. 2014 Jul 31;158(3):534-48.

doi: 10.1016/j.cell.2014.07.002. Epub 2014 Jul 10.

Allosteric inhibition of the IRE1α RNase preserves cell viability and function during endoplasmic reticulum stress

Rajarshi Ghosh¹, Likun Wang², Eric S Wang³, B Gayani K Perera⁴, Aeid Igbaria², Shuhei Morita², Kris Prado², Maike Thamsen², Deborah Caswell³, Hector Macias⁵, Kurt F Weiberth², Micah J Gliedt⁶, Marcel V Alavi⁷, Sanjay B Hari⁴, Arinjay K Mitra⁴, Barun Bhhatarai⁸, Stephan C Schürer⁹, Erik L Snapp¹⁰, Douglas B Gould¹¹, Michael S German⁵, Bradley J Backes⁶, Dustin J Maly⁴, Scott A Oakes¹², Feroz R Papa¹³

Affiliations

¹ Department of Medicine, University of California, San Francisco, San Francisco, CA 94143, USA; Department of Pathology, University of California, San Francisco, San Francisco, CA 94143, USA; Diabetes Center, University of California, San Francisco, San Francisco, CA 94143, USA; Lung Biology Center, University of California, San Francisco, San Francisco, CA 94143, USA; California Institute for Quantitative Biosciences, University of California, San Francisco, San Francisco, CA 94143, USA.
² Department of Medicine, University of California, San Francisco, San Francisco, CA 94143, USA; Diabetes Center, University of California, San Francisco, San Francisco, CA 94143, USA; Lung Biology Center, University of California, San Francisco, San Francisco, CA 94143, USA; California Institute for Quantitative Biosciences, University of California, San Francisco, San Francisco, CA 94143, USA.
³ Department of Pathology, University of California, San Francisco, San Francisco, CA 94143, USA.
⁴ Department of Chemistry, University of Washington, Seattle, WA 98195, USA.
⁵ Department of Medicine, University of California, San Francisco, San Francisco, CA 94143, USA; Diabetes Center, University of California, San Francisco, San Francisco, CA 94143, USA.
⁶ Department of Medicine, University of California, San Francisco, San Francisco, CA 94143, USA; Lung Biology Center, University of California, San Francisco, San Francisco, CA 94143, USA.
⁷ Department of Ophthalmology, University of California, San Francisco, San Francisco, CA 94143, USA.
⁸ Department of Molecular and Cellular Pharmacology,, Miller School of Medicine, University of Miami, FL 33136, USA.
⁹ Center for Computational Science, Miller School of Medicine, University of Miami, FL 33136, USA; Department of Molecular and Cellular Pharmacology,, Miller School of Medicine, University of Miami, FL 33136, USA.
¹⁰ Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY 10461, USA.
¹¹ Department of Ophthalmology, University of California, San Francisco, San Francisco, CA 94143, USA; Department of Anatomy, University of California, San Francisco, San Francisco, CA 94143, USA.
¹² Department of Pathology, University of California, San Francisco, San Francisco, CA 94143, USA; Diabetes Center, University of California, San Francisco, San Francisco, CA 94143, USA. Electronic address: [email protected].
¹³ Department of Medicine, University of California, San Francisco, San Francisco, CA 94143, USA; Diabetes Center, University of California, San Francisco, San Francisco, CA 94143, USA; Lung Biology Center, University of California, San Francisco, San Francisco, CA 94143, USA; California Institute for Quantitative Biosciences, University of California, San Francisco, San Francisco, CA 94143, USA. Electronic address: [email protected].

PMID: 25018104
PMCID: PMC4244221
DOI: 10.1016/j.cell.2014.07.002

Allosteric inhibition of the IRE1α RNase preserves cell viability and function during endoplasmic reticulum stress

Rajarshi Ghosh et al. Cell. 2014.

. 2014 Jul 31;158(3):534-48.

doi: 10.1016/j.cell.2014.07.002. Epub 2014 Jul 10.

