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Atomic-scale view of bacterial proteins offers path to new tuberculosis drugs

Source: Rockefeller University, Newswire
Date: 02/03/17
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With the first detailed analysis of a cellular component from a close relative of the pathogen that causes tuberculosis, Rockefeller scientists are suggesting strategies for new drugs to curb this growing health problem. Each year, nearly half a million people around the world are infected with mutant TB strains capable of evading existing antibiotics.

The research, conducted by a Rockefeller team led by Elizabeth Campbell, in collaboration with scientists at Memorial Sloan Kettering, focuses on a cluster of interacting proteins called RNA polymerase. Crucial to all cells, this protein machine carries out a fundamental process in which genes within the DNA blueprint are copied into RNA. The RNA polymerase is the target of the antibiotic rifampicin—a lynchpin of modern TB treatment, which relies on a combination of drugs. Some bacteria become resistant to rifampicin by acquiring RNA polymerase mutations.

“Now that we can visualize the molecular machinery of the bacteria that the drug targets, we can use a structure-guided approach to better understand how the drug works, how bacteria become resistant to it, and how to potentially improve the drug’s action,” says Campbell, a senior research associate in Seth A. Darst’s Laboratory of Molecular Biophysics. She is one of the senior authors of a report published in the online journal eLife.

A molecular map

To visualize the structure the researchers used an imaging method known as x-ray crystallography. By crystallizing enzymes and other molecules interacting with each other—essentially freezing them in action—investigators are able to see how they fit together, much like keys fitting into locks. This ability to visualize what’s going on can point the way toward more effective drugs, which may be able to latch more securely onto enzymes and other molecules.

“Based on the findings reported in this study,” Campbell says, “we’re already investigating new compounds with new mechanisms of action that appear to inhibit the rifampicin-resistant version of TB. Our eventual goal is to get them into clinical trials investigating new treatments for TB, including rifampicin-resistant TB.”

Rockefeller chemist Sean F. Brady, who was not directly involved in the study, provided the team with these new compounds. He is now working together with Campbell, Darst, and other colleagues to further develop them into antibiotics and characterize the basis of their activity.

Not all bacteria are alike

It’s estimated that up to one-third of the world’s population is infected with M. tuberculosis, the bacterium that causes TB. In the study, the researchers worked with a closely related strain called M. smegmatis. “We needed hundreds of liters of cells to get enough of the material to do the crystallization,” says Elizabeth Hubin, a former Rockefeller graduate student who carried out much of the work. “M. tuberculosis grows too slowly to be able to collect the volume that’s needed, and it’s very dangerous to work with in the lab.”

But M. smegmatis relies on an RNA polymerase that is almost identical in sequence, structure, and behavior to the M. tuberculosis RNA polymerase, which led to another important finding in the study: The RNA polymerase from Escherichia coli, the bacterium most commonly used in lab research, is not. This means there may be a drawback to relying on E. coli as a model when developing certain types of antibiotics for bacteria that cause TB or other diseases.

“Most of the studies previously done with RNA polymerase were done using E. coli,” Campbell says. “We’ve always assumed that the enzyme works the same way in all bacteria, but our study shows we can’t assume what’s found in one bacteria applies to all bacteria.

“Every pathogen needs to be studied individually,” she adds, “so the field has a lot of work to do.”

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Study Uncovers Hidden Risk for Breast Cancer in Some Women

Source: Memorial Sloan Kettering blog, On Cancer
Date: 03/17/17
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The connection between obesity and some kinds of cancer, including breast cancer, is well established. Not only is obesity a risk factor for developing the disease, it’s also a poor prognostic indicator for people who are diagnosed — those who are obese tend to have worse outcomes than those who are not.A handful of recent studies have suggested that it’s possible to be physically lean but metabolically obese — meaning that certain conditions in your body may bring about the risks that come with obesity even if you have a normal body mass index (BMI).

Now, a new study led by teams from Memorial Sloan Kettering and Weill Cornell Medicine indicates for the first time that this so-called metabolic obesity in normal-weight women can increase the likelihood that they will develop breast cancer.

“We call these women the walking wounded. They’re currently not recognized as being unhealthy,” says MSK breast medical oncologist and researcher Neil Iyengar, the first author of the study, published in the journal Cancer Prevention Research.Andrew Dannenberg, senior author of the study and Associate Director of Cancer Prevention at the Meyer Cancer Center of Weill Cornell Medical College, adds, “Physicians currently cannot reliably detect which patients who have a normal BMI may have underlying metabolic obesity that could be putting them at risk for cancer.”

Linking Fat and Cancer

Drs. Iyengar and Dannenberg investigate the biological mechanisms by which obesity promotes cancer. Earlier studies from their group have looked at breast cancer and tongue cancer.

One of the things the team studies is inflammation in the fat tissue and how it may drive cancer. The condition can lead to the release of various substances including hormones and growth factors that stimulate the development of cancer cells. These factors also serve as fuel for the growth of existing cancer cells.

Several cancers have been linked to obesity, including uterine cancer, colorectal cancer, and prostate cancer, though breast cancer has one of the strongest connections. “The breast is an organ that could be particularly susceptible to fat tissue dysfunction because the tissue where breast cancer most commonly arises — the milk ducts — is surrounded by fat,” Dr. Iyengar notes.

