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  • richardmitnick 5:18 pm on August 13, 2016 Permalink | Reply
    Tags: , , , , Protein Studies,   

    From Rosetta@home: “Designed Protein Containers Push Bioengineering Boundaries” 



    Rosetta@home has posted in their forum a new (July 21, 2016) article, Designed Protein Containers Push Bioengineering Boundaries
    from U Washington’s Institute for Protein Design which I highly recommend for anyone interested in Protein Studies.


    This forum article cites Designed Protein Containers Push Bioengineering Boundariess which goes on to cite Icosahedral protein nanocage – new paper and podcast published in Nature, and “Accurate design of megadalton-scale multi-component icosahedral protein complexes”, published in Science.

    Of this second paper, they write, “In this paper, former Baker lab graduate student Jacob Bale, Ph.D. and collaborators describe the computational design and experimental characterization of ten two-component protein complexes that self-assemble into nanocages with atomic-level accuracy. These nanocages are the largest designed proteins to date with molecular weights of 1.8-2.8 megadaltons and diameters comparable to small viral capsids. The structures have been confirmed by X-ray crystallography (see figure). The advantage of a multi-component protein complex is the ability to control assembly by mixing individually prepared subunits. The authors show that in vitro mixing of the designed subunits occurs rapidly and enables controlled packaging of negatively charged GFP by introducing positive charges on the interior surfaces of the two copmonents.

    The ability to design, with atomic-level precision, these large protein nanostructures that can encapsulate biologically relevant cargo and that can be genetically modified with various functionalities opens up exciting new opportunities for targeted drug delivery and vaccine design.”

    Also referenced in the forum is an article in Science, Jul. 21, 2016 by Robert F. Service This protein designer aims to revolutionize medicines and materials, about Dr David Baker.

    From this Science article, David Baker shows off models of some of the unnatural proteins his team has designed and made.© Rich Frishman

    included also is this video from Science.

    See the full article here.

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    Rosetta@home needs your help to determine the 3-dimensional shapes of proteins in research that may ultimately lead to finding cures for some major human diseases. By running the Rosetta program on your computer while you don’t need it you will help us speed up and extend our research in ways we couldn’t possibly attempt without your help. You will also be helping our efforts at designing new proteins to fight diseases such as HIV, Malaria, Cancer, and Alzheimer’s (See our Disease Related Research for more information). Please join us in our efforts! Rosetta@home is not for profit.

    About Rosetta

    One of the major goals of Rosetta is to predict the shapes that proteins fold up into in nature. Proteins are linear polymer molecules made up of amino acid monomers and are often refered to as “chains.” Amino acids can be considered as the “links” in a protein “chain”. Here is a simple analogy. When considering a metal chain, it can have many different shapes depending on the forces exerted upon it. For example, if you pull its ends, the chain will extend to a straight line and if you drop it on the floor, it will take on a unique shape. Unlike metal chains that are made of identical links, proteins are made of 20 different amino acids that each have their own unique properties (different shapes, and attractive and repulsive forces, for example), and in combination, the amino acids exert forces on the chain to make it take on a specific shape, which we call a “fold.” The order in which the amino acids are linked determines the protein’s fold. There are many kinds of proteins that vary in the number and order of their amino acids.

    To predict the shape that a particular protein adopts in nature, what we are really trying to do is find the fold with the lowest energy. The energy is determined by a number of factors. For example, some amino acids are attracted to each other so when they are close in space, their interaction provides a favorable contribution to the energy. Rosetta’s strategy for finding low energy shapes looks like this:

    Start with a fully unfolded chain (like a metal chain with its ends pulled).
    Move a part of the chain to create a new shape.
    Calculate the energy of the new shape.
    Accept or reject the move depending on the change in energy.
    Repeat 2 through 4 until every part of the chain has been moved a lot of times.

    We call this a trajectory. The end result of a trajectory is a predicted structure. Rosetta keeps track of the lowest energy shape found in each trajectory. Each trajectory is unique, because the attempted moves are determined by a random number. They do not always find the same low energy shape because there are so many possibilities.

    A trajectory may consist of two stages. The first stage uses a simplified representation of amino acids which allows us to try many different possible shapes rapidly. This stage is regarded as a low resolution search and on the screen saver you will see the protein chain jumping around a lot. In the second stage, Rosetta uses a full representation of amino acids. This stage is refered to as “relaxation.” Instead of moving around a lot, the protein tries smaller changes in an attempt to move the amino acids to their correct arrangment. This stage is regarded as a high resolution search and on the screen saver, you will see the protein chain jiggle around a little. Rosetta can do the first stage in a few minutes on a modern computer. The second stage takes longer because of the increased complexity when considering the full representation (all atoms) of amino acids.

    Your computer typically generates 5-20 of these trajectories (per work unit) and then sends us back the lowest energy shape seen in each one. We then look at all of the low energy shapes, generated by all of your computers, to find the very lowest ones. This becomes our prediction for the fold of that protein.

    To join this project, download and install the BOINC software on which it runs. Then attach to the project. While you are at BOINC, look at some of the other projects to see what else might be of interest to you.

    Rosetta screensaver



  • richardmitnick 12:20 pm on August 13, 2016 Permalink | Reply
    Tags: , Discrete Molecular Imaging (DMI) technology, DNA-PAINT technologies, From super to ultra-resolution microscopy, Protein Studies,   

    From Wyss: “From super to ultra-resolution microscopy” 

    Harvard bloc tiny
    Wyss Institute bloc
    Wyss Institute

    Jul 5, 2016 [Just showed up in social media.]

    Wyss Institute for Biologically Inspired Engineering at Harvard University
    Benjamin Boettner, benjamin.boettner@wyss.harvard.edu, +1 917-913-8051


    Wyss Institute for Biologically Inspired Engineering at Harvard University
    Seth Kroll, seth.kroll@wyss.harvard.edu, +1 617-432-7758

    The image shows how the Discrete Molecular Imaging (DMI) technology visualizes densely packed individual targets that are just 5 nanometer apart from each other in DNA origami structures (see schematics on the left). The image on the top right shows a DMI-generated super-resolution image of a clear pattern of individual signals. In the image on the bottom right, three different target species within the same origami structure have been visualized using Exchange-PAINT-enhanced DMI method. Credit: Wyss Institute at Harvard University.

    Proteins mostly do not work in isolation but rather make up larger complexes like the molecular machines that enable cells to communicate with each other, move cargo around in their interiors or replicate their DNA. Our ability to observe and track each individual protein within these machines is crucial to our ultimate understanding of these processes. Yet, the advent of super-resolution microscopy that has allowed researchers to start visualizing closely positioned molecules or molecular complexes with 10-20 nanometer resolution is not powerful enough to distinguish individual molecular features within those densely packed complexes.

