Development of a homogeneous AlphaLISA ubiquitination assay using ubiquitin binding matrices as universal components for the detection of ubiquitinated proteins

The Ubiquitin Proteasome Pathway (UPP) has become a target rich pathway for therapeutic intervention as its role in pathogenic disease is better understood. In particular many E3 ligases within this pathway have been implicated in cancer, inflammation and metabolic diseases. It has been of great interest to develop biochemical assays to identify inhibitors of catalytic E3 ubiquitination activity as a means of abrogating the disease mechanism. Here we describe a homogeneous biochemical assay that utilizes native ubiquitin and Tandem-repeated Ubiquitin-Binding Entities (TUBEs) as a detection technology for ubiquitination activity. We developed a TUBEs based proximity AlphaLisa® assay for Mdm2, which is an E3 ligase that negatively regulates p53 tumor suppressor protein. We further demonstrate that this assay strategy or design can also be applied to the development of assays to detect autoubiquitination of other E3 ligases that are also of interest for therapeutic intervention. This article is part of a Special Issue entitled: Ubiquitin Drug Discovery and Diagnostics.

► A homogenous autoubiquitination AlphaLisa assay was developed.
► Tandem Ubiquitin Binding Entities (TUBES) allows for the use of wild type ubiquitin.
► We demonstrate the universality of this approach with 3 E3s from 2 ligase classes.