The IRE1β-mediated unfolded protein response is repressed by the chaperone AGR2 in mucin producing cells
Abstract
Synopsis
Introduction
Results
Divergent-regulatory machineries for IRE1α and β
AGR2 binds IRE1β’s luminal domain
AGR2 binding favours IRE1β luminal domain monomers
IRE1β repression is affected by the state of AGR2’s active site and involves destabilisation of luminal domain dimers
The IRE1β-AGR2 pair responds to AGR2’s client, MUC2
IRE1β specialisation extends to its kinase–endonuclease extension domain
Discussion
Methods
Unique | Plasmid name | Figure | Reference | Description |
---|---|---|---|---|
UK1903 | CHO_IRE1_guideC15.1_pSpCas9(BB)-2A-mCherry | Kono et al, 2017 | Cas9 and guide targeting IRE1 in CHO-K1 ∆LD clone 15 (mCherry-tagged) | |
UK1968 | CHO_IRE1_hIRE1-LD_reptemp4_pCR-Blunt2-TOPO | Kono et al, 2017 | Repair template for wild-type hIRE1a LD reconstitution in CHO-K1 cells | |
UK2757 | CHO_ mIRE1b_LD_39-426_IRE1a_3xFLAG_reptemp4 | This study | Repair template for wild-type mIRE1b LD reconstitution in CHO-K1 cells | |
UK2881 | pBABEpu_FLAGM1_mAGR2 | This study | Plasmid used for retroviral transduction of FLAG-M1 AGR2 | |
UK40 | pL_VSVG | Bartz and Vodicka, 1997 | CMV_VSVG (RV packaging), for retroviral transduction of FLAG-M1 AGR2 | |
UK41 | pJK3 | Bartz and Vodicka, 1997 | GAG-POL, for retroviral transduction of FLAG-M1 AGR2 | |
UK42 | pCMV_TAT_HIV | Bartz and Vodicka, 1997 | Transactivator, for retroviral transduction of FLAG-M1 AGR2 | |
UK1314 | pCEFL_mCherry_3XFLAG_C | Sekine et al, 2015 | pCEFL with 3XFLAG_C tagged from mCherry-tagged plasmid, empty vector of UK2708 | |
UK2708 | muAGR2_pCEFL_mCherry_FLAG_M1 | This study | Mammalian expression of FLAG-M1-tagged muAGR2 from mCherry-tagged plasmid | |
UK2709 | muAGR2_pCEFL_mCherry | This study | Mammalian expression of muAGR2 from mCherry-tagged plasmid | |
UK1610 | pSpCas9(BB)-2A-mCherry_V2 | Amin-Wetzel et al, 2017 | Modified pSpCas9(BB)-2A vector to express mCherry together with guide RNA & Cas9 | |
UK2771 | FLAGM1_muAGR2_guide1_pSpCas9(BB)-2A-mCherry_V2_MP2 | This study | pSpCas9(BB)-2A vector to express mCherry together with guide RNA & Cas9 to knock out FLAG-M1-tagged muAGR2 | |
UK2772 | FLAGM1_muAGR2_guide2_pSpCas9(BB)-2A-mCherry_V2_MP5 | This study | pSpCas9(BB)-2A vector to express mCherry together with guide RNA & Cas9 to knock out FLAG-M1-tagged muAGR2 | |
UK2246 | IRE1a_LD_∆C_24-443_AviTag_H6_pET30a(+) | Amin-Wetzel et al, 2019 | C-terminally-tagged AviTag-H6 human IRE1a LD ∆C | |
UK2796 | pMAL-hIRE1bNLD_35-428_AT_His7 | This study (hIRE1b LD originating from Oikawa et al, 2012 | Bacterial expression MBP-hIRE1b_LD_(35-428)-AviTag-His7 | |
UK1107 | mIRE1b_pCDNA5_FRT_TO | This study | Full-length mIRE1b | |
UK916 | pCDNA5_FRT_TO | This study | Empty vector for UK1107 | |
UK2758 | CHO_ mIRE1b_LD_39-426_dC_IRE1a_3xFLAG_reptemp4 | This study | Repair template for mIRE1b LD dC reconstitution in CHO-K1 cells | |
UK3028 | pMAL-hIRE1bNLD_∆C-mGL-AT-His | This study | Bacterial expression hIRE1b-∆C version of UK2986 | |
