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Front Page

Finding NEMO:
Latest Crohn’s Disease Clue

The damp tunnels of the gut are a breeding ground for millions of microbes, some dangerous, but many harmless and even beneficial. Normally, immune cells patrolling the intestines keep a cool head, attacking only pathogens and leaving the others alone. But in people with Crohn’s disease, these cellular police lose control. They begin targeting even harmless microbes so vigorously and persistently that they create small hotbeds of inflammation. The irritation can create pits, ulcers, and actual holes, or fistulas, all of which may be exceedingly painful.

Clockwise from bottom, Derek Abbott, Lewis Cantley (inset), John Asara, and Andrew Wilkins have discovered that a genetic defect associated with Crohn's disease may result in an inability to modulate the activity of the NF-kappa B pathway. (Photo by Liza Green, HMS Media Services)

Ever since the American gastroenterologist Burrill Crohn characterized the illness in 1932, the molecular underpinnings of the disease have been a mystery. In 2000, researchers found that NOD2, a protein used by immune cells to detect bacterial fragments, was defective in some Crohn’s patients. But they did not know how that defect might be causing the disease, in large part because it was unclear how NOD2 normally functions. In a recent study, a team of HMS researchers hit upon a surprising answer.

Ubiquitin Tale

Derek Abbott, Lewis Cantley, and their colleagues report in the Dec. 29 Current Biology that NOD2, working with another protein, targets a central member of a major inflammatory avenue, the NF-kappa B pathway, essentially turning it into a molecular thermostat. What is surprising is the way NOD2 and its partner transform the protein, called NEMO (short for NF-kappa B essential modulator). Usually, proteins activate one another by adding a phosphate group. NOD2 and its partner change the function of NEMO by adding ubiquitins—the same tags that the cell uses to target proteins for destruction. The researchers believe that the defective NOD2 proteins found in Crohn’s patients are unable to efficiently ubiquitinate NEMO and consequently may compromise NEMO’s ability to regulate the NF-kappa B pathway. “We think that without NOD2 ubiquitination, you get persistent activation of NF-kappa B,” said Cantley, HMS professor of medicine at Beth Israel Deaconess Medical Center and also of systems biology.

This is not the first time that the NF-kappa B pathway has been implicated in Crohn’s disease, which is estimated to affect a half million Americans. To quell inflammation—and the pain and diarrhea that accompany the disease—patients are often prescribed a course of anti–NF-kappa B therapy. One of the most recent and promising of these drugs, infliximab (Remicade), works by blocking tumor necrosis factor-alpha (TNF-alpha), a main trigger of the Nf-kappa B pathway. Yet for all its success, infliximab remains a blunt instrument. To begin, TNF-alpha plays a wider role in the body, signaling inflammatory agents not just in the gut but in many other areas. Indeed, patients taking infliximab are more susceptible to secondary infections. Nor has TNF-alpha been shown to be defective in Crohn’s patients, which is why the existence of NOD2 variants has excited Crohn’s researchers. “You want to get as close as possible to the defect,” said Cantley. “The exciting part of this is it now reveals an aspect of NOD2 signaling unique to Crohn’s disease that is distinct from other pathways. It is potentially a novel site for targeting the disease that is more specific than TNF-alpha.”

Go Fish

NOD2’s story has been full of idiosyncracies. For example, after its discovery, researchers transfected NOD2 into cells and found that on its own, NOD2 activated the NF-kappa B pathway. On this basis, Crohn’s patients who harbor defective versions might be expected to exhibit a decrease in inflammatory activity, yet the reverse was true. “There was this paradox in that the genetics were telling us one thing and the biochemistry was telling us another. Clearly something more was going on,” said Abbott, an HMS fellow in pathology.

Of course, NOD2 is not the only protein to signal the NF-kappa B pathway. Thinking that the relationship between NOD2 and NF-kappa B might be more complex—and that NOD2 might have a different effect, depending on what other signals were being received—Abbott and his colleagues decided to go back and trace how exactly the two linked up. They knew that a group of kinases, the receptor interacting proteins (RIPs), played a role in NF-kappa B regulation. One of them, RIP2, had even been implicated in Crohn’s disease. Kinases typically work by adding phosphate groups. “So I began by looking for what RIP2 liked to phosphorylate,” said Abbott, who came to Cantley’s lab in 2000. “That was a horrible failure. But I also had this strange finding, that mutant forms of NOD2 would not bind RIP2.” The plot turned more favorable when he placed RIP2 and components of the NF-Kappa B pathway on an electrophoretic gel. One of them, NEMO, exhibited a huge band shift.

It did not take him long to determine the reason for the shift—NEMO was gaining ubiquitin groups. What is more, the kinase RIP2 appeared to play a key role in the ubiquitination process. When Abbott and his colleagues knocked out RIP2 using RNA interference, the NEMO band shift did not occur. Abbott set out to map where on NEMO the ubiquitins were being added. He knew that NEMO was ubiquitinated by two other signals, TNF-alpha and the T cell receptor (TCR), and that ubiquitination by both of them occurred at a particular spot, lysine 399. Abbott tried mutating this site, but he still saw the striking band shift, so RIP2 was involved in adding ubiquitins to another spot. “At this point the work really moved fast because it became clear that I was looking at a novel ubiquitination site,” he said. Using mass spectrometry, he found the site at lysine 285.

Putting the pieces together, Abbott, Cantley, and their colleagues believe that NOD2 binds RIP2 and that the two then somehow signal the cell’s ubiquitin machinery, which includes an E3 ligase, to add ubiquitins to NEMO. “What we hope is that the crosstalk between, say, TNF activation, which causes 399 ubiquitination, and NOD2, which causes 285 ubiquitination, maybe alters NF-Kappa B function in some way. What we are trying to show now is that NEMO acts as a molecular thermostat to turn up or down NF-kappa B activation,” said Abbott.

In this light, his initial strange finding—that mutant forms of NOD2 do not effectively bind RIP2—is telling. What it suggests is that although they may sense the presence of intracellular bacteria, the Crohn’s-associated versions are unable to effectively bind RIP2 and, conseqently, cannot carry out NEMO ubiquitination. This means the NF-kappa B inflammatory pathway is not properly modulated. But there are still gaps in the story. It is not clear why inflammation occurs in the presence of harmless bacteria. Nor is it clear how the inflammation might be stopped therapeutically. “One question we are asking here is how do we connect NOD2 with the ubiquitin machinery, because that machinery will probably have druggable targets. If we can discover the E3 ligase that does this, the enzyme might be a good target for activation or inhibition,” said Cantley.

—Misia Landau