    A team at Harvard’s Wyss Institute for Biologically Inspired Engineering led by Core Faculty member Peng Yin, Ph.D., has, for the first time, been able to tell apart features distanced only 5 nanometers from each other in a densely packed, single molecular structure and to achieve the so far highest resolution in optical microscopy. Reported on July 4 in a study in Nature Nanotechnology, the technology, also called “discrete molecular imaging” (DMI), enhances the team’s DNA nanotechnology-powered super-resolution microscopy platform with an integrated set of new imaging methods

    Last year, the opportunity to enable researchers with inexpensive super-resolution microscopy using DNA-PAINT-based technologies led the Wyss Institute to launch its spin-off Ultivue Inc.

    “The ultra-high resolution of DMI advances the DNA-PAINT platform one step further towards the vision of providing the ultimate view of biology. With this new power of resolution and the ability to focus on individual molecular features, DMI complements current structural biology methods like X-ray crystallography and cryo-electron microscopy. It opens up a way for researchers to study molecular conformations and heterogeneities in single multi-component complexes, and provides an easy, fast and multiplexed method for the structural analysis of many samples in parallel” said Peng Yin, who is also Professor of Systems Biology at Harvard Medical School.

    DNA-PAINT technologies, developed by Yin and his team are based on the transient binding of two complementary short DNA strands, one being attached to the molecular target that the researchers aim to visualize and the other attached to a fluorescent dye. Repeated cycles of binding and unbinding create a very defined blinking behavior of the dye at the target site, which is highly programmable by the choice of DNA strands and has now been further exploited by the team’s current work to achieve ultra-high resolution imaging.

    “By further harnessing key aspects underlying the blinking conditions in our DNA-PAINT-based technologies and developing a novel method that compensates for tiny but extremely disruptive movements of the microscope stage that carries the samples, we managed to additionally boost the potential beyond what has been possible so far in super-resolution microscopy,” said Mingjie Dai, who is the study’s first author and a Graduate Student working with Yin.

    In addition, the study was co-authored by Ralf Jungmann, Ph.D., a former Postdoctoral Fellow on Yin’s team and now a Group Leader at the Max Planck Institute of Biochemistry at the Ludwig Maximilian University in Munich, Germany.

    In this image the “Wyss!” name has been visualized in a DNA origami display with the so-far highest resolution possible in optical imaging using Discrete Molecular Imaging (DMI) technology. Credit: Wyss Institute at Harvard University.

    The Wyss Institute’s scientists have benchmarked the ultra-high resolution of DMI using synthetic DNA nanostructures. Next, the researchers plan to apply the technology to actual biological complexes such as the protein complex that duplicates DNA in dividing cells or cell surface receptors binding their ligands.

    “Peng Yin and his team have yet again broken through barriers never before possible by leveraging the power of programmable DNA, not for information storage, but create nanoscale ‘molecular instruments’ that carry out defined tasks and readout what they analyze. This new advancement to their DNA-powered super-resolution imaging platform is an amazing feat that has the potential to uncover the inner workings of cells at the single molecule level using conventional microscopes that are available in common biology laboratories,” said Donald Ingber, M.D., Ph.D., who is the Judah Folkman Professor of Vascular Biology at Harvard Medical School and the Vascular Biology Program at Boston Children’s Hospital, and also Professor of Bioengineering at the Harvard John A. Paulson School of Engineering and Applied Sciences.

    See the full article here .

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    Wyss Institute campus

    The Wyss (pronounced “Veese”) Institute for Biologically Inspired Engineering uses Nature’s design principles to develop bioinspired materials and devices that will transform medicine and create a more sustainable world.

    Working as an alliance among Harvard’s Schools of Medicine, Engineering, and Arts & Sciences, and in partnership with Beth Israel Deaconess Medical Center, Boston Children’s Hospital, Brigham and Women’s Hospital, Dana Farber Cancer Institute, Massachusetts General Hospital, the University of Massachusetts Medical School, Spaulding Rehabilitation Hospital, Tufts University, and Boston University, the Institute crosses disciplinary and institutional barriers to engage in high-risk research that leads to transformative technological breakthroughs.

  • richardmitnick 2:49 pm on July 31, 2016 Permalink | Reply
    Tags: , , Protein Studies,   

    From Technion: “Lethal Sequences” 

    Technion bloc


    No writer credit found

    Underrepresented sequences (URSs) inhibit protein translation and can be lethal. Top, The genetic information is transferred to the protein synthesis machine, the ribosome, by messenger RNA. Amino acids (colored circles) are added one by one and the newly synthesized protein is pushed out of the ribosome. On the left is a normal sequence. On the right is a sequence with the strong URS – CMYW, which slows translation and prevents ribosome recycling. Middle, left, proteins fold and assemble correctly. On the right, fewer full-length proteins are produced and truncated proteins are also produced due to the URS effect on the ribosome. Bottom, E. coli cells grown on plates in the absence (-) or presence (+) of the protein translation signal molecule IPTG. On the left, cells grow normally with or without IPTG. On the right, cells grow normally when the URS containing protein is not synthesized (no IPTG). When IPTG is added, the URS containing protein synthesis is initiated (+), ribosomes are inhibited, and fewer colonies of the bacteria grow. No image credit

    Prof. Noam Adir, Dean of the Schulich Faculty of Chemistry.

    A study from the lab of Prof. Noam Adir of the Schulich Faculty of Chemistry at Technion – Israel Institute of Technology: natural evolutionary processes prevent the presence of dangerous and potentially lethal molecular interactions by avoiding the presence of specific protein sequences in microorganisms. They found these sequences by a novel method – looking for what is missing in biological data sets. The group then experimentally showed that when these sequences are present in a protein, bacterial growth is indeed inhibited. The study was recently published in the Proceedings of the National Academy of Sciences, USA.

    Evolution is an ongoing process, whereby those individuals of species that are the most fit for their environment have more offspring and thus out-compete less fit individuals. The individual’s fitness is a product of the quality of its cellular biochemistry, made possible by the thousands of enzymes that allow its physiology to perform all of the necessary chemical reactions that allow the cell to live. Deficiency in these molecular functions can lead to disease, loss of adaptability to environmental changes, or weakness against other organisms. The molecular machines that make life possible are large polymers made up of linear sequences of building blocks that contain different chemical functions: proteins, DNA, and RNA. Biological variety is a result of the evolutionary changes in these polymers, first and foremost the result of the astronomic number of possible permutations in the order of the 20 naturally occurring amino acid (AA) residues that are the building blocks of proteins. There are 8,000 possible sequences of three AAs, 160,000 sequences of four AAs, over 3 million sequences of five AAs and so on. Since proteins can contain between hundreds to thousands of AAs, the possibilities are endless.

    The millions of different protein sequences found in all organisms determine the three-dimensional structures that give proteins the ability to function correctly. Proteins in cells can work alone or associate correctly with other cellular components, while avoiding incorrect and harmful associations with other components. Changes to the sequences naturally occur due to mutations (single site, or larger changes due to more dramatic sequence shuffling) of an organism’s DNA – the genetic material. Changes due to mutations can lead to new positive characteristics, or they may have negative consequences to the organism’s viability. A mutation that has a negative effect may prevent the organism from competing with other organisms in its environment, eventually leading to its demise. One could predict that over time, evolutionary pressure would work against the presence of organisms containing these internally lethal sequences and they would disappear.

