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Investigations Into Noncanonical Ubiquitination
In order to distinguish essays and pre-prints from academic theses, we have a separate category. These are often much longer text based documents than a paper.
The modification of proteins by the covalent attachment of ubiquitin is a natural process that crucially regulates a wide range of eukaryotic signaling outcomes. This process has been understood as the linking of the C-terminus of ubiquitin to the lysine residue of a target protein via an isopeptide linkage, catalyzed by the coordinated effort by E1, E2, and E3 enzymes. Importantly, ubiquitination has only been observed to be a eukaryotic phenomenon. In recent years though, intracellular bacteria, including human pathogens, have been observed to possess ubiquitin-interacting proteins in their genomes. These proteins serve to subdue and manipulate their hosts’ ubiquitin signaling for their own benefit. While some of these proteins act within the eukaryotic context, more recent findings reveal the existence of prokaryotic enzymes that catalyze ubiquitination using mechanisms never before seen in nature. These remarkable processes utilize different cofactors and target different amino acid residues of both ubiquitin as well as substrate protein. The findings reported in this Thesis involve structural and biochemical studies on two new ubiquitinating proteins, the only two proteins known to catalyze ubiquitination outside of the canonical pathway. Both proteins are present in the genome of the intracellular human pathogen Legionella pneumophila: the SidE family, which catalyzes ubiquitination via a mechanism combining ADP-ribosylation and phosphodiesterase activities, and MavC, which utilizes a mechanism reminiscent of transglutaminases. Key insights provided in this document include the discovery that SidE enzymes can modify multiple ubiquitin moieties within a ubiquitin chain, and that modified ubiquitin chains are resistant to hydrolytic cleavage from many deubiquitinating enyzmes. Also, the development of a robust, continuous assay for SidE-catalyzed ubiquitination using a synthetic substrate is described. The catalytic action of MavC, which differs from both canonical E1/E2/E3 ubiquitination and SidE ubiquitination is also here elucidated. The crystal structure of MavC in complex with its ubiquitinated product is presented and provides an atomic view into the basis of substrate recognition. These findings bring to light a new dimension of host-pathogen interactions, where pathogenic ubiquitinating enzymes have appeared to arise from convergent evolution. The regulation of these pathogenic enzymes by other effectors is also discussed, as well as biochemical studies of these regulators. Further, these findings describe possible new drug discovery strategies, as well as possible techniques for discovering similar enzymes in organisms besides Legionella.