Caltech researchers increase the potency of HIV-battling proteins

Posted by Biomechanism 

If one is good, two can sometimes be better. Researchers at the California Institute of Technology (Caltech) have certainly found this to be the case when it comes to a small HIV-fighting protein.

The protein, called cyanovirin-N (CV-N), is produced by a type of blue-green algae and has gained attention for its ability to ward off several diseases caused by viruses, including HIV and influenza. Now Caltech researchers have found that a relatively simple engineering technique can boost the protein’s battling prowess.

Daniel Kavanagh of Massachusetts General Hospital in the U.S. will test a class of molecular probes for their ability to identify cells latently infected with HIV. If successful, these probes could be used to target and eliminate these HIV-infected cells.

“By linking two cyanovirins, we were able to make significantly more potent HIV-fighting molecules,” says Jennifer Keeffe, a staff scientist at Caltech and first author of a new paper describing the study in the Proceedings of the National Academy of Sciences (PNAS). “One of our linked molecules was 18 times more effective at preventing infection than the naturally occurring, single protein.”

The team’s linked pairs, or dimers, were able to neutralize all 33 subtypes of HIV that they were tested against. The researchers also found the most successful dimer to be similar or more potent than seven well-studied anti-HIV antibodies that are known to be broadly neutralizing.

CV-N binds well to certain carbohydrates, such as the kind found in high quantities connected to the proteins on the envelope that surrounds the HIV virus. Once attached, CV-N prevents a virus from infecting cells, although the mechanism by which it accomplishes this is not well understood.

What is known is that each CV-N protein has two binding sites where it can bind to a carbohydrate and that both sites are needed to neutralize HIV.

Once the Caltech researchers had linked two CV-Ns together, they wanted to know if the enhanced ability of their engineered dimers to ward off HIV was related to the availability of additional binding sites. So they engineered another version of the dimers—this time with one or more of the binding sites knocked out—and tested their ability to neutralize HIV.

It turns out that the dimers’ infection-fighting potency increased with each additional binding site—three sites are better than two, and four are better than three. The advantages seemed to stop at four sites, however; the researchers did not see additional improvements when they linked three or four CV-N molecules together to create molecules with six to eight binding sites.

Although CV-N has a naturally occurring dimeric form, it isn’t stable at physiological temperatures, and thus mainly exists in single-copy form. To create dimers that would be stable under such conditions, the researchers covalently bound together two CV-N molecules in a head-to-tail fashion, using flexible polypeptide linkers of varying lengths.

Interestingly, by stabilizing the dimers and locking them into a particular configuration, it seems that the group created proteins with distances between binding sites that are very similar to those between the carbohydrate binding sites in a broadly neutralizing anti-HIV antibody.

“It is possible that we have created a dimer that has its carbohydrate binding sites optimally positioned to block infection,” says Stephen Mayo, Bren Professor of Biology and Chemistry, chair of the Division of Biology, and corresponding author of the new paper.

Because it is active against multiple disease-causing viruses, including multiple strains of HIV, CV-N holds unique promise for development as a drug therapy. Other research groups have already started investigating its potential application in prophylactic gels and suppositories.

“Our hope is that those who are working to make prophylactic treatments using cyanovirin will see our results and will use CVN2L0 instead of naturally occurring cyanovirin,” Keeffe says. “It has higher potency and may be more protective.”

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The paper, entitled “Designed oligomers of cyanovirin-N show enhanced HIV neutralization,” was published in the online edition of PNAS. In addition to Keeffe and Mayo, other authors on the paper include research technician Priyanthi N.P. Gnanapragasam, former biology graduate student Sarah K. Gillespie, biology graduate student John Yong, and Pamela J. Bjorkman, the Max Delbruck Professor of Biology at Caltech and a Howard Hughes Medical Institute investigator.

