Preventing Cancer Development Inside the Cell Cycle

Researchers from the NYU Cancer Institute, an NCI-designated cancer center at NYU Langone Medical Center, have identified a cell cycle-regulated mechanism behind the transformation of normal cells into cancerous cells. The study shows the significant role that protein networks can play in a cell leading to the development of cancer. The study results, published in the October 21 issue of the journal Molecular Cell, suggest that inhibition of the CK1 enzyme may be a new therapeutic target for the treatment of cancer cells formed as a result of a malfunction in the cell's mTOR signaling pathway.

In the study, NYU Cancer Institute researchers examined certain multi-protein complexes and protein regulators in cancer cells. Researchers identified a major role for the multi-protein complex called SCF^βTrCP. It assists in the removal from cancer cells the recently discovered protein DEPTOR, an inhibitor of the mTOR pathway. SCF (Skp1, Cullin1, F-box protein) ubiquitin ligase complexes are responsible for the removal of unnecessary proteins from a cell. This degradation of proteins by the cell's ubiquitin system controls cell growth and prevents malignant cell transformation. Researchers show that inhibiting the ability of SCF^βTrCPto degrade DEPTOR in cells can result in blocking the proliferation of cancer cells. In addition, researchers discovered that the activity of CK1 (Casein Kinase 1), a protein that regulates signaling pathways in most cells, is needed for SCF^βTrCP to successfully promote the degradation of DEPTOR.

"Low levels of DEPTOR and high levels of mTOR activity are found in many cancers, including cancers of the breast, prostate, and lung," said senior study author Michele Pagano, MD, the May Ellen and Gerald Jay Ritter Professor of Oncology and Professor of Pathology at NYU Langone Medical Center and a Howard Hughes Medical Institute Investigator. "It is critical for researchers to better understand how the protein DEPTOR is regulated.Our study shows it would be advantageous to increase the levels of DEPTOR in many types of cancer cells to inhibit mTOR and prevent cell proliferation."

The mTOR pathway (mammalian Target Of Rapamycin) regulates the growth, proliferation, and survival of a cell, and its proper regulation is essential to prevent the formation of cancer cells. DEPTOR interrupts the mTOR pathway by binding to mTOR protein complexes and blocking their enzymatic activities, restraining cell growth. This helps support the proliferation and survival of cancer cells.

Study experiments showed that a reduction of SCF^βTrCP and CK1 proteins in cells resulted in accumulation of DEPTOR. Also, DEPTOR was destroyed in cells only when SCF^βTrCP and CK1 were both present. Thus, inhibition of SCF^βTrCP or CK1 represents a novel and promising way to inhibit the mTOR pathway. A pharmacologic inhibitor of CK1 was tested by researchers and shown to successfully stabilize DEPTOR in cells, while other pharmacological agents had no effect.

"Our study findings demonstrate that DEPTOR is regulated by the SCF^βTrCPprotein complex in cells reentering the cell cycle, and deregulation of this event could contribute to the aberrant activation of the mTOR pathway in cancer," said lead author Shanshan Duan, PhD, a post-doctoral fellow in the Department of Pathology at NYU School of Medicine in Dr. Pagano's Laboratory. "This study suggests a novel approach to stop the deregulation of the mTOR pathway in cancer cells with promising small molecule inhibitors of CK1.This study is another step forward in the translation of laboratory findings into more effective approaches to cancer prevention and treatment."

Journal References:

1. Shanshan Duan, Jeffrey R. Skaar, Shafi Kuchay, Alfredo Toschi, Naama Kanarek, Yinon Ben-Neriah, Michele Pagano. mTOR Generates an Auto-Amplification Loop by Triggering the βTrCP- and CK1α-Dependent Degradation of DEPTOR. Molecular Cell, 2011; 44 (2): 317-324 DOI: 10.1016/j.molcel.2011.09.005
2. Shanshan Duan, Jeffrey R. Skaar, Shafi Kuchay, Alfredo Toschi, Naama Kanarek, Yinon Ben-Neriah, Michele Pagano. mTOR Generates an Auto-Amplification Loop by Triggering the βTrCP- and CK1α-Dependent Degradation of DEPTOR. Molecular Cell, 21 October 2011; 44(2) pp. 317 - 324 DOI: 10.1016/j.molcel.2011.09.005

Courtesy: ScienceDaily

Antiviral Drugs May Slow Alzheimer's Progression

Antiviral drugs used to target the herpes virus could be effective at slowing the progression of Alzheimer's disease (AD), a new study shows.

