Posts tagged protein
Articles and news from the latest research reports.
Research yields first detailed view of morphing Parkinson’s protein
Researchers have taken detailed images and measurements of the morphing structure of a brain protein thought to play a role in Parkinson’s disease, information that could aid the development of medications to treat the condition.
The protein, called alpha synuclein (pronounced sine-yoo-cline), ordinarily exists in a globular shape. However, the protein morphs into harmful structures known as amyloid fibrils, which are linked to protein molecules that form in the brains of patients with neurodegenerative diseases.
"The abnormal protein formation characterizes a considerable number of human diseases, such as Alzheimer’s, Parkinson’s and Huntington’s diseases and type II diabetes," said Lia Stanciu, an associate professor of materials engineering at Purdue University.
Until now, the transition from globular to fibrils had not been captured and measured.
Researchers incubated the protein in a laboratory and then used an electron microscope and a technique called cryoelectron microscopy to snap thousands of pictures over 24 hours, capturing its changing shape. The protein was frozen at specific time intervals with liquid nitrogen.
Findings reveal that the protein morphs from its globular shape into “protofibril” strands that assemble into pore-like rings. These rings then open up, forming pairs of protofibrils that assemble into fibrils through hydrogen bonds.
"We found a correlation between protofibrils in these rings and the fibrils, for the first time to our knowledge, by measuring their true sizes and visualizing the aggregation steps," Stanciu said. "A better understanding of the mechanism yields fresh insight into the pathogenesis of amyloid-related diseases and may provide us the opportunity to develop additional therapeutic strategies."
Parkinson’s disease affects 1 percent to 2 percent of people older than 60, and an increase in its prevalence is anticipated in coming decades.
The findings were detailed in a research paper appearing in the June issue of the Biophysical Journal. The paper was authored by doctoral student Hangyu Zhang; former postdoctoral research associate Amy Griggs; Jean-Christophe Rochet, an associate professor of medicinal chemistry and molecular pharmacology; and Stanciu.
The researchers caused the protein to morph into fibrils by exposing it to copper, mimicking what happens when people are exposed to lead and other heavy metals. The contaminants interfere with the protein, changing the oxidation states of ions in its structure.
Reference:
Hangyu Zhang, Amy Griggs, Jean-Christophe Rochet, and Lia A. Stanciu. In Vitro Study of a-Synuclein Protofibrils by Cryo-EM Suggests a Cu2D-Dependent Aggregation Pathway. Biophysical Journal, 2013 (in press)
Little less protein may be answer in neurodegenerative disorders
In some neurodegenerative diseases, and specifically in a devastating inherited condition called spinocerebellar ataxia 1 (SCA1), the answer may not be an “all-or-nothing,” said a collaboration of researchers from Baylor College of Medicine, the Jan and Dan Duncan Neurological Research Institute at Texas Children’s Hospital and the University of Minnesota in a report that appears online in the journal Nature. The problem might be solved with just a little less.
"If you can only decrease the levels of ataxin-1 (the protein involved in SCA1) by 20 percent, you can reduce many symptoms of the disease," said Dr. Huda Zoghbi, professor of molecular and human genetics and pediatrics at BCM and director of the Neurological Research Institute. She is also a Howard Hughes Medical Institute Investigator.
Her long-time colleague Dr. Harry Orr, director of the University of Minnesota Institute for Translational Neuroscience, echoed that sentiment: “Perhaps, if you decrease the levels of the protein, you will decrease the severity of the disease.” In this report, the laboratories of Zoghbi, Dr. Juan Botas, also of BCM and the Neurological Researcher Institute, Dr. Thomas Westbrook, assistant professor of molecular and human genetics at BCM, and Orr identified a molecular pathway in the cell (RAS/MAPK/MSK1) with components that can be modulated slightly to reduce the levels of defective ataxin-1, the protein that causes disease in patients with the disorder.
Spinocerebellar ataxia 1 occurs when the ataxin-1 gene is mutated, with three letters of the DNA alphabet repeating many, many times. The abnormal protein that results cannot fold correctly and piles up in the cell, eventually killing it. As with many neurodegenerative disorders, the process can take over a decade. A person usually does not develop symptoms of this form of ataxia until he or she is 30 years old or older. The person develops gait problems, eventually loses the ability to speak and function and dies. Zoghbi and Orr teamed to find the gene associated with the disorder in 1993. Their work on the disease has spanned 20 years.
