Posts tagged amino acids
Articles and news from the latest research reports.
Dietary Amino Acids Relieve Sleep Problems after Traumatic Brain Injury in Animals
Scientists who fed a cocktail of key amino acids to mice improved sleep disturbances caused by brain injuries in the animals. These new findings suggest a potential dietary treatment for millions of people affected by traumatic brain injury (TBI)—a condition that is currently untreatable.
“If this type of dietary treatment is proved to help patients recover function after traumatic brain injury, it could become an important public health benefit,” said study co-leader Akiva S. Cohen, Ph.D., a neuroscientist at The Children’s Hospital of Philadelphia (CHOP).
Cohen is the co-senior author of the animal TBI study appearing today in Science Translational Medicine. He collaborated with two experts in sleep medicine: co-senior author Allan I. Pack, M.D., Ph.D., director of the Center for Sleep and Circadian Neurobiology in the Perelman School of Medicine at the University of Pennsylvania; and first author Miranda M. Lim, M.D., Ph.D., formerly at the Penn Sleep Center, and now on faculty at the Portland VA Medical Center and Oregon Health and Science University.
Every year in the U.S., an estimated 2 million people suffer a TBI, accounting for a major cause of disability across all age groups. Although 75 percent of reported TBI cases are milder forms such as concussion, even concussion may cause chronic neurological impairments, including cognitive, motor and sleep problems.
“Sleep disturbances, such as excessive daytime sleepiness and nighttime insomnia, disrupt quality of life and can delay cognitive recovery in patients with TBI,” said Lim, a neurologist and sleep medicine specialist. Although physicians can relieve the dangerous swelling that occurs after a severe TBI, there are no existing treatments to address the underlying brain damage associated with neurobehavioral problems such as impaired memory, learning and sleep patterns.
Cohen and team investigate the use of selected branched chain amino acids (BCAA)—precursors of the neurotransmitters glutamate and GABA, which are involved in communication among neurons and help to maintain a normal balance in brain activity. His research team previously showed that a BCAA diet restored cognitive ability in brain-injured mice. The current study was the first to analyze sleep-wake patterns in an animal model.
Comparing mice with experimentally induced mild TBI to uninjured mice, the scientists found the injured mice were unable to stay awake for long periods of time. The injured mice had lower activity among orexin neurons, which help to maintain the animals’ wakefulness. This is similar to results in human studies showing decreased orexin levels in the spinal fluid after TBI.
In the current study, the dietary therapy restored the orexin neurons to a normal activity level and improved wakefulness in the brain-injured mice. EEG recordings also showed improved brain wave patterns among the mice that consumed the BCAA diet.
“These results in an animal model provide a proof-of-principle for investigating this dietary intervention as a treatment for TBI patients,” said Cohen. “If a dietary supplement can improve sleeping and waking patterns as well as cognitive problems, it could help brain-injured patients regain crucial functions.” Cohen cautioned that current evidence does not support TBI patients medicating themselves with commercially available amino acids.
(Source: chop.edu)
Scientists expand the genetic code of mammals to control protein activity in neurons with light
With the flick of a light switch, researchers at the Salk Institute for Biological Studies can change the shape of a protein in the brain of a mouse, turning on the protein at the precise moment they want. This allows the scientists to observe the exact effect of the protein’s activation. The new method, described in the Oct. 16, 2013, issue of the journal Neuron, relies on specially engineered amino acids—the molecules that make up proteins—and light from an LED. Now that it has been shown to work, the technique can be adapted to give researchers control of a wide variety of other proteins in the brain to study their functions.
"What we are now able to do is not only control neuronal activity, but control a specific protein within a neuron," says senior study author Lei Wang, an associate professor in Salk’s Jack H. Skirball Center for Chemical Biology and Proteomics and holder of the Frederick B. Rentschler Developmental Chair.
If a scientist wants to know what set of neurons in the brain is responsible for a particular action or behavior, being able to turn the neurons on and off at will gives the researcher a targeted way to test the neurons’ effects. Likewise, if they want to know the role of a certain protein inside the cells, the ability to activate or inactivate the protein of interest is key to studying its biology.