The University of Manchester scientists have previously shown that the herpes simplex virus type 1 (HSV1) is a risk factor for Alzheimer's when it is present in the brains of people who have a specific genetic risk to the disease.

AD is an incurable neurodegenerative condition affecting about 18 million people worldwide. The causes of the disease or of the abnormal protein structures seen in AD brains -- amyloid plaques and neurofibrillary tangles -- are completely unknown.

The Manchester team has established that the herpes virus causes accumulation of two key AD proteins -- β-amyloid (Aβ) and abnormally phosphorylated tau (P-tau) -- known to be the main components of plaques and tangles respectively. Both proteins are thought by many scientists to be involved in the development of the disease.

"We have found that the viral DNA in AD brains is very specifically located within amyloid plaques," said Professor Ruth Itzhaki, who led the team in the University's Faculty of Life Sciences. "This, together with the production of amyloid that the virus induces, suggests that HSV1 is a cause of toxic amyloid products and of plaques.

"Our results suggest that HSV1, together with the host genetic factor, is a major risk for AD, and that antiviral agents might be used for treating patients to slow disease progression."

Currently available antiviral agents act by targeting replication of HSV1 DNA, and so the researchers considered that they might be successful in treating AD only if the accumulation of β-amyloid and P-tau accumulation caused by the virus occurs at or after the stage at which viral DNA replication occurs.

"If these proteins are produced independently of HSV1 replication, antivirals might not be effective," said Professor Itzhaki. "We investigated this and found that treatment of HSV1-infected cells with acyclovir, the most commonly used antiviral agent, and also with two other antivirals, did indeed decrease the accumulation of β-amyloid and P-tau, as well as decreasing HSV1 replication as we would expect.

"This is the first study investigating antiviral effects on AD-like changes and we conclude that since antiviral agents reduce greatly β-amyloid and P-tau levels in HSV1-infected cells, they would be suitable for treating Alzheimer's disease. The great advantage over current AD therapies is that acyclovir would target only the virus, not the host cell or normal uninfected cells. Further, these agents are very safe and are relatively inexpensive.

"Also, by targeting a cause of Alzheimer's disease, other viral damage, besides β-amyloid and P-tau, which might be involved in the disease's pathogenesis, would also be inhibited.

"The next stage of our research -- subject to funding -- will focus on finding the most suitable antiviral agent -- or combination of two agents that operate via different mechanisms -- for use as treatment. We then need to investigate the way in which the virus and the genetic risk factor interact to cause the disease, as that might lead to further novel treatments.

"Eventually, we hope to begin clinical trials in humans but this is still some way off yet and again will require new funding."

The study, carried out with Dr Matthew Wozniak and other colleagues in the Faculty of Life Sciences, is published in the Public Library of Science (PLoS) One journal.

Journal Reference:

1. Matthew A. Wozniak, Alison L. Frost, Chris M. Preston, Ruth F. Itzhaki. Antivirals Reduce the Formation of Key Alzheimer's Disease Molecules in Cell Cultures Acutely Infected with Herpes Simplex Virus Type 1. PLoS ONE, 2011; 6 (10): e25152 DOI: 10.1371/journal.pone.0025152

Courtesy: ScienceDaily

Newly Discovered Reservoir of Antibiotic Resistance Genes

Waters polluted by the ordure of pigs, poultry, or cattle represent a reservoir of antibiotic resistance genes, both known and potentially novel. These resistance genes can be spread among different bacterial species by bacteriophage, bacteria-infecting viruses, according to a paper in the October Antimicrobial Agents and Chemotherapy.

"We found great quantities of bacteriophages carrying different antibiotic resistance genes in waters with fecal pollution from pigs, cattle, and poultry," says Maite Muniesa of the University of Barcelona, Spain, an author on the study. "We demonstrated that the genes carried by the phages were able to generate resistance to a given antibiotic when introduced into other bacteria in laboratory conditions," says Muniesa.

