STANFORD, Calif. — Investigators at the Stanford University School ofMedicine have found a way to quickly and reversibly fine-tune theactivity of individual proteins in cells and living mammals, providing apowerful new laboratory tool for identifying — more precisely than everbefore — the functions of different proteins.
The new technique also could help to speed the development of therapiesin which cancer-fighting proteins are selectively delivered to tumors.
The procedure, described in a Nature Medicine paper to be publishedonline Sept. 28, appears to be broadly applicable to efforts tounderstand the biological roles of all kinds of proteins, includingthose that are secreted by cells. This category includes many potentintercellular signaling proteins that can influence the immune system,for example by attracting its attention to an existing tumor.
"We have yet to find a protein the system doesn't work with," saidsenior author Steve Thorne, PhD, an assistant professor at theUniversity of Pittsburgh who was involved in the work while a researchassociate at Stanford. The work was conducted under the direction ofChris Contag, PhD, associate professor of pediatrics, of radiology andof microbiology and immunology; and Tom Wandless, PhD, assistantprofessor of chemical and systems biology.
This technique, which was tested in mice, involves pairing speciallybioengineered proteins with a drug, aptly named Shield-1, that preventsthe proteins from being degraded.
This approach stands in contrast to current ways of learning aboutproteins' functions, which are largely based on impeding a cell'sproduction of the protein. Unfortunately, that cellular process can beslow and cumbersome, meaning that scientists get a sluggish response tosuch manipulations. In addition, current methods to perturb proteinfunction are either irreversible — once a protein's production isknocked out, it can't be turned back on — or difficult to execute.
The new technique, instead, influences the level of speed with which theprotein is broken down—a much faster process than its production.Moreover, it is reversible and works like a dimmer switch for anoverhead light. The rate of a protein's degradation — and, thus, thelevel of its biological activity — can be increased or decreased bysupplying more or less of Shield-1, permitting scientists to study thebiological effects of slightly increasing or diminishing a protein'sactivity inside a cell over short time frames: for example, during aparticular period in an organism's development.
The Stanford team succeeded in controlling levels of proteins by arelatively simple method pioneered by Wandless and his then-graduatestudent, Laura Banaszynski, PhD. They created special, bioengineeredversions of several different proteins, in each case altering theprotein by adding a small extra piece that didn't interfere with itsbiological function, but flagged it for rapid degradation. Thisdegradation can be halted in its tracks, however, by Shield-1, whichbinds to the bioengineered protein, shielding it from destruction by thecell's breakdown machinery. The drug thus can enhance the bioengineeredprotein's intracellular concentration and activity; withdrawing the drughas the opposite effect.
"The process is tunable, and fast. As soon as you remove the drug, youaffect the degradation time of the protein," said Mark Sellmyer, agraduate student at the School of Medicine, who shares lead authorshipof the study with Banaszynski.
The degradation-vulnerable bioengineered proteins were each produced byattaching the gene coding for a protein to another DNA sequence codingfor the small extra piece that flags the protein for rapid degradation.The scientists then inserted the altered gene into a virus capable ofinfecting cells and introducing the altered gene into the cells' genomes.
In experiments demonstrating for the first time that the new techniquecan be used to effectively regulate a physiologically active protein inlive mice, cultured tumor cells were grafted under the skin ofimmunologically impaired mice. As expected, the mice developed numeroustumors. The investigators had altered these cultured tumor cells so thatthey produced a degradation-prone bioengineered version of the proteinIL-2 that, when secreted by cells, sends potent signals drawing theimmune system's attention to those cells. When these altered tumor cellswere grafted subcutaneously in the absence of Shield-1, the tumors grewjust as before.
But if the tumor cells were first pretreated with Shield-1 they secretedIL-2, preventing any initial tumor growth. If Shield-1 was withheld atfirst and then administered to the mice five days after the grafts,tumors that had developed in those first few days regressed. By day 14,the tumors were gone.
Another set of experiments employed a mutant virus that had beenpreviously developed by Thorne as a cancer therapy. The investigatorsinserted the gene for a bioengineered, degradation-prone form of acell-killing protein into the specialized virus. They then administeredit intravenously to live, tumor-bearing mice. When no Shield-1 wasprovided, the tumor growth was only slightly diminished. But if Shield-1was supplied three days after infection, when the virus had establisheda solid foothold in the tumors but been cleared from normal cells,tumors were completely eradicated in 90 percent of the mice. Meanwhile,normal cells were spared the substance's lethal effects.