    Over the past few years, there has been a world-wide effort to obtain the entire DNA sequences (the entire genomes) of many organisms. These data have given us the ability to predict all of the possible protein sequences (the proteome) that might exist in organisms as simple as bacteria or as complicated as humans. Prof. Adir and his students, Dr. Sharon Penias-Navon and Ms. Tali Schwartzman, hypothesized that the huge amount of data made available by modern genomics would allow them to look for short sequences that occur less often than expected or are completely missing in the organism’s proteome. They developed a computer program that searched the many existing data sets to identify short sequences that are underrepresented (URSs). While they found that most of the sequences of three or four AAs indeed do exist at their expected frequency in the proteins of different organisms, URSs do exist. They used the program to search for URSs in the proteomes of many different organisms (especially pathogenic microorganisms) and found that different organisms have different URSs. Adir and Penias-Navon wanted to prove that these URSs are indeed harmful, and they hypothesized that protein synthesis (translation) by the ribosome is the function that URSs might harm.

    They embedded bacterial URSs (identified in the proteome of the gut bacterium E. coli) comprised of three or four AAs in a normal protein sequence, and showed that no matter where they put the URS, protein translation was inhibited. They showed that these same E. coli URSs had no effect on protein translation in human cells, showing that the effect is species specific. They further showed that one four-AA URS was powerful enough to inhibit translation completely to the point where the growth of the bacterial cells was significantly reduced: these are indeed lethal sequences. Adir and Navon suggested that URSs could be used as highly specific anti-microbial agents, and a patent, together with the Technion, was submitted.

    In order to obtain even more precise molecular details on the action of the URS, they initiated a collaboration with Prof. Joseph Puglisi and his student Dr. Guy Kornberg of Stanford University, who are experts in following protein translation in single ribosomes, thereby obtaining direct information on the translation reaction mechanism. Using these single molecule methods, the inhibitory effect of the existence of a URS on translation was confirmed. Their methods enabled a precise determination of the site of inhibition. They found that as soon as the URS AAs enter the entrance to the ribosomal nascent protein exit tunnel, translation is inhibited.

    See the full article here .

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    Technion Campus

    A science and technology research university, among the world’s top ten,
    dedicated to the creation of knowledge and the development of human capital and leadership,
    for the advancement of the State of Israel and all humanity.

  • richardmitnick 1:26 pm on July 30, 2016 Permalink | Reply
    Tags: Alzheimer’s Parkinson’s and Huntington’s, , , Need for new approaches, Protein Studies   

    From SA: “Could Trashing Junk Proteins Quash Alzheimer’s, Parkinson’s, ALS and Huntington’s?” 

    Scientific American

    Scientific American

    July 26, 2016
    Esther Landhuis

    Credit: MaryLB/Getty Images

    Although clutter can be a nuisance, it does not typically pose a health threat—unless you’re an aging neuron. As brain cells get older, some proteins within and around the cell misfold. They twist into the wrong shape, unable to do their routine job. Then they glom together to form menacing clumps. If left to accumulate, this “junk” can overwhelm nerve cells’ quality control systems, triggering incurable brain disorders such as Alzheimer’s, Parkinson’s and Huntington’s.

    So whereas these diseases produce distinct symptoms and billions of dollars have been spent researching potential drugs that target their unique molecular culprits, some scientists are placing their bets on cross-cutting approaches that might work across multiple disorders. Rather than going after proteins such as amyloid beta for Alzheimer’s or alpha-synuclein for Parkinson’s, one researcher has set on a different approach: “I settled on the idea that perhaps we should just get rid of as many abnormally folded, nasty-looking proteins as possible,” says Karen Duff, a neuroscientist at Columbia University. Strategies that boost the cell’s quality control programs, rather than disarm specific pathologic proteins, have looked promising in lab animals that serve as models for human neurodegenerative disorders including Alzheimer’s, Parkinson’s, Huntington’s, amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. Several molecules have entered human testing. It is still a long road to approved therapies but a growing body of basic research is fueling a search for drugs that interact with cellular cleanup processes to provide one-size-fits-all approaches for treating a megaclass of brain disorders.

    Cells have two main systems for clearing excess or damaged proteins. Most of the quick cleanup occurs in cylindrical waste-disposal units called proteasomes, which chop unneeded material into smaller bits that can be recycled into new proteins. Proteasomes also keep cellular trash under control by breaking up misfolded proteins. When these scoundrels band together and the gang gets too large, the cell calls on a second degradation process—autophagy. Derived from Greek terms meaning “self-eating,” the autophagy system sends protein aggregates and malfunctioning cellular components into acidic compartments called lysosomes, where enzymes chew them up.

    In the early stages of Alzheimer’s and other so-called proteinopathies—disorders caused by a malformed protein- particular proteins adopt the wrong shape and join with similar misfits to form conglomerates that pile up in the brain. For a while the cell’s cleanup crews keep the junk at bay, sending protein aggregates for degradation as soon as they start to pile up. Rates of autophagy, however, slow with age. Over time, growing heaps of rogue proteins overwhelm the system and the cell gets sick and dies—or, at least, that has been the conventional thinking.

    But the problem goes much deeper; it is not simply that freak proteins aggregate and clog the brain. Scientists are discovering that many of the proteins that twist out of shape normally carry out important jobs in the very disposal systems that are supposed to help cells get rid of them, says neurobiologist Ralph Nixon of New York University. The problem has been traced down to the level of specific genes. Some disease mutations turn regular proteins awry by making them fold into the wrong shape. Misfolded proteins often misbehave, which can muck up the cell’s cleaning system and make the organism more susceptible to any number of proteinopathies.

    One famous misfit is presenilin-1. This protein is part of the enzymatic engine that churns out amyloid beta—a key molecular culprit in Alzheimer’s disease—by snipping it out of a larger precursor protein called APP. Glitches in the presenilin-1 gene can cause the rare inherited form of Alzheimer’s that strikes at a younger age. Apart from amyloid, though, presenilin-1 has a critical, beneficial function. Working with Ana Maria Cuervo, a professor at Albert Einstein College of Medicine, Nixon and co-workers found that presenilin-1 helps control the acidity of lysosomes. Neurons with abnormal presenilin-1 clear waste poorly and accumulate harmful protein aggregates. More recently Nixon’s team discovered that APP hampers waste disposal systems in nerve clusters that falter and trigger memory decline in the early stages of Alzheimer’s. Along with the earlier study on presenilin-1, these findings highlight the potential for therapies that target protein degradation pathways to help cells deal with buildup of harmful pathologic molecules.

    The need for new approaches comes into stark relief as the Alzheimer’s Association begins its annual conference this week in Toronto. Alzheimer’s is a disease for which there are still no treatments that fundamentally alter the course of the disease, as trial after trial of drug candidates have ended in failure.

