For the first time, scientists have developed a means of observing at the molecular level, real-time movies of key structural processes within the ribosome underpinning the synthesis of protein. They were able to do it in a manner that should someday allow for screening of novel drugs that inhibit the ribosome or for the improvement of existing antibiotics that are sometimes toxic."This is really a proof-of-principle of what we hoped we could achieve -- that we can make these types of measurements in a very robust and high-throughput manner," says study senior author Dr. Scott Blanchard, assistant professor of physiology and biophysics at Weill Cornell Medical College.His team published their findings in Molecular Cell.The ribosome is home to about 60 different molecules. These molecules work together in complex ways, using instructions found in RNA to churn out the proteins that cells need to live and grow. In the world of drug development, it is hard to overestimate the importance of the ribosome -- for example, more than half of today's antibiotics target this key cellular machine."Genetic instructions are presented to the ribosome in the form of messenger RNA," explains Dr. Blanchard. "The process of translating those mRNA instructions into proteins involves the selection by the ribosome of RNA molecules called transfer RNA (tRNA)."This selection process is the determining factor linking gene sequences with their end-product proteins.However, for years, much of what scientists know about ribosomal activity -- and how drugs might affect it -- has been guesswork, since it has been nearly impossible to view molecular activity within this tiny structure firsthand.But Dr. Blanchard's work has yielded a breakthrough: a cutting-edge technology in microscopy called single-molecule Fluorescence Resonance Energy Transfer (smFRET) that uses long-established technologies in a whole new way. "Using this technique, we're able to collect photons of light coming from many single molecules simultaneously," he explains. "This information reports on a biomolecule's location, its interaction with other molecules and tiny motions within the molecule itself. Before this technology, investigations of molecular processes were like trying to figure out how cars worked by watching traffic on the freeway from a satellite high up in space. Now, it's as if we're able to able to inspect the individual cars directly. In fact, both are important but having this new perspective should go a long way towards improving our understanding of how these machines operate."Observing one molecule at a time is fine, of course, but efficient drug discovery requires the rapid analysis of hundreds or even thousands of compounds in a "high-throughput" way.In their latest research, Dr. Blanchard's team used smFRET to track sub-nanometer movements in tRNA positions deep within the ribosome (that's a distance change of about one hundred millionths of an inch). "We observed three discrete configurations of tRNA within the ribosome that interconvert on the 100 millisecond timescale, including an intermediate 'hybrid' configuration that no one had ever had evidence of before," the researcher says.The ability of scientists to make nearly real time measurements of how the ribosome's structure changes during function may be the study's biggest accomplishment."Not only can you make exquisitely sensitive measurements on ribosome structure over time, you can also detect how the system changes in response to ligands, including antibiotics," Dr. Blanchard says. Those measurements should be able to be performed in a high-throughput manner, he notes."Our next step is to watch how specific drugs affect the dynamic processes within the ribosome," adds Dr. Blanchard. "We hypothesize that drugs activities that block normal ribosomal functions can be detected by measuring how they change the cadence of structural events in the molecule. As my graduate advisor coined it, it's like a molecular EKG. This new, close-up look at how molecular machines work, and how they are affected by therapeutic agents should open the door to better opportunities in drug discovery."This work was funded by the U.S. National Institute of General Medical Sciences and the Alice Baumfalk Charitable Trust-researchers include lead researcher James B. Munro as well as Roger B. Altman and Nathan O'Connor -- all of the Department of Physiology, Biophysics and Systems Biology at Weill Cornell.
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"What's really exciting for us is that NALP1 hasn't been specifically implicated in autoimmune diseases before," said Richard Spritz, MD, director of the Human Medical Genetics Program at UCDHSC and lead investigator for this study. "Since NALP1 appears to be part of our body's early-warning system for viral or bacterial attack, this gives us ideas about how to try to discover the environmental triggers of these diseases. This finding may also open up new approaches to treatment, possibly for many different autoimmune diseases."
As a group of approximately 80 disorders that can involve almost any tissue, organ or system, autoimmune and autoinflammatory diseases affect 15 million to 25 million people in the United States. In women, they rank among the top ten causes of death.
Dr. Spritz and his team hope to soon begin organising a clinical trial of a new treatment for vitiligo, based on their NALP1 discovery. Spritz foresees research labs using the information from the UCDHSC study to replicate or test the results in patients with other autoimmune diseases to see how broad potential applications might be. His hope is that the gene NALP1 is also found to be involved in autoimmune and autoinflammatory diseases such as Type 1 diabetes, Addison's disease, thyroid disease and lupus, among others.
"All diseases are complex, the result of different genes and environmental risk factors acting together in concert. But if NALP1 turns out to be one of the major genes involved in numerous autoimmune diseases, and if we can interrupt its negative effects, we may have the chance to treat many different chronic autoimmune disorders like vitiligo, lupus and psoriasis and perhaps eventually eliminate them altogether," said Dr. Spritz.
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