended to pull into a messy bun on the top of her head. She spent her time trying to figure out what cryptochromes did, which involved studying protein interactions in cells. She was in the lab so much that her jeans were stained with bleach and blue dye from coloring proteins.

On one typical day, Partch left her pipettes and protein dyes on the bench and decided to tackle cryptochromes with “sheer brute force,” she says. She had an idea: compare cryptochromes and photolyase from different species to see which areas of the cryptochromes were different from their photolyase ancestors, but exactly the same within the cryptochromes themselves. Although photolyases and cryptochromes are from the same family, they have different functions. The former repairs DNA, while the latter keeps time. Partch thought that although cryptochromes differ among species, if some regions were the same and didn’t appear in photolyase, they must be critical to the clock’s function.

Graphic: Yasemin Saplakoglu

By the time Partch stepped foot into the circadian rhythm field, she could go online on the PubMed database to find the identity and location of each amino acid, or protein building block, that made up the different cryptochromes. The string of amino acids that makes up a protein is called its protein sequence. She printed out the cryptochrome and photolyase protein sequences of species ranging from humans to insects and taped these printouts on a wall.

Partch peered through her thin-rimmed glasses as she spent hours highlighting similar areas of different proteins. Scotch-taped printouts of the protein sequences gathered dirt as they crinkled near her feet.

Partch highlighted every amino acid that matched up among mammalian cryptochromes. Using her highlighted pieces of paper and a base model of the structure of the most similar proteins at the time—the photolyases and plant cryptochromes, whose structures had been deciphered—she created a 3D mockup of a mammalian cryptochrome.

With its nooks and folds, the model looked like a rainbow-dyed oatmeal cookie. In a spattering of orange, yellow, green, blue, and pink, Partch highlighted five regions that remained the same across mammalian cryptochromes.

At the time, she had no bioinformatics experience, which would have allowed her to use computer programs to test her hypothesis about the structure of CRY proteins. So she shut the thick, black, leather-bound thesis notebook in which she’d drawn her colorful model. Partch then left Sancar’s lab to pursue postdoctoral research at the University of Texas Southwestern, where she studied the clock and similar proteins. In 2011 she began to set up her own lab at UC Santa Cruz, knowing that she wanted to understand what those five regions of the mammalian cryptochromes do.

The animal cryptochrome

While she was starting her lab, other researchers figured out what the blue, pink, and green regions did.

Five years ago, a lab at the University of Washington in Seattle first published the structure of a mammalian cryptochrome. Ning Zheng, a structural biologist, had been looking for something else altogether: a ligase, a protein that glues DNA strands together. The blue and pink regions of the mammalian cryptochrome turned out to be where the ligase attached to break down CRY. “We were more interested in the ligase without realizing that the cryptochrome was in fact more interesting,” Zheng says.

Zheng remembers how Partch sent him an excited email after he published his result. When he saw the diagram from her graduate thesis where she’d identified the blue region of cryptochrome as one of its more important components, Zheng was equally amazed. “She really had a lot of insight on the structure before it was even published,” he says.

Soon after, Eva Wolf, a crystallographer at the Institute of Molecular Biology in Munich, Germany, figured out how CRY communicates with a protein called PER, which helps CRY inactivate the clock complex. PER binds to a hotdog-like helix of CRY, corresponding to the pink area on Partch’s diagram. PER also gloms onto to the green area that Partch had identified on the backside of CRY and the blue area where the ligase binds. “So PER kind of gives CRY a big bear hug,” Partch says—a hug that keeps CRY from degrading at the wrong time.

 

All that remained was to find the final two regions that Partch had predicted could be important, the ones colored yellow and orange in her thesis. In 2015 Partch and her collaborator Andrew Liu, a circadian rhythm researcher at the University of Memphis, used a detailed imaging technique called nuclear magnetic resonance to find exactly where CRY binds to inhibit the molecular clock complex to prepare for slower hours of the day.

Then, this year, Partch’s lab solved the last piece of the puzzle she had outlined as a graduate student by using small x-ray scattering—a different imaging method that not only reveals the structure of a molecule, but also how it moves. This movement is hard to capture with other techniques, says Partch, but it is necessary to understand how a dynamic complex like the molecular clock actually changes.

Scientists had known that twice a day, the cryptochromes bind to the protein complex to slow down the clock. Partch found out how. A loop extends out of the molecular clock protein complex and fits into a pocket—formerly known to her as the yellow and orange area—on the cryptochrome. Almost a decade after Partch predicted that five colored regions of CRY would be important, she now finally understands why and how these proteins interact to help the body keep time.

In this podcast, Yasemin Saplakoglu follows a chronobiologist who is studying how a key protein keeps time inside the body. Illustration by Bailee DesRocher.

The binding pocket is ancient, Partch notes. Photolyase, CRY’s oldest relative, also has a pocket that binds a molecule to help it harvest light. “The pocket has been there since the origins of this family,” she says. “It has just gotten bigger, so that now it can hold a protein.” While her decade-old diagram correctly predicted that the region of CRY containing its pocket is important, she had underestimated how large the pocket would turn out to be.

