Tag: Biochemistry

Dutch researchers Roeland Nolte and Jeroen Cornelissen have successfully converted a virus into a unique drug distributor. They removed all of the dangerous material from the virus so that empty, semipermeable particles remained. They subsequently joined these particles together, yet even more important: they also succeeded in separating them again — a world first. With this discovery the researchers have blazed a trail for a new use of drugs. Their results were published in the journal Nature Chemistry. The researchers joined the viral particles together with light-sensitive polymers. The viral particle has a negative charge and the polymer a positive charge. Opposites attract and the two stick together. The polymers therefore act as a glue for which stuck means absolutely stuck. This being the case, the challenge lies in separating all the parts again.

The polymers used by the researchers are long chains of molecules that are joined together in a tree-like structure. This means that each end of the chain can bind to a different particle. The polymers used by the researchers can be manipulated with light and that is the secret of their success. If the polymers are very briefly illuminated with UV light or light with a low wavelength, they fall apart. This allowed the researchers to stick the viral particles together, inject this complex into the body and then to allow this to separate again in the body. The empty particle can be used in many ways. For example, it can be filled with drugs or growth promoters. After injection into the body you can use a pulse of light to ensure that the complex separates at a specific location, from which the drug can gradually spread. In addition to their use as drug distributors, virus complexes can also be used to construct chips. Then instead of drugs, a viral particle is filled with magnetic particles to make an integrated electronic switch. The researchers will now attempt to apply layers of polymer virus complexes of different compositions to a surface and then, just like for conventional chips, use light to make patterns and structures in this. The research was carried out at Radboud University Nijmegen.

Science Daily
July 6, 2010

Original web page at Science Daily

Transcription is the first step of gene expression and is characterized by a high fidelity of RNA synthesis. During transcription, the RNA polymerase active centre discriminates against not just non-complementary ribo NTP substrates but also against complementary 2′- and 3′-deoxy NTPs. A flexible domain of the RNA polymerase active centre, the Trigger Loop, was shown to play an important role in this process, but the mechanisms of this participation remained elusive. Here we show that transcription fidelity is achieved through a multi-step process. The initial binding in the active centre is the major discrimination step for some non-complementary substrates, although for the rest of misincorporation events discrimination at this step is very poor. During the second step, non-complementary and 2′-deoxy NTPs are discriminated against based on differences in reaction transition state stabilization and partly in general base catalysis, for correct versus non-correct substrates. This step is determined by two residues of the Trigger Loop that participate in catalysis. In the following step, non-complementary and 2′-deoxy NTPs are actively removed from the active centre through a rearrangement of the Trigger Loop. The only step of discrimination against 3′-deoxy substrates, distinct from the ones above, is based on failure to orient the Trigger Loop catalytic residues in the absence of 3’OH. We demonstrate that fidelity of transcription by multi-subunit RNA polymerases is achieved through a stepwise process. We show that individual steps contribute differently to discrimination against various erroneous substrates. We define the mechanisms and contributions of each of these steps to the overall fidelity of transcription.

BioMed Central
June 8, 2010

Original web page at BioMed Central

Almost every human protein has segments that can form amyloids, the sticky aggregates known for their role in disease. Yet cells have evolved some elaborate defences, finds Jim Schnabel. Of all the ways that proteins can go bad, becoming an amyloid is surely one of the worst. In this state, sticky elements within proteins emerge and seed the growth of sometimes deadly fibrils. Amyloids riddle the brain in Alzheimer’s disease and Creutzfeldt–Jakob disease. But until recently it has seemed that this corrupt state could threaten only a tiny fraction of proteins. Research is now hinting at a more unsettling picture. In work reported in February, a team led by David Eisenberg at the University of California, Los Angeles, sifted through tens of thousands of proteins looking for segments with the peculiar stickiness needed to form amyloid. They found, says Eisenberg, that “effectively all complex proteins have these short segments that, if exposed and flexible enough, are capable of triggering amyloid formation”. Not all proteins form amyloids, however. The ‘amylome’, as Eisenberg calls it, is restricted because most proteins hide these sticky segments out of harm’s way or otherwise keep their stickiness under control. His results and other work suggest that evolution treats amyloids as a fundamental threat. Amyloids have been found in some of the most common age-related diseases, and there is evidence that ageing itself makes some amyloid accumulation inevitable. It now seems as though the human body is perched precariously above an amyloidal abyss.

“The amyloid state is more like the default state of a protein, and in the absence of specific protective mechanisms, many of our proteins could fall into it,” says Chris Dobson, a structural biologist at the University of Cambridge, UK. Several laboratories are now trying to find ways to supplement or boost these protective mechanisms, in the hope of treating or preventing a host of amyloid-linked diseases. “Advances in understanding amyloids could lead to a powerful new class of medicines for many age-related conditions,” says Sam Gandy, a neurobiologist and clinician at Mount Sinai School of Medicine in New York. The recent work on amyloids has partially confirmed a prediction made 75 years ago by the British biophysicist William Astbury. Proteins start as linear chains of amino acids, but most then fold into complex, three-dimensional, ‘globular’ shapes. Astbury proposed that almost any globular protein could be made to form dysfunctional fibrils by damaging — or ‘denaturing’ — it with heat or chemicals. By the 1980s, researchers had come to understand that these artificially induced fibrils had the same peculiar structure seen in disease-linked amyloids, such as the amyloid-β deposits in the brains of people with Alzheimer’s disease. But the wider potential of proteins to naturally form this basic structure was not seen right away. “The previous paradigm was that the whole protein unfolded and then refolded into a fibrous structure,” says Eisenberg.

