NMR advance brings proteins into the open

A key protein interaction, common across all forms of life, had eluded scientists’ observation until a team of researchers cracked the case by combining data from four different techniques of nuclear magnetic resonance spectroscopy.

When working a cold case, smart investigators try something new. By taking a novel approach to nuclear magnetic resonance spectroscopy — a blending of four techniques — scientists have been able to resolve a key interaction between two proteins that could never be observed before. They report on their findings the week of June 24, 2013, in Proceedings of the National Academy of Sciences (PNAS).

The interaction, which the team first described, is nearly universal across all of life. A protein machine called a chaperone takes hold of a disordered smaller protein to help it find its proper folded conformation. In this case, the team set up test-tube experiments where they hoped to watch the capsule-shaped bacterial chaperone GroEL capture a disordered amyloid β (Aβ) protein, a molecule that in humans is central in Alzheimer’s disease.

The two proteins are well studied, but the motions they go through when they first meet — when the open GroEL capsule captures its target — have been invisible to scientists. Electron microscopy and X-ray crystallography are only good for taking snapshots of easily frozen moments in time. NMR is capable of sensing the interactions and kinetics of protein handshakes as they occur, but in some cases any single technique can provide only hints and whispers of what’s going on.

Brown University biologist Nicolas Fawzi, who was a postdoctoral researcher in the group of Marius Clore at the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) within the National Institutes of Health (NIH), worked with co-authors and NIDDK researchers David Libich, Jinfa Yang and Marius Clore to piece together the story of the proteins by combining four different NMR techniques. They figured out what each one could tell them about the interaction and built the case presented in PNAS.

“None of the four techniques alone gave us sufficient information,” said Fawzi, assistant professor of medical science in Brown’s Department of Molecular Pharmacology, Physiology, and Biotechnology. “Only by using them all together would we be able to figure out the structure and motions of Aβ when it was bound to GroEL. By having four indirect measurements together, that was able to give us a complete picture.”

The researchers acted like a team of detectives working on a case in which no single witness saw everything. Instead they found three witnesses, each with something different to contribute, and then one more that could corroborate some of what the others revealed and rule out other possibilities. The NMR techniques they used were lifetime line broadening, Carr-Purcell-Meinboom-Gill (CPMG) relaxation dispersion spectroscopy, and exchange-induced chemical shifts.

“The fourth technique we employed was Dark-state Exchange Saturation Transfer (DEST) spectroscopy, which we had developed in my lab at the NIH in 2011,” said Clore, also the paper’s corresponding author. “We were able to more effectively conduct our research by using that tool to corroborate and extend the information afforded by the other three measurements.”

Bouncing with the chaperone

The mystery debated among molecular biologists was what the GroEL chaperone requires of its captives at the moment they engage. Does it force them into a particular conformation? Does it hold on tightly while it closes its capsule lid around the smaller protein, or does the captive stay in motion at all?

What the team observed is that the GroEL is a permissive captor. It bound Aβ at just two “hydrophobic” sites, leaving the smaller protein to otherwise dangle in a variety of conformations. It also didn’t keep it bound the entire time, letting it instead detach and re-bind. Essentially Aβ would bounce off and on within GroEL’s binding cavity.

“By using these four techniques together we were able to extract information about the structure of the protein while it binds as well as how fast it comes on and off and what it’s doing at each position,” Fawzi said. “Instead of forming more particular structure upon binding it appears to retain great conformational heterogeneity.”

The lifetime line broadening technique, for example, told them that the Aβ was interacting with something big (GroEL), while the CPMG and chemical shift observations combined to show the length of time Aβ spent on GroEL before unbinding, as well as the structural details of Aβ when it was bound to GroEL. DEST provided information that could confirm much of the story of the other techniques.

Fawzi said GroEL’s laid-back approach could be a matter of being able to bind many different proteins in disordered conformations, but also of saving energy. Forcing proteins into a specific conformation just to make and sustain the initial capture would require more energy than it’s worth.

Eventually, in moments after those the team resolved in this study, GroEL closes its lid and encapsulates its target proteins fully, Fawzi said. That’s when it invests in forcing them to fold the right way.