The work was funded by the National Security Science and Engineering Faculty Fellowship program, the Defense Advanced Research Projects Agency Protein Design Processes program, and the Bill and Melinda Gates Foundation through the Grand Challenges in Global Health Initiative.

Cancer researchers look to dogs to better understand intricacies of bone cancer

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CANCER RESEARCH: A team led by Dr. Jaime Modiano, a College of Veterinary Medicine and Masonic Cancer center expert in comparative medicine, discovered a gene pattern that distinguishes the more severe form of bone cancer from a less aggressive form in dogs. Dogs are the only other species besides humans that develops this disease spontaneously with any frequency.

Dogs are much more likely to develop bone cancer than humans, but according to Modiano – who specializes in the relationship between animal and human disease – human and canine forms of bone cancer are very similar and the gene pattern is an exact match.

“Our findings pave the way to develop laboratory tests that can predict the behavior of this tumor in dogs and children at the time of diagnosis,” said Dr. Jaime Modiano, College of Veterinary Medicine and Masonic Cancer Center expert in comparative medicine. “This allows us to tailor individualized therapy to meet the patient’s needs.”

“Patients with less aggressive disease could be treated conservatively, reducing the side effects and the risks associated with treatment, while patients with more aggressive disease could be treated with more intense therapy,” said Modiano.

This new University of Minnesota discovery may help bone cancer patients fight their disease more effectively, according to new research published in the September issue of Bone.

Bone cancer typically affects children; the course and aggressiveness of the disease can vary from patient to patient and is very difficult to predict. Some patients respond remarkably well to conventional therapies. Their disease shows less aggressive behavior and they can survive for decades without recurrence. Others respond poorly to treatment or their disease comes back rapidly. Often, these patients survive less than five years.

Recently, a team led by Dr. Jaime Modiano, a College of Veterinary Medicine and Masonic Cancer Center expert in comparative medicine, discovered a gene pattern that distinguishes the more severe form of bone cancer from a less aggressive form in dogs. Dogs are the only other species besides humans that develops this disease spontaneously with any frequency.

In fact, dogs are much more likely to develop bone cancer than humans, but according to Modiano – who specializes in the relationship between animal and human disease – human and canine forms of bone cancer are very similar and the gene pattern is an exact match. The discovery of this key differentiating signature may be beneficial in the treatment planning of human bone cancer patients.

“Our findings pave the way to develop laboratory tests that can predict the behavior of this tumor in dogs and children at the time of diagnosis,” said Modiano. “This allows us to tailor individualized therapy to meet the patient’s needs.”

The downstream impact of the findings

University of Minnesota researchers hope to use their findings to develop practical and useful lab tests for humans and for companion animals that will help clinical care providers determine the type of cancer a patient faces, and how aggressive that cancer may be.

Then, depending on which type of cancer a patient has, clinicians could adjust interventions and treatment plans accordingly.

“Patients with less aggressive disease could be treated conservatively, reducing the side effects and the risks associated with treatment, while patients with more aggressive disease could be treated with more intense therapy,” said Modiano.

The study was funded by the National Cancer Institute, the AKC Canine Health Foundation and the Kate Koogler Canine Cancer Fund.

Scientists map attack tactics of plant pathogens

Posted by Biomechanism 

(“Biomechanism.com“) — Every year, plant diseases wipe out millions of tons of crops, lead to the waste of valuable water resources and cause farmers to spend tens of billions of dollars battling them.

Now a new discovery from a University of North Carolina at Chapel Hill-led research team may help tip the war between plants and pathogens in favor of flora.

A leaf infected by a pathogen called an oomycete. The oomycete gains entry into the leaf's intracellular spaces through natural openings and then grows by extending hyphae (filaments) between cells. When the hyphae fill up the leaf, the oomycete releases the next generation of infectious spores (the white tree-like structures emerging from the leaf surface. Oomycetes cause downy mildew diseases of many plants; one was responsible for the Irish Potato Famine and another for Sudden Oak Death Syndrome. Credit: Petra Epple, Dangl Lab, UNC-Chapel Hill.

