A bouncer stands at the door of a bar, allowing patrons to enter until the facility reaches capacity. He counts the guests while checking their IDs, but he forgets about a back door, where some of them exit. His tally is off. Scientists make a similar mistake if they use transcription and translation as a proxy for protein levels in the cell while ignoring degradation rates. Now they can monitor the back door, too, by measuring mammalian protein stability on a grand scale.“Labs can use pulse–chase to determine the stability of particular proteins, but those experiments are labor intensive,” said Stephen Elledge, a Howard Hughes investigator and the Gregor Mendel professor of genetics and of medicine at HMS and Brigham and Women’s Hospital. “We’ve developed a simple construct that enables global protein stability profiling.”
Two papers in the Nov. 7 Science detail the new approach and demonstrate how it can be applied.
Each construct in the new method (see figure) contains two parts—one coding for a GFP fusion protein that fluoresces green and the other coding for a protein called DsRed, which fluoresces red. A gene for an internal ribosome entry site links the two parts, ensuring they get translated in tandem (except under extreme conditions).
After controlling for differences in synthesis, the researchers focused on protein stability by comparing green-to-red ratios across cells that contain the constructs. Although each GFP fusion protein degrades at a different rate, depending on its structure and the context, long-lived DsRed always breaks down at the same rate, making comparisons possible. If one cell contains GFP-X and another contains GFP-Y, for example, their green-to-red ratios will be different, revealing the stability of X relative to Y.
“Elegant genetic screens allow you to sort data from complex mixtures, so although this was a step in the right direction, we still had more work ahead of us,” said postdoc Hsueh-Chi Sherry Yen, first author on both papers.
Using a library of protein-coding genes, Yen built approximately 8,000 unique constructs, copied each hundreds of times, placed them in cells, and pooled the cells together in a single dish. Each cell carried just one construct.
Borrowing a page from Julius Caesar, she pursued a divide and conquer strategy. She used a machine for fluorescence-activated cell sorting (FACS) to separate the cells into seven bins, based on their green-to-red ratios. The machine directed cells with unstable green fusion protein to Bin 1, cells with stable green fusion protein to Bin 7, and cells with moderately stable green fusion protein to Bins 2 through 6.
Sometimes, cells with the same construct ended up in different bins. This happened when a green fusion protein’s stability varied across the cell cycle. The FACS machine, for example, might catch one cell during anaphase and another with the same construct during interphase and direct them to different bins.
Yen could then probe the seven bins to understand their contents. She isolated the genomic DNA from cells floating in Bin 1, for example, and labeled it with one color dye. As a control, she isolated genomic DNA from the original unsorted dish—pre-FACS analysis—and labeled it with a different color. She hybridized both sets of DNA to a microarray and calculated the ratio of color 1 to color 2 at each spot (representing a gene) on the array. After repeating this procedure for the other six bins, she mapped the distribution of each GFP fusion protein across the bins.
This method allowed her to calculate the half-life of each protein. She was also able to identify proteins that might play a role in cell division, based on their presence in different bins at different stages of the cell cycle. Most importantly, Yen’s experiment provided proof of principle. Her method shows that global protein stability profiling is possible.
“This is a totally new technique by which you can measure the stability of thousands of proteins at the same time,” said New York University professor and HHMI investigator Michele Pagano, who was not involved in the study. “You can calculate the half-lives of proteins when the cell is in a particular state and make comparisons. For example, you can synchronize cells in a particular phase of the cell cycle, measure protein stability in that population, and then compare that to a control.”
Yen, Elledge, and colleagues demonstrated one application of the new technique. They knocked down SCF ubiquitin ligase in a population of cells, created a global protein stability profile, and compared it with a control profile to identify novel substrates.
“That’s just the beginning,” said Elledge.
Students may contact Stephen Elledge at selledge@genetics.med.harvard.edu for more information on this or other lab projects.
Conflict Disclosure: The authors report no conflicts.
Funding Sources: The Elledge lab is supported by the National Institutes of Health, Howard Hughes Medical Institute, the Department of Defense, and the Gates Foundation.
