Scientists have determined the detailed structure of an essential piece of the telomerase enzyme, an important contributor to the vast majority of human cancers. Understanding the physical shape of the protein has led to a better understanding of how it acts to immortalize cells - and should help scientists design broadly effective cancer drugs.
Until now, the lack of detailed structural information about the enzyme has hindered progress in developing agents to inhibit it, say the researchers, who published their findings in Nature Structural & Molecular Biology. Howard Hughes Medical Institute President Thomas R. Cech, whose laboratory is at the University of Colorado at Boulder, led the study, conducted with colleagues Steven A. Jacobs and Elaine R. Podell.
Cancer researchers have long sought a way to subdue telomerase, an enzyme whose excessive activity contributes to the unchecked growth of as many as 90 percent of human tumors. The enzyme is vital for some rapidly dividing cells - such as those in a developing embryo - where it extends telomeres, the regions of highly repetitive DNA found at the ends of chromosomes. In most healthy adult cells, telomerase is shut off, and telomeres slowly shrink during cell division - a normal process that helps limit cells' lifespan. Cancer cells, however, usually find a way to turn telomerase back on, achieving a dangerous immortality.
"Getting telomeres replicated again is required for carcinogenesis to proceed," Cech explained. "It's an essential step in the development of cancer, and that makes it of a lot of interest therapeutically, because it is a target that could impact a wide variety of cancers."
Telomerase inhibitors have been in clinical development for many years, but, Cech said, progress has been slow. "The development of anti-telomerase chemotherapeutics has been challenged by the fact that there was no structural basis for thinking about the problem," he said. "There was no picture in any detail of what any part of this protein looks like."
Many labs have been working toward developing that picture, but the task has proven challenging. That's because the enzyme tends to clump together once outside of cells, preventing it from forming the ordered crystals necessary for structural studies. Scientists from Cech's lab and others had tried to simplify matters by crystallizing a portion of the protein, but the segments that they selected clumped together just as stubbornly as the whole enzyme.
Jacobs, a Damon Runyon Cancer Research Foundation fellow in Cech's lab and the first author of the paper, developed a new approach. With the help of bacteria and a protein that emits green fluorescent light, Jacobs randomly screened tens of thousands of fragments of the enzyme for one that would lend itself to successful structural analysis. His strategy took advantage of the fact that when multiple copies of the fluorescent protein clump together, the fluorescence is quenched, or extinguished. So Jacobs engineered bacteria to produce fragments of the telomerase enzyme fused to the fluorescent protein. Since telomerase fragments that clustered together would drag along - and quench - their associated fluorescent protein, Jacobs knew that any bright green bacterial colonies were producing protein fragments that remained free. Those rare colonies would be the best candidates for further analysis.
Jacobs performed these experiments on fragments of the telomerase enzyme from a variety of organisms, and found that only a fragment from Tetrahymena -- the single-celled organism in which telomerase was first discovered -- would work. The researchers named the protein fragment "telomerase essential N-terminal" (TEN) domain, in reference to its position within the complete enzyme. It took a few more biochemical tricks, but eventually Jacobs crystallized the protein fragment and analyzed it using x-ray diffraction.