Toxic compound opens potentially important cell gates

June 05, 2001

A compound previously noted mainly for its role in disease and the self-destruction of sick cells is more than just a jobless, toxic transient in healthy cells, researchers have found.

Scientists from The Johns Hopkins University, Harvard University and The University of Hawaii have reported new evidence rehabilitating the reputation of this biological pariah, known as adenosine diphosphate ribose, or ADP-ribose. They found that as cells work to break the compound down, a pause in the disposal process allows ADP-ribose to open a gate for transporting calcium through cell membranes in the brain, lungs, heart, spleen, liver, kidney, and elsewhere.

"It's important to know what opens and closes calcium channels, because calcium transport into and out of cells is a significant step in a wide variety of physiological processes," says Maurice Bessman, professor of biology in the Krieger School of Arts and Sciences at Hopkins and an author of a paper in the May 31 issue of "Nature." "Examples include nerve cell signaling processes, regulation of the heartbeat and muscle contraction, immune system responses, olfaction, and energy metabolism."

Researchers found evidence that a protein originally used solely to break down ADP-ribose had become fused with a protein that creates a calcium channel known as LTRPC2. A key segment of the disposal protein apparently was incorporated through evolution into the larger protein that creates the calcium channel.

"Two different processes -- two old functions -- have been very tightly tied together, but they're both still carrying out their own individual enzymatic reactions," Bessman says. "You get a whole that is greater than the sum of its parts. We don't think this is the last example of this type of fusion, and will be looking for other similar proteins usurped for other functions in a comparable fashion."

Discovery of the link began when Andrew Scharenberg's lab at Harvard Medical School, which specializes in calcium channels' roles in the immune system, identified two genes that they thought were linked to calcium channels.

Scharenberg noted that both genes contained a section of DNA similar to a DNA segment that is the defining characteristic of a family of genes Bessman's group discovered and calls "Nudix" hydrolases. The characteristic section of these genes is known as a "Nudix box."

"We think this family of genes has been around for a long time," says Christopher Dunn, a research technician at Hopkins and an author on the "Nature" paper. "Although the proteins we worked with in this particular project are from humans, the Nudix hydrolases are found in the most primitive to the highest organisms, with nearly 600 members in 200 species identified so far."

Cells most commonly use Nudix proteins as "housecleaners"that break apart potentially harmful derivatives of nucleoside diphosphates, but Bessman's group has also shown Nudix proteins have been adapted to a variety of other functions.

Scharenberg sent clones of the Nudix genes he'd found to Bessman, and Dunn transferred the shorter gene into E. coli bacteria. When the bacteria used the gene to make proteins, Dunn tested them against a series of compounds that bind to Nudix boxes. The protein would only bind to ADP-ribose, a toxic biochemical that is used by some organisms including diptheria to disable cells.

"ADP-ribose is also involved in apoptosis, a self-destruct mechanism used by sick or genetically damaged cells," says Dunn. "Free ADP-ribose is released all the time in healthy cells as a byproduct of other reactions, but it's always cleaned up."

The second, larger gene identified by Scharenberg had a Nudix box at one end. When Bessman and Dunn isolated that segment of the larger protein and repeated the experiment they'd performed on the shorter gene, they found that it also would only bind to ADP-ribose.

Suspecting that the remainder of the second protein was involved in the formation of a calcium ion channel, Scharenberg and Bessman asked Reinhold Penner of the University of Hawaii at Honolulu to use a special "patch-clamp" technique to test whether LTRPC2 created a channel and whether that channel could be regulated with ADP-ribose. He found that the answer to both questions was yes.

"In fact, Reinhold found that the mechanism was very specific; other compounds can't open the channel," Bessman says. "This effort, in which three labs with different expertises worked toward a common goal, was a paradigm of scientific cooperation," Bessman concludes. "It's a bit like the fusion of the proteins we found; the whole was greater than the sum of its parts."
-end-
Other authors on the paper were Anne-Laure Perraud, Pierre Launay, Carsten Schmitz, Alexander Stokes, Qiqin Zhu, and Jean-Pierre Kinet, of Harvard; and Andrea Fleig and Leigh Ann Bagley of the University of Hawaii.

This research was supported by the National Institutes of Health and by a grant from the Beth Israel Pathology Foundation.

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