Posts tagged proteins
Articles and news from the latest research reports.
New front in war on Alzheimer’s, other protein-folding diseases
A surprise discovery that overturns decades of thinking about how the body fixes proteins that come unraveled greatly expands opportunities for therapies to prevent diseases such as Alzheimer’s and Parkinson’s, which have been linked to the accumulation of improperly folded proteins in the brain.
“This finding provides a whole other outlook on protein-folding diseases; a new way to go after them,” said Andrew Dillin, the Thomas and Stacey Siebel Distinguished Chair of Stem Cell Research in the Department of Molecular and Cell Biology and Howard Hughes Medical Institute investigator at the University of California, Berkeley.
Dillin, UC Berkeley postdoctoral fellows Nathan A. Baird and Peter M. Douglas and their colleagues at the University of Michigan, The Scripps Research Institute and Genentech Inc., will publish their results in the Oct. 17 issue of the journal Science.
Cells put a lot of effort into preventing proteins – which are like a string of beads arranged in a precise three-dimensional shape – from unraveling, since a protein’s activity as an enzyme or structural component depends on being properly shaped and folded. There are at least 350 separate molecular chaperones constantly patrolling the cell to refold misfolded proteins. Heat is one of the major threats to proteins, as can be demonstrated when frying an egg – the clear white albumen turns opaque as the proteins unfold and then tangle like spaghetti.
Heat shock
For 35 years, researchers have worked under the assumption that when cells undergo heat shock, as with a fever, they produce a protein that triggers a cascade of events that field even more chaperones to refold unraveling proteins that could kill the cell. The protein, HSF-1 (heat shock factor-1), does this by binding to promoters upstream of the 350-plus chaperone genes, upping the genes’ activity and launching the army of chaperones, which originally were called “heat shock proteins.”
Injecting animals with HSF-1 has been shown not only to increase their tolerance of heat stress, but to increase lifespan.
Because an accumulation of misfolded proteins has been implicated in aging and in neurodegenerative diseases such as Alzheimer’s, Parkinson’s and Huntington’s diseases, scientists have sought ways to artificially boost HSF-1 in order to reduce the protein plaques and tangles that eventually kill brain cells. To date, such boosters have extended lifespan in lab animals, including mice, but greatly increased the incidence of cancer.
Dillin’s team found in experiments on the nematode worm C. elegans that HSF-1 does a whole lot more than trigger release of chaperones. An equal if not more important function is to stabilize the cell’s cytoskeleton, which is the highway that transports essential supplies – healing chaperones included – around the cell.
“We are suggesting that, rather than making more of HSF-1 to prevent diseases like Huntington’s, we should be looking for ways to make the actin cytoskeleton better,” Dillin said. Such tactics might avoid the carcinogenic side effects of upping HSF-1.
Dillin is codirector of the Paul F. Glenn Center for Aging Research, a new collaboration between UC Berkeley and UC San Francisco supported by the Glenn Foundation for Medical Research. Center investigators will study the many ways that proteins malfunction within cells, ideally paving the way for novel treatments for neurodegenerative diseases.
A cell at war
Dillin compares a cell experiencing heat shock to a country under attack. In a war, an aggressor first cuts off all communications, such as roads, train and bridges, which prevents the doctors from treating the wounded. Similarly, heat shock disrupts the cytoskeletal highway, preventing the chaperone “doctors” from reaching the patients, the misfolded proteins.
“We think HSF-1 not only makes more chaperones, more doctors, but also insures that the roadways stay intact to keep everything functional and make sure the chaperones can get to the sick and wounded warriors,” he said.
The researchers found specifically that HSF-1 up-regulates another gene, pat-10, that produces a protein that stabilizes actin, the building blocks of the cytoskeleton.
By boosting pat-10 activity, they were able to cure worms that had been altered to express the Huntington’s disease gene, and also extend the lifespan of normal worms.
Dillin suspects that HSF-1’s main function is, in fact, to protect the actin cytoskeleton. He and his team mutated HSF-1 so that it no longer boosted chaperones, demonstrating, he said, that “you can survive heat shock with the normal level of heat shock proteins, as long as you make your cytoskeleton work better.”