Although we often think of antibiotic resistance genes as evolving into existence in response to the antibiotics that doctors use to fight human disease and that agribusiness uses to fatten farm animals, microbes had undoubtedly been using both antibiotics and resistance genes to compete with each other for millions of years before antibiotics revolutionized human medicine and resistance genes threatened their efficacy to the point where the World Health Organization considers them to be one of the biggest risks to human health.

Thus, the Spanish researchers suspect, based on their study, that these resistance gene reservoirs are the product of microbial competition, rather than pressure from human use of antibiotics. They note that the pasture-fed cattle in their study are not fed antibiotics, and they suggest that even if antibiotic feed additives were banned, new resistance genes might emerge while old ones spread from these reservoirs into bacteria that infect humans.

And if resistance genes are being mobilized from these reservoirs, it becomes important to understand how the resistance genes are transmitted from phage to new bacterial species, in order to develop strategies that could hinder this transmission, limiting the emergence of new resistance genes, says Muniesa.

Journal Reference:

1. M. Colomer-Lluch, L. Imamovic, J. Jofre, M. Muniesa. Bacteriophages Carrying Antibiotic Resistance Genes in Fecal Waste from Cattle, Pigs, and Poultry. Antimicrobial Agents and Chemotherapy, 2011; 55 (10): 4908 DOI: 10.1128/AAC.00535-11

Courtesy: ScienceDaily

Hidden Genetic Influence On Cancer Discovered

In findings with major implications for the genetics of cancer and human health, researchers at Beth Israel Deaconess Medical Center (BIDMC) and two other science teams in New York City and Rome have uncovered evidence of powerful new genetic networks and showed how it may work to drive cancer and normal development.

Four papers published online Oct. 14 in the journal Cell describe aspects of what may be a fundamentally new dimension of genetic activity that involves a vast posse of RNA molecules interacting and manipulating the molecular endgame behind the scenes. Each paper used a different approach, strengthening the basic discovery of the new RNA network.

In the half-century old central dogma of molecular biology, DNA issues its genetic blueprint to messenger RNA, which relays the orders to the protein-making machinery of the cell. The new studies suggest a significant new role for RNA on top of its traditional middle-management job: The RNA of one gene can control and be controlled by dozens or hundreds of RNAs of other genes.

In the case of a major tumor suppressor gene, PTEN, a shift in the associated RNA network appears to be as malevolent as a mutation in the gene itself in human prostate and colon cancer cells, in glioblastoma cells, and in a mouse model of melanoma, according to three of the papers.

The findings may enlarge the framework for investigating how tumors form and progress, who is at risk for cancer, and how to find and disable the essential misbehaving molecules that drive the growth and spread of cancer.

"For instance, we now know that the PTEN tumor suppressor gene is talking to a vast unrecognized RNA network," said Pier Paolo Pandolfi MD PhD, director of the Cancer Genetics Program at BIDMC and George C. Reisman Professor of Medicine at Harvard Medical School, and the senior author of two of the papers. "The RNAs talk through a new language. If this language is broken and the RNA network is perturbed, PTEN goes down, and this has devastating consequences. But it's incredibly exciting for therapeutic possibilities. You may be able to rewire the crosstalk between the RNAs for cancer prevention and therapy."

Scientists typically use genetic studies to probe how changes in the DNA code influence the action of the proteins. Targeted therapies have arisen from efforts to counteract the effect of problematic proteins, yet most of the genetic determinants of cancer remain a vexing puzzle. The newly discovered RNA network could explain much of the elusive genetic variation underlying cancer and other diseases, say authors of the papers.

The new RNA regulatory network also appears to extend into the massive non-protein-coding region of the human genome and plays an important role in normal muscle development, suggests another related paper in Cell. Because humans share so many protein-coding genes with other organisms, including worms and yeast, this large portion that is transcribed into non-coding RNA makes the human genome distinctive. Much of the function of that non-coding RNA has been a mystery.

