Homologous to E6-AP C-terminus (HECT) E3 ubiquitin ligases are associated with a range of human diseases and cellular processes. Identification of HECT E3 - substrate pairs is an essential step in understanding functions of the implicated ligases. In general, finding E3 ubiquitin ligase - substrate pairs by immunoprecipitation and in vitro ubiquitination/binding assay is a daunting task since the interactions between HECT E3 and their substrates are transient, weak, and sometimes require cofactors. In this report, we introduce a potentially novel approach to identify substrates through selective enrichment of the ubiquitinated substrates downstream of the HECT ligases. The approach is based on engineering HECT ligase ~ ubiquitin thioester intermediates directly from activated ubiquitin and HECT ligases, bypassing E1, E2, and ATP in the native enzymatic cascade. Thorough biochemical analysis showed that the engineered thioester intermediate is still compliant with the intrinsic mechanism of the native HECT ligase in catalyzing isopeptide ligation between ubiquitin and the substrates. Thus, this system recapitulates the final conjugation step which is sequestered from the preceding steps. We will show how this power of the simplification can be exploited to identify substrates of HECT ligases as well as to study the mechanism of the protein ubiquitination.

Engineering HECT ligase~ubiquitin thioester intermediates to identify HECT ligase-substrate pairs: Bypassing E1 and E2

246th National Meeting (2013)

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