UK2986 | pMAL-hIRE1bNLD-mGL-AT-His | This study | C-term mGL inserted in MBP-hIRE1bLD in UK2796 | |
UK2794 | H6pSUMO3_mAGR2_21_175 | This study | Bacterial expression of H6pSUMO3_mAGR2 WT | |
UK2048 | pET22b_H7_Smt3_Ire1a_LD∆C_24_444 R234C | Amin-Wetzel et al, 2017 | Bacterial expression of Smt3-tagged cysteine-free IRE1a LD∆C R234C, for labelling with Oregon Green | |
UK3053 | pMAL-hIRE1bNLD_35-428_∆C_AT_His7 | This study | Bacterial expression hIRE1b-∆C version of 2796 | |
UK13 | pCEFL.puro | Sekine et al, 2015 | Empty vector of UK2718 | |
UK2718 | SP_FLAGM1_muAGR2_pCEFL-puro_constr_MP1 | This study | Mammalian expression of FLAG-M1-tagged muAGR2 with puro resistance for intracellular staining and insertion of AGR2 as a transgene | |
UK2865 | SP_FLAGM1_muAGR2_C81S_pCEFL-puro_MP2 | This study | Mammalian expression of muAGR2 C81S with puro resistance for intracellular staining | |
UK2866 | SP_FLAGM1_muAGR2_E60A_pCEFL-puro_MP5 | This study | Mammalian expression of muAGR2 E60A with puro resistance for intracellular staining | |
UK2871 | SP_FLAGM1_muAGR2_C81W_pCEFL-puro_MP6 | This study | Mammalian expression of muAGR2 C81W with puro resistance for intracellular staining | |
UK2873 | SP_FLAGM1_muAGR2_C81E_pCEFL-puro_MP10 | This study | Mammalian expression of muAGR2 C81E with puro resistance for intracellular staining | |
UK2795 | H6pSUMO3_mAGR2_21_175_C81S | This study | Bacterial expression of H6pSUMO3_mAGR2 C81S | |
UK2835 | H6pSUMO3_mAGR2_C81E_21_175 | This study | Bacterial expression of H6pSUMO3_mAGR2 C81E | |
UK2833 | H6pSUMO3_mAGR2_C81W_21_175 | This study | Bacterial expression of H6pSUMO3_mAGR2 C81W | |
UK2816 | H6pSUMO3_mAGR2_21_175_E60A | This study | Bacterial expression of H6pSUMO3_mAGR2 E60A | |
UK3155 | pMAL-hIRE1bNLD_35-428_Nanoluc_H6 | This study | NanoLuc (complete, WT)-tagged MBP-hIRE1bLD (35-428) bacterial expression vector, a BRET donor | |
UK3060 | H6_pSUMO3_MGL-MP4 | This study | Bacterial expression of mGreen lantern | |
UK2805 | huMUC2_21-1259_pCEFL_mCherry (MP23) | This study, MUC2 originating from Javitt et al, 2019 | Mammalian expression N-term of human MUC2 fragment (from Debbie Fass) | |
UK2804 | huMUC2_21-1397_pCEFL_mCherry (MP21) | This study, MUC2 originating from Javitt et al, 2019 | Mammalian expression N-term of human MUC2 fragment (from Debbie Fass) | |
UK2947 | FLAGM1_huMUC2_4198-5179_pCEFL_mCherry_MP1 | This study, MUC2 originating from Lidell et al, 2003 | Mammalian expression FLAG-M1-tagged human MUC2 C-term. in pCEFL-mCherry, from Gunnar Hannsson | |
UK2952 | cgIRE1a_Int12_g2_pSpCas9(BB)-2A-mCherry | Appendix Fig. S2A–C | This study | CRISPR guide vector targeting intron 12 to create IRE1a ∆KEN deletion |
UK2876 | cgIRE1a_Ex22_g1_pSpCas9(BB)-2A-mCherry | Appendix Fig. S2A–C | This study | CRISPR guide vector targeting exon 22 to create IRE1a ∆KEN deletion |
UK3022 | endogenous_5’HA_mIRE1b_Ken-bGHT_repair_V1 | This study | Repair template for IRE1b KEN reconstitution in CHO-K1 cells | |
