Genome-wide siRNA screening reveals that DCAF4-mediated ubiquitination of optineurin stimulates autophagic degradation of Cu,Zn-superoxide dismutase

TR-FRET–based genome-wide siRNA screen for the regulators of SOD1 proteostasis

We have previously generated two antibodies that can specifically recognize DBR-exposed SOD1 in the immunoprecipitation assay (MS785 and MS27) (12, 21). During the analysis of the conformational change of SOD1^WT with these antibodies (MS antibodies), we noticed that a portion of the SOD1^WT population took the DBR-exposed conformation even in the absence of zinc deficiency (Fig. 1A, see long exposure panel). Previously, we have reported that even endogenous SOD1 adopted the DBR-exposed conformation during zinc deficiency, and several reports have indicated the involvement of conformationally-disordered SOD1^WT in SOD1 mutation-negative sporadic ALS (SALS). Taken together, we assumed the presence of an equilibrium state between the DBR-masked and DBR-exposed conformation even in SOD1^WT. Because SOD1^WT mainly takes the DBR-masked conformation and unknown factors appear to be required for the zinc deficiency–dependent conformational change, there should be a regulator(s) of SOD1 proteostasis. To reveal the molecular mechanism of SOD1 proteostasis, we attempted to identify the factors that were required for sequestering DBR-exposed SOD1^WT through a genome-wide siRNA screen.

The high-throughput assay system used to detect the SOD1 conformational change was established by utilizing time-resolved FRET (TR-FRET) technology (Fig. 1B). Here, an anti-tag antibody and anti-mutant SOD1-specific antibody (MS785) labeled with either a donor fluorophore (europium cryptate: Eu) or an acceptor fluorophore (d₂) were used (12). We searched the SOD1^WT construct that efficiently generates FRET signal in a zinc depletion-dependent manner. HEK293A cells were transfected with several SOD1^WT constructs and stimulated with the cell-permeable zinc chelator N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN). Then, the cell lysates were mixed with the fluorophore-labeled antibodies (Fig. S1A). The combination of SOD1^WT–Flag, an anti–Flag antibody labeled with Eu (Flag-Eu), and MS785 labeled with d₂ (MS785-d₂) efficiently generated a TPEN-dependent FRET signal (Fig. S1B).

Next, we established HeLa cells stably expressing SOD1^WT–Flag and checked the induction of a FRET signal with TPEN stimulation. By using these HeLa cells, we could obtain a robust signal increase in a high-throughput manner, which was sufficient for siRNA screens (Z′-factor >0.7) (Fig. 1C). Using this assay system, we carried out a genome-wide siRNA screen to identify factors required for depleting DBR-exposed SOD1^WT. In our screen, we explored genes whose suppression caused an increase in the FRET signal, indicating accumulation of DBR-exposed SOD1^WT even in the absence of zinc deficiency. In the 1st screening, we selected the genes that showed a score higher than 1.96 either in the robust Z-score or B-score as positive genes (Fig. 1D and Fig. S1C). In the 2nd screen, we reassessed these positive genes in duplicate. To narrow down the candidate genes, genes that showed a Z-score higher than 2.58 in duplicate were regarded as positive (Fig. 2A and Fig. S1D). Finally, we obtained 30 candidate genes that may be regulators of SOD1 proteostasis (Fig. 1D and Table S1).