“Most proteins have evolved to fold in a way that effectively conceals their amyloid-prone segments.” By 1999, it was clear that numerous proteins could be made to form amyloids. Dobson proposed that unfolding exposes an essential stickiness in a protein’s backbone of amino-acid chains. Researchers were also linking more and more amyloid-forming proteins to disease, including tau proteins in Alzheimer’s disease, α-synuclein in Parkinson’s disease, polyglutamine in Huntington’s disease, prion protein in Creutzfeldt-Jakob disease and amylin in type 2 diabetes. Eisenberg and his colleagues studied such proteins using fibril-forming assays and X-ray diffraction techniques and found that their tendency to form amyloids came from specific segments within them. These segments are typically about six amino acids long, and can be exposed when a protein partly unfolds. These ‘amyloidogenic’ segments, Eisenberg’s team found, have a self-complementary ‘steric zipper’ structure that lets them mesh very tightly with an identical segment exposed on another protein. Several of these segments are needed to seed, or nucleate, an amyloid. Segments stack atop one another to form sheets, two of which zip together to form the spine of the fibril. As it grows, the fibril is fringed by the remnants of the segments’ host proteins. Eventually, this sprouting fibril breaks to form two smaller fibrils, each of which will grow from both ends again — and so on. “The nucleation event may be rare,” Eisenberg says, “but once it starts, you can see how it would spread.”

In their study, Eisenberg’s team used a computer algorithm to determine when any short protein segment has sufficient steric-zipper-forming potential, based on its predicted three-dimensional structure. After calibrating against known amyloid segments, the team applied the algorithm to the genomes of human, budding yeast and the bacterium Escherichia coli and found that about 15% of the short segments coded by genes in these organisms had this property. “At that rate most proteins contain at least several of these amyloid-prone segments,” says Eisenberg. The work helps to clarify in a rigorous way why denaturing a protein often pushes it into the amyloid state, says Jeffery Kelly, a structural biologist and amyloid expert at the Scripps Research Institute in La Jolla, California. “It gives us a better idea of why some proteins have to partially unfold before they can start forming amyloids.”

Eisenberg, Dobson and others have speculated that the self-complementary stickiness of these short segments might have made them useful building blocks in the earliest stages of life on Earth. Moreover, reports have started to emerge of proteins that function normally in the amyloid state, for example some pituitary hormones. “We know by now of over two dozen native amyloids, so this state is clearly used by biology in a functional way as well as a dysfunctional way,” says Eisenberg. Protein segments with a ‘steric zipper’ structure mesh tightly to form the spine of amyloid fibrils. Even so, says Kelly, these native amyloids “are all highly regulated” by, for example, being tucked away inside membrane-bound compartments called vesicles. “That’s why biology can use them and not suffer the consequences.” Most modern proteins fold into globular structures. But their folding patterns are so complex that they couldn’t have evolved by accident. “If you had a machine that could generate protein sequences randomly, you would only rarely get one that can remain stable in the globular, soluble state,” Dobson says. Underlying that stability are a variety of evolved mechanisms. When proteins are first synthesized and start to fold, ‘chaperone’ proteins and related molecules are there to guard against amyloid formation. Other systems are in place to recognize, sequester and destroy amyloids when they do form.

The native folded state offers its own strong protection. Eisenberg’s group examined more than 12,000 proteins whose folded, three-dimensional structures are already known. They found that 95% of the predicted amyloid-prone segments within them are buried within the structures of their host proteins, and that those that are exposed are too twisted and inflexible to zip up with partner segments. “It seems that most proteins have evolved to fold in a way that effectively conceals their amyloid-prone segments,” says Eisenberg. So it may have been unnecessary for evolution to get rid of the segments outright. Yet all these safeguards amount to a defence line that will inevitably be breached. Some mutations and toxins, and the cellular wear and tear associated with ageing, can result in proteins that are less well folded and less protected by chaperoning and disposal mechanisms — and thus more liable to become amyloids. “The 40 or 50 amyloid-associated diseases we’ve found so far are probably only the ones in which our proteins are the most vulnerable,” says Dobson. “If we were to live longer, we might have to contend with more of these conditions.” By the same token, even a subtle hindrance of amyloidogenesis with drugs might have a major effect on disease and even on ageing in general. “If we could just enhance the natural protective mechanisms that stabilize a protein,” says Dobson, “we could take it back over to the side of the line where it’s soluble and stable.”

