Browsing by Subject "Unfolded Protein Response"
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Item Fic-Mediated AMPylation in Bacterial Infection and Endoplasmic Reticulum Stress(2015-04-14) Woolery, Andrew Ryan; Liu, Qinghua; Orth, Kim; Cobb, Melanie H.; Sternweis, Paul C.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 NFB, 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.Item Translational Control by the Ribosome-Associated Complex in the Unfolded Protein Response(2020-12-01T06:00:00.000Z) Wu, I-Hui; Shay, Jerry W.; Thomas, Philip J.; Mendell, Joshua T.; Tu, BenjaminRibosome-associated chaperones are ubiquitous and highly conserved. There are two classes of ribosome-associated chaperones in eukaryotes, the nascent polypeptide-associated complex (NAC) and the ribosome-associated complex (RAC). Mammalian RAC consists of Hsp70L1, an Hsp70 chaperone homologue, and Mpp11, a DnaJ cofactor. RAC interacts with the nascent chain near the polypeptide exit tunnel and the decoding center on the 60S and 40S ribosomal subunits, respectively. Its unique position on the ribosome implies the coordinating role of de novo protein folding with translation. Deletion of RAC causes growth defects and sensitizes to osmotic, cold, and aminoglycoside stresses in yeast. Furthermore, studies have shown that Mpp11 is over-expressed in head and neck squamous cell cancer and leukemia. However, the function of RAC in stress responses and its role in oncogenesis remain obscure. The current hypothesis predicts that RAC supports co-translational folding of nascent cytosolic polypeptides. To directly test this hypothesis, I altered levels of RAC components and monitored the cytosolic heat shock response (HSR) and the unfolded protein response (UPR) in the ER, two stress pathways known to be activated by accumulation of misfolded proteins. Contrary to its presumptive role in cytosolic protein folding, the reduction of RAC expression did not activate the cytosolic HSR. Unexpectedly, reduction of RAC sensitizes cells to ER stress by selectively attenuating activation of the IRE1 branch of UPR. When RAC is reduced, Xbp1 mRNA splicing is inhibited upon ER stress. Consistent with this activity, ER stress induces changes in the subcellular distribution of RAC, which coincides with the localization of Xbp1 mRNA. Mechanistically, reduction of RAC affects the pathway at a very early step, as IRE1 self-association is inhibited. Additionally, this study shows that the reduction of RAC enhances cellular mRNA translation, including Xbp1 mRNA translation. Interestingly, reduction of Pelo, a protein involved in recognizing stalled ribosomes, counters the inhibition of Xbp1 mRNA splicing, and IRE1 foci formation due to RAC knockdown. Collectively, these results suggest that RAC plays a central role in the IRE1 branch of the UPR tuning IRE1 clustering and mRNA translation.Item Uncovering Reversible AMPylation of BiP Mediated by dFic During ER Homeostasis(2015-01-16) Ham, Hyeilin; Tu, Benjamin; Orth, Kim; Krämer, Helmut; Liu, QinghuaAMPylation is a posttranslational modification involving a covalent attachment of an AMP moiety from ATP to hydroxyl side chains of target substrates. Fic domain which mediates AMPylation is highly conserved across species, including higher eukaryotes, implicating an essential role of this modification in cellular function. Despite the recent discoveries and characterization of a number of bacterial AMPylators and their targets during pathogenesis, the knowledge of AMPylation in eukaryotic system is still elusive. Therefore, the goal of my thesis is to determine the eukaryotic function of AMPylation and identifying the endogenous substrates of this novel modification. In an attempt to understand the physiological function of AMPylation in eukaryotes, we used Drosophila melanogaster as our genetic model organism and created mutant flies lacking functional Drosophila Fic (dFic). We found that the flies without enzymatic function of dFic exhibit blind phenotype due to impaired synaptic transmission. dFic enzymatic activity is required in glial cells for the normal visual neurotransmission. This suggests that a target of dFic may be a component of the visual signaling pathway. dFic was observed in the cell surface of the glial cells particularly enriched in capitate projections. However, dFic is localized to the ER in a number of fly tissues and also in the S2 cells, indicating that there may be another target of dFic in the ER that plays a more general role in the cellular function. In this study, we identified an ER molecular chaperone BiP/GRP78 as a novel substrate for dFic-mediated AMPylation. BiP was predominantly labeled with AMP by dFic in S2 cell lysate. AMPylation of BiP decreases during ER stress but increases upon the reduction of unfolded proteins. Both dFic and BiP are transcriptionally activated upon ER stress induction, implicating a role for dFic in the UPR. We identified a conserved threonine residue, Thr366, as the AMPylation site, which is in close proximity to the ATP binding site of BiP's ATPase domain. Our study presents the first substrate of AMPylation by a eukaryotic protein and proposes a new mode of posttranslational regulation of BiP, which is likely to serve a crucial role in maintaining ER protein homeostasis.