Article

Phosphorylation switches protein disulfide isomerase activity to maintain proteostasis and attenuate ER stress

Jiaojiao Yu1,2,†, Tao Li1,2,†, Yu Liu3, Xi Wang1, Jianchao Zhang1,2 ,Xi’e Wang1, Guizhi Shi4, Jizhong Lou2,5, Likun Wang1,2, Chih-chen Wang1,2 & Lei Wang1,2,*

Abstract Introduction

Accumulated unfolded proteins in the endoplasmic reticulum (ER) The endoplasmic reticulum (ER) is the main cellular organelle for trigger the unfolded protein response (UPR) to increase ER protein protein folding and secretion, calcium storage, and lipid synthesis. folding capacity. ER proteostasis and UPR signaling need to be regu- Numerous folding enzymes and molecular chaperones in the ER lated in a precise and timely manner. Here, we identify phosphory- guide the secretion of properly folded proteins while retaining lation of protein disulfide isomerase (PDI), one of the most misfolded proteins or targeting their degradation (Sitia & Braakman, abundant and critical folding catalysts in the ER, as an early event 2003). An accumulation of unfolded/misfolded proteins or alterna- during ER stress. The secretory pathway kinase Fam20C phosphory- tion of redox and Ca2+ states in the ER results in imbalanced lates Ser357 of PDI and responds rapidly to various ER stressors. proteostasis and ER stress (Balch et al, 2008; Walter & Ron, 2011; Phosphorylation of Ser357 induces an open conformation of PDI and Karagoz et al, 2019; Ushioda & Nagata, 2019). Chronic ER stress turns it from a “foldase” into a “holdase”, which is critical for plays a central role in various human pathologies, including cancer, preventing protein misfolding in the ER. Phosphorylated PDI also diabetes, cardiovascular diseases, and neurodegenerative diseases binds to the lumenal domain of IRE1a, a major UPR signal trans- (Oakes & Papa, 2015; Wang & Kaufman, 2016). ducer, and attenuates excessive IRE1a activity. Importantly, PDI- Endoplasmic reticulum proteostasis is governed by a dynamic S359A knock-in mice display enhanced IRE1a activation and liver signaling network, the unfolded protein response (UPR), which damage under acute ER stress. We conclude that the Fam20C-PDI consists of three distinct arms in mammals defined by ER transmem- axis constitutes a post-translational response to maintain ER brane sensors—IRE1a, PERK, and ATF6 (Walter & Ron, 2011). The proteostasis and plays a vital role in protecting against ER stress- output of UPR signaling is to induce the expression of genes involved induced cell death. in ER protein quality control and/or translational repression of global protein synthesis to counteract proteostatic perturbations in Keywords endoplasmic reticulum; Fam20C; IRE1a; phosphorylation; protein the ER. The IRE1a branch is the most conserved arm of the UPR. ER disulfide isomerase stress induces the dimerization/oligomerization of IRE1a and its Subject Categories Membranes & Trafficking; Post-translational Modifi- autophosphorylation, which leads to XBP1 mRNA splicing and cations & Proteolysis; Translation & Protein Quality generates the functional spliced XBP1 (XBP1s) transcription factor DOI 10.15252/embj.2019103841 | Received 28 October 2019 | Revised 7 regulating multifarious targets (Yoshida et al, 2001; Calfon et al, February 2020 | Accepted 11 February 2020 | Published online 9 March 2020 2002; Lee et al, 2003; Shoulders et al, 2013). Nevertheless, hyperac- The EMBO Journal (2020) 39:e103841 tivation of IRE1a under prolonged ER stress results in the transition from an adaptive UPR to a terminal proapoptotic program by surpass- See also: JPL Coelho & MJ Feige (May 2020) ing the oligomerization threshold that expands the RNase substrate repertoire to many other mRNAs or precursors of apoptosis-inhibitory microRNAs (Han et al, 2009; Hollien et al, 2009; Upton et al, 2012; Ghosh et al, 2014). Moreover, IRE1a can trigger cellular apoptosis through the TRAF2-ASK1-JNK pathway (Urano et al, 2000; Nishitoh

1 National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China 2 College of Life Sciences, University of Chinese Academy of Sciences, Beijing, China 3 CAS Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China 4 Laboratory Animal Center of Institute of Biophysics, Chinese Academy of Sciences, Aviation General Hospital of Beijing, University of Chinese Academy of Sciences, Beijing,China 5 Key Laboratory of RNA Biology, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China *Corresponding author. Tel: +86 10 64888501; E-mail: [email protected] †These authors contributed equally to this work

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et al,2002).Therefore,IRE1a functions as a central adjustor of cell a higher amplitude of XBP1 mRNA splicing (Figs 1B and EV1A). As fate under ER stress, and its activity is precisely and timely regulated the duration of splicing was similar (Fig EV1A), Fam20C may partici- by multiple regulatory elements (Hetz et al,2006;Lisbonaet al, 2009; pate in the early response to ER stress. Ectopic expression of Fam20C Rodriguez et al, 2012; Eletto et al, 2014; Sepulveda et al, 2018). wild-type (WT) decreased the XBP1 mRNA splicing level in Tg- However, the inherent latency of the UPR limits its responsiveness treated HepG2 cells compared with its inactive D478A mutant (DA) to the fluctuation of protein status in the ER, particularly in dedicated (Fig 1C). Similar results were observed when HepG2 cells were secretory cells with heavy protein folding burden. The post-transla- treated with another ER stress inducer tunicamycin (Tm), which tional regulation of BiP, a key chaperone in the ER, emerges as a inhibits protein N-glycosylation (Fig 1D and E). Again, we showed rapid and an economic way to regulate protein folding capacity that Fam20C can also negatively regulate IRE1a activity in HeLa cells during the early stage of ER stress (Preissler & Ron, 2018). However, by using Fam20C knockout (KO) cells generated by CRISPR/Cas9 whether post-translational regulation of other preexisting compo- technology previously reported (Zhang et al, 2018; Fig EV1B and C). nents contributes to the early response to ER stress remains unclear. To explore the mechanism by which Fam20C controls IRE1a The reversible phosphorylation of proteins is central to the regula- signaling, we set to identify the Fam20C interactome by co-immuno- tion of most aspects of cell function. Fam20C, a secretory pathway precipitation (co-IP) and mass spectrum (MS) analysis (Fig EV1D protein kinase, which recognizes S-x-E/pS motifs within the substrate and E). Three independent experiments led to the identification of a proteins, generates the majority of the secreted phosphoproteome total of 173 ER and Golgi proteins, which were based on DAVID GO (Tagliabracci et al, 2012, 2015; Cui et al, 2015). Functional mutations term analysis (Dataset EV1 and Fig EV1F). Among these Fam20C- in Fam20C cause a rare and often lethal osteosclerotic bone dysplasia interacting proteins, five proteins, including PDI (P4HB), P4HA1, called Raine syndrome (Raine et al, 1989). Recently, the critical roles P4HA2, TRIM68, and FKBP9, showed statistically significant of Fam20C in fine-tuning ER redox homeostasis (Zhang et al, 2018) increased binding with Fam20C after Tg treatment, whereas two and Ca2+ homeostasis (Pollak et al, 2018) have been revealed. proteins, SERCA2 and ERGIC2, showed statistically significant However, it is still an open question whether Fam20C is involved in decreased binding (Fig 1F). The increased binding between PDI and ER proteostasis regulation and UPR signaling under ER stress. Fam20C under ER stress was further confirmed by co-IP and In this study, unbiased proteomics analysis allows the identifi- immunoblotting (Fig 1G). We thereafter focused on PDI because it is cation of protein disulfide isomerase (PDI) as a substrate of Fam20C the most enriched protein among the Fam20C interactome under ER kinase under ER stress. PDI, also known as collagen prolyl 4-hydro- stress and is critical for protein folding and quality control in the ER. xylase subunit b (P4HB), is one of the most abundant enzymes in the ER. PDI is a versatile protein acting as both a thiol-disulfide PDI Ser357 is a genuine phosphosite of Fam20C oxidoreductase and a molecular chaperone (Hatahet & Ruddock, 2009; Wang et al, 2015). We show that Fam20C phosphorylates To determine whether PDI is phosphorylated by Fam20C during ER serine 357 (Ser357) of PDI and responds rapidly to various ER stres- stress, HepG2 cells were treated with Tg, and endogenous PDI was sors. Importantly, phosphorylation of Ser357 in the x-linker region immunoprecipitated and subjected to MS/MS analysis (Fig EV2A). induces an open conformation of PDI and turns it from a “foldase” to Three phosphorylation sites (Ser357, Ser331, and Ser427) were iden- a “holdase”, which is critical for preventing protein misfolding in the tified upon Tg treatment (Figs 2A and EV4D and E). Since phospho- ER. Furthermore, we demonstrate that phosphorylated PDI directly rylation of Ser331 and Ser427 had little effect on the conformation interacts with IRE1a to attenuate its signaling amplitude, which is and activity of PDI (see below), in this study, we focused on the critical for protecting against ER stress-induced liver damage, as phosphorylation effect of PDI Ser357. illustrated in a mouse model. Overall, our results unravel Fam20C- Protein disulfide isomerase comprises four thioredoxin (Trx) induced PDI phosphorylation as a rapid post-translational mecha- domains: two catalytic domains a and a’, both containing a CGHC nism for integrating ER proteostasis sensing and cell fate control. active site, separated by two homologous noncatalytic domains, b and b’, and a C-terminal tail. In addition, there is a short linker region “x” between the b’ and a’ domains. Ser357 is located in the x-linker of PDI, Results is well conserved from yeast to human in eukaryotes, and lies within a Fam20C S-x-E/pS recognition motif (Fig 2B). Using a generated anti- Fam20C depletion sensitizes the IRE1a branch of the UPR under body specifically recognizing phosphorylated Ser357 of PDI (pS357- ER stress PDI) (Fig EV2B), in vitro kinase assays showed that recombinant PDI was phosphorylated by purified Fam20C protein in a time-dependent To assess the role of Fam20C in the UPR, the activation of three UPR manner (Fig 2C). Phosphorylation of PDI WT by Fam20C resulted in a sensors, IRE1a, PERK, and ATF6, was evaluated in control (shCtrl) band migrating slightly slower on a regular gel, and this phosphoryla- and Fam20C stable knockdown (shFAM20C) HepG2 cells. When ER tion was confirmed to be pS357-PDI using dual-color immunoblotting stress was induced by the sarco/endoplasmic reticulum Ca2+- (Fig 2D). Notably, p-PDI migrated drastically slower on a phostag gel, ATPase (SERCA) calcium pump inhibitor thapsigargin (Tg), deple- which can be recognized by anti-pS357-PDI antibody. Fam20C also tion of Fam20C significantly elevated the phosphorylation of IRE1a caused the slow migration of PDI S357A mutant on both the regular but had little effect on the phosphorylation of PERK and its down- and phostag gels and was consistent with MS results showing that PDI stream substrate eIF2a or the nuclear localization of the N-terminal can be phosphorylated at other sites (Fig 2C). Interestingly, it seems cleavage product of ATF6 (Fig 1A). These results indicated that that S357A is a better substrate for Fam20C than the WT PDI, and the Fam20C could modulate the IRE1a branch of the UPR under ER lack of intermediate forms on the phostag gel suggests that the phos- stress. Consistent with this notion, Fam20C-depleted cells displayed phorylation of PDI’s multiple sites is cooperative.

