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. 2020 Mar 6;295(10):3148-3158.
doi: 10.1074/jbc.RA119.010239. Epub 2020 Feb 3.

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

Kengo Homma  1 , Hiromitsu Takahashi  2 , Naomi Tsuburaya  2 , Isao Naguro  2 , Takao Fujisawa  2 , Hidenori Ichijo  2
Affiliations

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

Kengo Homma et al. J Biol Chem. .

Abstract

Cu, Zn superoxide dismutase (SOD1) is one of the genes implicated in the devastating neurodegenerative disorder amyotrophic lateral sclerosis (ALS). Although the precise mechanisms of SOD1 mutant (SOD1mut)-induced motoneuron toxicity are still unclear, defects in SOD1 proteostasis are known to have a critical role in ALS pathogenesis. We previously reported that the SOD1mut adopts a conformation that exposes a Derlin-1-binding region (DBR) and that DBR-exposed SOD1 interacts with Derlin-1, leading to motoneuron death. We also found that an environmental change, i.e. zinc depletion, induces a conformational change in WT SOD1 (SOD1WT) to the DBR-exposed conformation, suggesting the presence of an equilibrium state between the DBR-masked and DBR-exposed states even with SOD1WT Here, we conducted a high-throughput screening based on time-resolved FRET to further investigate the SOD1WT conformational change, and we used a genome-wide siRNA screen to search for regulators of SOD1 proteostasis. This screen yielded 30 candidate genes that maintained an absence of the DBR-exposed SOD1WT conformation. Among these genes was one encoding DDB1- and CUL4-associated factor 4 (DCAF4), a substrate receptor of the E3 ubiquitin-protein ligase complex. Of note, we found that DCAF4 mediates the ubiquitination of an ALS-associated protein and autophagy receptor, optineurin (OPTN), and facilitates autophagic degradation of DBR-exposed SOD1. In summary, our screen identifies DCAF4 as being required for proper proteostasis of DBR-exposed SOD1, which may have potential relevance for the development of therapies for managing ALS.

Keywords: DDB1 and CUL4 associated factor 4 (DCAF4); amyotrophic lateral sclerosis (ALS) (Lou Gehrig disease); autophagy; genome-wide siRNA screen; protein aggregation; protein misfolding; proteostasis; superoxide dismutase 1 (SOD1).

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Conflict of interest statement

The authors declare that they have no conflicts of interest with the contents of this article

Figures

Figure 1.
Figure 1.
Screens of the genes involved in SOD1 proteostasis. A, immunoprecipitation–immunoblotting (IP–IB) assay. HeLa cells transfected with the indicated constructs were treated with 15 μm TPEN for 5 h. The cell lysates were analyzed with an IP–IB assay using the indicated antibodies. The amount of DBR-exposed SOD1 was analyzed. MS Abs: mixture of MS785 antibody and MS27 antibody. B, model of the TR-FRET–based conformational change assay showing the case of the Eu-labeled anti-tag antibody and the d2-labeled MS785 antibody. C, FRET signals of HeLa cells stably expressing SOD1WT—Flag with Flag–Eu and MS785–d2 are shown as fold changes relative to nonstimulated cells. The cells were stimulated with 10 μm TPEN for 4 h. The data are shown as the mean ± S.D. (n = 8). D, flow chart of the screens.
Figure 2.
Figure 2.
DCAF4 specifically interacts with DBR-exposed SOD1 through the DBR. A, results of the 2nd screen. The dots indicate the Z-score of the duplicate. The replicates that showed a higher Z-score are described as replicate 1. Gray dots indicate negative genes; black dots indicate positive genes; red dot indicates DCAF4. Red lines indicate 2.58. B–E, IP–IB analysis of each cell lysate with the indicated antibodies is shown. B, HeLa cells stably expressing SOD1WT–Flag were transfected with the indicated siRNAs. The amount of DBR-exposed SOD1 was analyzed. C, HeLa cells were transfected with siRNAs and treated with 10 μm TPEN for 5 h. The amount of DBR-exposed endogenous SOD1 was analyzed. D, HEK293A cells were transfected with the indicated constructs. The interaction between SOD1 and DCAF4 was analyzed. E, HEK293A cells were transfected with the indicated constructs and treated with 5 μm MG132 to stabilize SOD1 fragments for 8 h. The interaction between SOD1 and DCAF4 was analyzed.
Figure 3.
Figure 3.
DCAF4 facilitates autophagic degradation of DBR-exposed SOD1. A–C, IP–IB analysis of each cell lysate with the indicated antibodies. A, HeLa cells were transfected with Flag–SOD1WT or Flag–SOD1G93A and treated with MG132 (5 μm, 9 h), wortmannin (1 μm, 9 h), or bafilomycin (50 nm, 3 h). The amount of DBR-exposed SOD1 and p62 was analyzed. B, HeLa cells were transfected with the indicated siRNAs and Flag–SOD1G93A and then treated with 1 μm wortmannin. The amount of DBR-exposed SOD1 was analyzed. C, HeLa cells stably expressing SOD1WT–Flag were transfected with the indicated siRNAs. Then, the cells were treated with 2 μm wortmannin for 9 h. The amount of DBR-exposed SOD1 was analyzed. D and E, pulse–chase assay of DBR-exposed SOD1. HeLa cells were transfected with the indicated siRNAs and Flag–SOD1mut and then were metabolically labeled with [35S]methionine and -cysteine and chased in HBSS (+). The cell lysates were immunoprecipitated with MS785 and MS27. The relative level of radioactivity in each sample was calculated. The decrease is shown as a percentage of the intensity observed at 0 h. The data are the mean ± S.D.; *, p < 0.05; n = 3 (ANOVA with a post hoc Dunnett's test).
Figure 4.
Figure 4.
OPTN is involved in degradation of DBR-exposed SOD1. A–E, IP–IB analysis of each of the cell lysates with the indicated antibodies. A, HeLa cells were transfected with the indicated constructs. The interaction between SOD1 and endogenous OPTN or p62 was analyzed. B, HeLa cells stably expressing SOD1WT–Flag were stimulated with 10 μm TPEN for 5 h. The interaction with endogenous OPTN was analyzed. C, HeLa cells were transfected with the indicated constructs, and the interaction with endogenous OPTN was analyzed. D, HeLa cells stably expressing SOD1WT–Flag were transfected with the indicated siRNAs. The amount of DBR-exposed SOD1 was analyzed. E, HeLa cells were transfected with the indicated constructs. The amount of DBR-exposed SOD1 was analyzed.
Figure 5.
Figure 5.
DCAF4-mediated ubiquitination of OPTN facilitates the degradation of DBR-exposed SOD1. A–E, IP–IB analysis of each of the cell lysates with the indicated antibodies. A, HeLa cells were transfected with the indicated constructs, and the interaction between OPTN and DCAF4 was analyzed. B, HeLa cells were transfected with the indicated siRNAs and Flag–OPTN. The ubiquitination of OPTN was evaluated. C and D, HeLa cells were transfected with the indicated Flag–OPTN constructs. The ubiquitination of OPTN was evaluated. E, left, HeLa cells were transfected with the indicated constructs. The amount of DBR-exposed SOD1 was analyzed. Right, DBR-exposed/total SOD1 ratios are shown as the mean ± S.D. (fold to nontreated SOD1G93A, n = 3). **, p < 0.01 (ANOVA with a post hoc Bonferroni). *, nonspecific band.
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