By David N. Leff

Q: Why is the black widow spider (Lacrodectus mactans) called a black widow?

A: Because she is black and murders her mate after mating.

This seemingly counterproductive practice has given rise among life scientists to "the black widow phenomenon."

Thus, biochemist John Collier of Harvard University, in Boston, cited in today's Science, dated May 1, 1998: "Unlike many microbial pathogens, Bacillus anthracis [the anthrax bug] seems to depend on the death of its host to propagate. As the animal decays, the bacteria are exposed to oxygen; they turn to spores and repopulate the soil."

Molecular biologist George Vande Woude, director of basic research at the NIH National Cancer Institute, in Frederick, Md., defined this anthrax black widow phenomenon at the molecular level.

"What we think happens," he told BioWorld Today, "is that a key cellular enzyme, which is a mitogen-activated protein kinase — MEK for short — sees the anthrax lethality factor [LF] and embraces it. Then the LF itself clips the amino terminus of MEK, and inactivates it".

Vande Woude characterizes this metabolic Greek tragedy as "important new information," reported in a paper of which he is senior author, also in today's Science. It bears the title: "Proteolytic inactivation of MAP-kinase-kinase by anthrax lethal factor."

His research laboratory at NCI focuses on the molecular basis of cancer, and Vande Woude stumbled by serendipity into his current concentration on anthrax toxicity.

Among the 100,000 or more compounds that NCI/Frederick has screened over recent years for potential anti-tumor activity, a chemical called PD9859 had caught the Vande Woude co-authors' interest. It was a known inhibitor of that MAP-kinase-kinase cellular pathway. They arranged for a search of similar compounds in the agency's massive database.

Of all 100,000-plus agents, it turned up LF, the most potent component of the anthrax toxin's triple-protein structure. This 776-amino acid protein, the Science paper noted, "is thought to be one of the principal causes of death in infected individuals." (See BioWorld Today, Feb. 3, 1998, p. 1.)

"For a long time," Vande Woude recalled, "LF was considered to be a protease — a protein-cleaving enzyme. That was based upon its having certain structural similarities to known proteases. But its substrate, or target, was not known. So for us it became a matter of demonstrating that LF did indeed have some activity in inhibiting the MAPK pathway."

To make this case, they turned to the African clawed frog, Xenopus laevis. This South African tree toad made a name for itself long ago in early human pregnancy testing. When a few drops of urine from a pregnant woman were injected into a virgin female frog, she produced fertile eggs by parthenogenesis, which grew up into fatherless adult frogs.

Preventing Unfertilized Birth In Frogs

"Our lab has worked for a long time in oocyte maturation," Vande Woude recounted, "how unfertilized eggs are produced from immature eggs. That pathway requires MEK. When we added LF, the egg maturation process failed to go forward — presumably because that lethality factor blocked the pathway.

"But," he added, "of course LF is the toxin of anthrax that has been shown to result in the death of a rat in something like 38 minutes. So, preventing an egg from undergoing meiosis [cell division] really didn't say very much.

"But when we began looking at it in detail, we found it was the MAPK pathway that was not working. So from there, in in vitro systems, we uncovered the fact that LF was inactivating the MEK enzyme.

"This showed us that the stage at which an egg is ready to be fertilized is regulated by MAPK. If MEK kinase is not there, the egg moves forward and undergoes parthenogenesis.

"MAPK," Vande Woude explained, "is part of the signaling pathway that cells use to transfer messages from outside their world to the nucleus. It effects the regulation of how cells grow, divide and differentiate."

In tracking LF to its toxic target, the co-authors found that it wreaked its proteolytic cleavage "at the very amino terminus of this MEK molecule, which has been implicated in a site where MEK binds MAPK to get phosphorylated.

"It's really quite dramatic, " Vande Woude went on. "When we removed the first seven amino-terminal amino acids of MAPK, it inactivated the MEK enzyme, probably the way LF does. And even when you generate such a construct de novo, which we did, it is inactive to begin with."

Beating Biowar Arms Into Toy Pistols

Looking now toward generating a vaccine or antidote against anthrax infection, Vande Woude observed: "I think that's the new promise that this work develops. LF is clearly a protease, and now that we know its substrate, the paradigm established with HIV proteases can be brought to bear on this anthrax problem. One should be able to develop inhibitors that inactivate downstream where this thing is causing its lethal effect.

"Conceivably," he suggested, "we could find a drug that would make anthrax as a weapon of destruction as powerful as a water pistol."

Vande Woude hopes "to be collaborating with whomever wishes to jump on this, and we would give them whatever we can provide so that we can move forward. A number of conversations are already in the planning stage with people in government and in industry — the biotechnology community included."

But Vande Woude isn't turning away from his laboratory's original raison d'etre, which is to explore the molecular bases of cancer.

"For many years," he observed, "the MEK enzyme has been implicated in many different kinds of cancers. So this LF correlation is a new tool, we hope, that will give us some idea how to make targets that would prevent that pathway from activating tumor cells. That is our long-range goal." *

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