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Fo For as long as living things have been building proteins based on the code of messenger RNA molecules, there have been aminoacyl-tRNA synthetases. These enzymes, abbreviated as AARS, link transfer RNAs (tRNAs) with the corresponding amino acids. That seems like a tall enough task for a class of enzymes, and when protein-based life began, it was. However, as organisms have become more complex, AARS have acquired additional domains that allow them to do much more.
"In humans, the synthase is already highly decorated" with these additional domains, he says.paulo mold, a biochemist at the Scripps Research Institute who is studying this additional work.
Living things have at least one type of AARS molecule for each of the 20 proteinogenic amino acids. For some amino acids there are two variants with a separate enzyme for use in protein translation, which takes place in the mitochondria. All of these synthetases have a core segment involved in tRNA and amino acid binding, and all but one harbor one or more additional accessory domains. In addition, cells can produce more than 300 different protein variants from AARS genes through alternative splicing of their mRNAs or post-translational protein fragmentation. Some of these variants function as inflammatory cytokines. Others orchestrate the formation of blood vessels. The AARS for glutamic acid and proline are fused into a bipartite protein; The connector between them appears to control immune activity and lipid metabolism, and may even affect lifespan. Many AARS have been linked to human diseases caused not by defects in protein assembly but by these other non-canonical functions.
I heard how skeptical the department was about these results. I'm not blaming you. I would also be confused.
– Xiang-Lei Yang, Scripps-Forschung Institute
Some researchers now view enzymes as drug targets for cancer, immune disorders, and other diseases. The company that Schimmel co-foundedaTyr Pharmain San Diego, AARS proteins envision themselves as an entirely new class of drugs, distinct from small molecules or other biologics. The company currently operates aclinical studyare testing a part of the histidine enzyme HisRS for the treatment of inflammatory lung disease.
Alternative functions of SAAR in lower organisms such as bacteria have been known since the 1980s, but its activities are not extensive, Schimmel says. Then, beginning in the 1990s, Schimmel and others began to discover non-canonical functions in higher eukaryotes, beginning with unexpected roles in angiogenesis. The discovery of new functions for these ancient proteins was "a big surprise," he says.worthy of David, biochemist from the University of Toledo. But given the various features that researchers studying AARS have discovered, many of which relate to important pathways in the disease, Dignam says he thinks aTyr's approach makes sense. "Arguing that you can make medicine based on that is very logical in my opinion."
While other proteins have taken on supporting roles, the number and variety of byproducts found in AARS are remarkable, Schimmel says. And he doesn't think it's a coincidence. These special synthetases have been around since the dawn of protein-based life and are available for evolutionary modification. Given their essential role in protein synthesis, they occur regularly and are unlikely to disappear from a viable genome. This makes them a stable substrate for new functional domains. Furthermore, they have specific amino acid binding sites ready to interact with other proteins.
"It's a lock and key," says Schimmel. "Any protein that stands out on a good side chain that matches a synthase can eventually become a partner."
buildup and blockage of blood vessels
Schimmel says he's long been fascinated by AARS's original function: interpreting the genetic code. In the 1990s, Schimmel's lab, then at MIT, sequenced the AARS genes. "We were interested in developing small molecules that would target them and stop their activities in specific ways," he says. For example, if the AARS of a pathogen is different enough from that of humans, an antibiotic could be developed that disrupts protein synthesis in the infectious agent.
Schimmel was a postdoc at the time.Keisuke WakasugiI was curious about the sequence of the gene that codes for TyrRS, the AARS for tyrosine. In humans, TyrRS contains an extra segment at the carboxy terminus of the enzyme, a feature not present in prokaryotes or lower eukaryotes. The amino acid sequence for this part of the protein resembled that of a human cytokine, EMAP II, which recruits circulating immune cells in tissues to promote inflammation. Wakasugi decided to test this carboxyl domain for cytokine-like activity.
"That's a stupid idea," recalls Schimmel. But Wakasugi went further and, in fact, the TyrRS carboxyl domain behaved exactly like EMAP II, inducing cultured phagocytes and leukocytes to migrate and release inflammatory signals. Full-length TyrRS, on the other hand, did not affect cell behavior. This raised the possibility that the carboxyl domain in TyrRS might be defective for immune functions. No one in the lab believed the discovery at first, so Wakasugi repeated the experiments with the same results.
