<p>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 <i>Legionella pneumophila</i>: 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 <i>Legionella</i>.</p>
| Identifer | oai:union.ndltd.org:purdue.edu/oai:figshare.com:article/12192432 |
| Date | 24 April 2020 |
| Creators | Kedar Puvar (8762877) |
| Source Sets | Purdue University |
| Detected Language | English |
| Type | Text, Thesis |
| Rights | CC BY 4.0 |
| Relation | https://figshare.com/articles/Investigations_Into_Noncanonical_Ubiquitination/12192432 |
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