Volume 52, Issue 1, Pages 75-86 (October 2013) SOD1 as a Molecular Switch for Initiating the Homeostatic ER Stress Response under Zinc Deficiency  Kengo Homma, Takao Fujisawa, Naomi Tsuburaya, Namiko Yamaguchi, Hisae Kadowaki, Kohsuke Takeda, Hideki Nishitoh, Atsushi Matsuzawa, Isao Naguro, Hidenori Ichijo  Molecular Cell  Volume 52, Issue 1, Pages 75-86 (October 2013) DOI: 10.1016/j.molcel.2013.08.038 Copyright © 2013 Elsevier Inc. Terms and Conditions

Molecular Cell 2013 52, 75-86DOI: (10.1016/j.molcel.2013.08.038) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 1 Characterization of the Serum Factor that Inhibits the Starvation-Induced SOD1WT-Derlin-1 Interaction (A–F) IP immunoblotting (IP-IB) analysis of HEK 293A cells. (A) Transfected cells were serum starved for the indicated times. FBS was reintroduced after 60 hr of serum starvation for 12 hr. (B–F) Cells transfected with FLAG-SOD1 and Venus-Derlin-1(CT4)-HA were serum starved for 60 hr. Each sample was prepared as described in the Supplemental Experimental Procedures and added to the culture medium as indicated for 12 hr. The lanes were removed in (D). Molecular Cell 2013 52, 75-86DOI: (10.1016/j.molcel.2013.08.038) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 2 Zinc Deficiency Induces a Mutant-like Conformation in SOD1WT and Exposes the DBR (A and B) IP-IB analysis of the SOD1-Derlin-1 interaction. Transfected HEK 293A cells were incubated either with or without the indicated metals for 12 hr after 60 hr of FBS starvation. Mn, MnCl2; Ni, NiCl2; Mg, MgCl2; Ca, CaCl2; Zn, ZnCl2; Cu, CuSO4; and Fe, K4[Fe(CN)6]. (C) IP-IB analysis of the SOD1 conformational change. HeLa cells were incubated with or without zinc for the indicated times after 36 hr of FBS starvation. LC, light chain. (D) FBS was fractionated by Superdex 75 column in PBS. HEK 293A cells transfected with FLAG-SOD1 and Venus-Derlin-1(CT4)-HA were subjected to 60 hr FBS starvation and reintroduced with these fractions for 12 hr. IP-IB analysis of SOD1-Derlin-1 interaction (top). The zinc concentration of each fraction (bottom) (mean ± SD, n = 3). (E–G) IP-IB analysis of the SOD1-Derlin-1 interaction. Transfected HEK 293A cells were treated with 10 μM TPEN (E–G) or 1 mM EDTA (E) for the indicated times. (H and I) HEK 293A (H) and HeLa (H and I) cells were stimulated with TPEN for the indicated times. C, control rat IgG (H) or control rabbit IgG (I). LC, light chain. See also Figure S1. Molecular Cell 2013 52, 75-86DOI: (10.1016/j.molcel.2013.08.038) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 3 Loss of Zinc Coordination Induces a Mutant-like Conformation in SOD1WT (A–E) IP-IB analysis in transfected HEK 293A cells. Cells were stimulated with TPEN after pretreatment with or without 50 μg/ml CHX and 10 μM MG132 for 3 hr (A). Transfected cells were stimulated with 10 μM TPEN for 4 hr. Then, the culture medium was exchanged for FBS-free medium containing 25 μg/ml CHX and 2.5 μM MG132 with FBS or the indicated zinc concentration for 12 hr (B). Transfected cells were stimulated with TPEN as indicated (C–E). Blue boxes, zinc binding site SOD1 mutants; orange boxes, copper binding site SOD1 mutants. The lanes were removed in (C). (F) IP-IB analysis of SOD1 conformation. Lysates from HEK 293A cells transfected with FLAG-SOD1WT or FLAG-SOD1 mutants were immunoprecipitated with the FLAG antibody. Purified proteins were incubated with EDTA in an acidic buffer (pH 3.7) for 1 hr. After neutralization by dialysis, the samples were incubated with ZnCl2 for 6 hr. Molecular Cell 2013 52, 75-86DOI: (10.1016/j.molcel.2013.08.038) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 4 Zinc Deficiency Induces ER Stress through the SOD1WT-Derlin-1 Interaction (A) Gene expression analysis of HEK 293A cells incubated with 10 μM TPEN through qRT-PCR (n = 3). (B) IB analysis in HeLa cells stimulated with the indicated TPEN concentrations, 5 μg/ml tunicamycin (Tu) or 2 μM thapsigargin (Tg). Asterisks, nonspecific bands; arrows, Herp-specific bands. (C) Gene expression analysis in HEK 293A cells. The cells were