NBR1-mediated p62-liquid droplets enhance the Keap1-Nrf2 system.
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ABSTRACT:
SUBMITTER: Sanchez-Martin P
PROVIDER: S-EPMC7054683 | biostudies-literature | 2020 Mar
REPOSITORIES: biostudies-literature
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NBR1-mediated p62-liquid droplets enhance the Keap1-Nrf2 system.
Sánchez-Martín Pablo P Sou Yu-Shin YS Kageyama Shun S Koike Masato M Waguri Satoshi S Komatsu Masaaki M
EMBO reports 20200109 3
p62/SQSTM1 is a multivalent protein that has the ability to cause liquid-liquid phase separation and serves as a receptor protein that participates in cargo isolation during selective autophagy. This protein is also involved in the non-canonical activation of the Keap1-Nrf2 system, a major oxidative stress response pathway. Here, we show a role of neighbor of BRCA1 gene 1 (NBR1), an autophagy receptor structurally similar to p62/SQSTM1, in p62-liquid droplet formation and Keap1-Nrf2 pathway acti ...[more]
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Project description:p62/SQSTM1 is a multivalent protein that has the ability to cause liquid-liquid phase separation and serves as a receptor protein that participates in cargo isolation during selective autophagy. This protein is also involved in the non-canonical activation of the Keap1-Nrf2 system, a major oxidative stress response pathway. Here, we show a role of Neighbor of BRCA1 gene 1 (NBR1), an autophagy receptor structurally similar to p62/SQSTM1, in p62-liquid droplet formation and Keap1-Nrf2 pathway activation. Overexpression of NBR1 blocks selective degradation of p62/SQSTM1 through autophagy and promotes the accumulation and phosphorylation of p62/SQSTM1 in liquid-like bodies, which is required for the activation of Nrf2. NBR1 is induced in response to oxidative stress, which trigegrs p62-mediated Nrf2 activation. Conversely, loss of Nbr1 suppresses not only the formation of p62/SQSTM1-liquid droplets, but also of p62-dependent Nrf2 activation during oxidative stress. Taken together, our results show that NBR1 mediates p62/SQSTM1-liquid droplet formation to activate the Keap1-Nrf2 pathway.
| S-SCDT-EMBOR-2019-48902V1 | biostudies-other
Project description:BackgroundIdiopathic pulmonary fibrosis is a persistent disease of the lung interstitium for which there is no efficacious pharmacological therapy. Protodioscin, a steroidal saponin, possesses diverse pharmacological properties; however, its function in pulmonary fibrosis is yet to be established. Hence, in this investigation, it was attempted to figure out the anti-pulmonary fibrosis influences of protodioscin and its pharmacological properties related to oxidative stress.MethodsA mouse lung fibrosis model was generated using tracheal injections of bleomycin, followed by intraperitoneal injection of different concentrations of protodioscin, and the levels of oxidative stress and fibrosis were detected in the lungs. Multiple fibroblasts were treated with TGF-β to induce their transition to myofibroblasts. It was attempted to quantify myofibroblast markers' expression levels and reactive oxygen species levels as well as Nrf2 activation after co-incubation of TGF-β with fibroblasts and different concentrations of protodioscin. The influence of protodioscin on the expression and phosphorylation of p62, which is associated with Nrf2 activation, were detected, and p62 related genes were predicted by STRING database. The effects of Nrf2 inhibitor or silencing of the Nrf2, p62 and NBR1 genes, respectively, on the activation of Nrf2 by protodioscin were examined. The associations between p62, NBR1, and Keap1 in the activation of Nrf2 by protodioscin was demonstrated using a co-IP assay. Nrf2 inhibitor were used when protodioscin was treated in mice with pulmonary fibrosis and lung tissue fibrosis and oxidative stress levels were detected.ResultsIn vivo, protodioscin decreased the levels of fibrosis markers and oxidative stress markers and activated Nrf2 in mice with pulmonary fibrosis, and these effects were inhibited by Nrf2 inhibitor. In vitro, protodioscin decreased the levels of myofibroblast markers and oxidative stress markers during myofibroblast transition and promoted Nrf2 downstream gene expression, with reversal of these effects after Nrf2, p62 and NBR1 genes were silenced or Nrf2 inhibitors were used, respectively. Protodioscin promoted the binding of NBR1 to p62 and Keap1, thereby reducing Keap1-Nrf2 binding.ConclusionThe NBR1-p62-Nrf2 axis is targeted by protodioscin to reduce oxidative stress and inhibit pulmonary fibrosis.
