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The two pieces of the puzzle snapped into place for Goldberg and Cowman when a third group published its results in 2008. Michael Marletta's laboratory at the University of California, Berkeley, showed that nearly all exported malaria proteins were cleaved at the PEXEL motif and the process took place in the endoplasmic reticulum.
“We said, ‘Aha! We've got a protease of unknown function in the right place. We bet it's the processing enzyme,’” says Goldberg. They set about to test that hypothesis.
Both groups isolated PMV from parasites and added it to PEXEL-containing proteins in a test tube. “It cleaved 100 percent and we knew straight away—we've got it!” says Cowman, a molecular parasitologist.
Goldberg wanted to test whether the chopping activity of PMV was critical for protein export and, ultimately, the survival of the parasite. His postdoc Ilaria Russo made a catalytically dead version of PMV—it could bind PEXEL proteins but did not cut them. She flooded live parasites with the mutant enzyme to compete with normal PMV. Through the microscope, she saw that in cells containing only parasites with the dead enzyme, the parasites appeared sick. In cells that carried a parasite with dead enzyme and another parasite with normal PMV, both parasites were healthy. The normal PMV exported enough proteins to maintain parasite health. Clearly, PMV's protein-chopping action was essential for the parasite's growth.
Cowman's group also did a clever experiment to test whether PMV's cleavage of PEXEL was required for proper export of a protein. They created proteins that “looked” like they had been cleaved properly by PMV but were actually cleaved by another enzyme, called signal peptidase. These proteins built up inside the parasite.
“It has to be PMV handing off the cleaved protein to something else for export,” says Cowman. “It was one of the most clear-cut experiments I've ever seen.”
An inhibitor of PMV, which would shut down protein export and kill the parasite, has potential to be a powerful drug against malaria. “PMV is similar to the HIV protease for which very good drugs already exist,” says Cowman. “We should be able to develop very good drugs against this as well, which would inhibit the parasite at a final point in its life cycle.”
About six months after the Bangkok meeting, Goldberg got in touch with Cowman to let him know his group was wrapping things up. The two decided to submit their manuscripts simultaneously—even getting on the phone together to hit the “send” button. “It was a pleasure to do this alongside Alan Cowman's group,” says Goldberg. “This is the way I think science should work. We both had discovered something and we wanted both groups to get credit.”