    Research in Parkinson’s and Huntington’s has uncovered additional examples of disease proteins that, when mutated, stymie protein clearance pathways in neurons. Cuervo’s group found that a mutant protein associated with a heritable version of Parkinson’s gums up lysosomal channels. That leads the protein alpha-synuclein to build up and form toxic clumps in brain areas that control motor function. Last year Cuervo collaborated with Sheng Zhang, a professor at The University of Texas Health Science Center at Houston on experiments showing that huntingtin—the Huntington’s disease protein—helps the cell’s autophagy system identify what it should eliminate. Researchers had focused so much on huntingtin’s toxicity that it was surprising to discover this molecule has a regular day job, Cuervo notes. Rather than being the bad guy that ties up the cleaning system, huntingtin “also happens to be part of the cleaning crew,” she says. “That changes the way we have to approach the problem.”

    In recent years Cuervo and co-workers have discovered that giving the cleaning crew a little boost can go a long way. The tough part is figuring out which parts to tweak. “There are many ways to clean the house—a vacuum, a broom,” Cuervo says. Similarly, in cells “there are many ways to bring proteins to the lysosome.” Lysosomes are the final destination for misshapen proteins that get degraded in the autophagy system. One type of autophagy traps chunks of cellular material into “bags” that fuse with lysosomes. A different branch of autophagy—a specialty of Cuervo’s lab—involves molecular chaperones that escort misbehaving proteins through special tunnels into the lysosome.

    Several years ago her team designed a chemical that helps cells produce more tunnel components. When tested in cultured cells, the compound specifically activated chaperone-mediated autophagy without touching other pathways. More importantly, in recent studies yet to be published the chemical appears to improve anxiety, depression and memory in mice that mimic some features of Alzheimer’s. The researchers also plan to test the compound in mice modeling Parkinson’s disease.

    Tampering with chaperones can be tricky, though. Sometimes chaperones hang onto a bad protein for too long. The major chaperones are not very discriminating. They recognize all unfolded proteins—“anything that’s disordered or stretched out”—and cover those exposed, sticky regions to prevent clumping, says neuroscientist Chad Dickey of the University of South Florida. So it is easy for chaperones to get confused with tau, a protein that accumulates in the brains of people with Alzheimer’s. Normally tau binds to microtubules—molecular conveyor belts that move chromosomes and vesicles within cells. In the early stages of the disease, however, tau proteins undergo changes that nudge them off microtubules. Because tau has a loose molecular structure, chaperones treat free-floating tau as a misfolded protein. They hold onto it, trying to put it back onto microtubules, rather than sending it for degradation. As a result, tau accumulates inside cells to form the infamous clumps that are considered hallmark pathology in neurodegenerative disorders such as Alzheimer’s and progressive supranuclear palsy.

    Once a protein lands with a chaperone, any of a number of molecular co-factors swoop in to decide the protein’s fate. In separate studies published in June research teams identified two chaperone complexes that appear to work by ushering disease-linked proteins out of cells. The Florida group identified a co-factor that hooks up with tau, alpha-synuclein and other disease-associated molecules to evict them. “We think it’s a last-ditch effort by neurons to get rid of bad proteins,” Dickey says. Meanwhile a team led by Yihong Ye, a cell biologist at the National Institutes of Health, discovered another pathway that uses different protein workhorses to accomplish a similar off-load. The latter mechanism seems to only dispatch alpha-synuclein whereas the other system can dump several neurodegenerative disease–associated proteins.

    The scientists are not sure if the newly discovered pathways are connected or if they relate to a previously identified system that helps clear amyloid beta and other toxins out of the brain. Still, researchers are intrigued by the possibility that cells may use these clearance mechanisms to propagate misfolded proteins throughout the brain—in which case targeting the mechanisms could conceivably slow disease progression.

    Compounds that target autophagy or inhibit chaperones have been tested in many clinical trials, mostly in cancer patients. Cancer was the first disease researchers connected with autophagy. Generally scientists have thought autophagy protects against cancer, although some evidence suggests it can help tumor cells cope with nutrient scarcity and other stresses. Because the impact of autophagy on cancer seems to go both ways, cancer trials have tested therapies that enhance autophagy as well as drugs that block it. For neurodegenerative diseases, such research is still in its early phase. One problem is that many of the experimental molecules are too big to enter the brain, Dickey says. Another challenge: the drugs are not very selective. They may influence other processes within the cell.

    Nevertheless several autophagy-enhancing compounds have entered human testing for treatment of brain disorders. One, rilmenidine, is a prescription medicine for treating high blood pressure. Recently scientists completed a safety trial of rilmenidine in 16 U.K. adults with early Huntington’s disease. Data analyses are ongoing, says University of Cambridge molecular geneticist David Rubinsztein, one of the trial investigators. Bioblast Pharma—an Israel-based biotech company focused on rare diseases—is launching a phase I trial of trehalose, a sugar found in plants, fungi and invertebrates. The study will enroll healthy volunteers to receive the autophagy-inducing compound intravenously. Last month researchers led by Thomas Kukar, a professor at Emory University, published a paper showing that trehalose can reverse lysosomal deficiencies in mouse models of frontotemporal dementia. And Duff’s lab has unpublished data suggesting that trehalose lessens tau pathology and improves behavior in mouse models of neurodegeneration.

    “It seems you just want to clear out all the garbage in the brain,” Duff says.

    See the full article here .

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  • richardmitnick 8:47 am on July 25, 2016 Permalink | Reply
    Tags: , , Protein Studies,   

    From Hopkins: “Johns Hopkins biologists find protein that bolsters growth of damaged muscle tissue” 

    Johns Hopkins
    Johns Hopkins University

    Jul 19, 2016
    Arthur Hirsch

    Johns Hopkins University biologists have found that a protein that plays a key role in the lives of stem cells can bolster the growth of damaged muscle tissue, a step that could potentially contribute to treatments for muscle degeneration caused by old age and diseases such as muscular dystrophy.

    The results, published online by the journal Nature Medicine, show that a particular type of protein called integrin is present on the stem cell surface and used by stem cells to interact with, or “sense,” their surroundings. How stem cells sense their surroundings, also known as the stem cell “niche,” affects how they live and last for regeneration. The presence of the protein β1-integrin was shown to help promote the transformation of those undifferentiated stem cells into muscle after the tissue has degraded and improve regenerated muscle fiber growth as much as 50 percent.

    While the presence of β1-integrin in adult stem cells is apparent, “its role in these cells has not been examined,” especially its influence on the biochemical signals promoting stem cell growth, wrote the three authors—Chen-Ming Fan, an adjunct biology professor at Johns Hopkins; Michelle Rozo, who completed her doctorate in biology at Hopkins this year; and doctoral student Liangji Li.

