Daily Archives: 03.02.2016

Scientists Solve 40-Year-Old Salt Mystery

Report from The Scripps Research Institute

Scientists have discovered how the element sodium influences the signaling of a major class of brain cell receptors, known as opioid receptors. The discovery, from The Scripps Research Institute (TSRI) and the Univ. of North Carolina (UNC), suggests new therapeutic approaches to a host of brain-related medical conditions.

Researchers have unraveled the mystery of how the element sodium influences the signaling of opioid receptors, opening the door to new kinds of therapies. Image: The Scripps Research Institute

“It opens the door to understanding opioid related drugs for treating pain and mood disorders, among others,” says lead author Gustavo Fenalti, a postdoctoral fellow in the laboratory of Prof. Raymond Stevens of TSRI’s Department of Integrative Structural and Computational Biology.

“This discovery has helped us decipher a 40-year-old mystery about sodium’s control of opioid receptors,” says Stevens, who was senior author of the paper with UNC pharmacologist Prof. Bryan Roth. “It is amazing how sodium sits right in the middle of the receptor as a co-factor or allosteric modulator.”

The findings appear in an advanced online publication in the journal Nature.

A Sharper Image

The researchers revealed the basis for sodium’s effect on signaling with a high-resolution 3D view of an opioid receptor’s atomic structure. Opioid receptors are activated by peptide neurotransmitters (endorphins, dynorphins and enkephalins) in the brain. They can also be activated by plant-derived and synthetic drugs that mimic these peptides: among them morphine, codeine, oxycodone and heroin.

Despite these receptors’ crucial importance in health and disease, including pain disorders and addictions, scientists have only begun to understand in detail how they work. Opioid receptors are inherently flimsy and fragile when produced in isolation, and thus have been hard to study using X-ray crystallography, the usual structure-mapping method for large proteins.

In recent years, the Stevens laboratory has helped pioneer the structure determination of G protein-coupled receptors. Although the first crystallographic structures of opioid receptors were determined in 2012, these structural models weren’t fine-grained enough to solve a lingering mystery, particularly for the human delta opioid receptor.

That mystery concerned the role of sodium. The element is perhaps best known to biologists as one of the key “electrolytes” needed for the basic workings of cells. In the early 1970s, researchers in the laboratory of neuroscientist Solomon Snyder at Johns Hopkins Univ., who had helped discover opioid receptors, found evidence that sodium ions also act as a kind of switch on opioid receptor signaling. They noted that at concentrations normally found in brain fluid, these ions reduced the ability of opioid peptides and drugs like morphine to interact with opioid receptors.

How sodium could exert this indirect (“allosteric”) effect on opioid receptor activity was unclear — and has remained an unsolved puzzle for decades. Now that scientists have discovered the mechanism of sodium’s effect, then in principle they can exploit it to develop better opioid-receptor-targeting drugs.

A Switch Controlling Pain, Depression and Mood Disorders

For the new study, the team constructed a novel, fusion-protein-stabilized version of one of the main opioid receptors in the human brain, known as the delta opioid receptor, and managed to form crystals of it for X-ray crystallography. The latter revealed the receptor’s 3D atomic structure to a resolution of 1.8 Angstroms (180 trillionths of a meter) — the sharpest picture yet of an opioid receptor.

“Such a high resolution is really necessary to be able to understand in detail how the receptor works,” says Stevens.

The analysis yielded several key details of opioid receptor structure and function, most importantly the details of the “allosteric sodium site,” where a sodium ion can slip in and modulate receptor activity.

The team was able to identify the crucial amino acids that hold the sodium ion in place and transmit its signal-modulating effect. “We found that the presence of the sodium ion holds the receptor protein in a shape that gives it a different affinity for its corresponding neurotransmitter peptides,” Fenalti says.

With the structural data in hand, the researchers designed new versions of the receptor, in which key sodium-site amino-acids were mutated, to see how this would affect receptor signaling. Co-lead author Research Associate Patrick Giguere and colleagues in Roth’s Laboratory at UNC, which has long collaborated with the Stevens laboratory, tested these mutant receptors and found that certain amino-acid changes cause radical shifts in the receptor’s normal signaling response.

The most interesting shifts involved a little-understood secondary or “alternative” signaling route, known as the beta-arrestin pathway, whose activity can have different effects depending on the type of brain cell involved. Some drugs that normally bind to the delta opioid receptor and have little or no effect on the beta-arrestin pathway turned out to strongly activate this pathway in a few of these mutant receptors.