Totally eliminating the protein would not work. Mice that lack the gene have problems with learning and memory, indicating that ataxin-1 plays a role in those activities. Reducing the levels of ataxin-1 does not cure the disease, but it can significantly delay onset.
A Collaborative Innovation Award from the Howard Hughes Medical Institute enabled Zoghbi to put together the team that could screen for the genes or the gene pathway that could be manipulated to result in less ataxin-1.
"Harry and I had studied the disease and we had animal models. Botas, professor of molecular and human genetics at BCM, had a fruit fly model and Dr. Westbrook had a nice technology that enabled us to monitor ataxin-1 levels."
They began with a screen for genes that could affect the levels of ataxin-1 produced in the cell, said Dr. Ismail Al-Ramahi, a postdoctoral fellow in the lab of Botas. Dr. Jeehye Park, a post-doctoral fellow in Zoghbi’s laboratory, and Al-Ramahi are co-first authors of the report. Park and her colleagues carried out the screen in human cell lines and Al-Ramahi and his colleagues carried out the screen in fruit flies (Drosophila melanogaster).
The screen in human cells focused on forms of enzymes called kinases because they are susceptible to the effects of drugs. Using a special technique called RNA silencing, they targeted each known human kinase. At the same, Botas and Al-Ramahi screened kinase genes in fruit flies with a form of SCA1. When the two laboratories compared results, they found 10 genes in common that when inhibited could reduce the levels of ataxin-1 as well as the toxicity associated with it. The genes were part of the RAS/MAPK/MSKI signaling cascade within the cell.
Then the researchers focused on one protein in this pathway called MSK1 and found that when its levels were decreased in mice that were laboratory models of SCA1, the levels of ataxin-1 dropped and the animals improved. That was the final experiment that proved that reducing levels of the protein could stave off the disease.
"We want to look for more pathways," said Zoghbi. If they find more pathways, they may be able to reduce toxicity. "If you have a pain and you take acetaminophen all the time, you have a risk of toxicity. Similarly, if you took a nonsteroidal anti-inflammatory all the time, you would have another toxicity. If you alternate between them, there is less toxicity. If we hit only one pathway with a big inhibition, we risk some toxicity. If we find two or three pathways and hit each only a little, the rest of the body should not be hurt. Each little hit should help us reduce ataxin-1 by a respectable amount."
"I think what is novel about this paper is the integration of the screen in cells that was done in Huda’s lab and the screen in fruit flies done in our lab to look for targets for genes about which we knew nothing ahead of time," said Botas.
While the finding in spinocerebellar ataxia 1 is exciting, its potential application in other diseases is even more provocative.
"Now that we know that it works with ataxin-1, we can revisit many proteins whose levels drive neurodegeneration in sporadic and inherited diseases such as Alzheimer’s, Parkinson’s, Huntington’s and other neurological disorders," said Zoghbi. "This is a pilot study and the results from it are as important as a new pathway in neurodegenerative disease research."
"These are diseases that take a long time to develop," said Park. "Most Alzheimer’s occurs after the age of 85. If we could delay it until age 95, that would be very helpful."
"This is getting us really close, not only for SCA1, but I think it’s going to be a guidepost for work on a lot of other neurodegenerative diseases," said Orr. "It sets us a beautiful research strategy to get at that goal."
(Source: bcm.edu)
Preventing ‘traffic jams’ in brain cells
Imagine if you could open up your brain and look inside.
What you would see is a network of nerve cells called neurons, each with its own internal highway system for transporting essential materials between different parts of the cell.
When this biological machinery is operating smoothly, tiny motor proteins ferry precious cargo up and down each neuron along thread-like roadways called microtubule tracks. Brain cells are able to receive information, make internal repairs and send instructions to the body, telling the fingers to flex or the toes to curl.
But when the neuron gets blocked, this delicate harmony deteriorates. One result: diseases like Alzheimer’s.
Understanding such blockages and how traffic should flow normally in healthy brain cells could offer hope to people with neurodegenerative diseases.
Toward that end, a research team led by University at Buffalo biologist Shermali Gunawardena, PhD, has shown that the protein presenilin plays an important role in controlling neuronal traffic on microtubule highways, a novel function that previously was unknown.