Amyloids may not be the prime causes of all the diseases in which they have been found, but, typically, some by-product of the amyloid process is suspected. In Alzheimer’s disease, many scientists now believe that small and still-soluble forms of amyloid are the most toxic to brain cells. By contrast, the larger, insoluble fibrils “might even be protective to the extent that they sequester more toxic forms”, says Dobson. The general hope is that by preventing or slowing the initial cascade of amyloid formation, the true ‘toxic species’ of amyloid will be stopped at its source. “There’s a group of 500–600 genes that protect us when we’re young.” One anti-amyloid strategy is to use small molecules as extra chaperones to lower the probability that a protein will expose its amyloidogenic segments. FoldRx, a biotech company based in Cambridge, Massachusetts and founded by Kelly and Susan Lindquist of the Massachusetts Institute of Technology in Cambridge, recently demonstrated this principle in a clinical trial against familial amyloid polyneuropathy, a fatal neurodegenerative disease. Eisenberg says that this strategy is unlikely to work well against most amyloid-prone disease proteins, such as amyloid-β, because they are typically too small to stay tightly folded. “For those I think there would be no hope of stabilizing the native structure, because they don’t have one,” he says. Instead, his group is trying to develop compounds to ‘cap’ the steric zippers of amyloid fibrils, slowing down their formation in the hope that innate clearance mechanisms can then keep up. A third strategy is to boost the activity of these clearance mechanisms — which, according to work by Kelly’s lab, includes enzymes that specifically disaggregate amyloids. “There’s a group of 500–600 genes that protect us when we’re young, even if we’ve been so unlucky as to inherit, for example, a predisposing Parkinson’s or Alzheimer’s mutation,” he says. Finding ways to rejuvenate that system “is what almost our whole lab is working on these days,” says Kelly.

Nature
April 27, 2010

Original web page at Nature

Researchers at Lund University, Sweden, have taken a snapshot of proteins changing shape, sticking together and creating structures that are believed to trigger deadly processes in the nervous system. The discovery opens the possibility of designing drugs for a devastating neurological disease, ALS. Research indicates that ALS, in common with other neurological disorders, such as Alzheimer’s and Parkinson’s disease, is caused by our own proteins, which form aberrant aggregates that are fatally toxic to our nerve cells. However, it has not been known what causes these proteins to aggregate. Researchers at Lund University have now revealed what happens with proteins during the very first, critical step towards forming larger aggregates. It turns out that the protein superoxide dismutase interchanges between its normal structure and a misfolded form. During a brief moment the structure becomes partially misfolded to expose sticky patches that normally are hidden in the interior. These patches cause two or several protein molecules to stick together, thereby forming the cornerstone of the larger structures that are believed to underlie ALS.

The research team headed by Mikael Akke at the Center for Molecular Protein Science of Lund University used NMR spectroscopy to create a snapshot of the misfolded structure, which had not previously been seen. Knowledge of the misfolded protein structure potentially makes possible future efforts to rationally design drugs that prevent the misfolding event and hence the development of ALS. Amyotrophic lateral sclerosis (ALS) is a dreaded and incurable disease that affects nerve cells in the brain and spinal cord, leading to muscle atrophy and respiratory failure. Worldwide, 2 per 100,000 people are diagnosed with ALS each year and 6 per 1 million people suffer from ALS.

Science Daily
November 17, 2009

Original web page at Science Daily

Researchers have developed an improved version of an enzyme that degrades the dense scar tissue that forms when the central nervous system is damaged. By digesting the tissue that blocks re-growth of damaged nerves, the improved enzyme — and new system for delivering it — could facilitate recovery from serious central nervous system injuries. The enzyme, chrondroitinase ABC (chABC), must be supplied to the damaged area for at least two weeks following injury to fully degrade scar tissue. But the enzyme functions poorly at body temperature and must therefore be repeatedly injected or infused into the body. In a paper published November 2 in the early edition of the journal Proceedings of the National Academy of Sciences, researchers describe how they eliminated the thermal sensitivity of chABC and developed a delivery system that allowed the enzyme to be active for weeks without implanted catheters and pumps. This work was supported by the National Institutes of Health. “This research has made digesting scar clinically viable by obviating the need for continuous injection of chABC by thermally stabilizing the enzyme and harnessing bioengineered drug delivery systems,” said the paper’s lead author Ravi Bellamkonda, a professor in the Wallace H. Coulter Department of Biomedical Engineering at Georgia Tech and Emory University.

At physiological body temperature, chABC enzyme loses half of its enzymatic activity within one hour and their remaining functionality within three to five days. To thermostabilize the enzymes, Bellamkonda, Emory University cell biology associate professor Robert McKeon and Georgia Tech graduate student Hyun-Jung Lee mixed the enzyme with the sugar trehalose. The result — the enzyme’s activity was stabilized at internal body temperature for up to four weeks during in vitro tests. The researchers then used a lipid microtube-hydrogel scaffold system to deliver the thermostabilized enzymes into animals via a single injection. The scaffold provided sustained delivery of the enzyme for two weeks, with the microtubes enabling slow release and the hydrogel localizing the tubes to the lesion site. This delivery system also allowed the enzyme to diffuse deeper into the tissue than did catheter delivery. In animal studies, the enzyme’s ability to digest the scar was retained for two weeks post-injury and scar remained significantly degraded at the lesion site for at least six weeks. The researchers also observed enhanced axonal sprouting and recovery of nerve function at the injury site when the thermostabilized enzyme was delivered.

The delivery system also enabled the combination of therapies. Animals treated with thermostabilized chABC in combination with sustained delivery of neurotrophin-3 — a protein growth factor that helps to support the survival and differentiation of neurons — showed significant improvement in locomotor function and enhanced growth of sensory axons and sprouting of fibers for the neurotransmitter serotonin. “These results bring us a step closer to repairing spinal cord injuries, which require multiple steps including minimizing the extent of secondary injury, bridging the lesion, overcoming inhibition due to scar, and stimulating nerve growth,” added Bellamkonda, who is also deputy director of research for GTEC, a regenerative medicine center based at Georgia Tech and Emory University, and a Georgia Cancer Coalition Distinguished Cancer Scholar.