"Almost all of the scientific analysis of cancer genes focuses on the protein-coding genes," Pandolfi said, referring to the two percent of the human genome where instructions are passed from DNA to RNA to proteins. "We know that nearly half of the genome is transcribed into RNA that doesn't code for protein. Through this new 'language' of RNA, we can functionalize this space."

How it works

The newly discovered network of RNA molecules converse through tiny targeted molecules called microRNAs, Pandolfi and his colleagues have found. RNAs share a vocabulary composed of specific sequences along their strands called microRNA response elements (MREs). RNAs compete for certain matching microRNAs. Once attached, microRNAs disable their host RNA molecules. It works through simple math: An increase in RNA can sponge up more microRNA, allowing other RNA to go about their business unhindered.

Scientists have known for a decade that microRNA can block RNA and prevent it from being translated into proteins. Some research has advanced to harnessing specific small microRNA molecules as experimental therapeutic tools to block individual protein-coding genes. What's new in the Cell papers is the idea of reverse logic -- that a large RNA network uses microRNA as a regulatory language.

Tantalizing hints of this newfound regulatory network have shown up in recent studies from several labs. Last year, Pandolfi's group reported that both PTEN and its nemesis, the common cancer-promoting gene KRAS, have doppelgangers known as pseudogenes in the non-coding regions of the genome, which act as decoys for targeted microRNA, greatly influencing the activity of the two cancer genes.

This August, Pandolfi and his co-authors named this RNA language and network activity "competing endogenous RNA" (ceRNA, pronounced SIR-na) in a Cell essay. The paper synthesized the emerging experimental evidence in a new theory. They proposed that ceRNA activity greatly expanded the functional genetic information in the human genome and played important roles in diseases, including cancer.

The ceRNA hypothesis adds a major new layer to the highly regulated basic players defined by the central dogma of molecular biology -- DNA, RNA and proteins. Other more established regulatory networks that keep cells healthy -- and break down in disease -- include small molecules added to proteins, such as the recycling label called ubiquitin. Another layer called epigenetics acts on the DNA and its packaging to lock or unlock certain genes.

The findings in the papers

Two of the Cell papers use a combination of bioinformatics and experimental evidence to connect the PTEN tumor suppressor gene to a network of several hundred RNA molecules in close communication.

One of the new papers from the Pandolfi lab linked about 150 new genes to the tumor suppressor PTEN in human prostate and colon cancer cell lines. Working with a collaborator at Jefferson Medical College in Philadelphia, postdoctoral fellow Yvonne Tay and her co-authors scanned the RNA transcripts of protein-coding genes based on their MRE sequences and then tested a few of the results. "Surprisingly, PTEN can be regulated by a lot of other genes through the ceRNA network," Tay said.

In an independent paper, a team in the lab of Andrea Califano at Columbia University in New York evaluated glioblastoma RNA and microRNA expression data from The Cancer Genome Atlas, a public database. They found a network in which more than 500 genes regulate PTEN. Of these, 13 are frequently deleted in glioblastoma and seem to work together through the microRNA language to squelch the tumor suppressor activity as if the tumors had mutations or deletions of PTEN itself.

"All these papers address different aspects of this compelling story and reinforce each other," said Califano, who also found RNA networks that appeared to communicate by other means. "PTEN is just an example. In each cell, different cliques of genes are connected by this microRNA-mediated network, including all the established oncogenes and tumor suppressors. This layer explains a significant amount of genetic variability in cancer. It allows genes that have nothing to do with the typical oncogene or tumor suppressor to gang up and regulate it. The discovery of this network allows us to discover genes never before associated with a tumor type or disease."

In a second paper from the Pandolfi group, mutations in the PTEN RNA network speeded up the growth of cancer in a mouse model of melanoma. Postdoctoral fellow Florian Karreth and his co-authors discovered possible new PTEN ceRNAs in a mutagenesis screen of a mouse model of melanoma. With the help of a bioinformatics team from the University of Turin, they did an in-depth analysis of one ceRNA (ZEB2) that is reduced in human cancer and verified that its reduction accelerated cancer progression in the mice. Interestingly, while the ZEB2 ceRNA opposes melanoma by sponging the microRNAs that would otherwise repress PTEN's tumor suppression activity, the ZEB2 protein is known to promote other cancers. "It is astonishing that RNA and protein molecules encoded by the same gene can take part in opposing biological processes," Karreth said.