The research results were published online on May 24 in the journal Human Molecular Genetics. Gunawardena’s co-authors are Ge Yang of Carnegie Mellon University and Lawrence S. B. Goldstein of the Howard Hughes Medical Institute and the University of California, San Diego.
Inside the nerves of fruit fly larvae, presenilin helped to control the speed at which molecular motors called kinesins and dyneins moved along neurons. When the scientists halved the amount of presenilin present in the highway system, the motors moved faster; they paused fewer times and their pauses were shorter.
Given this data, Gunawardena thinks that tweaking presenilin levels may be one way to free up traffic and prevent dangerous neuronal blockages in patients with Alzheimer’s disease.
“Our major discovery is that presenilin has a novel role, which is to control the movement of motor proteins along neuronal highways,” said Gunawardena, an assistant professor of biological sciences. “If this regulation/control is lost, then things can go wrong. This is the first time a protein that functions as a controller of motors has been reported.
“In Alzheimer’s disease, transport defects occur well before symptoms, such as cell death and amyloid plaques, are seen in post-mortem brains,” she added. “As a result, developing therapeutics targeted to defects in neuronal transport would be a useful way to attack the problem early.”
The findings are particularly intriguing because scientists have known for several years that presenilin is involved in Alzheimer’s disease.
Presenilin rides along neuronal highways in tiny organic bubbles called vesicles that sit atop the kinesin and dynein motors, and also contain a second protein called the amyloid precursor protein (APP). Presenilin participates in cutting APP into pieces called amyloid beta, which build up to form amyloid plaques in patients with Alzheimer’s disease.
Such buildups can lead to cell death by preventing the transport of essential materials—like proteins needed for cell repair—along neurons.
The findings of the new study mean that presenilin may contribute to Alzheimer’s disease in at least two ways: not just by cleaving APP, but also by regulating the speed of the molecular motors that carry APP along neuronal highways.
“More than 150 mutations in presenilin have been identified in Alzheimer’s disease,” Gunawardena said. “Thus, understanding its function is important to understanding what goes wrong in Alzheimer’s disease.”
To track the movement of the kinesins and dyneins, the team tagged their cargo with a yellow fluorescent protein. This enabled the scientists to view the molecular motors chugging along inside the neuron under a microscope in a living animal. A special computer program then analyzed the motors’ paths, revealing more details about the nature of their movement and how often they paused.
Thanks to Rare Alpine Bacteria, Researchers Identify One of Alcohol’s Key Gateways to the Brain
Thanks to a rare bacteria that grows only on rocks in the Swiss Alps, researchers at The University of Texas at Austin and the Pasteur Institute in France have been the first to identify how alcohol might affect key brain proteins.
It’s a major step on the road to eventually developing drugs that could disrupt the interaction between alcohol and the brain.
“Now that we’ve identified this key brain protein and understand its structure, it’s possible to imagine developing a drug that could block the binding site,” said Adron Harris, professor of biology and director of the Waggoner Center for Alcohol and Addiction at The University of Texas at Austin.
Harris and his former postdoctoral fellow Rebecca Howard, now an assistant professor at Skidmore College, are co-authors on the paper that was recently published in Nature Communications. It describes the structure of the brain protein, called a ligand-gated ion channel, that is a key enabler of many of the primary physiological and behavioral effects of alcohol.
Harris said that for some time there has been suggestive evidence that these ion channels are important binding sites for alcohol. Researchers couldn’t prove it, however, because they couldn’t crystallize the brain protein well enough, and therefore couldn’t use X-ray crystallography to determine the structure of the protein with and without alcohol present.
“For many of us in the alcohol field, this has been a Holy Grail, actually finding a binding site for alcohol on the brain proteins and showing it with X-ray crystallography,” said Harris. “But it hasn’t been possible because it is not possible to get a nice crystal.”
The breakthrough came when Marc Delarue and his colleagues at the Pasteur Institute sequenced the genome of cyanobacteria Gloeobacter violaceus. They noted a protein sequence on the bacteria that is remarkably similar to the sequence of a group of ligand-gated ion channels in the human brain. They were able to crystallize this protein. Harris saw the results and immediately got in touch.
“This is something you never would have found with any sort of logical approach,” he said. “You never would have guessed that this obscure bacterium would have something that looks like a brain protein in it. But the institute, because of Pasteur’s fascination with bacteria, has this huge collection of obscure bacteria, and over the last few years they’ve been sequencing the genomes, keeping an eye out for interesting properties.”