| S-EPMC11003024 | biostudies-literature
Project description:Nrf2 signaling is vital for protecting cells against oxidative stress. However, its hyperactivation is frequently found in liver cancer through excessive build-up of p62/SQSTM1 bodies that sequester Keap1, an adaptor of the E3-ubiquitin ligase complex for Nrf2. Here we report that the Bax-binding protein MOAP-1 regulates p62-Keap1-Nrf2 signaling through disruption of p62 bodies. Upon induction of cellular stresses that stimulate formation of p62 bodies, MOAP-1 is recruited to p62 bodies and reduces their levels independent of the autophagy pathway. MOAP-1 interacts with the PB1-ZZ domains of p62 and interferes with its self-oligomerization and liquid-liquid phase separation, thereby disassembling the p62 bodies. Loss of MOAP-1 can lead to marked upregulation of p62 bodies, enhanced sequestration of Keap1 by p62 and hyperactivation of Nrf2 antioxidant target genes. MOAP-1 deficient mice exhibit an elevated tumor burden with excessive levels of p62 bodies and Nrf2 signaling in a diethylnitrosamine (DEN)-induced hepatocarcinogenesis model. Together, our data define MOAP-1 as a negative regulator of Nrf2 signaling via dissociation of p62 bodies.
| S-SCDT-EMBOR-2020-50854V1 | biostudies-other
Project description:BackgroundMacro-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.ResultsIn 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.ConclusionsThese 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.
| S-EPMC9945626 | biostudies-literature
Project description:Nrf2 signaling is vital for protecting cells against oxidative stress. However, its hyperactivation is frequently found in liver cancer through excessive build-up of p62/SQSTM1 bodies that sequester Keap1, an adaptor of the E3-ubiquitin ligase complex for Nrf2. Here, we report that the Bax-binding protein MOAP-1 regulates p62-Keap1-Nrf2 signaling through disruption of p62 bodies. Upon induction of cellular stresses that stimulate formation of p62 bodies, MOAP-1 is recruited to p62 bodies and reduces their levels independent of the autophagy pathway. MOAP-1 interacts with the PB1-ZZ domains of p62 and interferes with its self-oligomerization and liquid-liquid phase separation, thereby disassembling the p62 bodies. Loss of MOAP-1 can lead to marked upregulation of p62 bodies, enhanced sequestration of Keap1 by p62 and hyperactivation of Nrf2 antioxidant target genes. MOAP-1-deficient mice exhibit an elevated tumor burden with excessive levels of p62 bodies and Nrf2 signaling in a diethylnitrosamine (DEN)-induced hepatocarcinogenesis model. Together, our data define MOAP-1 as a negative regulator of Nrf2 signaling via dissociation of p62 bodies.
| S-EPMC7788458 | biostudies-literature
Project description:Hepatic lipotoxicity is a crucial factor in nonalcoholic steatohepatitis resulting from excessive saturated fatty acid-induced reactive oxygen species (ROS)-mediated cell death, which is associated with the accumulation of endoplasmic reticulum (ER) stress in the liver. The unfolded protein response (UPR) alleviates ER stress by restoring ER protein folding homeostasis. However, whether UPR contributes ROS elimination under lipotoxicity remains unclear. The Kelch like ECH-associated protein 1 (KEAP1)-nuclear factor, erythroid 2 like 2 (Nrf2) pathway provides antioxidant defense against lipotoxic stress by eliminating ROS and can be activated by the p62-Unc-51 like autophagy activating kinase 1 (ULK1) axis. However, the upstream molecular regulator of the p62-ULK1 axis-induced KEAP1-Nrf2 pathway in the same context remains unidentified. Here, we demonstrated that PKR-like ER kinase (PERK), a UPR sensor, directly phosphorylates p62 and ULK1, thereby activating the noncanonical KEAP1-Nrf2 pathway. We also elucidated the molecular mechanism underlying the PERK-mediated p62-ULK1 axis-dependent noncanonical KEAP1-Nrf2 pathway, which could represent a promising therapeutic strategy against hepatic lipotoxicity.
