Forum for Science, Industry and Business

Sponsored by:     3M 
Search our Site:

 

Forty's a crowd

29.06.2012
'Paper of the week' shows that a master regulator protein brings plethora of coactivators to gene expression sites

Molecular geneticists call big boss proteins that switch on broad developmental or metabolic programs "master regulators," as in master regulators of muscle development or fat metabolism. One such factor, the Activating Transcription Factor 6£ (ATF6£) protein, takes charge following a cellular crisis known as endoplasmic reticulum (ER) stress, which is triggered by the accumulation of misfolded and aggregated proteins.

Molecularly, the ER stress pathway is always poised for action. Inactive ATF6£ is normally embedded in cellular membranes, but at the first hint of protein overload, its working end springs superman-like into the nucleus, binds DNA and kicks on a host of target genes whose job is to clear a protein logjam.

Now, in a study published in the June 29 issue of The Journal of Biological Chemistry, and selected as "Paper of the Week" by the journal's editors, a team led by Stowers investigators Ron and Joan Conaway reveal that unlike the real superman ATF6£ does not work solo. Using the ATF6£ target gene HSPA5 as a probe, they apply mass spectrometry analysis to show that ATF6£ recruits a fleet of coactivators to assist in target activation.

"We knew that as a master regulator, ATF6£ was needed to turn on downstream genes in the ER stress response," says Ron Conaway, Ph.D., who with Joan Conaway, Ph.D., is co-corresponding author of the study. "Our goal was to determine what ATF6£ was bringing with it to these genes' control elements."

"By devising a clever mix of state-of-the-art mass spectrometry and good old-fashioned biochemistry, this study has revealed that ATF6£ is a virtual magnet for a wide range of 'A-list' co-regulators," said Michael K. Reddy, Ph.D., who oversees transcription mechanism grants at the National Institutes of Health's National Institute of General Medical Sciences, which partly supported the work. "These co-regulators offer a large array of proteins to target in efforts to control the ER stress response and to treat diseases that result from misfolded proteins."

That task of identifying co-regulators was challenging: labor-intensive molecular techniques the group applied to identify candidate interactors early on were not sensitive enough. At that point, the Conaways turned to their frequent collaborators Proteomics' Center director, Michael Washburn, Ph.D., and Laurence Florens, Ph.D., who heads the Stowers proteomics cores. Both had helped develop a sensitive mass spectrometry approach that can detect protein-protein interactions in highly complex mixtures, a technology known as MudPIT.

The group then set up a test-tube comparison. They genetically engineered a strand of DNA flanking the HSPA5 target gene, the so-called "enhancer" region recognized by ATF6£. They then dipped two identical DNA test strips into respective pots of cellular extracts¡Xone containing ATF6£ and one not¡Xreasoning that factors in the ATF6£ entourage would be recruited to the first but not the second. They then applied a single run of MudPIT to identify each ATF6£-specific partner.

In short, they found that it takes not a village but a metropolis to activate an ATF6£ target. Many proteins bound the enhancer in both samples, meaning either that they're just background, or else that they must bind DNA even when the gene is inactive. But more than 40 were present in about 5-fold excess only in ATF6£ƒ{spiked samples, suggesting they are tethered to the enhancer by ATF6£.

Among the latter were components of a multi-subunit behemoth protein known as Mediator, which bridges specific genetic switches (like ATF6£) and the catalytic machinery that copies a gene. Other proteins recruited by ATF6£ through overlapping but not identical domains belonged to other large complexes known as SAGA and ATAC, which enzymatically relax chromosome structure to allow gene expression.

Researchers know that all DNA-binding factors partner with other proteins to switch genes on or off. What is remarkable here is their sheer number. "It would be very interesting to find out whether this is the norm," says Ron Conaway. "This work raises a ton of little questions about mechanism."

Among them is how do ATF6£-interacting factors arrange themselves on the test strip, and does a single ATF6£ bind to all of them at once? "There are three separate ATF6£ binding sites on the HSPA5 enhancer and ATF6£ itself forms a dimer," explains Dotan Sela, Ph.D., a Conaway lab postdoc and the study's first author, "So potentially within this region there could be as many six activation domains," he explains.

Solving these puzzles could reveal molecular targets for seemingly unrelated diseases. While a little ATF6£ signaling is absolutely essential for cellular housekeeping, unrelieved ER stress is a hallmark of neurodegenerative conditions like Alzheimer's and Huntington's Diseases and is correlated with insulin insensitivity and type II diabetes.

A direct role for ATF6£ in what some now call "misfolded protein diseases" is unclear. Nonetheless, the study suggests ways to dampen ER stress signaling molecularly. "We show that the Mediator is relevant to HSPA5 expression," says Sela. "So one way to keep ATF6£ from turning on a gene might be to devise ways to block binding of the Mediator to ATF6£."

Joan Conaway also points out that MudPIT data analysis does not require previous identification of a "suspect." "Our approach complements methods that test candidate interactors one by one," says Joan Conaway. "Because the analysis is unbiased, it could reveal novel proteins interacting with a particular enhancer, which then could be confirmed using other methods."