stimulated with 10 μM TPEN in FBS-free medium for 4 hr; TPEN was then removed for 8 hr. FBS or the indicated metals were added, and the samples were incubated for another 12 hr (n = 3, ANOVA). (D) Gene expression analysis in HEK 293A cells transfected with a control, SOD1, or Derlin-1 siRNA stimulated with 10 μM TPEN for 12 hr (n = 4, ANOVA). (E) IB analysis in HeLa cells. Cells were transfected with a control, SOD1, or Derlin-1 siRNA and incubated either with or without 8 μM TPEN (TP) or 5 μg/ml tunicamycin (Tu) for 9 hr. All data are the mean ± SD. ∗p < 0.05, ∗∗p < 0.01. See also Figure S2 and Table S1. Molecular Cell 2013 52, 75-86DOI: (10.1016/j.molcel.2013.08.038) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 5 The SOD1-Derlin-1 Interaction Induces a Homeostatic ER Stress Response (A) IB analysis in HeLa cells stimulated with either 10 μM TPEN or 20 μM Tg for 30 min. p-PERK, phosphorylated PERK. (B) IB analysis in HeLa cells. Cells transfected with either control or Derlin-1 siRNA were stimulated as in (A). (C and D) Protein synthesis assay for HeLa cells transfected with control or Derlin-1 siRNA. After stimulation with 5 μM TPEN for the indicated times, the cells were metabolically labeled with [35S] methionine and cysteine (C). The level of radioactivity relative to the total protein in each sample was calculated. The decrease is shown as a percentage of the intensity observed at 0 hr TPEN stimulation (D) (n = 4, ANOVA). (E and F) Viability was measured for HeLa cells transfected with control or Derlin-1 siRNA. The cells were stimulated with either TPEN or staurosporine as indicated for 24 hr (E) (n = 3, ANOVA). The cells were pretreated with 50 μg/ml CHX for 15 min, washed out and then stimulated with TPEN as indicated for 24 hr (F) (n = 3, t test). The decrease is shown as a percentage of the viability observed in the nonstimulated condition. The data are the mean ± SD. ∗p < 0.05, ∗∗p < 0.01. See also Figure S3. Molecular Cell 2013 52, 75-86DOI: (10.1016/j.molcel.2013.08.038) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 6 The UPR Stimulated by Zinc-Deficient Conditions Induced ZIP14 (A) Gene expression analysis for mice livers that were intraperitoneally injected with either 20 mg/kg TPEN or 2 mg/kg tunicamycin for 12 hr via quantitative RT-PCR (control: n = 4, TPEN and Tu: n = 3, ANOVA). (B) Gene expression analysis in HepG2 stimulated with either 5 μM TPEN or 5 μg/ml tunicamycin for 24 hr (n = 3). (C) IB analysis of ZIP14 in HepG2 cells stimulated with 5 μM TPEN for 20 hr. (D) IP-IB analysis of SOD1 conformational change. HepG2 cells were stimulated with 8 μM TPEN for 12 hr. C, control rat IgG. (E and F) IB analysis of ZIP14 in HepG2 cells. The cells were infected with either a GFP or ATF6 1-380-FLAG-expressing adenovirus. Lysates were treated either with or without PNGaseF. Asterisks, nonspecific bands; parenthesis, glycosylated ZIP14; and arrow, deglycosylated ZIP14 (E). Cells infected with either Venus-HA or ATF6 171-380-FLAG-expressing lentiviruses were stimulated with 5 μM TPEN and 10 μM JNK inhibitor (SP600125) for 20 hr (F). The data are the mean ± SD. ∗p < 0.05, ∗∗p < 0.01. See also Figure S4. Molecular Cell 2013 52, 75-86DOI: (10.1016/j.molcel.2013.08.038) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 7 Schematic Representation of the Stress Response Induced by the SOD1-Derlin-1 Interaction The dissociation of zinc from SOD1 during zinc depletion induces a reversible conformational change in SOD1WT. The mutant-like SOD1 that exposes the DBR interacts with Derlin-1 and induces the homeostatic ER stress response. The UPR triggered by the zinc-depletion-induced SOD1-Derlin-1 interaction upregulate the UPR target genes, including chaperones, and attenuate the translation through the PERK-eIF2α pathway, preventing the accumulation of misfolded proteins. The activation of ATF6 also contributes to ZIP14 induction and facilitates zinc incorporation under zinc-deficient conditions. Molecular Cell 2013 52, 75-86DOI: (10.1016/j.molcel.2013.08.038) Copyright © 2013 Elsevier Inc. Terms and Conditions