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.
Highlights
Cu, Zn superoxide dismutase (SOD1) is one of the genes implicated in the devastating neurodegenerative disorder amyotrophic lateral sclerosis (ALS)
We conducted a high-throughput screening based on time-resolved FRET to further investigate the SOD1WT conformational change, and we used a genomewide small interfering RNA (siRNA) screen to search for regulators of SOD1 proteostasis
We found that damage-specific DNA-binding protein 1 (DDB1)- and CUL4-associated factor 4 (DCAF4) mediates the ubiquitination of an ALS-associated protein and autophagy receptor, optineurin (OPTN), and facilitates autophagic degradation of Derlin-1– binding region (DBR)-exposed SOD1
Summary
Zn superoxide dismutase (SOD1) is one of the genes implicated in the devastating neurodegenerative disorder amyotrophic lateral sclerosis (ALS). We conducted a high-throughput screening based on time-resolved FRET to further investigate the SOD1WT conformational change, and we used a genomewide 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. We previously reported that more than 100 different versions of SOD1mut interact with Derlin-1, which is a component of the ER-associated degradation (ERAD) machinery [11,12,13,14] This interaction causes a defect in the ERAD system, resulting in the induction of ER stress and eventually motoneuron death [11]. Inhibition of the SOD1–Derlin-1 interaction with a small-molecule compound ameliorated the ALS pathology in an in vitro model using patient-derived iPS motoneurons with SOD1 mutation and an in vivo model using ALS model mice expressing human SOD1mut [15]. Mutation in SOD1 causes a conformational change and exposure of the DBR, resulting in interaction of SOD1mut with Derlin-1 [12]
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