Although it took more than a decade to prove that such AARS fragments were actually present and relevant in a living animal, Wakasugi knew he was on the right track. "Paul and I were very excited to discover a new and unexpected function for human TyrRS," recalls Wakasugi, now a biochemist at the University of Tokyo. “Throughout this project, I felt like we opened the door to a new area of research.”
In the same study, Wakasugi also examined the amino-terminal catalytic domain of TyrRS and wondered if it might also affect cell migration. It behaved similarly to the cytokine interleukin-8 (IL-8). Both the amino-terminal fragment of TyrRS and IL-8 bind to the IL-8 receptor on certain leukocytes,make them migratein culture
The different functions of synthetases.
Aminoacyl-tRNA synthetases play a crucial role in protein synthesis by linking tRNAs with the amino acids dictated by the codon sequence. All AARS have also been found to perform non-protein synthetic functions in different body systems in different in vitro and in vivo systems. This table contains a selection of the best studied examples.
Vascular system and angiogenesis
CELL CYCLE AND TUMORIGENSIS
recruited moldXiang-Lei Yang, a postdoctoral researcher specializing in structural biology, to his lab at Scripps in La Jolla, California, to study how TyrRS might handle alternative functions. Yang focused on a specific amino acid sequence, glutamic acid-leucine-arginine, which is required for the cytokine activity of the fragment synthetase. The same sequence was also found in IL-8 and related cytokines. In crystal structures he found the complete TyrRS.bury this reason, but was exposed on the cytokine-like fragment.
IL-8 was known to promote blood vessel formation and growth, so Wakasugi also tested its amino-terminal TyrRS fragment for angiogenic activity. When he injected the mice with a small gel containing the fragment,The blood vessels have grown.and impregnate the gel. To further investigate this action, Schimmel called his colleague ScrippsMartin Friedlander, an ophthalmologist and cell and developmental biologist, and asked him to test the TyrRS fragment in his mouse models of ocular vascularity. Friedlander agreed, but also asked for a check. Therefore, together with the human TyrRS fragment, Wakasugi provided a natural variant of the tryptophan enzyme, TrpRS, which lacks the glutamic acid-leucine-arginine motif.
The results, Friedlander recalls, were not exactly what he expected. TrpRS, the putative control, "had a much stronger effect," says Friedlander, who is also president of the Lowy Institute for Medical Research in La Jolla. But this effect was the opposite of what TyrRS did: instead of promoting angiogenesis, as Wakasugi had seen in the gel, the TrpRS fragmentblockedin mammalian cell cultures, chick embryos, and mouse eyes. "TyrRS and TrpRS may have evolved as opposing regulators of angiogenesis," says Wakasugi.
Scientists initially resisted the idea that an AARS could have functions beyond protein synthesis. Yang remembers attending a conference shortly after Wakasugi published her paper on angiogenesis, where others were unaware that she was a Schimmel acolyte. Undercover, "I heard how skeptical the field was about these results," she recalls. "I don't blame them. I would be confused too.
Aminoacyl-tRNA synthetases play a key role in protein translation by linking transfer RNAs to their cognate amino acids. But in the hundreds of millions of years that they've been around, these synthetases (AARS) have done a lot of parallel work. One is to control the development of vertebrate vascularity.
© Thom Graves
Several AARS play a role in the evolution of the vertebrate circulatory system. During development, the serine enzyme SerRS downregulates the expression ofvascularendothelial growth factor A (VEGF-A),Avoid overvascularization.
In addition, a combination of glutamic acid and proline synthetase, GluProRS, binds to other proteins to form the interferon-γ-activated inhibitor of translation (GAIT) complex to block VEGF-A translation.
A part of the tryptophan synthase TrpRS also helps dampen angiogenesis by binding to and blocking VE-cadherin receptors on endothelial cells, preventing them from attaching to form the lining of blood vessels.
Meanwhile, a fragment of the TyrRS tyrosine synthase appears to promote blood vessel growth by stimulating the migration of these endothelial cells.
When did these functions arise in evolution?
