Real time monitoring of Ubiquitination on living cells by BRET assay's

Ubiquitin has emerged as an important regulator of protein stability and function in organisms ranging from yeast to mammals. The ability to detect in situ changes in protein ubiquitination without perturbing the physiological environment of cells would be a major step forward in understanding the ubiquitination process and its consequences. A new method has been created to study this dynamic post-translational modification in intact human embryonic kidney cells. Using bioluminescence resonance energy transfer (BRET) a measure of the ubiquitination of Beta-arrestin 2, a regulatory protein implicated in the modulation of G protein-coupled receptors. In addition to allowing the detection of basal and GPCR-regulated ubiquitination of Beta- arrestin 2 in living cells, real time BRET measurements permitted the recording of distinct ubiquitination kinetics that are dictated by the identity of the activated receptor. The ubiquitination BRET assay should prove useful as a tool for studying the dynamic ubiquitination of proteins and for understanding which cellular functions are regulated by the post-translational events.

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Ubiquitination is a rapid cellular and reversible post-translational modification that is involved in numerous aspects of biology. The 76-residue polypeptide ubiquitin fulfils essential functions in eukaryotes through its covalent attachment to other intracellular proteins. Historically, the best characterized role for this modification is the targeting of the proteins for degradation by the 26S proteasome after the transfer of an ubiquitin chain of at least four units, referred to as polyubiquitination. More recently, the addition of a single ubiquitin to one (monoubiquitination) ot multiple (polyubiquitination) protein sites has been described. In these cases, the post-translational modification serves as a reversible signal involved in a variety of key cellular functions including intracellular signaling, sub cellular localization and protein-protein interactions.
Despite the growing interest in ubiquitination, inspired by the diversity of cellular functions that appear to be influenced by this process, the lack of tools for monitoring its dynamic regulation limits studies in this field. Western blot analysis using antibodies to ubiquitin allows the detection of protein ubiquitination. Although this assay can be used to determine whether a protein is ubiquinated, it cannot be easily adapted to study the dynamic nature of the ubiquitination and deubiquitination cycles. To monitor the ubiquitination process in living cells, they took advantage of a bioluminescence resonance energy transfer (BRET) method, which allowed real-time detection of the protein-protein interactions in vivo. This technique is based on the nonradiative transfer of energy between a luminescent energy donor (here they used, Renilla luciferase, Rluc) and a fluorescent energy acceptor (Here it was green fluorescent protein, GFP). This is a system of choice monitoring for both constitutive and regulated intermolecular interactions because of the strict dependence on molecular proximity (<100 Angstroms) between the donor and acceptor molecules for energy transfer. It was recently proposed that Beta-arrestin 2 (Beta-arrestin), a versatile protein involved in the regulation of G protein-coupled receptor (GPCR) signaling, is ubiquitinated in response to receptor activation. Based on the stability of their interaction with Beta-arrestin, two broad classes of GPCRs are distinguishable: GPCRs known as class A receptors (for example, the Beta2adrenergic receptor, Beta­2AR) interact only transiently with Beta-arrestin after activation, whereas activated class B receptors (for example the V2-vasopressin receptor, V2R) form stable complexes with beta-arrestin. Recent findings are suggestive of a link between the dynamics of Beta-arrestin ubiquitination and the nature of its interaction with the activated receptor. A stable interaction correlates with sustained Beta-arrestin ubiquitination. Using Beta-arrestin as a model ubuiquitinated protein, we report here that BRET allows monitoring of the changes in the ubuiquitination state of a protein in real time.
To read on the procedural methods please see articles written by the University of Montréal’s Biochemical department, Laboratory of functional genomics, Montpellier France.