Over the past decade, researchers have developed a handful of ways of activating or inactivating neurons using light, as part of the burgeoning field of so-called optogenetics. In optogenetic experiments, mice are genetically engineered to have a light-sensitive channel from algae integrated into their neurons. When exposed to light, the channel opens or closes, changing the flow of molecules into the neuron and altering its ability to pass an electrochemical message through the brain. Using such optogenetic approaches, scientists can pick and choose which neurons in the brain they want turned on or off at any given time and observe the resulting change in the engineered mice.
"There’s no question that this is a great way to control neuronal activity, by borrowing light-responsive channels or pumps from other organisms and putting them in neurons," says Wang. "But rather than put a stranger into neurons, we wanted to control the activity of proteins native to neurons."
To make proteins respond to light, Wang’s team harnessed a photo-responsive amino acid, called Cmn, which has a large chemical structure. When a pulse of light shines on the molecule, Cmn’s bulky side chain breaks off, leaving cysteine, a smaller amino acid. Wang’s group realized that if a single Cmn was integrated into the right place in the structure of a protein, the drastic change in the amino acid’s size could activate or inactivate the entire protein.
To test their idea, Wang and his colleagues engineered new versions of a potassium channel in neurons, adding Cmn to their sequence.
"Basically the idea was that when you put this amino acid in the pore of the channel, the bulky side chain entirely blocks the passage of ions through the channel," explains Ji-Yong Kang, a graduate student who works in Wang’s group, and first author of the new paper. "Then, when the bond in the amino acid breaks in response to light, the channel is opened up."
The method worked in isolated cells: after trial and error, the scientists found the ideal spot in the channel to put Cmn, so that the channel was initially blocked, but opened when light shone on it. They were able to measure the change to the channel’s properties by recording the electrical current that flowed through the cells before and after exposure to light.
But to apply the technique to living mice, Wang and his colleagues needed to change the animals’ genetic code—the built-in instructions that cells use to produce proteins based on gene sequences. The normal genetic code doesn’t contain information on Cmn, so simply injecting Cmn amino acids into mice wouldn’t lead to the molecules being integrated into proteins. In the past, the Wang group and others have expanded the genetic codes of isolated cells of simple organisms like bacteria, or yeast, inserting instructions for a new amino acid. But the approach had never been successful in mammals. Through a combination of techniques and new tricks, however, Wang’s team was able to provide embryonic mice with the instructions for the new amino acid, Cmn. With the help from Salk Professor Dennis O’Leary and his research associate Daichi Kawaguchi, they then integrated the new Cmn-containing channel into the brains of the developing mice, and showed that by shining light on the brain tissue they could force the channel open, altering patterns of neuron activity. It was not only a first for expanding the genetic code of mammals, but also for protein control.
At the surface, the new approach has the same result as optogenetic approaches to studying the brain—neurons are silenced at a precise time in response to light. But Wang’s method can now be used to study a whole cadre of different proteins in neurons. Aside from being used to open and close channels or pores that let ions flow in and out of brain cells, Cmn could be used to optically regulate protein modifications and protein-protein interactions.
"We can pinpoint exactly which protein, or even which part of a protein, is crucial for the functioning of targeted neurons," says Wang. "If you want to study something like the mechanism of memory formation, it’s not always just a matter of finding what neurons are responsible, but what molecules within those neurons are critical."
Earlier this year, President Obama announced the multi-billion dollar Brain Research through Advancing Innovative Neurotechnologies (BRAIN) Initiative, a ten-year project to map the activity of the human brain. Creating new ways to study the molecules in the brain, such as using light-responsive amino acids to study neuronal proteins, will be key to moving forward on this initiative and similar efforts to understand the brain, says Wang. His lab is now working to develop ways to not only activate proteins, but inactive them using light-sensitive amino acids, and applying the technique to proteins other than Kir2.1.
Scientists view ‘protein origami’ to help understand, prevent certain diseases
Scientists using sophisticated imaging techniques have observed a molecular protein folding process that may help medical researchers understand and treat diseases such as Alzheimer’s, Lou Gehrig’s and cancer.