He noted that the team’s results – that boosting chaperones is not essential to surviving heat stress – were so contradictory to current thinking that “I made my post-docs’ lives hell for three years” insisting on more experiments to rule out errors. Yet, when Dillin presented the results recently to members of the protein-folding community, he said the first reaction of many was, “That makes perfect sense.”
Researchers find new mechanism for neurodegeneration
A research team led by Jackson Laboratory Professor and Howard Hughes Investigator Susan Ackerman, Ph.D., has pinpointed a surprising mechanism behind neurodegeneration in mice, one that involves a defect in a key component of the cellular machinery that makes proteins, known as transfer RNA or tRNA.
The researchers report in the journal Science that a mutation in a gene that produces tRNAs operating only in the central nervous system results in a “stalling” or pausing of the protein production process in the neuronal ribosomes. When another protein the researchers identified, GTPBP2, is also missing, neurodegeneration results.
“Our study demonstrates that individual tRNA genes can be tissue-specifically expressed in vertebrates,” Ackerman says, “and mutations in such genes may cause disease or modify other phenotypes. This is a new area to look for disease mechanisms.”
Neurodegeneration—the process through which mature neurons decay and ultimately die—is poorly understood, yet it underlies major human diseases, such as Alzheimer’s disease, Parkinson’s disease, Huntington’s disease and ALS (amyotrophic lateral sclerosis, also known as Lou Gehrig’s disease).
While the causes of neurodegeneration are still coming to light, there is mounting evidence that neurons are exquisitely sensitive—much more so than other types of cells—to disruptions in how proteins are made and how they fold.
tRNAs are critical in translating the genetic code into proteins, the workhorses of the cell. tRNAs possess a characteristic cloverleaf shape with two distinct “business” ends—one that reads out the genetic code in three-letter increments (or triplets), and another that transports the protein building block specified by each triplet (known as an amino acid).
In higher organisms, tRNAs are strikingly diverse. For example, while there are 61 distinct triplets that are recognized by tRNAs in humans, the human genome contains roughly 500 tRNA genes. To date little is known about why they are so numerous, whether they carry out overlapping or redundant functions, or whether they possibly have roles beyond the making of proteins.
“Multiple genes encode almost all tRNA types,” Ackerman says. “In fact, AGA codons are decoded by five tRNAs in mice. Until now, this apparent redundancy has caused us to completely overlook the disease-causing potential of mutations in tRNAs, as well as other repetitive genes.”
Ackerman and her colleagues at The Jackson Laboratory in Bar Harbor, Maine, and Farmington, Conn., The Scripps Research Institute in LaJolla, Calif., and Kumamoto University in Japan pinpointed a mutation in the tRNA gene n-Tr20 as a genetic culprit behind the neurodegeneration observed in mice lacking GTPBP2.
Remarkably, the tRNA’s activity is confined to the brain and other parts of the central nervous system, in both mice and humans. The tRNA encoded by n-Tr20 recognizes the triplet code, AGA (which specifies the amino acid arginine).
The n-Tr20 defect disrupts how proteins are made. Specifically, it causes the “factories” responsible for synthesizing proteins, called ribosomes, to stall when they encounter an AGA triplet.
Such stalling can be largely overcome, thanks to the work of a partner protein called GTPBP2. But when this partner is missing—as it is in the mutant mice that Ackerman and her colleagues studied—the stalling intensifies. This is thought to be a driving force behind the neurodegeneration seen in these mice.
(Source: jax.org)
Researchers find clue to stopping Alzheimer’s-like diseases
Tiny differences in mice that make them peculiarly resistant to a family of conditions that includes Alzheimer’s, Parkinson’s and Creutzfeldt-Jakob Disease may provide clues for treatments in humans.
Amyloid diseases are often incurable because drug designers cannot identify the events that cause them to start.
