29 November 2004. Ubiquitination, sumoylation (tagging with small ubiquitin-like modifiers), ubiquitinated aggregates, and problems with ubiquitin-mediated proteasomal degradation have all been linked to pathological processes that underlie various neurodegenerative diseases, including Alzheimer, Parkinson, and Huntington diseases. Researchers studying these processes may be interested in a new technique that can measure ubiquitination—and deubiquitination—in real time. In the December issue of Nature Methods (first published online Nov. 18), Michel Bouvier and colleagues from the University of Montreal, Quebec, describe a way to measure this covalent modification process using bioluminescence resonance energy transfer, commonly known as BRET.
BRET, first described by Carl Johnson and colleagues at Vanderbilt University, Tennessee, is a bit like its cousin FRET (florescence resonance energy transfer—see ARF related news story) in that it measures molecular proximity by relying on transfer of light from one molecule to another. But BRET uses a luciferase enzyme as the electron donor, thus eliminating the need to excite samples with light. In their study, Bouvier and colleagues used two hybrid proteins (think of the classical two-hybrid protein-protein interaction assay) in which a bait, in this case a protein fused to Renilla luciferase (RLuc), was used to lure in a prey—ubiquitin fused to green fluorescent protein (GFP).
Joint first authors Julie Perroy and Stephanie Pontier used a β-arrestin fusion as the bait. β-arrestin is a well-known substrate for ubiquitination. When the authors expressed the two hybrids, along with the luciferase substrate DeepBlueC coelenterazine, in human kidney cells, they found that the energy transfer, as judged by GFP fluorescence, dramatically increased as the ubiquitin-GFP concentration was raised. This indicates that the GFP fluorescence was the result of a direct interaction between the arrestin and ubiquitin hybrids.
But this does not necessarily mean that the authors were measuring ubiquitination. They may have just been measuring contact between arrestin and ubiquitin. So to confirm that the BRET signal was actually a reflection of a covalent modification, Perroy and Pontier used a mutant ubiquitin that cannot be covalently attached to substrate protein. Using this prey, no GFP emissions were observed that were above baseline, indicating that for BRET to take place in this system, the ubiquitin hybrid must covalently bind to its substrate.
To show that their detection system is sensitive enough to detect real biological changes, the authors used G-protein coupled receptor (GPCR) activation to induce β-arrestin ubiquitination. When the authors challenged cells with isoproterenol (ISO) or arginine-vasopressin (AVP), agonists for the V2-vasopressin receptor (V2R) and β2 adrenergic receptor (β2AR) GPCRs, respectively, they found significant increases in the BRET signal, indicating that β-arrestin had been ubiquitinated. But because activated β2ARs are known to interact only transiently with arrestin, the authors were able to take this experiment one step further and measure, in real time, the delicate dynamics of the ubiquitination process.
While both GPCR agonists caused a rapid and similarly robust increase in the BRET signal (over fivefold), peaking after about two minutes, the isoproterenol-induced signal persisted for over 10 minutes, whereas the vasopressin-induced signal began to fade as quickly as it peaked, and was all but absent after 10 minutes. “This reduction in BRET signal most likely reflects a deubiquitination process and not a degradation of β-arrestin,” suggest the authors.
One caveat of the method is that to avoid quenching, the GFP-ubiquitin reporter must be designed to prevent polyubiquitination. The authors circumvented this problem by changing ubiquitin lysines 48 and 63, which can bind additional ubiquitin chains, to alanines.
As compared to Western blot analysis, which is typically used to measure ubiquitination of substrates, the BRET assay has several advantages, suggest Bouvier and colleagues: It avoids possible signal alterations that could result from cell lysis; it can capture the dynamic nature of the process; it can be used to monitor changes in specific cells given a specific treatment; and, because of the nature of the BRET measurements, it can distinguish deubiquitination from the simple loss of signal due to degradation (baseline BRET acts as an internal control). It will be of interest to see if this technique could easily be adapted for use in neurons.—Tom Fagan.
Perroy J, Pontier S, Charest PG, Aubry M, Bouvier M. Real-time monitoring of ubiquitination in living cells by BRET. Nat. Methods 2004 November 18. Advanced online publication.