By studying the intricate mechanisms at work in protein production, a Princeton-led team has discovered why certain kinds of antibiotics are so effective. In doing so, they also have discovered how one protein protects against cell death, shedding light on a natural cancer-fighting process.

In a study appearing in the Aug. 7 edition of the journal Science, Thomas Silhavy, Princeton’s Warner-Lambert Parke-Davis Professor of Molecular Biology, and Johna van Stelten, a graduate student, working with two Swiss researchers have uncovered how some antibiotics in common use for 50 years — tetracycline and chloramphenicol — can be so lethal against certain strains of bacteria.

Simply put, these drugs plug things up.

Silhavy and van Stelten had been studying the mechanism by which proteins — from antibodies to hormones — are produced in bacteria’s cytoplasm, the gooey substance that makes up the cell’s interior, and then transported where they are needed. The spaghetti-like proteins exit the bacteria’s cytoplasm through microscopic tubes known as translocators.

Sometimes, proteins fold up accidentally and jam the translocator. “Proteins go through the translocator, like a piece of spaghetti through a hole,” Silhavy said. “But if you can imagine if you were to tie knots in the spaghetti, it wouldn’t be able to get through; it gets stuck.”

What happens then is ugly, according to Silhavy and van Stelten, who were the first ever to observe the event.

The bacterial cell actually attacks the jammed translocator, decimating it.

The researchers wondered what might happen in a more complex scenario, such as if antibiotics were introduced into the cell cytoplasm to purposely thwart bacteria.

The scientists found that the antibiotics tetracycline and chloramphenicol cause the ribosomes, a cell’s protein-producing machines, to stop midway through the process of making proteins, leaving partially constructed proteins stuck to the ribosome, jamming the translocator in the bacteria.

“This is very similar to plugging the translocator with a folded protein and, sure enough, this also causes translocator destruction,” Silhavy said. “It’s like putting an anchor on the spaghetti instead of a knot. They are stuck and dead forever.”

Researchers had been confused as to why these antibiotics seemed to be so adept at killing some kinds of bacteria more quickly than others. These experiments provide an explanation. Translocators are essential for life and, if some bacteria have fewer translocators from the start, then they are more vulnerable to such an attack.

“While it has been known for many years that these antibiotics work by inhibiting bacterial protein synthesis, it was not clear why some bacteria in a population appeared more susceptible than others,” van Stelten said. “Our work has identified a new reason why these antibiotics are lethal to bacteria that may help explain these earlier findings.”

The researchers made their discovery not because of a new piece of equipment or a new technique. “Like the vast majority of advancements in science and medicine, we happened upon this remarkable answer through basic research,” van Stelten said.

The finding could have important implications for medicine.

“If we are to have any hope of outpacing the antibiotic resistance obtained by bacteria, it is paramount that we fully understand the mechanism of action of the antibiotics we currently use,” van Stelten said. “Unfortunately, this is often very difficult as evidenced by the fact that, 50 years on, we are still learning new things about them.”

Their work also produced another important result. When the translocators in bacteria became jammed by errant proteins, the researchers observed that the translocators emitted a molecular signal — a stress response — that called in a destructive enzyme known as the FtsH protease. Under normal circumstances, the FtsH protease chops up the jammed translocators, contributing to cell death.

The scientists found, however, that when they increased the amount of YccA, a protein that is present in the bacterial cell, YccA proteins protected the translocators from the FtsH attackers. YccA, it turns out, is very similar to a human protein known as Bax Inhibitor-1 (BI-1) that is of great interest to cancer researchers because cancer proliferates when it malfunctions.

“We have determined how YccA works in preventing stress-induced death in bacteria,” van Stelten said. “We hope this new information will shed light on the mechanism of BI-1 in humans.”

Other researchers on the paper included Filo Silva and Dominique Belin from the University of Geneva in Switzerland.

The work was supported by the National Institute of General Medical Sciences of the National Institutes of Health, the New Jersey Commission on Cancer Research, the Canton de Geneve and the Swiss National Science Foundation.

Source:
Kitta MacPherson

Princeton University

A research team led by U of T Mississauga scientists has developed a new way to split up a dangerous pair of cancer proteins, a finding that could ultimately lead to chemotherapy that is more effective and has fewer side effects.

Working with scientists at the University of Central Florida and the Princess Margaret Hospital, Professor Patrick Gunning of the Department of Chemical and Physical Sciences has created several molecules that inhibit Stat3, a protein that–in cancer cells–pairs with another copy of itself and goes haywire. The findings appear in the September issue of the journal ChemBioChem: A European Journal of Chemical Biology.

