AlphaScreen®-Based Characterization of the Bifunctional Kinase/RNase IRE1α: A Novel and Atypical Drug Target

Abstract

IntroductIon Large-scale technologies used within the life science industry for drug discovery have recently proven useful in academic research laboratories, facilitating chemical genomics and the investigation of specific biological functions. these technologies, including homogeneous luminescence (e.g., alphascreen ® and aequorin), fluorescence intensity (fi) or polarization (fP), time-resolved fluorescence (trf), laser scanning cytometry, or microscopy-based approaches, have become enabling tools to characterize complex functions of biological targets.in the present study, we used alphascreen ® to investigate cell signaling pathways 1 and in particular those emanating from the endoplasmic reticulum (er) under stress conditions. the er ensures proper protein folding and export to later compartments of the secretory pathway. this is achieved through complex machineries, including protein synthesis, translocation/ folding, quality control, er-associated degradation (erad), and export.2 in addition to these functional attributes, the er has evolved a highly conserved adaptive signaling pathway, referred to as the unfolded protein response (uPr), whose activation occurs upon accumulation of improperly folded proteins in the er. 3 uPr signaling is mediated by 3 er resident transmembrane proteins-the PKr-like er kinase (PerK), the activating transcription factor 6 (atf6), and the inositol requiring enzyme 1 alpha (ire1α).3,4 our work has focused on ire1α, which is a transmembrane sensor of er stress. the luminal domain of ire1α contains binding sites for the chaperone BiP, whereas the cytosolic region has 2 main catalytic elements: a serine/threonine kinase and an endoribonuclease domain.4-6 under basal conditions, ire1α is thought to exist as a monomer, and in response to the accumulation of misfolded proteins in the er, ire1 proteins oligomerize, resulting in its trans-autophosphorylation and triggering of its endoribonuclease activity. 4 however, structural studies in Saccharomyces cerevisiae showed that the luminal domain of ire1 exists as a dimer/oligomer and suggested that it could potentially bind directly to unfolded peptides similar to Mhc class i molecules.7 this was confirmed by structural studies on the cytosolic domain of ire1 in S. cerevisiae, which revealed a dimer conformation that is subjected to conformational changes upon er stress, leading to activation of its kinase and rnase activities.8 recently, the cytosolic domain of ire1 was crystallized in an oligomeric form, which was promoted by the presence of the kinase inhibitor sunitinib.9,10 this highly ordered molecular structure was found assay technologies that were originally developed for high-throughput screening (hts) have recently proven useful in drug discovery for activities located upstream (target identification and validation) and downstream (adMet) of hts. here the authors investigated and characterized the biological properties of a novel target, ire1α, a bifunctional kinase/rnase stress sensor of the endoplasmic reticulum (er). they...

The Role of ER Stress-Induced Apoptosis in Neurodegeneration

“…In one very important study, Lin et al (2007), showed that prolonging IRE1 signalling improved cellular viability and delayed the onset of apoptosis, suggesting that developing drug targets that stimulate IRE1 signalling could be a viable strategy for preventing cell loss. The importance of IRE1 as a target was also recognised by Bouchecareilh et al, who developed a high-throughput screening assay to monitor the dimerization/oligomerization and phosphorylation properties of the cytosolic domain of IRE1 [129]. They show in vitro that dimerization/ oligomerization of the cytosolic domain of IRE1 correlates with the autophosphorylation ability of this domain and its endoribonuclease activity toward XBP1 mRNA.…”

Posttranscriptional Regulation of PER1 Underlies the Oncogenic Function of IREα

“…Bacteria were then collected by centrifugation, lysed, and recombinant proteins purified as recommended by the manufacturer (Gibco BRL). The resulting purified proteins were concentrated and dialyzed using Amicon ultra centrifugal filters (cutoff = 20,000 Da; Millipore Corp.), followed by functional testing as previously described (12, 13). …”

“…Total RNA (10 μg) from U87 or HepG2 was incubated with the cytoplasmic domain of human GST-IRE1α (5 μg) at 37°C for the indicated amounts of time in a buffer containing 250 mmol/L Tris pH 7.5, 600 mmol/L NaCl, 5 mmol/L MgCl2, 5 mmol/L MnCl2, 25 mmol/L β-mercaptoethanol, supplemented with or without 10 mmol/L ATP as previously described (12). As control, we used heat-denatured GST-IRE1α.…”

ERN1 and ALPK1 inhibit differentiation of bi-potential tumor-initiating cells in human breast cancer

“…Its role in cancer progression was also documented along this axis, showing an activation of the pathway inducing proliferation in colorectal carcinoma cells [26]. It was identified as a druggable target and potential therapeutic option in multiple myeloma [27, 28] and seems to be amplified in 8% of invasive breast cancers [18, 19]. Nevertheless most of these studies focused on the role of Xbp1 alternative splicing.…”