The molecular control of inflammation and epidermal thickening in skin lesions of patients with atopic dermatitis (AD) is not known. Sequestosome 1/p62 is a multifunctional adapter protein implicated in the control of key regulators of cellular homeostasis, such as proinflammatory and mechanistic target of rapamycin signaling.

Inhaled diacetyl vapors are associated with flavorings-related lung disease, a potentially fatal airway disease. The reactive α-dicarbonyl group in diacetyl causes protein damage in vitro. Dicarbonyl/l-xylulose reductase (DCXR) metabolizes diacetyl into acetoin, which lacks this α-dicarbonyl group. To investigate the hypothesis that flavorings-related lung disease is caused by in vivo protein damage, we correlated diacetyl-induced airway damage in mice with immunofluorescence for markers of protein turnover and autophagy. Western immunoblots identified shifts in ubiquitin pools. Diacetyl inhalation caused dose-dependent increases in bronchial epithelial cells with puncta of both total ubiquitin and K63-ubiquitin, central mediators of protein turnover. This response was greater in Dcxr-knockout mice than in wild-type controls inhaling 200 ppm diacetyl, further implicating the α-dicarbonyl group in protein damage. Western immunoblots demonstrated decreased free ubiquitin in airway-enriched fractions. Transmission electron microscopy and colocalization of ubiquitin-positive puncta with lysosomal-associated membrane proteins 1 and 2 and with the multifunctional scaffolding protein sequestosome-1 (SQSTM1/p62) confirmed autophagy. Surprisingly, immunoreactive SQSTM1 also accumulated in the olfactory bulb of the brain. Olfactory bulb SQSTM1 often congregated in activated microglial cells that also contained olfactory marker protein, indicating neuronophagia within the olfactory bulb. This suggests the possibility that SQSTM1 or damaged proteins may be transported from the nose to the brain. Together, these findings strongly implicate widespread protein damage in the etiology of flavorings-related lung disease.

This study evaluated the protective role of p38 mitogen-activated protein kinase (p38 MAPK) inhibitors and sequestosome 1 (Sqstm1/A170/p62), a stress-induced signal modulator, in acoustic injury of the cochlea in mice. Two weeks after the exposure of mice to acoustic stress, threshold shifts of the auditory brainstem response (ABR) from the pre-exposure level and hair cell loss were evaluated. The activation of p38 MAPK was observed in cochlea by immunostaining 4 h after acoustic stress. To examine the role of p38 MAPK in tissue injury, its inhibitors were i.p. injected into male wild-type C57BL mice before the acoustic overexposure. The inhibitors SB202190 and SB203580 but not the inactive analogue SB202474 dose-dependently decreased the auditory threshold shift and outer hair cell loss induced by acoustic overexposure, suggesting the involvement of p38 MAPK in ototoxicity. We found that acoustic overexposure induced the up-regulation of Sqstm1 mRNA expression in the cochlea of wild-type mice and that SQSTM1-deficient mice exhibited an enhanced ABR threshold shift and hair cell loss, suggesting a role of SQSTM1 in the protection of tissue from acoustic stress.

The atypical protein kinase C-interacting protein p62/sequestosome-1 (p62) has emerged as a crucial molecule in a variety of cellular functions due to its involvement in various signaling mechanisms. p62 has been implicated in the activation of NF-κB in TNFα-stimulated cells and has been shown to be activated in response to interleukin-1β (IL-1β). Here we demonstrate that p62 interacts with NEMO, the regulatory subunit of the complex responsible for activation of NF-κB transcription factor. Depletion of p62 obtained through a short interfering RNA targeting p62 mRNA abrogated TRAF6 capacity to promote NEMO ubiquitination and severely impairs NF-κB activation following IL-1β stimulation. Together, these results indicate that p62 is an important intermediary in the NF-κB activation pathways implemented through non-degradative ubiquitination events.