DCAF4 specifically interacts with DBR-exposed SOD1 through DBR

The Z score of the hit gene is relatively low in the screen, and we focused on the genes with high Z score (Table S1). There were genes related to the nucleotide metabolism, transcription, and cytoskeleton. However, we estimated that it is unlikely that these gene products directly regulate SOD1 proteostasis. The cullin–RING E3 ubiquitin ligases (CRLs) are the largest E3 ligase family and regulate many physiological processes, including proteostasis. Thus, among the candidate genes, we focused on damage-specific DNA-binding protein 1 (DDB1)–Cullin4 (CUL4)-associated factor 4 (DCAF4), which is speculated to function as a cellular substrate receptor of the CRL4 complex (Fig. 2A) (20). Suppression of DCAF4 with two independent siRNAs that differ from those used in the screens caused an accumulation of DBR-exposed SOD1^WT even in the absence of zinc deficiency, confirming the result of the screens (Fig. 2B and Fig. S2, A and B). To evaluate the involvement of DCAF4 in endogenous SOD1 proteostasis, the amount of zinc deficiency–induced DBR-exposed endogenous SOD1 was investigated in DCAF4 knockdown cells (Fig. 2C and Fig. S2C). Suppression of DCAF4 induced an enhanced accumulation of DBR-exposed SOD1. These results demonstrate that DCAF4 is required to inhibit the accumulation of DBR-exposed SOD1. Because DCAF4 is predicted to be a substrate receptor of the CRL4 complex, we also investigated the involvement of CUL4A and CUL4B. Suppression of both CUL4A and CUL4B also induced accumulation of DBR-exposed SOD1^WT, suggesting the involvement of the CRL4 complex (Fig. S2D).

To reveal the molecular mechanisms by which DCAF4 mediated the reduction in DBR-exposed SOD1, we first investigated their interaction. DCAF4 specifically interacts with SOD1^mut but not with SOD1^WT (Fig. 2D). This interaction was diminished with DBR-deleted SOD1^G93A(21–153) (Fig. 2E). In addition, SOD1(1–20), which includes the DBR, was sufficient for interaction with DCAF4. Because SOD1^WT possesses a masked DBR, which is exposed under zinc deficiency, we investigated the interaction between SOD1^WT and DCAF4 during zinc depletion. As expected, zinc deficiency induced the association of DCAF4 with SOD1^WT (Fig. S2E). These data indicate that DCAF4 specifically recognizes and interacts with DBR-exposed SOD1.

DCAF4 facilitates autophagic degradation of DBR-exposed SOD1

We next investigated the possibility that DCAF4 facilitates the degradation of DBR-exposed SOD1. To reveal the degradation pathway of DBR-exposed SOD1, we assessed the effect of MG132, a proteasome inhibitor, wortmannin and bafilomycin A1, autophagy inhibitors, on the amount of DBR-exposed SOD1. Treatment with wortmannin and bafilomycin A1 increased the amount of DBR-exposed SOD1 in both SOD1^WT and SOD1^G93A, although there was little effect with MG132 treatment (Fig. 3A and Fig. S3A). These data indicate that DBR-exposed SOD1 was predominantly degraded through the autophagic pathway, as suggested previously in studies using SOD1^mut (22, 23).

To further evaluate the involvement of DCAF4 in autophagic degradation of DBR-exposed SOD1, we assessed the effect of autophagy inhibition in DCAF4 knockdown cells. Wortmannin treatment failed to increase the level of DBR-exposed SOD1^G93A and DBR-exposed SOD1^WT in DCAF4 knockdown cells compared with control cells (Fig. 3, B and C, and Fig. S3, B and C). To confirm that DCAF4 contributes to the degradation of DBR-exposed SOD1, the degradation rate of DBR-exposed SOD1 was assessed with a pulse–chase experiment. The DCAF4-suppressed cells showed an attenuated rate of DBR-exposed SOD1^mut degradation compared with the control cells (Fig. 3, D and E). Collectively, these data indicate that DCAF4 contributes to autophagic degradation of DBR-exposed SOD1.

OPTN is involved in degradation of DBR-exposed SOD1

We speculated that DCAF4 mediates the ubiquitination of SOD1^mut that leads to the autophagic degradation of DBR-exposed SOD1. However, the accumulation of ubiquitinated SOD1 could not be detected even in the presence of wortmannin (Fig. S4A). Thus, we assumed that the substrate of DCAF4 is not DBR-exposed SOD1 itself. One of the candidate substrates was the autophagic receptor. Among the autophagic receptors, OPTN and SQSTM1/p62 are reported to be ALS-causative genes (24, 25). Therefore, we investigated their potential involvement in the DBR-exposed SOD1 degradation.

