Negative regulation of the Keap1-Nrf2 pathway by a p62/Sqstm1 splicing variant.
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ABSTRACT:
SUBMITTER: Kageyama S
PROVIDER: S-EPMC5854834 | biostudies-other | 2018 Jan
REPOSITORIES: biostudies-other
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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.
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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.
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Project description:p62/SQSTM1 (p62) is a scaffolding protein that facilitates the formation and degradation of ubiquitinated aggregates via its self-interaction and ubiquitin binding domains. The regulation of this process is unclear but may relate to the post-translational modification of p62. In the present study, we find that Keap1/Cullin3 ubiquitinates p62 at lysine 420 within its UBA domain. Substitution of lysine 420 with an arginine diminishes p62 sequestration and degradation activity similar what is seen when the UBA domain is deleted. Overexpression of Keap1/Cullin3 in p62-WT-expressing cells increases ubiquitinated inclusion formation and p62's association with LC3 and rescues proteotoxicity. This effect is not seen in cells expressing a mutant p62 that fails to interact with Keap1. Interestingly, p62 disease mutants have diminished or absent UBA domain ubiquitination. These data suggest that the ubiquitination of p62's UBA domain at lysine 420 may regulate p62's function and be disrupted in p62-associated disease.
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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.
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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.
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Project description:UnlabelledFerroptosis is a recently recognized form of regulated cell death caused by an iron-dependent accumulation of lipid reactive oxygen species. However, the molecular mechanisms regulating ferroptosis remain obscure. Here, we report that nuclear factor erythroid 2-related factor 2 (NRF2) plays a central role in protecting hepatocellular carcinoma (HCC) cells against ferroptosis. Upon exposure to ferroptosis-inducing compounds (e.g., erastin, sorafenib, and buthionine sulfoximine), p62 expression prevented NRF2 degradation and enhanced subsequent NRF2 nuclear accumulation through inactivation of Kelch-like ECH-associated protein 1. Additionally, nuclear NRF2 interacted with transcriptional coactivator small v-maf avian musculoaponeurotic fibrosarcoma oncogene homolog proteins such as MafG and then activated transcription of quinone oxidoreductase-1, heme oxygenase-1, and ferritin heavy chain-1. Knockdown of p62, quinone oxidoreductase-1, heme oxygenase-1, and ferritin heavy chain-1 by RNA interference in HCC cells promoted ferroptosis in response to erastin and sorafenib. Furthermore, genetic or pharmacologic inhibition of NRF2 expression/activity in HCC cells increased the anticancer activity of erastin and sorafenib in vitro and in tumor xenograft models.ConclusionThese findings demonstrate novel molecular mechanisms and signaling pathways of ferroptosis; the status of NRF2 is a key factor that determines the therapeutic response to ferroptosis-targeted therapies in HCC cells.
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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:Programmed cell death 4 (PDCD4) suppresses tumorigenesis, tumor progression, and invasion by inhibiting transcription and translation of oncogenes. However, the role of PDCD4 in lung tumorigenesis is unclear. Sequestosome1/p62 mediates cell proliferation, survival, and death through multiple signaling pathways, including autophagy and cell metabolism. p62/SQSTM1 is transcriptional target of Nrf2 and an important regulator of tumor growth. The aim of this study was to clarify whether and how PDCD4 regulates the p62-Nrf2 pathway, and how this regulation relates to tumorigenesis in human lung cancer cells. We established two stable human lung cancer cell lines, A549 and H460 that each overexpressed PDCD4. We found that PDCD4 overexpression decreased p62 expression levels and inhibited cell proliferation, and also increased the expression levels of cleaved PARP and cleaved caspase 3. Knockdown of p62 markedly increased the apoptotic rate of A549 and H460 cells overexpressing PDCD4. Furthermore levels of the epithelial-mesenchymal transition-related markers Slug, Snail, Twist1 and Vimentin were decreased and expression level of E-cadherin was increased in PDCD4-overexpressing cells. We also found that PDCD4 suppressed transcriptional activation of Nrf2 (an upstream regulator of p62) and increased endogenous levels of Keap1 (a negative regulator of Nrf2). Upregulation of Keap1 induced apoptosis and inhibited cell proliferation by suppressing activity of the p62-Nrf2 pathway in PDCD4-overexpressing cells. As anticipated, results from a mouse xenograft model showed that PDCD4 overexpression in xenografts inhibited cell proliferation and tumorigenesis. Taken together, our results demonstrate that PDCD4 overexpression, which increased Keap1 expression, reduces the levels and activity of the p62-Nrf2 pathway, thereby inhibiting tumorigenesis. Our findings suggest that PDCD4 may be a potential target for lung cancer therapies.
| S-EPMC7061761 | biostudies-literature
Project description:Background and aimsTRIM21 is a ubiquitin E3 ligase that is implicated in numerous biological processes including immune response, cell metabolism, redox homeostasis, and cancer development. We recently reported that TRIM21 can negatively regulate the p62-Keap1-Nrf2 antioxidant pathway by ubiquitylating p62 and prevents its oligomerization and protein sequestration function. As redox homeostasis plays a pivotal role in many cancers including liver cancer, we sought to determine the role of TRIM21 in hepatocarcinogenesis.MethodsWe examined the correlation between TRIM21 expression and the disease using publicly available data sets and 49 cases of HCC clinical samples. We used TRIM21 genetic knockout mice to determine how TRIM21 ablation impact HCC induced by the carcinogen DEN plus phenobarbital (PB). We explored the mechanism that loss of TRIM21 protects cells from DEN-induced oxidative damage and cell death.ResultsThere is a positive correlation between TRIM21 expression and HCC. Consistently, TRIM21-knockout mice are resistant to DEN-induced hepatocarcinogenesis. This is accompanied by decreased cell death and tissue damage upon DEN treatment, hence reduced hepatic tissue repair response and compensatory proliferation. Cells deficient in TRIM21 display enhanced p62 sequestration of Keap1 and are protected from DEN-induced ROS induction and cell death. Reconstitution of wild-type but not the E3 ligase-dead and the p62 binding-deficient mutant TRIM21 impedes the protection from DEN-induced oxidative damage and cell death in TRIM21-deficient cells.ConclusionsIncreased TRIM21 expression is associated with human HCC. Genetic ablation of TRIM21 leads to protection against oxidative hepatic damage and decreased hepatocarcinogenesis, suggesting TRIM21 as a preventive and therapeutic target.
| S-EPMC8024979 | biostudies-literature