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Browsing by Subject "Unfolded Protein Response"
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Item Characterizing the Unfolded Protein Response by Changes in Protein Thermal Stability(2023-09) McCracken, Neil Andrew; Mosley, Amber; Wek, Ron; Evans-Molina, Carmella; Georgiadis, Millie; Quinney, SaraThe Unfolded Protein Response (UPR) protects eukaryotic cells from the threat of excessive protein flux into the Endoplasmic Reticulum (ER). UPR sentries PERK, Ire1 and ATF6 detect unfolded protein in the ER and alert the cell of the condition. Downstream pathways increase translation of select responders while simultaneously decreasing the global protein load in order that toxic protein aggregates do not form in the cell. While this warning system has been characterized over several decades through extensive reporting of UPR impact on transcript and protein abundance, little is known about the biophysical changes that occur to proteins as part of the UPR in the context of the cellular environment. An understanding of how the UPR affects the folding, stability and protein oligomerization is vital for describing subtle but important changes that occur and contribute to maladaptive physiology in diseases including diabetes, cancer, and neurodegeneration. I propose that deficiencies in characterizing the UPR can be overcome by using thermal shifts assays (TSA) that quantify changes in protein stability post stimuli. Findings described herein show the utility of the biophysical thermal shift assay in characterizing the UPR. Thermal shift assays (TSA) measure susceptibility of proteins to denature upon heat treatment and consequently detect changes in protein structure, modification, and interactions in the cellular environment. Previously unobserved protein relationships related to the UPR were detected using TSA. These workflows were improved through more strategic upstream sampling and downstream data analysis through creation of the publicly available InflectSSP program. Observed UPR phenomena during N-linked glycosylation inhibition and UPR induction include protein degradation, changes in stability of N-linked glycosylation enzymes, and transcriptional targets canonical to the UPR. Stability changes in proteins downstream of PERK were also observed in experiments where PERK genetic ablation was combined with UPR induction. Finally, the thermal shift assay was used to develop a “signature” for the UPR that holistically describes the ER stress response. Results described in this dissertation provide an improved perspective of the UPR along with an approach that can be used to identify novel targets for therapeutic intervention of the UPR.Item Maintenance of Pdx1 mRNA translation in islet β-cells during the unfolded protein response(The Endocrine Society, 2014-11) Templin, Andrew T.; Maier, Bernhard; Tersey, Sarah A.; Hatanaka, Masayuki; Mirmira, Raghavendra G.; Department of Pediatrics, IU School of MedicineIn type 1 diabetes, proinflammatory cytokines secreted by infiltrating immune cells activate the unfolded protein response (UPR) in islet β-cells, which leads to attenuation of global mRNA translation. Under such conditions, privileged mRNAs required for adaptation to the prevailing stress are maintained in an actively translated state. Pdx1 is a β-cell transcription factor that is required for the adaptive UPR, but it is not known how translation of its mRNA is maintained under these conditions. To study translation, we established conditions in vitro with MIN6 cells and mouse islets and a mixture of proinflammatory cytokines (IL-1β, TNF-α, and IFN-γ) that mimicked the UPR conditions seen in type 1 diabetes. Cell extracts were then subjected to polyribosome profiling to monitor changes to mRNA occupancy by ribosomes. Similar to other privileged mRNAs (Atf4 and Chop), Pdx1 mRNA remained partitioned in actively translating polyribosomes under the UPR, whereas the mRNA encoding a proinsulin-processing enzyme (Cpe) and others partitioned into inactively translating monoribosomes. Bicistronic luciferase reporter analyses revealed that the distal portion of the 5'-untranslated region of mouse Pdx1 (between bp -105 to -280) contained elements that promoted translation under both normal and UPR conditions, and this region exhibited conserved sequences and secondary structure similar to those of other known internal ribosome entry sites. Our findings suggest that Pdx1 protein levels are maintained in the setting of the