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. 2015 Dec;17(12):1546-55.
doi: 10.1038/ncb3266. Epub 2015 Nov 9.

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

Shengyi Sun  1 , Guojun Shi  2 , Haibo Sha  2 , Yewei Ji  2 , Xuemei Han  3 , Xin Shu  2 , Hongming Ma  4 , Takamasa Inoue  5 , Beixue Gao  6 , Hana Kim  2 , Pengcheng Bu  7   8 , Robert D Guber  2 , Xiling Shen  7   8   9 , Ann-Hwee Lee  10 , Takao Iwawaki  11 , Adrienne W Paton  12 , James C Paton  12 , Deyu Fang  6 , Billy Tsai  5 , John R Yates 3rd  3 , Haoquan Wu  4 , Sander Kersten  13 , Qiaoming Long  14 , Gerald E Duhamel  15 , Kenneth W Simpson  16 , Ling Qi  1   2
Affiliations

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

Shengyi Sun et al. Nat Cell Biol. 2015 Dec.

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 (Sel1L(ΔIEC)) 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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Figures

Figure 1
Figure 1. IRE1α is a Sel1L-Hrd1 substrate in vitro
(a) Representative TEM images of Sel1Lf/f;ERCre+ MEFs treated with vehicle (WT) or 4-OHT (IKO) for 3 days. ER, endoplasmic reticulum (arrows); M, mitochondrion; N, nucleus. (b–c) Western blot analysis of Sel1Lf/f;ERCre+ (f/f;ERCre+) MEFs treated with 4-OHT for the indicated time (b), with quantitation shown in (c). (d) Western blot analysis of Sel1Lf/f;ERCre− MEFs treated with 4-OHT. (e) Western blot analysis of IRE1α in WT and Hrd1−/− MEFs, with quantitation shown in Supplementary Fig. 1e. (f) Western blot analysis of IRE1α in MEFs treated with 50 µg/ml cycloheximide (CHX) for the indicated time, with quantitation shown below. (g) Western blot analysis of IRE1α in CHX-treated WT and SEL1L-deficient (SEL1LCRISPR) HEK293T cells, with quantitation shown on the right. The ERAD substrate OS9 and the cytosolic proteins BAG6 and HSP90 are shown as controls. Representative data from three independent experiments shown. Unprocessed original scans of blots are shown in Supplementary Fig. 9.
Figure 2
Figure 2. The role of OS9 and intramembrane hydrophilic residues of IRE1α in Sel1L-Hrd1-mediated IRE1α degradation
(a) Western blot analysis of immunoprecipitates of Flag-agarose in transfected HEK293T, showing the ubiquitination of IRE1α by HRD1 in vitro. (b) Western blot analysis of IRE1α in WT and IKO MEFs stably expressing HRD1-myc, with quantitation shown in Supplementary Fig. 2b. (c–e) Western blot analysis of IRE1α decay in CHX-treated WT and OS9-deficient (OS9CRISPR) HEK293T cells, with quantitation of total protein at basal state and protein decay shown in (d) and (e), respectively. (f) Alignment of IRE1α transmembrane domain (444–464aa) across species showing three hydrophilic residues. (g–i) Western blot analysis of IRE1α in CHX-treated HEK293T cells transfected with WT and T3A Flag-IRE1α, with quantitation of total protein at basal state and protein decay shown in (h) and (i), respectively. (j) Western blot analysis of immunoprecipitates of Flag-IRE1α in transfected HEK293T cells, showing reduced ubiquitination of T3A IRE1α and interaction with Hrd1. (a), (b) and (j) are representative of 2 independent experiments, while others are representative of 3 independent experiments. (d) and (h), data represent sem of n=3 independent experiments. **, p<0.01, ***, p<0.001 Student’s two-tailed t test. Unprocessed original scans of blots are shown in Supplementary Fig. 9.
Figure 3
Figure 3. Sel1L-Hrd1-mediated IRE1α degradation is regulated by ER stress
(a–b) Western blot analysis of immunoprecipitates of Flag-agarose in transfected HEK293T treated with 200 nM thapsigargin (Tg) and/or 10 µM MG132. (c) Western blot analysis of immunoprecipitates of HA-agarose in IRE1α−/− MEFs stably expressing HA-IRE1α. Tm, 2.5 µg/ml tunicamycin for 2 h. (d) Pulse-chase analysis of IRE1α decay in HEK293T cells transfected with HA-IRE1α. Cells were treated with 300 nM Tg during the chase. Quantitation shown below. Phos-tag-based (P–T) Western blot analysis on the right indicated strong IRE1α phosphorylation and activation in response to Tg. (e) Western blot analysis of immunoprecipitates in WT and SEL1L-deficient (SEL1LCRISPR) HEK293T cells transiently transfected with NHK-GFP. (f) Sucrose gradient followed by Western blot analyses under non-reducing and reducing conditions in mock- or Tg-treated MEFs. (g) Limited proteolysis digestion of IRE1α in lysates from HEK293T cells treated with or without 100 nM Tg for 3 h, showing conformational change of IRE1α. *, degradation intermediates. Representative data from two independent experiments shown. Unprocessed original scans of blots are shown in Supplementary Fig. 9.