Harris and Howard asked their French colleagues to collaborate, got the cyanobacteria, changed one amino acid to make it sensitive to alcohol, and then crystallized both the original bacteria and the mutated one. They compared the two to see whether they could identify where the alcohol bound to the mutant. With further tests they confirmed that it was a meaningful site.
“Everything validated that the cavity in which the alcohol bound is important,” said Harris. “It doesn’t account for all the things that alcohol does, but it appears to be important for a lot of them, including some of the ‘rewarding’ effects and some of the negative, aversive effects.”
Going forward, Harris and his lab plan to use mice to observe how changes to the key protein affect behavior when the mice consume alcohol.
They’re also hoping to identify other important proteins from this family of ligand-gated ion channels. In the long term, he hopes to be involved in developing drugs that act on these proteins in ways that help people diminish or cease their drinking.
“So why do some people drink moderately and some excessively?” he said. “One reason lies in that the balance between the rewarding and the aversive effects, and that balance is different for different people, and it can change within an individual depending on their drinking patterns. Some of those effects are determined by the interactions of alcohol and these channels, so the hope is that we can alter the balance. Maybe we can diminish the reward or increase the aversive effects.”
Ever since the appetite-regulation hormone called leptin was discovered in 1994, scientists have sought to understand the mechanisms that control its action. It was known that leptin was made by fat cells, reduced appetite and interacted with insulin , but the precise molecular details of its function —details that might enable the creation of a new treatment for obesity — remained elusive.
Now, University of Texas Medical Branch at Galveston researchers have revealed a significant part of one of those mechanisms, identifying a protein that can interfere with the brain’s response to leptin. They’ve also created a compound that blocks the protein’s action — a potential forerunner to an anti-obesity drug.
In experiments with mice fed a high-fat diet, scientists from UTMB and the University of California, San Diego explored the role of the protein, known as Epac1, in blocking leptin’s activity in the brain. They found that mice genetically engineered to be unable to produce Epac1 had lower body weights, lower body fat percentages, lower blood-plasma leptin levels and better glucose tolerance than normal mice.
When the researchers used a specially developed “Epac inhibitor” to treat brain-slice cultures taken from normal laboratory mice, they found elevated levels of proteins associated with greater leptin sensitivity. Similar results were seen in the genetically engineered mice that lacked the Epac1 gene. In addition, normal mice treated with the inhibitor had significantly lower levels of leptin in their blood plasma — an indication that Epac1 also affected their leptin levels.
“We found that we can increase leptin sensitivity by creating mice that lack the genes for Epac1 or through a pharmacological intervention with our Epac inhibitor,” said UTMB professor Xiaodong Cheng, lead author of a paper on the study that recently appeared on the cover of Molecular and Cellular Biology. “The knockout mice gave us a way to tease out the function of the protein, and the inhibitor served as a pharmacological probe that allowed us to manipulate these molecules in the cells.”
Cheng and his colleagues suspected a connection between Epac1 and leptin because Epac1 is activated by cyclic AMP, a signaling molecule linked to metabolism and leptin production and secretion. Cyclic AMP is tied to a multitude of other cell signaling processes, many of which are targeted by current drugs. Cheng believes that understanding how it acts through Epac1 (and another form of the protein called Epac2) will also generate new pharmaceutical possibilities — possibly including a drug therapy that will help fight obesity and diabetes.
“We refer to these Epac inhibitors as pharmacological probes, and while they are still far away from drugs, pharmaceutical intervention is always our eventual goal,” Cheng said. “We were the first to develop Epac inhibitors, and now we’re working very actively with Dr. Jia Zhou, a UTMB medicinal chemist, to modify them and improve their properties. In addition, we are collaborating with colleagues at the NIH National Center for Advancing Translational Sciences in searching for more potent and selective pharmacological probes for Epac proteins.”
![A Sleep Aid Without the Side Effects
Insomniacs desperate for some zzzs may one day have a safer way to get them. Scientists have developed a new sleep medication that has induced sleep in rodents and monkeys without apparently impairing cognition, a potentially dangerous side effect of common sleep aids. The discovery, which originated in work explaining narcolepsy, could lead to a new class of drugs that help people who don’t respond to other treatments.
