Fluorescence Reveals Ubiquitination Dynamics

Ubiquitination, the enzymatic tagging of a protein with one or more copies of the 76-aa protein ubiquitin, has long been perceived as the biochemical equivalent of posting a "Condemned" sign, dooming the recipient to rapid degradation. However, recent evidence shows that this modification need not be a death sentence, and can play an important part in a variety of protein functions and signaling pathways. As scientists come to grips with a broadening range of ubiquitin-modulated activities, interest has grown in developing accurate and non-disruptive methods for tracking dynamic changes in ubiquitination levels.

In the December 2004 issue of Nature Methods, a team of Canadian and French researchers presents a novel strategy for the real-time detection of protein ubiquitination in living cells. Their system relies on a technique called BRET (bioluminescence resonance energy transfer), wherein a luminescent energy 'donor' molecule transfers energy to a proximal 'acceptor' molecule, which responds by fluorescing at a distinctive wavelength. In this particular approach, a protein of interest fused to luciferase is co-expressed with a GFP-ubiquitin fusion - if ubiquitination occurs, a readily detectable BRET signal is produced. This system proved capable of revealing the distinctive kinetics of ubiquitination for different proteins, and even detecting protein-protein interactions concomitant with an ubiquitination event. Importantly, this method also offers the possibility of monitoring highly transient ubiquitination changes, modifications that are considerably more difficult to observe by conventional immunochemical approaches. In an accompanying News and Views piece, noted signal transduction researcher Robert Lefkowitz concludes, "It seems likely that BRET assays such as those described by Perroy et al. will greatly expedite the unraveling of the biological roles of... post-translational modifications related to ubiquitination."

CONTACT:

Michel Bouvier (Université de Montréal, Montréal, QC, Canada)
Tel: +1 514 343 6372, E-mail: michel.bouvier@umontreal.ca

Robert Lefkowitz (Duke University, Durham, NC, USA)
Tel: +1 919 684 2974, E-mail: lefki001@receptor-biol.duke.edu

(C) Nature Methods press release.

Message posted by: Trevor M. D'Souza