The study, reported this month in the journal Cell, verifies a process that scientists knew existed but with a mechanism they had never been able to observe, according to Dr. Hays Rye, Texas A&M AgriLife Research biochemist.
“This is a step in the direction of understanding how to modulate systems to prevent diseases like Alzheimer’s. We needed to understand the cell’s folding machines and how they interact with each other in a complicated network,” said Rye, who also is associate professor of biochemistry and biophysics at Texas A&M.
Rye explained that individual amino acids get linked together like beads on a string as a protein is made in the cell.
“But that linear sequence of amino acids is not functional,” he explained. “It’s like an origami structure that has to fold up into a three-dimensional shape to do what it has to do.”
Rye said researchers have been trying to understand this process for more than 50 years, but in a living cell the process is complicated by the presence of many proteins in a concentrated environment.
"The constraints on getting that protein to fold up into a good ‘origami’ structure are a lot more demanding,” he said. “So, there are special protein machines, known as molecular chaperones, in the cell that help proteins fold.”
But how the molecular chaperones help protein fold when it isn’t folding well by itself has been the nagging question for researchers.
“Molecular chaperones are like little machines, because they have levers and gears and power sources. They go through turning over cycles and just sort of buzz along inside a cell, driving a protein folding reaction every few seconds,” Rye said.
The many chemical reactions that are essential to life rely on the exact three-dimensional shape of folded proteins, he said. In the cell, enzymes, for example, are specialized proteins that help speed biological processes along by binding molecules and bringing them together in just the right way.
“They are bound together like a three-dimensional jigsaw puzzle,” Rye explained. “And the proteins — those little beads on the string that are designed to fold up like origami — are folded to position all these beads in three-dimensional space to perfectly wrap around those molecules and do those chemical reactions.
“If that doesn’t happen — if the protein doesn’t get folded up right – the chemical reaction can’t be done. And if it’s essential, the cell dies because it can’t convert food into power needed to build the other structures in the cell that are needed. Chemical reactions are the structural underpinning of how cells are put together, and all of that depends on the proteins being folded in the right way.”
When a protein doesn’t fold or folds incorrectly it turns into an “aggregate,” which Rye described as “white goo that looks kind of like a mayonnaise, like crud in the test tube.
“You’re dead; the cell dies,” he said.
Over the past 20 years, he said, researchers have linked that aggregation process “pretty convincingly” to the development of diseases — Alzheimer’s disease, Lou Gehrig’s disease, Huntington’s disease, to name a few. There’s evidence that diabetes and cancer also are linked to protein folding disorders.
“One of the main roles for the molecular chaperones is preventing those protein misfolding events that lead to aggregation and not letting a cell get poisoned by badly folded or aggregated proteins,” he said.
Rye’s team focused on a key molecular chaperone — the HSP60.
“They’re called HSP for ‘heat shock protein’ because when the cell is stressed with heat, the proteins get unstable and start to fall apart and unfold,” Rye said. “The cell is built to respond by making more of the chaperones to try and fix the problem.
“This particular chaperone takes unfolded protein and goes through a chemical reaction to bind the unfolded protein and literally puts it inside a little ‘box,’” Rye said.
He added that the mystery had long been how the folding worked because, while researchers could see evidence of that happening, no one had ever seen precisely how it happened.
Rye and the team zeroed in on a chemically modified mutant that in other experiments had seemed to stall at an important step in the process that the “machine” goes through to start the folding action. This clued the researchers that this stalling might make it easier to watch.
They then used cryo-electron microscopy to capture hundreds of thousands of images of the process at very high resolutions which allowed them to reconstruct from two-dimensional flat images a three-dimensional model. A highly sophisticated computer algorithm aligns the images and classifies them in subcategories.
“If you have enough of them you can actually reconstruct and view a structure as a three-dimensional model,” Rye said.
What the team saw was this: The HSP60 chaperone is designed to recognize proteins that are not folded from the ones that are. It binds them and then has a separate co-chaperone that puts a “lid” on top of the box to keep the folding intermediate in the box. They could see the box move, and parts of the molecule moved to peel the chaperone box away from the bound protein — or “gift” in the box. But the bound protein was kept inside the package where it could then initiate a folding reaction. They saw tiny tentacles, “like a little octopus in the bottom of the box rising up and grabbing hold of the substrate protein and helping hold it inside the cavity.”