Between 10% and 15% of Americans chronically struggle with getting to or staying asleep. Many of them turn to sleeping pills for relief, and most are prescribed drugs, such as zolpidem (Ambien) and eszopiclone (Lunesta), that slow down the brain by binding to receptors for GABA, a neurotransmitter that’s involved in mood, cognition, and muscle tone. But because the drugs target GABA indiscriminately, they can also impair cognition, causing amnesia, confusion, and other problems with learning and memory, along with a number of strange sleepwalking behaviors, including wandering, eating, and driving while asleep. This has led many researchers to seek out alternative mechanisms for inducing sleep.
Neuroscientist Jason Uslaner of Merck Research Laboratories in West Point, Pennsylvania, and colleagues decided to tap into the brain’s orexin system. Orexin (also known as hypocretin) is a protein that controls wakefulness and is missing in people with narcolepsy. Past studies successfully induced sleep by inhibiting orexin, but had not looked into its effects on cognition. The researchers developed a new orexin-inhibiting compound called DORA-22 and confirmed that it could induce sleep in rats and rhesus monkeys as effectively as the GABA-modulating drugs.
Then the researchers went about testing the drugs’ effects on the animals’ cognition. They measured the rats’ cognition and memory by assessing the rodents’ ability to recognize objects. They presented the rats with a new object—say, a cone or a sphere—that the rats then sniffed and explored. Then they took the object away for an hour. After that hour, the rats were exposed to a new object and the one they’d already gotten to know; if the rats remembered, they spent less time checking out the familiar object. With the primates, Uslaner’s team tested their ability to match colors on a touchscreen and to pay attention to and identify the origin of a flashing light. In all the cases, the researchers found the GABA-modulating sleeping pills caused both the rats and the primates to respond more slowly and less accurately. Monkeys taking the memory and attention tests, for example, were 20% less accurate on the highest dose of each of the GABA-modulating drugs. But DORA-22 had no such effect on cognition, the team reports today in Science Translational Medicine.
"We were very excited," Uslaner says. "Folks who take sleep medications need to be able to perform cognitive tasks when they awake, and this [compound] could help them do so without impairment."
Although DORA-22 has not yet been tested in humans, it holds tremendous promise for helping people suffering from sleep disorders, says Emmanuel Mignot, a sleep researcher with the Stanford University School of Medicine in Palo Alto, California. “This study is encouraging and exciting, because there’s good reason to believe it would work differently from what we’ve used in the past,” says Mignot, who helped discover the link between orexin (or its absence) and narcolepsy. “Not every drug works for everyone, so it’s really, really good news to have a potential new drug on the horizon.”](http://41.media.tumblr.com/ec7d58bff47f9cfe77e5a11284a658a0/tumblr_mks52vgAk71rog5d1o1_500.jpg)
A Sleep Aid Without the Side Effects
Insomniacs desperate for some zzzs may one day have a safer way to get them. Scientists have developed a new sleep medication that has induced sleep in rodents and monkeys without apparently impairing cognition, a potentially dangerous side effect of common sleep aids. The discovery, which originated in work explaining narcolepsy, could lead to a new class of drugs that help people who don’t respond to other treatments.
Between 10% and 15% of Americans chronically struggle with getting to or staying asleep. Many of them turn to sleeping pills for relief, and most are prescribed drugs, such as zolpidem (Ambien) and eszopiclone (Lunesta), that slow down the brain by binding to receptors for GABA, a neurotransmitter that’s involved in mood, cognition, and muscle tone. But because the drugs target GABA indiscriminately, they can also impair cognition, causing amnesia, confusion, and other problems with learning and memory, along with a number of strange sleepwalking behaviors, including wandering, eating, and driving while asleep. This has led many researchers to seek out alternative mechanisms for inducing sleep.
Neuroscientist Jason Uslaner of Merck Research Laboratories in West Point, Pennsylvania, and colleagues decided to tap into the brain’s orexin system. Orexin (also known as hypocretin) is a protein that controls wakefulness and is missing in people with narcolepsy. Past studies successfully induced sleep by inhibiting orexin, but had not looked into its effects on cognition. The researchers developed a new orexin-inhibiting compound called DORA-22 and confirmed that it could induce sleep in rats and rhesus monkeys as effectively as the GABA-modulating drugs.
