Chinese Medicine Yields Secrets: Atomic Mechanism of Two-Headed Molecule Derived from Chang Shan, a Traditional Chinese Herb

Chinese Medicine Yields Secrets: Atomic Mechanism of Two-Headed Molecule Derived from Chang Shan, a Traditional Chinese Herb

  9:49:00 AM    Science Lover

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The mysterious inner workings of Chang Shan -- a Chinese herbal medicine used for thousands of years to treat fevers associated with malaria -- have been uncovered thanks to a high-resolution structure solved at The Scripps Research Institute (TSRI).

Scripps Research Institute scientists have determined a molecular 
 structure that helps explain how the Chinese herbal medicine 
Chang Shan works. (Credit: Image courtesy of the Schimmel lab.)

Described in the journal Nature this week, the structure shows in atomic detail how a two-headed compound derived from the active ingredient in Chang Shan works. Scientists have known that this compound, called halofuginone (a derivative of the febrifugine), can suppress parts of the immune system -- but nobody knew exactly how.

The new structure shows that, like a wrench in the works, halofuginone jams the gears of a molecular machine that carries out "aminoacylation," a crucial biological process that allows organisms to synthesize the proteins they need to live. Chang Shan, also known as Dichroa febrifuga Lour, probably helps with malarial fevers because traces of a halofuginone-like chemical in the herb interfere with this same process in malaria parasites, killing them in an infected person's bloodstream.

"Our new results solved a mystery that has puzzled people about the mechanism of action of a medicine that has been used to treat fever from a malaria infection going back probably 2,000 years or more," said Paul Schimmel, PhD, the Ernest and Jean Hahn Professor and Chair of Molecular Biology and Chemistry and member of The Skaggs Institute for Chemical Biology at TSRI. Schimmel led the research with TSRI postdoctoral fellow Huihao Zhou, PhD.

Halofuginone has been in clinical trials for cancer, but the high-resolution picture of the molecule suggests it has a modularity that would make it useful as a template to create new drugs for numerous other diseases.

The Process of Aminoacylation and its Importance to Life

Aminoacylation is a crucial step in the synthesis of proteins, the end products of gene expression. When genes are expressed, their DNA sequence is first read and transcribed into RNA, a similar molecule. The RNA is then translated into proteins, which are chemically very different from DNA and RNA but are composed of chains of amino acid molecules strung together in the order called for in the DNA.

Necessary for this translation process are a set of molecules known as transfer RNAs (tRNAs), which shuttle amino acids to the growing protein chain where they are added like pearls on a string. But before the tRNAs can move the pearls in place, they must first grab hold of them.

Aminoacylation is the biological process whereby the amino acid's pearls are attached to these tRNA shuttles. A class of enzymes known as aminoacyl-tRNA synthetases is responsible for attaching the amino acids to the tRNAs, and Schimmel and his colleagues have been examining the molecular details of this process for years. Their work has given scientists insight into everything from early evolution to possible targets for future drug development.

Over time what has emerged as the picture of this process basically involves three molecular players: a tRNA, an amino acid and the aminoacyl-tRNA synthetase enzyme that brings them together. A fourth molecule called ATP is a microscopic form of fuel that gets consumed in the process.

The new work shows that halofuginone gets its potency by interfering with the tRNA synthetase enzyme that attaches the amino acid proline to the appropriate tRNA. It does this by blocking the active site of the enzyme where both the tRNA and the amino acid come together, with each half of the halofuginone blocking one side or the other.

Interestingly, said Schimmel, ATP is also needed for the halofuginone to bind. Nothing like that has ever been seen in biochemistry before.

"This is a remarkable example where a substrate of an enzyme (ATP) captures an inhibitor of the same enzyme, so that you have an enzyme-substrate-inhibitor complex," said Schimmel.

