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Scientists Report New Lead in How Anthrax Kills Cells

For years scientists have known that anthrax bacillus produces a toxin containing a deadly protein called lethal factor. However, researchers have never been able to identify how lethal factor kills cells.

In this week's issue of Science*, a team of researchers report an exciting new lead. The group found, through a combination of luck and elegant science, that lethal factor goes after a signaling protein, known by the tongue-twisting name of MAP kinase kinase (MAPKK), and snips it in two.

Nicholas Duesbery, Ph.D., lead author of the study, and George Vande Woude, Ph.D., senior author, both with ABL-Basic Research Program in Frederick, Md., which is operated by a National Cancer Institute contract, said they think that this finding has important implications for developing new therapies for anthrax. They believe that knowing where lethal factor strikes provides a new focal point to develop rational strategies that block the enzyme from ever reaching MAPKK. Until now, scientists have relied on traditional vaccine approaches of teaching the immune system to recognize anthrax as an invader.

The scientists said their work establishes that lethal factor is a protease, an enzyme that clips proteins in two. They noted this finding could prove to be important because scientists have learned a great deal about proteases over the last decade while developing drugs that inhibit these enzymes in HIV. "This could be another example of how an investment in the basic sciences can pay off in unexpected ways," said George Vande Woude, Ph.D., director of the ABL-Basic Research Program.

"This work provides important biological insights with potential practical applications," said Richard Klausner, M.D., director of the National Cancer Institute. "It is exciting that, in this case, we now have a molecular target in sight that we might be able to exploit to thwart the deadly effects of anthrax."

Anthrax is a naturally occurring disease caused by the bacterium Bacillus anthracis. Spores from the bacterium enter the bodies of animals or humans, causing an infection. As the spores germinate, they release a toxin that can be extremely deadly, particularly if the spores are inhaled or ingested.

In the United States, about five new cases of anthrax are reported each year, with this figure rising to about 2,000 to 5,000 per year worldwide. Most of these cases involve the less-lethal cutaneous form of the disease, in which the spores enter the body via cuts or abrasions on the skin. However, in recent years, the emergent threat of a mass-produced, aerosol form of anthrax being employed as a biological weapon has been recognized. Though an anthrax vaccine exists, experts say new approaches to combat the disease would be invaluable.

Duesbery and Vande Woude set out to find a compound that worked in ways similar to a commercially available inhibitor of the MAP kinase pathway, called PD09859. Following a screen of the National Cancer Institute's 60 cell line in vitro anti-neoplastic drug database, the late NCI scientist Kenneth Paull, Ph.D., came up with lethal factor as a match. This unexpected piece of information suggested that lethal factor might also selectively inhibit the MAP kinase pathway.

The team's next task was to see if this was the case. They contacted Stephen Leppla, Ph.D., an author on the paper and an internationally recognized anthrax expert at the National Institute of Dental Research, part of the National Institutes of Health, to obtain purified lethal factor. Then, in a series of experiments, the scientists were able to demonstrate that lethal factor interacted with the MAPKK1 protein--a key component of the MAP kinase pathway--in frog, mouse, and human cell lines.

But they also noticed something interesting. Their data suggested that lethal factor might be a protease that inactivates the MAP kinase pathway by cleaving MAPKK. Duesbery and Vande Woude then contacted a long-time collaborator, Natalie Ahn, Ph.D., an author on the paper and a scientist at the University of Colorado, and asked for a purified protein that could be used in assays to follow up on this lead. Ahn sent what is known as a MAPKK1 fusion protein that was tagged with a known sequence of amino acids near the protein's so-called NH-2 terminus. Here, the group got lucky--the tag was close to where lethal factor cleaves the protein. "It turned out that having the tag at the NH-2 terminus made it much clearer that MAPKK1 was being cut into two pieces," said Duesbery. "If by chance, the tag had been placed on the C terminus, we would have had a tougher time detecting the change in the protein's molecular weight that told us this was the case."

Knowing approximately where lethal factor clips MAPKK1, the group was able to pinpoint in a series of experiments the precise cleavage points on both MAPKK1 and a related protein, MAPKK2. "The 60 cell database and Ken Paull's work were essential in making the first step," said Duesbery. "And once we made the first step, everything flowed after that. It really shows the power of databases in providing unexpected leads that make serendipitous discoveries like this possible."

* The study is titled, "Proteolytic Inactivation of MAP-Kinase-Kinase by Anthrax Lethal Factor." The authors are Nicholas S. Duesbery, Craig P. Webb, Stephen H. Leppla, Valery M. Gordon, Kurt R. Klimpel, Terry D. Copeland, Natalie G. Ahn, Marriane K. Oscarsson, Kenji Fukasawa, Ken D. Paull, and George F. Vande Woude.

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