According to biochemists at the Scripps Research Institutepaulo mold, the addition of accessory domains that perform such tasks tracks important events in the evolution of the circulation. The first blood vessel system, which lacked the endothelium present in modern vertebrates, probably arose in a common ancestor of vertebrates and arthropods around 700 to 600 million years ago. Around the same time, TyrRS acquired a glutamic acid-lysine-arginine motif that is now believed to promote angiogenesis. Then, around 540 to 510 million years ago, a vertebrate ancestor evolved a closed vascular system that pumped blood through endothelium-lined vessels. Sometime during the same period half a billion years ago, TrpRS incorporated a WHEP domain that now regulates its ability to block angiogenesis. Furthermore, SerRS acquired a unique domain for this enzyme, which now prevents supervascularization in developing zebrafish and probably other vertebrates.
On the other hand, the role of GluProRS in angiogenesis does not appear to be closely synchronized with the evolution of the vasculature. A linker protein connected AARS to glutamic acid and proline enzymes about 800 million years ago, before circulatory systems existed.
© THOM GRAVES
700-600 million years ago
540-510 million years ago
See the full infographic:RED|pdf
vascular system and beyond
While the functions of TyrRS and TrpRS discovered by Wakasugi and colleagues were interesting, it was not clear whether the enzyme fragments actually performed these functions in vivo. Yang realized that in order for her and other scientists to trust the non-canonical features of AARS, she needed to find evidence that they were present in animals.
The team hasn't done this yet for TrpRS or TyrRS, but Wakasugi has found his opportunity with the SerRS serine enzyme. Several published genetic screens in zebrafish have identified defects in vascular development when SerRS is mutated. But the mutations that eliminated the enzyme's ability to bind tRNA and amino acidshas not caused such defects, indicates thatAnything elsehappened.
To find out what, Yang turned to a sequence called UNE-S, which is found in vertebrates but not invertebrates, SerRS. Yang's team, who joined the Scripps faculty in 2005 and now shares a lab with Schimmel, quickly identified a nuclear localization sequence within UNE-S and found that mutations that disrupted this signal caused the vascular defects in the cells. zebrafish embryos. They discovered that SerRS resembles at its coreminimize expressionof vascular endothelial growth factor A (VEGFA). The study, published in 2012, was the first to demonstrate an essential and natural role for an AARS accessory domain in a living animal. Soon after, the team reported that SerRS was jamming nuclear blocks.VEGFAfor competitions andInterventions in c-Myc, a transcription factor that normally promotes gene expression.
Meanwhile, Schimmel and Yang's groups continued to try to explain the non-canonical functions of TrpRS and TyrRS, even as they found more parallels for these enzymes. Yang led studies on the structure and mechanism of the TrpRS fragment. He found that full TrpRS does not affect angiogenesis, because it doesbounded by a WHEP domain– so named because this domain is found in the aminoacyl-tRNA synthetases for tryptophan (W), histidine (H), glutamic acid (E), and proline (P), as well as the enzymes glycine and methionine. Yang's team found that when TrpRS is exposed by proteases in the extracellular space, it binds to a cellular receptor called VE-cadherin. In particular, tryptophans in the receptor appeared to enter the TrpRS active site.create the link. Therefore, Wakasugi saw that only the fragment, not all of the TrpRS, blocked angiogenesis.
More recently, mold has also taken an interest in plant-derived amino acid-like compounds, such as resveratrol, the substance in red wine thought to combat oxidative stress. Resveratrol and tyrosine are similar in that they both contain a phenolic ring, and this is important for resveratrol's ability to affect pro and antioxidant gene expression. In 2015, Schimmel's team reported that under stress conditions, TyrRS moves into the nucleus of cultured human cells or living mice, where the present resveratrol fits neatly into the TyrRS active site. This disables the normal catalytic activity of TyrRS to bind tyrosine molecules to the corresponding tRNAs. Instead of TyrRSstimulates PARP-1 activation, an enzyme involved in DNA repair.
A few years later, the team discovered that an alternatively spliced version of TyrRSstimulates platelet proliferationin mice and cultured cells and could be used to treat people with low platelet counts.
Schimmel hopes that AARS' non-canonical roles will keep the group busy for a long time. "We're just scratching the surface of what needs to be learned," she says. "I am just as excited or even more excited about these enzymes than when I started decades ago."