    The experiment shows that β1-integrin—one of 28 types of integrin—maintains a link between the stem cell and its environment, and interacts biochemically with a growth factor called fibroblast growth factor (FGF) to promote stem cell growth and restoration after muscle tissue injury. Aged stem cells do not respond to FGF, and the results also show that β1-integrin restores aged stem cell’s ability to respond to FGF to grow and improve muscle regeneration.

    By tracking an array of proteins inside the stem cells, the researchers tested the effects of removing β1-integrin from the stem cell. This is based on the understanding that the activities of stem cells—undifferentiated cells that can become specialized—are dependent on their environment and supported by the proteins found there.

    “If we take out β1-integrin, all these other [proteins] are gone,” Fan, the study’s senior author and a staff member at the Carnegie Institution for Science in Washington and Baltimore, said in an interview.

    Why that is the case is not clear, but the experiment showed that without β1-integrin, stem cells could not sustain growth after muscle tissue injury.

    By examining β1-integrin molecules and the array of proteins that they used to track stem cell activity in aged muscles, the authors found that all of these proteins looked like they had been removed from aged stem cells. They injected an antibody to boost β1-integrin function into aged muscles to test whether this treatment would enhance muscle regeneration. Measurements of muscle fiber growth with and without boosting the function of β1-integrin showed that the protein led to as much as 50-percent more regeneration in cases of injury in aged mice.

    When the same β1-integrin function-boosting strategy was applied to mice with muscular dystrophy, the muscle was able to increase strength by about 35 percent.

    Fan said the team’s research will next try to determine what is happening inside the stem cells as they react with their immediate environment, as a step to understanding more about the interaction of the two. That, in turn, could help refine the application of integrin as a therapy for muscular dystrophy and other diseases, and for age-related muscle degeneration.

    “We provide here a proof-of-principle study that may be broadly applicable to muscle diseases that involve [stem cell] niche dysfunction,” the authors wrote. “But further refinement is needed for this method to become a viable treatment.”

    See the full article here .

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    Johns Hopkins Campus

    The Johns Hopkins University opened in 1876, with the inauguration of its first president, Daniel Coit Gilman. “What are we aiming at?” Gilman asked in his installation address. “The encouragement of research … and the advancement of individual scholars, who by their excellence will advance the sciences they pursue, and the society where they dwell.”

    The mission laid out by Gilman remains the university’s mission today, summed up in a simple but powerful restatement of Gilman’s own words: “Knowledge for the world.”

    What Gilman created was a research university, dedicated to advancing both students’ knowledge and the state of human knowledge through research and scholarship. Gilman believed that teaching and research are interdependent, that success in one depends on success in the other. A modern university, he believed, must do both well. The realization of Gilman’s philosophy at Johns Hopkins, and at other institutions that later attracted Johns Hopkins-trained scholars, revolutionized higher education in America, leading to the research university system as it exists today.

  • richardmitnick 7:48 am on July 25, 2016 Permalink | Reply
    Tags: An engineered protein can disrupt tumor-promoting ‘messages’ in human cells, , Protein Studies,   

    From U Washington: “An engineered protein can disrupt tumor-promoting ‘messages’ in human cells “ 

    U Washington

    University of Washington

    July 21, 2016
    James Urton

    Over a century of research has shined light on the once-murky innards of our cells, from the genes that serve as our “blueprints” to the proteins and other molecules that are our cellular taskmasters.

    Building on this basic knowledge, the search is underway for cellular mechanisms that could serve as gateways for new therapies. These could lead to precise treatments for disease — targeting a specific cellular function or gene with fewer unintended side effects. Ideally, these effects would also be temporary, returning cells to normal operation once the underlying condition has been treated.

    A team of researchers from the University of Washington and the University of Trento in Italy announced findings that could pave the way for these therapies. In a paper published July 18 in Nature Chemical Biology, they unveiled an engineered protein that they designed to repress a specific cancer-promoting message within cells.

    And that approach to protein design could be modified to target other cellular messages and functions, said senior author and UW chemistry professor Gabriele Varani.

    “What we show here is a proving ground — a process to determine how to make the correct changes to proteins,” he said.

    A schematic of the RNA-binding region of Rbfox2, shown in grey, attached to part of its natural RNA target, depicted in orange, green, blue and red.Yu Chen, Fan Yang, Gabriele Varani

    For their approach, Varani and his team modified a human protein called Rbfox2, which occurs naturally in cells and binds to microRNAs. These aptly named small RNA molecules adjust gene expression levels in cells like a dimmer switch. Varani’s group sought to engineer Rbfox2 to bind itself to a specific microRNA called miR-21, which is present in high levels in many tumors, increases the expression of cancer-promoting genes and decreases cancer suppressors. If a protein like Rbfox2 could bind to miR-21, the researchers hypothesized, it could repress miR-21’s tumor growth effects.

    But for this approach to be successful, the protein must bind to miR-21 and no other microRNA. Luckily, all RNA molecules, including microRNAs, have an inherent property that imbues them with specificity. They consist of a chain of chemical “letters,” each with a unique order or sequence. To date, no other research team had ever successfully altered a protein to bind to microRNAs.

    “That is because our knowledge of protein structure is much better than our knowledge of RNA structure,” said Varani. “We historically lacked key information about how RNA folds up and how proteins bind RNA at the atomic level.”

    UW researchers relied on high-quality data on Rbfox2’s structure to understand, down to single atoms, how it binds to the unique sequence of “letters” in its natural RNA targets. Then they predicted how Rbfox2’s sequence would have to change to make it bind to miR-21 instead. Elegantly, altering just four carefully selected amino acids made Rbfox2 shift its attachment preference to miR-21, preventing the microRNA from passing along its tumor-promoting message.

    The UW team spent several years proving this, since they had to test each change individually and in combination. They also had to make sure that the modified Rbfox2 protein would bind strongly to miR-21 but not other microRNAs. Since microRNAs have many functions in cells, it would be counterproductive to repress miR-21 while disrupting other normal microRNA-mediated functions.

    A 3-D ‘ribbon’ depiction of the Dicer-Rbfox2 hybrid. The RNA-binding portion of Rbfox2 is in purple. The green regions slice are the regions of Dicer that cleave RNA molecules.Fan Yang, Gabriele Varani

    The researchers also engineered a second protein that should clear miR-21 from cells entirely. They did this by grafting the regions of Rbfox2 that bound to miR-21 onto a separate protein called Dicer. Dicer normally chops RNAs into small chunks and generates functional microRNAs. But the hybrid Rbfox2-Dicer protein displayed a specific affinity to slice miR-21 into oblivion.

    Varani and his team believe that Rbfox2 could be redesigned to bind to microRNA targets other than miR-21. There are thousands of microRNAs to choose from, and many have been implicated in diseases. The key to realizing this potential would be in streamlining and automating the painstaking methods the team used to model Rbfox2’s atomic-level interactions with RNA.

    “This method relies on knowledge of high-quality structures,” said Varani. “That allowed us to see which alterations would change binding to the microRNA target.”

    Not only would these be useful laboratory tools to study microRNA functions, but they could — in time — form the basis of new therapies to treat disease.

