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IRE1α is an endogenous substrate of endoplasmic-reticulum-associated degradation

Abstract

Endoplasmic reticulum (ER)-associated degradation (ERAD) represents a principle quality control mechanism to clear misfolded proteins in the ER; however, its physiological significance and the nature of endogenous ERAD substrates remain largely unexplored. Here we discover that IRE1α, the sensor of the unfolded protein response (UPR), is a bona fide substrate of the Sel1L–Hrd1 ERAD complex. ERAD-mediated IRE1α degradation occurs under basal conditions in a BiP-dependent manner, requires both the intramembrane hydrophilic residues of IRE1α and the lectin protein OS9, and is attenuated by ER stress. ERAD deficiency causes IRE1α protein stabilization, accumulation and mild activation both in vitro and in vivo. Although enterocyte-specific Sel1L-knockout mice (Sel1LΔIEC) are viable and seem normal, they are highly susceptible to experimental colitis and inflammation-associated dysbiosis, in an IRE1α-dependent but CHOP-independent manner. Hence, Sel1L–Hrd1 ERAD serves a distinct, essential function in restraint of IRE1α signalling in vivo by managing its protein turnover.

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Figure 1: IRE1α is a Sel1L–Hrd1 substrate in vitro.
Figure 2: The role of OS9 and the intramembrane hydrophilic residues of IRE1α in Sel1L–Hrd1-mediated IRE1α degradation.
Figure 3: Sel1L–Hrd1-mediated IRE1α degradation is regulated by ER stress.
Figure 4: Sel1L–Hrd1-mediated IRE1α degradation requires BiP.
Figure 5: IRE1α is an endogenous Sel1L–Hrd1 substrate in vivo.
Figure 6: Consequences of Sel1L–Hrd1 ERAD deficiency on IRE1α activation and inflammation.
Figure 7: Sel1LΔIEC (EKO) mice are susceptible to experimental colitis.
Figure 8: IRE1α protein accumulation is critical for the pathogenesis of colitis in Sel1LΔIEC (EKO) mice.

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Acknowledgements

We thank K.-L. Tung for help with the organoid culture; and other members of the Qi laboratory for comments and technical assistance. S.S. is an International Student Research Fellow of the Howard Hughes Medical Institute (59107338). L.Q. is the recipient of the Junior Faculty and Career Development Awards from American Diabetes Association (ADA). This work was supported by R21AI085332 (G.E.D.), 1R03AI114344 (H.W.), Chinese National Natural Science Foundation Grant 31371391 (Q.L.), National Heart, Lung, and Blood Institute Proteomics Center Award HHSN268201000035C, R01 MH067880 and 8P41GM103533-17 (J.R.Y. III), NIH R01DK105393, R01GM113188, UL1TR000457 of the Clinical and Translation Science Center at Weill Cornell Medical College, ADA 1-12-CD-04 and Cornell VERG seed grant (L.Q.).

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Authors and Affiliations

Authors

Contributions

S.S. designed and performed most in vivo experiments; G.S. designed and performed most in vitro experiments; X.H. and J.R.Y. III performed the proteomic analysis; S.S., P.B. and X.S. performed the organoid culture; Y.J. assisted in some in vivo experiments; H.S. generated various enterocyte cell lines; H.S., S.S. and X.S. cultured organoids. H.K. and R.D.G. repeated some in vitro experiments; B.G. and D.F. generated inducible Hrd1 knockout mice; H.M. and H.W. generated SEL1L-knockout HEK293T cells; T. Inoue and B.T. performed limited proteolysis analysis; T. Iwawaki, A.-H.L., A.W.P., J.C.P. and Q.L. provided key reagents; S.K. performed microarray analyses; G.E.D. performed histological examination of the tissue; K.W.S. performed FISH and designed the experiments; L.Q. conceived the project and supervised the project. L.Q. and S.S. wrote the manuscript, S.S. and G.S. wrote the methods and figure legends, and all authors edited and approved the manuscript.

Corresponding authors

Correspondence to Qiaoming Long or Ling Qi.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Identification of IRE1α as an ERAD substrate in vitro.

