? The objective of this application is to develop a new method for identifying substrates of E3 ubiquitin ligases. In the last few years it has become apparent that ubiquitination regulates almost all aspects of cellular function. Ubiquitin is attached to proteins by a cascade of enzymatic reactions involving the E1 ubiquitin-activating enzyme, the E2 ubiquitin-conjugating enzymes, and the E3 ubiquitin ligases. The substrate specificity of the pathway is conveyed by the E3s, of which there are hundreds in the human genome. Many of the medical disorders associated with the ubiquitin pathway involve mutations to a particular E3 ubiquitin ligase. In these cases the identification of the E3 substrates is crucial to understanding the molecular basis of the disease. We will create a procedure by which a particular E3 can be redesigned so that instead of attaching ubiquitin to its substrates, it modifies the substrates with an epitope-tagged version of the ubiquitin-like molecule NEDD8. Given that only a single family of proteins, the cullins, are normally modified with NEDD8 in the cell it will not be difficult to pick out the new set of proteins (potential substrates of the redesigned E3) that have been tagged with NEDD8. The proposed method is innovative because unlike other methods for identifying E3 substrates (two-hybrid analysis, immunoprecipitation), it does not require the direct detection of a transient and potentially weak complex, the E3/substrate complex. In order to transform an E3 so that it modifies substrate with NEDD8, we will redesign the E3 so that it binds and functions with UBC12, the E2 enzyme for NEDD8. The mutations needed to induce this transformation will be identified using a molecular modeling program, RosettaDesign, developed by the principal investigator. Initially, we will focus on redesigning two HECT domain ubiquitin ligases, E6AP and RSP5, to function with UBC12. We will test the redesigns in vitro and in vivo to monitor if known substrates are being tagged with NEDD8 instead of ubiquitin. Favorable results will indicate that this new method should be an excellent approach for identifying substrates of other E3 ubiquitin ligases important to health and disease. ? ? ?
| Guntas, Gurkan; Kuhlman, Brian (2012) Redesigning the NEDD8 pathway with a bacterial genetic screen for ubiquitin-like molecule transfer. J Mol Biol 418:161-6 |
| Eletr, Ziad M; Kuhlman, Brian (2007) Sequence determinants of E2-E6AP binding affinity and specificity. J Mol Biol 369:419-28 |
| Eletr, Ziad M; Huang, Danny T; Duda, David M et al. (2005) E2 conjugating enzymes must disengage from their E1 enzymes before E3-dependent ubiquitin and ubiquitin-like transfer. Nat Struct Mol Biol 12:933-4 |
