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Project description:Squestosome 1 (SQSTM1), also known as p62, is a multi-functional adaptor protein known for its pleotropic roles in autophagy, proteostasis, inflammation and cancer. Recently, p62 has emerged as an important modulator of protein quality control and aging. However, its role in the heart is not well understood. Our understanding of the role of p62 in the heart has been limited to the indirect assessment of its function in the setting of autophagy inhibition or proteotoxic stress. However, whether p62 is required to maintain cardiac function at rest or in response to stress has not been explored. Here we investigated the functional consequence of cardiac p62 deletion in the absence of other contributing phenotypic and systemic factors observed in the whole-body p62 deleted mice. Lack of cardiomyocytes p62 precipitated cardiac aging in mice and was associated with reduced contractile function and a progressive development of cardiac hypertrophy and fibrosis. Transcriptomic analysis of p62-deleted heart revealed a selective impairment in Nrf2 transcription, which was confirmed in the hearts of p62cKO mice. We further showed that absence of p62 in adult mice resulted in excessive oxidative stress and cell death when mice were rendered hypoxic. To gain mechanistic insights, we employed loss and gain of p62 function in H9c2 cardiomyoblasts and showed a sustained reduction in Nrf2 protein expression, nuclear translocation and transcriptional activity in p62-deficient cells. Mechanistically, p62-deficient cells exhibited an increase in proteasome-mediated Nrf2 degradation. In contrast, gain of p62 function led to Nrf2 stabilization and transcriptional activity.
2023-12-31 | GSE196019 | GEO
Project description:NRF2 is a transcription factor responsible for antioxidant stress responses that is usually regulated in a redox-dependent manner. p62 bodies formed by liquid-liquid phase separation contain Ser349-phosphorylated p62, which participates in the redox-independent activation of NRF2. However, the regulatory mechanism and physiological significance of p62 phosphorylation remain unclear. Here, we identify ULK1 as a kinase responsible for the phosphorylation of p62. ULK1 co-localizes with p62 bodies, directly interacting with p62. ULK1-dependent phosphorylation of p62 allows KEAP1 to be retained within p62 bodies, thus activating NRF2. p62S351E/+ mice are phosphomimetic knock-in mice in which Ser351, corresponding to human Ser349, is replaced by Glu. These mice, but not their phosphodefective p62S351A/S351A counterparts, exhibit NRF2 hyperactivation and growth retardation. This retardation is caused by malnutrition and dehydration due to obstruction of the esophagus and forestomach secondary to hyperkeratosis, a phenotype also observed in systemic Keap1-knockout mice. Our results expand our understanding of the physiological importance of the redox-independent NRF2 activation pathway and provide new insights into the role of phase separation in this process.
| S-SCDT-10_15252-EMBJ_2022113349 | biostudies-other
Project description:p62/SQSTM1 is a ubiquitin-binding autophagy receptor and signaling protein that accumulates in premalignant liver diseases and most hepatocellular carcinomas (HCC). Although p62 was proposed to participate in formation of benign adenomas in autophagy-deficient livers, its role in HCC initiation was not explored. Here we show that p62 is necessary and sufficient for HCC induction in mice and that its high expression level in non-tumor human liver predicts rapid HCC recurrence after curative ablation. High p62 expression is needed for activation of NRF2 and mTORC1, c-Myc induction and protection of HCC-initiating cells from oxidative stress-induced death.
2016-11-23 | GSE77323 | GEO
Project description:Neurodegenerative disorders are an increasingly common and irreversible burden on society, often affecting the ageing population, but their aetiology and disease mechanisms are poorly understood. Studying monogenic neurodegenerative diseases, with known genetic cause, provides an opportunity to understand cellular mechanisms also affected in more complex disorders. We recently reported that loss-of-function mutations in the autophagy adaptor protein, SQSTM1/p62, lead to a slowly progressive neurodegenerative disease presenting in childhood. To further elucidate the neuronal involvement, we studied the cellular consequences of loss of p62 in a neuroepithelial stem (NES) cell model and differentiated neurones, derived from reprogrammed p62 patient cells, or by CRISPR/Cas9-directed gene editing in NES cells. Transcriptomic and proteomic analyses suggest that p62 is essential for neuronal differentiation by controlling the metabolic shift from aerobic glycolysis to oxidative phosphorylation required for neuronal maturation. This shift is blocked by the failure to sufficiently downregulate lactate dehydrogenase expression due to the loss of p62, possibly through impaired Hif-1α downregulation and increased sensitivity to oxidative stress. The findings implicate an important role for p62 in neuronal energy metabolism and particularly in the regulation of the shift between glycolysis and oxidative phosphorylation, required for normal neurodifferentiation.
2019-01-31 | GSE99559 | GEO
Project description:These are the results of the iCLIP experiment for p62/SQSTM1 in Human Huh-7 cells treated with DMSO. We used iCLIP method to identify the RNA targets of p62 and nucleotide positions of the p62 interaction on RNA. We used 2 replicates and 2 different antibodies against endogenous p62 to enrich protein/RNA complexes. cDNAs were tagged with iCLIP composite barcodes (e.g. NNNTTGTNN) which contain 4 sample-encoding bases (e.g. TTGT) and and 5 random bases (noted with N in NNNTTGTNN example) which serve as unique molecular identifiers to post-filter PCR duplicates. These composite barcodes are found in the read headers (after last colon ':' character) of submitted fastq files.