In practical terms, these findings suggests a number of ways in which new drugs could target these receptors — and not only delta opioid receptors but also the other two “classical” opioid receptors, mu and kappa opioid receptors. “The sodium site architecture and the way it works seems essentially the same for all three of these opioid receptor types,” says Fenalti.

No More Insulin Injections?

by Anne Trafton, MIT News Office

In patients suffering from Type 1 diabetes, the immune system attacks the pancreas, eventually leaving patients without the ability to naturally control blood sugar. These patients must carefully monitor the amount of sugar in their blood, measuring it several times a day and then injecting themselves with insulin to keep their blood sugar levels within a healthy range. However, precise control of blood sugar is difficult to achieve, and patients face a range of long-term medical problems as a result.

A better diabetes treatment, many researchers believe, would be to replace patients’ destroyed pancreatic islet cells with healthy cells that could take over glucose monitoring and insulin release. This approach has been used in hundreds of patients, but it has one major drawback — the patients’ immune systems attack the transplanted cells, requiring patients to take immunosuppressant drugs for the rest of their lives.

Now, a new advance from MIT, Boston Children’s Hospital, and several other institutions may offer a way to fulfill the promise of islet cell transplantation. The researchers have designed a material that can be used to encapsulate human islet cells before transplanting them. In tests on mice, they showed that these encapsulated human cells could cure diabetes for up to six months, without provoking an immune response.

Although more studies are needed, this approach “has the potential to provide diabetics with a new pancreas that is protected from the immune system, which would allow them to control their blood sugar without taking drugs. That’s the dream,” says Daniel Anderson, the Samuel A. Goldblith Associate Professor in MIT’s Department of Chemical Engineering, a member of MIT’s Koch Institute for Integrative Cancer Research and Institute for Medical Engineering and Science (IMES), and a research fellow in the Department of Anesthesiology at Boston Children’s Hospital.

Anderson is the senior author of two studies describing this method in the Jan. 25 issues of Nature Medicine and Nature Biotechnology. Researchers from Harvard University, the University of Illinois at Chicago, the Joslin Diabetes Center, and the University of Massachusetts Medical School also contributed to the research.

Encapsulating cells

Since the 1980s, a standard treatment for diabetic patients has been injections of insulin produced by genetically engineered bacteria. While effective, this type of treatment requires great effort by the patient and can generate large swings in blood sugar levels.

At the urging of JDRF director Julia Greenstein, Anderson, Langer, and colleagues set out several years ago to come up with a way to make encapsulated islet cell transplantation a viable therapeutic approach. They began by exploring chemical derivatives of alginate, a material originally isolated from brown algae. Alginate gels can be made to encapsulate cells without harming them, and also allow molecules such as sugar and proteins to move through, making it possible for cells inside to sense and respond to biological signals.

However, previous research has shown that when alginate capsules are implanted in primates and humans, scar tissue eventually builds up around the capsules, making the devices ineffective. The MIT/Children’s Hospital team decided to try to modify alginate to make it less likely to provoke this kind of immune response.

“We decided to take an approach where you cast a very wide net and see what you can catch,” said Arturo Vegas, a former MIT and Boston Children’s Hospital postdoc who is now an assistant professor at Boston University. Vegas is the first author of the Nature Biotechnology paper and co-first author of the Nature Medicine paper. “We made all these derivatives of alginate by attaching different small molecules to the polymer chain, in hopes that these small molecule modifications would somehow give it the ability to prevent recognition by the immune system.”

After creating a library of nearly 800 alginate derivatives, the researchers performed several rounds of tests in mice and nonhuman primates. One of the best of those, known as triazole-thiomorpholine dioxide (TMTD), they decided to study further in tests of diabetic mice. They chose a strain of mice with a strong immune system and implanted human islet cells encapsulated in TMTD into a region of the abdominal cavity known as the intraperitoneal space.

The pancreatic islet cells used in this study were generated from human stem cells using a technique recently developed by Douglas Melton, a professor at Harvard University who is an author of the Nature Medicine paper.

Following implantation, the cells immediately began producing insulin in response to blood sugar levels and were able to keep blood sugar under control for the length of the study, 174 days.

