A Microbead-Based Flow Cytometry Assay to Assess the Activity of Ubiquitin and Ubiquitin-Like Conjugating Enzymes

Abstract

The peptidic posttranslational modifiers of the ubiquitin (Ub) family (ubiquitin-like, UbLs) are conjugated to thousands of proteins to modify their function and fate. Dysregulation of their conjugation/deconjugation pathways is associated with a variety of pathological disorders. However, the techniques currently available to monitor the levels of target modification by UbLs as well as the activity of UbL-conjugating enzymes are limited and generally not quantitative. Here, we describe a microbead-based flow cytometry assay to accurately quantify UbL conjugation activity. It measures the capacity of UbL-conjugating enzymes, either purified or present in cell extracts, to transfer their respective UbL onto target substrates immobilized on color-coded microbeads. Although this protocol describes its use to study protein modification by Ub, SUMO-1 to SUMO-3, and NEDD8, this assay may be applicable to investigating conjugation of any other UbLs. It should therefore prove a precious tool for both screening UbL-conjugating enzymes inhibitors and following UbL pathway dysregulations in both physiological and pathological settings.

Keywords: Enzymatic assay; NEDD8; SUMO-1; SUMO-2; Ubiquitin; Ubiquitin-like protein modifiers (UbLs).