Authors

Affiliations

¹ Department of Medicine, University of California, San Francisco, San Francisco, CA 94143, USA; Department of Pathology, University of California, San Francisco, San Francisco, CA 94143, USA; Diabetes Center, University of California, San Francisco, San Francisco, CA 94143, USA; Lung Biology Center, University of California, San Francisco, San Francisco, CA 94143, USA; California Institute for Quantitative Biosciences, University of California, San Francisco, San Francisco, CA 94143, USA.
² Department of Medicine, University of California, San Francisco, San Francisco, CA 94143, USA; Diabetes Center, University of California, San Francisco, San Francisco, CA 94143, USA; Lung Biology Center, University of California, San Francisco, San Francisco, CA 94143, USA; California Institute for Quantitative Biosciences, University of California, San Francisco, San Francisco, CA 94143, USA.
³ Department of Pathology, University of California, San Francisco, San Francisco, CA 94143, USA.
⁴ Department of Chemistry, University of Washington, Seattle, WA 98195, USA.
⁵ Department of Medicine, University of California, San Francisco, San Francisco, CA 94143, USA; Diabetes Center, University of California, San Francisco, San Francisco, CA 94143, USA.
⁶ Department of Medicine, University of California, San Francisco, San Francisco, CA 94143, USA; Lung Biology Center, University of California, San Francisco, San Francisco, CA 94143, USA.
⁷ Department of Ophthalmology, University of California, San Francisco, San Francisco, CA 94143, USA.
⁸ Department of Molecular and Cellular Pharmacology,, Miller School of Medicine, University of Miami, FL 33136, USA.
⁹ Center for Computational Science, Miller School of Medicine, University of Miami, FL 33136, USA; Department of Molecular and Cellular Pharmacology,, Miller School of Medicine, University of Miami, FL 33136, USA.
¹⁰ Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY 10461, USA.
¹¹ Department of Ophthalmology, University of California, San Francisco, San Francisco, CA 94143, USA; Department of Anatomy, University of California, San Francisco, San Francisco, CA 94143, USA.
¹² Department of Pathology, University of California, San Francisco, San Francisco, CA 94143, USA; Diabetes Center, University of California, San Francisco, San Francisco, CA 94143, USA. Electronic address: [email protected].
¹³ Department of Medicine, University of California, San Francisco, San Francisco, CA 94143, USA; Diabetes Center, University of California, San Francisco, San Francisco, CA 94143, USA; Lung Biology Center, University of California, San Francisco, San Francisco, CA 94143, USA; California Institute for Quantitative Biosciences, University of California, San Francisco, San Francisco, CA 94143, USA. Electronic address: [email protected].

PMID: 25018104
PMCID: PMC4244221
DOI: 10.1016/j.cell.2014.07.002

Abstract

Depending on endoplasmic reticulum (ER) stress levels, the ER transmembrane multidomain protein IRE1α promotes either adaptation or apoptosis. Unfolded ER proteins cause IRE1α lumenal domain homo-oligomerization, inducing trans autophosphorylation that further drives homo-oligomerization of its cytosolic kinase/endoribonuclease (RNase) domains to activate mRNA splicing of adaptive XBP1 transcription factor. However, under high/chronic ER stress, IRE1α surpasses an oligomerization threshold that expands RNase substrate repertoire to many ER-localized mRNAs, leading to apoptosis. To modulate these effects, we developed ATP-competitive IRE1α Kinase-Inhibiting RNase Attenuators-KIRAs-that allosterically inhibit IRE1α's RNase by breaking oligomers. One optimized KIRA, KIRA6, inhibits IRE1α in vivo and promotes cell survival under ER stress. Intravitreally, KIRA6 preserves photoreceptor functional viability in rat models of ER stress-induced retinal degeneration. Systemically, KIRA6 preserves pancreatic β cells, increases insulin, and reduces hyperglycemia in Akita diabetic mice. Thus, IRE1α powerfully controls cell fate but can itself be controlled with small molecules to reduce cell degeneration.