(a) Western blot analysis of Bag6 and Calnexin in ER and cytosolic fractions from Sel1Lf/f;ERCre+MEFs treated with vehicle (WT) or 4-OHT (IKO) for 4 days. (b) IRE1α abundance shown in spectral counts in the purified microsomes of WT and IKO MEFs measured by quantitative LC-MS/MS. (c) Western blot analysis of whole cell lysates from WT and IKO MEFs. (d) Q-PCR analysis of WT and IKO MEFs. (e) Quantitation of IRE1α protein levels in Fig. 1e. (f) Q-PCR analysis of WT and Hrd1−/− MEFs. (g) Q-PCR analysis of IRE1a and OS9 in SEL1L-deficient HEK293T cells. (h) Autoradiography of immunoprecipitates of Flag-agarose in Flag-IRE1α-transfected WT and SEL1L −/− HEK293T cells pulsed for 30 min with 35S-methionine/cysteine. All but (b) are representative of n = 3 independent experiments. (b), a total of 107 cells for each cell type in one experiment. (dg), error bars represent sem from n = 3 independent experiments. , p < 0.05; , p < 0.001 by Student’s two-tailed t test. Unprocessed original scans of blots are shown in Supplementary Fig. 9.

Supplementary Figure 2 Molecular mechanism underlying IRE1α degradation.

(a) Western blot analysis of immunoprecipitates of Flag-agarose in transfected HEK293T under denaturing condition, confirming that ubiquitination occurs directly on IRE1α protein. (b) Quantitation of IRE1α protein levels in Fig. 2b. (c) Western blot analysis of immunoprecipitates of HA- or Flag-agarose in transfected HEK293T cells showing OS9 interaction with endogenous and transfected IRE1α. (d) Western blot analysis of IRE1α in WT and OS9-deficient (OS9CRISPR) HEK293T cells, with quantification shown in (e). (f) Immunofluorescent staining of Flag-IRE1α and endogenous BiP in transfected HEK293T cells. (g) RT-PCR analysis of Xbp1 splicing in HEK293T cells transfected with control (CON), WT or T3A IRE1α. (h) Western blot analysis of immunoprecipitates of Flag-agarose in transfected HEK293T treated with thapsigargin (Tg) for the indicate time. (i) Western blot analysis of IRE1α in WT and IKO MEFs treated with 300 nM Tg for the indicated time, with quantitation shown on the right. (j) Western blot analysis of immunoprecipitates of HA-agarose in transfected HEK293T treated with MG132 in the presence or absence of 0.5 μg ml−1 SubAB for 3 h, showing reduced IRE1α ubiquitination in the absence of BiP. (k) Q-PCR analysis of Ire1a in MEFs with ectopic expression of BiP. Representative data from 3 independent experiments shown. (b), (e) and (k), error bars represent sem from n = 3 independent experiments. , p < 0.05; , p < 0.01 by Student’s two-tailed t test. Unprocessed original scans of blots are shown in Supplementary Fig. 9.

Supplementary Figure 3 IRE1α is degraded by the Sel1L-Hrd1 ERAD in various tissues.

(a) Diagram illustrating the breeding scheme for the generation of Sel1LΔIEC mice. Expected and observed genotypes in the offsprings from the last breeding shown below. (b) Immunohistochemical staining of OS9 in colon. (c) Western blot analysis of IRE1β in the gut. Kidney tissue from Sel1Lf/f;ERCre − (WT) and Sel1Lf/f;ERCre+ (IKO) mice 13 days post-tamoxifen injection is used as a negative control for IRE1β. Quantitation is shown on the right (n = 2 mice each genotype). Below, Q-PCR analysis of Ire1β in the gut. Error bars represent sem from n = 3 mice each genotype. (d) Sequence alignment of IRE1β transmembrane domain between different species and with human IRE1α. h, human; z, zebra fish. (e) Western blot analysis of IRE1α in the pancreas of Sel1Lf/f;ERCre − (WT) and Sel1Lf/f;ERCre+ (IKO) mice 4 and 8 days post-tamoxifen injection, with quantitation shown on the right (n = 2 mice each genotype at a given timepoint). (fg) Western blot and Q-PCR analyses (g) of IRE1α in white adipose tissue of Sel1LΔadipo (AKO) mice, with quantitation shown below the blot. (f), values, mean ± sem from n = 3 mice of each genotype. Error bars in (g) represent sem from n = 6 mice of each genotype. All experiments were repeated twice. , p < 0.05; , p < 0.01 by Student’s two-tailed t test. n.s., not significant. Unprocessed original scans of blots are shown in Supplementary Fig. 9.