Paget's disease of bone (PDB) is a late-onset disorder characterised by focal areas of increased bone resorption, with osteoclasts that are increased in size, multinuclearity, number and activity. PDB-causing missense and nonsense variants in the gene encoding Sequestosome-1/p62 (SQSTM1) have been identified, all of which cluster in and around the ubiquitin-associated (UBA) domain of the protein. SQSTM1 is ubiquitously expressed and there is, as yet, no clear reason why these mutations only appear to cause an osteoclast-related phenotype. Using co-immunoprecipitation and tandem mass spectrometry, we identified a novel interaction in human osteoclast-like cells between SQSTM1 and Autophagy-Linked FYVE domain-containing protein (ALFY/WDFY3). Endogenous ALFY and SQSTM1 both localised within the nuclei of osteoclasts and their mononuclear precursors. When osteoclasts were starved to induce autophagy, SQSTM1 and ALFY relocated to the cytoplasm where they formed large aggregates, with cytoplasmic relocalisation appearing more rapid in mature osteoclasts than in precursors in the same culture. Overexpression of wild-type SQSTM1 in HEK293 cells also resulted in the formation of cytoplasmic aggregates containing SQSTM1 and endogenous ALFY, as did overexpression of a PDB-causing missense mutant form of SQSTM1, indicating that this mutation does not impair the formation of SQSTM1- and ALFY-containing aggregates. Expression of ALFY in bone cells has not previously been reported, and the process of autophagy has not been studied with respect to osteoclast activity. We have identified a functional interaction between SQSTM1 and ALFY in osteoclasts under conditions of cell stress. The difference in response to starvation between mature osteoclasts and their precursors may begin to explain the cell-specific functional effects of SQSTM1 mutations in PDB.

p62/Sequestosome 1 (p62) is a stress-induced protein that is involved in several different intracellular pathways, including regulation of aspects of protein degradation. p62 levels are elevated in several types of cataracts. We investigated whether levels of p62 and its phosphorylation were altered in the lenses of Cx50D47A mice, which express a mutant of connexin50 (Cx50) that leads to cataracts and impaired lens differentiation. To evaluate the importance of p62 in the lens defects caused by a connexin50 mutant, we also examined the effect of deleting p62 in homozygous Cx50D47A mice.

Sequestosome 1 (SQSTM1) also known as ubiquitin-binding protein p62 (p62) is a cargo protein involved in the degradation of misfolded proteins via selective autophagy. Disruption of autophagy and resulting accumulation of misfolded proteins in the endoplasmic reticulum (ER) leads to ER stress. ER stress is implicated in several neurodegenerative diseases and obesity. As knockout of p62 (p62KO) reportedly induces obesity in mice, we examined how p62 contributes to ER stress and the ensuing unfolded protein response (UPR) in hypothalamus using mouse organotypic cultures in the present study. Cultures from p62KO mice showed significantly reduced formation of LC3-GFP puncta, an index of autophagosome formation, in response to the chemical ER stressor thapsigargin compared to wild-type (WT) cultures. Hypothalamic cultures from p62KO mice exhibited higher basal expression of the UPR/ER stress markers CHOP mRNA and ATF4 mRNA than WT cultures. Thapsigargin enhanced CHOP, ATF4, and BiP mRNA as well as p-eIF2α protein expression in both WT and p62KO cultures, but all peak values were greater in p62KO cultures. A proteasome inhibitor increased p62 expression in WT cultures and upregulated the UPR/ER stress markers CHOP mRNA and ATF4 mRNA in both genotypes, but to a greater extent in p62KO cultures. Therefore, p62 deficiency disturbed autophagosome formation and enhanced both basal and chemically induced ER stress, suggesting that p62 serves to prevent ER stress in mouse hypothalamus by maintaining protein folding capacity.