UK3093 | endogenous_5’HA_mIRE1a_Ken_repair_bGHT_pBSKS | This study | Repair template for IRE1a KEN reconstitution in CHO-K1 cells | |
UK2668 | SENP2_364-589_pET-28a MP9 | This study | Bacterial expression of human SUMO3 protease SENP2 active fragment to cleave SUMO-tag | |
UK1452 | SubA_22-347_3xFLAG_KDEL_pCEFL_mCherry | Amin-Wetzel et al, 2019 | Mammalian expression of SubA protease | |
UK1459 | SubA_22-347_S272A_3xFLAG_KDEL_pCEFL_mCherry | Amin-Wetzel et al, 2019 | Mammalian expression of SubA protease mutant variant | |
UK 2557 | haBiP_pCEFL_mCherry | This study | Mammalian expression of BiP | |
UK887 | hPDI1_WT_mCherry_KDEL | Avezov et al, 2015 | Mammalian expression of PDI1 |
Gene | Primer | Sequence (5’ → 3’) | E (%) | Use |
---|---|---|---|---|
XBP1 | 1470 hamXBP1.19 S 5 mXBP1.14AS | GGCCTTGTAATTGAGAACCAGGAG GAATGCCCAAAAGGATATCAGACTC | — | RT-PCR |
Scara3 | 3237 cgScara3.1 S 3238 cgScara3.1AS | GGCTCTGCTCCTTGTGGCCG CAGGGCTTTTGGGTCCAGTCCT | 95.2 | qPCR |
Pmp22 | 3243 cgPmp22.1 S 3244 cgPmp22.1AS | TCGTCAGCGAGTGAATGGCTACA GCTGCTGCACTCATCACGCA | 102.5 | qPCR |
Hgsnat | 3235 cgHgsnat.1 S 3236 cgHgsnat.1AS | CTCCACCGTCCTTTATCACACCCAG CACCAGGCAGTGAATCTCATCAGG | 107.0 | qPCR |
Bloc1s1 | 52 MmBlos1.1 S 53 MmBlos1.2AS | CAAGGAGCTGCAGGAGAAGA GCCTGGTTGAAGTTCTCCAC | 109.9 | qPCR |
Pdgfrb | 3241 cgPdgfrb.2 S 3242 cgPdgfrb.2AS | AAACCCCCTACAGCTGTCTT CAATCCCCATGGCGTCTGCG | 108.1 | qPCR |
Rpl27 | 3249 cgRpl27.2 S 3250 cgRpl27.2AS | ACAATCACCTCATGCCCACAAG GCGTTTCAGGGCTGGGTCTC | 105.3 | qPCR |
GAPDH | 3255 cg.GAPDH.1 S 3256 cg.GAPDH.1AS | TTTCCGTGCAGTGCCAGCCT CCAGGCGTCCAATACGGCCA | 106.8 | qPCR |
Mammalian cell culture
Transfection
Gene manipulation and allele analysis
Flow cytometry and fluorescence-activated cell sorting (FACS)
Intracellular FLAG-M1 staining of AGR2 variants for flow cytometry analysis
Genetic complementation of AGR2
Mammalian cell lysis
Immunoprecipitation (IP)
Reducing/non-reducing SDS-PAGE and immunoblotting
Mass spectrometry
Protein purification
Human IRE1β luminal domain variants
Human IRE1α luminal domain variants
Mouse AGR2 variants
Analytical size-exclusion chromatography (SEC)
TCA precipitation
Bio-Layer interferometry
BRET
Mass photometry by interferometric scattering microscopy (ISCAT)
Fluorescence detection system sedimentation velocity analytical ultracentrifugation (FDS-SV-AUC)
RNA isolation and reverse transcription
PCR analysis of XBP1 mRNA splicing
qPCR
Data availability
Author contributions
Disclosure and competing interests statement
Acknowledgements
Supporting Information
References
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This cover highlights the article Weakened APC/C activity at mitotic exit drives cancer vulnerability to KIF18A inhibition by Colin R. Gliech, Andrew J. Holland and colleagues. A large portion of cancer cells are dependent on kinesis KIF18A function because of alterations in the ratio of APC/C and spindle assembly checkpoint (SAC) signals.
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