“One observation that caught our attention in our initial studies was that there was a group of patients with breast cancer who were not considered obese by BMI, but who had the same biological changes in their tissues that we see in obese patients,” he adds.

He and his collaborators set out to determine how common this problem was.

A Surprising Discovery

The current study included 72 women with normal BMIs who were undergoing mastectomy at MSK. Most had breast cancer, but some were having preventive surgery. The women donated blood samples as well as their breast tissue, so the researchers could look for changes in their bodies at the time of surgery.

The investigators were surprised to find that 39% of these women had inflammation in the fat tissue of their breasts. They also had higher-than-normal levels of aromatase, the enzyme that makes estrogen, in their breast tissue. Increased levels of estrogen in the breast could directly stimulate the development of breast cancer, Dr. Iyengar says.

“Interestingly, when we looked at the blood, we found that these women with fat inflammation and elevated aromatase had insulin resistance, which can be thought of as a precursor to diabetes. They also had higher triglyceride levels and other inflammatory changes,” he adds. “These are the kinds of changes we normally see in obese patients.”

These findings suggest that the obesity epidemic is much greater than currently recognized and includes some women of normal weight. The investigators believe that a link is also likely to exist between metabolic obesity and cancer in men.

Developing a New Method of Diagnosis

So now the question is, how would a woman know if she falls into this category?

Currently, the only way to detect inflammation in fat tissue is by looking at tissue that comes from a biopsy or surgery, Dr. Iyengar explains, procedures that are invasive and not typically employed on a regular basis.

His team is currently undertaking new studies to look for other ways to diagnose metabolic obesity. These methods may include scans that could measure a person’s body composition or blood tests that could be conducted during a routine physical. He says these tests eventually could become a part of regular wellness checkups and health screenings, and not just for those with cancer.

The researchers feel a sense of urgency about developing these new tests. “Right now these patients go to the doctor, who looks at their normal BMI and says, ‘Keep up the good work,’” Dr. Dannenberg says. “But in fact they may have undetected metabolic obesity that could put them at risk not only for cancer but also for diabetes, high blood pressure, heart disease, and other kinds of diseases.”

Dr. Iyengar adds that a lack of physical activity or eating an unhealthy diet may contribute, but it’s too early to know for sure. “This is an active area of research and is reshaping the way we think about who is healthy and who is not,” he says.

“Once we can more easily and reliably detect who has metabolic obesity, our goal is to ultimately develop interventions — such as specific diets, exercise programs, or even medications — that are tailored to decrease the risk of cancer and other disorders related to obesity,” he concludes.

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Tardigrades use unique protein to protect themselves from desiccation

Source: Cell Press
Date: 03/16/17
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Tardigrades, the microscopic animals also known as water bears and moss piglets, have captured the imagination of scientists for almost 250 years, thanks to their Muppet-like appearance and their ability to survive extreme environments that would destroy most other living things. One of these skills is the ability to endure being dried out for up to a decade or longer. In Molecular Cell on March 16, a team of scientists report that this knack for survival is due to a unique set of proteins they dubbed tardigrade-specific intrinsically disordered proteins (TDPs).

“The big takeaway from our study is that tardigrades have evolved unique genes that allow them to survive drying out,” says Thomas Boothby, the Life Sciences Research Foundation Postdoctoral Fellow at the University of North Carolina, Chapel Hill, and the study’s first author. “In addition, the proteins that these genes encode can be used to protect other biological material–like bacteria, yeast, and certain enzymes–from desiccation.”

For a long time, it was assumed that a sugar called trehelose gave tardigrades the ability to tolerate desiccation. Trehelose is found in a number of other organisms that can survive being dried out, including yeast, brine shrimp, and some nematodes. But biochemical studies of tardigrades have found trehelose at low levels or not at all, and sequencing has not revealed the gene for the enzyme required to make this sugar.

“The question has been, ‘If tardigrades aren’t relying on trehelose to survive desiccation, what do they use instead?'” Boothby says. He and his team set out to discover how they do it.

The first step of the research was to look at which genes were active under various conditions: unstressed, drying out, and frozen. The researchers identified genes that were upregulated and expressed at high levels when the animals began to dry out. The proteins that these genes encode, the TDPs, are in a class of proteins called intrinsically disordered proteins (IDPs). Unlike most proteins, IDPs have no fixed three-dimensional structure.

After they found the TDP genes expressed at high levels during the drying-out period in one species of tardigrade, the team looked at two other species and found the same genes. One species, which had the genes turned on all the time, is able to survive drying out much more quickly that the others. “We think it can do this because it has so many of these proteins around already and doesn’t need time to make them,” Boothby says.

To verify that these TDPs were what gave tardigrades their unique abilities, the researchers put the genes encoding them into yeast and bacteria, and confirmed that the TDPs protected these other organisms.

Trehelose helps other organisms to survive drying out by forming glass-like solids when they dry, rather than crystals. Boothby and his colleagues found that TDFs form similar glass-like solids, and showed that when the glassiness of TDPs was disrupted, it correlated with a loss of their protective abilities.

Boothby says TDPs have a number of potential uses, including protecting crops from drought and safeguarding medications that normally require cold storage. “Being able to stabilize sensitive pharmaceuticals in a dry state is very important to me personally,” he says. “I grew up in Africa, where lack of refrigeration in remote areas is a huge problem. These real-world applications are one of the things that led me to study tardigrades.”