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BIOCHEMICAL AND STRUCTURAL STUDIES OF PATHOGEN EFFECTORS ASSOCIATED WITH UBIQUITIN ADP-RIBOSYLATION
Ubiquitination and ADP-ribosylation are reversible post-translational modifications involved in various cellular activities. Pathogens like Legionella pneumophila and Chromobacterium violaceum target host ubiquitin system via modifications involving ADP-ribosylation. Specifically, Legionella pneumophila mediates atypical ubiquitination of host targets using the SidE effector family in a process that involves ubiquitin ADP-ribosylation on arginine 42 as an obligatory step. On the other hand, Chromobacterium violaceum effector CteC ADP-ribosylates threonine 66 of ubiquitin and causes overall blocking of host ubiquitin signaling. Removal of ADP-ribosylation requires (ADP-ribosyl)hydrolases, with macrodomain enzymes being a major family in this category. In the current study, a proteome-wide screening of ubiquitin interactors in the Legionella secreted proteome was performed, which led to the Legionella macrodomain effector MavL as a regulator of the SidE-mediated ubiquitination pathway by reversing the ubiquitin arginine ADP-ribosylation, likely to minimize potential detrimental effects caused by modified ubiquitin. Crystal structure of ADP-ribose-bound MavL was determined, providing structural insights into substrate recognition and catalytic mechanism. Further bioinformatical analyses reveal DUF4804 as a class of MavL-like macrodomain enzymes uniquely selective for mono-ADP-ribosylated arginine residue. The arginine-specific macrodomains are also present in eukaryotes, as exemplified by two previously uncharacterized (ADP-ribosyl)hydrolases in Drosophila melanogaster. Crystal structures of several proteins in this class provide insights into arginine specificity and a shared mode of ADP-ribose interaction distinct from previously characterized macrodomains. The crystal structure of NAD+-bound CteC was also determined, which provided insights into its ADP-ribosylation activity and its ubiquitin specificity. Collectively, the studies described here provide biochemical and structural characterizations and mechanistic insights into bacterial effectors associated with ubiquitin ADP-ribosylation.
- Doctor of Philosophy
- West Lafayette