Chemical and biochemical studies of ubiquitin conjugation machinery

Abstract

The post-translational modification of proteins is a major mechanism employed in eukaryotic cells to expand the functional diversity of the proteome. Covalent modification of amino acid side chains confers new or altered functionality to the modified protein by creating new recognition surfaces on the protein for the interaction of nucleic acids or other proteins, modulating enzymatic activity, or altering cellular localization or half-life. The post-translational modification of proteins with ubiquitin (Ub) is an important mechanism of regulating protein function. Ub is a 76-residue protein that is primarily attached to lysine residues in target proteins through an enzymatic cascade catalyzed by E1, E2, and E3 enzymes. Ub conjugation is important for fundamental cellular processes, including transcription, DNA repair, endocytosis, apoptosis, and signal transduction. Ub conjugation is reversible. Proteases termed deubiquitinating enzymes (DUBs) function to remove Ub from target proteins. Genome sequencing efforts have uncovered the existence of many predicted enzymes with unknown function. Many enzymes have been assigned function based on sequence homology to proteins with known function without confirmation of enzymatic activity. A powerful chemical approach to determine enzyme function from a complex mixture of proteins is activity-based protein profiling. This method makes use of chemical probes that are active site-directed for the assignment of function to proteins. We describe here the design and generation of an expanded set of Ub-based chemical probes with which we identified and recovered E1, E2, and E3 Ub ligases from cell lysates. Furthermore, we describe the biochemical and structural characterization of the catalytic domain of one E3 Ub ligase we recovered, HUWE1, and the identification of a structural element within the catalytic domain of HUWE1 that modulates its activity. Finally, we discuss a protein engineering method that we are applying to the HUWE1 catalytic domain to understand how the conformational flexibility of this domain is important to its function.