Then the researchers went about testing the drugs’ effects on the animals’ cognition. They measured the rats’ cognition and memory by assessing the rodents’ ability to recognize objects. They presented the rats with a new object—say, a cone or a sphere—that the rats then sniffed and explored. Then they took the object away for an hour. After that hour, the rats were exposed to a new object and the one they’d already gotten to know; if the rats remembered, they spent less time checking out the familiar object. With the primates, Uslaner’s team tested their ability to match colors on a touchscreen and to pay attention to and identify the origin of a flashing light. In all the cases, the researchers found the GABA-modulating sleeping pills caused both the rats and the primates to respond more slowly and less accurately. Monkeys taking the memory and attention tests, for example, were 20% less accurate on the highest dose of each of the GABA-modulating drugs. But DORA-22 had no such effect on cognition, the team reports today in Science Translational Medicine.
"We were very excited," Uslaner says. "Folks who take sleep medications need to be able to perform cognitive tasks when they awake, and this [compound] could help them do so without impairment."
Although DORA-22 has not yet been tested in humans, it holds tremendous promise for helping people suffering from sleep disorders, says Emmanuel Mignot, a sleep researcher with the Stanford University School of Medicine in Palo Alto, California. “This study is encouraging and exciting, because there’s good reason to believe it would work differently from what we’ve used in the past,” says Mignot, who helped discover the link between orexin (or its absence) and narcolepsy. “Not every drug works for everyone, so it’s really, really good news to have a potential new drug on the horizon.”
The good side of the prion: A molecule that is not only dangerous, but can help the brain grow
A few years ago it was found that certain proteins, the prions, when defective are dangerous, as they are involved in neurodegenerative syndromes such as the Creutzfeldt-Jakob and the Alzheimer diseases. But now research is showing their good side, too: when performing well, prions may be crucial in the development of the brain during childhood, as observed by a study carried out by a team of neuroscientists at Trieste’s SISSA which appeared yesterday in the Journal of Neuroscience.
Doctor Jekyll and Mr. Hyde: the metaphor of the good man who hides an evil side suits well the prion (PrPC in its physiological cellular form), a protein which abounds in our brain. Unlike Doctor Jekyll, the prion was at first considered for its upsetting properties: if the molecule abnormally folds over itself it unfortunately plays a crucial role in neurodegenerative processes that lead to dreadful syndromes such as the mad cow disease.
Prions, however, in their normal form abound in synapses, the contact points where the nervous signal is passed from a neuron to the next. Such protein relatively abounds in the brain of very young children, and this is the reason why scientists have assumed it may play a role in the
nervous system development, and in particular in neurogenesis, in the development of new synaptic connections and in plasticity.
More in detail
Maddalena Caiati, Victoria Safiulina, Sudhir Sivakumaran, Giuseppe Legname, Enrico Cherubini, all researchers at SISSA, and Giorgia Fattorini of the Università Politecnica delle Marche have verified at the molecular level the effects of PrPC on the cell plasticity of the hippocampus, a brain structure which has important functions related to memory. Maddalena Caiati and her colleagues have demonstrated that PrPC controls synaptic plasticity (the growth capacity of the nervous tissue) through a transduction pathway which involves also another protein, the protein kinase A enzyme (PKA). The recently published research is only the starting point. As for the future, it will be interesting to get a closer look at the role played by the prion protein in the development of neuronal circuits both under physiological and pathologic conditions in neurodegenerative diseases.
Scientists Discover Structure of Protein Essential for Quality Control, Nerve Function
Using an innovative approach, scientists at The Scripps Research Institute (TSRI) have determined the structure of Ltn1, a recently discovered “quality-control” protein that is found in the cells of all plants, fungi and animals.
Ltn1 appears to be essential for keeping cells’ protein-making machinery working smoothly. It may also be relevant to human neurodegenerative diseases, for an Ltn1 mutation in mice leads to a motor-neuron disease resembling amyotrophic lateral sclerosis (ALS, also known as Lou Gehrig’s disease).
“To better understand Ltn1’s mechanism of action, we needed to solve its structure, and that’s what we’ve done here,” said TSRI Associate Professor Claudio Joazeiro.
“In addition, this project has brought us a set of structural analysis techniques that we can apply to other exciting problems in biology,” said TSRI Professor Bridget Carragher.
Joazeiro and Carragher, along with Clint Potter, also a TSRI professor, are senior authors of the new report, which appears in the online Early Edition of the Proceedings of the National Academy of Sciences the week of January 14, 2013.
