Ultraspeed Resolution Reveals that Ubiquitins Bind Sequentially

An innovative reaction quenching protocol with millisecond resolution has revealed that cells attach ubiquitin chains to proteins marked for destruction link by link rather than all at once.

The addition of a chain of four or more ubiquitin molecules to a target protein marks that protein for destruction by protein-degrading complexes in the cell. Heretofore, it was not known whether these molecules were added to the target protein sequentially or as a pre-formed chain.

In the current study, investigators at California Institute of Technology (Pasadena, USA) developed new methodology to study the action of ubiquitin ligase, the enzyme complex that attaches ubiquitin to the target protein at short time intervals that had not previously been possible. For this purpose, they adapted an instrument called a "quench-flow" machine, a machine that allows for extreme precision in the stopping, or "quenching," of a reaction. This instrument allowed them to follow changes in structure at intervals as short as 10 milliseconds in both yeast and human proteins.

"We devised methods to take snapshots of ubiquitin ligase reactions at a rate of up to 100 "pictures" every second," said senior author Dr. Raymond Deshaies, professor of biology at the California Institute of Technology. "This enables us to see things that would normally evade detection. Prior methods did not have sufficient time resolution to see what was going on. It is as if you gave an ice-cream cone to a kid and took pictures 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 child ate the ice cream one bite at a time, or swallowed the entire scoop in one gulp."

Results published in the December 3, 2009, issue of the journal Nature revealed that the three ubiquitin ligase enzymes, E1, E2, and E3 work as a team to build polyubiquitin chains on substrates by sequential transfers of single ubiquitins.

"The new method revealed the biological equivalent of small, single bites of ice cream," said Dr. Deshaies. "Using our approach we could see that our ubiquitin ligase builds ubiquitin chains one ubiquitin at a time. Gaining these kinds of insights into the ubiquitin system is important because ubiquitin ligases play a critical role in a number of human diseases, including cancer, due to their role in the regulation of the cell cycle."

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California Institute of Technology