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. 2014 Feb 20;53(4):562-576.
doi: 10.1016/j.molcel.2014.01.004. Epub 2014 Feb 6.

Protein disulfide isomerase A6 controls the decay of IRE1α signaling via disulfide-dependent association

Davide Eletto #  1 , Daniela Eletto #  1 , Devin Dersh  1 , Tali Gidalevitz  2 , Yair Argon  1
Affiliations

Protein disulfide isomerase A6 controls the decay of IRE1α signaling via disulfide-dependent association

Davide Eletto et al. Mol Cell. .

Abstract

The response to endoplasmic reticulum (ER) stress relies on activation of unfolded protein response (UPR) sensors, and the outcome of the UPR depends on the duration and strength of signal. Here, we demonstrate a mechanism that attenuates the activity of the UPR sensor inositol-requiring enzyme 1α (IRE1α). A resident ER protein disulfide isomerase, PDIA6, limits the duration of IRE1α activity by direct binding to cysteine 148 in the lumenal domain of the sensor, which is oxidized when IRE1 is activated. PDIA6-deficient cells hyperrespond to ER stress with sustained autophosphorylation of IRE1α and splicing of XBP1 mRNA, resulting in exaggerated upregulation of UPR target genes and increased apoptosis. In vivo, PDIA6-deficient C. elegans exhibits constitutive UPR and fails to complete larval development, a program that normally requires the UPR. Thus, PDIA6 activity provides a mechanism that limits UPR signaling and maintains it within a physiologically appropriate range.