The final study extends functional evidence of the new RNA network phenomenon to the normal differentiation of human muscle cells and to the large realm of human non-coding RNAs. Irene Bozzoni's group at the Sapienza University of Rome found that a long non-protein coding RNA works similarly as a decoy for microRNAs in normal muscle differentiation in mice and humans. In Duchenne muscular dystrophy, the decoy RNA is missing at a crucial time, preventing muscle cells from maturing.

"This explains in part why Duchenne cells have trouble, and it gives us another circuitry to attack in order to cure the disease," said Bozzoni, who heard about Pandolfi's ceRNA hypothesis at a meeting last year. "We have been working on noncoding RNA and microRNA for quite a long time. This cross-talk of RNAs through microRNAs is a revolutionary idea."

Journal References:

1. Yvonne Tay, Lev Kats, Leonardo Salmena, Dror Weiss, Shen Mynn Tan, Ugo Ala, Florian Karreth, Laura Poliseno, Paolo Provero, Ferdinando Di Cunto, Judy Lieberman, Isidore Rigoutsos, Pier Paolo Pandolfi. Coding-Independent Regulation of the Tumor Suppressor PTEN by Competing Endogenous mRNAs. Cell, 2011; 147 (2): 344-357 DOI: 10.1016/j.cell.2011.09.029
2. Florian A. Karreth, Yvonne Tay, Daniele Perna, Ugo Ala, Shen Mynn Tan, Alistair G. Rust, Gina DeNicola, Kaitlyn A. Webster, Dror Weiss, Pedro A. Perez-Mancera, Michael Krauthammer, Ruth Halaban, Paolo Provero, David J. Adams, David A. Tuveson, Pier Paolo Pandolfi. In Vivo Identification of Tumor- Suppressive PTEN ceRNAs in an Oncogenic BRAF-Induced Mouse Model of Melanoma. Cell, 2011; 147 (2): 382-395 DOI: 10.1016/j.cell.2011.09.032
3. Pavel Sumazin, Xuerui Yang, Hua-Sheng Chiu, Wei-Jen Chung, Archana Iyer, David Llobet-Navas, Presha Rajbhandari, Mukesh Bansal, Paolo Guarnieri, Jose Silva, Andrea Califano. An Extensive MicroRNA-Mediated Network of RNA-RNA Interactions Regulates Established Oncogenic Pathways in Glioblastoma. Cell, 2011; 147 (2): 370-381 DOI: 10.1016/j.cell.2011.09.041
4. Marcella Cesana, Davide Cacchiarelli, Ivano Legnini, Tiziana Santini, Olga Sthandier, Mauro Chinappi, Anna Tramontano, Irene Bozzoni. A Long Noncoding RNA Controls Muscle Differentiation by Functioning as a Competing Endogenous RNA. Cell, 2011; 147 (2): 358-369 DOI: 10.1016/j.cell.2011.09.028

Courtesy: ScienceDaily

Understanding the Beginnings of Embryonic Stem Cells Helps Predict the Future

Scientists have shown that laboratory-grown cells express a protein called Blimp1, which represses differentiation to somatic or regular tissue cells during germ cell development. Studies of these cells show that they also express other genes associated with early germ cell specification.

Ordinarily, embryonic stem cells exist only a day or two as they begin the formation of the embryo itself. Then they are gone.

In the laboratory dish, however, they act more like perpetual stem cells -- renewing themselves and exhibiting the ability to form cells of almost any type, a status called totipotency.

Dr. Thomas Zwaka, associate professor in the Stem Cell and Regenerative Medicine Center at Baylor College of Medicine, and his colleagues here and abroad showed that laboratory-grown cells express a protein called Blimp1, which represses differentiation to somatic or regular tissue cells during germ cell development. Studies of these cells show that they also express other genes associated with early germ cell specification.

A report on their work published online October 13 in the journal Current Biology. It will appear in the October 25 print edition of the journal.

