The E3 Ligase Smurf1 Regulates Wolfram Syndrome Protein Stability At The Endoplasmic Reticulum

Ubiquitin-mediated proteasomal degradation represents the most critical pathway to control the stability and quality of cellular proteins in eukaryotes. Ubiquitin ligases (also called E3) are responsible for substrate recognition and are divided into two major classes:RING finger-type and HECT domain-type. Smurfl belongs to the Nedd4 (Neuronal precursor cell-expressed developmentally down-regulated 4) family of HECT-type E3 ligases and plays a critical role in the regulation of embryonic development, cell polarity and bone homeostasis by targeting the degradation of Smadl/5, TGFβR, RhoA, MEKK2, Prickle 1 and JunB. Here, we show that treatment of cells with different ER stress inducer result in the proteasome-mediated degradation of Smurfl, implicating a possible role for Smurfl in the ER stress response. Furthermore, we identify the endoplasmic reticulum (ER)-localized Wolfram syndrome protein (WFS1) as a specific degradation substrate of Smurfl. Wolfram syndrome is an autosomal recessive disorder leading to juvenile-onset insulin-dependent diabetes mellitus, optic atrophy, sensorineural deafness and diabetes, which was first reported in 1938, Mutations in the WFS1 gene are the most frequent genetic cause of Wolfram syndrome, and the first mutations in the WFS1 gene were identified in Wolfram syndrome patients in 1998. WFS1 negatively regulates ER stress signaling by stabilizing the E3 ligase HRD1, recruiting ATF6a (activating transcription factor 6a, a key transcription factor to activate unfolded protein response target genes) to HRD1 and enhancing its ubiquitination and proteasomal degradation. WFS1-deficient mice exhibit impaired glucose homeostasis, defective insulin secretion, increased apoptosis of pancreatic islet cells due to enhanced ER stress and an increase in the unfolded protein response. Therefore, WFS1 is critical in maintaining ER homeostasis. To date, little is known about how the stability of WFS1 is controlled, especially how it is downregulated.We show that Smurfl interacts with WFS1 at the ER independent its E3 ligase activity. Both the N-terminal cytoplasmic part and the C-terminal ER luminal part of WFS1 contain the Smurf1-interacting information. Knockdown of endogenous Smurfl protein resulted in a significant increase in WFS1 protein levels, and overexpression of Smurfl promotes the ubiquitination and proteasomal degradation of WFS1. We also show a C-terminal luminal region in WFS1, including residues 667-700, is involved in this degradation. Wild-type WFS1, as well as a subset of WFS1 mutants that include this degron region, are susceptible to Smurfl-mediated degradation. By contrast, pathophysiological deletion mutants of WFS1 lacking the degron, including W648X, Y660X and Q667X, exhibit resistance against Smurfl-mediated degradation and possess longer half-lives. Depletion of Smurfl by RNA interference results in increased WFS1 and decreased ATF6a levels. Smurfl overexpression upregulates ATF6 levels, and as a target of ATF6, expression of CHOP was increased significantly.These findings reveal for the first time that Smurfl targets an ER-localized protein for degradation and that Smurfl is regulated by ER stress.