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Figures

Figure 1
Figure 1. Loss of PDIA6 causes an augmented unfolded protein response
(A-B) 3T3 cells expressing a non-targeting (shCtrl) or a PDIA6-targeting shRNA (clone 1) were treated with the indicated dose of TM for 6hrs. GRP94 (A) and BiP (B) mRNA levels were detected by RT-qPCR, normalized to β-actin, and their expression in shCtrl cells without stress was set at 1. Plots are means±SD, n=3. Significant differences between shPDIA6 and shCtrl conditions are indicated by asterisks. (*, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001 (student t test)). (C) shCtrl or shPDIA6 3T3 cells were exposed to the indicated dose of TG for 6hrs and labeled with [35S]Met/Cys for 30 min. Relative GRP94 synthesis was determined by immunoprecipitation and normalized to untreated shCtrl cells. (D) shCtrl or shPDIA6 (clone 1) 3T3 cells were treated with varying concentrations of TM for 16 hrs and levels of GRP94, BiP or PDIA6 were determined with αKDEL antibody. 14-3-3 was used as a loading control. *, an unknown KDEL-positive protein whose expression is unchanged. (E) Quantitation of GRP94 and BiP levels from panel D. Values are means±SD relative to DMSO-treated shCtrl cells (*, p ≤ 0.05; n=3). (F-G) shCtrl or shPDIA1 3T3 cells were treated with TM and analysed as in panels D-E. (H-J) Akita-INS cells were transduced with shCtrl or shPDIA6 (clone 1) lentiviruses and Akita insulin expression was induced with 2μg/mL doxycycline (Dox). The relative levels of indicated proteins were measured by immunoblotting and normalized to the loading control, 14-3-3 (numbers below bands). The induction of GRP94 (I) or OS9.1/OS9.2 (J) was quantified as in panels D-E. See also Figs. S1-S2.
Figure 2
Figure 2. Depletion of PDIA6 leads to slower growth and hyper-sensitivity to ER stress
(A) HeLa cells, stably expressing shPDIA6 (clone 3) or shCtrl were plated at 5×103 cells/35mm dish, treated with 50nM TG for the indicated time, then returned to growth medium for 6 days. Plates were assessed for colony formation by staining with crystal violet. HeLa cells were used because they form distinct colonies. (B) Quantitation of crystal violet-stained colonies from HeLa cells expressing shCtrl, shPDIA6 and a shRNA clone targeting BiP (shBiP). Plates from experiments such as in A were lysed in 2% w/v SDS and absorbance at 570 nm was measured. Values are means±SD (*, p ≤ 0.05; n=4). (C) ShCtrl or shPDIA6 (clone 3) 293T cells were selected for 5 days with 2μg/mL puromycin, and then transfected with either an empty vector (EV) or a plasmid expressing V5-tagged PDIA6 (PDIA6-V5). The proliferation rates were determined by an XTT assay. (D) Immunoblots of protein extracts from the 24h time point in (C), to measure PDIA6 expression (both endogenous and exogenous). See also Fig. S2.
Figure 3
Figure 3. PDIA6 affects the decay of IRE1 signaling
(A-B) shCtrl or shPDIA6 3T3 cells were incubated with 200 ng/mL TM for the indicated times and unspliced (u) and spliced (s) XBP1 mRNA was amplified by RT-PCR. β-actin served as control for RNA recovery. Means±SD are plotted in B (*, p ≤ 0.05; n=3). (C-D) shCtrl or shPDIA6 3T3 cells were pulsed with 100nM TG for 4hrs, then “chased” for the indicated times in medium without TG. XBP1 splicing was assayed as in A. Separate experiments were quantified in (D). (*, p ≤ 0.05; n=3). (E-F) 3T3 cells expressing shCtrl or shPDIA1 were subjected to continuous TM stress and analysed as in A. Percentage of XBP1 splicing is plotted in (F). (G) ShCtrl or shPDIA3 3T3 cells were first exposed to TM, then chased in fresh growth medium for the indicated times. Total RNA was analysed for XBP1 splicing as in A. (H) Protein extracts from shCtrl, shBiP or shPDIA3 cells were immunoblotted to determine the level of expression of PDIA3 and PDIA6. 14-3-3 served as loading control. Note that: 1) PDIA3 depletion is not accompanied by induction of PDIA6; 2) both PDIA3 and PDIA6 are over-expressed in BiP-depleted cells, presumably via general UPR. (I) shCtrl or shPDIA6 3T3 cells were pre-treated (or left untreated) with the Bcl-2 inhibitor ABT-737 (10μM for 30 min). Cells were then pulsed with 50μg/mL TM for 2hrs and chased for the indicated times in fresh medium. XBP1 splicing was assayed as in A. Under such modest ER stress, XBP1 splicing in PDIA6-sufficient cells is minimal and the difference with PDIA6-deficient cells is more dramatic (*, p ≤ 0.05; n=3). (J) ShCtrl or shPDIA6 (clone 3) 293T cells were transfected with the indicated V5-tagged PDIA6 constructs (EV, empty vector; WT, wild-type PDIA6; Trap, C58A-C193A PDIA6; Cys-less, C55A-C58A-C190A-C193A PDIA6). Cells were incubated with 100 nM TG for 2hrs and then “chased” in fresh medium for 4 or 8 hrs. Phosphorylated IRE1 was resolved by Phos-tag gels. Blots with α-PDIA6 are given below each Phos-tag panel, to show the relative levels of endogenous and exogenous PDIA6. Numbers under the panels, percentages of phosphorylated IRE1 as determined by densitometry. This calculation is conservative, given the variability of the unphosphorylated band across the samples. See also Figs. S3-S4.