Supplementary Figure 4 Regulatory circuitry of ERAD and IRE1α and elevated inflammation in Sel1L-deficient cells.

(a) Q-PCR analysis of m-ICcl2 enterocytes stably expressing XBP1s. Quantitation shows an average of two independent experiments. (bc) IRE1α-XBP1 regulates expression of ERAD components. (b) Sequences of mutated Xbp1 or Ire1a alleles identified from 74 clonal amplicons by CRISPR in m-ICcl2 cells. For each allele, sequence on top is WT and mutant amplicons are shown below. Green, protospacer adjacent motif sequence; dashes, deletion; blue, insertion; red, mutation. (c) qPCR analysis in WT and knockout m-ICcl2 cell colonies treated with 300 nM Tg for 4 h. (d) Model showing the regulatory circuitry between IRE1α and Sel1L-Hrd1 ERAD. (e) Phos-tag-based (P-T) Western blot analysis of IRE1α phosphorylation in the gut of Sel1Lf/f;ERCre− (WT) and Sel1Lf/f;ERCre+ (IKO) mice 13 days post-tamoxifen injection. (fg) P-T Western blot analysis of IRE1α phosphorylation (f) and RT-PCR analysis of Xbp1 mRNA splicing (g) in white adipose tissue (WAT) of WT and Sel1LΔadipo (AKO) mice. Each lane represents an independent sample. (hj) Knockdown of Sel1L enhances inflammatory tone of enterocytes. (h) Q-PCR (left) and Western blot analyses (right) of Sel1L in m-ICcl2 enterocytes stably expressing Sel1L knockdown (Sel1Li) and control (CONi). Quantitation shows an average of two independent experiments. (i) Western blot analysis of p-JNK in m-ICcl2 enterocytes treated with 50 ng/mL murine TNFα for the indicated time, with quantitation shown in (j). All experiments were repeated twice. , p < 0.05; , p < 0.01; , p < 0.001 by Student’s two-tailed t test. Unprocessed original scans of blots are shown in Supplementary Fig. 9.

Supplementary Figure 5 The status of UPR and IRE1α signaling in colonic epithelium and sensitivity to experimental colitis of Sel1LΔIEC (EKO) mice.

(a) Colon length of adult WT and Sel1LΔIEC mice. Error bars represent sem from n = 3 WT and n = 4 EKO mice. (bc) Microarray analysis of colon epithelium of WT and EKO mice under basal conditions. Samples from n = 3 mice of each genotype were analyzed. (b) Top upregulated pathways in colon epithelium of EKO mice by gene set enrichment analysis (GSEA). (c) Volcano plot of microarray analysis highlighting up-regulated UPR and ERAD genes. (d) TUNEL analysis of colon 4 days after 3% DSS feeding. Representative images of 3 mice studied. (ef) Assessment of stool consistency and stool blood on day 6 post-DSS as a blind study. Score for stool consistency: 0, normal; 1, soft but still formed; 2, very soft; 3, diarrhea. Score for stool blood: 0, negative; 1, positive hemoccult; 2, blood traces in stool visible; 3, rectal bleeding. Error bars represent sem from n = 3 mice. (f) Average daily water intake per mouse during the 5-day DSS treatment. Error bars represent sem from n = 6 WT and n = 4 EKO mice. (g) FISH staining showing the distribution of Eubacteria(red) in colonic mucosa of DSS-treated mice at day 9. Bacteria detected in the crypts of WT mice (Panel 2). More severe bacteria invasion in Sel1LΔIEC mice, including patchy bacteria invasion into epithelium (panel 3), intralumenal bacteria proliferation (panel 4) and thick mat of superficial invasive bacteria (panel 5). Green, non-specific signal; blue, DAPI-stained nucleus. Representative images of 3 mice studied. (h) Cluster diagram-based principal coordinates analyses (PCoA) using unweighted UniFrac of fecal microbiota from 8-week-old co-housed littermates (WT, n = 8; EKO, n = 6 mice). (i) Quantitation of flow cytometric analyses of activated CD69 + CD8 + T cells in spleen and mesenteric lymph nodes (MLN) of water (CON) or DSS-treated WT and Sel1LΔIEC mice on day 7. Error bars represent sem from n = 3 mice. (j) Serum cytokine levels in water (CON) or DSS-treated WT and Sel1LΔIEC mice at day 7. Error bars represent sem from n = 9 WT CON, n = 8 WT DSS, n = 7 EKO DSS mice. , p < 0.05; , p < 0.01; , p < 0.001 by Student’s two-tailed t test.