Dealing with inflammation and metabolism.
When the evidence for non-canonical functions for the AARS came out of Schimmel's lab,Pablo Fuchs, a biochemist at the Cleveland Clinic's Lerner Research Institute, studied the control of inflammation in macrophages. In particular, his team studied a complex that forms when cells are exposed to the cytokine interferon-γ. A protein complex called GAIT (interferon-γ activated inhibitor of translation) produced in macrophages binds to and blocks inflammation-related mRNAs. Within the complex, the researchers found GluProRS, an enzyme that contains the AARS of glutamic acid and proline.
"We stumbled across it by chance," Fox recalls. "I didn't think it was an interesting enzyme." But he knew about Schimmel's work and picked up the phone to call Scripps.
Within a minute of starting the call, Schimmel interrupted Fox to greet Fox on what Schimmel called the most exciting area of AARS research: non-canon features. Mold has also promised to help, says Fox. "He's been a great support and friend ever since."Kim Sunghoon, a former postdoctoral fellow in Schimmel's lab, now at Yonsei University in South Korea, Fox's team discovered that interferon-γ causes GluProRS to be phosphorylated, leaving its place in translation and binding to the other members of the GAIT.stop productionof inflammatory proteins.
It's not clear why glutamic acid and proline synthetase paired up around 800 million years ago, but Fox has one.hypothesis, which he published in 2018. Proline is synthesized from glutamic acid, and during this evolutionary phase, emerging animal proteins began to contain more proline. This may have resulted in increased production of ProRS, which took up all available proline, requiring more to be produced from glutamic acid. This may have led to a deficit in glutamic acid levels, which impairs protein synthesis. "The solution was to merge the two synthetases into a single gene so that they were produced in equal amounts," says Fox. "No one steals from the other."
The linker between the two synthetases is crucial for the activity of the GAIT complex; is outthree WHEP domainsthat bind to target RNAs. Fox speculates that sometime after the linker appeared, the cell co-opted it to regulate inflammation.
Fox's team recently wondered if the GAIT signaling pathway might work in cells other than macrophages. When the researchers looked at the fat cells, they saw that the insulin treatment caused GluProRS to become phosphorylated, bypassing the protein synthesis machinery. But he did not join the other GAIT partners. Instead, it paired with a normally cytosolic protein called fatty acid transport protein 1 (FATP1). Together, the molecular duo reached the fat cell membrane, where the transporter carried the fatty acids into the cell.
I am just as excited or even more excited about these enzymes than I was decades ago when I started using them.
– Paul Schimmel, Scripps Research Institute
The researchers engineered a mouse that lacked the phosphorylation site necessary to release GluProRS in order to find FATP1. With reduced fatty acid storage capacity, the mice were lean and weighed 15% to 20% less than controls. Furthermore, they were almost alive.four more months, giving them about 15% more lifespan. A similar increase in people would equate to a decade or more. "If we could target this phosphorylation site, maybe we could extend the lifespan," Fox says. His lab is in the early stages of finding a small molecule to inhibit this phosphorylation event.
In the many jobs that AARS has taken on beyond its traditional role, Schimmel and his colleagues see a problem: They keep cells and bodies stable. "They seem to be something that plays a modulatory role and restores more homeostasis," she says.Leslie Nangle, a graduate of the Schimmel Laboratory who is now a Senior Director of Research at aTyr Pharma. Many researchers think tinkering with these essential enzymes is risky, Kim says, but he and Schimmel see potential in using AARS to treat disease. Schimmel's company aTyr, which Kim and Yang also co-founded, hopes to turn the enzymes themselves into biologic therapies. In addition, Kim runs the non-profit drug discovery organization in Seoul.Biocon, where researchers are developing several small molecules that interact with AARS and biologics based on natural variants of AARS.
Biocon is currently testing molecules to treat cardiac fibrosis, alopecia areata (an autoimmune disease that causes hair loss), and inflammation. One fibrosis treatment, currently in phase 1, targets the site of proline synthetase, which binds the amino acid to its tRNA. Fibrosis results from an accumulation of collagen, which is two-thirds proline. The Biocon researchers found that a drug can target this active site and shut down canonical function in healthy cultured cells by more than 90% without severely affecting other protein synthesis or cell proliferation, Kim says. At first, he and his colleagues did not believe the results, but he began to understand them. "A normal cell doesn't necessarily carry out high-level protein synthesis all the time," he says. "As long as there is some residual activity, a normal cell can be perfectly happy."