Professor Sheena Radford, Astbury Professor of Biophysics at the University of Leeds, said: “Amyloid diseases are associated with the build-up of fibrous plaques out of long strings of ‘misfolding’ proteins, but it is not clear what kicks the process off. That means the normal approach of designing a drug to destroy or disable the species that start the disease process does not work.
“We have to take a completely different tack: instead of targeting the cause of the disease, we need to disrupt the plaque building process.”
The University of Leeds-led team’s study, published in the journal Molecular Cell, looked to mice for a way forward.
“We already knew that mice were not prone to the build up of some of these plaques. This study, for the first time, observed the building happening and saw the differences between the mice proteins and their almost identical human equivalents,” Professor Radford said.
She added: “We mixed the mice and human proteins and found that the mice protein actually stopped the formation of the plaque-forming fibrils by the human protein.”
The research was conducted completely in the test-tube using human and mice beta-2 microglobulin proteins produced in the laboratory. Plaques made up of beta-2 microglobulin are associated with Dialysis Related Amyloidosis (DRA). Instead of being a neurodegenerative condition like Alzheimer’s or Parkinson’s, DRA primarily affects the joints of people on kidney dialysis.
The team observed differences in the formation of the plaque-forming fibrils in samples containing only mice protein, samples with only the human protein and samples containing mixtures of the two.
The lead researcher, Dr Theodoros Karamanos, said: “These two versions of the proteins are almost exactly the same, with very slight differences in structure, but the outcomes are completely different. If I put a misfolding-prone protein in the human sample, I see the formation of fibrils in two days in the right conditions. If I do the same in the mouse sample, I can leave it for weeks and there are no fibrils.
Dr Karamanos added: “The exciting thing is that if you mix the proteins—with only one mouse protein for every five human proteins—you see a significant disruption of the formation of fibrils.”
The study used Nuclear Magnetic Resonance spectroscopy to look at a molecular level at the interactions of the different proteins and identified tiny differences in the physical and chemical properties of the surfaces that made a great difference to whether plaques are formed.
The results showed that the mouse protein binds to the human protein more tightly than the human protein binds to its misfolded form. Interestingly, subtle differences in the driving forces of binding (i.e. the balance of hydrophobic and charge-charge interactions) in the binding interface govern the outcome of assembly.
Dr Karamanos said: “We can’t just load up a syringe and inject mouse protein into patients. But if we know the properties of the interface between the two proteins that are responsible for the inhibition effect, we can ask the chemists to design small molecule drugs which mimic what the mouse protein does to the human protein. That may be a key insight into how to stop the plaque building process.”
New Method Reveals Key Protein Interaction For Embryonic Stem Cell Differentiation
Proteins are responsible for the vast majority of the cellular functions that shape life, but like guests at a crowded dinner party, they interact transiently and in complex networks, making it difficult to determine which specific interactions are most important.
Now, researchers from the University of Chicago have pioneered a new technique to simplify the study of protein networks and identify the importance of individual protein interactions. By designing synthetic proteins that can only interact with a pre-determined partner, and introducing them into cells, the team revealed a key interaction that regulates the ability of embryonic stem cells to change into other cell types. They describe their findings June 5 in Molecular Cell.
“Our work suggests that the apparent complexity of protein networks is deceiving, and that a circuit involving a small number of proteins might control each cellular function,” said senior author Shohei Koide, PhD, professor of biochemistry & molecular biophysics at the University of Chicago.
For a cell to perform biological functions and respond to the environment, proteins must interact with one another in immensely complex networks, which when diagrammed can resemble a subway map out of a nightmare. These networks have traditionally been studied by removing a protein of interest through genetic engineering and observing whether the removal destroys the function of interest or not. However, this does not provide information on the importance of specific protein-to-protein interactions.
To approach this challenge, Koide and his team pioneered a new technique that they dub “directed network wiring.” Studying mouse embryonic stem cells, they removed Grb2, a protein essential to the ability of the stem cell to transform into other cell types, from the cells. The researchers then designed synthetic versions of Grb2 that could only interact with one protein from a pool of dozens that normal Grb2 is known to network with. The team then introduced these synthetic proteins back into the cell to see which specific interactions would restore the stem cell’s transformative abilities.