For molecular and structural biologists, the newly proven blend of NMR techniques could open a number of other cold cases of elusive interactions.

“We can now look at how these big machines can do their job while they are working,” Fawzi said. “This is not just limited to this GroEL machine.”

Dream of regenerating human body parts gets a little closer

Damage to vital organs, the spinal cord, or limbs can have an enormous impact on our ability to move, function – and even live. But imagine if you could restore these tissues back to their original condition and go on with life as normal.

Well, this is the dream for regenerative medicine. And while humans missed out on these abilities in the evolutionary lottery, a recent study in mice shows we’re making small progress to achieving this dream.

Learning from animals

Nature has provided the animal kingdom with many different ways to achieve perfect regeneration. Some amphibians – such as salamanders – are famous for their superhero-like ability to regenerate heart, brain, spinal cord, tail and can even whole limb tissue throughout their life.

Although organ and spinal cord regeneration are clinically important and worthy of intense research investment, regrowing whole limbs provides a flagship example of perfect regeneration in the salamander.

It has been known for more than a hundred years that if a salamander loses a limb, it grows right back. This process is extremely precise and removal of the limb at the shoulder regrows a full limb, but removal at the wrist only regrows the missing hand portion.

Interestingly, there does not seem to be a limit on how many times they can perform this clever trick and each time the limb comes back perfect.

But mammals (including humans and mice) seem to have missed out on this important skill. The question of how to enhance the regenerative capabilities in humans, either by adding the missing ingredients, or activating these latent abilities currently lies wide open.

Extending regeneration to mammals

Mammals currently only have the capacity to regenerate the very tip of their finger. But the result is far from perfect. A range of studies in mice have shown the digit-tip regrowth is severely restricted. Removal of the very tip of the mouse digit will be replaced, but removal of the tissue a small distance further up the digit and closer to nail bed (the equivalent to a human cuticle), will fail to regrow.

Last week, a group of researchers from the United States and Japan published work extending our understanding of the mechanism by which a resident stem cell population within the mouse digit tip nail bed can be activated to induce digit tip regeneration. In other words, we can now grow more of the digit back in mice and possibly more of the human finger.

Resident stem cells are specialised cells found at various locations within the body. When activated, these cells multiply and then transform into other cell types required to replace worn out cells under conditions of normal tissue maintenance.

This work builds on previous studies identifying the stem cell population in the nail bed by unveiling a signalling mechanism that could be exploited to enhance the amount of tissue that could be regrown. The potential for repair after injury appears very limited in many tissues and organs. Understanding how to enhance stem cell activation in these tissues may stimulate repair not previously thought possible.

The ability to switch on and mobilise resident stem cells in regeneration will be important in a wide range of new therapies, particularity for organs affected by injury or disease. On a world stage, momentum is currently growing for these types of strategies. It is clear that once refined, these approaches are sure to have a profound influence on many different aspects of clinical medicine, opening up the possibility of replacing diseased or injured tissues.

We may be some way off from the dream of replacing whole limbs in humans but recent progress confirms that by deepening our understanding of stem cell activation, we can directly unlock more regeneration in mammals than normally possible.

Sugar solution makes tissues see-through

Japanese researchers have developed a new sugar and water-based solution that turns tissues transparent in just three days, without disrupting the shape and chemical nature of the samples. Combined with fluorescence microscopy, this technique enabled them to obtain detailed images of a mouse brain at an unprecedented resolution.

The team from the RIKEN Center for Developmental biology reports their finding today in Nature Neuroscience.

Over the past few years, teams in the USA and Japan have reported a number of techniques to make biological samples transparent, that have enabled researchers to look deep down into biological structures like the brain.

“However, these clearing techniques have limitations because they induce chemical and morphological damage to the sample and require time-consuming procedures,” explains Dr. Takeshi Imai, who led the study.

SeeDB, an aqueous fructose solution that Dr. Imai developed with colleagues Drs. Meng-Tsen Ke and Satoshi Fujimoto, overcomes these limitations.

Using SeeDB, the researchers were able to make mouse embryos and brains transparent in just three days, without damaging the fine structures of the samples, or the fluorescent dyes they had injected in them.