"The first thing we saw was a large amount of an unfolded protein inside of this cavity,” he said. “Even though we knew from lots and lots of other studies that it had to go in there, nobody had ever seen it like this before. We can also see the non-native protein interacting with parts of the box that no one had ever seen before. It was exciting to see all of this for the first time. I think we got a glimpse of a protein in the process of folding, which we actually can compare to other structures.”
“By understanding the mechanism of these machines, the hope is that one of the things we can learn to do is turn them up or turn them off when we need to, like for a patient who has one of the protein folding diseases,” he said.
(Source: today.agrilife.org)
Brain Cancer: Hunger for Amino Acids Makes It More Aggressive
An enzyme that facilitates the breakdown of specific amino acids makes brain cancers particularly aggressive. Scientists from the German Cancer Research Center (DKFZ) discovered this in an attempt to find new targets for therapies against this dangerous disease. They have reported their findings in the journal “Nature Medicine”.
To fuel phases of fast and aggressive growth, tumors need higher-than-normal amounts of energy and the molecular building blocks needed to build new cellular components. Cancer cells therefore consume a lot of sugar (glucose A number of tumors are also able to catabolize the amino acid glutamine, an important building block of proteins. A key enzyme in amino acid decomposition is isocitrate dehydrogenase (IDH). Several years ago, scientists discovered mutations in the gene coding for IDH in numerous types of brain cancer. Very malignant brain tumors called primary glioblastomas carry an intact IDH gene, whereas those that grow more slowly usually have a defective form.
“The study of the IDH gene currently is one of the most important diagnostic criteria for differentiating glioblastomas from other brain cancers that grow more slowly,” says Dr. Bernhard Radlwimmer from the German Cancer Research Center (Deutsches Krebsforschungszentrum, DKFZ). “We wanted to find out what spurs the aggressive growth of glioblastomas.” In collaboration with scientists from other institutes including Heidelberg University Hospital, Dr. Martje Tönjes and Dr. Sebastian Barbus from Radlwimmer’s team compared gene activity profiles from several hundred brain tumors. They aimed to find out whether either altered or intact IDH show further, specific genetic characteristics that might help explain the aggressiveness of the disease.
The researchers found a significant difference between the two groups in the highly increased activity of the gene for the BCAT1 enzyme, which in normal brain tissue is responsible for breaking down so-called branched-chain amino acids. However, Radlwimmer’s team discovered, only those tumor cells whose IDH gene is not mutated produce BCAT1. “This is not surprising, because as IDH breaks down amino acids, it produces ketoglutarate – a molecule which BCAT1 needs. This explains why BCAT1 is produced only in tumor cells carrying intact IDH. The two enzymes seem to form a kind of functional unit in amino acid catabolism,” says Bernhard Radlwimmer.
Glioblastomas are particularly dreaded because they aggressively invade the healthy brain tissue that surrounds them. When the researchers used a pharmacological substance to block BCAT1’s effects, the tumor cells lost their invasive capacity. In addition, the cells released less of the glutamate neurotransmitter. High glutamate release is responsible for severe neurological symptoms such as epileptic seizures, which are frequently associated with the disease. When transferred to mice, glioblastoma cells in which the BCAT1 gene had been blocked no longer grew into tumors.
“Altogether, we can see that overexpression of BCAT1 contributes to the aggressiveness of glioblastoma cells,” Radlwimmer says. The study suggests that the two enzymes, BCAT1 and IDH, cooperate in the decomposition of branched-chain amino acids. These protein building blocks appear to act as a “food source” that increases the cancer cells’ aggressiveness. Branched-chain amino acids also play a significant role in metabolic diseases such as diabetes. This is the first time that scientists have been able to show the role of these amino acids in the growth of malignant tumors.
“The good news,” sums up Radlwimmer, “is that we have found another target for therapies in BCAT1. In collaboration with Bayer Healthcare, we have already started searching for agents that might be specifically directed against this enzyme.” The researchers also plan to investigate whether BCAT1 expression may serve as an additional marker to diagnose the malignancy of brain cancer.