"What are embryonic stem cells?" said Zwaka, who is also part of the Center for Cell and Gene Therapy at BCM, Texas Children's Hospital and The Methodist Hospital. "It is quite a surprise that we have them. In the embryo, there is a mass of cells that eventually form the embryo, but they do not persist. They do not have a program built in that allows them to persist."

To study this, he examined mice. If you put the mass of cells in a Petri dish in the laboratory, they act as thought they are stem cells with the ability for self renewal and totipotency -- the ability to become almost any kind of cell.

Understanding what happens early in development of embryonic stem cells in the laboratory might help make the process of growing them and another, new kind of stem cell called induced pluripotent stem cells -- cells with the potential of becoming many different kinds of tissues that are derived from somatic or adult cells.

"These induced pluripotent stem cells are poorly understood," said Zwaka. "If we know what is happening when we derive embryonic stem cells in the laboratory, it will inform us when we make induced pluripotent stem cells. The end product is similar."

The process of making the induced pluripotent stem cells is noisy and random, he said.

"Every time, the clones look different and emerge at different time points," said Zwaka. By contrast, embryonic development is like clockwork, with events occurring at the same point with each embryo. However, development of embryonic stem cells in the laboratory becomes more disorganized as time goes on.

In the laboratory dish, the mouse embryo continues to develop at a fairly organized rate for two or three days, but when the single cells are separated and grown singly, the embryonic stem cells begin to emerge. Only a tiny subset -- roughly 1 percent -- of the cells become an embryonic stem cell in the laboratory."

"We found that these cells (from the embryonic stem cells come) resemble in almost every feature an early germ cell (primordial germ cell)," he said. (Primordial germ cells are the source of gametes -- eggs and sperm.)

"It seems that these seeming germ cells are the cells that make the embryonic stem cells in culture," he said.

"Germ cells in the embryo are unique and pluripotent (able to become many different kinds of cells) and have a very sophisticated program in them that protects the from becoming somatic cells (specific tissue cells)," he said. "They retain their primitive state." Blimp1 is a master regulator of germ cells.

In the future, he said, he hopes that investigators in both fields can collaborate and learn from one another.

Others who took part in this research include Li-Fang Chua of BCM, M. Azim Surani of the Wellcome Trust Cancer Research UK Gurdon Institute at the University of Cambridge, and Rudolf Jaenisch of Whitehead Institute for BiomedicaI Research at the Massachusetts Institute of Technology in Cambridge.

Funding for this work came from the Huffington Foundation and the National Institutes of Health.

For more information on basic science research at Baylor College of Medicine, please go to From the Lab at Baylor College of Medicine.

Journal Reference:

1. Li-Fang Chu, M. Azim Surani, Rudolf Jaenisch, Thomas P. Zwaka. Blimp1 Expression Predicts Embryonic Stem Cell Development In Vitro. Current Biology, 13 October 2011 DOI: 10.1016/j.cub.2011.09.010

Courtesy: ScienceDaily

Uncharted Territory: Scientists Sequence the First Carbohydrate Biopolymer

DNA and protein sequencing have forever transformed science, medicine, and society. Understanding the structure of these complex biomolecules has revolutionized drug development, medical diagnostics, forensic science, and our understanding of evolution and development. But, one major molecule in the biological triumvirate has remained largely uncharted: carbohydrate biopolymers.

Today, for the first time ever, a team of researchers led by Robert Linhardt of Rensselaer Polytechnic Institute has announced in the October 9 Advanced Online Publication edition of the journal Nature Chemical Biology the sequence of a complete complex carbohydrate biopolymer. The surprising discovery provides the scientific and medical communities with an important and fundamental new view of these vital biomolecules, which play a role in everything from cell structure and development to disease pathology and blood clotting.

The paper is titled "The proteoglycan bikunin has a defined sequence."

"Carbohydrate biopolymers, known as glycosaminoglycans, appear to be really important in how cells interact in higher organisms and could explain evolutionary differences and how development is driven. We also know that carbohydrate chains respond to disease, injury, and changes in the environment," said Linhardt, who is the Ann and John H. Broadbent Jr. '59 Senior Constellation Professor of Biocatalysis and Metabolic Engineering at Rensselaer. "In order to understand how and why this all happens, we first need to know their structure. And today, at least for the simplest glycosaminoglycan structure, we can now do this."

