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Characterization of a Small‐molecule Modulator of IRE1α Activity
Author(s) -
SalamiOyenuga Jemilat,
Raina Kanak,
Crews Craig
Publication year - 2015
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.29.1_supplement.723.2
Subject(s) - unfolded protein response , hek 293 cells , endoplasmic reticulum , microbiology and biotechnology , xbp1 , signal transduction , kinase , rna splicing , chemistry , biology , biochemistry , receptor , gene , rna
Endoplasmic Reticulum (ER) stress due to the accumulation of unfolded/misfolded proteins in the ER lumen is resolved by a series of cellular processes, known collectively as the Unfolded Protein Response (UPR). UPR signaling is responsible for maintaining cell health under ER stress conditions and its misregulation has been implicated in the pathogenesis of several diseases. To remediate misregulated UPR, new ways to probe/modulate UPR signaling have to be developed. Along these lines, our lab has synthesized a novel small molecule (NA14) that interacts with and modulates the downstream activity of Inositol Requiring Enzyme 1 (IRE1α), a central player in one of the three signaling branches responsible for ER stress response. We have characterized the interaction between NA14 and recombinant human IRE1α protein using thermal shift assays and studied its effects on IRE1α kinase and ribonuclease enzymatic activities. We have also tested the toxicity of NA14 to HEK293T human kidney cells and treated these cells with NA14 to study its effects on downstream IRE1α and UPR signaling. We show that NA14 binds to recombinant human IRE1α and also inhibits its kinase and ribonuclease enzymatic activities. We also show that NA14 is not cytotoxic to HEK293T cells. We are currently working on understanding the effects of NA14 on downstream human IRE1α and UPR signaling. Till date, we have used a luciferase reporter assay to show that NA14 downregulates the splicing of X‐Box Binding Protein 1 (XBP1) mRNA, one of the key consequence of IRE1α activation. Our aim is to have a clear understanding of the mechanism of regulation of IRE1α activity by NA14. This would enable its use as a chemical probe for studying ER stress pathways in normal and disease contexts.