The article, "ATP-Directed Capture of Bioactive Herbal-Based Medicine on Human tRNA Synthetase," by Huihao Zhou, Litao Sun, Xiang-Lei Yang and Paul Schimmel was published in the journal Nature on Dec. 23, 2012.

This work was supported by the National Institutes of Health through grants #GM15539, #23562 and #88278 and by a fellowship from the National Foundation for Cancer Research.

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Strange Behavior: New Study Exposes Living Cells to Synthetic Protein

Strange Behavior: New Study Exposes Living Cells to Synthetic Protein

  10:22:00 AM    Science Lover

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One approach to understanding components in living organisms is to attempt to create them artificially, using principles of chemistry, engineering and genetics. A suite of powerful techniques -- collectively referred to as synthetic biology -- have been used to produce self-replicating molecules, artificial pathways in living systems and organisms bearing synthetic genomes.

The depletion of ATP in cells of the bacterium Escherichia coli causes them to transition to a filamentous state and form dense lipid structures known as endoliposomes. The structures can be clearly observed in these transmission electron micrographs of increasing magnification. (Credit: Image courtesy of Arizona State University)

In a new twist, John Chaput, a researcher at Arizona State University's Biodesign Institute and colleagues at the Department of Pharmacology, Midwestern University, Glendale, AZ have fabricated an artificial protein in the laboratory and examined the surprising ways living cells respond to it.

"If you take a protein that was created in a test tube and put it inside a cell, does it still function," Chaput asks. "Does the cell recognize it? Does the cell just chew it up and spit it out?" This unexplored area represents a new domain for synthetic biology and may ultimately lead to the development of novel therapeutic agents.

The research results, reported in the advanced online edition of the journal ACS Chemical Biology, describe a peculiar set of adaptations exhibited by Escherichia coli bacterial cells exposed to a synthetic protein, dubbed DX. Inside the cell, DX proteins bind with molecules of ATP, the energy source required by all biological entities.

"ATP is the energy currency of life," Chaput says. The phosphodiester bonds of ATP contain the energy necessary to drive reactions in living systems, giving up their stored energy when these bonds are chemically cleaved. The depletion of available intracellular ATP by DX binding disrupts normal metabolic activity in the cells, preventing them from dividing, (though they continue to grow).

After exposure to DX, the normally spherical E. coli bacteria develop into elongated filaments. Within the filamentous bacteria, dense intracellular lipid structures act to partition the cell at regular intervals along its length. These unusual structures, which the authors call endoliposomes, are an unprecedented phenomenon in such cells.

"Somewhere along the line of this filamentation, other processes begin to happen that we haven't fully understood at the genetic level, but we can see the results phenotypically," Chaput says. "These dense lipid structures are forming at very regular regions along the filamented cell and it looks like it could be a defense mechanism, allowing the cell to compartmentalize itself." This peculiar adaptation has never been observed in bacterial cells and appears unique for a single-celled organism.

Producing a synthetic protein like DX, which can mimic the elaborate folding characteristics of naturally occurring proteins and bind with a key metabolite like ATP is no easy task. As Chaput explains, a clever strategy known as mRNA display was used to produce, fine-tune and amplify synthetic proteins capable of binding ATP with high affinity and specificity, much as a naturally occurring ATP-binding protein would.

First, large libraries of random sequence peptides are formed from the four nucleic acids making up DNA, with each strand measuring around 80 nucleotides in length. These sequences are then transcribed into RNA with the help of an enzyme -- RNA polymerase. If a natural ribosome is then introduced, it attaches to the strand and reads the random sequence RNA as though it was a naturally-occurring RNA, generating a synthetic protein as it migrates along the strand. In this way, synthetic proteins based on random RNA sequences can be generated.

Exposing the batch of synthetic proteins to the target molecule and extracting those that bind can then select for ATP-binding proteins. But as Chaput explains, there's a problem: "The big question is how do you recover that genetic information? You can't reverse transcribe a protein back into DNA. You can't PCR amplify a protein. So we have to do all these molecular biology tricks."

