Misregulation of the ubiquitin proteasome system (UPS) causes many human diseases. In the UPS, ubiquitin (Ub) is relayed across three enzyme families (E1�>E�>E3) before conjugating to a substrate protein. In this cascade, the E2-E3 interactions specify which substrates are ubiquitinated and the residues through which Ub is bound to the substrate or to other bound Ub. From the ~40 E2s and >600 E3s known to exist in humans are ~24,000 possible E2-E3 interactions. Unfortunately, weak binding association, complex stoichiometry, and potential accessory proteins have made the mapping of E2-E3 interactions exceedingly difficult with established techniques. To reliably detect the protein-protein interactions of any given E2, we have developed a site-specific photo-cross-linking technique that employs selective alkylation of an E2 cysteine point mutant by a sterically efficient iodoacetamide diazirine (Figure 1A). To diazirine modified mutants of the E2 UbcH7, we have successfully photo-cross-linked the HECT E3 ligase E6AP as a dimer (UbcH7-UbcH7-E6AP) in vitro. We are currently investigating the nature of this dimerization and the residues that are involved. Concurrently, we are developing a novel chemical method to produce a stable mimic of the reactive E2~Ub intermediate that functions with E3s in vivo (Figure 1B). Through thiol-ene conjugation, we can form an E2-Ub scaffold that will serve as a potent in vitro activity-based probe once it is alkylated with a diazirine.Figure 1  .  Photo-cross-linking (A) Ub-E2 thiol-ether (B).

Activity-based probes for ubiquitin ligases

246th National Meeting (2013)

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