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Figures

**Figure 1. IRE1α’s kinase uses homo-oligomerization as a rheostat to control RNase activity and apoptosis**
(A) Anti-phospho-IRE1α and anti-Myc immunoblots (ratiometric quantitation, normalized to GAPDH). (B) Agarose gel of PstI-digested XBP1 cDNA amplicons (ratiometric quantitation of spliced to total XBP1 cDNAs). (C) Q-PCR for Insulin1 (Ins1) and TXNIP mRNAs. (D) %Annexin-V positive staining. (A-C) utilized INS-1::IRE1α (WT) cells under increasing [Dox] at 24hr, whereas (D) is at 72 hr. (E) Model of how IRE1α promotes both adaptive and apoptotic outputs. (F) Immunoblots of increasing concentrations of IRE1α*(WT), dP-IRE1α*(WT), and IRE1α*(I642G) −/+ 1NM-PP1 (10 μM) followed by disuccinimidyl suberate (DSS) (250 μM) crosslinking, with oligomer/monomer quantification (G). (H) Time course urea PAGE of cleavage of α³²P-labeled XBP1 RNA and Insulin2 (Ins2) RNA by IRE1α*(WT) and IRE1α*(I642G) −/+ 1NM-PP1 (10 μM), with quantification (I). (J) Model of oligomerization-dependence of RNase activity against XBP1 and Ins2 RNAs by IRE1α*(WT) and IRE1α*(I642G). Three independent biological samples were used for XBP1 splicing, Q-PCR and Annexin V experiments. Data plotted as mean value ± SD. P-values: *<0.05 and ** <0.01, ns=not significant. Also see Figure S1.

**Figure 2. IRE1α cancer mutants are disabled for apoptosis**
(A) Cancer-associated mutations in human IRE1α. (B) Time course Annexin-V staining of INS-1 cells stably expressing human IRE1α (WT), (L474R), (R635W), (S765F), (Q780*), and (P830L) under saturating Dox (1μg/ml). (C) Anti-phospho-IRE1α and anti-Myc immunoblots, and (D) agarose gel of PstI-digested XBP1 cDNA amplicons from INS-1 cells expressing human IRE1α (WT) and mutants with Dox (1μg/ml) for 24hr. (E) XBP1 splicing from (D) as a function of IRE1α phosphorylation from (C). (F) Time course Q-PCR of Ins1 mRNA from INS-1 cells expressing IRE1α (WT) and mutants under Dox (1μg/ml). (G) Cartoon of monomeric human IRE1α (P830L) (right panel) and IRE1α (Q780*) dimerized with a IRE1α (WT) subunit (left panel) based on PDB: 3P23. (H) Time course MTT staining of INS-1 cells expressing IRE1α (WT), IRE1α (P830L) or IRE1α (Q780*) −/+ Dox (1μg/ml), or parental INS-1 cells −/+ 100 nM Tg. (I,J) Time-course Q-PCR for p21 mRNA, and Ki67 staining, from INS-1 IRE1α (WT), IRE1α (P830L) or IRE1α (Q780*) cells under Dox (1μg/ml). Three independent biological samples were used for Q-PCR, Ki67, and Annexin V experiments. Data plotted as mean +/− SD. Also see Figure S2.

**Figure 3. Divergent modulation of IRE1α RNase activity using distinct classes of kinase inhibitors**
(A) Phosphorimager analysis of human IRE1α* (25 nM) and IRE1α* (P830L) (25 nM) kinase activity against peptide substrate (PAKtide, 2 μM) in the presence of ³²Pγ-ATP. (B) Autoradiogram of IRE1α* (P830L) autophosphorylation under increasing [APY29]. (C) 5’FAM-3’BHQ XBP1 minisubstrate to measure RNase activity. (D) RNase activities of IRE1α* and IRE1α* (P830L) −/+ APY29 (20 μM) per (C). (E) Urea PAGE of XBP1 cleavage products from (D). (F) Immunoblots of increasing IRE1α* (P830L) after incubation with DMSO or APY29 (200 μM) and DSS, with oligomer/monomer quantification. (G) Model of APY29 rescue of oligomerization and RNase activity in IRE1α* (P830L). (H) Structure of KIRA6. (I) KIRA6 inhibition of IRE1α* kinase activity. IC₅₀ values by fitting percent kinase activity per assay in (A) (n = 3). (K) Urea PAGE of competition cleavage by IRE1α* of XBP1 RNA mini-substrate (J) and α³²P-labeled Ins2 RNA (K), under indicated [KIRA6]; IC₅₀s by fitting in-gel fluorescence intensities (XBP1) and phosphorimager (Ins2). (L) Immunoblots of increasing [IRE1α*] incubated with DMSO or KIRA6 (10 μM) and DSS, with oligomer/monomer quantification. (M) Left: cartoon of sfGFP-IRE1α reporter. Right: Images of sfGFP-IRE1α induced with (sub-apoptotic) 1ng/ml Dox for 24hr in INS-1 cells −/+ DTT (5 mM) for 1hr −/+ KIRA6 (1 μM). Scale bar is 5 μm. (N) Model for how KIRA6 lowers oligomeric status and RNase activity of IRE1α*. Data plotted as mean +/− SD. Also see Figure S3.