They could then visualize the neuronal circuitry inside a mouse brain, at the whole-brain scale, under a customized fluorescence microscope without making mechanical sections through the brain.

They describe the detailed wiring patterns of commissural fibers connecting the right and left hemispheres of the cerebral cortex, in three dimensions, for the first time. They also report that they were able to visualize in three dimensions the wiring of mitral cells in the olfactory bulb, which is involved the detection of smells, at single-fiber resolution.

“Because SeeDB is inexpensive, quick, easy and safe to use, and requires no special equipment, it will prove useful for a broad range of studies, including the study of neuronal circuits in human samples,” explain the authors.

Getting to grips with migraine

Researchers identify some of the biological roots of migraine from large-scale genome study

In the largest study of migraines, researchers have found 5 genetic regions that for the first time have been linked to the onset of migraine. This study opens new doors to understanding the cause and biological triggers that underlie migraine attacks.

The team identified 12 genetic regions associated with migraine susceptibility. Eight of these regions were found in or near genes known to play a role in controlling brain circuitries and two of the regions were associated with genes that are responsible for maintaining healthy brain tissue. The regulation of these pathways may be important to the genetic susceptibility of migraines.

Migraine is a debilitating disorder that affects approximately 14% of adults. Migraine has recently been recognised as the seventh disabler in the Global Burden of Disease Survey 2010 and has been estimated to be the most costly neurological disorder. It is an extremely difficult disorder to study because no biomarkers between or during attacks have been identified so far.

"This study has greatly advanced our biological insight about the cause of migraine," says Dr Aarno Palotie, from the Wellcome Trust Sanger Institute. "Migraine and epilepsy are particularly difficult neural conditions to study; between episodes the patient is basically healthy so it’s extremely difficult to uncover biochemical clues.

"We have proven that this is the most effective approach to study this type of neurological disorder and understand the biology that lies at the heart of it."

The team uncovered the underlying susceptibilities by comparing the results from 29 different genomic studies, including over 100,000 samples from both migraine patients and control samples.

They found that some of the regions of susceptibility lay close to a network of genes that are sensitive to oxidative stress, a biochemical process that results in the dysfunction of cells.

The team expects many of the genes at genetic regions associated with migraine are interconnected and could potentially be disrupting the internal regulation of tissue and cells in the brain, resulting in some of the symptoms of migraine.

"We would not have made discoveries by studying smaller groups of individuals," says Dr Gisela Terwindt, co-author from Leiden University Medical Centre. "This large scale method of studying over 100,000 samples of healthy and affected people means we can tease out the genes that are important suspects and follow them up in the lab."

The team identified an additional 134 genetic regions that are possibly associated to migraine susceptibility with weaker statistical evidence. Whether these regions underlie migraine susceptibility or not still needs to be elucidated. Other similar studies show that these statistically weaker culprits can play an equal part in the underlying biology of a disease or disorder.

"The molecular mechanisms of migraine are poorly understood. The sequence variants uncovered through this meta-analysis could become a foothold for further studies to better understanding the pathophysiology of migraine" says Dr Kári Stefánsson, President of deCODE genetics.

"This approach is the most efficient way of revealing the underlying biology of these neural disorders," says Dr Mark Daly, from the Massachusetts General Hospital and the Broad Institute of MIT and Harvard. "Effective studies that give us biological or biochemical results and insights are essential if we are to fully get to grips with this debilitating condition.

"Pursuing these studies in even larger samples and with denser maps of biological markers will increase our power to determine the roots and triggers of this disabling disorder."

Addiction Relapse Might Be Thwarted By Turning Off Brain Trigger

Researchers at the Ernest Gallo Clinic and Research Center at UC San Francisco have been able to identify and deactivate a brain pathway linked to memories that cause alcohol cravings in rats, a finding that may one day lead to a treatment option for people who suffer from alcohol abuse disorders and other addictions.

In the study, researchers were able to prevent the addicted animals from seeking alcohol and drinking it, the equivalent of relapse.

“One of the main causes of relapse is craving, triggered by the memory by certain cues – like going into a bar, or the smell or taste of alcohol,” said lead author Segev Barak, PhD, at the time a postdoctoral fellow in the lab of co-senior author Dorit Ron, PhD, a Gallo Center investigator and UCSF professor of neurology.