“The really exciting part of this was being able to show, in an immune-competent mouse, that when encapsulated these cells do survive for a long period of time, at least six months,” said Omid Veiseh, a senior postdoc at the Koch Institute and Boston Children’s hospital, co-first author of the Nature Medicine paper, and an author of the Nature Biotechnology paper. “The cells can sense glucose and secrete insulin in a controlled manner, alleviating the mice’s need for injected insulin.”

The researchers also found that 1.5-millimeter diameter capsules made from their best materials (but not carrying islet cells) could be implanted into the intraperitoneal space of nonhuman primates for at least six months without scar tissue building up.

“The combined results from these two papers suggests that these capsules have real potential to protect transplanted cells in human patients,” said Robert Langer, the David H. Koch Institute Professor at MIT, a senior research associate at Boston’s Children Hospital, and co-author on both papers.  “We are so pleased to see this research in cell transplantation reach these important milestones.”

Cherie Stabler, an associate professor of biomedical engineering at the University of Florida, said this approach is impressive because it tackles all aspects of the problem of islet cell delivery, including finding a source of cells, preventing an immune response, and developing a suitable delivery material.

“It’s such a complex, multipronged problem that it’s important to get people from different disciplines to address it,” said Stabler, who was not involved in the research. “This is a great first step towards a clinically relevant, cell-based therapy for Type I diabetes.”

Insulin independence

The researchers now plan to further test their new materials in nonhuman primates, with the goal of eventually performing clinical trials in diabetic patients. If successful, this approach could provide long-term blood sugar control for such patients. “Our goal is to continue to work hard to translate these promising results into a therapy that can help people,” Anderson said.

“Being insulin-independent is the goal,” Vegas said. “This would be a state-of-the-art way of doing that, better than any other technology could. Cells are able to detect glucose and release insulin far better than any piece of technology we’ve been able to develop.”

The researchers are also investigating why their new material works so well. They found that the best-performing materials were all modified with molecules containing a triazole group — a ring containing two carbon atoms and three nitrogen atoms. They suspect this class of molecules may interfere with the immune system’s ability to recognize the material as foreign.

The work was supported, in part, by the JDRF, the Leona M. and Harry B. Helmsley Charitable Trust, the National Institutes of Health, and the Tayebati Family Foundation.

Other authors of the papers include MIT postdoc Joshua Doloff; former MIT postdocs Minglin Ma and Kaitlin Bratlie; MIT graduate students Hok Hei Tam and Andrew Bader; Jeffrey Millman, an associate professor at Washington University School of Medicine; Mads Gürtler, a former Harvard graduate student; Matt Bochenek, a graduate student at the University of Illinois at Chicago; Dale Greiner, a professor of medicine at the University of Massachusetts Medical School; Jose Oberholzer, an associate professor at the University of Illinois at Chicago; and Gordon Weir, a professor of medicine at the Joslin Diabetes Center.

Queen Bees ‘Sterilize’ Daughters to Keep them Working

Original Report by Australian National University

Queen bees and ants emit a chemical that alters the DNA of their daughters and keeps them as sterile and industrious workers, scientists have found.

“When deprived of the pheromone that queens emit, worker bees and ants become more self-centered and lazy, and they begin to lay eggs,” said lead researcher Luke Holman from The Australian National University (ANU).

“Amazingly, it looks like the queen pheromone works by chemically altering workers’ genes,” said Dr Holman, a biologist in the ANU Research School of Biology.

Queen bees and ants can have hundreds of thousands of offspring and live for many years, while workers are short-lived and mostly sterile, even though they have the same DNA as the queen.

Recent research suggests that a chemical modification to a baby bee or ant’s DNA, called DNA methylation, helps determine whether the baby develops into a queen or a worker.

Holman collaborated with biologists from the University of Helsinki to investigate whether the queen’s pheromone altered DNA methylation in workers.

The team found evidence that indeed, workers exposed to pheromones tag their DNA with methylation differently, which might suppress queenly characteristics in the workers.

Surprisingly, the queen pheromone of honeybees seemed to lower methylation, while the queen pheromone of ants seemed to increase it, suggesting things work differently in bees and ants.

“Bees and ants evolved their two-tier societies independently. It would be confusing but cool if they had evolved different means to the same end,” Holman said.

Holman said he was looking forward to studying Australian bees next, which evolved sociality independently from the European species in this study.

“It brings us one step closer to understanding how these animals evolved their amazing cooperative behavior, which in many ways is a step beyond human evolution,” he said.