The main trick involves an earlier step in the process. A molecular linker is chemically attached to the RNA templates, such that each RNA strand forms a bond with its newly translated protein. The mRNA-protein hybrids are exposed to selection targets (like ATP) over consecutive rounds of increasing stringency. After each round of selection, those library members that remain bound to the target are reverse-transcribed into cDNA (using their conveniently attached RNA messages), and then PCR amplified.

In the current study, E. coli cells exposed to DX transitioned into a filamentous form, which can occur naturally when such cells are subject to conditions of stress. The cells display low metabolic activity and limited cell division, presumably owing to their ATP-starved condition.

The study also examined the ability of E. coli to recover following DX exposure. The cells were found to enter a quiescent state known as viable but non-culturable (VBNC), meaning that they survived ATP sequestration and returned to their non-filamentous state after 48 hours, but lost their reproductive capacity. Further, this condition was difficult to reverse and seems to involve a fundamental reprogramming of the cell.

In an additional response to DX, the filamentous cells form previously undocumented structures, which the authors refer to as endoliposomes. These dense lipid concentrations, spanning the full width of the filamented E. coli, segment the cells into distinct compartments, giving the cells a stringbean-like appearance under the microscope.

The authors speculate that this adaptation may be an effort to maintain homeostasis in regions of the filamentous cell, which have essentially been walled off from the intrusion of ATP-depleting DX. They liken endoliposomes to the series of water-tight compartments found in submarines which are used to isolate damaged sections of the ship and speculate that DX-exposed cells are partitioning their genetic information into regions where it can be safely quarantined. Such self-compartmentalization is known to occur in some eukaryotic cells, but has not been previously observed in prokaryotes like E. coli.

The research indicates that there is still a great deal to learn about bacterial behavior and the repertoire of responses available when such cells encounter novel situations, such as an unfamiliar, synthetic protein. The study also notes that many infectious agents rely on a dormant state, (similar to the VBNC condition observed in the DX-exposed E. coli), to elude detection by antibiotics. A better understanding of the mechanisms driving this behavior could provide a new approach to targeting such pathogens.

The relative safety of E. coli as a model organism for study may provide a fruitful tool for more in-depth investigation of VBNC states in pathogenic organisms. Further, given ATP's central importance for living organisms, its suppression may provide another avenue for combating disease. One example would be an engineered bacteriophage capable of delivering DX genes to pathogenic organisms.

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Researchers Watch Tiny Living Machines Self-Assemble

Researchers Watch Tiny Living Machines Self-Assemble

  1:05:00 PM    Science Lover

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Enabling bioengineers to design new molecular machines for nanotechnology applications is one of the possible outcomes of a study by University of Montreal researchers that was published in Nature Structural and Molecular Biology June 10. The scientists have developed a new approach to visualize how proteins assemble, which may also significantly aid our understanding of diseases such as Alzheimer's and Parkinson's, which are caused by errors in assembly.

Vallée-Bélisle and Michnick have developed a new 
approach to visualize how proteins assemble, which may 
also significantly aid our understanding of diseases such 
as Alzheimer’s and Parkinson’s, which are caused by errors 
in assembly. Here shown are two different assembly stages 
(purple and red) of the protein ubiquitin and the fluorescent 
probe used to visualize these stage (tryptophan: see yellow).

"In order to survive, all creatures, from bacteria to humans, monitor and transform their environments using small protein nanomachines made of thousands of atoms," explained the senior author of the study, Prof. Stephen Michnick of the university's department of biochemistry. "For example, in our sinuses, there are complex receptor proteins that are activated in the presence of different odor molecules. Some of those scents warn us of danger; others tell us that food is nearby." Proteins are made of long linear chains of amino acids, which have evolved over millions of years to self-assemble extremely rapidly -- often within thousandths of a split second -- into a working nanomachine. "One of the main challenges for biochemists is to understand how these linear chains assemble into their correct structure given an astronomically large number of other possible forms," Michnick said.