The Conaways began their pioneering studies of mammalian gene expression over three decades ago, when only laborious biochemical techniques were available. As a result, both deeply appreciate what a technological leap the current work represents. "This study provides proof of principle for the utility of mass spectrometry in defining novel transcriptional activators," says Ron Conaway. "We want to compare this data with that from other activators¡Xit's what we will be working on in the future."

In addition to Washburn and Florens, Lu Chen of the Conaway lab and Skylar Martin-Brown of the Washburn lab also contributed to the work.

Funding for the study came from the Stowers Institute for Medical Research, the National Institute of General Medical Science (GM041628) and the Helen Nelson Medical Research Fund at the Greater Kansas City Community Foundation.

About the Stowers Institute for Medical Research

The Stowers Institute for Medical Research is a non-profit, basic biomedical research organization dedicated to improving human health by studying the fundamental processes of life. Jim Stowers, founder of American Century Investments, and his wife, Virginia, opened the Institute in 2000. Since then, the Institute has spent over 900 million dollars in pursuit of its mission.

Currently, the Institute is home to nearly 550 researchers and support personnel; over 20 independent research programs; and more than a dozen technology-development and core facilities.

Kristin Kessler | EurekAlert!
Further information:
http://www.stowers.org

More articles from Life Sciences:

nachricht Rising water temperatures could endanger the mating of many fish species
03.07.2020 | Alfred-Wegener-Institut, Helmholtz-Zentrum für Polar- und Meeresforschung

nachricht Moss protein corrects genetic defects of other plants
03.07.2020 | Rheinische Friedrich-Wilhelms-Universität Bonn

All articles from Life Sciences >>>

The most recent press releases about innovation >>>

Die letzten 5 Focus-News des innovations-reports im Überblick:

Im Focus: Electrons in the fast lane

Solar cells based on perovskite compounds could soon make electricity generation from sunlight even more efficient and cheaper. The laboratory efficiency of these perovskite solar cells already exceeds that of the well-known silicon solar cells. An international team led by Stefan Weber from the Max Planck Institute for Polymer Research (MPI-P) in Mainz has found microscopic structures in perovskite crystals that can guide the charge transport in the solar cell. Clever alignment of these "electron highways" could make perovskite solar cells even more powerful.

Solar cells convert sunlight into electricity. During this process, the electrons of the material inside the cell absorb the energy of the light....

Im Focus: The lightest electromagnetic shielding material in the world

Empa researchers have succeeded in applying aerogels to microelectronics: Aerogels based on cellulose nanofibers can effectively shield electromagnetic radiation over a wide frequency range – and they are unrivalled in terms of weight.

Electric motors and electronic devices generate electromagnetic fields that sometimes have to be shielded in order not to affect neighboring electronic...

Im Focus: Gentle wall contact – the right scenario for a fusion power plant

Quasi-continuous power exhaust developed as a wall-friendly method on ASDEX Upgrade

A promising operating mode for the plasma of a future power plant has been developed at the ASDEX Upgrade fusion device at Max Planck Institute for Plasma...

Im Focus: ILA Goes Digital – Automation & Production Technology for Adaptable Aircraft Production

Live event – July 1, 2020 - 11:00 to 11:45 (CET)
"Automation in Aerospace Industry @ Fraunhofer IFAM"

The Fraunhofer Institute for Manufacturing Technology and Advanced Materials IFAM l Stade is presenting its forward-looking R&D portfolio for the first time at...

Im Focus: AI monitoring of laser welding processes - X-ray vision and eavesdropping ensure quality

With an X-ray experiment at the European Synchrotron ESRF in Grenoble (France), Empa researchers were able to demonstrate how well their real-time acoustic monitoring of laser weld seams works. With almost 90 percent reliability, they detected the formation of unwanted pores that impair the quality of weld seams. Thanks to a special evaluation method based on artificial intelligence (AI), the detection process is completed in just 70 milliseconds.

Laser welding is a process suitable for joining metals and thermoplastics. It has become particularly well established in highly automated production, for...

All Focus news of the innovation-report >>>

Anzeige

Anzeige

VideoLinks
Industry & Economy
Event News

International conference QuApps shows status quo of quantum technology

02.07.2020 | Event News

Dresden Nexus Conference 2020: Same Time, Virtual Format, Registration Opened

19.05.2020 | Event News

Aachen Machine Tool Colloquium AWK'21 will take place on June 10 and 11, 2021

07.04.2020 | Event News

 
Latest News

Rising water temperatures could endanger the mating of many fish species

03.07.2020 | Life Sciences

Risk of infection with COVID-19 from singing: First results of aerosol study with the Bavarian Radio Chorus

03.07.2020 | Studies and Analyses

Efficient, Economical and Aesthetic: Researchers Build Electrodes from Leaves

03.07.2020 | Power and Electrical Engineering

VideoLinks
Science & Research
Overview of more VideoLinks >>>