Supplementary Figure 6 Dissecting the role of IRE1α heterozygosity and inflammation in the pathogenesis of experimental colitis in Sel1LΔIEC (EKO) mice.

(a) Breeding scheme for the generation of EKO; Ire1a+/− mice and their littermates. (b) Body weight of EKO; Ire1a+/− mice and their littermates. Error bars represent sem from n = 5 mice per genotype. (c) Western blot analysis of m-ICcl2 enterocytes with either control (CONi) or Sel1L knockdown (Sel1Li) treated with 50 μM JNK inhibitor SP600125 (SP) for 1 h followed by 50 ng/mL murine TNFα for 10 min. (df) 6 week-old mice were injected intraperitoneally with 30 mg/kg SP600125 (SP) every other day with concurrent treatment of 3% DSS for 5 days followed by fresh water: body weight change (d), colon length (e) and representative H&E images of colon at day 7 (f). Error bars represent sem from n = 5 mice per genotype pooled from two independent experiments. , p < 0.05; , p < 0.01; , p < 0.001 comparing EKO SP to EKO Veh by Student’s two-tailed t test. Unprocessed original scans of blots are shown in Supplementary Fig. 9.

Supplementary Figure 7 CHOP is dispensable for the disease pathogenesis of Sel1LΔIEC (EKO) mice.

(ab) Western blot and Q-PCR analyses of PERK in colon epithelium of WT and Sel1LΔIEC (EKO) mice. Error bars represent sem from n = 3 mice per genotype. (c) Western blot analysis in colon epithelium. (d) Q-PCR analysis of Chop mRNA levels in the colon. Note that PERK protein level and activity in the gut is not affected by IRE1α protein levels in EKO vs. EKO; Ire1a+/− mice. Error bars represent sem from n = 3 mice per genotype. (e) Growth curve of 4 littermates: WT, Chop−/−, Sel1LΔIEC (EKO) and EKO;Chop−/− mice. Error bars represent sem from n = 6 mice per genotype. (fg) Western blot analysis of protein levels in the gut, with quantitation shown in (g). Error bars represent sem from n = 3 (WT and Chop−/− ) or n = 4 (EKO and EKO;Chop−/− ) mice per genotype. (h) Phos-tag-based (P-T) Western blot analysis of IRE1α phosphorylation in the gut. n.s., not significant. , p < 0.05; , p < 0.01; , p < 0.001 by Student’s two-tailed t test. Unprocessed original scans of blots are shown in Supplementary Fig. 9.

Supplementary Figure 8 Epithelial Sel1L heterozygosity has no effect on IRE1α activity and the susceptibility to experimental colitis.

(a) Breeding scheme for the generation of Sel1LΔIEC/+ (HET) and their littermates. (b) Growth curve of the littermates. Error bars represent sem from n = 5 mice per genotype. (cd) Western blot analysis of the colon, with quantitation of an average of data from two mice per genotype (d). (e) Q-PCR analysis of UPR genes in the colon. Error bars represent sem from n = 3 mice per genotype. (fh) 12 week-old mice were treated with 5-day 3% DSS followed by fresh water: (f) body weight change, (g) colon length and (h) representative H&E images of colon on day 9. Error bars represent sem from n = 5 mice per genotype. , p < 0.05; , p < 0.01; , p < 0.001 by Student’s two-tailed t test. Unprocessed original scans of blots are shown in Supplementary Fig. 9.

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Sun, S., Shi, G., Sha, H. et al. IRE1α is an endogenous substrate of endoplasmic-reticulum-associated degradation. Nat Cell Biol 17, 1546–1555 (2015). https://doi.org/10.1038/ncb3266

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