'Moonlighting' Molecules Discovered; Researchers Uncover New Kink In Gene Control

Since the completion of the human genome sequence, a question has baffled researchers studying gene control: How is it that humans, being far more complex than the lowly yeast, do not proportionally contain in our genome significantly more gene-control proteins?Now, a collaborative effort at the Johns Hopkins School of Medicine to examine protein-DNA interactions across the whole genome has uncovered more than 300 proteins that appear to control genes, a newly discovered function for all of these proteins previously known to play other roles in cells. The results, which appear in the October 30 issue of Cell, provide a partial explanation for human complexity over yeast but also throw a curve ball in what we previously understood about protein functions.

"Everyone knows that transcription factors bind to DNA and everyone knows that they bind in a sequence-specific manner," says Heng Zhu, Ph.D., an assistant professor in pharmacology and molecular sciences and a member of the High Throughput Biology Center. "But you only find what you look for, so we looked beyond and discovered proteins that essentially moonlight as transcription factors."

The team suspects that many more proteins encoded by the human genome might also be moonlighting to control genes, which brings researchers to the paradox that less complex organisms, such as plants, appear to have more transcription factors than humans. "Maybe most of our genes are doing double, triple or quadruple the work," says Zhu. "This may be a widespread phenomenon in humans and the key to how we can be so complex without significantly more genes than organisms like plants."

The team set out to figure out which proteins encoded by the genome bind to which DNA sequences. It had been predicted by examining the human genome sequence that about 1,400 to 1,700 of encoded proteins are so-called transcription factors -- proteins that bind to specific sequences in DNA to turn a gene on or off. The researchers also included in their study, in addition to these proteins, other types that are known to maintain chromosome structure and bind to structurally different RNA. Also included were proteins that normally relay information within a cell and are not thought to directly come in contact with DNA. In total, they collected nearly 4,200 human proteins together on a protein microarray, or protein "chip."

To identify proteins on that chip that bound DNA directly, the group first reviewed previously published scientific literature and catalogued 460 different, short sequences of DNA that are known or predicted to bind proteins.

One at a time, the team tested each of the 460 DNA sequences against the 4,200 protein-containing chip. In addition to finding many protein-DNA interactions for transcription factors, some confirming previously known interactions, the team found 367 new unconventional DNA binding proteins -- proteins known to do other cellular jobs.

"This nearly doubled the number of known protein-DNA interactions," says Jiang Qian, Ph.D., an assistant professor of ophthalmology at Hopkins. "But we only looked at about a fifth of all the proteins in the human genome -- there could be hundreds, even thousands more of these unconventional transcription factors that we don't yet know about."

One of the unconventional transcription factors discovered was the protein MAP Kinase 1, also known as ERK2, a protein long studied for its ability to control cell growth and development via its ability to add phosphate groups to other molecules.

"It's one of the best studied proteins out there, but no one ever thought ERK2 could directly regulate gene expression by actually binding to DNA," says Seth Blackshaw, Ph.D., an assistant professor of neuroscience and a member of the High Throughput Biology Center and the Neuroregeneration Program at the Institute for Cell Engineering.

To be certain that ERK2 really does bind DNA and control genes in living cells, the team tested the protein in human cells. They found that ERK2 mutated to no longer bind DNA causes specific genes to be turned on, while both normal ERK2 and ERK2 that's no longer able to chemically modify proteins turn off those same genes. "It clearly acts to repress specific genes," says Blackshaw. "Maybe this will help clear up some of the puzzles that have arisen in ERK2 experiments over the years."

A central question in understanding how genes are controlled is hich of the 20,000 proteins encoded by our genome act on which segments of DNA. "It's not possible to predict this a priori," Blackshaw says. "Someone has to do the experiment -- because we just don't know enough about how proteins bind to DNA -- patterns have surfaced in this field's 45 year history, but not enough yet to establish any rules."

This study was funded by the National Institutes of Health, a National Eye Institute Vision Core grant, a W. M. Keck Foundation Distinguished Young Investigator in Medical Research Award, a grant from the Ruth and Milton Steinbach Fund and a generous gift from Mr. and Mrs. Robert and Clarice Smith

Authors on the paper are Shaohui Hu, Zhi Xie, Akishi Onishi, Xueping Yu, Lizhi Jiang, Jimmy Lin, Hee-Sool Rho, Crystal Woodard, Hong Wang, Jun-Seop Jeong, Shunyou Long, Xiaofei He, Herschel Wade, Blackshaw, Qian, and Zhu, all of Johns Hopkins.

Bodybuilding With Steroids Damages Kidneys

Athletes who use anabolic steroids may gain muscle mass and strength, but they can also destroy their kidney function, according to a paper being presented at the American Society of Nephrology's 42nd Annual Meeting and Scientific Exposition in San Diego, CA. The findings indicate that the habitual use of steroids has serious harmful effects on the kidneys that were not previously recognized.Reports of professional athletes who abuse anabolic steroids are increasingly common. Most people know that using steroids is not good for your health, but until now, their effects on the kidneys have not been known. Leal Herlitz, MD (Columbia University Medical Center) and her colleagues recently conducted the first study describing injury to the kidneys following long-term abuse of anabolic steroids. The investigators studied a group of 10 bodybuilders who used steroids for many years and developed protein leakage into the urine and severe reductions in kidney function. Kidney tests revealed that nine of the ten bodybuilders developed a condition called focal segmental glomerulosclerosis, a type of scarring within the kidneys. This disease typically occurs when the kidneys are overworked. The kidney damage in the bodybuilders has similarities to that seen in morbidly obese patients, but appears to be even more severe.