“The name, ‘directed network wiring,’ comes from the fact that we create minimalist networks,” Koide said. “We first remove all communication lines associated with a protein of interest and add back a single line. It is analysis by addition.”
Despite the complexity of the protein network associated with stem cell development, the team discovered that restoring only one interaction—between Grb2 and a protein known as Ptpn11/Shp2 phosphatase—was enough to allow stem cells to again change into other cell types.
“We were really surprised to find that consolidating many interactions down to a single particular connection for the protein was sufficient to support development of the cells to the next stage, which involves many complicated processes,” Koide said. “Our results show that signals travel discrete and simple routes in the cell.”
Koide and his team are now working on streamlining directed network wiring and applying it to other areas of study such as cancer. With the ability to dramatically simplify how scientists study protein interaction networks, they hope to open the door to new research areas and therapeutic approaches.
“We can now design synthetic proteins that are far more sophisticated than natural ones, and use such super-performance proteins toward advancing science and medicine,” he said.
Protein researchers closing in on the mystery of schizophrenia
Seven per cent of the adult population suffer from schizophrenia, and although scientists have tried for centuries to understand the disease, they still do not know what causes the disease or which physiological changes it causes in the body. Doctors cannot make the diagnosis by looking for specific physiological changes in the patient’s blood or tissue, but have to diagnose from behavioral symptoms.
In an attempt to find the physiological signature of schizophrenia, researchers from the University of Southern Denmark have conducted tests on rats, and they now believe that the signature lies in some specific, measurable proteins. Knowing these proteins and comparing their behaviour to proteins in the brains of not-schizophrenic people may make it possible to develop more effective drugs.
It is extremely difficult to study brain activity in schizophrenic people, which is why researchers often use animal models in their strive to understand the mysteries of the schizophrenic brain. Rat brains resemble human brains in so many ways that studying them makes sense if one wants to learn more about the human brain.
Schizophrenic symptoms in rats
The strong hallucinogenic drug phenocyclidine (PCP), also known as “angel’s dust”, provides a range of symptoms in people which are very similar to schizophrenia.
“When we give PCP to rats, the rats become valuable study objects for schizophrenia researchers,” explains Ole Nørregaard Jensen, professor and head of the Department of Biochemistry and Molecular Biology.
Along with Pawel Palmowski, Adelina Rogowska-Wrzesinska and others, he is the author of a scientific paper about the discovery, published in the international Journal of Proteome Research.
Among the symptoms and reactions that can be observed in both humans and rats are changes in movement and reduced cognitive functions such as impaired memory, attention and learning ability.
"Scientists have studied PCP rats for decades, but until now no one really knew what was going on in the rat brains at the molecular level. We now present what we believe to be the largest proteomics data set to date," says Ole Nørregaard Jensen.
PCP is absorbed very quickly by the brain, and it only stays in the brain for a few hours. Therefore, it was important for researchers to examine the rats’ brain cells soon after the rats were injected with the hallucinogenic drug.
"We could see changes in the proteins in the brain already after 15 minutes. And after 240 minutes, it was almost over," says Ole Nørregaard Jensen.
The University of Southern Denmark holds some of the world’s most advanced equipment for studying proteins, and Ole Nørregaard Jensen and his colleagues used the university’s so-called mass spectrometres for their protein studies.
352 proteins cause brain changes
"We found 2604 proteins, and in 352 of them, we saw changes that can be associated with the PCP injections. These 352 proteins will be extremely interesting to study in closer detail to see if they also alter in people with schizophrenia - and if that’s the case, it will of course be interesting to try to develop a drug that can prevent the protein changes that lead to schizophrenia," says Ole Nørregaard Jensen about the discovery and the work that now lies ahead.
The 352 proteins in rat brains responded immediately when the animals were exposed to PCP. Roughly speaking, the drug made proteins turn on or off when they should not turn on and off. This started a chain reaction of other disturbances in the molecular network around the proteins, such as changes in metabolism and calcium balance.