Links to Neurodegenerative Disease
Ltn1 first turned up on biologists’ radar screens several years ago when a joint Novartis-Phenomix research team noted that mice with an unknown gene mutation were born normal but suffered from progressive paralysis. The scientists dubbed the animals lister mice, because they listed to one side as they walked. Collaborating with Joazeiro, the Novartis team reported in a 2009 paper that the mutated gene normally codes for a type of enzyme known as an E3 ubiquitin ligase, and that the mouse phenotype was due to a neurodegenerative syndrome resembling ALS.
In a study published in the journal Nature the following year, Joazeiro and his postdoctoral research associate Mario H. Bengtson found that the enzyme serves as a crucial quality-control manager for the cellular protein-making factories called ribosomes. Occasionally a ribosome receives miscoded genetic instructions and produces certain types of abnormal proteins, known as “nonstop proteins”— jamming the ribosomal machinery like a wrinkled sheet of paper in an office printer. Bengtson and Joazeiro found that Ltn1 fixes jammed ribosomes by tagging nonstop proteins with ubiquitin molecules, thereby marking them for quick destruction by roving cellular garbage-disposers called proteasomes.
“The question for us then was, ‘How does Ltn1 do this?’” said Joazeiro.
Pushing the Boundaries of Electron Microscopy
To help find out, he began a collaboration with Carragher and Potter, who run the National Resource for Automated Molecular Microscopy (NRAMM), an advanced electron microscope facility at TSRI that is funded by the National Institutes of Health’s National Center for Research Resources.
Ltn1 was deemed too large for its structure to be determined by current nuclear magnetic resonance (NMR) technology, and, as the scientists know now, too flexible to allow the highly regular crystalline packing needed by X-ray crystallographers. “It’s a very floppy molecule, so it would be hard to crystallize,” said Potter.
Advanced electron microscopy offered a way, however. Dmitry Lyumkis, a graduate student in the NRAMM laboratory and first author of the study, took high-resolution images of yeast Ltn1 with an electron microscope. He then used sophisticated image and data processing software to align and average individual images. The technique eliminates much of the random “noise” that obscures single images and produces a sharp 3D picture of the protein.
No one has ever used electron microscopy to distinguish so many—more than 20—conformations of such a small protein. “Usually electron microscopists determine no more than two or three conformational states, and they work with protein complexes whose size is in the megadalton range, but Ltn1 is only 180 kilodaltons, an order of magnitude smaller,” Lyumkis said.
An Unusually Flexible Structure
The analysis revealed that Ltn1 has an elongated, double-jointed and extraordinarily flexible structure with two working ends—the N-terminus and C-terminus. “We anticipate that the N-terminus is responsible for association with the ribosome and know that the C-terminus is responsible for the ubiquitylation of nonstop proteins,” said Lyumkis. “We suspect that the high flexibility of this structure is needed for it to work on the variety of nonstop proteins that can get stuck in ribosomes.”
One of the next steps for the team is to evaluate Ltn1’s individual segments, which appear to be more rigid, using X-ray crystallography, in order to develop a piece-by-piece atomic-resolution model of the enzyme. Another is to determine the structure of Ltn1 when it is attached to a ribosome and operating on a nonstop protein. Joazeiro notes that a typical yeast cell has nearly 200,000 ribosomes but requires only 200 Ltn1 copies for adequate quality control under normal growth conditions. “Somehow this enzyme can efficiently sense which ribosomes are jammed, and we expect that by solving the joint structure of Ltn1 and a ribosome, we’ll be able to understand how it does this,” he says.
Lyumkis, Carragher, Potter and their colleagues at NRAMM also plan to use a similar electron microscopy-based approach to find the structures of other important proteins with highly variable “heterogeneous” conformations. “Heterogeneity has been a big challenge,” said Potter, “and being able to collect this large dataset and do all of this data processing successfully has been a critical breakthrough.”
Study uncovers protein key to fighting and preventing obesity
University of Florida researchers and colleagues have identified a protein that, when absent, helps the body burn fat and prevents insulin resistance and obesity. The findings from the National Institutes of Health-funded study were published online ahead of print Sunday, Jan. 6, in the journal Nature Medicine.
The discovery could aid development of drugs that not only prevent obesity, but also spur weight loss in people who are already overweight, said Dr. Stephen Hsu, one of the study’s corresponding authors and a principal investigator with the UF Sid Martin Biotechnology Development Institute.
