SMURF1 controls the PPP3/calcineurin complex and TFEB at a regulatory node for lysosomal biogenesis.

Macroautophagy/autophagy is a homeostatic process in response to multiple signaling, such as the lysosome-dependent recycling process of cellular components. Starvation-induced MTOR inactivation and PPP3/calcineurin activation were shown to promote the nuclear translocation of TFEB. However, the mechanisms via which signals from endomembrane damage are transmitted to activate PPP3/calcineurin and orchestrate autophagic responses remain unknown. This study aimed to show that autophagy regulator SMURF1 controlled TFEB nuclear import for transcriptional activation of the lysosomal biogenesis. We showed that blocking SMURF1 affected lysosomal biogenesis in response to lysosomal damage by preventing TFEB nuclear translocation. It revealed galectins recognized endolysosomal damage, and led to recruitment of SMURF1 and the PPP3/calcineurin apparatus on lysosomes. SMURF1 interacts with both LGALS3 and PPP3CB to form the LGALS3-SMURF1-PPP3/calcineurin complex. Importantly, this complex further stabilizes TFEB, thereby activating TFEB for lysosomal biogenesis. We determined that LLOMe-mediated TFEB nuclear import is dependent on SMURF1 under the condition of MTORC1 inhibition. In addition, SMURF1 is required for PPP3/calcineurin activity as a positive regulator of TFEB. SMURF1 controlled the phosphatase activity of the PPP3CB by promoting the dissociation of its autoinhibitory domain (AID) from its catalytic domain (CD). Overexpression of SMURF1 showed similar effects as the constitutive activation of PPP3CB. Thus, SMURF1, which bridges environmental stress with the core autophagosomal and autolysosomal machinery, interacted with endomembrane sensor LGALS3 and phosphatase PPP3CB to control TFEB activation.Abbreviations: ATG: autophagy-related; LLOMe: L-Leucyl-L-Leucine methyl ester; ML-SA1: mucolipin synthetic agonist 1; MTOR: mechanistic target of rapamycin kinase; PPP3CB: protein phosphatase 3 catalytic subunit beta; RPS6KB1/p70S6K: ribosomal protein S6 kinase B1; SMURF1: SMAD specific E3 ubiquitin protein ligase 1; TFEB: transcription factor EB.

SMURF1 attenuates endoplasmic reticulum stress by promoting the degradation of KEAP1 to activate NRF2 antioxidant pathway.

Cancer cells consistently utilize the unfolded protein response (UPR) to encounter the abnormal endoplasmic reticulum (ER) stress induced by the accumulation of misfolded proteins. Extreme activation of the UPR could also provoke maladaptive cell death. Previous reports have shown that NRF2 antioxidant signaling is activated by UPR and serves as noncanonical pathway to defense and reduce excessive ROS levels during ER stress. However, the mechanisms of regulating NRF2 signaling upon ER stress in glioblastoma have not been fully elucidated. Here we identify that SMURF1 protects against ER stress and facilitates glioblastoma cell survival by rewiring KEAP1-NRF2 pathway. We show that ER stress induces SMURF1 degradation. Knockdown of SMURF1 upregulates IRE1 and PERK signaling in the UPR pathway and prevents ER-associated protein degradation (ERAD) activity, leading to cell apoptosis. Importantly, SMURF1 overexpression activates NRF2 signaling to reduce ROS levels and alleviate UPR-mediated cell death. Mechanistically, SMURF1 interacts with and ubiquitinates KEAP1 for its degradation (NRF2 negative regulator), resulting in NRF2 nuclear import. Moreover, SMURF1 loss reduces glioblastoma cell proliferation and growth in subcutaneously implanted nude mice xenografts. Taken together, SMURF1 rewires KEAP1-NRF2 pathway to confer resistance to ER stress inducers and protect glioblastoma cell survival. ER stress and SMURF1 modulation may provide promising therapeutic targets for the treatment of glioblastoma.

Enhanced liquidity of p62 droplets mediated by Smurf1 links Nrf2 activation and autophagy.

BACKGROUND: Macro-autophagy/Autophagy is an evolutionarily well-conserved recycling process to maintain the balance through precise spatiotemporal regulation. However, the regulatory mechanisms of biomolecular condensates by the key adaptor protein p62 via liquid-liquid phase separation (LLPS) remain obscure. RESULTS: In this study, we showed that E3 ligase Smurf1 enhanced Nrf2 activation and promoted autophagy by increasing p62 phase separation capability. Specifically, the Smurf1/p62 interaction improved the formation and material exchange of liquid droplets compared with p62 single puncta. Additionally, Smurf1 promoted the competitive binding of p62 with Keap1 to increase Nrf2 nuclear translocation in p62 Ser349 phosphorylation-dependent manner. Mechanistically, overexpressed Smurf1 increased the activation of mTORC1 (mechanistic target of rapamycin complex 1), in turn leading to p62 Ser349 phosphorylation. Nrf2 activation increased the mRNA levels of Smurf1, p62, and NBR1, further promoting the droplet liquidity to enhance oxidative stress response. Importantly, we showed that Smurf1 maintained cellular homeostasis by promoting cargo degradation through the p62/LC3 autophagic pathway. CONCLUSIONS: These findings revealed the complex interconnected role among Smurf1, p62/Nrf2/NBR1, and p62/LC3 axis in determining Nrf2 activation and subsequent clearance of condensates through LLPS mechanism.