First, we examined the interaction of these receptors with SOD1 and found that OPTN preferentially interacted with SOD1^G93A and TPEN-stimulated SOD1^WT rather than SOD1^WT, whereas the interaction between SOD1^mut and p62 could not be detected under the tested conditions (Fig. 4, A and B, and Fig. S4B). Next, we tested whether OPTN also recognizes and interacts with the exposed DBR. In contrast with DCAF4, DBR-deleted SOD1^G93A(21–153) showed a weak but substantial interaction with OPTN even though its expression level was modest (Fig. 4C). In addition, the SOD1(1–20) fragment did not interact with OPTN (comparison of Flag–SOD1(1–20)–CFP with Flag–CFP). Thus, it is unlikely that OPTN interacts with the exposed DBR. OPTN may recognize the other region of conformationally disordered SOD1 (see “Discussion”).

To examine the contribution of OPTN to SOD1 proteostasis, we suppressed the expression of OPTN and assessed the amount of DBR-exposed SOD1^WT. The suppression of OPTN expression induced accumulation of DBR-exposed SOD1^WT, suggesting that OPTN is required for autophagic degradation of DBR-exposed SOD1 (Fig. 4D and Fig. S4C). Several OPTN mutants have been reported to be associated with ALS (26). We speculated that these mutants lost the ability to maintain SOD1 proteostasis. Although the interactions between SOD1^G93A and two representative ALS-related OPTN mutants (OPTN^Q398X and OPTN^E478G) were preserved (Fig. S4D), they failed to reduce the amount of DBR-exposed SOD1^G93A by co-expression, suggesting that they were inactive in terms of autophagic degradation of DBR-exposed SOD1 (Fig. 4E and Fig. S4E).

DCAF4-mediated ubiquitination of OPTN facilitates degradation of DBR-exposed SOD1

Because OPTN contributed to the degradation of DBR-exposed SOD1 and an interaction between DCAF4 and OPTN could be observed (Fig. 5A), we now assumed that DCAF4-mediated OPTN ubiquitination could be involved in the autophagic degradation of DBR-exposed SOD1. Thus, we first examined whether OPTN is ubiquitinated or not by the immunoprecipitation analysis of OPTN in a denatured condition to prevent a contamination of ubiquitinated substrates of OPTN followed by the detection of ubiquitinated proteins. Ubiquitinated OPTN was clearly observed (Fig. 5B). Then, the ubiquitination of OPTN was assessed in the DCAF4 knockdown cells. Suppression of DCAF4 partially but significantly attenuated the ubiquitination of OPTN, suggesting that the CRL4 complex, including DCAF4, ubiquitinates OPTN (Fig. 5B and Fig. S5). Next, we aimed to identify the ubiquitination site in OPTN. Because we found that the OPTN^Q398X mutant was at least less ubiquitinated compared with the OPTN^WT (Fig. 5C), all the lysine residues after 399 were mutated to arginine. Among these mutants, OPTN^K501R showed resistance to ubiquitination (Fig. 5C). This is consistent with a previous report that identified the lysine 501 residue as a ubiquitination site in OPTN via MS (27). Notably, ubiquitination was also absent in the OPTN^E478G mutant (Fig. 5D). These data demonstrate that DCAF4-including CRL4 complex mediates OPTN ubiquitination at lysine 501, and this ubiquitination is absent in the ALS-related OPTN^mut.

To evaluate the functional importance of this ubiquitination in SOD1 proteostasis, the ability of OPTN^K501R to reduce the amount of DBR-exposed SOD1 was assessed. Exogenous expression of OPTN^WT but not OPTN^K501R reduced the amount of DBR-exposed SOD1^G93A and SOD1^G37R (Fig. 5E), demonstrating a requirement of the ubiquitination for facilitating the degradation of DBR-exposed SOD1.

Antibodies

Anti-tag antibodies labeled with either Eu or d₂ (Fig. S1A) were purchased from Cisbio. The antibodies against Flag tag (Wako, 012-22384), HA tag (Roche Applied Science, 1867431), SOD1 (Enzo Life Science, ADI-SOD-100), ubiquitin (Santa Cruz Biotechnology, sc-8017), OPTN (Proteintech, 10837-1-AP), Cullin4 (Abcam, ab76470), and p62 (Progen Biotechnik, GP62-C) were purchased from the indicated suppliers. Control rat immunoglobulin G1 mAb was purchased from BioGenesis. MS785 and MS27 were prepared as described previously (12, 21). The purified MS785 was labeled with either Eu or d₂ using a d₂-labeling kit or europium cryptate labeling kit (Cisbio) according to the manufacturer's instructions.