Figure 4
Figure 4. PDIA6 and IRE1 interact directly through mixed disulfide bonds
(A) HA-tagged IRE1 was expressed in 293T cells from either weak (tyrosine kinase, Tk-basal) or strong (cytomegalovirus enhancer chicken β-actin, Cax) promoter. PDIA6, BiP, and PDIA1 co-precipitating with IRE1-HA were detected by immunoblotting. 14-3-3 served as a control for non-specific interactions with IRE1. h.c., heavy chain of the αHA antibody. (B) 293T cells expressing HA-tagged IRE1 (Tk-basal promoter) were either not untreated or acutely exposed to 2 μg/mL TM for 3hrs. The indicated samples were then chased for 16hrs in fresh medium. Lysates were analysed as in A. (C) 293T cells were either not transfected (−) or transfected with WT, a trapping (Trap) or Cys-less versions of V5-tagged PDIA6 cDNA. IRE1 and BiP co-precipitated with PDIA6-V5 were detected by immunoblotting. (D) Cell lysates from panel C were analysed by non-reducing gels, followed by dual-color immunoblots with αIRE (green) and αV5 (red) antibodies to detect mixed disulfide between IRE1 and PDIA6. The yellow signal depicts the co-migrated IRE1-PDIA6 bands. 14-3-3 (green) served as a loading control. (E) The trapping PDIA6-V5 mutant was expressed in 293T cells alongside the indicated IRE1 mutants. Cell lysates were analysed as in (C). (F) Cell lysates, generated as in (E), were resolved on a non-reducing gel and immunoblotted to detect IRE1-PDIA6-V5 mixed disulfides. The arrows point at the same co-migrating bands. See Fig. S4B for a color version of the same blot. (G) WT or C148S IRE-HA were expressed in 293T cells with WT PDIA6-V5 and immunoprecipitated with a αHA antibody. IRE1-bound PDIA6 was detected by immunoblotting. See also Fig. S4.
Figure 5
Figure 5. The reactivity of Cys148 is important for the activation kinetics of IRE1
(A) IRE1 knockout (KO) MEFs stably complemented with WT or C148S IRE1-HA were treated with 300nM TG for 2hrs. RNA samples were then assayed for XBP1 splicing. Percentage of splicing is indicated in each lane. TG-treated 3T3 served as positive controls. EV, empty cDNA vector. ND, not detectable. (B) Cell lysates from the same samples used in (A) were immunoblotted to determine the level of expression of the rescuing IRE1-HA. 3T3 lysates served as reference for endogenous IRE1 expression. Note that the expression of each rescuing protein as well as the endogenous IRE1 are similar. *, unknown product of the expression plasmid, presumably a cleaved IRE protein. (C) Extent of XBP1 splicing in IRE1-HA-rescued IRE1 KO MEFs was determined upon persistent exposure to 300nM TG for the indicated times. β-actin served as loading control. (D) Means±SD of experiments as in (C) are plotted (*, p ≤ 0.05; n=3). (E) A C148-dependent disulfide forms during activation of IRE1. 293T cells were transfected with either empty vector (EV) or with the indicated IRE1-HA cDNAs (under control of the pCAX promoter). Cells were treated with 100nM TG for 2hrs, followed by 16hr chase in fresh medium where indicated. Reactive cysteines were alkylated with NEM prior to cell lysis and endogenous or HA-tagged exogenous IRE1 were detected by non-reducing gel. α-tubulin (which does not contain reactive cysteines) was used as control for the electrophoretic shift. (F) 293T cells were transiently transfected with full length IRE1 or with FLAG-tagged IRE1 luminal domain (NLD) that was either WT, C148S or lacked all three cysteines (C109/148/332S). Expression of all NLDs was limited to the ER with a C-terminal KDEL peptide. Protein extracts were resolved by non-reducing PAGE and analysed by immunoblot. EV, empty vector transfection. The predicted sizes of monomeric NLD or multimeric complexes are indicated at the sides of the blots. (G) shCtrl and shPDIA6 293T cells were transfected with WT IRE1 NLD or mock transfected. Protein extracts were analysed as in F. Co-expressed PDIA6-V5 complemented the silenced enzyme in shPDIA6 cells in reducing multimer formation. See also Fig. S5.