| S-EPMC8801383 | biostudies-literature
Project description:Cancer-derived loss-of-function mutations in the KEAP1 tumor suppressor gene stabilize the NRF2 transcription factor, resulting in a prosurvival gene expression program that alters cellular metabolism and neutralizes oxidative stress. In a recent genotype-phenotype study, we classified 40% of KEAP1 mutations as ANCHOR mutants. By immunoprecipitation, these mutants bind more NRF2 than wild-type KEAP1 and ubiquitylate NRF2, but they are incapable of promoting NRF2 degradation. BioID-based protein interaction studies confirmed increased abundance of NRF2 within the KEAP1 ANCHOR mutant complexes, with no other statistically significant changes to the complexes. Discrete molecular dynamic simulation modeling and limited proteolysis suggest that the ANCHOR mutations stabilize residues in KEAP1 that contact NRF2. The modeling supports an intramolecular salt bridge between the R470C ANCHOR mutation and E493; mutation of the E493 residue confirmed the model, resulting in the ANCHOR phenotype. In live cells, the KEAP1 R320Q and R470C ANCHOR mutants colocalize with NRF2, p62/SQSTM1, and polyubiquitin in structured spherical droplets that rapidly fuse and dissolve. Transmission electron microscopy coupled with confocal fluorescent imaging revealed membraneless phase-separated biomolecular condensates. We present a model wherein ANCHOR mutations form p62-dependent biomolecular condensates that may represent a transitional state between impaired proteasomal degradation and autophagy.
| S-EPMC6206457 | biostudies-literature
Project description:An important pathophysiological consequence of pressure overload-induced cardiac hypertrophy is adverse cardiac remodeling, including structural changes in cardiomyocytes and extracellular matrix. Diosmetin (DIO), a monomethoxyflavone isolated from citrus fruits, had antioxidative stress effects in multiple organs. The purpose of this study was to examine the biological effect of diosmetin on pathological cardiac hypertrophy. In mice, diosmetin treatment reduced cardiac hypertrophy and dysfunction in an aortic banding- (AB-) induced pressure overload model and reducing myocardial oxidative stress by increasing antioxidant gene expression. In vitro, diosmetin (10 or 50 μm, 12 h or 24 h) protected PE-induced cardiomyocyte hypertrophy in neonatal rat cardiomyocytes. Mechanistically, diosmetin inhibited autophagy by activating the PI3K/Akt pathway. In particular, diosmetin induced the accumulation of p62 and its interaction with Keap1, promoted the nuclear translocation of Nrf2, and increased the expression of antioxidant stress genes in the process of cardiac hypertrophy. Furthermore, knockdown of p62 in rat primary cardiomyocytes abrogate the protective effect of diosmetin on cardiomyocyte hypertrophy. Similarly, the Nrf2 inhibitor ML385 obviously abolished the above effects by diosmetin treatment. In conclusion, our results suggest that diosmetin protects cardiac hypertrophy under pressure overload through the p62/Keap1/Nrf2 signaling pathway, suggesting the potential of diosmetin as a novel therapy for pathological cardiac hypertrophy.
| S-EPMC8888112 | biostudies-literature
Project description:Lipotoxicity, induced by saturated fatty acid (SFA)-mediated cell death, plays an important role in the pathogenesis of nonalcoholic fatty liver disease (NAFLD). The KEAP1 (kelch like ECH associated protein 1)-NFE2L2/NRF2 (nuclear factor, erythroid 2 like 2) pathway is a pivotal defense mechanism against lipotoxicity. We previously reported that SQSTM1/p62 has a cytoprotective role against lipotoxicity through activation of the noncanonical KEAP1- NFE2L2 pathway in hepatocytes. However, the underlying mechanisms and physiological relevance of this pathway have not been clearly defined. Here, we demonstrate that NFE2L2-mediated induction of SQSTM1 activates the noncanonical KEAP1-NFE2L2 pathway under lipotoxic conditions. Furthermore, we identified that SQSTM1 induces ULK1 (unc-51 like autophagy activating kinase 1) phosphorylation by facilitating the interaction between AMPK (AMP-activated protein kinase) and ULK1, leading to macroautophagy/autophagy induction, followed by KEAP1 degradation and NFE2L2 activation. Accordingly, the activity of this SQSTM1-mediated