“The molecules we have created are particularly nice because they’re showing selectivity against cancer cells but not against healthy cells,” says senior author Gunning. “This molecule could be used in conjunction with typical chemotherapeutics, and it could mean that drugs will have less resistance-so you could use lower dosages and cause fewer side effects.”

The Stat3 protein is involved in almost all cancers, and is known to contribute to the resistance of cancer cells to current drug therapies. “Most currently available therapeutics aim to induce cell death,” says Gunning. “We wanted to make small molecules that could try and stop this protein.”

In cancerous cells, Stat3 proteins bind together to work as a lethal pair, and inhibitors work to prevent this. This type of protein-protein interaction is notoriously difficult to counter. Gunning’s team targeted binding “hotspots” on a known Stat3 inhibitor called S3I-201. They chemically altered the inhibitor to produce several new variants, which they then tested on Stat3.

In in vitro studies, some variants proved to be even more powerful than S3I-201, and showed activity against prostate, breast and acute myeloid leukemia cancer cell lines. “These are some of the most potent inhibitors in the literature so far for this particular protein,” says Gunning. “In some cases, they were more than twice as effective as the existing inhibitor.”

When the team used more complex cancer cell models, they found the inhibitors survived the passage across the cell membrane and still targeted the Stat3 cancer proteins inside. Gunning and his colleagues are working to make the new inhibitors even more effective, as well as more metabolically stable, meaning that they can survive the chemical defense mechanisms within the cell.

The Leukemia and Lymphoma Society of Canada, the University of Toronto and the National Institutes of Health funded the study. Gunning’s team is currently studying the use of their new inhibitors alongside traditional chemotherapy drugs.

Other researchers at Rush involved in the study were Lynda Powell, PhD, Dr. Rasa Kazlauskaite and Dr. Sheila Dugan.

The study was funded by the National Institutes of Health and the Charles J. and Margaret Roberts Trust.

Source:
Nicolle Wahl

University of Toronto

Wei Zheng, M.D., Ph.D., M.P.H., cancer epidemiologist with Vanderbilt-Ingram Cancer Center, Nashville, Tenn., has received a prestigious MERIT Award from the National Institutes of Health (NIH) for his research on women and cancer. The MERIT (Method to Extend Research in Time) awards provide long-term support to investigators with impressive records of scientific achievement in research areas of special importance or promise. Fewer than five percent of NIH-funded investigators are selected to receive MERIT awards, which provide financial support for up to 10 years without competitive review.

“I am very excited to receive this award and the award is recognition of the teamwork involved in our research,” said Zheng, Ingram Professor of Cancer Research and director of the Vanderbilt Epidemiology Center. “Epidemiological studies require a multidisciplinary team and I am privileged to work with so many talented, dedicated people at Vanderbilt and many other institutions.”

The MERIT award will support continuation of the Shanghai Women’s Health Study, a population-based study of 75,000 women who were recruited between 1997 and 2000 with a major focus to identify associations between diet and lifestyle and diseases such as cancer.

Zheng and his team are studying the impact of soy foods, tea, ginseng and cruciferous vegetables on cancer risk and health. In addition to answering detailed surveys, the women provide blood and urine samples for identification of exposure to dietary influences as well as potential disease biomarkers.

“Many studies have looked at diet and lifestyle factors that are bad for individuals but we are trying to find things that are protective against disease,” said Zheng. “These observational studies may allow us to gather enough evidence to launch a large trial that tests the effects of some of these compounds.”

Zheng and his investigative team also are conducting genome-wide association studies, scanning the entire genome for disease susceptibility biomarkers. They are studying telomeres, DNA copy number variations, prostaglandin metabolites and other biomarkers that may be important in cancer and other disease processes.

“Wei Zheng is an outstanding cancer investigator and mentor to many of our young researchers,” said Bill Blot, Ph.D., associate director of Cancer Prevention, Control and Population-Based Research at Vanderbilt-Ingram. “The MERIT award recognizes the importance of this high-impact research on international health.”

Zheng said the award also validates the contributions of the thousands of women in the study who donate their time as well as biological samples to provide answers to important health questions. He hopes this and other related studies will have a major impact on cancer and disease prevention.

“One of our goals is to build a risk assessment model for breast cancer that will allow us to identify those women at high risk for the disease for cost-effective prevention,” Zheng said.

Source
The Vanderbilt-Ingram Cancer Center