Cell culture

HEK293A cells were purchased from Invitrogen and cultured in Dulbecco's modified Eagle's medium (DMEM) containing 4.5 mg/ml glucose (Sigma, D5796), 10% FBS, and 100 units/ml penicillin (Meiji Seika, 6111400D2039). HeLa cells were purchased from ATCC and cultured in DMEM containing 1.0 mg/ml glucose (Sigma, D6046) supplemented with 10% FBS and 100 units/ml penicillin G. HeLa cells stably-expressing SOD1^WT C-terminally tagged with Flag (SOD1^WT–Flag stable HeLa cells) were previously generated (21) and cultured in DMEM containing 1.0 mg/ml glucose, 10% FBS, 100 units/ml penicillin, and 2 μg/ml puromycin (Gibco) in 5% CO₂ at 37 °C. All cells were cultured in 5% CO₂ at 37 °C and verified to be negative for mycoplasma.

Plasmids and transfection

The cDNAs encoding SOD1^WT, OPTN^WT, OPTN^mut, and DCAF4 were cloned into a pcDNA3.0 plasmid (Invitrogen) using standard molecular biology techniques, and all constructs were verified by sequencing. The pcDNA3.0–Flag–SOD1^WT, pcDNA3.0–Flag–SOD1^mut, Flag–SOD1^G93A(21–153), pcDNA3.0–Flag–CFP, and pcDNA3.0–Flag–SOD1(1–20)-CFP plasmids have been previously constructed (11, 12, 21).

Transfection was performed using polyethyleneimine-Max (Polysciences, 24765) according to the manufacturer's instructions. Transfections with siRNA were carried out by forward or reverse transfection using Lipofectamine RNAiMAX (Invitrogen, 133778-150) according to the manufacturer's instructions. When cells were transfected with both plasmid and siRNA, the transfection with siRNA was performed 12–24 h before the transfection with plasmid. The siRNAs used are listed in Table S2.

Immunoblotting analysis

Cells were lysed on ice in a lysis buffer (20 mm Tris-HCl, pH 7.5, 150 mm NaCl, 10 mm EDTA, 1% sodium deoxycholate, and 1% Triton X-100) supplemented with 1 mm phenylmethylsulfonyl fluoride, and 5 g/ml leupeptin. After centrifugation, supernatants were sampled by adding 2× SDS sample buffer (80 mm Tris-HCl, pH 8.8, 80 μg/ml bromphenol blue, 28.8% glycerol, 4% SDS, and 10 mm DTT). The samples were resolved by SDS-PAGE and electroblotted onto polyvinylidene difluoride membranes. After blocking with 5% skim milk in TBS-T (50 mm Tris-HCl, pH 8.0, 150 mm NaCl, and 0.05% Tween 20), the membranes were probed with each primary antibody. After incubation with the appropriate secondary antibodies, the proteins were detected in an ECL system using a ChemiDoc imaging system and Image Lab software (Bio-Rad). All immunoblotting analyses were repeated at least three times, and almost the same results were obtained.

Immunoprecipitation analysis

The supernatants of centrifuged cell lysates were immunoprecipitated with anti-Flag antibody (Sigma, A2220, or Wako Pure Chemicals Industries, 016-22784), MS785, MS27, or control rat immunoglobulin G1 mAb (BioGenesis) using protein G–Sepharose (GE Healthcare, 17061802). The beads were washed with washing buffer 1 (20 mm Tris-HCl, pH 7.5, 500 mm NaCl, 5 mm EGTA, and 1% Triton X-100) and washing buffer 2 (20 mm Tris-HCl, pH 7.5, 150 mm NaCl, and 5 mm EGTA) or with lysis buffer. The proteins were eluted with 2× SDS sample buffer or 3× Flag peptide (Sigma) in lysis buffer.