Science Daily
November 17, 2009

Original web page at Science Daily

Scientists at the Singapore Immunology Network (SIgN) have determined how the master gene regulator p53 could switch a gene in a cell “on” or “off” by recognizing specific sequences of nucleotides in the gene’s DNA. Their findings provide a missing piece about p53 gene repression that has eluded researchers investigating the master regulator, which undergoes mutations or deletions in over 50 percent of all cancers. “The precise interaction of p53 with its response elements has been studied for some 20 years, and while we have a good understanding of how p53 turns on genes, no clear answer as to the equally important question of how p53 turns off or ‘represses’ genes has emerged,” said Sir David Lane, Ph.D., a co-discoverer of p53 and now Chief Scientist at Singapore’s A*STAR (Agency for Science, Technology and Research), which oversees SIgN. “The SIgN group’s identification of a bona fide ‘repressive’ response element has provided the missing piece which has eluded p53 researchers for a long time, as well as a definitive key with which to perform future studies,” Dr. Lane added.

The findings, highlighted in the October issue of Nature Reviews Cancer and published in the Proceedings of the National Academy of Sciences in August, may allow scientists to confirm the many genes involved in the complex pathways of p53 and, potentially, to uncover new p53 pathways. The findings also clarify scientists’ understanding about the cellular pathways damaged by p53 mutations and may point to areas in the pathways where new cancer targets might be discovered. The specific sequences of nucleotides, known as response elements, that are recognized by p53 have been very difficult to decipher because they could total over one million possible combinations. In fact, predicting whether p53 actually switched a gene “on” or “off” had been an elusive goal until this recent discovery. “The findings are truly intriguing,” added Dr. Lane, who attributed the SIgN group’s success to a combination of sound thinking and the right opportunities. “I expect their findings to have very positive and significant impact on the progress of biomedical research and to help define this vital tumor-fighting pathway.”

By applying a systematic approach to analyzing known p53 response elements, the Singapore scientists succeeded in identifying a simpler two-nucleotide core sequence that was sufficient to provide an accurate prediction. Interestingly, the discovery was made by a SIgN research group, led by Ren Ee Chee, Ph.D., that focuses on immunology rather than molecular biology or genetics. “We had been studying a metastasis gene which is upregulated in liver cancer called Lasp-15,” said Dr. Ren. “As it happened to be under the control of p53, we wanted to determine in detail the role of p53. However we quickly realized that the existing literature was not helpful enough as there were ambiguities over how p53 exerts control over specific genes.” This led to the SIgN researchers’ identification of the definitive two-nucleotide sequence, and subsequent establishment of a general set of rules to predict the roles of nucleotides within a response element, which enabled them to correct those of 20 response elements (out of 162 assessed). Dr. Ren added, “Our findings illustrate how exciting science can be, when innovative discoveries can arise from unexpected sources. They are also proof that frequently in nature, what may seem very complicated at first eventually turns out to be simple and elegant.”

Science Daily
November 17, 2009

Original web page at Science Daily

A collaboration between scientists at Vanderbilt University and the University of California, San Francisco has led to the first direct information about the molecular structure of prions. In addition, the study has revealed surprisingly large structural differences between natural prions and the closest synthetic analogs that scientists have created in the lab. Prions are the infectious proteins responsible for human Creutzfeldt-Jakob disease, bovine spongiform encephalopathy, or “mad cow” disease, scrapie in sheep and several other related nervous system disorders in mammals. For a number of years, scientists have been using the tools of genetic engineering to create synthetic versions of these particles so they could study them more easily. Although researchers have made particles that appear identical to natural prions, they have had trouble duplicating their infectious behavior. “We expected to find subtle differences, but we found major differences instead,” says Gerald Stubbs, professor of biological sciences at Vanderbilt University. “Although we cannot say for certain that the differences we’ve seen can explain why natural prions are so infectious, there is a good chance that they are closely related.”

The study, which was recently published online in the Proceedings of the National Academy of Sciences, was a joint effort by the Stubbs laboratory and that of Stanley Prusiner at the University of California, San Francisco, who received the Nobel prize for the discovery of prions. “Our results will aid in attempts to create the infectious synthetic prions that are needed to figure out how prions work and ultimately to find cures for the diseases that they cause,” says the lead author of the study, Holger Wille, assistant adjunct professor of neurology in the Institute for Neurodegenerative Diseases, which is based at UCSF and directed by Prusiner. Prusiner’s group was the first one that succeeded in making infectious prions in the test tube. However, they are not nearly as infectious as the real thing. Six years ago, Prusiner contacted Stubbs, who is a world authority on determining the molecular structures of fibrous materials, and asked if he was interested in collaborating on an effort to characterize the detailed structure of prions. It didn’t take much convincing. “I’ve always been interested in prions, so I readily agreed,” says Stubbs.

