NBR1-mediated p62-liquid droplets enhance the Keap1-Nrf2 system
Ontology highlight
ABSTRACT:
SUBMITTER: Dr. Masaaki Komatsu
PROVIDER: S-SCDT-EMBOR-2019-48902V1 | biostudies-other |
REPOSITORIES: biostudies-other
Similar Datasets
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 triggers 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-EPMC7054683 | 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: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: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: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:A key anti-oxidant pathway, the Keap1-Nrf2 system, is regulated by p62/Sqstm1 via multiple mechanisms, including gene expression, post-translational modifications such as ubiquitination and phosphorylation, and autophagic degradation of p62/Sqstm1 and Keap1. Herein, we demonstrate a novel mode of regulation of the Keap1-Nrf2 system, mediated by a splicing variant of p62/Sqstm1 pre-mRNA. Ensembl database search and subsequent biochemical analyses in mice revealed the presence of an mRNA that encodes p62/Sqstm1 protein lacking the Keap1-interacting region (KIR), which is essential for the interaction with Keap1. Like full-length p62, the variant was induced under conditions in which Nrf2 was activated (e.g., impairment of autophagy), oligomerized with itself and/or full-length protein, and was degraded by autophagy. However, the variant failed to interact with Keap1 and sequester it into variant-positive aggregates. Remarkably, while full-length p62 stabilized Nrf2 and induced the gene expression of Nrf2 targets, the variant increased the amount of Keap1 and enhanced ubiquitination of Nrf2, thereby suppressing the induction of Nrf2 targets. Hepatocytes isolated from genetically modified mice that express full-length p62, but not the variant, were susceptible to activation of Nrf2 in response to stress. Collectively, our results suggest that splicing of p62/Sqstm1 pre-mRNA negatively regulates the Keap1-Nrf2 pathway.
| S-EPMC5854834 | biostudies-other
Project description:Currently, most antioxidants do not show any favorable clinical outcomes in reducing myocardial ischemia-reperfusion (I/R) injury, suggesting an urgent need for exploring a new regulator of redox homeostasis in I/R hearts. Here, using heart-specific transgenic (TG) and knockdown (KD) mouse models, tumor susceptibility gene 101 (Tsg101) is defined as a novel cardiac-protector against I/R-triggered oxidative stress. RNA sequencing and bioinformatics data surprisingly reveal that most upregulated genes in Tsg101-TG hearts are transcribed by Nrf2. Accordingly, pharmacological inhibition of Nrf2 offsets Tsg101-elicited cardio-protection. Mechanistically, Tsg101 interacts with SQSTM1/p62 through its PRR domain, and promotes p62 aggregation, leading to recruitment of Keap1 for degradation by autophagosomes and release of Nrf2 to the nucleus. Furthermore, knockout of p62 abrogates Tsg101-induced cardio-protective effects during I/R. Hence, our findings uncover a previously unrecognized role of Tsg101 in the regulation of p62/Keap1/Nrf2 signaling cascades and provide a new strategy for the treatment of ischemic heart disease.
| S-EPMC7264471 | biostudies-literature
Project description:Isodeoxyelephantopin (ESI), isolated from Elephantopus scaber L. has been reported to exert anticancer effects. In this study, we aimed to investigate whether and how cancer cells exert protective responses against ESI treatment. Confocal fluorescence microscopy showed that ESI significantly induced autophagy flux in the lung cancer cells expressing mCherry-EGFP-LC3 reporter. Treatment of the cells with ESI increased the expression levels of the autophagy markers including LC3-II, ATG3 and Beclin1 in a dose-dependent manner. Pretreatment with autophagy inhibitor 3-methyladenine (3-MA) not only attenuated the effects of ESI on autophagy, but also enhanced the effects of ESI on cell viability and apoptosis. Mechanistically, the SILAC quantitative proteomics coupled with bioinformatics analysis revealed that the ESI-regulated proteins were mainly involved in Nrf2-mediated oxidative stress response. We found that ESI induced the nuclear translocation of Nrf2 for activating the downstream target genes including HO-1 and p62 (SQSTM1). More importantly, ESI-induced p62 could competitively bind with Keap1, and releases Nrf2 to activate downstream target gene p62 as a positive feedback loop, therefore promoting autophagy. Furthermore, knockdown of Nrf2 or p62 could abrogate the ESI-induced autophagy and significantly enhanced the anticancer effect of ESI. Taken together, we demonstrated that ESI can sustain cell survival by activating protective autophagy through Nrf2-p62-keap1 feedback loop, whereas targeting this regulatory axis combined with ESI treatment may be a promising strategy for anticancer therapy.
| S-EPMC5584574 | biostudies-literature
Project description:Sqstm1/p62 functions in the non-canonical activation of nuclear factor (erythroid-derived 2)-like 2 (Nrf2). However, its physiological relevance is not certain. Here, we show that p62(-/-) mice exhibited an accelerated presentation of ageing phenotypes, and tissues from these mice created a pro-oxidative environment owing to compromised mitochondrial electron transport. Accordingly, mitochondrial function rapidly declined with age in p62(-/-) mice. In addition, p62 enhanced basal Nrf2 activity, conferring a higher steady-state expression of NAD(P)H dehydrogenase, quinone 1 (Nqo1) to maintain mitochondrial membrane potential and, thereby, restrict excess oxidant generation. Together, the p62-Nrf2-Nqo1 cascade functions to assure mammalian longevity by stabilizing mitochondrial integrity.
| S-EPMC3271336 | biostudies-literature
Project description:Upregulation of neuronal oxidative stress is involved in the progression of secondary brain injury (SBI) following intracerebral hemorrhage (ICH). In this study, we investigated the potential effects and underlying mechanisms of luteolin on ICH-induced SBI. Autologous blood and oxyhemoglobin (OxyHb) were used to establish in vivo and in vitro models of ICH, respectively. Luteolin treatment effectively alleviated brain edema and ameliorated neurobehavioral dysfunction and memory loss in vivo. Also, in vivo, we found that luteolin promoted the activation of the sequestosome 1 (p62)/kelch-like enoyl-coenzyme A hydratase (ECH)-associated protein 1 (Keap1)/nuclear factor erythroid 2-related factor 2 (Nrf2) pathway by enhancing autophagy and increasing the translocation of Nrf2 to the nucleus. Meanwhile, luteolin inhibited the ubiquitination of Nrf2 and increased the expression levels of downstream antioxidant proteins, such as heme oxygenase-1 (HO-1) and reduced nicotinamide adenine dinucleotide phosphate (NADPH): quinine oxidoreductase 1 (NQO1). This effect of luteolin was also confirmed in vitro, which was reversed by the autophagy inhibitor, chloroquine (CQ). Additionally, we found that luteolin inhibited the production of neuronal mitochondrial superoxides (MitoSOX) and alleviated neuronal mitochondrial injury in vitro, as indicated via tetrachloro-tetraethylbenzimidazol carbocyanine-iodide (JC-1) staining and MitoSOX staining. Taken together, our findings demonstrate that luteolin enhances autophagy and anti-oxidative processes in both in vivo and in vitro models of ICH, and that activation of the p62-Keap1-Nrf2 pathway, is involved in such luteolin-induced neuroprotection. Hence, luteolin may represent a promising candidate for the treatment of ICH-induced SBI.
| S-EPMC6985769 | biostudies-literature