**Figure 4. KIRA6 inhibits IRE1α Terminal UPR outputs and apoptosis**
(A) Anti-total JNK, anti-phospho-JNK, and anti-Pro- and Cleaved Caspase-3 immunoblots of INS-1 IRE1α (WT) cells treated with Dox (5 ng/ml) −/+ 1μM KIRA6 for 72hr. (B) JNK2α1 phosphorylation under indicated [KIRA6] by *in vitro* ELISA-based anti-phospho-JNK assay. (C) Anti-total and phospho-JNK immunoblots of INS-1 cells pretreated for 1hr with indicated [KIRA6], then 1μM Tg for 2hr. (D) Q-PCR for Ins1 mRNA in INS-1 IRE1α (WT) cells treated with Dox (5 ng/ml) −/+ KIRA6 (1 μM). (E) Anti-Proinsulin immunoblot of samples in (A). (F) % Annexin V staining in INS-1 IRE1α (WT) cells after 72hr in Dox (5 ng/ml) and indicated [KIRA6]. (G) Competition between indicated [1NM-PP1] and KIRA6 (1 μM) for IRE1α* (I642G) RNase. (H) Agarose gel of PstI-digested XBP1 cDNA amplicons from INS-1 cells IRE1α (I642G) cells induced by Dox (1 μg/ml) for 24hr, then 1NM-PP1 (0.5 μM) −/+ indicated [KIRA6] for 3hr, with quantitation. (I) Annexin V staining of INS-1 IRE1α (I642G) cells after 72hr with Dox (1 μg/ml), Tm (0.5 μg/ml), 1NM-PP1 (1 μM) and indicated [KIRA6]. (J) Model of 1NM-PP1 and KIRA6 competition of oligomerization and RNase activity in IRE1α* (I642G). Data plotted as mean +/− SD. P-values: *<0.05, ** <0.01. Also see Figure S4.

**Figure 5. KIRA6 reduces ER stress-induced death of cultured cells and in pancreatic islet explants**
(A) Immunoblots for total and phospho-IRE1α in INS-1 cells pretreated for 1hr with indicated [KIRA6], or 10 μM (NMe)KIRA6, then Tg (1 μM) for 2hr. (B) Agarose gel of XBP1 cDNA amplicons from INS-1 cells pre-treated with indicated [KIRA6] for 1hr, or 10 μM (NMe)KIRA6, followed by 0.5 μg/ml Tm for 8hr. (C) Ratios of XBP1^S over (XBP1^S + XBP1^U) from (B). (D) Q-PCR for Ins1 mRNA (normalized to no Tm) in INS-1 cells pretreated for 1hr with indicated [KIRA6], then 12hr in Tm (0.5 μg/ml). (E) Immunofluorescence: Insulin (green) and TXNIP (red) in islets of C57BL/6 mice under 0.5μg/ml Tm −/+ 0.5 μM KIRA6 for 16hr. (F) IL-1β secretion from THP-1 cells after 4hr −/+ 0.5 μM KIRA6, 5 μg/ml Tm, 1 μM Tg, or 5 mM ATP. (G) Ki67+ INS1 cells under 0.25 μg/ml BFA −/+ 0.5 μM KIRA6 for 48hr. (H) Proliferating mouse islet β-cells under 0.5 μg/mL Tm −/+ 0.5 μM KIRA6 for 48hr (nuclei double-positive for EdU and β-cell nuclear marker, Nkx6.1, over total Nkx6.1 positive nuclei). (I) Annexin-V staining of INS-1 cells treated with 0.25 μg/ml BFA and indicated [KIRA6] for 72hr. (J) Immunofluorescence images of C57BL/6 islets treated with 0.5 μg/mL Tm −/+ 0.5 μM KIRA6 for 16 hr. Co-stained for DAPI (blue), insulin (green), and TUNEL (red). Quantification of TUNEL+ β-cells (white arrows) normalized to DAPI+ cells. (K) Glucose-stimulated insulin secretion (GSIS) by C57BL/6 islets after 0.5μg/mL Tm −/+ 0.5 μM KIRA6 for 16hr; [Glucose] was 2.5 mM or 16.7 mM for 60 min. (L) Immunoblots for alpha-1 anti-trypsin in HEK293 cells transfected with pCDNA3.1-α1hAT-NHK, then treated with KIRA6 (1 μM) −/+ Tm (0.5 μg/ml) for 20hr. Three independent biological samples were used for XBP1 splicing, Q-PCR, Annexin V and immunofluorescence experiments. Data plotted as mean +/− SD. P-values: *<0.05, ** <0.01. Also see Figure S5.