"These 352 proteins are what causes the rats to change their behaviour - and the events are probably comparable to the devastating changes in a schizophrenic brain," explains Ole Nørregaard Jensen.
The protocol for studying rat brain proteins with mass spectrometry, developed by Ole Nørregaard Jensen and his colleagues, is not limited to schizophrenia studies - it can also be used to explore other diseases.
The research was a collaboration between the University of Southern Denmark, the Danish Technological Institute and NeuroSearch A/S.
Details about the experiment
Twelve rats were used for the experiment. Six received an injection with the hallucinogenic drug PCP (10 mg/kg body weight), and six were injected with a saline solution to serve as controls. After 15 minutes, the first two animals in each group were killed and within less than two minutes, samples of their brains (temporal lobes) were taken and quickly frozen in liquid nitrogen.
After 30 and 240 minutes, respectively, the same was done to other rats. All experiments were conducted in accordance with Danish and EU guides for the handling of laboratory animals. The collected tissue samples were then subjected to various mass spectrometric protein analyses. The analyses revealed differences in the phosphorylation of proteins indicating which proteins had been affected by the drug PCP.
Interpretation of the complex protein data set suggest that PCP affects a number of processes in brain cells and leads to changes in calcium balance in the brain cells, changes in the transport of substances into and out of cells, changes in cell metabolism and changes in the structure of the cell’s internal skeleton, the cytoskeleton.
Watching how the brain works
University of Miami researchers develop a method to visualize protein interactions in a living organism’s brain
There are more than a trillion cells called neurons that form a labyrinth of connections in our brains. Each of these neurons contains millions of proteins that perform different functions. Exactly how individual proteins interact to form the complex networks of the brain still remains as a mystery that is just beginning to unravel.
For the first time, a group of scientists has been able to observe intact interactions between proteins, directly in the brain of a live animal. The new live imaging approach was developed by a team of researchers at the University of Miami (UM).
(Image caption: Photonic resonance energy transfer described by Förster, or FRET, occurs when two small proteins come within a very small distance of each other — eight nanometers or less. The fluorescence lifetime of the donor molecule will become shorter — from 3 nanosecond to, perhaps, 2.5 nanoseconds. We then interpret this as evidence that the two proteins of interest are physically interacting with each other — a molecular signaling event. Credit: Akira Chiba/University of Miami)
"Our ultimate goal is to create the systematic survey of protein interactions in the brain," says Akira Chiba, professor of Biology in the College of Arts and Sciences at UM and lead investigator of the project. "Now that the genome project is complete, the next step is to understand what the proteins coded by our genes do in our body."
The new technique will allow scientists to visualize the interactions of proteins in the brain of an animal, along different points throughout its development, explains Chiba, who likens protein interactions to the way organisms associate with each other.
"We know that proteins are one billionth of a human in size. Nevertheless, proteins make networks and interact with each other, like social networking humans do," Chiba says. "The scale is very different, but it’s the same behavior happening among the basic units of a given network."
The researchers chose embryos of the fruit fly (Drosophila melanogaster) as an ideal model for the study. Because of its compact and transparent body, it is possible to visualize processes inside the Drosophila cells using a fluorescence lifetime imaging microscope (FLIM). The results of the observations are applicable to other animal brains, including the human brain.
The Drosophila embryos in the study contained a pair of fluorescent labeled proteins: a developmentally essential and ubiquitously present protein called Rho GTPase Cdc42 (cell division control protein 42), labeled with green fluorescent tag and its alleged signaling partner, the regulatory protein WASp (Wiskot-Aldrich Syndrome protein), labeled with red fluorescent tag. Together, these specialized proteins are believed to help neurons grow during brain development. The proteins were selected because the same (homolog) proteins exist in the human brain as well.
Previous methods required chemical or physical treatments that most likely disturb or even kill the cells. That made it impossible to study the protein interactions in their natural environment.