“We learned that when rats were exposed to the smell or taste of alcohol, there was a small window of opportunity to target the area of the brain that reconsolidates the memory of the craving for alcohol and to weaken or even erase the memory, and thus the craving” he said.

The study, also supervised by co-senior author Patricia H. Janak, PhD, a Gallo Center investigator and UCSF professor of neurology, was published online on June 23 in Nature Neuroscience.

Neural Mechanism That Triggers Alcohol Memory

In the first phase of the study, rats had the choice to freely drink water or alcohol over the course of seven weeks, and during this time developed a high preference for alcohol.

In the next phase, they had the opportunity to access alcohol for one hour a day, which they learned to do by pressing a lever. They were then put through a 10-day period of abstinence from alcohol.

Following this period, the animals were exposed for five minutes to just the smell and taste of alcohol, which cued them to remember how much they liked drinking it. The researchers then scanned the animals’ brains, and identified the neural mechanism responsible for the reactivation of the memory of the alcohol – a molecular pathway mediated by an enzyme known as mammalian target of rapamycin complex 1 (mTORC1).

They found that just a small drop of alcohol presented to the rats turned on the mTORC1 pathway specifically in a select region of the amygdala, a structure linked to emotional reactions and withdrawal from alcohol, and cortical regions involved in memory processing.

They further showed that once mTORC1 was activated, the alcohol-memory stabilized (reconsolidated) and the rats relapsed on the following days, meaning in this case, that they started again to push the lever to dispense more alcohol.

“The smell and taste of alcohol were such strong cues that we could target the memory specifically without impacting other memories, such as a craving for sugar,” said Barak, who added that the Ron research group has been doing brain studies for many years and has never seen such a robust and specific activation in the brain.

Drug that Erases the Memory of Alcohol

In the next part of the study, the researchers set out to see if they could prevent the reconsolidation of the memory of alcohol by inhibiting mTORC1, thus preventing relapse. When mTORC1 was inactivated using a drug called rapamycin, administered immediately after the exposure to the cue (smell, taste), there was no relapse to alcohol-seeking the next day.

Strikingly, drinking remained suppressed for up to 14 days, the end point of the study. These results suggest that rapamycin erased the memory of alcohol for a long period, said Ron.

The authors said the study is an important first step, but that more research is needed to determine how mTORC1 contributes to alcohol memory reconsolidation and whether turning off mTORC1 with rapamycin would prevent relapse for more than two weeks.

The authors also said it would be interesting to test if rapamycin, an FDA-approved drug currently used to prevent organ rejection after transplantation, or other mTORC1 inhibitors that are currently being developed in pharmaceutical companies, would prevent relapse in human alcoholics.

“One of the main problems in alcohol abuse disorders is relapse, and current treatment options are very limited.” Barak said. “Even after detoxification and a period of rehabilitation, 70 to 80 percent of patients will relapse in the first several years. It is really thrilling that we were able to completely erase the memory of alcohol and prevent relapse in these animals. This could be a revolution in treatment approaches for addiction, in terms of erasing unwanted memories and thereby manipulating the brain triggers that are so problematic for people with addictions.”

Trying to Learn a Foreign Language? Avoid Reminders of Home

Something odd happened when Shu Zhang was giving a presentation to her classmates at the Columbia Business School in New York City. Zhang, a Chinese native, spoke fluent English, yet in the middle of her talk, she glanced over at her Chinese professor and suddenly blurted out a word in Mandarin. “I meant to say a transition word like ‘however,’ but used the Chinese version instead,” she says. “It really shocked me.”

Shortly afterward, Zhang teamed up with Columbia social psychologist Michael Morris and colleagues to figure out what had happened. In a new study, they show that reminders of one’s homeland can hinder the ability to speak a new language. The findings could help explain why cultural immersion is the most effective way to learn a foreign tongue and why immigrants who settle within an ethnic enclave acculturate more slowly than those who surround themselves with friends from their new country.