Figure 4
Figure 4. Sel1L-Hrd1-mediated IRE1α degradation requires BiP
(a) Western blot analysis of immunoprecipitates in mock- or Tg-treated HEK293T cells expressing HA-IRE1α. (b) Western blot analysis of immunoprecipitates in HEK293T cells treated with 1 µg/mL WT SubAB or mutant SubAA272B (mut) for 1 h, showing that BiP links IRE1α to Sel1L. (c) Western blot analysis of IRE1α in MEFs pre-treated with 1 µg/mL WT SubAB or mutant SubAA272B (mut) for 1 h followed by cycloheximide (CHX) for the indicated time with quantitation shown on the right. Error bars represent sem, n=3 independent experiments. (d) Western blot analysis of IRE1α in MEFs with ectopic expression of BiP, with or without Tg treatment. (e) Western blot analysis of immunoprecipitates in IRE1α−/− MEFs stably expressing either WT or D123P HA-IRE1α, with or without Tg treatment. (f) Western blot analysis of IRE1α in IRE1α−/− MEFs stably expressing either WT or D123P HA-IRE1α, pre-treated with 300 nM Tg for 2 h followed by CHX treament. Quantitation shown below. (a) and (c) are representative of 3 independent experiments, while others are representative of 2 independent experiments. P–T, phos-tag-based Western blot to visualize IRE1α phosphorylation. (g) Model for ERAD-mediated IRE1α degradation under basal and stress conditions. Unprocessed original scans of blots are shown in Supplementary Fig. 9.
Figure 5
Figure 5. IRE1α is an endogenous Sel1L-Hrd1 substrate in vivo
(a) Growth curve of adult WT and Sel1LΔIEC (EKO) mice (showing the mean for 5 mice of each genotype at each time point). (b–c) Western blot analysis of IRE1α and Sel1L-Hrd1 ERAD protein levels in isolated colonic epithelium of WT and EKO mice, with quantitation shown in (c). Each lane represents an independent sample. HSP90, a loading control. Error bars represent sem, n=3 mice of each genotype. (d) Q-PCR analysis of Sel1l, Hrd1 and Ire1α mRNA levels in the gut. Error bars represent sem, n=3 mice of each genotype. (e) Immunohistochemical staining of IRE1α in colon. Representative images of 3 mice studied. (f–g) Western blot and Q-PCR analyses of Sel1L and IRE1α in the gut of Sel1Lf/f;ERCre− (WT) and Sel1Lf/f;ERCre+ (IKO) mice 13 days post-tamoxifen injection. Error bars represent sem, n=5 mice of each genotype (only two shown for Western blots). (h–i) Western blot and Q-PCR analyses of Hrd1 and IRE1α in the gut of Hrd1+/+;ERCre+ (WT) and Hrd1f/f;ERCre+ (Hrd1−/−) mice 10 days post-tamoxifen injection. n=2 mice of each genotype. (j) Representative images of primary crypt organoids in culture. (k) Western blot analysis of primary intestinal crypt organoids treated with 25 µg/ml cycloheximide (CHX) for 2 h, with quantitation shown below the blot. (l) Western blot analysis of IRE1α in primary adipocytes from WT or adipocyte-specific Sel1L-deficient (AKO) mice treated with 50 µg/ml cycloheximide (CHX) for the indicated time, with quantitation shown on the right. Representative data from two independent repeats shown. *, p<0.05; **, p<0.01 by Student’s two-tailed t test. Unprocessed original scans of blots are shown in Supplementary Fig. 9.
Figure 6
Figure 6. Consequences of Sel1L-Hrd1 ERAD deficiency on IRE1α activation and inflammation
(a) Phos-tag-based (P-T) Western blot analysis of IRE1α phosphorylation in isolated colonic epithelium of WT and Sel1LΔIEC (EKO) mice. N=3 mice of each genotype. (b–c) RT-PCR analysis of Xbp1 mRNA splicing, with quantitation shown in (c). Error bars represent sem, n=3 mice of each genotype. (d–e) Western blot and RT-PCR analyses of IRE1α activation in WT and IKO MEFs, with quantitation of spliced Xbp1 shown in (e). Error bars represent sem, n=3 independent experiments. (f) Western blot analysis of IRE1α in the NP-40 soluble (S) and insoluble (P) fractions of the colon using lysis buffer containing 0.5% NP-40. The distribution of Bag6 and H2A marks the soluble and insoluble pellet fractions, respectively. (g–h) Western blot analysis of IRE1α activation in WT and IKO MEFs treated with thapsigargin (Tg) at the indicated concentration for 2 h, with quantitation of total p-IRE1α and percent of p-IRE1α shown in (h). In (g), data was derived from the same blot at the same exposure, with irrelevant lanes in the middle cut off. (i) Q-PCR