(Image caption: FRET (Förster resonance energy transfer) between the two interacting protein partners occurs, Cdc42 and WASp, within neurons, during the time and space that coincides with the formation of new synapses in the brain of the baby insect. Synapses connect individual neurons in the brain. Credit: Akira Chiba / University of Miami)
The current study addresses these challenges by using the occurrence of a phenomenon called Förster resonance energy transfer, or FRET. It occurs when two small proteins come within a very small distance of each other, (eight nanometers). The event is interpreted as the time and place where the particular protein interaction occurs within the living animal.
(Image caption: Proteins are one billionth of a human in size. Nevertheless, proteins make networks and interact with each other, like social networking humans do,” says Akira Chiba, professor of Biology in the College of Arts and Sciences at the University of Miami. “The scale is very different, but it’s the same behavior happening among the basic units of a given network.” Credit: Akira Chiba / University of Miami)
The findings show that FRET between the two interacting protein partners occurs within neurons, during the time and space that coincides with the formation of new synapses in the brain of the baby insect. Synapses connect individual neurons in the brain.
"Previous studies have demonstrated that Cdc42 and WASp can directly bind to each other in a test-tube, but this is the first direct demonstration that these two proteins are interacting within the brain," Chiba says.
(Source: eurekalert.org)
Study in Fruitflies Strengthens Connection Among Protein Misfolding, Sleep Loss, and Age
Pulling an “all-nighter” before a big test is practically a rite of passage in college. Usually, it’s no problem: You stay up all night, take the test, and then crash, rapidly catching up on lost sleep. But as we age, sleep patterns change, and our ability to recoup lost sleep diminishes.
Researchers at the Perelman School of Medicine, University of Pennsylvania, have been studying the molecular mechanisms underpinning sleep. Now they report that the pathways of aging and sleep intersect at the circuitry of a cellular stress response pathway, and that by tinkering with those connections, it may be possible to alter sleep patterns in the aged for the better – at least in fruit flies.
Nirinjini Naidoo, PhD, associate professor in the Center for Sleep and Circadian Neurobiology and the Division of Sleep Medicine, led the study with postdoctoral fellow Marishka Brown, PhD, which was published online before print in the journal Neurobiology of Aging.
Increasing age is well known to disrupt sleep patterns in all sorts of ways. Elderly people sleep at night less than their younger counterparts and also sleep less well. Older individuals also tend to nap more during the day. Naidoo’s lab previously reported that aging is associated with increasing levels of protein unfolding, a hallmark of cellular stress called the “unfolded protein response.”
Protein misfolding is also a characteristic of several age-related neurodegenerative diseases, such as Alzheimer’s and Parkinson’s diseases, and as it turns out, also associated with sleep deprivation. Naidoo and her team wanted to know if rescuing proper protein folding behavior might counter some of the detrimental sleep patterns in elderly individuals.
Using a video monitoring system to compare the sleep habits of “young” (9–12 days old) and “aged” (8 weeks old) fruit flies, they found that aged flies took longer to recover from sleep deprivation, slept less overall, and had their sleep more frequently interrupted compared to younger control animals. However, adding a molecule that promotes proper protein folding – a molecular “chaperone” called PBA — mitigated many of those effects, effectively giving the flies a more youthful sleep pattern. PBA (sodium 4-phenylbutyrate) is a compound currently used to treat such protein-misfolding-based diseases as Parkinson’s and cystic fibrosis.
The team also asked the converse question: Can protein misfolding induce altered sleep patterns in young animals. Another drug, tunicamycin, induces protein misfolding and stress, and when the team fed it to young flies, their sleep patterns shifted towards those of aged flies, with less sleep overall, more interrupted sleep at night, and longer recovery from sleep deprivation.
Molecular analysis of sleep-deprived and PBA-treated flies suggested that PBA acts through the unfolded protein response. PBA, Naidoo says, had two effects on aged flies: it “consolidated” baseline sleep, increasing the total amount of time slept and shifted recovery sleep, after sleep deprivation, to look more like that of a young fly.
“It rescued the sleep patterns in the older flies,” she explains.