Prions, because of their association with mad cow disease, are the most notorious of the amyloids, which are insoluble clumps of fibrous protein that play a role in a number of neurodegenerative diseases, including Alzheimer’s, Parkinson’s and Lou Gehrig disease, as well as some other common illnesses, including type II diabetes. “It is particularly difficult to determine the molecular structure of fibrous materials like these because they have an intrinsically high level of disorder,” Stubbs explains. When viewed with an electron microscope, which can magnify images up to one million times, the natural and synthetic prions look nearly identical. They both clump together to form microscopic filaments. At a magnification of approximately one hundred thousand times, the only visible difference is the width of the filaments: the synthetic material shows a wider distribution of widths than the natural material. The Stubbs lab used unconventional X-ray diffraction methods to get the first details of the molecular structures of natural prions and Prusiner’s synthetic prions. The researchers found that the synthetic prions were shaped something like a ladder. Based on electron microscopic images, the Prusiner lab had proposed that the natural prions have a more complex, three-sided cylindrical shape, and the X-ray experiments supported this proposal.

“The natural, infectious prions are folded into a much more complicated shape,” says Stubbs. Proteins are molecules that are folded into shapes that determine their biological properties. Prions and the other amyloids are cases in which proteins are misfolded into shapes that interfere with normal biological processes. “Normally, the cellular systems deal with misfolded proteins but, for some reason, these slip through the cracks,” he says. Prions don’t have any DNA in their make-up so they don’t reproduce in a normal fashion. Instead, they spread by transforming proteins they come into contact with into prions by causing them to misfold. “Our data on prion structure is an important step toward understanding prion infection,” says Stubbs, “and understanding the process is essential before people can design drugs that restrict or prevent it.”

Science Daily
October 20, 2009

Original web page at Science Daily

A University of Oregon research team has found that evolution can never go backwards, because the paths to the genes once present in our ancestors are forever blocked. The findings — the result of the first rigorous study of reverse evolution at the molecular level — appeared in the Sept. 24 issue of Nature. The team used computational reconstruction of ancestral gene sequences, DNA synthesis, protein engineering and X-ray crystallography to resurrect and manipulate the gene for a key hormone receptor as it existed in our earliest vertebrate ancestors more than 400 million years ago. They found that over a rapid period of time, five random mutations made subtle modifications in the protein’s structure that were utterly incompatible with the receptor’s primordial form. The discovery of evolutionary bridge burning implies that today’s versions of life on Earth may be neither ideal nor inevitable, said Joe Thornton, a professor in the UO’s Center for Ecology and Evolutionary Biology and the Howard Hughes Medical Institute.

“Evolutionary biologists have long been fascinated by whether evolution can go backwards,” Thornton said, “but the issue has remained unresolved because we seldom know exactly what features our ancestors had, or the mechanisms by which they evolved into their modern forms. We solved those problems by studying the problem at the molecular level, where we can resurrect ancestral proteins as they existed long ago and use molecular manipulations to dissect the evolutionary process in both forward and reverse directions.” Thornton’s team, which included UO research scientist Jamie Bridgham and collaborator Eric A. Ortlund, a biochemist at Atlanta’s Emory University, focused on the evolution of a protein called the glucocorticoid receptor (GR), which binds the hormone cortisol and regulates the stress response, immunity, metabolism and behavior in humans and other vertebrates. “This fascinating study highlights the value of studying evolutionary processes,” said Irene Eckstrand, who oversees evolution grants at the National Institutes of Health’s National Institute of General Medical Sciences. “By showing how molecular structures are finely tuned by evolution, Dr. Thornton’s research will have a broad impact on basic and applied sciences, including the design of drugs that target specific proteins.”

In previous work, Thornton’s group showed that the first GR evolved more than 400 millions ago from an ancestral protein that was also sensitive to the hormone aldosterone. They then identified seven ancient mutations that together caused the receptor to evolve its new specificity for cortisol. Once Thornton’s team knew how the GR’s modern function evolved, they wondered if it could be returned to its ancestral function. So they resurrected the GR as it existed soon after cortisol specificity first evolved — in the common ancestor of humans and all other vertebrates with bones — and then reversed the seven key mutations by manipulating its DNA sequence. ‘We expected to get a promiscuous receptor just like the GR’s ancestor, but instead we got a completely dead, non-functional protein,” Thornton said. “Apparently other mutations that occurred during early GR evolution acted as a sort of evolutionary ratchet, rendering the protein unable to tolerate the ancestral features that had existed just a short time earlier.” To identify the mutations, Thornton’s team prepared crystals of resurrected ancient GR proteins and took them to the particle accelerator at the Advanced Photon Source outside Chicago, where they used powerful X-rays to determine the protein’s atomic structure before and after the shift in function. By comparing the precise atomic maps of each protein, they identified five specific mutations in the later version of the GR that clashed with the architecture of the earlier protein.

“Suppose you’re redecorating your bedroom — first you move the bed, then you put the dresser where the bed used to be,” Thornton said. “If you decide you want to move the bed back, you can’t do it unless you get that dresser out of the way first. The restrictive mutations in the GR prevented evolutionary reversal in the same way.” When Thornton’s group set the five mutations back to their ancestral state, the protein could now tolerate having the seven key changes reversed, which then transformed it into a promiscuous receptor just like the its ancestor. Despite their powerful role as a ratchet preventing reversal, the five restrictive mutations had little or no direct effect on the protein’s function when they occurred. And although they must be reversed before the protein can tolerate the ancestral state, reversing them first does absolutely nothing to enhance the ancestral function. “This means that even if the ancestral function were suddenly to become optimal again, there’s no way natural selection could drive the protein directly back to its ancestral form,” Thornton said.

