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dc.contributor.advisorMaly, Dustin J
dc.contributor.authorFeldman, Hannah C
dc.date.accessioned2019-05-02T23:18:06Z
dc.date.submitted2019
dc.identifier.otherFeldman_washington_0250E_19764.pdf
dc.identifier.urihttp://hdl.handle.net/1773/43647
dc.descriptionThesis (Ph.D.)--University of Washington, 2019
dc.description.abstractFaithful folding of proteins in the endoplasmic reticulum (ER) is essential for cell survival and proper cellular function. Understandably, unfolded or misfolded proteins contribute to a myriad of human diseases. Numerous cellular perturbations can overwhelm the homeostatic capacity of the ER, causing the accumulation of unfolded proteins, and activation of the of the multi-domain protein sensor IRE1α as part of the unfolded protein response (UPR). Clustering of IRE1α lumenal domains in the presence of unfolded proteins promotes kinase trans-autophosphorylation in the cytosol and subsequent RNase domain activation. Depending on the duration and severity of ER stress, the kinase and RNase domains of IRE1α can have various outputs; exhibiting both adaptive/pro-survival and pro-apoptotic activities. IRE1α’s role in cell fate decisions has consequently made it an attractive target for pharmacological modulation. Interestingly, there is an allosteric relationship between the kinase and RNase domains of IRE1α, which allows ATP-competitive inhibitors to modulate the activity of the RNase domain. Here, we use kinase inhibitors to study how ATP-binding site conformation affects the activity of the RNase domain of IRE1α. In this study we find that diverse ATP-competitive inhibitors of IRE1α promote dimerization and activation of RNase activity despite blocking kinase autophosphorylation. In contrast, a subset of ATP-competitive ligands, which we call KIRAs, allosterically inactivate the RNase domain through the kinase domain by stabilizing monomeric IRE1α. Structural studies of IRE1α and integration of our extensive structure activity relationship (SAR) data has led us to formulate a model to rationalize how ATP-binding site ligands are able to control the IRE1α oligomeric state and subsequent RNase domain activity through conformational control of a motif known at the αC-helix. Furthermore we show that αC-helix conformation can be rheostatically tuned using ATP-competitive ligands, which led to the development of a new class of IRE1α kinase inhibitors known as partial KIRAs (pKIRAs). Unlike previously characterized RNase inhibitors, pKIRAs only partially weaken IRE1α dimerization and incompletely inhibit RNase activities. Most notably, pKIRAs are unique in their ability to selectively modulate the RNase functional outputs of IRE1α − preserving pro-survival outputs but preventing pro-apoptotic outputs in cells experiencing ER stress.
dc.format.mimetypeapplication/pdf
dc.language.isoen_US
dc.rightsnone
dc.subjectallostery
dc.subjectATP-competitive inhibitor
dc.subjectER stress
dc.subjectIRE1
dc.subjectprotein kinase
dc.subjectunfolded protein response
dc.subjectBiochemistry
dc.subjectChemistry
dc.subjectCellular biology
dc.subject.otherChemistry
dc.titlePharmacological Modulation and Functional Characterization of the Protein Kinase-Endoribonuclease IRE1α
dc.typeThesis
dc.embargo.termsRestrict to UW for 1 year -- then make Open Access
dc.embargo.lift2020-05-01T23:18:06Z


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