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Figure 1.Fam20C depletion sensitizes the IRE1a branch of the UPR under ER stress. A Detection of the activation of IRE1a, PERK, and ATF6 UPR branches by immunoblotting in control (shCtrl) and Fam20C stable knockdown (shFAM20C) HepG2 cells treated with or without 5 lM Tg for 30 min. ATF6-FL: full-length ATF6; ATF6-N: N-terminal cleavage product of ATF6; Asterisk: unspecific background bands. Fam20C knockdown was verified by protein immunoblotting of Concanavalin A-Sepharose (Con A)-enriched culture medium. Ponceau staining was shown as a loading control. B(Left) Detection of spliced XBP1 (S) and unspliced XBP1 (U) mRNA in shCtrl and shFAM20C HepG2 cells treated with or without 5 lM Tg for 1 h. (Right) Quantification of XBP1 mRNA splicing levels. C(Left) Detection of XBP1 mRNA splicing in shFAM20C HepG2 cells expressing RNAi-resistant codon-altered Fam20C wild-type (WT) or its inactive D478A mutant (DA) treated with or without 5 lM Tg for 1 h. Fam20C expression levels were shown by protein immunoblotting. (Right) Quantification of XBP1 mRNA splicing levels. D, E (Left) Detection of XBP1 mRNA splicing in shCtrl and shFAM20C HepG2 cells (D) or shFAM20C HepG2 cells expressing Fam20C WT or DA (E) treated with or without 2 lg/ml Tm for 8 h. (Right) Quantification of XBP1 mRNA splicing levels. F Volcano plot depicting the log2 of fold change versus – log10 (P-value) for Fam20C-interacting proteins in Tg-treated HepG2 cells. The red dots represent proteins showing statistically significant increase of binding with Fam20C (log2 fold change ≥ 1; log10 (P-value) ≥ 1.3); the blue dots represent proteins showing statistically significant decrease of binding with Fam20C (log2 fold change ≤ 1; log10 (P-value) ≥ 1.3); the green dot is Fam20C. G HepG2 cells expressing FLAG-tagged Fam20C were treated with or without 5 lM Tg for 30 min. FLAG immunoprecipitates were analyzed by PDI immunoblotting. Data information: In (B–E), data were shown as mean SEM of three independent experiments. *P < 0.05, **P < 0.01 (two-tailed Student’s t-test). Source data are available online for this figure.

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The pS357-PDI signal was detected in untreated HepG2 cells and HepG2 cells (Fig EV2D–F), the pS357-PDI signal diminished and was was remarkably weakened when treated with k-protein phosphatase restored by expressing HA-tagged PDI WT but not PDI S357A (Fig EV2C). Moreover, in two CRISPR/Cas9-edited clones of PDI KO (Fig EV2G). By using two clones of Fam20C KO HeLa cells

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◀ Figure 2. PDI Ser357 is phosphorylated by Fam20C during ER stress. A Representative MS/MS fragmentation spectrum of tryptic phosphorylated PDI peptide (Ile351-Lys370) depicting PDI Ser357 phosphorylation enriched from HepG2 cells treated with 5 lM Tg for 10 min. B(Upper) Domain organization of the human PDI molecule. The residue numbering is for human PDI with signal sequence. The “CGHC” motifs in the a and a’ domains are active sites; carboxyl-terminal KDEL motif is the ER retention sequence. Ser357 located in the x-linker region is marked in red. (Lower) Amino acid sequence alignments of PDI x-linker in several species. Residue positions are indicated by numbering with signal sequences. Human PDI Ser357 and its counterparts are shown in red. C Time-dependent incorporation of phosphate group into PDI WT or S357A catalyzed by recombinant Fam20C. Phosphorylated PDI was determined by anti-pS357-PDI immunoblotting on both regular and phostag gels. D Phosphorylated PDI catalyzed by Fam20C was analyzed by dual-color immunoblotting with anti-PDI (red) and anti-pS357-PDI (green) antibodies. The yellow signal depicts the phosphorylated PDI species. E–G Detection of pS357-PDI in HepG2 cells treated with 5 lM Tg (E), 2.5 lg/ml Tm (F), or 1 mM DTT (G) for indicated times. H, I (Left) Detection of p-PDI in WT and PDI KO HepG2 cells (H) or in WT and Fam20C KO HeLa cells (I) treated with 5 lM Tg for indicated times by protein immunoblotting on both regular and phostag gels. (Right) Quantification of p-PDI percentage based on band intensities on phostag gels. Data information: Data were shown as mean SEM of three (H) or four (I) independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 (one-way ANOVA, the post hoc Tukey’s HSD test). Source data are available online for this figure.

(Fig EV2H), we showed that phosphorylation of PDI Ser357 was lumen or the extracellular space (Tagliabracci et al, 2013; Zhang strictly dependent on Fam20C kinase activity (Fig EV2I). Convinc- et al, 2018). To decipher the mechanism of Fam20C-catalyzed phos- ingly, Fam20C is the bona fide kinase catalyzing PDI Ser357 phos- phorylation of PDI, we constructed PDI-mEmerald-KDEL and phorylation. Fam20C-mApple to detect the spatiotemporal interaction between PDI and Fam20C. As expected, in unstressed cells, PDI displayed ER Phosphorylation of PDI Ser357 is induced by ER stress localization, and Fam20C colocalized with the cis-Golgi resident protein GM130. Interestingly, after a Tg treatment of 30 min, the ER Next, we used different ER stress inducers to investigate the correla- distribution was observed for a significant portion of Fam20C, tion between ER stress and the phosphorylation of PDI. Tg, Tm, and which showed colocalization with PDI (Pearson correlation coeffi- dithiothreitol (DTT, a reducing reagent that reduces protein disulfide cient = 0.51 0.02; Fig 3A and B). Importantly, most of the bonds) induced PDI phosphorylation in a dose-dependent manner Fam20C was relocated from the ER to the Golgi at 60 min after Tg (Fig EV3A–C). Notably, PDI phosphorylation was triggered by phar- washout (Fig 3A) and was accompanied by the disappearance of the macological ER stress within minutes, while the total amount of PDI pS357-PDI signal (Fig 3C). Similarly, the pS357-PDI signal disap- protein did not change (Fig 2E–G). The rapid phosphorylation of peared as quickly as 30 min after the washout of CPA, a reversible PDI under ER stress preceded the upregulation of the BiP chaperone, SERCA inhibitor, suggesting that PDI is highly sensitive to SERCA the canonical UPR target, suggesting that PDI phosphorylation is an activity (Fig EV3G). Thus, it seems that Fam20C could sense ER early response to ER stress. Similarly, Brefeldin A (BFA, an inhibitor stress and be retained in the ER to phosphorylate PDI. To further of ER-Golgi traffic) and MG132 (an inhibitor of protein degradation), test this concept, we pretreated HepG2 cells with cycloheximide which also trigger ER stress, can induce PDI phosphorylation (CHX, an inhibitor of protein synthesis) to reduce the protein folding (Fig EV3D and E). We also used the reversible SERCA inhibitor load in the ER and then introduced Tg. Tg-induced pS357-PDI levels cyclopiazonic acid (CPA) and showed that CPA induces PDI Ser357 gradually decreased with the extension of CHX treatment (Fig 3D). phosphorylation in a dose-dependent manner (Fig EV3F). Simultaneously, Fam20C no longer showed ER distribution in CHX- The Tg-induced pS357-PDI signal was detected neither in PDI KO pretreated cells upon Tg introduction (Fig 3E). nor in Fam20C KO cells by immunoblotting, further indicating that To provide further evidence that Fam20C can catalyze PDI phos- phosphorylation of PDI under ER stress conditions was specifically phorylation in the ER lumen, we engineered a KDEL ER retrieval dependent on Fam20C kinase (Fig 2H and I). PDI phosphorylation sequence to the C-terminus of Fam20C-mApple, as this sequence stoichiometry was quantified by phostag gels, which showed that can be sequestrated by the cis-Golgi-anchored KDEL receptor. more than half of the total PDI had been phosphorylated after Tg Fam20C-mApple-KDEL displayed perfect colocalization with PDI- treatment for 30 min, either in HepG2 cells or in HeLa cells (Fig 2H mEmerald-KDEL in the ER at basal state (Fig 3F). pS357-PDI and I). Considering that PDI is one of the most abundant folding immunoblotting showed that Fam20C-KDEL was more efficient at catalysts in the ER lumen, the high stoichiometry of PDI phosphory- catalyzing PDI phosphorylation than Fam20C WT (Fig 3G, compare lation suggests that this post-translational modification could be lane 5 to lane 3). Interestingly, Fam20C-mApple-KDEL formed gran- functionally important to regulate ER stress. ular structures 30 min after Tg treatment, and at least some of the PDI proteins were recruited into these condensates (Fig 3F). Accord- Phosphorylation of PDI depends on the retention of Fam20Cin ingly, the phosphorylation level of PDI was largely increased after the ER Tg treatment in cells expressing Fam20C-KDEL (Fig 3G, compare lane 6 to lane 4 and lane 2), supporting the notion that the Fam20C Approximately 90% of Fam20C is secreted to the extracellular and PDI-containing condensates formed during ER stress facilitate space, and the intracellular protein is mainly localized in the cis- the phosphorylation process. Altogether, these results suggested that Golgi apparatus (Tagliabracci et al, 2012). Therefore, Fam20C-cata- during ER stress, Fam20C is retained in the ER to catalyze efficient lyzed protein phosphorylation is believed to occur in the Golgi phosphorylation of PDI.

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Phosphorylation of PDI induces its functional switch from an simulation using the previously solved crystal structure of PDI (PDB oxidoreductase to a molecular chaperone code 4EL1). After the introduction of phosphorylated Ser357, PDI underwent a significant domain rearrangement and showed a more To address how phosphorylated Ser357 of PDI regulates the confor- open conformation (Movie EV1). When the last snapshot of the mational change, we performed molecular dynamics (MD) simulation was superimposed with the crystal structure based on

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◀ Figure 3. Phosphorylation of PDI depends on the retention of Fam20C in the ER. A Fluorescent photomicrographs of HepG2 cells co-transfected with PDI-mEmerald-KDEL (green) and Fam20C-mApple (red). The cells were treated with DMSO (Upper) or 5 lMTg(Middle) for 30 min. After Tg treatment, the cells were washed with PBS and then returned to culture medium for 60 min (Lower). GM130 was immunostained (blue) as a cis-Golgi marker. Scale bar = 10 lm. B Pearson correlation coefficient calculated from (A). Data were shown as mean SEM of N > 80 cells from four independent experiments. ***P < 0.001 (one-way ANOVA, the post hoc Tukey’s HSD test). C Detection of pS357-PDI and UPR signaling in HepG2 cells after Tg treatment and washout for indicated times. D HepG2 cells were treated with 25 ng/ll CHX for indicated times and analyzed by immunoblotting for pS357-PDI and UPR signaling. 5 lM Tg or DMSO was added 30 min before cell harvest. E Fluorescent photomicrographs of HepG2 cells co-transfected with PDI-mEmerald-KDEL (green) and Fam20C-mApple (red) pretreated with 25 ng/ll CHX for 7.5 h, followed by introduction of DMSO (Upper)or5 lMTg(Lower) for 30 min, respectively. GM130 was immunostained (blue) as a cis-Golgi marker. Scale bar = 10 lm. F Fluorescent photomicrographs of HepG2 cells co-transfected with PDI-mEmerald-KDEL (green) and Fam20C-mApple-KDEL (red) treated with DMSO (Upper)or5 lM Tg (Lower) for 30 min, respectively. Scale bar = 10 lm. G HepG2 cells were transfected with empty vector (–), FLAG-tagged Fam20C, or Fam20C-KDEL followed by introduction without or with 5 lM Tg for 30 min, respectively. PDI phosphorylation was detected by protein immunoblotting on both regular and phostag gels. p-PDI percentage was quantified based on band intensities on phostag gels. Source data are available online for this figure.

