Chemical Biology Approaches to Ubiquitination

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Enzymes in the ubiquitin-dependent proteolysis pathway catalyze the removal of damaged proteins that are hallmarks of many diseases. Ubiquitination involves the transfer of ubiquitin (Ub) between a series of E1-activating, E2-conjugating and E3-ligating enzymes, ultimately forming polyubiquitin chains on target substrates that are recognized by receptors such as the 26S proteasome to control protein degradation. Recent proteomic experiments show that most enzymes in the Ub pathway are post-translationally modified (PTM) under different cellular stresses. In most cases, the protein kinases or lysine acetytransferases that specifically modify ubiquitination enzymes have not been identified. PTMs uncovered in the Ub cascade have the potential to alter protein interactions between the enzymes needed for Ub catalysis and modulate many cellular events. In our first NSERC Discovery grant we; (1) implemented orthogonal translation methods to efficiently synthesize specifically phosphorylated and acetylated Ub, (2) developed real-time FRET-based kinetic assays to measure E2~Ub conjugate formation and (3) identified how Ub acetylation alters the formation of E2~Ub conjugates. In the next 5 years we will build on these achievements to identify how phosphorylation and acetylation of E2 and E3 enzymes modulates their structures, interactions, kinetics of Ub transfer and formation of polyubiquitin chains. The following questions will be addressed: (1)How do phosphorylation or acetylation of E2 enzymes modify the conformations and stabilities of E2~Ub conjugates? (2)How does phosphorylation or acetylation of an E2 or E3 enzyme alter the transfer of Ub? (3)How does acetylation of Ub alter the dynamics and structures of polyubiquitin chains? Our aims and objectives are: (1)Identify and optimize the synthesis of phosphorylated and acetylated E2 and E3 proteins using orthogonal translation methods, (2)Determine how acetylation or phosphorylation modifies specific E2~Ub conformations needed for Ub transfer. Use structural methods to examine conformations of acetylated or phosphorylated E2~Ub conjugates and show how interactions with E3 enzymes are modified. (3)Determine how acetylation or phosphorylation alters the rate of Ub transfer from an E2 to an E3 or substrate. Kinetic FRET experiments will measure the rates of E2~Ub unloading with RING, HECT and RBR E3 enzymes, (4)Identify how lysine acetylation of Ub modifies the arrangement of a polyUb chain. Use NMR methods to determine conformations and interactions of acetylated diUb chains and interactions with a proteasomal subunit. This work will identify how PTMs of Ub, E2 and E3 enzymes impact the structures and kinetics of Ub transfer and provide insights into downstream cellular events. HQP will gain expertise that will prepare them for careers in the private-sector and academia. The methods used have the potential for future licensing opportunities to develop antibodies for specific PTMs.