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Chemical Genetics Identifies New Way of Disrupting Cell's Protein Recycling System
Small Molecules Block Ubiquitin-Proteasome PathwayThe study of genetic mutations has led to the discovery of thousands of genes and countless biological pathways. A slight variation on the theme is "chemical genetics," which was conceived to bypass the genome and directly "mutate" these pathways with small-molecule inhibitors. Chemical genetics aims to uncover molecular interactions that the more traditional approach may have missed. It also comes with a bonus: the chemicals used could lead to the next blockbuster drug.
Randall King (left) and colleagues have found a new type of inhibitor for the ubiquitin-proteasome pathway. Ubistatins, characterized by Greg Tochtrop, prevent ubiquitinated proteins from accessing the proteasome. Similar molecules could prevent cancer by interrupting mitosis. (Photo by Leah Gourley)
So far, the approach has paled in the shadow of true genetics, but reporting in the Oct. 1 Science, researchers led by HMS assistant professor of cell biology Randall King help bring it into the spotlight by revealing a new class of chemical "mutagen," one that inhibits the ubiquitin-proteasome system. Acting in a completely unexpected fashion, these inhibitors highlight a critical protein-protein interaction and may point to a new form of cancer drug.
The proteasome is a hollow, multiprotein complex that is lined with proteolytic enzymes called core proteases. The complete unit acts like a microscopic garbage disposal--sucking ubiquitinated proteins in one end, digesting them, and spitting small peptides and amino acids out the other. But it is much more than a recycling plant for unwanted protein. The proteasome is thought to be a major factor in neurodegenerative diseases, such as Huntington's, in which undegraded proteins accumulate and eventually poison neurons. And its action is necessary for key regulatory processes such as the chopping up of foreign antigens that is essential for keeping the immune system primed. The ubiquitin-proteasome pathway is also crucial for cell division because it removes proteins that put the brakes on mitosis. It is for this reason that proteasome inhibitors have recently been approved for the treatment of multiple myeloma.
"What we have discovered is a new way to drug the ubiquitin-proteasome pathway that would not have been predicted before we began this study," said King. The investigation sought chemicals that could prevent completion of mitosis by blocking removal of cyclin B, one of those mitotic brakes. By design, the properties of the chemicals used in the screen and the proteins they might inhibit were not known in advance. "The real novelty here is the mechanism of inhibition, which we were able to stumble upon precisely because the initial chemical screen was unbiased," King said. "We didn't make any assumptions about what might be a good target or a bad target." This is one of the strengths of the chemical genetics approach.
Cyclin B Blockers
This initial screen was carried out by Noël Peters, a research assistant in the King lab. Using a cell-free system from frog eggs to mimic cell cycle progression in the test tube, Peters tested more than 100,000 compounds for their ability to block mitotic completion. The screening, which took about a year, uncovered 22 inhibitors. Sixteen of these turned out to block mitotic entry, while the remaining six were found to block cyclin B degradation.
| "What we have discovered is a new way to drug the ubiquitin-proteasome pathway that would not have been predicted before we began this study." |
When Peters and colleagues tested the six compounds in assays for degradation of cyclin B and of another protein, beta-catenin, which is ubiquitinated by its own ligase, three of the compounds unexpectedly blocked degradation of both proteins. This indicated that ubiquitination was not the step they inhibited. Peters also found that these three compounds failed to inhibit the core catalytic activity of the proteasome. Together, these findings suggested that the three inhibitors must act somewhere between ubiquitination and degradation--but where?
The Break Point
To answer this question, King enlisted the help of Raymond Deshaies from the California Institute of Technology. Deshaies had developed a cell-free system for measuring proteasome activity that enables him to feed ubiquitinated proteins directly to highly purified proteasomes. With this system, King predicted, it might be easier to pinpoint how the inhibitors work. Rati Verma, a scientist in Deshaies's lab, found that in their system, two of the compounds, now called ubistatins, strongly inhibited the degradation of a ubiquitinated protein called Sic1.In order for Sic1 to be degraded, it must first bind to proteasome receptors that recognize ubiquitinated proteins, then the ubiquitin must be removed by a peptidase component on the proteasome surface. Only then does Sic pass into the proteasome proper, where it is chewed up by the core proteases.
When King and colleagues inhibited the core proteases, they found that ubistatins prevented the build-up of de-ubiquitinated Sic1 in the proteasome. This suggested that either ubiquitinated Sic1 failed to bind to the receptors on the proteasome surface or that the ubiquitin was not getting removed. Verma found that poor binding was the culprit because when he purified proteasomes in the presence of the ubistatins, ubiquitinated Sic1 failed to come along for the ride, as it does if ubistatins are not present.
Subsequently, in collaboration with David Fushman of the University of Maryland, King was able to show that ubistatins bind to the interface between two ubiquitin molecules, thereby masking the ability of ubiquitin chains to be recognized by the proteasome. "Even though we knew that the ubiquitin chain was important for targeting proteins for degradation, I don't think anyone would have predicted prior to this study that it would be straightforward to target that interaction with a small molecule. That's where the value of chemical genetics lies because it highlights new ways to disrupt complex biochemical systems," King said.
In this case, there may be additional value. Currently drugs are overwhelmingly small molecules that bind to small pockets in enzymes or receptors, but as David Bellows and Mike Tyers from Mount Sinai Hospital, Toronto, write in an accompanying Science perspective, "ubistatins demonstrate that it is indeed possible to disrupt protein-protein interactions with small molecules Š and thus get at the very heart of biological specificity, the regulation of protein interaction." Although ubistatins are not useful themselves as drugs because of solubility problems, they pinpoint an interaction that could be a useful drug target. Last year, the Food and Drug Administration approved bortezomib (Velcade), the first proteasome inhibitor for treatment of cancer, but this drug targets the catalytic core of the proteasome. The potential advantage of ubistatin-like drugs is that they would be far more selective, targeting only a subset of the proteins that the proteasome degrades.
--Tom Fagan