the bb’ domain, the a’ domain rotated around the x-linker outward We next investigated the effect of phosphorylation on PDI toward the other three thioredoxin domains, making p-PDI an activities. PDI S357E showed significantly higher potency than PDI extended “L”-shaped molecule compared with the previous “U”- WT in suppressing the aggregation of denatured GAPDH and shaped structure (Fig 4A). Time-resolved analysis showed that the rhodanese upon dilution (Fig 4G and H), in line with the previous distance between domains a and a’ increased to ~70 A˚ from the observation that open forms of PDI display a higher chaperone original ~50 A˚ , and the angle among domains b, b’, and a’ markedly activity (Wang et al, 2012). We also prepared genuine-phosphory- increased to ~170° from the original ~140° (Fig 4B, red). The open lated PDI by using the Fam20C kinase assay and observed that conformation of p-PDI is distinct from the previous PDI simulations PDI WT, but not the S357A mutant, exhibited dramatically with compact conformations, which most likely represent the intrin- increased chaperone activity as the phosphorylation level sic stable substrate-free state (Fig 4B, black; Yang et al, 2014). increased (Fig 4I and J). To our surprise, PDI S357E displayed a Therefore, this open form of p-PDI may have an important func- remarkable decrease in both reductase and isomerase activities tional influence. compared to PDI WT as measured by insulin reduction assay There is a continuous hydrophobic surface on the inner side of (Fig 4K) and scrambled RNase A reactivation assay (Fig 4L). the structure of PDI, which can be probed by 1-anilino-8-naphtha- Consistently, Fam20C-catalyzed phosphorylation largely lene sulfonate (ANS; Wang et al, 2010). The phosphorylation mimic suppressed the reductase (Fig 4M) and isomerase (Fig 4N) activi- PDI S357E showed much higher ANS fluorescence intensity ties of PDI WT but not PDI S357A. We also examined the effects compared to PDI WT, indicating that a more hydrophobic surface on PDI activities of the other two phosphosites identified by MS, was exposed (Fig 4C). In a limited proteolysis assay, full-length PDI Ser331 (Fig EV4D), and Ser427 (Fig EV4E), which are located in S357E was quickly digested by proteinase K around the x-linker the b’ domain and a’ domain, respectively (Fig EV4F). The Ser331 region, whereas PDI WT was more resistant, indicating that PDI and Ser427 phosphorylation mimicking mutant showed similar S357E tends to adopt an open conformation (Fig 4D). Similar results overall structural conformations (Fig EV4G and H), chaperone were observed when PDI WT and S357E were digested by trypsin activities (Fig EV4I and J), and enzymatic activities (Fig EV4K and and chymotrypsin (Fig EV4A and B). Moreover, we measured the L) to PDI WT. Taken together, phosphorylation of Ser357 induces intrinsic fluorescence of PDI by employing the PDI W128F/W407F PDI to be captured in an open conformation and triggers its func- mutant, in which the fluorescence is dominated by Trp364 in the tional switch from an oxidoreductase to a molecular chaperone x-linker as an intrinsic reporter of microenvironment changes (Wang (Fig 4O). et al, 2010). We observed a dramatic fluorescence intensity increase and a red shift of maximum emission in the intrinsic tryptophan flu- Phosphorylation of PDI Ser357 safeguards ER proteostasis orescence spectrum of PDI S357E compared to PDI WT, demonstrat- ing that conformational change occurs around the x-linker in this The fact that phosphorylation of PDI on Ser357 promotes its chap- phosphorylation mimic (Fig EV4C). To investigate the conforma- erone activity inspired us to test the possibility that pS357-PDI tional change driven by the phosphorylation of PDI Ser357 in cells, regulates ER proteostasis during ER stress. To visualize and detect DMSO- or Tg-treated HepG2 cells were lysed and subjected to protei- ER proteome stress in living cells, we generated a Halo-tag mutant nase K digestion. Clearly, PDI in Tg-treated cells was more suscepti- (K73T/L172Q) prone to ER-localized aggregation based on a previ- ble to proteolytic digestion (Fig 4E). The increased proteinase K ous screen (Liu et al, 2017), by introducing an ER targeting signal sensitivity on PDI is dependent on the presence of Fam20C, and Tg sequence and a KDEL ER retrieval sequence (hereafter referred to

washout can reverse this feature (Fig 4E). Moreover, along with the as AgHaloER). The AgHaloER sensor was labeled with our devel- increase in Fam20C expression, the phosphorylation level of PDI oped solvatochromic fluorogenic probe (P1), which turns on fluo- gradually increased and became more susceptible to attack by rescence only upon its misfolding and aggregation. In addition, the

proteinase K (Fig 4F). All these results indicated that the phosphory- AgHaloER probe can be combined with commercially available lated PDI adopts an open conformation both in vitro and in cells. always-fluorescent ligand (TMR) to enable dual-color imaging,

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◀ Figure 4. Phosphorylation of PDI induces its functional switch from an oxidoreductase to a molecular chaperone. A The last snapshot of the molecular dynamic simulation of pS357-PDI. PDI is composed of four thioredoxin-like domains a (orange), b (green), b’ (blue), a’ (cyan), and the x-linker (red). The initial structure of PDI (PDB code 4EL1) is shown in gray. pSer357 and Ser357 are shown in stick. B The time course of the distance between the geometrical centers of domains a and a0 and the angle among domains b, b0, and a0 from the simulation of pS357-PDI (red) and PDI (black; Yang et al, 2014). C(Left) ANS fluorescence spectra of PDI WT and S357E. Controls with ANS alone were shown. A.U.: arbitrary units. (Right) Enhancement factor was quantified. D Limited proteolysis of PDI WT (Left) and S357E(Right) proteins by proteinase K (Pro K) for indicated times. Main digested fragments were indicated on the right margin. Band abb’x* is a digested product with cleavage after Met356 in the x-linker region (Wang et al, 2010). E shCtrl and shFAM20C HepG2 cells were treated without or with 5 lM Tg for 30 min, and in some aliquots of cells, Tg was removed for 30 min. Cell lysates were subjected to proteinase K digestion for 3 min and immunoblotting analysis. F HepG2 cells expressing HA-tagged PDI and FLAG-tagged Fam20C with different ratios of plasmid concentrations were subjected to proteinase K digestion for 21 min and immunoblotting analysis. G–J Chaperone activities of PDI WT and S357E (G, H) or phosphorylated PDI WT and S357A by Fam20C for indicated times (I, J). Denatured and reduced GAPDH (G, I) and rhodanese (H, J) were used as substrates, respectively. The chaperone activity of PDI WT was taken as 100%. K, M Reductase activities of PDI WT and S357E (K) or phosphorylated PDI WT and S357A by Fam20C for indicated times (M) measured by insulin reduction assay. The activity of PDI WT was taken as 100%. L, N Isomerase activities of PDI WT and S357E (L) or phosphorylated PDI WT and S357A by Fam20C for indicated times (N) measured by scrambled RNase A reactivation assay. The activity of PDI WT was taken as 100%. O Schematic diagram illustrating phosphorylation of PDI Ser357 by Fam20C induces its functional switch from a “foldase” to a “holdase”. Data information: All data were shown as mean SEM of three independent experiments. In (C, G, H, K, L), **P < 0.01, ***P < 0.001 (two-tailed Student’s t-test). In (I, J, M, N), ***P < 0.001 (two-way ANOVA, the post hoc Tukey’s HSD test). Source data are available online for this figure.

allowing for direct visualization of the AgHaloER sensor in cells Phosphorylated PDI interacts with IRE1a and attenuates IRE1a both before and after stress conditions (Fig 5A). It is worth noting signaling during ER stress that fluorescence resonance energy transfer did not occur in two- color imaging of AgHalo labeled by P1 and TMR (Liu et al, 2018). Because Fam20C phosphorylates PDI and suppresses IRE1a signal- Confocal fluorescence microscopic imaging confirmed that AgHa- ing under ER stress, we decided to test the possibility that phospho-

loER was correctly localized in the ER lumen (Fig 5B). Live cell rylated PDI can directly modulate IRE1a signaling. We first

imaging of HepG2 cells expressing AgHaloER labeled by P1 (green) evaluated the levels of XBP1 mRNA splicing in WT and PDI KO

and TMR (red) demonstrated that the AgHaloER was well-folded HepG2 cells. Compared with control cells, PDI KO cells displayed a with little green fluorescence signal under unstressed conditions higher amplitude of XBP1 mRNA splicing after Tg treatment and formed granular green fluorescent structures after Tg treat- (Fig 6A). Expression of PDI WT or S357E, but not S357A, decreased

ment for 30 min (Fig 5C). Thus, the fluorogenic AgHaloER-P1 XBP1 mRNA splicing in PDI KO cells (Fig 6B). Similar results were conjugate provides a direct readout for the facile detection of ER obtained when cells were treated with Tm (Fig EV5A and B). Again, proteome stress. PDI S357E selectively suppressed the phosphorylation of IRE1a but To examine the role of PDI phosphorylation in regulating ER did not affect PERK or ATF6 signaling (Fig 6C).

proteostasis, we first detected AgHaloER signaling in PDI KO HepG2 Next, we performed co-IP assays to analyze the interaction cells. Depletion of PDI resulted in the accumulation of granular between PDI and IRE1a. The PDI-IRE1a interaction was detected

structures of AgHaloER with green fluorescence, suggesting that PDI by reciprocal co-IP in cells co-expressing HA-PDI and IRE1a-FLAG

is vital for the correct folding of the AgHaloER sensor. Replenish- (Fig EV5C). Importantly, the interaction between endogenous PDI ment with PDI WT or S357E, but not the S357A mutant, and IRE1a was also detected in cells and was induced under ER suppressed the granule formation in PDI KO cells, highlighting the stress (Fig 6D). Interestingly, the binding of PDI to IRE1a was first importance of Ser357 phosphorylation in maintaining ER proteosta- induced by Tg and then reduced after removal of Tg, correlating

sis (Fig 5D). Furthermore, AgHaloER-P1 fluorescence could also be with the release of BiP from IRE1a and rebinding (Fig 6E). The detected in shFAM20C HepG2 cells, albeit the puncta sizes were ER stress-related PDI-IRE1a association matches PDI phosphoryla- smaller than those observed in PDI-depleted cells (Fig 5E), in line tion dynamics (Fig 3C), and PDI S357E showed a stronger interac- with the observation that cells lacking Fam20C were more sensitive tion with IRE1a than PDI S357A (Fig EV5D), implying that to ER stress (Fig 1). Since the accumulation of misfolded proteins phosphorylation of PDI promotes its binding with IRE1a. Consis- in the ER will imbalance ER proteostasis and even induce cell tent with this idea, depletion of Fam20C greatly reduced the Tg- death if ER stress cannot be resolved, we further studied whether induced PDI-IRE1a interaction (Fig 6F). In addition, overexpres- phosphorylation of PDI Ser357 could affect cell viability. A crystal sion of Fam20C WT, but not the inactive D478A mutant,

violet staining assay showed that AgHaloER-expressing PDI KO enhanced the phosphorylation level of PDI and the interaction HepG2 cells were more vulnerable than WT cells when subjected between PDI and IRE1a (Fig 6G). Importantly, the enhancement of to long-term Tg treatment. Replenishment with PDI WT or S357E the PDI-IRE1a interaction relied on the phosphorylation of PDI in KO cells completely rescued cell viability to the extent of WT Ser357 by Fam20C (Fig 6H). PDI is an ER lumen protein; co-IP cells, whereas expression of S357A had little effect (Fig 5F). Alto- (Fig 6I) and GST-pulldown (Fig EV5E) assays supported that PDI gether, these results indicated that phosphorylation of PDI Ser357 directly binds to the N-terminal lumenal domain of IRE1a is critical to maintain ER proteostasis and reduce cell death under (IRE1aNLD). Furthermore, we generated phosphorylated GST-PDI ER stress. by the Fam20C kinase assay and found that the binding affinity

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◀ Figure 5. Phosphorylation of PDI Ser357 safeguards ER proteostasis. A(Upper) Schematic representation of ER-located fluorogenic probe Halo-tag K73T/L172Q mutant (AgHaloER). SP: signal peptide. The carboxyl-terminal KDEL motif is the

ER retention sequence. (Lower) Diagram of AgHaloER that detects ER proteostasis in live cells. B Fluorescent photomicrographs of HepG2 cells expressing AgHaloER (TMR-labeled, red). Endogenous PDI was immunostained (green) as an ER marker. Scale bar = 10 lm.