Previous studies have shown that cultural icons such as landmarks and celebrities act like “magnets of meaning,” instantly activating a web of cultural associations in the mind and influencing our judgments and behavior, Morris says. In an earlier study, for example, he asked Chinese Americans to explain what was happening in a photograph of several fish, in which one fish swam slightly ahead of the others. Subjects first shown Chinese symbols, such as the Great Wall or a dragon, interpreted the fish as being chased. But individuals primed with American images of Marilyn Monroe or Superman, in contrast, tended to interpret the outlying fish as leading the others. This internally driven motivation is more typical of individualistic American values, some social psychologists say, whereas the more externally driven explanation of being pursued is more typical of Chinese culture.

To determine whether these cultural icons can also interfere with speaking a second language, Zhang, Morris, and their colleagues recruited male and female Chinese students who had lived in the United States for a less than a year and had them sit opposite a computer monitor that displayed the face of either a Chinese or Caucasian male called “Michael Lee.” As microphones recorded their speech, the volunteers conversed with Lee, who spoke to them in English with an American accent about campus life.

Next, the team compared the fluency of the volunteers’ speech when they were talking to a Chinese versus a Caucasian face. Although participants reported a more positive experience chatting with the Chinese version of “Michael Lee,” they were significantly less fluent, producing 11% fewer words per minute on average, the authors report online today in the Proceedings of the National Academy of Sciences. “It’s ironic” that the more comfortable volunteers were with their conversational partner, the less fluent they became, Zhang says. “That’s something we did not expect.”

To rule out the possibility that the volunteers were speaking more fluently to the Caucasian face on purpose, thus explaining the performance gap, Zhang and colleagues asked the participants to invent a story, such as a boy swimming in the ocean, while simultaneously being exposed to Chinese and American icons rather than faces. Seeing Chinese icons such as the Great Wall also interfered with the volunteers’ English fluency, causing a 16% drop in words produced per minute. The icons also made the volunteers 85% more likely to use a literal translation of the Chinese word for an object rather than the English term, Zhang says. Rather than saying “pistachio,” for example, volunteers used the Chinese version, “happy nuts.”

Understanding how these subtle cultural cues affect language fluency could help employers design better job interviews, Morris says. For example, taking a Japanese job candidate out for sushi, although a well-meaning gesture, might not be the best way to help them shine.

"It’s quite striking that these effects were so robust," says Mary Helen Immordino-Yang, a developmental psychologist at the University of Southern California in Los Angeles. They show that "we’re exquisitely attuned to cultural context," she says, and that "even subtle cues like the ethnicity of the person we’re talking to" can affect language processing. The take-home message? "If one wants to acculturate rapidly, don’t move to an ethnic enclave neighborhood where you’ll be surrounded by people like yourself," Morris says. Sometimes, a familiar face is the last thing you need to see.

Repairing Bad Memories

It was a Saturday night at the New York Psychoanalytic Institute, and the second-floor auditorium held an odd mix of gray-haired, cerebral Upper East Side types and young, scruffy downtown grad students in black denim. Up on the stage, neuroscientist Daniela Schiller, a riveting figure with her long, straight hair and impossibly erect posture, paused briefly from what she was doing to deliver a mini-lecture about memory.

She explained how recent research, including her own, has shown that memories are not unchanging physical traces in the brain. Instead, they are malleable constructs that may be rebuilt every time they are recalled. The research suggests, she said, that doctors (and psychotherapists) might be able to use this knowledge to help patients block the fearful emotions they experience when recalling a traumatic event, converting chronic sources of debilitating anxiety into benign trips down memory lane.

And then Schiller went back to what she had been doing, which was providing a slamming, rhythmic beat on drums and backup vocals for the Amygdaloids, a rock band composed of New York City neuroscientists. During their performance at the institute’s second annual “Heavy Mental Variety Show,” the band blasted out a selection of its greatest hits, including songs about cognition (“Theory of My Mind”), memory (“A Trace”), and psychopathology (“Brainstorm”).

“Just give me a pill,” Schiller crooned at one point, during the chorus of a song called “Memory Pill.” “Wash away my memories …”

The irony is that if research by Schiller and others holds up, you may not even need a pill to strip a memory of its power to frighten or oppress you.

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