One-third of adults and about 17 percent of children in the United States are obese, according to the Centers for Disease Control and Prevention. Although unrelated studies have shown that lifestyle changes such as choosing healthy food over junk food and increasing exercise can help reduce obesity, people are often unable to maintain these changes over time, Hsu said.
“The problem is when these studies end and the people go off the protocols, they almost always return to old habits and end up eating the same processed foods they did before and gain back the weight they lost during the study,” he said. Developing drugs that target the protein, called TRIP-Br2, and mimic its absence may allow for the prevention of obesity without relying solely on lifestyle modifications, Hsu said.
First identified by Hsu, TRIP-Br2 helps regulate how fat is stored in and released from cells. To understand its role, the researchers compared mice that lacked the gene responsible for production of the protein, with normal mice that had the gene.
They quickly discovered that mice missing the TRIP-Br2 gene did not gain weight no matter what they ate — even when placed on a high-fat diet — and were otherwise normal and healthy. On the other hand, the mice that still made TRIP-Br2 gained weight and developed associated problems such as insulin resistance, type 2 diabetes and high cholesterol when placed on a high-fat diet. The normal and fat-resistant mice ate the same amount of food, ruling out differences in food intake as a reason why the mice lacking TRIP-Br2 were leaner.
“We had to explain why the animals eating so much fat were remaining lean and not getting high cholesterol. Where was this fat going?” Hsu said. “It turns out this protein is a master regulator. It coordinates expression of a lot of genes and controls the release of the fuel form of fat and how it is metabolized.”
When functioning normally, TRIP-Br2 restricts the amount of fat that cells burn as energy. But when TRIP-Br2 is absent, a fat-burning fury seems to occur in fat cells. Although other proteins have been linked to the storage and release of fat in cells, TRIP-Br2 is unique in that it regulates how cells burn fat in a few different ways, Hsu said. When TRIP-Br2 is absent, fat cells dramatically increase the release of free fatty acids and also burn fat to produce the molecular fuel called ATP that powers mitochondria — the cell’s energy source. In addition, cells free from the influence of TRIP-Br2 start using free fatty acids to generate thermal energy, which protects the body from exposure to cold.
“TRIP-Br2 is important for the accumulation of fat,” said Dr. Rohit N. Kulkarni, also a senior author of the paper and an associate professor of medicine at Harvard Medical School and the Joslin Diabetes Center. “When an animal lacks TRIP-Br2, it can’t accumulate fat.”
Because the studies were done mostly in mice, additional studies are still needed to see if the findings translate to humans.
“We are very optimistic about the translational promise of our findings because we showed that only human subjects who had the kind of fat (visceral) that becomes insulin-resistant also had high protein levels of TRIP-Br2,” Hsu said.
“Imagine you are able to develop drugs that pharmacologically mimic the complete absence of TRIP-Br2,” Hsu said. “If a patient started off fat, he or she would burn the weight off. If people are at risk of obesity and its associated conditions, such as type 2 diabetes, it would help keep them lean regardless of how much fat they ate. That is the ideal anti-obesity drug, one that prevents obesity and helps people burn off excess weight.”
(Source: news.ufl.edu)
The Nerve-Growth Factor: A New Tool for Manipulating Neurons
The human nervous system is a vast network of several billion neurons, or nerve cells, endowed with the remarkable ability to receive, store and transmit information. In order to communicate with one another and with non-neuronal cells the neurons rely on the long extensions called axons, which are somewhat analogous to electrically conducting wires. Unlike wires, however, the axons are fluid-filled cylindrical structures that not only transmit electrical signals but also ferry nutrients and other essential substances to and from the cell body. Many basic questions remain to be answered about the mechanisms governing the formation of this intricate cellular network. How do the nerve cells differentiate into thousands of different types? How do their axons establish specific connections (synapses) with other neurons and non-neuronal cells? And what is the nature of the chemical messages neurons send and receive once the synaptic connections are made?
This article will describe some major characteristics and effects of a protein called the nerve-growth factor (NGF), which has made it possible to induce and analyze under highly favorable conditions some crucial steps in the differentiation of neurons, such as the growth and maturation of axons and the synthesis and release of neurotransmitters: the bearers of the chemical messages. The discovery of NGF has also promoted an intensive search for other specific growth factors, leading to the isolation and characterization of a number of proteins with the ability to enhance the growth of different cell lines.