Abstract
Abstract Protein modifications by the covalent attachment of ubiquitin (Ub) or ubiquitin-like proteins (UBLs) such as SUMO, ISG15 or NEDD8 participate in many critical cellular processes. These include cell-cycle regulation, DNA repair, endocytosis, mitophagy, oncogenesis, post-membrane-protein trafficking, antiviral pathways and most notably, proteasomal degradation of target proteins. Ubiquitination and modification by UBLs share a similar catalytic cascade which requires the sequential action of three classes of enzymes: E1 activating enzymes, E2 conjugating enzymes and E3 ligases. While Ub/UBLs are activated by E1 through an ATP-dependent thioester bond formation and transferred to E2 from E1, the exact mechanism of how they are subsequently attached to a protein substrate depends on the type of E3's containing characteristic catalytic domain - RING, HECT or RBR. It has been generally accepted that E3's mediate substrate specificity and E2's are the main determinants for selection of the lysine to construct the modifier chains, which thereby directly control the cellular fate of the substrate. Recent research has linked dysregulation of the Ub/UBLs modification system to numerous diseases including cancer, immunological disorders and neurodegeneration. The high substrate specificity provided by combinations of over 30 E2's and over 600 E3's in the human genome makes these enzymes emerging drug targets. In response to a growing market demand, SignalChem has developed an extensive array of products encompassing enzymes, Ub/UBL modifiers and substrates in the ubiquitination, SUMOylation, ISGylation and NEDDylation processes. With Promega's AMP-GloTM reagents and an optimized assay protocol, we have identified and validated a variety of functionally competent combinations of these enzyme components. With the established protocol, each enzyme in the catalytic cascade has been assessed for their specific activity towards generation of free AMP. In addition, inhibition profiles of all three enzymes have been obtained using the assay system, further demonstrating their potential to be used in high-throughput screening to identify potential lead compounds for drug discovery and development programs. Citation Format: Eric Yao, Shenshen Lai, Jun Yan. Empowering research on ubiquitin and ubiquitin-like protein modification cascade using recombinant enzyme systems [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3862.
Published Version
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