GR’s evolutionary irreversibility suggests that the molecules that drive our biology today may not be inevitable products of the evolutionary process. “In the GR’s case, restrictive mutations erased the conditions that previously opened up the ancestral form as an evolutionary possibility. It’s likely that throughout history other kinds of restrictive mutations have taken place, closing off innumerable trajectories that evolution might otherwise have taken,” Thornton speculated. “If we could wind back the clock and allow history to unfold again, different sets of mutations, apparently inconsequential at the time, would almost certainly occur, opening up some potential paths and blocking others — including the one that leads to the present that actually evolved in our world,” he said. “If what we observed in GR evolution is a general phenomenon, then the biology we have is just one of many possible rolls of the evolutionary dice.”

Science Daily
October 6, 2009

Original web page at Science Daily

University of Illinois at Chicago College of Medicine researchers have discovered how some recently approved drugs act against cancer cells. The finding may lead to a more effectively targeted anti-cancer strategy. In a new study reported in the journal PLoS ONE online, UIC researchers show how a class of drugs inhibits a protein called FoxM1 and suggest that the drugs’ ability to inhibit FoxM1 may account for their anti-cancer activity. FoxM1 is one of the most highly over-produced proteins in cancer cells and is believed to play an important role is causing cells to become cancerous. Because production of the protein is not usually switched on in non-dividing cells, the protein may present a promising target for anti-cancer treatments. Andrei Gartel, UIC associate professor of molecular genetics, and his colleagues had previously shown that antibiotics called thiazoles kill cancer cells and inhibit FoxM1. When they went on to investigate whether the antibiotics attacked other proteins involved in cancer, they got a surprising result. “We found that these thiazole antibiotics actually stabilized other cancer-causing proteins,” Gartel said.

It was an unexpected hint suggesting that thiazole antibiotics may act as inhibitors of the proteasome, a molecular complex that acts as a trash collector in cells, degrading old proteins that the cell has marked for destruction. This inhibition of the proteasome was confirmed in later experiments, Gartel said. Recently, a number of proteasome inhibitors have shown promise against cancer, but no one understands why they have anti-cancer effects. “We decided to see if these proteasome inhibitors, including Velcade, were, like our antibiotics, targeting FoxM1,” he said. The researchers found that the proteasome inhibitors did inhibit FoxM1 and that they also caused cells to self-destruct in the same concentrations. It’s possible, Gartel suggested, that by using thiazole antibiotics in combination with well-known proteasome inhibitors, “we may see a synergy that allows us to markedly reduce the dose of any one of these drugs and still effectively kill the cancer cells.”

Science Daily
August 25, 2009

Original web page at Science Daily

The interaction represented produced the famous explanation of the structure of DNA, but the model pictured is a stiff snapshot of idealized DNA. As researchers from Baylor College of Medicine and the University of Houston note in a report that appears online in the journal Nucleic Acids Research, DNA is not a stiff or static. It is dynamic with high energy. It exists naturally in a slightly underwound state and its status changes in waves generated by normal cell functions such as DNA replication, transcription, repair and recombination. “Many models and experiments have been interpreted with the static model,” said Dr. Lynn Zechiedrich, associate professor of molecular virology and microbiology at BCM and a senior author of the report. “But this model does not allow for the fact that DNA in real life is transiently underwound and overwound in its natural state.” DNA appears a perfect spring that can be stretched and then spring back to its original conformation. How far can you stretch it before something happens to the structure and it cannot bounce back? What happens when it is exposed to normal cellular stresses involved in doing its job? That was the problem that Zechiedrich and her colleagues tackled.

Their results also addresses a question posed by another Nobel laureate, the late Dr. Linus Pauling, who asked how the information encoded by the bases could be read if it is sequestered inside the DNA molecular with phosphate molecules on the outside. It’s easy to explain when the cell divides because the double-stranded DNA also divides at the behest of a special enzyme, making its genetic code readily readable. “Many cellular activities, however, do not involve the separation of the two strands of DNA,” said Zechiedrich. To unravel the problem, former graduate student, Dr. Graham L. Randall, mentored jointly by Zechiedrich and Dr. B. Montgomery Pettitt of UH, simulated 19 independent DNA systems with fixed degrees of underwinding or overwinding, using a special computer analysis started by Pettitt. They found that when DNA is underwound in the same manner that you might underwind a spring, the forces induce one of two bases – adenine or thymine – to “flip out” of the sequence, thus relieving the stress that the molecule experiences. “It always happens in the underwound state,” said Zechiedrich. “We wanted to know if torsional stress was the force that accounted for the base flipping that others have seen occur, but for which we had no idea where the energy was supplied to do this very big job. When the base flips out, it relieves the stress on the DNA, which then relaxes the rest of the DNA not involved in the base flipping back to its “perfect spring” state. When the molecule is overwound, it assumes a “Pauling-like DNA” state in which the DNA turns itself inside out to expose the bases — much in the way Pauling had predicted.”

Zechiedrich and her colleagues theorize that the base flipping, denaturation, and Pauling-like DNA caused by under- and overwinding allows DNA to interact with proteins during processes such as replication, transcription and recombination and allows the code to be read. And back to the idea of the “perfect spring” behavior of the DNA helix – “This notion is entirely wrong,” said Zechiedrich. “Underwinding is not equal and opposite to overwinding, as predicted, not by a long shot, that’s really a cool result that Graham got.”