"To understand how a protein goes from a linear chain to a unique assembled structure, we need to capture snapshots of its shape at each stage of assembly said Dr. Alexis Vallée-Bélisle, first author of the study. "The problem is that each step exists for a fleetingly short time and no available technique enables us to obtain precise structural information on these states within such a small time frame. We developed a strategy to monitor protein assembly by integrating fluorescent probes throughout the linear protein chain so that we could detect the structure of each stage of protein assembly, step by step to its final structure."

The protein assembly process is not the end of its journey, as a protein can change, through chemical modifications or with age, to take on different forms and functions. "Understanding how a protein goes from being one thing to becoming another is the first step towards understanding and designing protein nanomachines for biotechnologies such as medical and environmental diagnostic sensors, drug synthesis of delivery," Vallée-Bélisle said.

This research was supported by the Natural Sciences and Engineering Research Council of Canada and Le fond de recherché du Québec, Nature et Technologie. The article, "Visualizing transient protein folding intermediates by tryptophan scanning mutagenesis," published in Nature Structural & Molecular Biology, was coauthored by Alexis Vallée-Bélisle and Stephen W. Michnick of the Département de Biochimie de l'Université de Montréal. The University of Montreal is known officially as Université de Montréal.

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Environs Prompt Advantageous Gene Mutations as Plants Grow; Changes Passed to Progeny

Environs Prompt Advantageous Gene Mutations as Plants Grow; Changes Passed to Progeny

  1:21:00 PM    Science Lover

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If a person were to climb a towering redwood and take a sample from the top and a sample from the bottom of the tree, a comparison would show that the two DNA samples are different.

Research shows that if a person were to climb a towering redwood and take a sample from the top and a sample from the bottom of the tree, a comparison would show that the two DNA samples are different. (Credit: © Galyna Andrushko / Fotolia)

Christopher A. Cullis, chair of biology at Case Western Reserve University, explains that this is the basis of his controversial research findings.

Cullis, who has spent over 40 years studying mutations within plants, most recently flax (Linum usitatissimum), has found that the environment not only weeds out harmful and useless mutations through natural selection, but actually influences helpful mutations.

Cullis published his findings in the International Journal of Genetics and Molecular Biology and repeated them in the Journal of Visualized Experiments, where he challenged other scientists to repeat his experiment themselves.

Specifically, Cullis focuses on mutations involving the appearance of a small sequence of DNA known as LIS-1 and how the environment affects these changes.

The controversy stems from the idea that the environment changes organisms as they grow and these changes are passed on.

While originally accepted, the theory was eventually thrown out because science revealed that animals pass along DNA through their gamete or sex cells, which are not affected by the environment. This concept was assumed to be the same for plants, but Cullis's research says otherwise.

In his second study, three separate strands (the plastic strand, short strand, and tall strand) of the Stormont Cirrus variety of flax were grown under three separate conditions.

Each of the strands had been bred over multiple generations under different conditions: The plastic strand's ancestors were grown under control conditions, the short strand's ancestors were grown under low-nutrient conditions, and the tall strand's ancestors were grown under high-nutrient conditions.



The experiment showed each strand responded to each condition in a different way, corresponding to the environment its ancestors were grown in. The plastic strand outgrew the other strands under control conditions, the short strand outgrew the other strands when few nutrients were available, and the tall strand grew best when nutrients were readily available.

All this information does not completely explain Cullis's assertion that the environment can in a single generation help sift out the useful mutations.

This is where polymerase chain reaction (PCR) amplification of DNA comes in. Through this process, the researchers could see when a specific DNA sequence (in this case LIS-1) appears or disappears.