For cancer and other diseases, Biocon is developing small molecule candidates that either bypass the tRNA amino acid binding site or target the extracellular activities of secreted AARS, meaning protein synthesis should not be compromised. In addition, aTyr researchers expect the company's therapeutics, based on AARS's own derivatives, to be relatively safe. "When you come from a world of natural physiology, you start to feel better," says aTyr CEOsanjay shukla.
In the retina of developing mice, fragments of tryptophan synthetase, TrpRS, lacking a restrictive protein coat (B, C) prevent vascularization of a secondary tissue layer (right). (The images on the right include the shadows from the neighboring primary layers shown on the left.)
PNAS, 99:178–83, 2002, © 2002 NATIONAL ACADEMY OF SCIENCES
Nangle and colleagues, along with a subsidiary of TyrPangu Biopharmacyin Hong Kong, began cataloging and subsequently evaluating natural AARS splice variantsbiological activitiesin a variety of human cell-based assays. They looked for effects on cell proliferation and protection, immune modulation and inflammation, cell differentiation, regulation of transcription, and cholesterol transport. "We think there must be a therapeutic benefit," says Schimmel.
aTyr is currently looking for an immunomodulator based on the WHEP domain of the histidine HisRS enzyme. Complete HisRS in human T cell culturessilenced activated cellsand reduced cytokine production. In subsequent experiments, the aTyr researchers found that the WHEP domain binds to receptors on these immune cells to decrease activity. The company hopes its modified version of the HisRS-WHEP peptide, which binds to part of an antibody so it stays in the bloodstream longer, will have the same calming effect on an inflammatory disease called sarcoidosis. This disease affects a variety of organs, most commonly the lungs, and can sometimes require lifelong treatment with immunosuppressive steroids. These drugs come with a list of dangerous and distressing side effects ranging from insomnia to glaucoma and infections.
aTyr presented results from multiple animal models of inflammatory lung disease at the American Thoracic Society meeting in 2017, 2018, and 2019, and these results suggest that the company's 1923 candidate appears promising. For example, the cancer drug bleomycin can damage the lungs, but HisRS or its WHEP domain reduced inflammation and fibrosis.19The bleomycin-treated mice breathed rapidly to compensate for their damaged lungs, but the 1923-treated mice returned to a normal respiratory rate.
aTyr's 1923 has already passed Phase 1 safety testing in healthy people with no red flags. Now, the company is running a phase 1/2 trial in people with sarcoidosis to confirm safety, find the right dose, and even see signs of efficacy. The patients are taking steroids in the study and the goal is to decrease their steroid dose during the study. Those taking 1923 are expected to have the same symptoms or improve, while those taking a placebo should see their symptoms worsen as their steroid doses are reduced.
It is proof of the need for a new treatment that volunteers are willing to risk their symptoms intensifying if they end up in the placebo arm, says the participating doctor.Daniel Kulver, a pulmonologist at the Cleveland Clinic. "[Steroids are] very toxic," says Culver, who notes that one of his patients calls his steroid prescription "the devil's drug" because it does almost as much harm as good. "People are very, very motivated to find something different."
The study is scheduled to enroll 36 participants, but has been delayed by the COVID-19 crisis. With such a small sample size, Culver isn't expecting a "home run," but he hopes the data will be good enough to start a larger Phase 3 study. aTyr is also planning a 30-person Phase 2 study starting in 1923 for Respiratory Complications Related to COVID-19.
If aTyr is successful, it will be the first instance of a therapy built around an AARS, but probably not the last. In Kim's view, SAARs are ready and waiting to respond to anything that challenges homeostasis, from cancer to the novel coronavirus. "I am renaming the synthetases 'Molecular 911'."
Correction (June 2, 2020): The original version of a table in this story states that during HIV infection, lysine synthetase (LysRS) is packaged into new virus particles that use their UUU sequence to initiate reverse transcription in newly infected cells. Instead, the viral particles use LysRS to deliver their cognate tRNA, which is used as a promoter for reverse transcription.The scientistsorry for the mistake