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    New insights into the cell’s cancer-suppressing machinery


    Richard Kriwacki, PhD

    The p53 protein plays an immensely important role in protecting the body from cancer. Dubbed the cell’s guardian of the genome, p53 is constantly poised to detect potentially tumor-causing DNA damage. When it detects such abnormalities, p53 either arrests cell proliferation or triggers a lethal cell suicide program that rids the body of the malignant cell before it can divide. The p53 machinery is so critical that its breakdown figures in more than half of all cancers, unleashing cancer cells to grow unchecked.

    Now St. Jude researchers have used sophisticated analytical techniques to discover important new details about the proteins that control the p53 machinery. The basic studies could yield treatments that reactivate blocked p53 machinery to trigger suicide in a broad range of cancer cells.

    Led by Richard Kriwacki, PhD, Structural Biology, the new studies explored the function of a key p53 activator, a protein called p14Arf (abbreviated Arf). Arf was originally discovered in 1997 by Kriwacki’s colleague, Charles Sherr, MD, PhD, a Howard Hughes Medical Institute Investigator and co-chair of Genetics and Tumor Cell Biology. In healthy cells, p53 is inhibited by a protein called Hdm2. But when a cell exhibits abnormal proliferation, Arf springs into action, attaching itself to Hdm2, thwarting its function and allowing p53 to either arrest or kill the cell.

    Studying Arf and Hdm2 is important because in many cancers, defects in either of these proteins cause failure of the p53 machinery. In some cancers overproduction of Hdm2 shuts down the p53 machinery. And in yet other cancers, Arf is damaged, preventing it from switching off Hdm2 and concomitantly switching on the p53 tumor suppression program.

    In their studies, Kriwacki and his colleagues have sought to solve a key biological problem—how Arf attaches itself to Hdm2 to inhibit its function. However, the scientists have been hampered by the disordered, floppy structure of the proteins, which makes structural analysis nearly impossible—like trying to deduce the shape of a wet noodle.

    When the two proteins link up, they fold together in an orderly embrace. But that combination still remains difficult for scientists to analyze because the joined proteins form insoluble structures. These structures are all but impossible to study using the powerful structural analysis technique called nuclear magnetic resonance (NMR) spectroscopy. NMR is a method of probing molecules with magnetic fields and radio signals to map their atomic structure.

    “We have been working on this problem since I started my lab at St. Jude almost 10 years ago, and we have gained many useful insights into this interaction,” Kriwacki said. “But the problem has been that—while these proteins on their own are highly soluble and easy to study—they are so disordered there really isn’t much to learn. But when we mixed them together, they would just precipitate from the solution, and studying them using NMR was frustrating.”

    However, in their new experiments, Kriwacki and his colleagues tackled this problem by figuring out how to synthesize just the right snippet of the Arf protein that latches on to Hdm2. Unlike the ungainly, full-size Arf-Hdm2 complex, the smaller combination was soluble enough to study using NMR.

    The result of their studies both in the test tube and in mouse cells, reported recently in the Journal of Molecular Biology, gave the scientists an unprecedented structural map of the linkage between Arf and Hdm2. The findings, Kriwacki said, confirmed theories from previous studies about how Arf and Hdm2 zip together and gave the researchers a pathway to future analyses. These insights have both scientific and clinical value, Kriwacki said.

    “Arf is a real enigma. It’s a bizarre protein,” he said. “There really are not other proteins in cells of higher organisms that exhibit similar structural features. And what we have reported on Arf are really the only things that are known about its structure and this unusual type of co-assembly mechanism.” Such understanding has broader significance because Arf appears to regulate many other cell mechanisms besides p53.

    Of the clinical value of their discoveries, Kriwacki said, “A key goal is to translate our structural and biophysical insights into the development of an Arf mimic. If we can understand what the minimal chemical elements of Arf are, we can stitch together a molecule that could recapitulate those elements. Such a molecule could be used in cancer treatments to activate the p53 pathway in tumor cells that have arisen because of Arf loss or Hdm2 over-expression.”

    Other authors of this paper include Sivashankar Sivakolundu, PhD, Brian Bothner, PhD, Chimere Ashley, MD, and John Satumba, PhD, all of Structural Biology; Amanda Nourse, PhD, of the Hartwell Center for Bioinformatics and Biotechnology; and Simon Moshiach PhD, and Jill Lahti, PhD, both of Tumor Cell Biology.

    The research was sponsored in part by the National Institutes of Health and ALSAC.

    March 2009