When the plastic strand is grown under low nutrient conditions, the LIS-1 sequence, which had been absent, appears and continues for future generations. Since the LIS-1 sequence helps plants survive when there is a shortage of nutrients, its presence helps confirm Cullis's belief that the environment can act on how a plant mutates and keep helpful mutations, even within one generation.

These findings help explain why the top of a redwood is genetically different from the bottom. Young redwoods grow by the tips of the existing branches budding into meristems. Each new meristem is different from the tree because the environment has affected its genetic makeup. And as the redwood grows, the top becomes more and more genetically different from the bottom.

Due to the controversy surrounding Cullis's findings, many scientists are hesitant to accept them as true. Cullis himself recalls at first being skeptical and thinking, "If this really works… [we can] get a plant that's better adapted to its environment in one generation."

These adapted plants have practical uses. Cullis hopes to identify the specific gene sequence responsible for flax's ability to withstand harsh environments and insert it into the DNA sequence of other plants so that they too can withstand trying environments.

This would bypass the current method of genetically engineering plants, which involves isolating specific DNA sequences that control heat-resistance, cold-resistance, pest-resistance, etc., and instead narrows the effort down to one DNA sequence.

By inserting this sequence into the plant and growing it in a specific trying environment, scientists could make the plant resistant to what they want. All of that plant's offspring would be adapted to the environment and ready to grow.

By making the plant do all the work, the price of producing better crops would be greatly reduced. This would greatly benefit developing nations that need a large supply of food in an otherwise harsh environment. The DNA sequence may no longer just help the plant survive, but can now help entire countries thrive.

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Brain's Master Switch Is Verified

Brain's Master Switch Is Verified

  9:20:00 PM    Science Lover

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The protein that has long been suspected by scientists of being the master switch allowing brains to function has now been verified by an Iowa State University researcher.

Yeon-Kyun Shin, professor of biochemistry, 
biophysics and molecular biology at ISU, has 
shown that the protein called synaptotagmin1 
(Syt1) is the sole trigger for the release of 
neurotransmitters in the brain using this 
instrument that allows a new technique called 
single vesicle fusion method. 
(Credit: ISU photo by Bob Elbert)

Yeon-Kyun Shin, professor of biochemistry, biophysics and molecular biology at ISU, has shown that the protein called synaptotagmin1 (Syt1) is the sole trigger for the release of neurotransmitters in the brain.

Prior to this research, Syt1 was thought to be a part of the protein structure (not the sole protein) that triggered the release of neurotransmitters at 10 parts per million of calcium.

Shin's research is published in the current issue of the journal Science.

"Syt1 was a suspect previously, but people were not able to pinpoint that it's the real one, even though there were lots and lots of different trials," said Shin.

"In this case, we are trying to show in the laboratory that it's the real one. So we excluded everything else, and included SNARE proteins -- that's the machinery of the release, and the Syt1 is a calcium-sensing timer."

Syt1 senses, at 10 ppm of calcium, and tells the SNARE complex to open the pore to allow the movement of the neurotransmitters.

Brain activity occurs when neurotransmitters move into a fusion pore.

"We are showing that this Syt1 senses the calcium at 10 ppm, and sends the signal to the SNARE complex to open the fusion pore. That is the process that we are showing right now," Shin said.

Shin and his researchers were able to pinpoint the protein using a new technique called single vesicle fusion method. Using this method, they were able to create and monitor a single fusion event.

Previous research didn't allow scientists to look at single events, and instead required detecting many events and then taking an average of those events, Shin says.

Shin, who has been looking at this brain activity for 15 years, is happy about the discovery.

"We are quite excited that for the first time we are showing that Syt1 is really what triggers the signal in the brain," he said. "This is a really important thing in terms of neurosciences. This is the heart of the molecular part of the brain function."

Shin believes his discovery may be useful in understanding brain malfunctions such as autism, epilepsy and others.

While researching brain function, Shin has previously shown that taking statin drugs to lower cholesterol may actually inhibit some brain function.

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