A bioluminescent assay for monitoring conjugation of ubiquitin and ubiquitin-like proteins

摘要: Post-translational modification of target proteins by ubiquitin (Ub) and ubiquitin-like (Ubl) is a critical mechanism for regulating protein functions affecting diverse cellular processes. Ub/Ubl are conjugated to lysine residues in substrate through an adenosine triphosphate (ATP)-dependent enzymatic cascade involving enzyme 1 (E1)-activating enzyme, E2-conjugating E3 ligase. The amount monophosphate (AMP) produced the first step, E1-mediated activation, represents accurate measure transfer during process. Here we describe novel bioluminescent assay platform, AMP-Glo, quantify conjugation measuring AMP generated. AMP-Glo performed two-step reaction. step terminates ubiquitination reaction, depletes remaining ATP, converts generated reaction diphosphate (ADP), second ADP converted which detected as signal using luciferase/luciferin, proportional concentration correlated with activity. We demonstrate use study screen chemical modulators enzymes involved Because there sequential enhancement light output presence E1, E2, E3, system can be used deconvolute inhibitor specificity.