Liu thinks Partch’s new paper improves the understanding of how these four proteins interact together to make the molecular clock tick. “The pocket provides an opportunity for fine-tuning,” he says. For instance, perhaps the pocket could serve as a dock for a drug that would allow precise control of the clock. “The idea is that you can make the clock work a little bit better,” Liu adds.

Other researchers, such as Zheng, plan on tailoring their own research based on these results. “I will have to make my models less static,” he says.

Stifling a CRY

Scientists in other fields are turning their attention to these studies as well, Liu says, because they provide a way to link clock function with physiology and disease.

Disruption of the body’s internal clock can happen with aging, taking on night shifts, traveling across time zones, and in physiological conditions such as obesity and cancer. Partch was the first to show that a unique protein found in some cancer cells was a time thief and could entirely stop the clock. This disruption is dangerous because the clock is interconnected with bodily processes. And it can wreak havoc; studies have linked a number of diseases to disturbed clocks.

Upsetting the clock can disrupt sleep, mood, the brain’s reward center, and learning, leading to sleeping disorders and depression. In the blood, it can increase the amount of fats, leading to high cholesterol. It can increase the amount of cytokines, small proteins secreted in the immune system, and contribute to autoimmune disorders. It can cause the body to store more fat, leading to obesity. It can even tamper with DNA repair proteins, which can lead to tumors. “The clock is not just something that sits in my office on my wall,” Liu says.

Meanwhile, Partch is racing against time. She is looking for a small molecule that could bind to a confused cryptochrome and keep it off the clock complex, allowing the molecular clock to return to its dawn-like state. Such a molecule might serve as a drug that travelers could take to synchronize their body clocks to the time zone to which they’re traveling, thus preventing the ill effects of jet lag.

Other labs are also working on developing possible drugs to control the clock, but the U.S. Patent Office has ruled that no other lab can patent such small molecules for one year.

“It is a model that is supported by a lot of evidence,” says Eva Wolf of Munich. “One could probably put a drug into that pocket and disrupt the main interaction between cryptochrome and [the other proteins].”

Reflecting on her years of research on CRY, Partch pulls out her thesis to a page showing her decade-old mammalian cryptochrome mock-up model—decorated with its blue, pink, yellow, orange, and green swatches. At the time, she noted the conserved areas of the protein. Though she didn’t know their function, she wrote that they were “striking.” “Yes, striking indeed,” she now says. These regions stayed constant across millions of years. Sure enough, they turned out to play major roles in the ticking of our internal clocks.

If Partch’s hunch about the pocket is right, there may come a time when we pack a time-zone-switch pill along with our passport, ibuprofen, and phone chargers—an era when we may have to remember to swallow the correct time.

© 2017 Yasemin Saplakoglu / UC Santa Cruz Science Communication Program

Yasemin Saplakoglu

Yasemin Saplakoglu

Author

B.S. (biomedical engineering) University of Connecticut

Internships: Princeton University Office of Research, Scientific American

The first time I felt like a science communicator, I had red cabbage in one hand and brussel sprouts in the other. Wearing a lab coat down to my toes and goggles too big for my face, I explained to my 7th grade classmates—as they all held their noses—why I was extracting colors from malodorous vegetables.

Numerous chromatography experiments later, science remained my muse and writing became my pastime. I was captivated both by observing the spontaneous beating of cardiac cells cultured together and by the elegant fall of words in a sentence. Inspired by my rediscovery of a box of stories I had written as a child and from a talk given by Rebecca Skloot on the interwoven fields of science, writing, and ethics, I found myself running toward the redwoods.

Yasemin’s website

Bailee DesRocher

Bailee DesRocher

Illustrator

B.F.A. (theatre arts) Valdosta State University

Internships: SUNY Buffalo, La Brea Tar Pits, StarTalk Podcast Network

Bailee is a science illustrator based in Los Angeles. Since receiving her B.F.A., she has been an educator and artist at the Natural History Museum of Los Angeles County, as well as an educator and fossil preparator at their sister institution, the La Brea Tar Pits & Museum. She truly enjoys working with scientists, writers, and educators to develop visual science communication and outreach materials, including comics and animation. Bailee is extremely enthusiastic about paleontology, minerals, space exploration, urban wildlife, and nachos.

Bailee’s website

Fiorella Ikeue

Fiorella Ikeue

Illustrator

B.A. (biology) Kalamazoo College

Internship: Patagonia

Fiorella is an artist and science illustrator based in the Bay Area. She works with clients to create visuals that communicate scientific information in a beautiful and compelling way. She is well-versed in the overlap between science and art. Her personal work consists of traditional paintings that explore connections in nature and give voice to the species and places that need protection. This summer she will intern with Patagonia to create large-scale graphics that showcase the company’s environmental initiatives.

Fiorella’s website

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