    Lead author on the paper is former UW researcher Yu Chen, who is now at the Seattle Children’s Research Institute. Other UW chemistry co-authors were Fang Yang, Tom Pavelitz, Wen Yang, Katherine Godin, Matthew Walker and Suxin Zheng. Co-authors from the University of Trento include Lorena Zubovic and Paolo Macchi. The research was funded by the National Institutes of Health, the University of Trento and the government of Trento province.

    See the full article here .

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    The University of Washington is one of the world’s preeminent public universities. Our impact on individuals, on our region, and on the world is profound — whether we are launching young people into a boundless future or confronting the grand challenges of our time through undaunted research and scholarship. Ranked number 10 in the world in Shanghai Jiao Tong University rankings and educating more than 54,000 students annually, our students and faculty work together to turn ideas into impact and in the process transform lives and our world. For more about our impact on the world, every day.

    So what defines us — the students, faculty and community members at the University of Washington? Above all, it’s our belief in possibility and our unshakable optimism. It’s a connection to others, both near and far. It’s a hunger that pushes us to tackle challenges and pursue progress. It’s the conviction that together we can create a world of good. Join us on the journey.

  • richardmitnick 5:44 am on July 23, 2016 Permalink | Reply
    Tags: , , Protein Studies,   

    From U Wisconsin: “New UW-Madison center offers ultra-speed protein analysis” 

    U Wisconsin

    University of Wisconsin

    July 22, 2016
    David Tenenbaum

    UW-Madison undergraduate Kyle Connors operates a mass spectrometer in the new NIH National Center for Quantitative Biology at UW–Madison. Photo: Nick Wilkes

    Three University of Wisconsin—Madison researchers have won a prestigious, five-year grant to establish the National Center for Quantitative Biology of Complex Systems, which will develop next-generation protein measurement technologies and offer them to biologists nationwide.

    It is proteins that do the work in the body: Hemoglobin, for example, holds oxygen for transport in the blood stream, while insulin helps regulate sugar in the blood. Knowing which protein forms are present in what quantities, their subcellular location and their function is critical to understanding health and disease.

    The scientific technique of mass spectrometry, or mass spec, can already recognize proteins, but the researchers are eying a speed-up akin to that which revolutionized genetics research over the past 20 years.

    Genes are vital carriers of information and templates for proteins, says co-investigator David Pagliarini, a UW–Madison professor of biochemistry. But genes alone don’t explain everything.

    There is lot of action between the gene, the protein it patterns, and the actual biological result,” he explains. “Mass spec technology allows you to measure the proteins, which are closer to action, and we plan to push the limits on pace, depth, throughput.”

    The center, funded at $6 million by the National Institutes of Health (NIH), will develop and make available advanced protein measurement technologies, says Josh Coon, a UW–Madison professor of biomolecular chemistry and an expert in mass spec. “These are complicated, high-end instruments that hundreds or thousands of biomedical researchers who are funded by the NIH need access to. There are many problems that are not solved with current technology, and that high-throughput mass spec can address.”

    A modified orbitrap mass spectrometer in the Coon Laboratory. The modifications illuminate trapped protein ions with infrared photons, providing the basis of a new protein sequencing technology. Photo: Nick Wilkes

    Two among the many areas of interest concern lung cancer and diabetes, Coon says. “We have researchers who want to examine proteins related to the function — or failure — of the pancreatic cells that make insulin.”

    The center will serve as a training ground in mass spec and a laboratory to invent new techniques and equipment. One tactic to be explored relies on parallel processing, an approach like the one that fed a revolution in gene sequencing.

    Co-investigator Lingjun Li, a UW–Madison professor of pharmacy, will develop chemical markers to identify individual samples after they are mixed for mass spec analysis. In a similar vein, Coon will explore “metabolic tags” composed of amino acids that enter proteins after being eaten by lab animals.

    “We are not developing technology in a vacuum,” says Li, “but with specific biomedical needs in mind. Our methods will be broadly available to NIH researchers, and they will be the test bed that validates our methods.”

    Pagliarini says he will serve as “a bridge between technology development and biological applications. Our future collaborators have told us there are certain problem out there waiting for a solution in new technology.”

    “The NIH wants the center to invent and disseminate technologies,” says Coon. “We hope to do for proteins what high-throughput sequencing has done for genomic studies.”

    See the full article here .

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    In achievement and prestige, the University of Wisconsin–Madison has long been recognized as one of America’s great universities. A public, land-grant institution, UW–Madison offers a complete spectrum of liberal arts studies, professional programs and student activities. Spanning 936 acres along the southern shore of Lake Mendota, the campus is located in the city of Madison.

  • richardmitnick 7:28 pm on June 8, 2016 Permalink | Reply
    Tags: , , Protein Studies,   

    From Caltech: “Solving Molecular Structures” 

    Caltech Logo


    Lori Dajose

    The various steps of the atomic structure determination by X-ray crystallography are shown from left to right: crystals of the green fluorescent protein variant mPlum; a single mPlum crystal X-ray diffraction pattern obtained at Caltech’s Molecular Observatory; the calculated electron density map (blue) interpreted with the the positioning of all mPlum polypeptide chain atoms (shown in ball-and-stick representation); and the entire atomic structure of mPlum shown in ribbon representation. Credit: Hoelz Laboratory/Caltech

    Determining the chemical formula of a protein is fairly straightforward, because all proteins are essentially long chains of molecules called amino acids. Each chain, however, folds into a unique three-dimensional shape that helps produce the characteristic properties and function of the protein. These shapes are more difficult to determine (or “solve”); scientists traditionally do so using a technique called X-ray crystallography, in which X-rays are shot through a crystallized sample and scatter off the atoms in a distinctive pattern.

    This spring, Caltech students had the opportunity to use the technique to solve protein structures themselves in a new course taught by Professor of Chemistry André Hoelz.

    Although the Institute has a long history in the fields of structural biology and X-ray crystallography, the chance to get hands-on experience with the technique is rare at most universities, Caltech included. Indeed, the method is more commonly performed at specialized facilities with high-energy X-ray beam lines, including the Stanford Synchrotron Radiation Laboratory (SSRL).


    However, in 2007, thanks to a gift from the Gordon and Betty Moore Foundation, Caltech opened the Molecular Observatory—a dedicated, completely automated radiation beam line at SSRL.

    Graeme Card examines the sample mount holder in SSRL’s Molecular Observatory for Structural Molecular Biology at Beamline 12. (Courtesy: SLAC)

    “The Molecular Observatory gives us lots of beam time,” notes Hoelz. “Recently, I also received a grant from the Innovation in Education Fund from the Provost’s Office that was matched by the Division of Chemistry and Chemical Engineering, and this allowed me the opportunity to develop this course and train students in a way not commonly found at universities.”

