Forum for Science, Industry and Business

Sponsored by:     3M 
Search our Site:

 

Commandeering cellular machinery: recognition mechanism to detect small molecules

04.10.2004


Researchers have learned how to commandeer the complex machinery that cells use to recognize and respond to such important molecules as steroid hormones, thyroid hormones and vitamin D.



The development could provide a foundation for a new family of biologically-based mechanisms able to detect common drugs, chemical weapons and other small molecules. By allowing manipulation of this cellular protein machinery – known as nuclear receptors – the technique could also lead to new methods for producing enzymes and important pharmaceutical compounds. "We are hijacking these nuclear receptors for a new set of purposes," explained Donald Doyle, assistant professor in the School of Chemistry and Biochemistry at the Georgia Institute of Technology. "We want to change the nuclear receptors themselves so they don’t recognize what they normally recognize, and instead recognize the small molecules we want to detect. That would allow us to develop a new type of sensing mechanism."

A paper published in the September 27 – October 1, 2004 issue of the journal Proceedings of the National Academy of Sciences describes how Doyle’s research team – which also included Lauren Schwimmer, Priyanka Rohatgi, Bahareh Azizi and Katherine Seley – modified one type of nuclear receptor to bind a drug compound to which it previously did not respond. Based on this success, the researchers hope to demonstrate broader application with other small molecules.


The work was sponsored by the Research Corporation, the Seaver Foundation and the National Science Foundation.

Nuclear receptors are ligand-activated transcription factors contained in cells. When activated by specific small molecules, the nuclear receptors initiate a complex process that results in gene expression. Because these receptors play a vital role in controlling cellular response to these small molecules, scientists have been attempting to understand them and solve their molecular structures – with a goal of creating pharmaceuticals able to turn them off or on. The research often involves placing nuclear receptors into yeast cells, which because they do not have nuclear receptors of their own, allow scientists to isolate the activities of specific receptors.

For their study, Doyle’s team chose the nuclear receptor retinoid x receptor (RXR) whose molecular structure has been well documented by other researchers. Using a technique known as structure-based codon randomization, the Georgia Tech researchers used their knowledge of RXR’s structure to modify the 20 different amino acids that make up the receptor pocket. The goal was to create a library containing 32,768 different variations in the hope of creating – and then finding – a few re-engineered receptors that would have the ability to bind to a molecule known as LG335. (LG335 is structurally similar to Targretin®, a pharmaceutical developed to bind RXR. However, LG335 does not effectively bind unmodified RXR.)

Though the goal was 32,768 variants, the method actually produced approximately 380,000 samples – which would have been impossible to test using conventional screening methods. However, Doyle and his research team used a new protein engineering technique known as chemical complementation that allowed the variants to be tested in parallel – a task akin to finding genetic needles in thousands of haystacks simultaneously.

Using the technique, the researchers placed each variant, which had been inserted into yeast cells, onto petri plates containing LG335. A small number of yeast cells (less than 0.1 percent of the total) grew into visible colonies, suggesting they might contain nuclear receptors for the LG335.

To verify that the colonies were growing in response to LG335 and not some other compound contained in the yeast, samples were then smeared onto another petri plate that did not contain LG335. Any colonies that grew there were discarded. From their initial 380,000 candidates, the researchers ended up with about a dozen nuclear receptors whose recognition pockets had been re-engineered to respond to the LG335.

"Using this technique, we don’t have to evaluate each member of our library," Doyle said. "The yeast actually do the work for us. If only one in a million respond to the compound on the plate, the yeast will form a colony around that one. This allows us to do highly parallel and rapid screening to find a few functional receptors in a large collection of nonfunctional receptors."

The receptors found by the researchers varied in their responsiveness, with some significantly more sensitive than others. Some performed the function of on-off switches, while others were more like dimmer switches, responding in proportion to the amount of LG335 bound to them.

Doyle’s team also studied their re-engineered receptors in mammalian cells – and found they behaved much the same as in yeast. The re-engineered receptors could be used in gene therapy against cancer, or as research tools to investigate gene function. But more importantly, the new receptors serve as proof of principle for the protein engineering technique used to produce them. Doyle envisions using that technique to produce other nuclear receptors that could be the basis for sensing arrays in which a variety of receptors, each sensitive to a different compound, could be used to quickly analyze an unknown agent. A sensor array might also be used in a hospital emergency room to rapidly test for chemical agents in the blood of an unconscious patient. "If we could take the receptors, express them and put them into a device where there is a color change or another signal produced, we could potentially detect small molecules in a robust way that could complement or replace other detection technologies," he explained. The same technology could also be used to produce enzymes, and to regulate metabolism in cells, Doyle said.