analysis of mRNA levels of Xbp1s in WT and Sel1L-deficient primary adipocytes that were either mock (CON) or treated with 150 nM Tg for 3 h. n=2 mice per genotype. (j–k) Sel1L deficiency enhances inflammatory tone of enterocytes. (j) Primary crypt organoids were treated with 50 ng/ml TNFα for 20 min and analyzed for JNK phosphorylation. Quantitation of the average of n=2 mice of each genotype shown below the blot. (k) m-ICcl2enterocytes stably expressing either control (CONi) or Sel1L knockdown (Sel1Li) were treated with 50 ng/mL murine TNFα for 60 min and analyzed for Tnfa gene expression. Error bars represent sem, n=3 independent experiments. (a–e) and (k) are representative of 3 independent experiments, while others are representative of 2 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.
Figure 7
Figure 7. Sel1LΔIEC (EKO) mice are susceptible to experimental colitis
(a) Gross colon morphology of 14-wk-old WT and Sel1LΔIEC mice. Representative images of 5 mice studied. (b–j)12-wk-old mice were treated with 3% DSS for 5 days followed by fresh water. (b–c) Western blot analysis of caspase-3 or -8 cleavage in colon epithelium before or on day 7 after DSS-treatment. Quantitation is shown in (d). CON, before DSS treatment. Error bars represent sem, n=3 mice of each genotype. (e) Body weight change. Error bars represent sem from n=11 mice of each genotype pooled from two independent experiments. (f) Survival curve of DSS-treated mice with or without antibiotics (Abx). ***, p<0.001 comparing EKO to the other cohorts by the log-rank Mantel–Cox test. Combined data from n=7 (with Abx) and 13 mice (without Abx) each genotype. (g) Colon length on day 9. Error bars represent sem from n=6 WT and n=5 EKO mice. (h) Inflammatory score of colon on day 9 as a blind study. 0, none; 1, low; 2, moderate; 3, high; 4, maximal. Error bars represent sem from n=11 mice of each genotype. (i) H&E images of colon on day 9 showing epithelial ulceration (arrow heads), severe edema (asterisks), and regenerative crypts (arrows). Representative images of 5 mice studied. (j) FISH staining showing the presence of E. coli (red), rarely found in WT mice, in the colonic mucosa of DSS-treated mice on day 9. Green, non-specific signal; blue, DAPI-stained nucleus. Panels 1 and 2 show invasive E. coli in colonic mucosa and sloughed-off epithelia, respectively, of EKO mice. Representative images of 3 mice studied. n.s., not significant; **, p<0.01; ***, p<0.001 by Student’s two-tailed t test unless otherwise indicated. Unprocessed original scans of blots are shown in Supplementary Fig. 9.
Figure 8
Figure 8. IRE1α protein accumulation is critical for the pathogenesis of colitis in Sel1LΔIEC (EKO) mice
(a–c) 7-week-old littermates were treated with 3% DSS for 5 days followed by fresh water: (a) body weight change. *, p<0.05; **, p<0.01 comparing EKO to EKO;Ire1a+/− by Student’s t test. (b) Colon length on day 7. (a-b), error bars represent sem from n=6 mice of each genotype pooled from three independent experiments. (c) Representative H&E images of colon on day 7 showing epithelial ulceration (arrows), severe edema (asterisks), and regenerative crypts (arrow heads). Representative images of 6 mice studied. (d) Western blot analysis of IRE1α and Sel1L protein levels in the gut, with quantitation shown on the right (n=3 mice for EKO;Ire1a+/− and n=2 for the rest). Each lane represents an independent sample. αTubulin, a loading control. Unprocessed original scans of blots are shown in Supplementary Fig. 9. (e) Q-PCR analysis of BiP mRNA levels in the gut. Error bars represent sem, n=3 mice of each genotype. (f) Q-PCR analysis of Chop mRNA levels in isolated colon epithelium of WT and Sel1LΔIEC mice. Error bars represent sem, n=3 mice of each genotype. (g–i) The experiments and data presentation are the same as those shown above in (a-c), with the exception that EKO;Chop−/− and their control cohorts were characterized. Error bars represent sem from n=6 mice of each genotype pooled from two independent experiments. n.s., not significant. *, p<0.05; **, p<0.01; ***, p<0.001 by Student’s two-tailed t test. (j) Our model: Sel1L-Hrd1 ERAD degrades IRE1α protein, thereby restraining IRE1α activation and signaling. Subsequently, IRE1α signaling modulates inflammation and the pathogenesis of experimental colitis. The finding reported in this study is shown in red.
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