Uncovering the structure of a "Rosetta Stone" protein may help scientists understand how cells are programmed to die, and in turn, the role loss of the process plays in cancer.
Researchers, lead by Charles Brenner, Ph.D., associate professor of microbiology and immunology at Jefferson Medical College of Thomas Jefferson University in Philadelphia, have determined the 3-dimensional structure of such a Rosetta Stone protein.
First proposed in 1999 by researchers at UCLA, Rosetta Stone proteins occur when two proteins that are separate in some forms of life are fused in another form of life. The fusion "event" almost always reveals a previously hidden interaction between the two nonrelated proteins.
"This may be the first example in cancer biology of separate proteins in one form of life fused in another," says Dr. Brenner, who is a member of Jefferson's Kimmel Cancer Center. One of the two proteins, Fhit, has been implicated in many common human cancers.
Dr. Brenner and his co-workers report their results July 27 in the journal Current Biology.
Jefferson researchers have been studying the human Fhit protein since 1996. Jefferson Medical College scientists Kay Huebner, Ph.D., professor of microbiology and immunology and Carlo Croce, M.D., professor and chair of microbiology and immunology and director of Jefferson's Kimmel Cancer Center and their co-workers discovered that the protein is encoded at the most fragile site in the human genome and is lost in many human cancers.
In 1998, Dr. Brenner's group, working with Drs. Huebner and Croce, determined the 3-dimensional structure of the Fhit protein in its active form. Later in 1998, the same researchers discovered that in the fruitfly Drosophila melanogaster and the flatworm Caenorhabditis elegans, the Fhit protein is naturally fused to an unrelated protein called Nit. Curiously, the NitFhit fusion protein is found in invertebrates, while vertebrates such as humans and mice and fungi such as baker's yeast contain separate Nit and Fhit proteins. Scientists believe that gene and protein fusions occur because pairs of proteins work in the same biological pathways. In the case of Nit and Fhit, he says, "if you found a Nit and Fhit sequence in the mouse and human, you would have no initial idea that they function in the same pathway," he says. "In finding them as part of the same polypeptide in invertebrates, there's an indication that they do."
Because the human Fhit protein is inactivated in many human cancers and loss of Fhit leads to cells with defects in programmed cell death, the scientists wanted to discover additional proteins in the Fhit pathway. When they examined the expression of Nit and Fhit in the mouse, they saw both proteins rise and fall in seven of eight tissues almost identically. They also found Nit in every organism in which they had found Fhit. These results made the case for NitFhit as a Rosetta Stone protein very strong.
Dr. Brenner's team purified the worm NitFhit protein expressed in bacteria, coaxed NitFhit into a crystalline form, and bombarded the crystals with x-rays at Cornell University's and Brookhaven National Laboratory's synchrotron sources. Post-doctoral fellow Helen Pace, Ph.D., working with Dr. Brenner, determined phases of the diffracted X-rays to obtain a three-dimensional map of the NitFhit protein.
"The structure clearly shows how Nit interacts with Fhit," he says. "A central Nittetramer binds a Fhit dimer on one side of the molecule and another Fhit dimer on the other side of the molecule. Specific Nit sequences invade Fhit sequences to make Nit fit."
The Jefferson scientists are particularly encouraged that the flatworm is a leading system in which to study cell death. According to Dr. Brenner, the structure of NitFhit tells us that Fhit is functioning in a large complex with Nit in the worm. Following the activity of Nit in worms and other organisms "ought to take us to the next vista point.
"This structure is leading us to the idea that regulation of Nit may be important for the cell death activity of Fhit."
Jefferson researchers are beginning to investigate the role of Nit and Fhit in the development of other organisms, such as zebrafish, which have separate proteins. They would also like to determine a potential role of NitFhit in cell death in the developing worm.
Dr. Brenner notes that Fhit is absent in tumors and is not a cancer drug target. "If Nit is a pro-survival enzyme, which ought to be inhibited by Fhit, then Nit itself may be the drug target."
Steve Benowitz or Phyllis Fisher
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Journal
Current Biology