The finding – published in the July 29, 2011, issue of the journal Science – suggests that while pathogens employ a diverse arsenal of weapons, they use these to attack plants by honing in on a surprisingly limited number of cellular targets.

“This is a major advance in understanding the biomechanisms involved in the ongoing evolutionary battle between plants and pathogens,” said Jeff Dangl, Ph.D., the study’s lead author and John N. Couch Professor of Biology in the College of Arts and Sciences.

The new finding is one of two studies published concurrently in Science related to the first comprehensive plant “interactomes” – maps of the tens of thousands of interactions that link a cell’s proteins. Those connections govern how proteins assemble into complex functional machines that dictate the tasks a cell can perform, such as growth, division and response to light, water and nutrients. And these same machines are often recruited into the battle against infectious agents.

One of the new studies mapped the interactome for about a third of the proteins encoded by the genome of the plant Arabidopsis thaliana, or thale cress. Arabidopsis is widely used for research purposes as a model organism – similar to the way mice are used in medical research – because of traits that make it useful for understanding the workings of many other plant species.

A leaf infected by an oomycete pathogen. Special feeding structures, called haustoria, bulge from the pathogen's hyphae (filaments) into the inside of the plant cells (the purple balloon-like structures inside the clear-colored individual cells). Credit: Petra Epple, Dangl Lab, UNC-Chapel Hill.

Dangl’s group led an additional study incorporating that interactome data with the construction of a second interactome. The second map focused on understanding how two very different pathogens (the bacteria Pseudomonas syringae and the oomycete parasite Hyaloperonospora arabidopsidis) infect plants and how plants fight back.

One method that these pathogens, which live in between cells, use for successful infection is to deploy virulence proteins (known as effectors) into the plant cell. The effectors muzzle the host’s defenses and allow the pathogen to hijack the plant’s cellular machinery.

In the new study, Dangl and his collaborators at institutions including Harvard University, the Salk Institute in La Jolla, Calif., and the University of Warwick, U.K., found that these two pathogens have evolved to focus their effectors onto a limited set of roughly 165 interconnected proteins that act in cellular machines in Arabidopsis cells – despite the fact that they last shared a common ancestor over 2 billion years ago, and use vastly different biomechanisms to colonize plants.

“This likely means that to suppress host plants’ defenses, all plant pathogens have evolved weapons that focus on a relatively small group of cellular machines,” Dangl said. “Knowing this should facilitate faster breeding for disease resistance and development of environmentally sustainable treatments for many devastating plant diseases.”

He said that neither interactome is a complete map, and more work needs to be done fully identify which protein networks are targeted by pathogens.

“We’ve found the needles in the haystack, but we still have to comb through another two-thirds of the hay,” said Dangl. “Our data suggest that there will be only a few hundred targets for effectors from all pathogens, out of the roughly 27,000 proteins encoded in the whole Arabidopsis genome.”

Academic scientists, seed breeders and biotech companies interested in these proteins will benefit from freely available data from both interactomes. The findings also could have implications for human health research.

“Professor Dangl and colleagues have used a powerful combination of network theory and laboratory experimentation to develop an approach to understanding the evolutionary logic by which pathogens and their hosts interact,” said James Anderson, Ph.D., who oversees regulatory biology grants at the National Institutes of Health. “While this study focused on plants, the results illustrate the value of model organisms in revealing fundamental principles that help us understand human responses to infectious diseases and provide the basis for devising new therapeutic strategies.”

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The study, “Independently Evolved Virulence Effectors Converge onto Hubs in a Plant Immune System Network,” was co-written by researchers from more than a dozen institutions.

The research in Dangl’s lab was funded by the National Institute of General Medical Sciences, part of the National Institutes of Health; the National Science Foundation’s Arabidopsis 2010 Program; and the Department of Energy.