A new intermediate step and unexpected enzymatic activity in a metabolic pathway in the body, which could lead to new drug design for psychiatric and neurodegenerative diseases, has been discovered by researchers at Georgia State University.

The research team has been studying a metabolic pathway called the tryptophan kynurenine pathway, which is linked to psychiatric and neurodegenerative disorders, including depression, anxiety, Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, AIDS dementia complex, asphyxia in newborns and epilepsy. The medical potential of this pathway warrants detailed study to provide information about the pathway’s enzymes and their regulation.

This pathway produces several neurotransmitter regulators and is responsible for metabolizing nearly 99 percent of the tryptophan in the body. Tryptophan is a precursor of serotonin, the neurotransmitter responsible for mood.

The researchers determined the structure and mechanism of an enzyme in the kynurenine pathway, AMSDH.

To better understand the rapid chemical reaction catalysed by this enzyme, Dr. Aimin Liu, professor in the Department of Chemistry and core member of the Center for Diagnostics and Therapeutics at Georgia State, organized a research team, including graduate students Lu Huo, Ian Davis, Fange Liu and Shingo Esaki, and researchers at Brookhaven National Laboratory and Kansai University in Osaka, Japan. They used new scientific techniques, including time-lapse crystallography and single-crystal spectroscopy, to slow down the reaction rate by nearly 10,000 times. This allowed them to observe a new intermediate step, the thiohemiacetal intermediate, and discover an unexpected isomerase activity in AMSDH.

‘By doing this, we find new chemistry, and we also open up avenues for others to design specific drugs to target this pathway,’ Liu said. ‘This pathway is highly associated with neurodegenerative diseases and depression.’

The researchers took a high concentration of the purified protein, grew single crystals, mixed them with their substrate and froze them at different time points in liquid nitrogen at 77 Kelvin to stop all molecular activity. They sent the crystals to Argonne National Laboratory for remote data collection. The X-ray diffraction patterns collected there were used to create an electron density map, a 3-D, atomic-level resolution of the molecule’s shape. The researchers used time-lapse crystallography and single-crystal spectroscopy to observe intermediate steps of the reaction.

‘This is the first absorbance spectrum of this intermediate,’ Davis said. ‘When we look for this in solution assays, we don’t see this absorbance band because this intermediate is very short-lived in solution. But by doing it in crystal and freezing it down, you can actually see it in the crystalline state.

‘Enzymes work by stabilizing reactive intermediates. Through this isomerization mechanism, we found a new reactive intermediate stabilized by this enzyme. So if you want to design a drug, your best bet is to try and make something that looks very similar to this so that it will bind to the enzyme. That’s a general strategy for drug design. You want to try and make drugs that look very similar to transition states. Basically, we found a new transition state in this work.’

Information from the study has been deposited in the protein database, which can be accessed by other scientists. EurekAlert

Researchers at UT Southwestern Medical Center have identified hyaluronon (HA) as a critical substance made by the body that protects against premature births caused by infection. Pre-term birth from infection is the leading cause of infant mortality in many countries according to the World Health Organization. The findings of the study are the first to identify the specific role that HA plays in the reproductive tract.

Dr. Yucel Akgul, first author and senior author Dr. Mala Mahendroo
“We found that HA is required to allow the epithelial lining of the reproductive tract to serve as the first line of defence against bacterial infections,” said senior author Dr. Mala Mahendroo, an Associate Professor in the Department of Obstetrics and Gynecology’s Cecil H. and Ida Green Center for Reproductive Biology Sciences. “Because of this action, HA offers cervical protection against the bacterial infections that cause 25 to 40 percent of pre-term births in women.”

Hyaluronon is a natural substance found in many tissues, and is both a lubricant and a beneficial component of eyes, joints, and skin. It has long been thought to play an essential role in increasing the cervix’s flexibility during the birth process; however, the study, which was conducted using mouse models, showed that HA is not essential for increased cervical pliability during late pregnancy. Rather, the substance plays an important barrier role in epithelial cells of the lower reproductive tract and in so doing protects against infection-related pre-term birth. The World Health Organization estimates that 1.09 million children under age 5 die from direct complications of being born prematurely, meaning before the 37th week of pregnancy.