C Confocal live cell imaging of HepG2 cells expressing AgHaloER labeled by P1 (green) and TMR (red), followed by introduction of DMSO or 5 lM Tg for 30 min. Scale bar = 10 lm.

D Confocal live cell imaging of WT and PDI KO HepG2 cells co-transfected with AgHaloER labeled by P1 (green) and TMR (red) and empty vector (–), PDI WT, S357E, or S357A. Scale bar = 10 lm.

E Confocal live cell imaging of shCtrl and shFAM20C HepG2 cells expressing AgHaloER labeled by P1 (green) and TMR (red). Scale bar = 10 lm. F(Upper) Cell viabilities of WT and PDI KO HepG2 cells co-transfected with AgHaloER and empty vector (–), PDI WT, S357E, or S357A. Cells were treated by 5 lM Tg for indicated times and visualized by crystal violet staining. (Lower) Quantification of surviving fraction of cells. Data were shown as mean SEM of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 versus WT HepG2 (two-way ANOVA, the post hoc Tukey’s HSD test).

of PDI and IRE1aNLD is largely increased by phosphorylation intraperitoneally with a concentration of 50 ng Tm per gram of (Fig 6J). animal (Sepulveda et al, 2018). Kinetic analysis of Xbp1 mRNA PDI can interact with client proteins through disulfide bridges or splicing in the liver revealed that both the strength and duration of hydrophobic interactions. To explore whether PDI interacts with IRE1a signaling were increased in the PdiS359A/S359A mice compared IRE1a in a cysteine-dependent manner, we substituted all three to the littermate Pdi+/+ mice (Fig 7C). Higher Xbp1 mRNA splicing cysteines in the lumenal domain of IRE1a with serine residues. The levels were also observed in isolated primary hepatocytes from IRE1a C109/148/332S mutations did not affect the interaction with PdiS359A/S359A mice after treatment with Tg (Fig 7D) and Tm PDI, suggesting that the PDI-IRE1a association is cysteine-indepen- (Fig 7E). In accordance, IRE1a was phosphorylated to a higher dent (Fig 6K, Left). By contrast, another PDI family member, P5, degree in primary hepatocytes from PDI S359A KI mice under ER interacted with IRE1a in a cysteine-dependent manner as previ- stress (Fig EV6B). ously reported (Eletto et al, 2014; Fig 6K, Right). There are three We also compared the expression of other downstream UPR binding sites in PDI domains, a, b’, and a’, contributing to efficient signaling genes in PdiS359A/S359A and Pdi+/+ mice. The mRNA collagen prolyl 4-hydroxylase tetramer assembly (Koivunen et al, levels of XBP1 targeting genes Bip (Fig 7F), Pdi (Fig 7G), Erdj4

2005) and platelet aIIbb3 integrin binding (Wang et al, 2019). We (Fig 7H), and Edem1 (Fig 7I) were less affected under basal condi- then employed PDI binding mutants that have been previously tions but were sharply induced after 24 h of Tm injection, con- used to study the molecular details of the PDI-IRE1a interaction. firming enhanced IRE1a signaling in PDI S359A KI mice. The The single mutants W128I and L403W (with the binding site in the proinflammatory cytokine Il6 (Fig 7J) and proapoptotic transcrip- a and a’ domains being disrupted, respectively) rather than I289A tion factor Chop (Fig 7K) were also upregulated in PDI S359A KI (with the binding site in the b’ domain being disrupted) impaired mice after Tm administration, implying that more cell apoptosis the interaction between PDI with IRE1a, and the W128I/L403W occurs. Indeed, the serum alanine transaminase (ALT) level, an double mutant almost completely abolished the PDI-IRE1a interac- indicator of liver injury, was significantly elevated in PdiS359A/S359A tion (Fig EV5F). Importantly, the PDI W128I/L403W double mice under ER stress (Fig 7L). Although the kidney injury biomark- mutant was still unable to interact with IRE1a when Fam20C was ers creatinine (Cr) (Fig EV6C) and blood urea nitrogen (BUN) co-expressed, albeit Ser357 in the double mutant was sufficiently (Fig EV6D) were not significantly different between PDI S359A KI phosphorylated (Fig 6L). Collectively, these results suggest that and WT mice, there seems to be a tendency of kidney injury in upon ER stress, phosphorylated PDI binds to the lumenal domain PdiS359A/S359A mice under ER stress. The extent of liver damage was of IRE1a via noncovalent hydrophobic interactions and suppresses further analyzed by hematoxylin and eosin staining of liver sections IRE1a activation. from Tm-challenged mice. Massive hepatocyte damage was observed, including edema and vacuolization (Fig EV6E), Phosphorylation of PDI protects against ER stress-induced liver inflammatory cell infiltration (Fig 7M), and hemorrhage (Fig EV6F). damage in vivo The levels of cell apoptosis were also monitored by using the TUNEL assay. As expected, only occasional apoptotic nuclei were The above finding that p-PDI negatively regulates IRE1a signaling seen in WT mice with the low dose of Tm administrated, whereas a could be physiologically important because excessive activity of significant increase in apoptotic cells (> 15%) was observed in the IRE1a under ER stress can lead to cellular apoptosis and diseases. livers of Tm-challenged PdiS359A/S359A mice (Fig 7N). In summary, To reveal the potential protective role of p-PDI under pathophysio- phosphorylation of PDI attenuates the excessive activity of IRE1a logical conditions, we generated a PdiS359A/S359A knock-in (KI) and plays a prosurvival role in protecting against ER stress-induced mouse by CRISPR/Cas9-mediated genome editing (Figs 7A and liver damage. EV6A). Ser359 of mouse PDI is homologous to Ser357 of human PDI (Fig 2B), and S359A KI was validated by DNA sequencing (Fig 7B). The PdiS359A/S359A homozygotes were born at Mendelian ratios and Discussion had no phenotypic abnormalities from birth through adulthood, in line with the results that the human PDI S357A is as active as the In this study, we demonstrate that PDI, a key folding catalyst in the WT protein (Fig 4). For acute ER stress, mice were injected ER, is rapidly phosphorylated by the secretory pathway kinase

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◀ Figure 6. Phosphorylated PDI interacts with IRE1a and attenuates IRE1a signaling during ER stress. A(Left) Detection of XBP1 mRNA splicing in WT and PDI KO HepG2 cells treated with or without 5 lM Tg for 1 h. PDI KO was verified by protein immunoblotting. (Right) Quantification of XBP1 mRNA splicing levels. Data were shown as mean SEM of three independent experiments. *P < 0.05 (two-tailed Student’s t-test). B (Left) Detection of XBP1 mRNA splicing in PDI KO HepG2 cells expressing PDI WT or its mutants treated with or without 5 lM Tg for 1 h. PDI expression levels were shown by protein immunoblotting. (Right) Quantification of XBP1 mRNA splicing levels. Data were shown as mean SEM of three independent experiments. *P < 0.05, **P < 0.01 (one-way ANOVA, the post hoc Tukey’s HSD test). C Detection of IRE1a, PERK, and ATF6 activation by immunoblotting in PDI KO HepG2 cells expressing PDI S537AorS357E treated with 5 lM Tg for 30 min. Asterisk indicates unspecific background band. D HepG2 cells were treated without or with 5 lM Tg for 1 h. Endogenous IRE1a immunoprecipitates were analyzed by PDI and BiP immunoblotting. E HepG2 cells expressing FLAG-tagged IRE1a were treated without or with 5 lM Tg for 1 h. An aliquot of Tg-treated cells was then washed with culture medium and chased for 4 h. FLAG immunoprecipitates were analyzed by PDI and BiP immunoblotting. F shCtrl and shFAM20C HepG2 cells were treated without or with 5 lM Tg for 1 h. Endogenous IRE1a immunoprecipitates were analyzed by PDI and BiP immunoblotting. G Co-immunoprecipitation of FLAG-tagged IRE1a and HA-tagged PDI in HepG2 cells expressing V5-tagged Fam20C WT or DA. H Co-immunoprecipitation of FLAG-tagged IRE1a, HA-tagged PDI WT, or S357A in HepG2 cells expressing V5-tagged Fam20C. I Co-immunoprecipitation of HA-tagged PDI and FLAG-tagged IRE1aNLD in HepG2 cells. J Recombinant GST-PDI was phosphorylated by Fam20C protein for 2 h and then used to pulldown His-IRE1aNLD protein, as visualized by Coomassie blue staining. Successful phosphorylation of GST-PDI was verified by pS357-PDI immunoblotting. K Co-immunoprecipitation of FLAG-tagged IRE1aNLD WT or its mutant C109/148/332S with HA-tagged PDI (Left) or MYC-tagged P5 (Right) in HepG2 cells. L Co-immunoprecipitation of FLAG-tagged IRE1a and HA-tagged PDI WT or its binding mutant W128I/L403WinHepG2 cells expressing V5-tagged Fam20C or not. Source data are available online for this figure.

Fam20C under ER stress, and the p-PDI with increased chaperone & Tsou, 1993; Wang et al, 2015). Nevertheless, the molecular activity is important for maintaining ER proteostasis and tuning the mechanism for the adjustment of the two activities is elusive. amplitude of IRE1a signaling (Fig 7O). In contrast to the canonical Here, we report that phosphorylation of Ser357 in the x-linker UPR pathways with inherent latency acting at transcriptional and region of PDI induces PDI from a “foldase” to a “holdase”. This translational levels, post-translational modification promptly functional switch is important to prevent proteotoxicity under ER responds to the proteostatic perturbations in the ER through preex- stress, probably because the prevention of misfolded protein aggre- isting components (Preissler & Ron, 2018). Recent reports on the gation is of higher priority than normal oxidative protein folding in regulation of the ER Hsp70 chaperone BiP at the post-translational the stressed ER. Indeed, similar mechanisms have been reported level have set such an example. BiP is AMPylated when the folding not only for phospho-regulated chaperones (Velasco et al, 2019) requirement in the ER is low, and the reversible deAMPylation but also for redox-regulated chaperones and acid-activated chaper- recruits BiP back into the chaperone cycle under ER stress (Preissler ones (Voth & Jakob, 2017). Our MD and biochemical analyses indi- et al, 2015, 2017a,b). Thus, both the phosphorylation of PDI and cate that the functional switch of PDI is based on the relocation of the deAMPylation of BiP are early responses to ER stress by simply the x-linker region and that p-PDI tends to adopt an open confor- increasing the protein folding capacity at the post-translational level. mation distinct from the relatively closed conformation of Because PDI and BiP are both high-abundance proteins in the ER, nonphosphorylated PDI. This open conformation has a more their post-translational regulation can well match the accumulating exposed hydrophobic surface that favors unfolded/misfolded load of misfolded/unfolded proteins. However, whether there is a substrate binding but may disrupt the complete catalytic cycle of crosstalk between post-translational regulation of PDI and BiP is still PDI. Indeed, MD simulation revealed that the distance between the an open question. two active sites in p-PDI increased to 70 A˚ from the original Phosphorylated PDI not only suppresses the aggregation of 40.3 A˚ , and this increased distance may impair the cooperation of misfolded proteins but also binds to the lumenal domain of IRE1a the two active sites during catalysis. The phosphorylation-induced and limits its activation. The amplitude and duration of IRE1a acti- conformational change is in line with previous studies that the x- vation determines cell fate under ER stress, and the precise control linker of PDI can be trapped in either a “capped” or an of IRE1a activity is of vital importance. Previous studies revealed “uncapped” conformation by mutagenesis (Nguyen et al, 2008; that IRE1a activity can be modulated either from the lumenal side Wang et al, 2010) or small molecule ligand (Bekendam et al, by BiP (Bertolotti et al, 2000; Kimata et al, 2003; Bakunts et al, 2016). Collectively, we propose that PDI is a novel phospho-regu- 2017), P5 (Eletto et al, 2014), and Hsp47 (Sepulveda et al, 2018) lated chaperone against ER stress. chaperones or from the cytosolic side by BAK and BAX (Hetz et al, In this study, we demonstrate that Fam20C plays an important 2006), BAX inhibitor-1 (Lisbona et al, 2009), PUMA and BIM (Rodri- role in regulating ER proteostasis and UPR signaling, in addition guez et al, 2012), ABL (Morita et al, 2017), and MITOL (Takeda to its previously identified regulatory role in regulating redox et al, 2019). Our results show that the recruitment of p-PDI to IRE1a homeostasis (Zhang et al, 2018) and Ca2+ homeostasis (Pollak is induced by Tg addition and then reduced after Tg removal, coin- et al, 2018). Thus, Fam20C-mediated phospho-regulation could be ciding with the release and binding between BiP and IRE1a. Thus, it a general mechanism to maintain ER homeostasis. Fam20C-cata- seems that p-PDI acts as a reservoir for the precise control of IRE1a lyzed protein phosphorylation was believed to occur in the lumen signaling from the lumenal side, adding an additional layer of of Golgi and/or extracellularly (Tagliabracci et al, 2013; Zhang complexity of IRE1a regulation. et al, 2018). However, we show that Fam20C is at least partially Protein disulfide isomerase has been recognized as both an retained in the ER during ER stress and could form condensates oxidoreductase and a molecular chaperone for a long time (Wang with PDI, resulting in higher local concentrations for catalyzing

