Biochemical and Biophysical Research Communications
A kinase inhibitor activates the IRE1α RNase to confer cytoprotection against ER stress
Section snippets
Materials and methods
Cell culture and retroviral transduction. Mouse embryonic fibroblasts (MEFs) were grown in DMEM 10% FCS, 50 U/mL penicillin and 50 μg/mL streptomycin. For retroviral expression, a cDNA encoding wild type or mutant mouse Ire1α was cloned into pBABEpuro, and site-directed mutagenesis carried out using Quickchange (Invitrogen). Virus production was carried out in the 293-GPG cell line. Briefly, retroviral expression constructs were transiently transfected into 293-GPG cells using Lipofectamine 2000
Results and discussion
To investigate the effects of inhibiting the IRE1α kinase, we reconstituted Ire1α−/− mouse embryonic fibroblasts (MEFs) using a retrovirus bearing a cDNA encoding mouse IRE1α mutated at Ile642 to Gly. This substitution is predicted to sensitize IRE1α to the ATP-competitive drug 1-tert-butyl-3-naphthalen-1-ylmethyl-1H-pyrazolo[3,4-d]pyrimidin-4-ylamine (1NM-PP1) by creating an enlarged active site pocket not found in wild-type kinases [17]. While this rational protein engineering method is
Acknowledgments
We thank Chao Zhang and Kevan Shokat for the gift of 1NM-PP1, and David Ron for providing Ire1α−/− MEFs. This work was supported by NIH Grants K08 AI054650 (S.A.O.), and K08 DK065671 (F.R.P.), an HHMI Physician-Scientist Early Career Award (S.A.O.), the Steward Trust Foundation (S.A.O.), the Sandler Program in Basic Sciences (S.A.O. and F.R.P.), and the UC Cancer Research Coordinating Committee (S.A.O. and F.R.P.), the Burroughs Wellcome Foundation (F.R.P.), the Hillblom Foundation (F.R.P.),
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The molecular mechanism and functional diversity of UPR signaling sensor IRE1
2021, Life SciencesCitation Excerpt :They used a kinase-dead mutant of IRE1 (I642G) and found that it can activate RNase in a way that it will only catalyze Xbp1 splicing but not RIDD. It has been established that 1NM-PP1 [1-tert-butyl-3-(naphthalen-1-ylmethyl)-1H-pyrazolo[3,4-d] pyrimidin-4-amine] bypass the auto-phosphorylation for Xbp1 splicing in I642G mutant [111,112]. It was proposed that a dimeric moderately active RNase pocket is sufficient to catalyze Xbp1 splicing as Xbp1 represents the preferred substrate for IRE1.
Protein aggregation and ER stress
2016, Brain ResearchCitation Excerpt :XBP1 expression and splicing was reported as neuroprotective in AD (Casas-Tinto et al., 2011), HD (Zuleta et al., 2012) and PD mouse models (Sado et al., 2009). IRE1 activation with a small molecule, which resulted in XBP1 splicing, also lead to transient protection (Han et al., 2008; Lin et al., 2007). Paradoxically, XBP1 deficiency was also found to be protective, as it increases autophagy of SOD1 and Htt in an ALS and HD models respectively (Hetz et al., 2009; Vidal et al., 2012).
Allosteric inhibition of the IRE1α RNase preserves cell viability and function during endoplasmic reticulum stress
2014, CellCitation Excerpt :Under low, manageable levels of ER stress, adaptive UPR signaling promotes secretory homeostasis, partly through IRE1α-mediated splicing of XBP1 mRNA and consequent XBP1s outputs. Likewise, pre-emptive PERK activation affords a measure of cytoprotection against subsequent ER stress by attenuating translation (Lu et al., 2004), as does preconditioning with 1NM-PP1-bound IRE1α (I642G) to transiently stabilize an intermediate activation mode of the RNase confined to XBP1 splicing (Han et al., 2008). However, under high ER stress, IRE1α acquires endonucleolytic activity against a large plethora of RNA targets, first identified in D. melanogaster and termed RIDD (Hollien and Weissman, 2006), including ER-localized mRNAs and noncoding RNAs in mammals (Han et al., 2009; Hollien et al., 2009; Lerner et al., 2012; Upton et al., 2012).
IRE1α induces thioredoxin-interacting protein to activate the NLRP3 inflammasome and promote programmed cell death under irremediable ER stress
2012, Cell MetabolismCitation Excerpt :To dissect the effects of IRE1α's catalytic activities on TXNIP upregulation, we tested two point mutants. The first, IRE1α (I642G), has an enlarged adenosine triphosphate (ATP)-binding pocket in its kinase domain that destroys phosphotransfer catalytic activity; the enlarged pocket can selectively bind 1NM-PP1, a cell-permeable adenosine nucleotide mimic with a bulky chemical head group (Figure 2C) (Han et al., 2008; Papa et al., 2003). Binding of 1NM-PP1 to IRE1α (I642G) allosterically activates the RNase domain, causing it to forcibly splice XBP1 mRNA, while bypassing the autophosphorylation requirement (Figures S4B and S4C).
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These authors contributed equally to this work.