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

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.

A small-molecule inhibitor of SOD1-Derlin-1 interaction ameliorates pathology in an ALS mouse model.

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disorder. Despite its severity, there are no effective treatments because of the complexity of its pathogenesis. As one of the underlying mechanisms of Cu, Zn superoxide dismutase (SOD1) gene mutation-induced ALS, SOD1 mutants (SOD1mut) commonly interact with an endoplasmic reticulum-resident membrane protein Derlin-1, triggering motoneuron death. However, the importance of SOD1-Derlin-1 interaction in in vitro human model and in vivo mouse model remains to be elucidated. Here, we identify small-molecular-weight compounds that inhibit the SOD1-Derlin-1 interaction by screening approximately 160,000 compounds. The inhibitor prevents 122 types of SOD1mut from interacting with Derlin-1, and significantly ameliorates the ALS pathology both in motoneurons derived from patient induced pluripotent stem cells and in model mice. Our data suggest that the SOD1-Derlin-1 interaction contributes to the pathogenesis of ALS and is a promising drug target for ALS treatment.

A systematic immunoprecipitation approach reinforces the concept of common conformational alterations in amyotrophic lateral sclerosis-linked SOD1 mutants.

Mutations in the Cu, Zn superoxide dismutase (SOD1) gene are one of the causative agents of amyotrophic lateral sclerosis (ALS). Although more than 100 different mutations in SOD1 have been identified, it is unclear whether all the mutations are pathogenic or just single nucleotide polymorphisms (SNPs) unrelated to the disease. Our previous systematic analysis found that all pathogenic SOD1 mutants (SOD1(mut)) have a common property, namely, an association with Derlin-1, a component of the endoplasmic reticulum-associated degradation machinery. For the proposed mechanism, we found that most pathogenic SOD1(mut) have a constitutively exposed Derlin-1-binding region (DBR), which is concealed in wild-type SOD1 (SOD1(WT)). Moreover, we generated MS785, a monoclonal antibody against DBR. MS785 distinguished most ALS-causative SOD1(mut) from both SOD1(WT) and non-toxic SOD1(mut). However, MS785 could not recognize SOD1(mut) that has mutations in the MS785 epitope region. Here, we developed a new diagnostic antibody, which could compensate for this shortcoming of MS785. We hypothesized that in ALS-causative SOD1(mut), the DBR-neighboring region [SOD1(30-40)] may also be exposed. We then generated MS27, a monoclonal antibody against SOD1(30-40). We found that MS27 could distinguish SOD1(WT) from the pathogenic SOD1(mut), which has mutations in the MS785 epitope region. Moreover, all pathogenic SOD1(mut), without exception, were immunoprecipitated with a combination of MS785 and MS27. The MS785-MS27 combination could be developed as a novel mechanism-based biomarker for the diagnosis of ALS.

SOD1 as a molecular switch for initiating the homeostatic ER stress response under zinc deficiency.

Zinc is an essential trace element, and impaired zinc homeostasis is implicated in the pathogenesis of various human diseases. However, the mechanisms cells use to respond to zinc deficiency are poorly understood. We previously reported that amyotrophic lateral sclerosis (ALS)-linked pathogenic mutants of SOD1 cause chronic endoplasmic reticulum (ER) stress through specific interactions with Derlin-1, which is a component of the ER-associated degradation machinery. Moreover, we recently demonstrated that this interaction is common to ALS-linked SOD1 mutants, and wild-type SOD1 (SOD1(WT)) comprises a masked Derlin-1 binding region (DBR). Here, we found that, under zinc-deficient conditions, SOD1(WT) adopts a mutant-like conformation that exposes the DBR and induces the homeostatic ER stress response, including the inhibition of protein synthesis and induction of a zinc transporter. We conclude that SOD1 has a function as a molecular switch that activates the ER stress response, which plays an important role in cellular homeostasis under zinc-deficient conditions.

A novel monoclonal antibody reveals a conformational alteration shared by amyotrophic lateral sclerosis-linked SOD1 mutants.

OBJECTIVE: Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that is characterized by the selective loss of upper and lower motoneurons. Although >100 different Cu, Zn superoxide dismutase (SOD1) mutations have been identified in ALS patients, it remains controversial whether all of them are disease-causative mutations. Therefore, it is necessary to develop molecular mechanism-based diagnosis and treatment of ALS caused by SOD1 mutations. METHODS: We previously reported that 3 pathogenic mutations of SOD1 cause chronic endoplasmic reticulum (ER) stress by inducing the binding of SOD1 to Derlin-1, a component of the ER homeostatic machinery. Here, we systematically analyzed 132 SOD1 mutants and found that most have a constitutively exposed Derlin-1-binding region (DBR) that is occluded in the wild-type protein. To develop the novel molecular mechanism-based antibody that can specifically recognize the aberrant structure of toxic SOD1 mutants, we generated the monoclonal antibody against the DBR. RESULTS: MS785, a monoclonal antibody generated against the DBR, distinguished most ALS-causative SOD1 mutants from both wild-type and nontoxic mutants. Moreover, MS785 recognized endogenous SOD1 in B lymphocytes derived from 14 ALS patients carrying SOD1 mutations but not from 11 healthy controls. INTERPRETATION: This is the first study to address the common property of all ALS-causative SOD1 mutants. MS785 is the first molecular mechanism-based antibody that was shown to be able to distinguish ALS-linked toxic SOD1 mutants from both wild-type and nontoxic mutants. MS785 may thus become an innovative tool for the diagnosis of ALS.