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◀ Figure 7. Phosphorylation of PDI protects against ER stress-induced liver damage in vivo. A Schematic diagram illustrating the generation of PdiS359A/S359A knock-in (KI) mice by CRISPR/Cas9-mediated genome editing. B Sanger sequencing results of Pdi+/+ (WT) and PdiS359A/S359A (KI) mice. C(Left) Detection of Xbp1 mRNA splicing in mice liver after injection of Tm (50 ng/g, i.p.) for indicated times. Each lane represents independent animals. (Right) Quantification of Xbp1 mRNA splicing levels at indicated times. D, E Detection of Xbp1 mRNA splicing in primary hepatocytes isolated from WT and KI mice treated with 0.2 lM Tg for 2 h (D) or 0.5 lg/ml Tm for 2 h (E). F–K Detection of Bip (F), Pdi (G), Erdj4 (H), Edem1 (I), Il6 (J), Chop (K) mRNA levels by real-time PCR in the liver of the same animals as in (C) after 24 h of Tm injection. L Detection of serum alanine transaminase (ALT) levels of the same animals as in (C) after 24 h of Tm injection. M Hematoxylin and eosin staining of liver tissue of the same animals as in (C) after 24 h of Tm injection (magnification × 400). Immune cell infiltration was indicated with a black arrow. Scale bar = 50 lm. Representative images were shown from five animals per group analyzed. N(Left) TUNEL assay of the same liver as in (M). Scale bar = 40 lm; zoom box, scale bar = 5 lm. (Right) Quantification of apoptotic cells. 10 random fields were counted for each TUNEL-stained tissue sample. O Proposed model for the role of p-PDI. (Left) Under basal conditions, PDI is responsible for the catalysis of oxidative protein folding, and IRE1a is maintained by BiP chaperone in a monomeric inactive state. (Middle) Upon ER stress, Fam20C kinase is retained in the ER for rapid phosphorylation of PDI. Phosphorylated Ser357 induces an open conformation of PDI and turns it from a “foldase” to a “holdase”, which is critical for preventing protein misfolding in the ER. Meanwhile, BiP dissociates and IRE1a is activated for XBP1 splicing. p-PDI directly binds to IRE1a to control the amplitude of IRE1a activity for adaptive UPR signaling. (Right)In PDI S357A mutant, protein aggregates form under ER stress due to incapable phosphorylation of PDI, and sustained ER stress induces hyperactivation of IRE1a, which leads to terminal UPR and apoptosis. Data information: All data were shown as mean SEM from five biological replicates. In (C, N), *P < 0.05, ***P < 0.001 (two-tailed Student’s t-test). In (F–L), *P < 0.05, ***P < 0.001 (two-way ANOVA, the post hoc Tukey’s HSD test). Source data are available online for this figure.

efficient phosphorylation processes. Moreover, ER stress also Materials and Methods induces an elevated ATP level in the ER via AXER (an ATP/ADP exchanger in the ER membrane; Klein et al, 2018), which may Antibodies, chemicals, peptides, recombinant proteins, and DNA also facilitate Fam20C-catalyzed phosphorylation. An unresolved question is how Fam20C senses the environmental changes in the The sources and identifiers of antibodies, chemicals, peptides, ER to phosphorylate certain clients. We notice from our Fam20C recombinant proteins, and DNA used in this paper can be found in interactome data that the interaction between Fam20C and Table EV1. ERGIC2 significantly decreased after Tg treatment. ERGIC2, together with ERGIC3, functions as a cargo receptor in protein Cell culture trafficking between the ER and Golgi (Otte et al, 2001; Orci et al, 2003). Thus, we speculate that the transport of Fam20C from the HepG2 cells were cultured in RPMI medium modified without ER to Golgi might be mediated by ERGIC2/3 and that Fam20C is calcium nitrate with 2.05 mM L-glutamine (HyClone) supplemented retained in the ER under ER stress, possibly due to its decreased with 10% fetal bovine serum (Gibco). HeLa cells were cultured in interaction with ERGIC2/3. Moreover, we observed that the ER- Dulbecco’s modified Eagle’s Medium (DMEM) (HyClone) supple- retained Fam20C is rapidly transported to the Golgi after Tg mented with 5% fetal bovine serum. All media were supplemented washout and that p-PDI diminishes simultaneously. The decrease with 100 lg/ml streptomycin and 100 U/ml penicillin (Invitrogen),

in p-PDI could either be catalyzed by an unidentified protein and the cells were cultured at 37°C with 5% CO2. Plasmid transfec- phosphatase in the ER or achieved by selective degradation tion was accomplished by using ViaFect (Promega) according to the through the ER-associated degradation or the autophagic pathway manufacturer’s instructions. (Cha-Molstad et al, 2015). Our finding that the Fam20C-PDI axis negatively regulates Construction of PDI S359A knock-in mice IRE1a signaling is physiologically relevant because terminal UPR is associated with many diseases, such as diabetes, cancer, and Mice harboring PDI S359A mutation were generated by CRISPR/ neurodegeneration (Oakes & Papa, 2015). Indeed, under Tm- Cas9-mediated genome editing (Biocytogen). In brief, the S359A induced acute ER stress, the PdiS359A/S359A mice had elevated levels mutation was introduced in Pdi exon 8 using an overlap exten- of the proinflammatory cytokine Il6 and the proapoptotic transcrip- sion-PCR method. Homology regions covering ~2 kb upstream of tion factor Chop as well as liver damage. In line with our results, Pdi exon 5 and ~2 kb downstream of exon 10 were subcloned into CHOP mRNA is robustly induced by Tg stimulation in Fam20C- the targeting vector. Two single-guide RNAs (sgRNAs) targeting depleted U2OS cells, and cardiomyocyte-specific Fam20c knockout intron 4 (50-TATGGTTTAGGCAATGACAA-30) and intron 10 (50-A causes accelerated deterioration of cardiac function in mice follow- GATCTATACCTAGGAAGCT-30) were designed. Cas9/sgRNA and ing various pathological stimuli (Pollak et al, 2018). Recently, targeting vector were microinjected into C57BL/6J oosperms and Fam20C has been reported to be significantly elevated in the islet implanted into pseudopregnant females. Mice (Pdi+/S359A) carrying b cells of diabetic mice (Kang et al, 2019). Thus, future efforts to the recombined allele containing the S359A mutation were geno- validate the function of the Fam20C-PDI axis under pathophysio- typed by PCR followed by sequencing using primers (50-GT logical conditions, such as diabetes, are particularly important for ACTAGCCTAGCCATGCACCAAGG-30,50-AAGGGGCATCTGAAAGA providing a new therapeutic strategy to combat ER stress-asso- GAGCAGTG-30). Southern blot was performed to identify the ciated diseases. proper integration of recombined loci in targeted mice.

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Heterozygotic mice (Pdi+/S359A) were intercrossed to generate RNA isolation, RT–PCR, and real-time PCR homozygotic mice (PdiS359A/S359A). All these mice were maintained in specific pathogen-free conditions and housed separately during Total RNA was isolated from cells and tissues using TRIzol (Invitro- the experiments. For acute ER stress, 16- to 20-week-old male gen), and RNA samples were then reverse-transcribed into cDNA mice were injected intraperitoneally with tunicamycin (Tm, using GoScript Reverse Transcription System (Promega). Quantita- Abcam) or dimethylsulfoxide (DMSO, Sigma-Aldrich) for different tive real-time PCRs employing SYBR Select Master Mix (Applied times. Blood was collected via the inferior vena cava (IVC) in Biosystems) were performed in the QuantStudio 7 Flex machine anesthetized mice for serum biochemical analysis. The liver was (Applied Biosystems), following the manufacturer’s instructions. frozen at 80°C for biochemical analysis, and the right major lobe The relative amounts of mRNAs were calculated from the values of of the live was fixed in 4% paraformaldehyde (NOVON) for 72 h comparative threshold cycle by using Actb as a control. The meth- for histological analyses. Animal experiments were conducted ods for the XBP1 mRNA splicing assay were performed as previ- with the approval of the Institutional Biomedical Research Ethics ously described (Upton et al, 2012). In brief, XBP1 mRNA was Committee of the Institute of Biophysics, Chinese Academy of reverse-transcribed, PCR-amplified, and then resolved on 2.5% Science. agarose (Sigma-Aldrich) gels. PCR primers were described in Table EV2. Primary hepatocyte isolation and culture Protein purification Mice were anesthetized, and abdominal cavity was dissected. The IVC was cannulated with a 24-gauge 3/4-inch angiocatheter (BD Recombinant human PDI protein and its mutants were expressed in Biosciences), and the portal vein was cut. The liver was perfused Escherichia coli BL21 (DE3) cells and purified as described (Wang via the IVC with 20 ml KRG buffer (120 mM NaCl, 20 mM et al, 2010). Recombinant IRE1aNLD (24–390) protein was expressed

NaHCO3, 20 mM glucose, 5 mM HEPES, pH 7.4, 5 mM KCl, 1 mM in Escherichia coli BL21 (DE3) cells and purified as described (Zhou

MgSO4, 1 mM KH2PO4) with 0.5 mM EGTA at 37°C, followed by et al, 2006). Human Fam20C (141–578) protein was expressed in perfusion with 20 ml of 25 mg/ml collagenase type 4 (Sigma- Hi5 insect cells and purified from the conditioned medium (Xiao Aldrich) in KRG buffer. After the liver was digested, it was cut to et al, 2013). Proteins were quantified spectrophotometrically at release the hepatocytes, passed through a 75-lm cell strainer, and 280 nm and stored at 80°C in aliquots. washed with ice-cold DMEM until the supernatant appear clear after spinning. The pellet was suspended with DMEM supple- In vitro kinase assay mented with 10% fetal bovine serum and cultured at 37°C with