Preparation of assay plate for siRNA screen

The genome-wide siRNA library used in the primary screen consisted of siRNAs targeting 18,023 human genes (Dharmacon siGENOME SMARTpool collections: human genome (G-005005-E2), human drug targets (G-004655-E2), and human druggable subsets (G-004675-E2)). The assay plates (Greiner, 784086) for the 1st screen were prepared as reported previously with minor modification in layout and the amount of siRNA by using Biomek FX^P (Beckman Coulter), POD automation (LABCYTE), and Echo liquid handler (LABCYTE) (32). In the 2nd screen, the siRNAs targeting positive genes in the 1st screen were spotted onto a 384-well plate with POD automation and Echo liquid handler. The individual siRNAs targeting the same gene were placed on the same plate.

TR-FRET–based siRNA screen

Lipofectamine RNAiMAX in Opti-MEM (Invitrogen) was added at 3 μl/well to the assay plates using a Multidrop Combi. After 15 min, 3.33 × 10⁵ cells/ml of HeLa cells expressing SOD1^WT–Flag were seeded at 15 μl/well using the Multidrop Combi, and the final siRNA concentration was set at 30 nm. After 48 h of incubation, DMSO or TPEN (final 10 μm in reference well) was overlaid using the Multidrop Combi and manually, respectively. After 4.5 h of stimulation, the medium was eliminated by centrifugation. Then, the fluorophore-labeled antibodies and MS27 antibody (for stabilization of the DBR-exposed SOD1 (21)) in FRET buffer (25 mm phosphate buffer, pH 7.0, 400 mm KF, 0.1% BSA, 0.5% Triton X-100, 10 mm EDTA, 5 μg/ml leupeptin, 1 mm phenylmethylsulfonyl fluoride) were added with the Multidrop Combi. After 1 h of incubation at room temperature, the FRET intensity (337-nm excitation filter/665-nm emission filter) and donor intensity (337-nm excitation filter/620-nm emission filter) were measured using PHERAstar (BMG Labtech), and the FRET ratio was calculated as FRET intensity/donor intensity. FRET signal was the FRET ratio of the samples divided by that of the DMSO-treated samples. The Z′-factor between the DMSO- and TPEN-treated reference groups in each plate was set at over 0.2 (1st screen) or 0.5 (2nd screen). Unsatisfactory plates were discarded and recreated. To compare between plates, the FRET ratio was normalized with a sample-based robust Z-score and B-score (1st screen) or a reference-based Z-score (2nd screen) (33–35).

Quantitative RT-PCR (qRT-PCR)

Total RNA was isolated from cells using Isogen reagent (Wako) and reverse-transcribed using ReverTra Ace qPCR RT Master Mix with gDNA Remover (Toyobo). Primers were designed using the Universal Probe Library Assay Design Center (Roche Applied Science). Quantitative RT-PCR was carried out using SYBR Green PCR Master Mix and a LightCycler 96 (Roche Applied Science). The levels were normalized to the S18 mRNA level. The primer sequences are listed in Table S3.

Pulse–chase analysis

HeLa cells were labeled with l-[³⁵S]Met and l-[³⁵S]Cys (PerkinElmer Life Sciences) for 1 h in medium lacking methionine and cysteine. After washing with PBS, the cells were chased in HBSS(+). Cells were lysed on ice in lysis buffer, and the supernatants of centrifuged cell lysates were immunoprecipitated with MS785 and MS27. The beads were washed with washing buffer 1 and washing buffer 2. Immunoprecipitated samples were eluted with 2× SDS sample buffer, resolved by SDS-PAGE, and analyzed with an image analyzer (Storm 840, GE Healthcare). Quantification was performed using ImageJ software.

Ubiquitination assay

Cells were lysed in PBS containing 0.5% SDS, 5 μg/ml leupeptin, and 1 mm phenylmethylsulfonyl fluoride. The lysates were diluted with PBS and sonicated. The supernatants of cell lysates were immunoprecipitated with anti–Flag antibody. The beads were washed with washing buffer 1 and washing buffer 2. The proteins were eluted with 2× SDS sample buffer or 3× Flag peptide in washing buffer 2.

Statistical analysis

Statistical analyses were performed via ANOVA with a post hoc Dunnett's test. The values are the mean ± S.D. calculated from at least three different experiments. Significance was assumed with *, p < 0.05.

Materials and data availability

The materials and data that support the findings of this study are available from the corresponding author on reasonable request.