Abstract 5335: Dissecting the ubiquitin pathway using homogenous and sensitive bioluminescent assay

Post-translational modification of target proteins by ubiquitin (Ub) and ubiquitin-like (Ubl) proteins is a fundamental mechanism for regulating protein functions and protein stability thus affecting diverse cellular processes. Several ubiquitin-like (Ubl) molecules have been identified including SUMO, NEDD8, ATG8/15 and ISG15 which share the ubiquitin fold Ub/Ubl proteins are conjugated to lysine residues in substrate proteins through an ATP-dependent enzymatic cascade involving E1-activating enzyme, E2-conjugating enzyme and E3-ligase. To mark proteins for degradation, multiple ubiquitins are covalently attached to produce a Lys48 linked poly-ubiquitin chain. The poly-ubiquitinated proteins are recognized by the 26S proteasome system and deubiquitinated and destroyed. Substrates with ubiquitin chains linked through Lys 6, 11, 27, 29, 33 also regulate protein degradation. Substrates marked with mono-ubiquitination or Lys63 ubiquitin chains are involved in cell signaling independent of degradative functions. Many diseases are associated with dysfunction of ubiquitin signaling, with the E3 ligase class of enzymes are considered to be the most important target. However, in recent years, evidence have accumulated demonstrating the significance of E2 class of enzymes where mutations and impairment of this class lead to severe diseases states including chromosomal instability, cancer predisposition, neurological syndromes, and immunological disorders. Thus, in addition to E3, E2 group of enzymes represent important class of therapeutic targets. To address the need for developing drugs targeting these enzymes, we have developed a novel assay that can be used to screen for modulators of these enzymes by taking advantage of the first ubiquitin activating enzyme E1 which activates free Ub forming a high-energy thioester bond between E1’s active site Cys and the C-terminus of Ub, generating AMP and pyrophosphate (PPi) as products of the reaction. We monitor the activity of these enzymes by determining the concentration of AMP generated in the reactions in a luminescence-based assay. Our assay interrogate not only E1 but also E2 and E3 in a very robust and sensitive manner. The assay is homogenous, bioluminescent, and show a very high signal to background in a very selective manner for each class of the ubiquitin pathway. The assay was successfully tested for ubiquitination, SUMOlyation and Neddylation reactions and thus can be used for screening of enzymes targeting these pathways. Citation Format: Said A. Goueli, Kevin Hsiao, Subhanjan Mondal. Dissecting the ubiquitin pathway using homogenous and sensitive bioluminescent assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5335. doi:10.1158/1538-7445.AM2017-5335