Figure 6
Figure 6. PDIA6 also affects PERK signaling but not ATF6 activity
(A) Extracts of 293T cells expressing the indicated versions of PDIA6-V5 (WT, client-trapping (Trap) or Cys-less mutants), along with a Myc-tagged PERK, were immunoprecipitated with αV5 antibody and blotted to detect co-precipitated Myc-tagged PERK. 14-3-3 served as specificity control. (B) shCtrl or shPDIA6 3T3 cells were exposed to 1μM TG for the indicated times. Cell lysates were then immunoblotted to detect total or Ser-51-phosphorylated eIF2α. Means±SD of the phospho-signal/total eIF2α ratios are plotted (*, p≤0.05; n=3). Untreated shCtrl samples served as internal reference. (C) 293T cells stably expressing HA-tagged ATF6 were infected with lentivirus containing shCtrl or shPDIA6 (clone 3). After exposure to TM for the indicated times, nuclear fractions were isolated as in (Li et al., 2000) and immunoblotted to detect the active, nuclear fragment of ATF6. LaminA/C and 14-3-3 were used as nuclear or cytosolic markers, respectively. Cells were pretreated with 1μM MG-132 to prevent proteasomal degradation of nuclear ATF6. F.L, full length ATF6. (D) The 5X ATF6 site luciferase reporter was expressed in shCtrl and shPDIA6 3T3 cells, which were then treated with the indicated doses of TM for 18hrs. Means±SD of relative Firefly luciferase activity, normalized to Renilla activity, are shown (n=4). (E) IRE1α+/+ and IRE1α−/− MEFs stably expressing shCtrl or shPDIA6 were transiently transfected with a luciferase reporter driven by a minimal BiP promoter, exposed to TM and analysed as in (D) (*, p ≤ 0.05). (F) Proposed model for the attenuation of IRE1 by PDIA6. The luminal domain of inactive IRE1 is occupied by BiP. Some fraction of IRE1 is independently bound reversibly by PDIA6. Upon ER stress BiP dissociates, IRE1 dimerizes, oligomerizes and becomes an active kinase and a ribonuclease, splicing XBP-1. We propose that during the activation sequence, IRE1 becomes disulfide-bonded via its luminal Cys148. This makes activated IRE1 a target for PDIA6, which then reduces the disulfide and converts oligomeric IRE1 back to the monomeric form, which is available for another cycle of activation. PDIA6 is also known to bind BiP, but this interaction is Cys-independent. See also Fig. S6.
Figure 7
Figure 7. The C. elegans PDIA6 homologue is an essential gene whose silencing induces UPR
(A) Sequence alignment of murine PDIA6 and C. elegans tag-320, using Lalign v.2.1.30. The CXXC motifs and their flanking residues are highlighted. Symbols under the alignment indicate identity/conservation of residues. (B) Phylogenetic analysis (Phylogeny.fr) of C. elegans PDI-1, -2, -3 and the PDIA6 homologue, and mouse PDIA1 and PDIA6. Line lengths and numbers indicate relative sequence relatedness. (C) Comparison of heterozygous pdi-6/Q40::YFP and pdi-6/pdi-6 homozygous larva at an L2 stage. Ph, pharynx. G, gonadal precursor. The pdi-6/pdi-6 homozygous larva is at the same age post-hatching as the heterozygous larva, without apparent morphological defects. Bars, 50μ. (D) Developmental arrest of pdi-6 homozygous larvae. Eggs laid by pdi-6/Q40::YFP adults were allowed to develop for 3 days and the number of L4 or older animals of each genotype was scored. The values are percentages of the starting numbers of embryos, means±SD (n=4). No pdi-6/pdi-6 animals developed to L4 stage. (E) Induction of a UPR reporter in larvae and adult animals by RNAi. YAR72 worms harboring the phsp-4::GFP reporter (zcIs4) on an rrf-3 (ok1426) RNAi-sensitized background were exposed from eggs to pdi-1, -2 or -6 RNAi and representative animals were imaged. Note reporter induction in worms depleted of pdi-6 and the distinct cellular pattern (intestine and pharynx for pdi-6 vs hypodermis for pdi-2 in L1 animals). The exposure of the adult pdi-2 panel was adjusted to show the individual vulval cells. Ph, pharynx. Sp, spermatheca. Hy, hypodermis. Int, intestine. (F) Fecundity of animals subjected to RNAi of the indicated genes. YAR72 animals (as in E) were grown on the indicated RNAi bacteria for 1-2 generations, singled and their progeny enumerated. L4440, empty vector control; L4440 ind, control animals with spontaneously induced reporter. Note the correspondence between low brood size on pdi-2, pdi-6 RNAi plates and in spontaneously induced animals and the induction of the UPR reporter. RNAi of pdi-1 does not induce this reporter significantly. Data are means±SD (n >7 individual animals per genotype). See also Fig. S7.
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