These results, Naidoo says, suggest three key messages. First, sleep loss leads to protein misfolding and cellular stress, and as we age, our ability to recover from that stress decreases. Second, aging and sleep apparently form a kind of negative “chicken-and-egg” feedback loop, in which sleep loss or sleep fragmentation lead to cellular stress, followed by neuronal dysfunction, and finally even poorer-quality sleep.
Sleep recharges neuronal batteries, Naidoo explains, and if a person is forced to stay awake, those batteries run down. Dwindling physiological resources must be devoted to the most critical cell functions, which do not necessarily include protein homeostasis. “Staying awake has a cost, and one of those costs is problems with protein folding.”
Finally, and most importantly, she says these results suggest — assuming they can be replicated in mice and humans – that it may be possible using drugs such as PBA to “fix sleep” in aged or mutant animals.
“People know that sleep deteriorates with aging,” Naidoo says, “But this might be able to be stopped or reversed with molecular chaperones.” Her team is now looking to determine if a similar situation exists in mammals and if better sleep translates into longer lifespan.
(Source: uphs.upenn.edu)
Surprising culprit found in cell recycling defect
To remain healthy, the body’s cells must properly manage their waste recycling centers. Problems with these compartments, known as lysosomes, lead to a number of debilitating and sometimes lethal conditions.
Reporting in the Proceedings of the National Academy of Sciences (PNAS), researchers at Washington University School of Medicine in St. Louis have identified an unusual cause of the lysosomal storage disorder called mucolipidosis III, at least in a subset of patients. This rare disorder causes skeletal and heart abnormalities and can result in a shortened lifespan. But unlike most genetic diseases that involve dysfunctional or missing proteins, the culprit is a normal protein that ends up in the wrong place.
Image caption: In normal cells, phosphotransferase (green) is shown overlapping with the Golgi apparatus (red), which indicates that phosphotransferase is located in the Golgi, where it should be (Credit: Eline van Meel, PhD)
“There is a lot of interest and study about how cells distribute proteins to the right parts of the cell,” said senior author Stuart A. Kornfeld, MD, PhD, the David C. and Betty Farrell Professor of Medicine. “Our study has identified one of the few examples of a genetic disease caused by the misplacement of a protein. The protein functions just fine. It just doesn’t stay in the right place.”
The right place, in this case, is the Golgi apparatus, the cell’s protein packaging center. The protein in question – phosphotransferase – normally resides in the Golgi, where its job is to attach address labels to proteins bound for the lysosome. There are 60 such lysosomal proteins, and all of them must be properly labeled if they are to end up in a lysosome, where they recycle waste.
Image caption: In mutant cells, the protein phosphotransferase (green) is spread beyond the Golgi (red). Outside the Golgi, this wayward phosphotransferase is no longer able to perform its job of properly addressing enzymes bound for the lysosome (Credit: Eline van Meel, PhD)
Kornfeld and his colleagues, including first author Eline van Meel, PhD, postdoctoral research associate, showed that the phosphotransferase protein responsible for adding the address label starts out in the Golgi as it should, but seems to lack the signal to keep it there.
“Under normal circumstances, the phosphotransferase moves up through the Golgi, but then it’s recaptured and sent back,” Kornfeld said. “Our study shows that the mutant phosphotransferase moves up but is not recaptured. Ironically, the phosphotransferase that escapes the Golgi ends up in the lysosomes, where it is degraded.”
Because phosphotransferase gradually wanders away from the Golgi, a low level of lysosomal enzymes end up being properly addressed, but at perhaps 20 percent of the normal amount.
“In many lysosomal storage disorders, such as Tay-Sachs or Gaucher’s disease, only one out of the 60 enzymes is missing from the lysosome,” Kornfeld said. “But the mislocalization of phosphotranferase causes the misdirection of all 60 lysosomal enzymes.”
While the errant phosphotransferase ends up being degraded in the lysosome, the resulting misdirected lysosomal proteins end up in the bloodstream. As a result, children with this disorder have lysosomal proteins in their blood at levels 10 to 20 times higher than normal. But because some get to the lysosome at a low level, people with mucolipidosis III don’t have the most severe form of the disease.
