Nuclear and cytoplasmic WDR-23 isoforms mediate differential effects on GEN-1 and SKN-1 substrates

Brett N Spatola,Bin Wang,Jacqueline Y Lo,Sean P Curran

doi:10.1038/s41598-019-48286-y

Abstract

Maintaining a healthy cellular environment requires the constant control of proteostasis. E3 ubiquitin ligase complexes facilitate the post-translational addition of ubiquitin, which based on the quantity and specific lysine linkages, results in different outcomes. Our studies reveal the CUL4-DDB1 substrate receptor, WDR23, as both a positive and a negative regulator in cellular stress responses. These opposing roles are mediated by two distinct isoforms: WDR-23A in the cytoplasm and WDR-23B in the nucleus. C. elegans expressing only WDR-23A display activation of SKN-1 and enhanced survival to oxidative stress, whereas animals with restricted WDR-23B expression do not. Additionally, we identify GEN-1, a Holliday junction resolvase, as an evolutionarily conserved WDR-23 substrate and find that the nuclear and cytoplasmic isoforms of WDR-23 differentially affect double-strand break repair. Our results suggest that through differential ubiquitination, nuclear WDR-23B inhibits the activity of substrates, most likely by promoting protein turnover, while cytoplasmic WDR-23A performs a proteasome-independent role. Together, our results establish a cooperative role between two spatially distinct isoforms of WDR-23 in ensuring proper regulation of WDR-23 substrates.

Highlights

Proteostasis plays an integral part in ensuring organismal survival, and numerous diseases can arise when the balance of the creation, maintenance, and degradation of proteins is dysregulated[1,2]
We investigated if WDR-23 isoforms differentially regulate SKN-1, a known substrate for WDR-23
The expression of gcs-1 was not restored in animals with expression of only WDR-23B; it is notable that the impact of SKN-1 activation on gst-4 and gcs-1 differs by an order of magnitude, perhaps indicating differences in the regulatory pathways that govern these genes beyond SKN-1

Summary

Introduction

Proteostasis plays an integral part in ensuring organismal survival, and numerous diseases can arise when the balance of the creation, maintenance, and degradation of proteins is dysregulated[1,2]. One of the most abundant, yet complex, group of E3 ubiquitin ligases are the cullin-RING ligases (CRLs), which form multi-protein complexes. Adaptor proteins form the bridge between the cullin and the substrate receptor, which directly bind to substrates. DDB1-CUL4 associated factors (DCAFs), are an important part of the CRL, but there is a gap in the identification of specific substrates[7]. WDR-23, the C. elegans homolog of mammalian DCAF11/WDR23, is a CRL substrate receptor that exists in two spatially distinct isoforms, a cytoplasmic WDR-23A and nuclear WDR-23B16–18. The possibility of proteasome-independent functions have been documented for DCAF11, as DCAF11 was found to ensure histone supply during DNA replication via ubiquitination of its substrate SLBP (stem-loop binding protein)[20]. Additional studies must be conducted to further examine the diverse roles of WDR23 in protein regulation

Methods

Results

Conclusion