Researchers at HMS have developed a new way to find interactions between proteins, beginning with those that relate with a type of protein called deubiquitinating enzymes, or Dubs. These enzymes are named for their ability to remove ubiquitin molecules from other proteins, an action that regulates the numbers of these proteins in a cell and, in turn, throttles many different biochemical processes. Until recently, though, most Dubs had not been studied in detail.
In a paper published in the July 24 Cell, co-authors Mat Sowa and Eric Bennett, postdoctoral fellows in the laboratory of Wade Harper, a professor in the Department of Pathology, use a semi-high-throughput method to analyze more than 75 percent of the known Dubs, uncovering sevenfold more Dub-binding proteins than previously reported.
Sowa and Bennett began by performing immunoprecipitation experiments on Dubs to collect the proteins that bound to them. To identify these proteins, the researchers then used mass spectroscopy in collaboration with Steven Gygi of the Department of Cell Biology. But because each immunoprecipitation yielded a complicated mixture of proteins, the mass spec results were almost indecipherable. The scientists devised a new computer algorithm to look for patterns in the data and pick out the proteins that were binding specifically to Dubs.
Compared with the traditional approach of analyzing each immunoprecipitation experiment by hand, the computerized method was much more productive and reliable. The researchers analyzed 75 of the 95 Dubs encoded by the human genome and found 774 proteins that bound to them. Many Dubs were placed into biological pathways for the first time. A case in point is the Dub USP13, which the scientists were able to place in the endoplasmic reticulum–associated degradation pathway (ERAD) using traditional cell biological studies.
The advantage of their method, according to Sowa and Bennett, is that it is simpler and more reliable than previous manual techniques. It is also remarkably accurate—using multiple independent tests, they confirmed the computerized predictions of interactions 68 percent of the time, a very high rate for this type of analysis.
Another benefit is that the software can be applied to other biological problems. Sowa and Bennett have combined their algorithms and data into a single package called CompPASS, or Comparative Proteomic Analysis Software Suite, and made it available online for other scientists. They continue to investigate the function of Dubs using more detailed biochemical techniques and hope to expand the applications of CompPASS through existing and future collaborations with other labs.
CompPASS is available at http://pathology.hms.harvard.edu/labs/harper/Welcome.html.
Students may contact Wade Harper at wade_harper@hms.harvard.edu for more information.
Conflict Disclosure: The authors declare no conflicts of interest.
Funding Sources: The National Institutes of Health, Paul Glenn Foundation, Stewart Trust, American Cancer Society, Damon Runyon Cancer Research Foundation; the content of this work is the responsibility solely of the authors.