Previous studies by UT Southwestern reproductive biology researchers showed that HA is present in both the cervix and cervical mucus of pregnant women. Next steps include determining the mechanism by which HA affects cervical protection against infection.

“This study demonstrates that HA plays a crucial role in the epithelial barrier as well as the cervix’s mucus,” said Dr. Yucel Akgul, first author of the study and research scientist in the Department of Obstetrics and Gynecology. “Our next step is to identify exactly how HA protects the cervix, which can have important clinical implications in the effort to reduce infection-mediated pre-term labour.” UT Southmwestern Medical Center

Genetic mutations may link smoking and alcohol consumption to destruction of the pancreas observed in chronic pancreatitis, according to a 12-year study led by researchers at the University of Pittsburgh School of Medicine. The findings provides insight into why some people develop this painful and debilitating inflammatory condition while most heavy smokers or drinkers do not appear to suffer any problems with it.

The process appears to begin with acute pancreatitis, which is the sudden onset of inflammation causing nausea, vomiting and severe pain in the upper abdomen that may radiate to the back, and is typically triggered by excessive drinking or gallbladder problems, explained senior investigator David Whitcomb, M.D., Ph.D., chief of gastroenterology, hepatology and nutrition, Pitt School of Medicine. Up to a third of those patients will have recurrent episodes of acute pancreatitis, and up to a third of that group develops chronic disease, in which the organ becomes scarred from inflammation.

“Smoking and drinking are known to be strong risk factors for chronic pancreatitis, but not everyone who smokes or drinks damages their pancreas,” Dr. Whitcomb said. “Our new study identifies gene variants that when combined with these lifestyle factors make people susceptible to chronic pancreatitis and may be useful to prevent patients from developing it.”

In the North American Pancreatitis Study II consortium, researchers evaluated gene profiles and alcohol and smoking habits of more than 1,000 people with either chronic pancreatitis or recurrent acute pancreatitis and an equivalent number of healthy volunteers. The researchers took a closer look at a gene called CTRC, which can protect pancreatic cells from injury caused by premature activation of trypsin, a digestive enzyme inside the pancreas instead of the intestine, a problem that has already been associated with pancreatitis.

They found that a certain variant of the CTRC gene, which is thought to be carried by about 10 percent of Caucasians, was a strong risk factor for alcohol- or smoking-associated chronic pancreatitis. It’s possible that the variant fails to protect the pancreas from trypsin, leaving the carrier vulnerable to ongoing pancreatic inflammation and scarring.

“This finding presents us with a window of opportunity to intervene in the diseases process,” Dr. Whitcomb said. “When people come to the hospital with acute pancreatitis, we could screen for this gene variant and do everything possible to help those who have it quit smoking and drinking alcohol, as well as test new treatments, because they have the greatest risk of progressing to end-stage chronic pancreatitis.” University of Pittsburgh Health Sciences

A new study identifies a gene that is especially active in aggressive subtypes of breast cancer. The research suggests that an overactive BCL11A gene drives triple-negative breast cancer development and progression.

The research, which was done in human cells and in mice, provides new routes to explore targeted treatments for this aggressive tumour type.

There are many types of breast cancers that respond differently to treatments and have different prognoses. Approximately one in five patients is affected by triple-negative breast cancer; these cancers lack three receptor proteins that respond to hormone therapies used for other subtypes of breast cancer. In recent years it has become apparent that the majority of triple-negative tumours are of the basal-like subtype.

Although new treatments are being explored, the prognosis for triple-negative cancer is poorer than for other types. To date, only a handful of genomic aberrations in genes have been associated with the development of triple-negative breast cancer.