“Type III patients live into adulthood, but they’re very impaired,” said Kornfeld. “They have joint and heart problems and have trouble walking. In the most severe form, type II, there is zero activity of phosphotransferase. None of the 60 enzymes are properly tagged, so these patients’ lysosomes are empty. Children with type II usually die by age 10.”
Having implicated wayward phosphotransferase in this lysosomal storage disorder, Kornfeld and his colleagues are investigating what goes wrong that allows it to escape the Golgi.
“We think there must be some protein in the cell that recognizes phosphotransferase when it gets to the end of the Golgi, binds it and takes it back,” said Kornfeld. “Now we’re trying to understand how that works.”
(Source: news.wustl.edu)
Molecular biology mystery unravelled
The nature of the machinery responsible for the entry of proteins into cell membranes has been unravelled by scientists, who hope the breakthrough could ultimately be exploited for the design of new anti-bacterial drugs.
Groups of researchers from the University of Bristol and the European Molecular Biology Laboratory (EMBL) used new genetic engineering technologies to reconstruct and isolate the cell’s protein trafficking machinery. Its analysis has shed new light on a process which had previously been a mystery for molecular biologists.
The findings, published this week in the Proceedings of the National Academy of Sciences (PNAS), could also have applications for synthetic biology - an emerging field of science and technology, for the development of novel membrane proteins with useful activities.
Proteins are the building blocks of all life and are essential for the growth of cells and tissue repair. The proteins in the membrane typically help the cell interact with its environment and conserve energy.
Researchers were able to identify the ‘holo-translocon’ as the machinery which inserts proteins into the membrane. It is a large membrane protein complex and is uniquely capable of both protein-secretion and membrane-insertion.
Professor Ian Collinson, from the School of Biochemistry at Bristol University, said: “These findings are important as they address outstanding questions in one of the central pillars of biology, a process essential in every cell in every organism. Having unravelled how this vital holo-translocon works, we’re now in a position to look at its components to see if they can help in the design or screening for new anti-bacterial drugs.”
Scientists discover two proteins that control chandelier cell architecture
Chandelier cells are neurons that use their unique shape to act like master circuit breakers in the brain’s cerebral cortex. These cells have dozens, often hundreds, of branching axonal projections – output channels from the cell body of the neuron – that lend the full structure of a chandelier-like appearance. Each of those projections extends to a nearby excitatory neuron. The unique structure allows just one inhibitory chandelier cell to block or modify the output of literally hundreds of other cells at one time.
Without such large-scale inhibition, some circuits in the brain would seize up, as occurs in epilepsy. Abnormal chandelier cell function also has been implicated in schizophrenia. Yet after nearly 40 years of research, little is known about how these important inhibitory neurons develop and function.
In work published today in Cell Reports, a team led by CSHL Professor Linda Van Aelst identifies two proteins that control the structure of chandelier cells, and offers insight into how they are regulated.
To study the architecture of chandelier cells, Van Aelst and colleagues first had to find a way to visualize them. Generally, scientists try to find a unique marker, a sort of molecular signature, to distinguish one type of neuron from the many others in the brain. But no markers are known for chandelier cells. So Van Aelst and Yilin Tai, Ph.D., lead author on the study, developed a way to label chandelier cells within the mouse brain.
Using this new method, the team found two proteins, DOCK7 and ErbB4, whose activity is essential in processes that give chandelier cells their striking shape. When the function of these proteins is disrupted, chandelier cells have fewer, more disorganized, axonal projections. Van Aelst and colleagues used a series of biochemical experiments to explore the relationship between the two proteins. They found that DOCK7 activates ErbB4 through a previously unknown mechanism; this activation must occur if chandelier cells are to develop their characteristic architecture.
Moving forward, Van Aelst says she is interested in exploring the relationship between structure and function of chandelier cells. “We envisage that morphological changes are likely to impact the function of chandelier cells, and consequently, alter the activity of cortical networks. We believe irregularities in these networks contribute to the cognitive abnormalities characteristic of schizophrenia and epilepsy. As we move forward, therefore, we hope that our findings will improve our understanding of these devastating neurological disorders.”