**Figure 6. Intravitreal KIRA6 preserves photoreceptor cell numbers and function under ER stress**
(A) % XBP1 splicing in SD rat retinas 72hr post-intravitreal—and Q-PCR for TXNIP mRNA (B) and Rhodopsin mRNA (C) 96hr post-intravitreal—injection of 20 μg/ml Tm −/+ 10 μM KIRA6. (D) Primer extension mapping of IRE1α cleavage site in Rhodopsin RNA with alignment of Rhodopsin and XBP1 mRNA (E). Urea PAGE of cleavage of ³²P-labeled Rhodopsin mRNA by IRE1α* with indicated [KIRA6], with IC₅₀ (F); black arrow: intact RNA; red arrow: cleaved RNA. (H) OCT images and histological sections of SD rats 7d post-intravitreal injection of 20 μg/ml Tm −/+ 10 μM KIRA6; bars and asterisks denote ONLs. (I) SD rats intravitreally injected at P21with 2 μl Tm or DMSO to achieve indicated [Tm]; ERG measurements at a light intensity of 0 dB recorded at P28. (J) Representative scotopic ERG at a light intensity of 0 dB from a SD rat treated with Tm (3μg/ml) +/− KIRA6 (10 μM) at P21 and analyzed at P28. (K) Quantified a- and b-wave amplitudes of 0 dB scotopic ERGs from SD rats treated with DMSO or Tm (3μg/ml) +/− KIRA6 (10 μM) at P21 and analyzed at P28. Also see Figure S6.

**Figure 7. Systemic KIRA6 attenuates β-cell functional loss, increases insulin levels, and ameliorates hyperglycemia in the Akita mouse**
(A) Random AM blood glucose (BG) levels in male *Ins2^+/Akita* mice intraperitoneally (i.p) injected for 37 days b.i.d. with KIRA6 (5 mg/kg)(n=6) or vehicle (n=6) starting at P21 (i.e., Day 1). BGs (mean +/− SEM), also analyzed by Two-way RM ANOVA; p-value = 0.0122. (B) Cohort body weights at Day 49. (C) Glucose tolerance tests on Day 49 (12d post injections) of O/N fasted *Ins2^+/Akita* mice (P53) after i.p. (2 g/kg) glucose (KIRA6 n=6, Vehicle n=3). (D-E) Random insulin and C-peptide levels in *Ins2^+/Akita* mice on Day 58 (21d post injections). KIRA6 (5 mg/kg)(n=5) and vehicle (n=4). (F) Whole pancreatic histological sections from *Ins2^+/Akita* mice on Day 53 (15d post injections). Islets delineated by dashed outline. Immunofluorescence micrographs of samples in (F): co-stained for DAPI (blue), insulin (green), with merge. (H) Total β-cell area as a percentage of total pancreas area on Day 55 (18d post injections). KIRA6 (5 mg/kg)(n=6) and vehicle (n=3). (I) Model of how KIRA6 prevents the terminal UPR by inhibiting IRE1α oligomers. Data plotted as mean +/− SD. P-values: *<0.05, ** <0.01. Also see Figure S7.

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Allosteric inhibition of the IRE1α RNase preserves cell viability and function during endoplasmic reticulum stress

Affiliations

Allosteric inhibition of the IRE1α RNase preserves cell viability and function during endoplasmic reticulum stress

Authors

Affiliations

Abstract

Figures

References

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MeSH terms

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LinkOut - more resources

Full Text Sources

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