5% CO2. 1 mg/ml recombinant PDI WT or S357A protein and 20 lg/ml

Fam20C protein were incubated in HEPES, pH 7.0, 10 mM MnCl2 at Generation of PDI knockout HepG2 cell lines 30°C. The reactions were initiated by adding ATP (Sigma-Aldrich) to a final concentration of 2 mM and were terminated at the indi- CRISPR/Cas9 genome editing was performed to generate PDI knock- cated time points by adding SDS-loading buffer containing 15 mM out HepG2 cell lines. Briefly, the guide sequence targeting human EDTA. Reaction products were separated by SDS–PAGE and visual- PDI (50-GCGGAAAAGCAACTTCGCGG-30) was designed using the ized by Coomassie blue staining and immunoblotting. CRISPR design tool (http://chopchop.cbu.uib.no/). HepG2 cells were transfected with pSpCas9 (BB)-2A-GFP vector (Laboratory of k-phosphatase assay Feng Zhang, Addgene) containing the sgRNA for 48 h, and GFP- positive cells were single-cell-sorted into a 96-well plate format HepG2 cell lysates were treated with indicated units k-protein phos- containing RPMI medium by fluorescence-activated cell sorting (BD phatase (NEB) in 50 mM HEPES, pH 7.5, 100 mM NaCl, 2 mM Influx). Expanded single clones were screened for PDI knockout by dithiothreitol (DTT, Sigma-Aldrich), and 0.01% Brij 35 with 10 mM protein immunoblotting and DNA sequencing. The following MnCl2 at 30°C for 1 h, followed by SDS–PAGE and immunoblotting. primers were used to amplify the region surrounding the proto- spacer adjacent motif (PAM): 50-CAGGATTTATAAAGGCGAGGC-30, Fluorescence spectra 50-CTCACAGAACTCCACCAGCA-30. Intrinsic fluorescence spectra of 5 lM PDI or its mutants in 50 mM RNA interference Tris–HCl, pH 7.6, 150 mM NaCl were recorded at 310–400 nm at 25°C with excitation at 290 nm. For ANS fluorescence spectra, To generate stable Fam20C knockdown HepG2 cells, cells were 50 lM ANS was incubated with or without 5 lM PDI proteins in transfected with pSUPER plasmid (OligoEngine) expressing the 50 mM Tris–HCl, pH 7.6, 150 mM NaCl for 20 min at 25°C in the short-hairpin RNA (shRNA) targeting FAM20C sequence dark. ANS fluorescence emission spectra at 400-650 nm were 50-GAGCTGTACTCCAGACACA-30 by ViaFect according to the measured with excitation at 370 nm. The concentration of ANS was manufacturer’s instructions. Puromycin (InvivoGen) was added determined using an extinction coefficient at 350 nm of into the culture medium to a final concentration of 2 lg/ml to 5,000 M 1 cm 1. Enhancement factor = [F (protein + ANS + buffer) – kill the negative cells. Cells and culture medium were harvested F (protein + buffer)]/[F (ANS + buffer) – F (buffer)], where F is the and analyzed by immunoblotting to confirm the knockdown fluorescence intensity at 480 nm. Fluorescence spectra were recorded efficiency. by using RF-5301PC Spectrofluorophotometer (SHIMADZU).

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Circular dichroism spectra 50 mM Tris–HCl, pH 7.6, for digestion at 25°C. The reactions were terminated at different times by adding 0.5 mM phenylmethanesul- Circular dichroism spectra of 2.5 lM PDI or its mutants in 20 mM fonylfluoride (PMSF) and analyzed by SDS–PAGE and Coomassie sodium phosphate buffer, pH 7.5, were measured at 190–260 nm at blue staining. 25°C by Chirascan Plus (Applied Photophysics). Scanning speed was 2.5 s/dot. In cells HepG2 cells were lysed by 50 mM Tris–HCl, pH 7.6, 150 mM NaCl, Chaperone activity assay 5 mM KCl, 1% NP-40 with phosphatase inhibitor cocktail (Roche) for 30 min on ice. Proteinase K was added to a final concentration Rabbit muscle glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of 100 lg/ml with 6 mg/ml total lysates. The reactions were termi- was purified as described before (Cai et al, 1994). Denaturation of nated at indicated times by adding 0.5 mM PMSF and analyzed by GAPDH was carried out by incubation of 0.14 mM GAPDH in 3 M SDS–PAGE and immunoblotting. guanidine hydrochloride with 1 mM DTT overnight at 4°C. Refold- ing was initiated by 50-fold dilution of the denatured GAPDH into Immunoblotting and phostag gels 100 mM sodium phosphate, pH 7.4, 2.5 mM EDTA with or without 7 lM PDI or its mutants at 25°C. GAPDH aggregation was moni- Harvested cells were washed with ice-cold phosphate-buffered tored by recording the light scattering at 488 nm for 30 min. Denat- saline (PBS) and then lysed in radioimmunoprecipitation assay uration of rhodanese (Sigma-Aldrich) was carried out by incubation (RIPA) lysis buffer (50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 0.25% of 45 lM rhodanese in 6 M guanidine hydrochloride with 10 mM deoxycholic acid, 1% NP-40, 1 mM EDTA) (Millipore) containing DTT for 1 h at room temperature (RT). Refolding was initiated by phosphatase and protease inhibitor cocktails (Roche) for 30 min on 100-fold dilution of the denatured rhodanese into 200 mM sodium ice. To remove cell debris, cell lysates were centrifuged at phosphate, pH 7.5, with or without 1 lM PDI or its mutants at 25°C. 16,000 × g for 15 min. Protein concentration of cell lysates was Rhodanese aggregation was monitored by recording the light scat- quantified by BCA method (Beyotime), and the same amount of tering at 350 nm for 30 min. Light scattering was recorded by using proteins was loaded and separated by SDS–PAGE and then trans- RF-5301PC Spectrofluorophotometer (SHIMADZU). The chaperone ferred to polyvinylidene fluoride (PVDF) membranes (pore size activities of PDI proteins were calculated as (I0 I)/(I0 I1) × 0.45 lm). The membranes were blocked in TBST buffer (50 mM

100%, where I0, light scattering intensity determined in the absence Tris pH 8.0, 150 mM NaCl, 0.05% Tween-20) containing 5% (w/v) of PDI; I and I1, the values determined in the presence of PDI skimmed milk or 5% (w/v) bovine serum albumin (BSA) (for phos- mutants and PDI WT, respectively. phor-protein immunoblotting). After blocking, the membranes were washed in TBST and incubated with various primary antibodies at Reductase activity assay 4°C overnight and followed by incubated with horseradish peroxi- dase (HRP) or fluorescent-labeled secondary antibodies at RT for 130 lM insulin (Sigma-Aldrich) was added to 0.1 M potassium 2 h and then visualized by using a ChemiScope Mini imaging phosphate buffer, pH 7.5, 2.5 mM EDTA, 0.1 mM DTT in the system (Clinx Science) with enhanced chemiluminescence (Thermo absence or presence of 0.5 lM PDI or its mutants to initiate the reac- Fisher) or by an Odyssey CLx infrared imager (LICOR). tion, and the absorbance at 650 nm that represents light scattering For phostag gels, 50 lM phostag acrylamide (NARD) and from reduced and precipitated insulin B chains was immediately 100 lM MnCl2 were included in the gel recipe according to the recorded at 25°C using UV-2700 Spectrophotometer (SHIMADZU). manufacturer’s instructions. Phostag gels were washed in transfer The reductase activity was calculated as described (Wang et al, buffer supplemented with 10 mM EDTA for three times before trans- 2010) and normalized to that of PDI WT. ferring to PVDF membranes.

Isomerase activity assay Immunofluorescence

Preparation of scrambled bovine pancreatic RNase A (Sigma- Transfected and treated HepG2 cells were harvested and seeded on Aldrich) (sRNase A) was according to Lyles and Gilbert (1991). 8 lM a glass bottom cell culture dish (NEST), washed with PBS for three sRNase A was reactivated in 100 mM Tris–acetate, pH 8.0, 50 mM times and fixed with 4% paraformaldehyde for 15 min, then perme- NaCl, 1 mM EDTA, 1 mM GSH, and 0.2 mM GSSG in the absence or abilized with 0.3% Triton X-100 for 5 min and blocked with 5% presence of 3 lM PDI proteins at 25°C. The reactivation of sRNase A BSA for 30 min at RT. Cells were incubated with primary antibodies was assayed quantitatively by monitoring the absorbance increase at at 4°C overnight and fluorescent-conjugated secondary antibodies in 296 nm due to the hydrolysis of 20,30-cyclic CMP (Sigma-Aldrich), dark at 37°C for 1 h. The cells were rinsed with Hank’s balanced and the concentration of reactivated RNase A was calculated as salt solution and analyzed by confocal laser scanning microscopy described (Wang et al, 2009) and normalized to that of PDI WT. (Zeiss, LSM710).

Limited proteolysis assay ER proteostasis assay

In vitro HepG2 transfectants expressing ER-localized aggregation-prone

Proteinase K, trypsin, or chymotrypsin (Amresco) was added to a Halo-tag mutant (K73T/L172Q) (AgHaloER) without or with PDI WT final concentration of 5 lg/ml with 1 mg/ml PDI or its mutants in or S357A or S357E were seeded on a glass bottom cell culture dish.

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After 36 h, the spent medium was replaced with fresh RPMI identification, HepG2 cells were treated with DMSO or 5 lM Tg for medium containing 5 lM P1 or TMR ligand to label AgHaloER 10 min. The cell extracts were incubated with anti-FLAG or mono- protein for 30 min. In case needed, cells were washed with PBS in clonal mouse anti-PDI antibody overnight at 4°C, followed by the three times, then cultured with RPMI medium, and further treated addition of protein A+G beads for 2 h. The immunoprecipitates with DMSO or 5 lM thapsigargin (Tg, Sigma-Aldrich) for additional were washed by PBS for five times, separated by SDS–PAGE, stained 30 min. Confocal images were obtained using an Olympus Fluo- by Coomassie blue, and immunoblotted with polyclonal rabbit anti- ViewTM FV1200 confocal microscope. The P1 signal was visualized PDI in parallel. with a blue argon laser (488 nm), and the TMR signal was visual- ized using a green HeNe laser (568 nm). In-gel digestion of proteins For Fam20C interactome identification, all bands below Fam20C Crystal violet staining excluding immunoglobulin chains were excised and cut into small plugs; for PDI phosphosite identification, the band corresponding to