Having demonstrated the ability to re-engineer one nuclear receptor to respond to a small molecule to which it previously did not bind, the research team next wants to demonstrate that the technique could apply to other small molecules. "Now we have to see how far we can push this and how many small molecules we can accommodate with this technique," Doyle said. "We are trying to generalize this approach to genetic selection. There is a lot of diversity we can work with in terms of different binding pockets and shapes, so this is only the first step."

John Toon | EurekAlert!
Further information:
http://www.gatech.edu

More articles from Life Sciences:

nachricht During HIV infection, antibody can block B cells from fighting pathogens
14.08.2018 | NIH/National Institute of Allergy and Infectious Diseases

nachricht First study on physical properties of giant cancer cells may inform new treatments
14.08.2018 | Brown University

All articles from Life Sciences >>>

The most recent press releases about innovation >>>

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

Im Focus: New interactive machine learning tool makes car designs more aerodynamic

Scientists develop first tool to use machine learning methods to compute flow around interactively designable 3D objects. Tool will be presented at this year’s prestigious SIGGRAPH conference.

When engineers or designers want to test the aerodynamic properties of the newly designed shape of a car, airplane, or other object, they would normally model...

Im Focus: Robots as 'pump attendants': TU Graz develops robot-controlled rapid charging system for e-vehicles

Researchers from TU Graz and their industry partners have unveiled a world first: the prototype of a robot-controlled, high-speed combined charging system (CCS) for electric vehicles that enables series charging of cars in various parking positions.

Global demand for electric vehicles is forecast to rise sharply: by 2025, the number of new vehicle registrations is expected to reach 25 million per year....

Im Focus: The “TRiC” to folding actin

Proteins must be folded correctly to fulfill their molecular functions in cells. Molecular assistants called chaperones help proteins exploit their inbuilt folding potential and reach the correct three-dimensional structure. Researchers at the Max Planck Institute of Biochemistry (MPIB) have demonstrated that actin, the most abundant protein in higher developed cells, does not have the inbuilt potential to fold and instead requires special assistance to fold into its active state. The chaperone TRiC uses a previously undescribed mechanism to perform actin folding. The study was recently published in the journal Cell.

Actin is the most abundant protein in highly developed cells and has diverse functions in processes like cell stabilization, cell division and muscle...

Im Focus: Lining up surprising behaviors of superconductor with one of the world's strongest magnets

Scientists have discovered that the electrical resistance of a copper-oxide compound depends on the magnetic field in a very unusual way -- a finding that could help direct the search for materials that can perfectly conduct electricity at room temperatur

What happens when really powerful magnets--capable of producing magnetic fields nearly two million times stronger than Earth's--are applied to materials that...

Im Focus: World record: Fastest 3-D tomographic images at BESSY II

The quality of materials often depends on the manufacturing process. In casting and welding, for example, the rate at which melts solidify and the resulting microstructure of the alloy is important. With metallic foams as well, it depends on exactly how the foaming process takes place. To understand these processes fully requires fast sensing capability. The fastest 3D tomographic images to date have now been achieved at the BESSY II X-ray source operated by the Helmholtz-Zentrum Berlin.

Dr. Francisco Garcia-Moreno and his team have designed a turntable that rotates ultra-stably about its axis at a constant rotational speed. This really depends...

All Focus news of the innovation-report >>>

Anzeige

Anzeige

VideoLinks
Industry & Economy
Event News

Within reach of the Universe

08.08.2018 | Event News

A journey through the history of microscopy – new exhibition opens at the MDC

27.07.2018 | Event News

2018 Work Research Conference

25.07.2018 | Event News

 
Latest News

'Building up' stretchable electronics to be as multipurpose as your smartphone

14.08.2018 | Information Technology

During HIV infection, antibody can block B cells from fighting pathogens

14.08.2018 | Life Sciences

First study on physical properties of giant cancer cells may inform new treatments

14.08.2018 | Life Sciences

VideoLinks
Science & Research
Overview of more VideoLinks >>>