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STUB1 regulates TFEB-induced autophagy-lysosome pathway.

ABSTRACT: TFEB is a master regulator for transcription of genes involved in autophagy and lysosome biogenesis. Activity of TFEB is inhibited upon its serine phosphorylation by mTOR The overall mechanisms by which TFEB activity in the cell is regulated are not well elucidated. Specifically, the mechanisms of TFEB turnover and how they might influence its activity remain unknown. Here, we show that STUB1, a chaperone-dependent E3 ubiquitin ligase, modulates TFEB activity by preferentially targeting inactive phosphorylated TFEB for degradation by the ubiquitin-proteasome pathway. Phosphorylated TFEB accumulated in STUB1-deficient cells and in tissues of STUB1-deficient mice resulting in reduced TFEB activity. Conversely, cellular overexpression of STUB1 resulted in reduced phosphorylated TFEB and increased TFEB activity. STUB1 preferentially interacted with and ubiqutinated phosphorylated TFEB, targeting it to proteasomal degradation. Consistent with reduced TFEB activity, accumulation of phosphorylated TFEB in STUB1-deficient cells resulted in reduced autophagy and reduced mitochondrial biogenesis. These studies reveal that the ubiquitin-proteasome pathway participates in regulating autophagy and lysosomal functions by regulating the activity of TFEB.

SUBMITTER: Sha Y

PROVIDER: S-EPMC5579343 | biostudies-literature | 2017 Sep

REPOSITORIES: biostudies-literature

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STUB1 regulates TFEB-induced autophagy-lysosome pathway.

Sha Youbao Y Rao Lang L Settembre Carmine C Ballabio Andrea A Eissa N Tony NT

The EMBO journal 20170728 17

TFEB is a master regulator for transcription of genes involved in autophagy and lysosome biogenesis. Activity of TFEB is inhibited upon its serine phosphorylation by mTOR The overall mechanisms by which TFEB activity in the cell is regulated are not well elucidated. Specifically, the mechanisms of TFEB turnover and how they might influence its activity remain unknown. Here, we show that STUB1, a chaperone-dependent E3 ubiquitin ligase, modulates TFEB activity by preferentially targeting inactive ...[more]

PMID: 28754656

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Project description:BACKGROUND & AIMS:Defects in lysosome function and autophagy contribute to the pathogenesis of alcoholic liver disease. We investigated the mechanisms by which alcohol consumption affects these processes by evaluating the functions of transcription factor EB (TFEB), which regulates lysosomal biogenesis. METHODS:We performed studies with GFP-LC3 mice, mice with liver-specific deletion of TFEB, mice with disruption of the transcription factor E3 gene (TFE3-knockout mice), mice with disruption of the Tefb and Tfe3 genes (TFEB and TFE3 double-knockout mice), and Tfebflox/flox albumin cre-negative mice (controls). TFEB was overexpressed from adenoviral vectors or knocked down with small interfering RNAs in mouse livers. Mice were placed on diets of regular ethanol feeding plus an acute binge to induce liver damage (ethanol diet); some mice also were given injections of torin-1, an inhibitor of the kinase activity of the mechanistic target of rapamycin (mTOR). Liver tissues were collected and analyzed by immunohistochemistry, immunoblots, and quantitative real-time polymerase chain reaction to monitor lysosome biogenesis. We analyzed levels of TFEB in liver tissues from patients with alcoholic hepatitis and from healthy donors (controls) by immunohistochemistry. RESULTS:Liver tissues from mice on the ethanol diet had lower levels of total and nuclear TFEB compared with control mice, and hepatocytes had decreased lysosome biogenesis and autophagy. Hepatocytes from mice on the ethanol diet had increased translocation of mTOR into lysosomes, resulting in increased mTOR activation. Administration of torin-1 increased liver levels of TFEB and decreased steatosis and liver injury induced by ethanol. Mice that overexpressed TFEB in the liver developed less severe ethanol-induced liver injury and had increased lysosomal biogenesis and mitochondrial bioenergetics compared with mice carrying a control vector. Mice with knockdown of TFEB and TFEB-TFE3 double-knockout mice developed more severe liver injury in response to the ethanol diet than control mice. Liver tissues from patients with alcohol-induced hepatitis had lower nuclear levels of TFEB than control tissues. CONCLUSIONS:We found that ethanol feeding plus an acute binge decreased hepatic expression of TFEB, which is required for lysosomal biogenesis and autophagy. Strategies to block mTOR activity or increase levels of TFEB might be developed to protect the liver from ethanol-induced damage.

Project description:The autophagy-lysosome system is an important cellular degradation pathway that recycles dysfunctional organelles and cytotoxic protein aggregates. A decline in this system is pathogenic in many human diseases including neurodegenerative disorders, fatty liver disease, and atherosclerosis. Thus there is intense interest in discovering therapeutics aimed at stimulating the autophagy-lysosome system. Trehalose is a natural disaccharide composed of two glucose molecules linked by a ɑ-1,1-glycosidic bond with the unique ability to induce cellular macroautophagy/autophagy and with reported efficacy on mitigating several diseases where autophagy is dysfunctional. Interestingly, the mechanism by which trehalose induces autophagy is unknown. One suggested mechanism is its ability to activate TFEB (transcription factor EB), the master transcriptional regulator of autophagy-lysosomal biogenesis. Here we describe a potential mechanism involving direct trehalose action on the lysosome. We find trehalose is endocytically taken up by cells and accumulates within the endolysosomal system. This leads to a low-grade lysosomal stress with mild elevation of lysosomal pH, which acts as a potent stimulus for TFEB activation and nuclear translocation. This process appears to involve inactivation of MTORC1, a known negative regulator of TFEB which is sensitive to perturbations in lysosomal pH. Taken together, our data show the trehalose can act as a weak inhibitor of the lysosome which serves as a trigger for TFEB activation. Our work not only sheds light on trehalose action but suggests that mild alternation of lysosomal pH can be a novel method of inducing the autophagy-lysosome system.Abbreviations: ASO: antisense oligonucleotide; AU: arbitrary units; BMDM: bone marrow-derived macrophages; CLFs: crude lysosomal fractions; CTSD: cathepsin D; LAMP: lysosomal associated membrane protein; LIPA/LAL: lipase A, lysosomal acid type; MAP1LC3: microtubule-associated protein 1 light chain 3; MFI: mean fluorescence intensity; MTORC1: mechanistic target of rapamycin kinase complex 1; pMAC: peritoneal macrophages; SLC2A8/GLUT8: solute carrier family 2, (facilitated glucose transporter), member 8; TFEB: transcription factor EB; TMR: tetramethylrhodamine; TREH: trehalase.

Dataset Information

STUB1 regulates TFEB-induced autophagy-lysosome pathway.

Publications

STUB1 regulates TFEB-induced autophagy-lysosome pathway.

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