Researchers Design Variant of Main Painkiller Receptor
Opioids, such as morphine, are still the most effective class of painkillers, but they come with unwanted side effects and can also be addictive and deadly at high doses. Designing new pain-killing drugs of this type involves testing them on their corresponding receptors, but access to meaningful quantities of these receptors that can work in experimental conditions has always been a limiting factor.
Now, an interdisciplinary collaboration between researchers at the University of Pennsylvania has developed a variant of the mu opioid receptor that has several advantages when it comes to experimentation. This variant can be grown in large quantities in bacteria and is also water-soluble, enabling experiments and applications that had previously been very challenging or impossible.
The study was led by Renyu Liu, an assistant professor in the Department of Anesthesiology and Critical Care at Penn’s Perelman School of Medicine, and Jeffery Saven, an associate professor in the Department of Chemistry in the School of Arts and Sciences. Jose Manuel Perez-Aguilar, then a graduate student in the Department of Chemistry, and Jin Xi, Felipe Matsunaga and Xu Cui, lab members in the Department of Anesthesiology and Critical Care, along with Bernard Selling of Impact Biologicals Inc., contributed significantly to this study.
Their research was published in the Journal PLOS ONE.
The mu opioid receptor belongs to a class of cellular membrane proteins called G protein-coupled receptors, or GPCRs. Involved in wide range of biological processes, these receptors bind to molecules in the environment, initiating cellular signaling pathways. In the case of this receptor, binding to opioid molecules leads to a profound reduction of pain but also to a variety of unpleasant and potentially fatal side-effects, a problem that researchers from multiple disciplines are attempting to address.
“There are two directions for solving this problem in basic science, either working on the opioid molecule or working on the receptor,” Liu said. “We’re doing the latter.”
Experimenting on the mu opioid receptor has been challenging for several reasons. The human receptor itself is relatively scarce, appearing in small quantities on only a few types of cells, making harvesting appreciable amounts impractical. Researchers have also been unable to grow it recombinantly — genetically engineering bacteria to express the protein en masse — as some parts of the protein are toxic to E.coli. Hydrophobic, or water-hating, amino acid groups on the exterior of the receptor that help it sit in the cell’s membrane also make it insoluble in water when isolated.
The researchers set out to address these challenges by computationally designing variants of the mu opioid receptor. This task had challenges of its own; their research was conducted long before the crystal structure of receptor was known.
“The problem with this receptor is that the native structure has only very recently been solved and only a significant re-engineered mouse model at that,” Liu said. “When we started this project, we were blind.”
Starting with only the gene sequence for the human version of the receptor, the researchers knew the order of the protein’s amino acids but not how they were folded together. The structures for other GPCRs, such as rhodopsin and the beta-2 adrenergic receptor, were known at the time, however.
“Based on the comparison of our sequence to the sequences of those GPCRs, we built a computer model of the protein,” Saven said. “When the structure of the mouse version of this receptor appeared, we were able to compare our model to that structure, and they matched up really well.”
From that comparison, the researchers were able to identify the hydrophobic amino acids on the exterior of the structure, as well as some of those that were potentially toxic to E. coli.
“The objective then was to redesign those exterior amino acids,” Saven said. “Based on the physical and chemical interactions these amino acids have with each other and with water, we were able to identify sequence combinations that are consistent with the model — where atoms don’t overlap in space — and preferentially occupy the exterior surface with ones that are water soluble.”
Replacing 53 of the protein’s 288 amino acids, the research team introduced the new gene sequence into E. coli, which were able to produce large quantities of the variant. Beyond looking like the now-available mouse mu opioid receptor, the researchers were able to show its value to future studies by performing functional tests.
“We showed that this water-soluble form of the protein can compete with the native, membrane-based form when binding with antagonists that are fluorescently labeled,” Saven said. “You can watch the fluorescence shift as more of these water-soluble variants are floating around in the solution.”
The team’s computational approach enables further iterations of the variant to be more easily designed, meaning it can be tweaked alongside experimental conditions.
“This is a great product that can do a lot of things,” Liu said. “You can use this variant to look at the structure-function relationship for the receptor, or even potentially use it as a screening tool.”