Fic-Mediated AMPylation in Bacterial Infection and Endoplasmic Reticulum Stress




Woolery, Andrew Ryan

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The post-translational modification AMPylation is emerging as a significant regulatory mechanism in both prokaryotic and eukaryotic biology. This process involves the covalent addition of an adenosine monophosphate to a protein resulting in a modified protein with altered activity. Proteins capable of catalyzing AMPylation, termed AMPylators, are comparable to kinases in that they both hydrolyze ATP and reversibly transfer a part of this primary metabolite to a hydroxyl side chain of the protein substrate. To date, all AMPylators discovered contain one of two domains: the Fic domain or the adenylyl transferase domain. All currently characterized AMPylators are bacterial in origin and are primarily Type III or Type IV secreted effector proteins, which are injected into a host cell to manipulate host signaling to the microbe's advantage. Examples of these are VopS (Vibrio parahaemolyticus), IbpA (Histophilus somni) and DrrA (Legionella pneumophila). The discovery of SidD, a deAMPylator also from L. pneumophila, shows that this modification is dynamic and could likely have a regulatory role in eukaryotic biology. Supporting this idea is the presence of a single copy of the Fic domain in most metazoans, including humans. The substrates, localization, and function of Fic proteins and other AMPylators in eukaryotic biology are perhaps the largest open questions in this rapidly expanding field. The goal of my dissertation work was to expand the understanding of the effects of AMPylation in eukaryotic signaling. I approached this goal in three ways: by examining the effects of an AMPylator (VopS) with known targets (Rho GTPases) on different aspects of cell signaling, developing screening tools for AMPylation and attempting to elucidate some of the functions of the human AMPylator, FicD, in which the targets are unclear. I found that VopS, in addition to collapsing the host actin cytoskeleton, also inhibits many aspects of host defense signaling including NFB, MAP kinases and the phagocytic NADPH oxidase system. I explored the possibility of other potential substrates of VopS by collaborating on an extensive protein microarray screen for AMPylation, determining that the entire Rho GTPase family is AMPylated. I also discovered that the human AMPylator FicD is induced during the unfolded protein response, is localized to the endoplasmic reticulum and is capable of AMPylating the ER chaperone BiP/GRP78. The progress made in these studies will contribute to understanding the role of this enigmatic modification in mammalian cell signaling.

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