research highlights

PROTEOMICS A day in the half-life of a protein

Researchers describe a method called Because bleach-chase can be used to accu- bleach-chase to quantitatively measure the rately quantify protein half-life, the method half-lives of fluorescently tagged proteins in can help clearly identify which proteins are human cancer cells. being actively degraded and which are being One of the ways that a cell regulates itself is diluted as the cell divides. Eden, co–first by removing proteins that it no longer needs author Naama Geva-Zatorsky, Alon and their or that are detrimental to its health. This hap- colleagues have been working with human pens by two major mechanisms: degradation cancer cells, which have moderate growth (such as via the proteasome) or dilution of rates, so the balance of degradation and dilu- the protein owing to cell growth. Rapidly tion is important. In determining half-lives dividing cells such as bacteria mainly rely on for 100 diverse proteins, they found that dilution to remove proteins, but terminally about 48% were regulated by degradation, differentiated mammalian cells mostly use 10% by dilution and the remaining 42% by a degradation. Because protein removal is mixture of the two processes. often dominated by one process or another They then applied stress to the cells in depending on the cell type, “the balance the form of a chemotherapy drug, which between degradation and dilution is often slowed the growth rate, and looked at the ignored,” says Eran Eden. “We wanted a way effect this had on the protein removal rate. to measure both.” Whereas the degradation rate of short- Eden, a recently minted Ph.D. from Uri lived proteins did not really change, the Alon’s group at the Weizmann Institute of half-lives of the long-lived proteins, which Science, Alon and their colleagues consid- are largely removed by dilution, became ered the traditional methods for measuring even longer. When they tested other protein degradation rates, which include stresses, which also slowed cell growth but pulse-chase and translation inhibition. at a different rate, they observed the same Although pulse-chase has been used for effect. “We think this is quite a general decades, this method requires radioactive phenomenon,” Eden says. “This is a sim- labeling and protein-specific antibodies ple principle that can help one understand for detection. Protein synthesis inhibitors, a lot about the removal rate of a protein in contrast, can greatly perturb the cell. in response to different stresses.” Notably, Nature America, Inc. All rights reserved. All rights Inc. America, Nature 1 The researchers wanted a non-radioactive this result seems to suggest that cells do method that would minimally perturb the not use protein degradation to compen-

© 201 cells and that could be scaled to follow the sate for changes in the dilution rate. fate of a large number of proteins. The bleach-chase method should be use- They came up with a method to measure ful to anyone who needs to measure protein protein half-lives that they call bleach-chase. half-lives. “Doing these experiments is very, They label the protein of interest with a fluo- very simple compared to pulse-chase,” says rescent YFP tag and then shine light on the Eden. Because the method allows one to gen- cells, just for long enough to bleach 10–60% erate results much more rapidly than with of the fluorophores; the short bleaching pulse-chase, “it can really open the ability to time does not substantially perturb the cells. measure protein half-life on a large scale,” he “Conceptually, we can think of this as an says. One drawback is that the proteins must ‘anti-tag’ that is generated at timepoint zero,” be fluorescently tagged, which could alter the explains Eden. “If you monitor the cells protein removal rate, but the researchers were through time you will see that the new pro- careful to rule out this possibility with pulse- tein that is produced is the YFP-tagged pro- chase control experiments. tein.” The problem with the anti-tag, however, As fluorescently tagged protein librar- is that it is invisible to fluorescence detection. ies are becoming available for more and Thus, to determine the protein removal rate, more organisms, the method is likely to be the researchers perform the experiment widely applicable. twice, once with bleaching and once with- Allison Doerr out bleaching, using fluorescence time-lapse RESEARCH PAPERS microscopy for detection. Then they simply Eden, E. et al. Proteome half-life dynamics subtract the fluorescence levels to obtain the in living human cells. Science advance online dynamics of the ‘invisible’ proteins. publication (13 January 2011).

nature methods | VOL.8 NO.3 | MARCH 2011 | 201