UPR, in part, through elements in the 5'-untranslated region that confer privileged mRNA translation in a 5'-7-methylguanylate cap-independent manner.Item Nmp4 Suppresses Osteoanabolic Potency(2023-07) Heim, Crystal Noelle; Bidwell, Joseph; Wek, Ronald; White, Kenneth; Robling, Alexander; Plotkin, LilianTreating severe osteoporosis is limited to two strategies: 1. Stimulation of the parathyroid hormone receptor with analogs for parathyroid hormone (PTH) or parathyroid hormone related peptide, and 2. Stimulation of Wnt signaling via neutralization of sclerostin, a natural inhibitor of this pathway, with a monoclonal antibody (romosozumab-aqqg, Scl-mAb). Despite mobilizing distinct molecular and cellular pathways to stimulate bone gain, all their efficacies rapidly diminish. Identifying the barrier to enhancing potency is a clinical priority. We recently reported that mice harboring the conditional loss of the transcription factor Nmp4 (Nuclear Matrix Protein 4) in mesenchymal stem/progenitor cells (MSPCs) exhibited no measurable baseline effect on the skeleton but showed a significantly enhanced increase in bone formation during PTH therapy. Remarkably, (and unexpectedly) skeletal response to PTH therapy was not improved when Nmp4 was conditionally disabled at the osteoblast or osteocyte stages. For the present study, we hypothesized that the potency of any osteoanabolic drug is pre-programmed (and can be re-programmed) in osteoprogenitors. To test this hypothesis, we treated our global Nmp4-/- mice, various conditional knockout mice, and their controls with Scl-mAb. We observed a similar pattern of improved bone response in our mouse models, which we previously observed with the PTH therapy. That is, removal of Nmp4 early in osteoblast differentiation (MSPC) was required for an exaggerated bone-formation response to Scl-mAb therapy. Disabling Nmp4 later in osteogenic differentiation did not increase the potency of Scl-mAb. These data suggest that Nmp4 is part of a common barrier to improving the efficacy of any osteoanabolic. Potential pathways and actors that comprise the re-programming of Nmp4-/- MSPCs to support the exaggerated osteoanabolic effect on the skeleton are discussed.Item Novel targets of eiF2 kinases determine cell fate during the integrated stress response(2014-12) Baird, Thomas; Wek, Ronald C.; Turchi, John J.; Anderson, Ryan; Liu, Yunlong; Quilliam, LawrenceEukaryotic cells rapidly modulate protein synthesis in response to environmental cues through the reversible phosphorylation of eukaryotic initiation factor 2 (eIF2α~P) by a family of eIF2α kinases. The eIF2 delivers initiator Met-tRNAiMet to the translational apparatus, and eIF2α~P transforms its function from a translation initiation factor into a competitive inhibitor of the guanine nucleotide exchange factor (GEF) eIF2B, which is responsible for the recycling of eIF2-GDP to the translationally-competent eIF2-GTP state. Reduced eIF2-GTP levels lower general protein synthesis, which allows for the conservation of energy and nutrients, and a restructuring of gene expression. Coincident with global translational control, eIF2α~P directs the preferential translation of mRNA encoding ATF4, a transcriptional activator of genes important for stress remediation. The term Integrated Stress Response (ISR) describes this pathway in which multiple stresses converge to phosphorylate eIF2α and enhance synthesis of ATF4 and its downstream effectors. In this study, we used sucrose gradient ultracentrifugation and a genome-wide microarray approach to measure changes in mRNA translation during ER stress. Our analysis suggests that translational efficiencies vary across a broad range during ER stress, with the majority of transcripts being either repressed or resistant to eIF2α~P, while a notable cohort of key regulators are subject to preferential translation. From this latter group, we identify IBTKα as being subject to both translational and transcriptional induction during eIF2α~P in both cell lines and a mouse model of ER stress. Translational regulation of IBTKα mRNA involves the stress-induced relief of two inhibitory uORFs in the 5’-leader of the transcript. Also identified as being subject to preferential translation is mRNA encoding the bifunctional aminoacyl tRNA synthetase EPRS. During eIF2α~P, translational regulation of EPRS is suggested to occur through the bypass of a non-canonical upstream ORF encoded by a CUG start codon, highlighting the diversity by which upstream translation initiation events