    In the new course, “Macromolecular Structure Determination with Modern X-ray Crystallography Methods” (BMB/Ch 230), Hoelz’s students have been using the Molecular Observatory and other on-campus crystallization resources to solve the structures of various proteins, in particular, variants of green fluorescent proteins (GFPs)—proteins that exhibit bright green fluorescence under certain wavelengths of light. “These proteins are crucial tools in biology because they can be visualized by fluorescence techniques. It’s important to know their physical structure, because it affects the intensity and wavelength at which the protein fluoresces,” says Anders Knight, a first-year graduate student studying protein engineering with Frances Arnold, the Dick and Barbara Dickinson Professor of Chemical Engineering, Bioengineering and Biochemistry, and one of nine students—including two undergraduates—in the inaugural class.

    During the first few weeks of the course, students determined the proper conditions—the pH levels and the mix of salts and buffer solutions—that are required to get a protein to crystallize. These conditions vary from protein to protein, making it tricky to “grow” perfect single crystals of the proteins. “Most of the ones we are working with have known 3-D structures, and they crystallize relatively easily, so they are a great place to start learning about the technique of X-ray crystallography,” Knight says. “But some of us were also given protein variants that had never been crystallized before.”

    Once the students crystallized their proteins, single crystals were mounted in tiny nylon loops that are attached to small metal bases, frozen in liquid nitrogen, and loaded into pucks that were shipped to SSRL. There, the pucks were loaded into a robotic machine—remotely controllable from Caltech and operated by the students—that placed them, one by one, into a powerful X-ray beam. X-rays are scattered at characteristic angles by the electrons within the crystallized samples, generating a diffraction pattern that can be converted into a so-called electron-density map, which is then used to determine the 3-D location of all of the atoms.

    “The electron density map doesn’t exactly show you what the protein’s structure is,” Knight says. “You do have to correctly interpret the electron density map to determine where the protein’s atoms will go. It’s difficult, but this class is designed to give us practice doing that. Collecting data at SSRL was a great learning experience. It was interesting to be able to accurately mount and position the crystals—each smaller than a millimeter—on the beamline from hundreds of miles away. The data collection went fairly quickly, taking around eight minutes.”

    For their final assignment, students will write a mock journal paper about their methods and the final protein structure. Most of the structures had been determined previously, but one student did solve a previously unknown GFP structure, a bright red fluorescent protein called dTomato.

    “dTomato is a product of directed evolution in protein engineering, created by subjecting its parent, DsRed, through several rounds of random genetic mutations,” says Phong Nguyen, a graduate student in the lab of Doug Rees, Roscoe Gilkey Dickinson Professor of Chemistry and Howard Hughes Medical Institute Investigator, and the student who solved the structure of dTomato in Hoelz’s class. “By solving its structure, we can see how directed evolution—a method developed by Frances Arnold to create new proteins using the principles of evolution—changed the protein from its parent. Specifically, we are able to explain how individual mutations contributed to the structural outcome of the protein and consequently to differing chemical and physical properties from the parent. We all are so excited to solve a new structure and contribute knowledge to the field of GFP protein engineering.”

    “Having the Molecular Observatory at Caltech allows us to train students with very sophisticated technology,” says Hoelz, who is now envisioning a second, related course. “Students would learn recombinant protein expression and purification, directly prior to this course, so they can purify the proteins themselves with cutting-edge technology and then go on to determine their 3D structure by X-ray crystallography,” he says.

    “In my opinion, learning by doing is the best way to master how to determine crystal structures and this new course will solidify the strong roots Caltech has in X-ray crystallography,” Hoelz adds. “Not only will this new course accelerate the otherwise slow learning process for this technique, but it will also allow non-structural biology laboratories on campus to determine crystal structures of their favorite proteins using Molecular Observatory, a unique and spectacular facility at Caltech.”

    See the full article here .

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    The California Institute of Technology (commonly referred to as Caltech) is a private research university located in Pasadena, California, United States. Caltech has six academic divisions with strong emphases on science and engineering. Its 124-acre (50 ha) primary campus is located approximately 11 mi (18 km) northeast of downtown Los Angeles. “The mission of the California Institute of Technology is to expand human knowledge and benefit society through research integrated with education. We investigate the most challenging, fundamental problems in science and technology in a singularly collegial, interdisciplinary atmosphere, while educating outstanding students to become creative members of society.”
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  • richardmitnick 11:58 am on May 19, 2016 Permalink | Reply
    Tags: , Protein Studies,   

    From U Illinois: “Scientists discover the evolutionary link between protein structure and function” 

    U Illinois bloc

    University of Illinois

    May 18, 2016
    Lauren Quinn

    Heme protein showing loops in orange near the bottom

    Proteins are more than a dietary requirement. This diverse set of molecules powers nearly all of the cellular operations in a living organism. Scientists may know the structure of a protein or its function, but haven’t always been able to link the two.

    “The big problem in biology is the question of how a protein does what it does. We think the answer rests in protein evolution,” says University of Illinois professor and bioinformatician Gustavo Caetano-Anollés.

    Geologists have found remnants of life preserved in rock billions of years old. In some cases, preservation of microbes and tissues has been so good that microscopic cellular structures that were once associated with specific proteins, can be detected. This geological record gives scientists a hidden connection to the evolutionary history of protein structures over incredibly long time periods. But, until now, it hasn’t always been possible to link function with those structures to know how proteins were behaving in cells billions of years ago, compared with today.

    “For the first time, we have traced evolution onto a biological network,” Caetano-Anollés notes.

    Caetano-Anollés and graduate students Fayez Aziz and Kelsey Caetano-Anollés used networks to investigate the linkage between protein structure and molecular function. They built a timeline of protein structures spanning 3.8 billion years across the geological record, but needed a way to connect the structures with their functions. To do that, they looked at the genetic makeup of hundreds of organisms.

    “It turns out that there are little snippets in our genes that are incredibly conserved over time,” Caetano-Anollés says. “And not just in human genomes. When we look at higher organisms, such as plants, fungi and animals, as well as bacteria, archaea, and viruses, the same snippets are always there. We see them over and over again.”

    The research team found that these tiny gene segments tell proteins to produce “loops,” which are the tiniest structural units in a protein. When loops come together, they create active sites, or molecular pockets, which give proteins their function. For example, hemoglobin, the protein that carries oxygen in blood, has two loops which create the active site that binds oxygen. The loops combine to create larger protein structures called domains.

    Remarkably, the new study shows that loops have been repeatedly recruited to perform new functions and that the process has been active and ongoing since the beginning of life.

    “This recruitment is important for understanding biological diversity,” Caetano-Anollés says.

    One important aspect of the study relates to the actual linkage between domain structure and functional loops. The researchers found that this linkage is characterized by an unanticipated property that unfolds in time, an “emergent” property known as hierarchical modularity.

    “Loops are cohesive modules, as are domains, proteins, cells, organs, and bodies.” Caetano-Anollés explains. “We are all made of cohesive modules, including our human bodies. That’s hierarchical modularity: the building of small cohesive parts into larger and increasingly complex wholes.”