2018-03-31 | E-MTAB-4894 | biostudies-arrayexpress
Project description:Cancer-derived loss-of-function mutations in the KEAP1 tumor suppressor gene stabilize the NRF2 transcription factor, resulting in a pro-survival gene expression program that alters cellular metabolism and neutralizes oxidative stress. In a previous study of KEAP1 mutations observed in lung cancer, we classified 40% of the mutations as ‘superbinders’ (superbinders). These mutants bind and ubiquitylate NRF2 but do not promote NRF2 degradation. Here, we further investigated the molecular mechanism(s) driving the superbinder phenotype. BioID-based quantitative proteomic analysis of the R320Q and R470C superbinder mutations revealed increased co-complexed NRF2 without significant alteration to other KEAP1-associated proteins, including CUL3, VCP, and several ubiquitin receptors within the proteasome lid. Dynamic simulation modeling and limited proteolysis analyses suggest that superbinder mutations stabilize residues in KEAP1 that contact NRF2. In cells, KEAP1 R320Q and R470C mutants co-localize with NRF2, p62/SQSTM1 and polyubiquitin in spherical clusters that rapidly fuse and dissolve; KEAP1-NRF2 localization to these clusters requires p62. Expression of R320Q and R470C in lung cancer cells provided resistance to the reactive oxygen species-inducing drug bleomycin. We present a model wherein superbinder mutations alter the conformational dynamics of the KEAP1-NRF2 complex to alter the cycling of KEAP1 between open and closed conformations, thus inhibiting NRF2 degradation.
2018-11-07 | PXD008411 | Pride
Project description:The purpose of this study is to identify the differential transcriptome profiles in WT, hepatic Atg5 KO, TSC1 KO, Atg5/TSC1 DKO, Atg5/TSC1/p62 TKO and Atg5/TSC1/Nrf2 TKO mouse livers. Hepatic mRNA from 2-month-old mice from 5 different mouse strains were extracted and performed for Nextseq analysis in quadruplicates.
2021-12-14 | GSE186783 | GEO
Project description:Autophagy deficiency caused by conditional knockout of Atg7 results in severe hepatitis accompanied by abundant accumulation of p62. p62 stablizes Nrf2 by disrupting the association between Keap1 and Nrf2. To understand the pathogenesis of hepatitis under the autophagy deficiency, we examined gene expression profiles of livers from Atg7-null, Nrf2-null and Atg7-Nrf2 double mutant mice. Eight week old Atg7F/F:Mx1-Cre mice and Atg7F/F:Mx1-Cre:Nrf2-/- together with control mice were injected with pIpC. At 4 weeks after pIpC injection, total RNAs were purified from each mouse liver.
2010-10-14 | E-GEOD-20415 | biostudies-arrayexpress
Project description:Huntington's disease (HD) is a dominantly inherited genetic disease caused by mutant huntingtin (htt) protein with expanded polyglutamine tracts. A neuropathological hallmark of HD is the presence of neuronal inclusions of mutant htt. p62 is an important regulatory protein in selective autophagy, a process by which aggregated proteins are degraded, and it is associated with several neurodegenerative disorders including HD. Here we investigated the effect of p62 depletion in three HD model mice: R6/2, HD190QG and HD120QG mice. We found that loss of p62 in these models led to longer lifespans and reduced nuclear inclusions, although cytoplasmic inclusions increased with polyglutamine length. In mouse embryonic fibroblasts (MEFs) with or without p62, mutant htt with a nuclear localization signal (NLS) showed no difference in nuclear inclusion between the two MEF types. In the case of mutant htt without NLS, however, p62 depletion increased cytoplasmic inclusions. Furthermore, to examine the effect of impaired autophagy in HD model mice, we crossed R6/2 mice with Atg5 conditional knockout mice. These mice also showed decreased nuclear inclusions and increased cytoplasmic inclusions, similar to HD mice lacking p62. These data suggest that the genetic ablation of p62 in HD model mice enhances cytoplasmic inclusion formation by interrupting autophagic clearance of polyQ inclusions. This reduces polyQ nuclear influx and paradoxically ameliorates disease phenotypes by decreasing toxic nuclear inclusions. Gene expression profiles were analyzed to examine the effects of p62 depletion in mouse with or without mutant huntingtin exon 1 To examine the effect of p62 depletion on the transcriptome of Huntington's disease model mice, we crossed p62 knockout mice with HD model mice. We extracted total RNA from the striatum of these mice at 8 weeks and used for a microaaray analysis. The samples are HD transgenic mice with p62 knockout (HD_p62KO), HD mice with normal p62 (HD_p62WT), non-HD-transgenic mice with p62 knockout (NT_p62KO), and non-HD-transgenic mice with normal p62 (NT_p62WT).
2014-10-10 | E-GEOD-62210 | biostudies-arrayexpress
Project description:Glucocorticoids are widely used to treat inflammatory disorders. Prolonged use results in side effects including osteoporosis, diabetes and obesity. The selective glucocorticoid receptor (GR) modulator Compound A (CpdA) exhibits an inflammation-suppressive effect, largely in absence of detrimental side effects. To understand the mechanistic differences between the classic glucocorticoid dexamethasone (DEX) and CpdA, we looked for proteins oppositely regulated using an unbiased proteomics approach. We found that the autophagy receptor p62 but not GR mediates the anti-inflammatory action of CpdA in macrophages. CpdA drives the upregulation of p62 by recruiting the NRF2 transcription factor to its promoter. Contrarily, the classic GR ligand dexamethasone recruits GR to p62 and other NRF2 controlled gene promoters, resulting in gene downregulation. Both DEX and CpdA are able to induce autophagy, albeit in a cell-type and time-dependent manner. Suppression of LPS-induced IL-6 and MCP1 genes in bone marrow-derived macrophages by CpdA is hampered upon p62 silencing, confirming that p62 is essential for the anti-inflammatory capacity of CpdA. Together, these results demonstrate how off-target mechanisms of selective GR ligands may establish a more efficient anti-inflammatory therapy
2022-06-03 | PXD007793 | Pride