Random Walk
Ubiquitin's Kiss of Death
Ubiquitin is a molecular stoolie, fingering other proteins for destruction by a cell’s molecular trigger men. But don’t feel bad for the victims—they all need to die. Some are molecules whose job is done, such as messengers whose signal has been sent. Others might be old and falling apart, or perhaps didn’t form properly in the first place. Ubiquitin fingers its marks for the whisper chipper by attaching a chain of four or more copies of itself to the soon-to-be-departed, but like any good hit, it all happens so fast that you can’t see it coming.
Until now, that is. Nathan Pierce, a grad student in the lab of Raymond Deshaies, a professor of biology at Caltech and a Howard Hughes Medical Institute investigator, has created a sort of biological stop-motion animation based on a piece of apparatus called a quench-flow machine that allows you to stop, or quench, a reaction after a very precise interval of time has elapsed—for these studies, in increments of 10 milliseconds. Previous studies had looked at the reaction on the scale of seconds or minutes, which “did not have sufficient time resolution to see what was going on,” says Deshaies. “It’s as if you gave an ice-cream cone to a child and took a picture every minute. You would see the ice cream disappear from the first photo to the next, but since the pictures are too far apart in time, you would have no idea whether the kid ate the ice cream one bite at a time, or swallowed the entire scoop in one gulp.”
In this case, the question was whether the ubiquitin molecules got added one at a time, or did the entire preassembled chain get added in one go? It was already known that three different enzymes, dubbed E1, E2, and E3, are involved in the process. E1 readies the ubiquitin for transfer, then hands it off to E2. A form of E3 called a RING ligase (RING stands for “really interesting new gene”) then binds to the E2 and the target protein simultaneously, causing the E2 to transfer the ubiquitin molecule to the target. “The process is so complicated and so fast,” Pierce notes, “that we weren’t able to see how the chain is actually built.”
It turns out that the ubiquitins are attached one by one, and a paper by Pierce announcing this appeared in the December 3, 2009, issue of Nature. In addition to Pierce and Deshaies, the other authors were postdoc Gary Kleiger and Assistant Professor of Chemistry Shu-ou Shan.
The next task was to figure out how the process works so quickly. One answer was provided in a parallel study spearheaded by Kleiger. He showed that E2 and E3 interact with each other at a blistering pace while building the chain—far faster than is commonly seen in protein trysts. As is often the case with humans, opposites attract, and the enzymatic speed dating is enabled by an acidic tail on E2 that nestles into an alkaline canyon on E3. Kleiger’s work appeared in the November 25, 2009, issue of Cell; the other authors included Caltech postdoc Anjanabha Saha, Steven Lewis and Brian Kuhlman of the University of North Carolina at Chapel Hill, and Deshaies.
Deshaies now wants to find the reaction sequence’s slowest step, the one that sets the speed limit for the entire process. By finding the slowest step and making it slower, he says, the enzymes “may become too slow to get their job done—to build chains—in the time available to them to do so. Being able to develop drugs to block their function would open up a new frontier in medicine.”
The work was funded by a Gordon Ross Fellowship, the National Institutes of Health, and the Howard Hughes Medical Institute. —LO