Science Daily
August 11, 2009

Original web page at Science Daily

What happens if the next big influenza mutation proves resistant to the available anti-viral drugs? This question is presenting itself right now to scientists and health officials this week at the World Health Assembly in Geneva, Switzerland, as they continue to do battle with H1N1, the so-called swine flu, and prepare for the next iteration of the ever-changing flu virus. Promising new research announced by Rensselaer Polytechnic Institute could provide an entirely new tool to combat the flu. The discovery is a one-two punch against the illness that targets the illness on two fronts, going one critical step further than any currently available flu drug. “We have been fortunate with H1N1 because it has been responding well to available drugs. But if the virus mutates substantially, the currently available drugs might be ineffective because they only target one portion of the virus,” said Robert Linhardt, the Ann and John H. Broadbent Jr. ’59 Senior Constellation Professor of Biocatalysis and Metabolic Engineering at Rensselaer. “By targeting both portions of the virus, the H and the N, we can interfere with both the initial attachment to the cell that is being infected and the release of the budding virus from the cell that has been affected.”

The findings of the team, which have broad implications for future flu drugs, will be featured on the cover of the June edition of European Journal of Organic Chemistry. The influenza A virus is classified based on the form of two of its outer proteins, hemagglutinin (H) and neuraminidase (N). Each classification – for example H5NI “bird flu” or H1N1 “swine flu” – represents a different mutation of hemagglutinin and neuraminidase or H and N. Flu drugs currently on the market target only the neuraminidase proteins, and disrupt the ability of the virus to escape an infected cell and move elsewhere to infect other healthy cells. The new process developed by Linhardt is already showing strong binding potential to hemagglutinin, which binds to sialic acid on the surface of a healthy cell, allowing the virus to entire the cell.

“We are seeing promising preliminary results that the chemistry of this approach will be effective in blocking the hemagglutinin portion of the disease that is currently not targeted by any drug on the market,” he said. In addition, Linhardt and his team have shown their compound to be just as effective at targeting neuraminidase as the most popular drugs on the market, according to Linhardt. The approach can also be modified to specifically target the neuraminidase or the hemagglutinin, or both, depending on the type of mutation that is present in the current version of the flu, according to Linhardt. In the next steps of his research, Linhardt will look at how their compounds bind to hemagglutinin, and he will test the ability to block the virus first in cell cultures and then in infected animal models. “It is still early in the process,” he said. “We are several steps away from a new drug, but this technique is allowing us to move very quickly in creating and testing these compounds.”

The technique that Linhardt used is the increasingly popular technique of “click chemistry.” Linhardt is among the first researchers in the world to utilize the technique to create new anti-viral agents. The process allows chemists to join small units of a substance together quickly to create a new, full substance. In this case, Linhardt used the technique to quickly build a new derivative of sialic acid. Because it is chemically very similar to the sialic acid found on the surface of a cell, the virus could mistake the compound as the real sialic acid and bind to it instead of the cell, eliminating the connections to hemagglutinin and neuraminidase that are required for initial infection and spread of the infection in the body. The currently available drugs are translation-state inhibitors whose chemical structure allows them to only effectively target the neuraminidase.

Science Daily
June 2, 2009

Original web page at Science Daily

This three-dimensional molecular model shows a prion protein with four extra octarepeat sequences as a result of an insertional mutation in the gene. The extra sequences change the copper-binding properties of the protein. Image from Stevens et al., PLoS Pathogens (2009). An investigation of a rare, inherited form of Creutzfeldt-Jakob disease suggests that disrupted regulation of copper ions in the brain may be a key factor in this and other prion diseases. Researchers at the University of California, Santa Cruz, discovered a striking relationship between changes in the copper-binding properties of abnormal prion proteins and the clinical features of prion disease in patients with certain rare, genetic mutations. They described their findings in a paper published by PLoS Pathogens on April 17. “The loss of copper regulation may play a very important role in prion disease progression,” said Glenn Millhauser, professor of chemistry and biochemistry at UCSC and corresponding author of the paper.

Prion diseases are fatal neurodegenerative brain disorders caused by a misfolded form of the normal cellular prion protein. Human prion diseases include classic and variant types of Creutzfeldt-Jakob disease (CJD). The vast majority of CJD cases are sporadic, meaning they are thought to arise from spontaneous misfolding of the prion protein. Infectious transmission of the prion accounts for a very small percentage of cases, while about 10 percent of cases are caused by inherited defects in the structure of the prion protein. Millhauser and his coauthors studied the effects of insertional mutations that cause extra sequences of eight amino acids (known as the octarepeat sequence) to be incorporated into the prion protein. Whereas the normal prion protein has four octarepeat segments, insertional mutations can result in as many as nine additional octarepeats. The extra octarepeats change the properties of the prion protein and eventually lead to the progressive brain damage characteristic of CJD. These insertional mutations are known from a small number of cases reported in the literature, involving about 30 families and 108 individuals. Reviews of these cases have suggested that higher numbers of inserts are associated with earlier-age onset of the disease.

The octarepeat domain takes up copper ions, which are essential for the proper functioning of neurons. Millhauser’s lab looked at the effects of insertional mutations on the prion protein’s ability to bind copper. Graduate student Daniel Stevens, lead author of the paper, and postdoctoral researcher Eric Walter performed experiments using magnetic resonance spectroscopy to study how prion proteins with different numbers of octarepeats interact with copper.