HepG2 transfectants expressing AgHaloER without or with PDI WT PDI was excised. After destaining, reduction (10 mM DTT in 25 mM or S357A or S357E were counted and seeded on 6-well cell culture NH4HCO3 for 45 min at 56°C), and alkylation (40 mM iodoac- plates, and treated with 5 lM Tg for indicated times. The cells were etamide in 25 mM NH4HCO3 for 45 min at RT in the dark), the washed gently with warmed PBS and then incubated with crystal plugs were washed twice with 50% acetonitrile, dried using a violet solution for 30 min at RT. The plates were washed by distilled SpeedVac, and digested with trypsin in 25 mM NH4HCO3 overnight water for three times and then imaged. at 37°C to allow complete digestion. The reaction was terminated by adding formic acid to a 1% final concentration. Immunoprecipitation and pulldown assay Liquid chromatography (LC)–MS/MS analysis Transfected and treated HepG2 cells were lysed in RIPA lysis The digested peptides were separated on an Acclaim PepMap buffer containing phosphatase and protease inhibitor cocktails. RSLC C18 capillary column (Reprosil-Pur C18-AQ, 3 lm; Dr. For immunoprecipitation of IRE1a-FLAG, HA-PDI, and endoge- Maisch GmbH). A linear acetonitrile gradient was used to elute nous IRE1a, cell lysates were incubated with anti-FLAG the bounded peptides at a flow rate of 300 nl/min. The eluate (Sigma-Aldrich), anti-HA (Sigma-Aldrich), and anti-IRE1a (CST) was electrosprayed at a 2.0 kV voltage directly into a Q Exactive antibodies at 4°C overnight, respectively, followed by incubation mass spectrometer (Thermo Fisher Scientific). In the data-depen- with protein A+G beads (Beyotime) for 1 h. Beads were washed dent acquisition mode, the MS data were acquired at a high reso- three times with RIPA buffer and then analyzed by SDS–PAGE lution of 70,000 (m/z 200) across a mass range of 300–1,600 m/ and immunoblotting. z. The top 20 precursor ions were selected from each MS full For GST-pulldown assay, 2 lM GST-PDI protein on glutathione scan with isolation width of 2 m/z for fragmentation in the HCD sepharose (GE Healthcare) was incubation with 4 lM IRE1aNLD collision cell. Subsequently, MS/MS spectra were acquired at a protein at RT for 2 h or 4°C overnight. Beads were washed three resolution of 17,500 (m/z 200). The dynamic exclusion time times with PBS and then analyzed by SDS–PAGE and Coomassie was 40 s. blue staining. Protein identification Histological evaluation and TUNEL assay The raw data from Q Exactive were analyzed with Proteome Discoverer 2.2.0.388 (Thermo Fisher Scientific) using SEQUEST Liver tissue specimens were fixed in 4% paraformaldehyde, embed- HT search engine for protein identification and Percolator for ded in paraffin, sectioned (5 lM thick), and stained with hema- false discovery rate (FDR, < 1%) analysis against a UniProt toxylin–eosin or fluorescein-dUTP by DeadEnd Fluorometric TUNEL human protein database (updated 10-2017). The peptide mass System (Promega). tolerance was set to 10 ppm and the MS/MS mass tolerance to 0.02 Da. The peptide confidence was set as high for peptide filter. Serum biochemical analysis Label-free quantification (LFQ) analysis was performed using consensus mode, and parameters were set as follows: unique and Blood samples were kept at RT for 1 h and centrifuged at 2,000 × g razor used for peptide quantification; precursor abundance based for 30 min to obtain sera. Sera were frozen at 80°C for further on intensity; total peptide amount used for normalization mode; assay. Serum alanine aminotransferase (ALT), creatinine (Cr), and pairwise ratio for ratio calculation; maximum allowed fold change blood urea nitrogen (BUN) were measured using commercial kits as 100. For Fam20C interactome identification, proteins with (Nanjing Jiancheng Bioengineering Institute, China) according to unique peptides ≥ 2 were selected and three independent experi- the manufacturer’s instructions. ments were performed. Based on DAVID GO term analysis, a total of 173 proteins localized in the ER and Golgi were identified Mass spectrometry (MS) in all three experiments. For PDI phosphosite identification, we selected phosphorylation for serine, threonine, or tyrosine and Sample preparation methionine oxidation as variable modifications and the cysteine To identify the Fam20C interactome during ER stress, HepG2 cells carbamidomethylation as a fixed modification. The tandem mass were transfected with FLAG-tagged Fam20C for 36 h and treated spectra of the matched phosphorylated peptides were manually with DMSO or 5 lM Tg for 30 min. For PDI phosphosite checked for their validity.

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Molecular dynamics simulation Author contributions JY and TL designed and performed experiments, analyzed the data, and

Molecular dynamics (MD) simulations follow similar procedures as prepared the figures. YL created the AgHaloER sensor and P1 ligand. XW and reported earlier (Yang et al, 2014). Briefly, the crystal structure of XEW generated PDI and Fam20C KO cells. JZ analyzed the MS data. GS contrib- hPDI in the oxidized (PDB code 4EL1) state (Wang et al, 2013) was uted to the histopathological analysis. JL performed the MD simulation. LKW used as the initial structure. This structure was processed with VMD participated in the designs and data analysis. C-CW and LW designed and (Humphrey et al, 1996) to generate the necessary files for MD simu- supervised the experiments. LW conceived the project, interpreted the data, lation, and phosphorylated Ser357 residue was used. The protein and wrote the manuscript with the input from JY and TL. All authors approved system was solvated in 128 × 96 × 96 A˚ 3 water box and maintained the final version of the manuscript. with 150 mM NaCl salt concentration. The energy minimization and simulations were performed with NAMD (Phillips et al, 2005) and Conflict of interest CHARMM forcefield for proteins (MacKerell et al, 1998). The simula- The authors declare that they have no conflict of interest. tion protocol is the same as that in previous study (Yang et al, 2014).

Quantification and statistical analysis References

Band intensities on gels and blots were quantified using the soft- Bakunts A, Orsi A, Vitale M, Cattaneo A, Lari F, Tade L, Sitia R, Raimondi A, ware ImageJ. All data were presented as mean, and error bars repre- Bachi A, Anken E (2017) Ratiometric sensing of BiP-client versus BiP levels sent standard error of mean (SEM) from ≥ 3 biological replicates. by the unfolded protein response determines its signaling amplitude. Elife Statistical analyses were done using GraphPad Prism (v.7.0) or 6:e27518 Origin 7.0. The two-tailed Student’s t-test was used to analyze data Balch WE, Morimoto RI, Dillin A, Kelly JW (2008) Adapting proteostasis for between two groups, and the one-way and two-way ANOVAs disease intervention. Science 319: 916 – 919 followed by the post hoc Tukey’s HSD test were used when more Bekendam RH, Bendapudi PK, Lin L, Nag PP, Pu J, Kennedy DR, Feldenzer A, than two groups were present. Statistical significance levels were Chiu J, Cook KM, Furie B et al (2016) A substrate-driven allosteric switch defined as *P < 0.05; **P < 0.01; ***P < 0.001. All statistical details that enhances PDI catalytic activity. Nat Commun 7: 12579 including number of biological or technical replicates can be found Bertolotti A, Zhang YH, Hendershot LM, Harding HP, Ron D (2000) Dynamic in each figure legend. interaction of BiP and ER stress transducers in the unfolded-protein response. Nat Cell Biol 2: 326 – 332 Cai H, Wang CC, Tsou CL (1994) Chaperone-like activity of protein disulfide Data availability isomerase in the refolding of a protein with no disulfide bonds. J Biol Chem 269: 24550 – 24552 The mass spectrometry proteomics data have been deposited to the Calfon M, Zeng HQ, Urano F, Till JH, Hubbard SR, Harding HP, Clark SG, Ron ProteomeXchange Consortium via the PRIDE (Perez-Riverol et al, D(2002) IRE1 couples endoplasmic reticulum load to secretory capacity 2019) partner repository with the dataset identifier PXD016619 by processing the XBP-1 mRNA. Nature 415: 92 – 96 (http://www.ebi.ac.uk/pride/archive/projects/PXD016619). Cha-Molstad H, Sung KS, Hwang J, Kim KA, Yu JE, Yoo YD, Jang JM, Han DH, Molstad M, Kim JG et al (2015) Amino-terminal arginylation targets Expanded View for this article is available online. endoplasmic reticulum chaperone BiP for autophagy through p62 binding. Nat Cell Biol 17: 917 – 929 Acknowledgements Cui J, Xiao J, Tagliabracci VS, Wen J, Rahdar M, Dixon JE (2015) A secretory We thank Hui Zhang, Qinyu Zhu, and Junyu Xiao (Peking University) for kinase complex regulates extracellular protein phosphorylation. Elife 4: providing the recombinant Fam20C protein; Jifeng Wang, Mengmeng Zhang, e06120 and Fuquan Yang (Laboratory of Proteomics, Institute of Biophysics) for assist- Eletto D, Eletto D, Dersh D, Gidalevitz T, Argon Y (2014) Protein disulfide ing in MS analysis; Hao Hu and Taotao Wei (Institute of Biophysics) for help in isomerase A6 controls the decay of IRE1alpha signaling via disulfide- primary hepatocyte isolation; Pingyong Xu (Institute of Biophysics) for provid- dependent association. Mol Cell 53: 562 – 576 ing mEmerald and mApple plasmids; and Yunpeng Zhai, Yan Teng, Jianhui Li, Ghosh R, Wang L, Wang ES, Perera BG, Igbaria A, Morita S, Prado K, Thamsen and Junying Jia (Institute of Biophysics) for technical assistance. We also thank M, Caswell D, Macias H et al (2014) Allosteric inhibition of the IRE1alpha staff from Laboratory Animal Research Center of Institute of Biophysics, partic- RNase preserves cell viability and function during endoplasmic reticulum ularly Zhuanzhuan Xing and Jiaang Zhao, for in vitro fertilization and embryo stress. Cell 158: 534 – 548 transfer; Yongxia Liu, Sai Yang, and Ximei Zhang for breeding and manage- Han D, Lerner AG, Vande Walle L, Upton JP, Xu W, Hagen A, Backes BJ, Oakes ment of laboratory animals; and Lei Zhou for providing veterinary care and SA, Papa FR (2009) IRE1alpha kinase activation modes control alternate technical support. This work was supported by the National Key R&D Program endoribonuclease outputs to determine divergent cell fates. Cell 138: of China (2016YFA0500200, 2017YFA0504000); the National Natural Science 562 – 575 Foundation of China (31771261, 31571163, 31870761, 31770877, 11672317); Hatahet F, Ruddock LW (2009) Protein disulfide isomerase: a critical the Strategic Priority Research Program of CAS (XDB37020303)§; and the Youth evaluation of its function in disulfide bond formation. Antioxid Redox Innovation Promotion Association, CAS, to LW and XW. Signal 11: 2807 – 2850

§Correction added online on 18 May 2020, after first online publication: the funding information has been updated.

ª 2020 The Authors The EMBO Journal 39:e103841 | 2020 19 of 21 The EMBO Journal Jiaojiao Yu et al