The team looked at breast cancers from almost 3000 patients. Their search had a particular focus: they examined changes to genes that affect the behaviour of stem cells and developing tissues, because other work they have done suggests that such genes, when mutated, can often drive cancer development. Among these was BCL11A.

‘Our understanding of genes that drive stem cell development led us to search for consequences when these genes go wrong,’ says Dr Pentao Liu, senior author on the study, from the Wellcome Trust Sanger Institute. ‘BCL11A activity stood out because it is so active in triple-negative cancers.

‘It had all the hallmarks of a novel breast cancer gene.’

Higher activity of the BCL11A gene was found in approximately eight out of ten patients with basal-like breast cancer and was associated with a more advanced grade of tumour. In cases where additional copies of the BCL11A gene were created in the cancer, the prospects for survival of the patient were diminished.

‘Our gene studies in human cells clearly marked BCL11A as a novel driver for triple-negative breast cancers,’ says Dr Walid Khaled, joint first author on the study from the Wellcome Trust Sanger Institute and University of Cambridge. ‘We also showed that adding an active human BCL11A gene to human or mouse breast cells in the lab drove them to behave as cancer cells. Wellcome Trust Sanger Institute

Despite a strong suspected link between genetics and asthma, commonly found genetic mutations account for only a small part of the risk for developing the disease — a problem known as missing heritability.

Rare and low frequency genetic mutations have been thought to explain missing heritability, but it appears they are unlikely to play a major role, according to a new study led by scientists from the University of Chicago. Analysing the coding regions of genomes of more than 11,000 individuals, they identified mutations in just three genes that were associated with asthma risk. Each was associated with risk in specific ethnicities.

‘Previous studies have likely overestimated the heritability of asthma,’ said study senior author Carole Ober, PhD, Blum-Riese Professor and chair of the Department of Human Genetics at the University of Chicago. ‘This could be because those estimates are based on correlations between family members that share environment as well as genes, which could inflate the heritability. Gene-environment interactions are not considered in these large scale association studies, and we know that these are particularly important in establishing individual risks for asthma.’

Asthma affects more than 25 million adults and children of all ages and ethnicities in the US. Due to the widespread nature of the disease, most studies of its genetic underpinnings have focused on commonly occurring mutations, referred to as genetic variants. However, while numerous such variants have been identified, they are able to account for only a small proportion of the risk for inheriting or developing asthma. Rare mutations, found in less than five percent of the population, have been hypothesized to explain this disparity.

Graduate student Catherine Igartua led the analysis under the supervision of co-senior author Dan Nicolae, PhD, Professor in the Departments of Medicine, Statistics and Human Genetics. She evaluated nearly 33,000 rare or low frequency mutations in more than 11,000 individuals of a variety of ethnicities representing European, African and Latino backgrounds. She analysed mutations jointly across subjects, using a technique that allowed them to study mutations common in one ethnicity, but rare in others.

Only mutations in the genes GRASP, GSDMB and MTHFR showed a statistical link to asthma risk. Mutations in the first two genes were found primarily in Latino individuals, and mutations in the last gene in those with African ancestry. These genes, involved in protein scaffolding, apoptosis regulation and vitamin B9 metabolism respectively, have as yet unknown roles in asthma. The rarity and ethnic-specificity of these genes is insufficient to account for the widespread prevalence of asthma.

Although rare mutations might not contribute much to population asthma risk, these genes still have the potential to serve as targets for therapeutic development. Ober points to the discovery of rare mutations in the LDL receptor that eventually led to the development of statins to treat high cholesterol. She also notes that it is possible, but unlikely, that there are mutations with large effects on asthma risk outside of their screen as it looked at approximate 60 percent of mutations in coding regions of the genome.

‘It was assumed that there would be rare mutations with larger effect sizes than the common variants we have been studying,’ Ober said. ‘Surprisingly, we found that low frequency mutations explain only a very small amount of asthma risk.’ The University of Chicago Medicine