The normal prion protein responds dynamically to varying concentrations of copper by changing the way it binds the metal, allowing it to soak up more copper ions at higher concentrations. When the researchers studied proteins with octarepeat inserts, however, they found that the protein loses this ability to switch binding modes as the number of inserts increases beyond four. “We got excited when we saw that the threshold in the effects on copper binding corresponds to the threshold for age of onset that was seen in the clinical studies,” Millhauser said. The average age of onset is 64 years for patients with one to four extra repeats, but for patients with five to nine inserts the average age of onset drops to 38 years. Similarly, Millhauser’s group found a transition in the copper-binding properties of the protein that occurred between four and five inserts, the same threshold observed for early-onset disease. For the statistical analysis of clinical cases, Millhauser enlisted the help of statisticians David Draper and Abel Rodriguez, professors of applied mathematics and statistics in the Jack Baskin School of Engineering at UCSC. Draper and Rodriguez used several approaches to analyze the pooled data from case studies in the literature. Their results are consistent with the existence of two groups of patients: a group with one to four extra octarepeats and late-onset disease, and a group with five or more inserts and early-onset disease.

The normal function of the prion protein remains uncertain, but the new findings support the idea that it plays a role in the regulation of copper ions in the brain, Millhauser said. The prion protein is anchored to the outside of the cell membranes of neurons and is concentrated at the synapses, the junctions between neurons where signals are transmitted. The concentration of copper in the synapses is dynamic, and as the copper concentration goes up and down the prion protein switches from one copper-binding mode to another. Millhauser suspects that the prion protein soaks up excess copper ions to protect brain cells from harmful reactions. “The prion protein goes into a neuroprotective mode at higher levels of copper, and that mode gets lost when extra octarepeats are added to the protein structure,” he said. While changes in copper binding begin to appear with four or more extra octarepeats, other changes in the molecular properties of the prion protein occur with as few as one insert. These changes include an increased propensity to clump together and form protein deposits in brain tissue. Research on prion diseases has tended to focus on these aggregates and deposits, which are thought to have toxic effects on brain cells. But the strong relationship between changes in copper binding and clinical progression of the disease suggests that more attention should be given to the normal function of the prion protein, Millhauser said. “The fundamental issue may be the loss of copper regulation, and excess copper may be what causes the cytotoxicity,” he said.

National Institute General Medical Sciences
May 4, 2009

Original web page at University California Santa Cruz

Medical physicists at the University of Virginia have created a novel way to kill tumor cells using nanoparticles and light. The technique, devised by Wensha Yang, an instructor in radiation oncology at the University of Virginia, and colleagues Ke Sheng, Paul W. Read, James M. Larner, and Brian P. Helmke, employs quantum dots. Quantum dots are semiconductor nanostructures, 25 billionths of a meter in diameter, which can confine electrons in three dimensions and emit light when exposed to ultraviolet radiation. Yang and his colleagues realized that quantum dots also give off light when exposed to megavoltage x-rays, such as those used in cancer radiotherapy. That property, the scientists realized, makes quantum dots an ideal mediator in therapies employing light-activated compounds to treat cancer. A compound called Photofrin is the only photosensitizer currently approved by the FDA. Photofrin is absorbed by cancer cells and, upon exposure to light, becomes active and kills cells.

It is currently used to treat certain kinds of shallowly located tumors, but Yang and his colleagues realized that combing Photofrin with quantum dots could create an efficient method to kill even deeply seated cancer cells. Upon exposure to high doses of radiation, the dots become luminescent and emit light; that light triggers the cancer-killing activity of the Photofrin. In theory, the process, which so far has been studied only in cancer cells grown in culture, could work on tumors located too deep within the body to be reached by an external light source. To prevent normal tissues from being affected by the treatment, the toxicity of the quantum dot-Photofrin conjugate is only activated when radiation is applied. Also, the area to be treated is targeted with conformal radiation, which is delivered with high precision within the three-dimensional contours of the tumor, with minimal spillover to surrounding healthy tissues. As a result, Yang says, “the toxicity of the drug is substantially lower in the lower radiation dose area” outside the boundaries of the tumor. In tests on human lung carcinoma cells, the process resulted in a 2-6 times lower tumor cell survival compared to radiation alone, but with minimal toxicity to nearby cells.

EurekAlert! Medicine
August 19, 2008

Original web page at EurekAlert! Medicine

Chemists in Japan report development of the world’s first DNA molecule made almost entirely of artificial parts. The finding could lead to improvements in gene therapy, futuristic nano-sized computers, and other high-tech advances, they say. As the genetic blueprint of all life forms, DNA uses the same set of four basic building blocks, known as bases, to code for a variety of proteins used in cell functioning and development. Until now, scientists have only been able to craft DNA molecules with one or a few artificial parts, including certain bases. The researchers used high-tech DNA synthesis equipment to stitch together four entirely new, artificial bases inside the sugar-based framework of a DNA molecule. This resulted in unusually stable, double-stranded structures resembling natural DNA. Like natural DNA, the new structures were right-handed and some easily formed triple-stranded structures. The unique chemistry of these structures and their high stability offer unprecedented possibilities for developing new biotech materials and applications, the researchers say.
Source: Journal of the American Chemical Society

Science Daily
July 22, 2008

Original web page at Science Daily