The first glycosaminoglycan sequenced was obtained from bikunin. Bikunin is a proteoglycan, a protein to which a single glycosaminoglycan chain is attached. Unlike less sophisticated carbohydrate biopolymers, such as starch and cellulose, the proteoglycans are decorated with structurally complex carbohydrates that enable them to perform more sophisticated and defined roles in the body. Bikunin, for example, is a natural anti-inflammatory that is used as a drug for the treatment of acute pancreatitis in Japan. It has the simplest chemical structure of any proteoglycan. Linhardt views the discovery of the structure of bikuin as the first step on the ladder to the discovery of the structure of more complex proteoglycans.

"The first genome sequences of DNA were on the simplest organisms such as bacteria. Once the technology was developed it ultimately led to the sequencing of the human genome," he said. "In our efforts to sequence carbohydrate biopolymers we don't yet know if the defined structure we observe for this simple protoglycan will hold for much more complex proteoglycans."

But, looking for structure in more complex proteoglycans will be among the next steps in the research for Linhardt and his team. The search for structure could help put to rest a long-running debate in the scientific community as to whether complex carbohydrate biopolymers require a defined structure to function.

"Despite all that is known about glycan formation, our understanding has not yet been deep enough to infer sequence or even determine if sequence occurs," Linhardt said. "These findings represent a new way of looking at these complex biomolecules as ordered structures."

Linhardt's research into carbohydrate sequencing began 30 years ago. In his previous work, he determined that some order existed in at least a portion of some carbohydrate biopolymers, but it did not represent the entire finished puzzle.

"Previously, we could see a pattern, but we could not see if all the chains were playing the same music. The tools did not yet exist. Now we can recognize it as a symphony."

To uncover the entire structure, Linhardt and his team, which was led by his doctoral student Mellisa Ly, borrowed a technique from the field of protein research called the proteomics top-down approach. As opposed to the bottom-up approach that first breaks apart a complex biopolymer into pieces and then rebuilds it piece by piece like a jigsaw puzzle, the top-down approach used by Linhardt and colleagues allows the researcher to picture the whole intact puzzle. This can only be accomplished with some of the most sophisticated technology available to the scientific community today, including very high-powered mass spectrometers.

Linhardt used a mass spectrometer located in the Rensselaer Center for Biotechnology and Interdisciplinary Studies (CBIS) to make his initial discoveries, and had these results independently confirmed on a separate and higher-level spectrometer at the University of Georgia. Mass spectrometers break down a molecule into separate charged particles or ions. These ions can then be categorized and analyzed based on their mass-to-charge ratio. These ratios then allow for sequencing of the entire molecule.

"This was truly the convergence of really sophisticated spectroscopy and its application to biology," Linhardt said. "We were fortunate to have a lot of time to play with the instrument at CBIS to understand its capabilities."

Beyond the technology it also took faith and determination. According to Linhardt, "It takes a student that is willing to try something even when the odds are pretty low. If it doesn't work, you make incremental progress. If it does work, you can make a great discovery. But, from the beginning you need to be a believer that it is worth taking the chance because it takes a lot of hard work in the lab."

And the odds weren't in Linhardt's favor. Despite being the most simple of proteoglycans, there were still 290 billion different possible sequences for the molecule.

"The first sample we looked at, we got the structure," Linhardt said. "In the end we did 15 chains and they all came back playing the same exact symphony."

The research is funded by the National Institutes of Health.

Linhardt and Ly were joined in the research by Tatiana Laremore of Rensselaer; Franklin Leach and Jonathan Amster of the University of Georgia; and Toshihiko Toida of Chiba University in Japan.

Journal Reference:

1. Mellisa Ly, Franklin E Leach, Tatiana N Laremore, Toshihiko Toida, I Jonathan Amster, Robert J Linhardt. The proteoglycan bikunin has a defined sequence. Nature Chemical Biology, 2011; DOI: 10.1038/nchembio.673

Courtesy: ScienceDaily