can regulate expression of a downstream coding sequence. This body of work provides for a better understanding of how translational control during stress is modulated genome-wide and for the processes by which this mode of gene regulation in the ISR contributes to cell fate.Item Transcription factor ATF4 directs basal and stress-induced gene expression in the unfolded protein response and cholesterol metabolism in the liver(American Society for Cell Biology, 2016-05-01) Fusakio, Michael E.; Willy, Jeffrey A.; Wang, Yongping; Mirek, Emily T.; Al Baghdadi, Rana J. T.; Adams, Christopher M.; Anthony, Tracy G.; Wek, Ronald C.; Department of Biochemistry & Molecular Biology, IU School of MedicineDisturbances in protein folding and membrane compositions in the endoplasmic reticulum (ER) elicit the unfolded protein response (UPR). Each of three UPR sensory proteins-PERK (PEK/EIF2AK3), IRE1, and ATF6-is activated by ER stress. PERK phosphorylation of eIF2 represses global protein synthesis, lowering influx of nascent polypeptides into the stressed ER, coincident with preferential translation of ATF4 (CREB2). In cultured cells, ATF4 induces transcriptional expression of genes directed by the PERK arm of the UPR, including genes involved in amino acid metabolism, resistance to oxidative stress, and the proapoptotic transcription factor CHOP (GADD153/DDIT3). In this study, we characterize whole-body and tissue-specific ATF4-knockout mice and show in liver exposed to ER stress that ATF4 is not required for CHOP expression, but instead ATF6 is a primary inducer. RNA-Seq analysis indicates that ATF4 is responsible for a small portion of the PERK-dependent UPR genes and reveals a requirement for expression of ATF4 for expression of genes involved in oxidative stress response basally and cholesterol metabolism both basally and under stress. Consistent with this pattern of gene expression, loss of ATF4 resulted in enhanced oxidative damage, and increased free cholesterol in liver under stress accompanied by lowered cholesterol in sera.Item The unfolded protein response regulates hepatocellular injury during the pathogenesis of nonalcoholic steatohepatitis(2016-08) Willy, Jeffrey Allen; Wek, Ronald C.Non-alcoholic steatohepatitis (NASH), which is characterized by the induction of hepatocellular death and inflammation, is associated with the activation of cellular stress pathways such as the Unfolded Protein Response (UPR), an adaptive response to disruptions in endoplasmic reticulum (ER) homeostasis. Because the role of the UPR in the progression of liver disease is not well understood, we established an in vitro model to evaluate the role of the UPR in NASH and translated results to clarify disease progression in human liver biopsy samples. Treating HepG2 cells and primary human hepatocytes with saturated, but not unsaturated free fatty acids (FFAs), at physiologic concentrations induced hepatotoxicity by inhibiting autophagic flux. Saturated FFA treatment activated the UPR, including the transcription factors CHOP (GADD153/DDIT3) and NF-κB, leading to increased expression and secretion of cytokines such as TNFα and IL-8 that contributed to hepatic cell death and inflammation. Depletion of either CHOP or the RELA subunit of NF-κB in hepatocytes alleviated autophagy and cytokine secretion, resulting in enhanced cell viability and lowered inflammatory responses during exposure to saturated FFAs. We carried out next generation sequencing on cells deleted for either CHOP or RELA and identified IBTKα as a novel UPR member directly regulated by CHOP and NF-κB. In response to saturated FFAs, loss of IBTKα increased cell survival through lowered phagophore formation and reduced cytokine secretion. We also identified binding partners of IBTKα by immunoprecipitation and LC/MS, indicating that that IBTKα is part of a protein complex which functions at ER exit sites to facilitate initiation of autophagy and protein secretion. Furthermore, we discovered that CHOP and RELA coordinately regulate proteasome activity through NRF2 as an adaptive response to an inhibition of autophagic flux following palmitate exposure. To validate our model, we utilized human liver biopsy samples and demonstrated up-regulation of the UPR coincident with accumulation of autophagy markers, as well as secretion of cytokines IL 8 and TNFα in serum of NASH patients. Our study provides a mechanistic understanding of the roles of the UPR and autophagy in regulating saturated FFA induced hepatotoxicity at the cellular level.