When the bodybuilders discontinued steroid use their kidney abnormalities improved, with the exception of one individual with advanced kidney disease who developed end-stage kidney failure and required dialysis. Also, one of the bodybuilders started taking steroids again and suffered a relapse of severe kidney dysfunction.

The researchers propose that extreme increases in muscle mass require the kidneys to increase their filtration rate, placing harmful levels of stress on these organs. It's also likely that steroids have direct toxic effects on the kidneys. "Athletes who use anabolic steroids and the doctors caring for them need to be aware of the potentially serious risks to the kidney," said Dr. Herlitz.

This study was conducted in the laboratory of Dr. Vivette D'Agati, MD at Columbia Univeristy Medical Center. Study co-authors include Glen Markowitz, MD, Joshua Schwimmer, MD, Michael Stokes, MD, Cheryl Kunis, MD, Vivette D'Agati, MD, (Columbia University Medical Center); Alton Farris, MD, and Robert Colvin, MD (Massachusetts General Hospital).

The study abstract, "Development of FSGS Following Anabolic Steroid Use in Bodybuilders," (TH-PO163) will be presented as part of a Poster Session during the American Society of Nephrology's 42nd Annual Meeting and Scientific Exposition on Oct. 29 in the Scientific Exposition Hall of the San Diego Convention Center in San Diego, CA.

Just One Cigarette Has Harmful Effect On Arteries Of Young Healthy Adults

Even one cigarette has serious adverse effects on young adults, according to research presented by Dr. Stella Daskalopoulou at the Canadian Cardiovascular Congress 2009, co-hosted by the Heart and Stroke Foundation and the Canadian Cardiovascular Society.Her study found that smoking one cigarette increases the stiffness of the arteries in 18 to 30 year olds by a whopping 25 per cent.

Arteries that are stiff or rigid increase resistance in the blood vessels, making the heart work harder. The stiffer the artery, the greater the risk for heart disease or stroke.

"Young adults aged 20-24 years have the highest smoking rate of all age groups in Canada," says Dr. Daskalopoulou, an internal medicine and vascular medicine specialist at McGill University Health Centre. "Our results are significant because they suggest that smoking just a few cigarettes a day impacts the health of the arteries. This was revealed very clearly when these young people were placed under physical stress, such as exercise."

The study compared the arterial stiffness of young smokers (five to six cigarettes a day) to non-smokers. The median age was 21 years. Arterial measurements were taken in the radial artery (in the wrist), the carotid artery (in the neck), and in the femoral artery (in the groin), at rest and after exercise.

Arterial stiffness in both smokers and non-smokers was measured using a new but well established method called applanation tonometry. Dr. Daskalopoulou introduced the 'arterial stress test' which measures the arteries' response to the stress of exercise. The test is comparable to a cardiac stress test, which measures the heart's response to the stress of exercise.

"In effect we were measuring the elasticity of arteries under challenge from tobacco," explains Dr. Daskalopoulou.

An initial arterial stress test was carried out to establish a baseline measurement for both the non-smokers and the smokers, who were asked not to smoke for 12 hours prior to the test. After the first meeting, smokers returned and smoked one cigarette each and then repeated the stress test. During the final meeting, smokers were asked to chew a piece of nicotine gum prior to the stress test.

Dr. Daskalopoulou found that after exercise the arterial stiffness levels in non-smokers dropped by 3.6 per cent. Smokers, however, showed the reverse: after exercise their arterial stiffness increased by 2.2 per cent. After nicotine gum, it increased by 12.6 per cent. After one cigarette, it increased by 24.5 per cent.

Interestingly, there was no difference in the arterial stiffness measurements between smokers and non-smokers at rest.

"In effect, this means that even light smoking in otherwise young healthy people can damage the arteries, compromising the ability of their bodies to cope with physical stress, such as climbing a set of stairs or running to catch a bus," says Dr. Daskalopoulou. "It seems that this compromise to respond to physical stress occurs first, before the damage of the arteries becomes evident at rest."

"More than 47,000 Canadians will die prematurely each year due to tobacco use, which often starts in the teen years," warns Heart and Stroke Foundation spokesperson Dr. Beth Abramson. "We know that over 90 per cent of teenagers who smoke as few as three to four cigarettes a day may be trapped into a lifelong habit of regular smoking, which typically lasts 35 to 40 years."

Smoking contributes to the build up of plaque in the arteries, increases the risk of blood clots, reduces the oxygen in the blood, increases blood pressure, and makes the heart work harder. Smoking also nearly doubles the risk of ischemic stroke.

Dr. Abramson says this study reinforces the importance of education, prevention programs, and legislation such as the recently passed Bill C-32, Cracking Down on Tobacco Marketing Aimed at Youth Act.