Hetz C, Bernasconi P, Fisher J, Lee AH, Bassik MC, Antonsson B, Brandt GS, stress-induced neuronal cell death triggered by expanded polyglutamine Iwakoshi NN, Schinzel A, Glimcher LH et al (2006) Proapoptotic BAX and repeats. Genes Dev 16: 1345 – 1355 BAK modulate the unfolded protein response by a direct interaction with Oakes SA, Papa FR (2015) The role of endoplasmic reticulum stress in human IRE1alpha. Science 312: 572 – 576 pathology. Annu Rev Pathol 10: 173 – 194 Hollien J, Lin JH, Li H, Stevens N, Walter P, Weissman JS (2009) Regulated Orci L, Ravazzola M, Mack GJ, Barlowe C, Otte S (2003) Mammalian Ire1-dependent decay of messenger RNAs in mammalian cells. J Cell Biol Erv46 localizes to the endoplasmic reticulum-Golgi intermediate 186: 323 – 331 compartment and to cis-Golgi cisternae. Proc Natl Acad Sci USA 100: Humphrey W, Dalke A, Schulten K (1996) VMD: visual molecular dynamics. 4586 – 4591 J Mol Graph 14: 27 – 38 Otte S, Belden WJ, Heidtman M, Liu J, Jensen ON, Barlowe C (2001) Erv41p Kang T, Boland BB, Alarcon C, Grimsby JS, Rhodes CJ, Larsen MR (2019) and Erv46p: new components of COPII vesicles involved in transport Proteomic analysis of restored insulin production and trafficking in obese between the ER and Golgi complex. J Cell Biol 152: 503 – 518 diabetic mouse pancreatic islets following euglycemia. J Proteome Res 18: Perez-Riverol Y, Csordas A, Bai JW, Bernal-Llinares M, Hewapathirana S, 3245 – 3258 Kundu DJ, Inuganti A, Griss J, Mayer G, Eisenacher M et al (2019) The Karagoz GE, Acosta-Alvear D, Walter P (2019) The unfolded protein response: PRIDE database and related tools and resources in 2019: improving detecting and responding to fluctuations in the protein-folding capacity of support for quantification data. Nucleic Acids Res 47:D442 – D450 the endoplasmic reticulum. Cold Spring Harb Perspect Biol 11:a033886 Phillips JC, Braun R, Wang W, Gumbart J, Tajkhorshid E, Villa E, Chipot C, Kimata Y, Kimata YL, Shimizu Y, Abe H, Farcasanu IC, Takeuchi M, Rose MD, Skeel RD, Kale L, Schulten K (2005) Scalable molecular dynamics with Kohno K (2003) Genetic evidence for a role of BiP/Kar2 that regulates Ire1 NAMD. J Comput Chem 26: 1781 – 1802 in response to accumulation of unfolded proteins. Mol Biol Cell 14: Pollak AJ, Liu C, Gudlur A, Mayfield JE, Dalton ND, Gu Y, Chen J, Heller Brown 2559 – 2569 J, Hogan PG, Wiley SE et al (2018) A secretory pathway kinase regulates Klein MC, Zimmermann K, Schorr S, Landini M, Klemens PAW, Altensell J, Jung sarcoplasmic reticulum Ca(2 + ) homeostasis and protects against heart M, Krause E, Nguyen D, Helms V et al (2018) AXER is an ATP/ADP exchanger failure. Elife 7:e41378 in the membrane of the endoplasmic reticulum. Nat Commun 9: 3489 Preissler S, Rato C, Chen R, Antrobus R, Ding S, Fearnley IM, Ron D (2015) Koivunen P, Salo KE, Myllyharju J, Ruddock LW (2005) Three binding sites in AMPylation matches BiP activity to client protein load in the endoplasmic protein-disulfide isomerase cooperate in collagen prolyl 4-hydroxylase reticulum. Elife 4:e12621 tetramer assembly. J Biol Chem 280: 5227 – 5235 Preissler S, Rato C, Perera LA, Saudek V, Ron D (2017a) FICD acts Lee AH, Iwakoshi NN, Glimcher LH (2003) XBP-1 regulates a subset of bifunctionally to AMPylate and de-AMPylate the endoplasmic reticulum endoplasmic reticulum resident chaperone genes in the unfolded protein chaperone BiP. Nat Struct Mol Biol 24: 23 – 29 response. Mol Cell Biol 23: 7448 – 7459 Preissler S, Rohland L, Yan Y, Chen R, Read RJ, Ron D (2017b) AMPylation Lisbona F, Rojas-Rivera D, Thielen P, Zamorano S, Todd D, Martinon F, Glavic targets the rate-limiting step of BiP’s ATPase cycle for its functional A, Kress C, Lin JH, Walter P et al (2009) BAX inhibitor-1 is a negative inactivation. Elife 6:e29428 regulator of the ER stress sensor IRE1alpha. Mol Cell 33: 679 – 691 Preissler S, Ron D (2018) Early events in the endoplasmic reticulum unfolded Liu Y, Fares M, Dunham NP, Gao Z, Miao K, Jiang X, Bollinger SS, Boal AK, protein response. Cold Spring Harb Perspect Biol 10:a037309 Zhang X (2017) AgHalo: a facile fluorogenic sensor to detect drug-induced Raine J, Winter RM, Davey A, Tucker SM (1989) Unknown syndrome: proteome stress. Angew Chem Int Ed Engl 56: 8672 – 8676 microcephaly, hypoplastic nose, exophthalmos, gum hyperplasia, Liu Y, Miao K, Li Y, Fares M, Chen S, Zhang X (2018) A HaloTag-based cleft palate, low set ears, and osteosclerosis. J Med Genet 26: 786 – multicolor fluorogenic sensor visualizes and quantifies proteome stress in 788 live cells using solvatochromic and molecular rotor-based fluorophores. Rodriguez DA, Zamorano S, Lisbona F, Rojas-Rivera D, Urra H, Cubillos-Ruiz Biochemistry 57: 4663 – 4674 JR, Armisen R, Henriquez DR, Cheng EH, Letek M et al (2012)BH3-only Lyles MM, Gilbert HF (1991) Catalysis of the oxidative folding of ribonuclease proteins are part of a regulatory network that control the sustained A by protein disulfide isomerase: pre-steady-state kinetics and the signalling of the unfolded protein response sensor IRE1alpha. EMBO J 31: utilization of the oxidizing equivalents of the isomerase. Biochemistry 30: 2322 – 2335 619 – 625 Sepulveda D, Rojas-Rivera D, Rodriguez DA, Groenendyk J, Kohler A, MacKerell AD, Bashford D, Bellott M, Dunbrack RL, Evanseck JD, Field MJ, Lebeaupin C, Ito S, Urra H, Carreras-Sureda A, Hazari Y et al (2018) Fischer S, Gao J, Guo H, Ha S et al (1998) All-atom empirical potential for Interactome screening identifies the ER luminal chaperone Hsp47 as a molecular modeling and dynamics studies of proteins. J Phys Chem B 102: regulator of the unfolded protein response transducer IRE1alpha. Mol 3586 – 3616 Cell 69: 238 – 252 Morita S, Villalta SA, Feldman HC, Register AC, Rosenthal W, Hoffmann- Shoulders MD, Ryno LM, Genereux JC, Moresco JJ, Tu PG, Wu C, Yates JR III, Petersen IT, Mehdizadeh M, Ghosh R, Wang L, Colon-Negron K et al Su AI, Kelly JW, Wiseman RL (2013) Stress-independent activation of XBP1s (2017) Targeting ABL-IRE1 alpha signaling spares ER- stressed pancreatic and/or ATF6 reveals three functionally diverse ER proteostasis beta cells to reverse autoimmune diabetes. Cell Metab 25: 883 environments. Cell Rep 3: 1279 – 1292 Nguyen VD, Wallis K, Howard MJ, Haapalainen AM, Salo KE, Saaranen MJ, Sitia R, Braakman I (2003) Quality control in the endoplasmic reticulum Sidhu A, Wierenga RK, Freedman RB, Ruddock LW et al (2008) Alternative protein factory. Nature 426: 891 – 894 conformations of the x region of human protein disulphide-isomerase Tagliabracci VS, Engel JL, Wen J, Wiley SE, Worby CA, Kinch LN, Xiao J, Grishin modulate exposure of the substrate binding b’ domain. J Mol Biol 383: NV, Dixon JE (2012) Secreted kinase phosphorylates extracellular proteins 1144 – 1155 that regulate biomineralization. Science 336: 1150 – 1153 Nishitoh H, Matsuzawa A, Tobiume K, Saegusa K, Takeda K, Inoue K, Hori S, Tagliabracci VS, Pinna LA, Dixon JE (2013) Secreted protein kinases. Trends Kakizuka A, Ichijo H (2002) ASK1 is essential for endoplasmic reticulum Biochem Sci 38: 121 – 130

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Tagliabracci VS, Wiley SE, Guo X, Kinch LN, Durrant E, Wen J, Xiao J, Cui J, Wang C, Yu J, Huo L, Wang L, Feng W, Wang CC (2012) Human protein- Nguyen KB, Engel JL et al (2015) A single kinase generates the majority of disulfide isomerase is a redox-regulated chaperone activated by oxidation the secreted phosphoproteome. Cell 161: 1619 – 1632 of domain a’. J Biol Chem 287: 1139 – 1149 Takeda K, Nagashima S, Shiiba I, Uda A, Tokuyama T, Ito N, Fukuda T, Wang C, Li W, Ren JQ, Fang JQ, Ke HM, Gong WM, Feng W, Wang CC Matsushita N, Ishido S, Iwawaki T et al (2019) MITOL prevents ER stress- (2013) Structural insights into the redox-regulated dynamic induced apoptosis by IRE1alpha ubiquitylation at ER-mitochondria conformations of human protein disulfide isomerase. Antioxid Redox contact sites. EMBO J 38:e100999 Signal 19: 44 – 53 Upton JP, Wang L, Han D, Wang ES, Huskey NE, Lim L, Truitt M, McManus Wang L, Wang X, Wang CC (2015) Protein disulfide-isomerase, a folding MT, Ruggero D, Goga A et al (2012) IRE1alpha cleaves select microRNAs catalyst and a redox-regulated chaperone. Free Radic Biol Med 83: during ER stress to derepress translation of proapoptotic Caspase-2. 305 – 313 Science 338: 818 – 822 Wang M, Kaufman RJ (2016) Protein misfolding in the endoplasmic reticulum Urano F, Wang XZ, Bertolotti A, Zhang YH, Chung P, Harding HP, Ron D as a conduit to human disease. Nature 529: 326 – 335 (2000) Coupling of stress in the ER to activation of JNK protein kinases by Wang L, Zhou J, Wang L, Wang CC, Essex DW (2019) The b’ domain of transmembrane protein kinase IRE1. Science 287: 664 – 666 protein disulfide isomerase cooperates with the a and a’ domains to Ushioda R, Nagata K (2019) Redox-mediated regulatory mechanisms of functionally interact with platelets. J Thromb Haemost 17: 371 – 382 endoplasmic reticulum homeostasis. Cold Spring Harb Perspect Biol 11: Xiao JY, Tagliabracci VS, Wen JZ, Kim SA, Dixon JE (2013) Crystal a033910 structure of the Golgi casein kinase. Proc Natl Acad Sci USA 110: Velasco L, Dublang L, Moro F, Muga A (2019) The complex phosphorylation 10574 – 10579 patterns that regulate the activity of Hsp70 and its cochaperones. Int J Yang S, Wang X, Cui L, Ding X, Niu L, Yang F, Wang C, Wang CC, Lou J (2014) Mol Sci 20: 4122 Compact conformations of human protein disulfide isomerase. PLoS One 9: Voth W, Jakob U (2017) Stress-activated chaperones: a first line of defense. e103472 Trends Biochem Sci 42: 899 – 913 Yoshida H, Matsui T, Yamamoto A, Okada T, Mori K (2001) XBP1 mRNA is Walter P, Ron D (2011) The unfolded protein response: from stress pathway induced by ATF6 and spliced by IRE1 in response to ER stress to produce to homeostatic regulation. Science 334: 1081 – 1086 a highly active transcription factor. Cell 107: 881 – 891 Wang C, Chen S, Wang X, Wang L, Wallis AK, Freedman RB, Wang CC (2010) Zhang L, Niu Y, Zhu L, Fang J, Wang X, Wang L, Wang CC (2014) Different Plasticity of human protein disulfide isomerase: evidence for mobility interaction modes for protein-disulfide isomerase (PDI) as an efficient around the X-linker region and its functional significance. J Biol Chem 285: regulator and a specific substrate of endoplasmic reticulum 26788 – 26797 oxidoreductin-1alpha (Ero1alpha). J Biol Chem 289: 31188 – 31199 Wang CC, Tsou CL (1993) Protein disulfide isomerase is both an enzyme and Zhang J, Zhu Q, Wang X, Yu J, Chen X, Wang J, Wang X, Xiao J, Wang CC, a chaperone. FASEB J 7: 1515 – 1517 Wang L (2018) Secretory kinase Fam20C tunes endoplasmic reticulum Wang L, Li SJ, Sidhu A, Zhu L, Liang Y, Freedman RB, Wang CC (2009) redox state via phosphorylation of Ero1alpha. EMBO J 37:e98699 Reconstitution of human Ero1-Lalpha/protein-disulfide isomerase Zhou J, Liu CY, Back SH, Clark RL, Peisach D, Xu Z, Kaufman RJ (2006) The oxidative folding pathway in vitro. Position-dependent differences in role crystal structure of human IRE1 luminal domain reveals a conserved between the a and a’ domains of protein-disulfide isomerase. J Biol Chem dimerization interface required for activation of the unfolded protein 284: 199 – 206 response. Proc Natl Acad Sci USA 103: 14343 – 14348

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