noncanonical KEAP1-NFE2L2 pathway conferred hepatoprotection against lipotoxicity in the livers of conventional sqstm1- and liver-specific sqstm1-knockout mice. Moreover, this pathway activity was evident in the livers of patients with nonalcoholic fatty liver. This axis could thus represent a novel target for NAFLD treatment. Abbreviations: ACACA: acetyl-CoA carboxylase alpha; ACTB: actin beta; BafA1: bafilomycin A1; CM-H2DCFDA:5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate; CQ: chloroquine; CUL3: cullin 3; DMSO: dimethyl sulfoxide; FASN: fatty acid synthase; GSTA1: glutathione S-transferase A1; HA: hemagglutinin; Hepa1c1c7: mouse hepatoma cells; HMOX1/HO-1: heme oxygenase 1; KEAP1: kelch like ECH associated protein 1; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3; MEF: mouse embryonic fibroblast; MTORC1: mechanistic target of rapamycin kinase complex 1; MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NAC: N-acetyl-L-cysteine; NAFLD: nonalcoholic fatty liver disease; NASH: nonalcoholic steatohepatitis; NFE2L2/NRF2: nuclear factor, erythroid 2 like 2; NQO1: NAD(P)H quinone dehydrogenase 1; PA: palmitic acid; PARP: poly (ADP-ribose) polymerase 1; PRKAA1/2: protein kinase AMP-activated catalytic subunits alpha1/2; RBX1: ring-box 1; ROS: reactive oxygen species; SESN2: sestrin 2; SFA: saturated fatty acid; siRNA: small interfering RNA; SQSTM1/p62: sequestosome 1; SREBF1: sterol regulatory element binding transcription factor 1; TBK1: TANK binding kinase 1; TUNEL: terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling; ULK1: unc-51 like autophagy activating kinase.
| S-EPMC7595589 | biostudies-literature
Project description:BackgroundCDGSH iron sulfur domain 2 (CISD2) is an iron-sulfur protein with a [2Fe-2S] cluster, which is critical for cell proliferation and iron homeostasis. It has been demonstrated that aberrant expression of CISD2 is associated with the progression of multiple cancers. However, the underlying mechanism of CISD2 in regulating tumorigenesis remains obscure.MethodsBioinformatics strategies were used to investigate the protein interaction network and functional annotation of CISD2. In the functional experiment, cell viability was measured by CCK-8 kit. The levels of cellular reactive oxygen species (ROS), intracellular free iron, lipid peroxides, and lysosomal activity were determined by DCF-DA, RPA, C11-BODIPY, and cathepsin B staining, respectively. The glutathione (GSH) content was determined using a GSH assay kit.ResultsWe showed that knockdown of CISD2 significantly accelerated the Erastin-induced ferroptotic cell death with excess lipid peroxidation, GSH exhaustion, and iron accumulation, while overexpression of CISD2 hindered the sensitivity to Erastin. Further assays via confocal microscopy and western blot exhibited that CISD2 knockdown markedly enhanced the lysosomal activity, and activated ferritinophagy under the exposure of Erastin. Pharmacological inhibition of lysosomal function could inhibit the degradation of ferritin heavy chain (FTH), and attenuate the phenotypes of ferroptosis, such as accelerated iron accumulation and lipid peroxidation. Notably, we found that Erastin-induced compensatory elevation of nuclear factor erythroid 2-related factor 2 (NRF2) could be eliminated in CISD2 depletion cells. Mechanically, CISD2 knockdown promoted the degradation of autophagy adaptor p62 and resulted in an increased binding affinity of Keap1 with NRF2, thus leading to the increased ubiquitination and subsequent degradation of NRF2. Enforced expression of NRF2 reversed the sensitivity of shCISD2 cells to ferroptosis both in vitro and in vivo. Conversely, enforced expression of Keap1 exacerbated the degradation of NRF2, reduced the transcriptional expression of FTH and heme oxygenase 1 (HO-1), increased the oxidative damage, and thus further facilitated ferroptosis.ConclusionTaken together, our current results illustrated two parallel mechanisms involved in the shCISD2-mediated ferroptosis. One was that shCISD2 enhanced the accumulation of free iron via ferritinophagy-dependent ferritin turnover; the other was that CISD2 depletion induced the inhibition of the p62-Keap1-NRF2 pathway, which resulted in oxidative stress and ferroptosis.
| S-EPMC9523958 | biostudies-literature