Radiologists at Washington University School of Medicine in St. Louis have developed a first-of-its-kind noninvasive imaging technique that allows them to watch two proteins interacting in live animals.
The technique genetically fuses proteins of interest with carefully cleaved sections of luciferase, the protein fireflies use to create light. When the target proteins interact, the sections of luciferase come together and create light that can be detected outside the body by a highly sensitive camera.
“Instead of looking at a protein by itself, this technique lets us see when two proteins come together and dance,” says David Piwnica-Worms, M.D., Ph.D., professor of molecular biology and pharmacology and of radiology. “Those kinds of interactions are very important for many different processes, and they’re also key to developing and evaluating new drugs.”
Piwnica-Worms and colleagues demonstrated the technique’s feasibility on human proteins that interact in the presence of the antibiotic rapamycin. The research appears in the online edition of the Proceedings of the National Academy of Sciences and in print in the Aug. 17 issue of the journal.
According to Piwnica-Worms, understanding protein interactions has become much more important to biologists in recent years.
“We’ve learned that the human genetic code only has a fraction of the genes we expected, and as a result it’s become clear that the context of protein-to-protein interactions significantly affects what proteins can do,” he explains. “That’s what lets us get away with so few genes -- the same protein can do different things based on when or where it’s used.”
Scientists have studied these interactions previously in cell cultures and in solutions obtained by carefully opening up cells. Luciferase has been used previously to identify the presence of molecules in the cell and in live animals, but this is the first time scientists have used it in the test tube or in live animals to detect the coupling of two proteins by a drug.
The biggest challenge of the project, according to Piwnica-Worms, was determining the best place to split luciferase.
“We were ideally looking for a split version of luciferase that had zero activity when separated but had very high light output when the partner proteins interacted,” he explains.
Researchers led by Kathryn E. Luker, Ph.D., a postdoctoral fellow in Piwnica-Worms’ lab, first divided luciferase into overlapping halves. They then produced many copies of the halves, and used an enzyme to chomp off varying lengths from the ends of the halves where the split was made. They packed the resulting library of luciferase fragments into phages, viruses that infect bacteria. Scientists allowed the phages to infect bacterial colonies, and then looked for bacteria that glowed.
Of 19,000 bacterial colonies, approximately 120 lit up. The three brightest were further tested to determine which pair of fragments worked best.
In a line of experimental mice, scientists genetically fused one member of the best pair of luciferase fragments onto the protein targeted by rapamycin, attaching the luciferase fragment to the specific portion of the protein where rapamycin establishes its bond. They attached the other luciferase fragment to a protein known through previous research to interact with rapamycin's target protein only in the presence of rapamycin.
Using a commercially available instrument known as an in vivo bioluminescence camera, they found they could detect light from the luciferase fragments only when they injected the mice with rapamycin, causing the two proteins to interact. In mutations that disabled rapamycin’s ability to bind to the target protein, scientists detected no light even after rapamycin injections.
According to Piwnica-Worms, the series of experiments proved the accuracy and selectivity of the new luciferase-based approach.
Scientists also successfully tested the new technique on two proteins linked to regulation of the cell life cycle. An anti-cancer drug is being developed to block the interaction of these proteins.
“We can now monitor the effects of that drug in live animals using the luciferase technique,” says Piwnica-Worms. “It’s been an exciting development – we’re already collaborating with several colleagues around campus to use this system to study interactions between seven or eight other important pairs of proteins.”