    Hierarchical modularity also exists in manmade networks, such as the internet. For example, each router represents a “node” that pushes information to different computers. When millions of computers interact with each other online, larger and more complex entities emerge. Caetano-Anollés suggests that the evolution of manmade networks could be mapped in the same way as the evolution of biological networks.

    “From a computer science point of view, few people have been exploring how to track networks in time. Imagine exploring how the internet grows and changes when new routers are added, are disconnected, or network with each other. It’s a daunting task because there are millions of routers to track and internet communication can be highly dynamic. In our study, we are showcasing how you can do it with a very small network,” Caetano-Anollés explains.

    The methods developed by Caetano-Anollés and his team now have the potential to explain how change is capable of structuring systems as varied as the internet, social networks, or the collective of all proteins in an organism.

    The article, The early history and emergence of molecular functions and modular scale-free network behavior, is published in Scientific Reports. M. Fayez Aziz and Kelsey Caetano-Anollés, also from the University of Illinois, co-authored the report. Full text of the article can be found at: http://www.nature.com/articles/srep25058.

    See the full article here .

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    The University of Illinois at Urbana-Champaign community of students, scholars, and alumni is changing the world.

    With our land-grant heritage as a foundation, we pioneer innovative research that tackles global problems and expands the human experience. Our transformative learning experiences, in and out of the classroom, are designed to produce alumni who desire to make a significant, societal impact.

  • richardmitnick 4:46 pm on January 25, 2016 Permalink | Reply
    Tags: , Crouching Protein Hidden Enzyme, Protein Studies,   

    From Scripps: “Crouching Protein, Hidden Enzyme” 

    Scripps Research Institute

    January 25, 2016
    Office of Communications
    Tel: 858-784-2666
    Fax: 858-784-8136

    The new research shows the workings of a crucial molecular enzyme. In this image, the green glow in the structure denotes the location of the Rpn11 enzymatic active site in its inhibited conformation at the heart of the isolated lid complex.

    A new study led by scientists at The Scripps Research Institute (TSRI) and the University of California (UC), Berkeley shows how a crucial molecular enzyme starts in a tucked-in somersault position and flips out when it encounters the right target.

    The new findings, published recently in the journal eLife, give scientists a clearer picture of the process through which cells eliminate proteins that promote diseases such as cancer and Alzheimer’s.

    “Having an atomic-resolution structure and a better understanding of this mechanism gives us the ability to someday design therapeutics to combat cancer and neurodegeneration,” said TSRI biologist Gabriel Lander, who was co-senior of author of the study with Andreas Martin of UC Berkeley.

    Keeping Cells Healthy

    The new study sheds light on the proteasome, a molecular machine that serves as a recycling center in cells. Proteasomes break down spent or damaged proteins and can even eliminate harmful misfolded proteins observed in many diseases.

    The new research is the first study in almost 20 years to solve a large component of the proteasome at near-atomic resolution. Lander said the breakthrough was possible with recent advances in cryo-electron microscopy (EM), an imaging technique in which a sample is bombarded with an electron beam, producing hundreds of thousands of protein images that can be consolidated into a high-resolution structure.

    Using cryo-EM, scientists investigated part of the proteasome that contains a deubiquitinase enzyme called Rpn11. Rpn11 performs a crucial function called deubiquitination, during which it cleaves molecular tags from proteins scheduled for recycling in the proteasome. This is a key step in proteasomal processing—without Rpn11, the protein tags would clog the proteasome and the cell would die.

    From previous studies, scientists knew Rpn11 and its surrounding proteins latch onto the proteasome to form a sort of lid. “The lid complex wraps around the proteasome like a face-hugger in the movie ‘Alien,’” said Lander.

    The lid complex can also exist separately from the proteasome—which poses a potential problem. If Rpn11 cleaves tags from proteins that haven’t gotten to the proteasome yet, those proteins could skip the recycling stage and cause disease. Scientists had wondered how nature had solved this problem.

    A Guide for Future Therapies

    The study provides an answer, showing the lid complex as it floats freely in cells. In this conformation, Rpn11 is carefully nestled in the crook of surrounding proteins, stabilized and inactive.

    “There’s a sophisticated network of interactions that pin the Rpn11 deubiquitinase against neighboring subunits to keep it inhibited in the isolated proteasome lid,” explained Corey M. Dambacher, a researcher at TSRI at the time of the study and now a senior scientist at Omniome, Inc., who was first author of the study with TSRI Research Associate Mark Herzik Jr. and Evan J. Worden of UC Berkeley.

    “In order for Rpn11 to perform its job, it has to flip out of this inhibited conformation,” said Herzik.

    The new study also shows that, to flip out of the conformation at the proteasome, the proteins surrounding deubiquitinase pivot and rotate—binding to the proteasome and releasing the deubiquitinase active site from its nook.

    Lander called the system “finely tuned,” but said there may be ways to manipulate it. The study collaborators at UC Berkeley made small mutations to the proteins holding Rpn11 in position, and found that any small change will release the deubiquitinase, even when the lid is floating freely.

    Lander said the new understanding of the mechanism that activates Rpn11 could guide future therapies that remove damaged or misfolded proteins.

    “Accumulation of these toxic proteins can lead to diseases such as Parkinson’s and Alzheimer’s, as well as a variety of cancers,” Lander said. “If we can harness the proteasome’s ability to remove specific proteins from the cell, this gives us incredible power over cellular function and improves our ability to target certain cells for destruction.”

    Going forward, the researchers hope to use the same cryo-EM techniques to investigate other components of the proteasome—and figure out exactly how it recognizes and destroys proteins. “There’s still a lot to learn,” said Lander.

    For more information on the study, “Atomic structure of the 26S proteasome lid reveals the mechanism of deubiquitinase inhibition,” see http://elifesciences.org/content/early/2016/01/08/eLife.13027

    This research was supported by the Damon Runyon Cancer Research Foundation (grant DFS-#07-13), the Pew Scholars program, the National Institutes of Health (grants DP2 EB020402 and R01-GM094497), the Searle Scholars Program, the National Science Foundation CAREER Program (grant NSF-MCB-1150288), the Howard Hughes Medical Institute and a National Science Foundation Graduate Research Fellowship.

    See the full article here .

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    The Scripps Research Institute (TSRI), one of the world’s largest, private, non-profit research organizations, stands at the forefront of basic biomedical science, a vital segment of medical research that seeks to comprehend the most fundamental processes of life. Over the last decades, the institute has established a lengthy track record of major contributions to the betterment of health and the human condition.

    The institute — which is located on campuses in La Jolla, California, and Jupiter, Florida — has become internationally recognized for its research into immunology, molecular and cellular biology, chemistry, neurosciences, autoimmune diseases, cardiovascular diseases, virology, and synthetic vaccine development. Particularly significant is the institute’s study of the basic structure and design of biological molecules; in this arena TSRI is among a handful of the world’s leading centers.

    The institute’s educational programs are also first rate. TSRI’s Graduate Program is consistently ranked among the best in the nation in its fields of biology and chemistry.

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