Autophagy is a homeostatic process that is important for degrading protein aggregates, nutrient deposits, dysfunctional organelles and several signaling molecules. p62/sequestosome-1 is a protein that binds to several autophagy substrates, such as ubiquitinated proteins, damaged mitochondria and signaling molecules such as an Nrf2 inhibitor Keap1, promoting their autophagic degradation. Sestrin2, a stress-inducible protein, has recently been shown to bind to p62 and promote autophagic degradation of such p62 targets. Because Sestrin2 is a metabolic regulator that suppresses diverse age- and obesity-associated pathologies, the autophagy-controlling function of Sestrin2 may be important for its other physiological functions. However, the molecular mechanism of how Sestrin2 can promote clearance of p62-associated proteins has been unclear. Here we show that Sestrin2 physically associates with Unc-51-like protein kinase 1 (ULK1) and p62 to form a complex in which both Sestrin2 and p62 become phosphorylated by ULK1 at multiple sites. Ser403 of p62, whose phosphorylation is known to promote autophagic degradation of p62 and its targets, is among the sites phosphorylated by ULK1. ULK1-mediated p62 phosphorylation was facilitated by Sestrin2 in cells as well as in in vitro kinase assays. Consistent with this finding, oligomycin-induced energy deprivation, which strongly activates ULK1, provoked a robust Ser403 phosphorylation of p62 in wild-type mouse embryonic fibroblasts. However, in ULK1/2- and Sestrin2-deficient mouse embryonic fibroblasts, oligomycin-induced p62 phosphorylation was dramatically attenuated, suggesting that endogenous Sestrin2-ULK1/2 mainly mediates p62 phosphorylation in response to energetic stress. Taken together, this study identifies ULK1 as a new p62 Ser403 kinase and establishes Sestrin2 as a promoter of ULK1-mediated p62 phosphorylation.

Deficiency in the signal adaptor protein sequestosome 1 (SQSTM1/A170/p62) in mice is associated with mature-onset obesity, accompanied by insulin and leptin resistance. We previously established that redox sensitive transcription factor Nrf2 up-regulates SQSTM1 expression in response to atherogenic stimuli or laminar shear stress in vascular cells, and here examine the role of SQSTM1 in neointimal hyperplasia and vascular remodelling in vivo following carotid artery ligation. Neointimal hyperplasia was markedly enhanced at ligation sites after 3 weeks in SQSTM1(-/-) compared with wild-type (WT) mice. The intimal area and stenotic ratio were, respectively, 2.1- and 1.7-fold higher in SQSTM1(-/-) mice, indicating enhanced proliferation of vascular smooth muscle cells (SMCs). When aortic SMCs were isolated from WT and SQSTM1(-/-) mice and cultured in vitro, we found that SQSTM1(-/-) SMCs proliferated more rapidly in response to foetal calf serum (FCS) and attained 2-3-fold higher cell densities compared to WT SMCs. Moreover, migration of SQSTM1(-/-) SMCs was enhanced compared to WT SMCs. Early and late phases of p38(MAPK) activation in response to FCS stimulation were also more enhanced in SQSTM1(-/-) SMCs, and inhibitors of p38 and ERK1/2 signalling pathways significantly attenuated SMC proliferation. In summary, SQSTM1(-/-) mice exhibit enhanced neointimal hyperplasia and vascular remodelling following arterial ligation in vivo. The enhanced proliferation of SQSTM1(-/-) aortic SMCs in vitro highlights a novel role for SQSTM1 in suppressing smooth muscle proliferation following vascular injury.

Mallory-Denk bodies (MDBs) are hepatocytic protein aggregates found in steatohepatitis and several other chronic liver diseases as well as hepatocellular carcinoma. MDBs are mainly composed of phosphorylated keratins and stress protein p62/Sequestosome-1 (p62), which is a common component of cytoplasmic aggregates in a variety of protein aggregation diseases. In contrast to the well-established role of keratins, the role of p62 in MDB pathogenesis is still elusive. We have generated total and hepatocyte-specific p62 knockout mice, fed them with 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) to induce MDBs and allowed the mice to recover from DDC intoxication on a standard diet to investigate the role of p62 in MDB formation and elimination. In the absence of p62, smaller, granular and less distinct MDBs appeared, which failed to mature to larger and compact inclusions. Moreover, p62 deficiency impaired the binding of other proteins such as NBR1 and Hsp25 to MDBs and altered the cellular defense mechanism by downregulation of Nrf2 target genes. Upon recovery from DDC intoxication on a standard diet, there was an enhanced reduction of p62-deficient MDBs, which was accompanied by a pronounced decrease in ubiquitinated proteins. Our data provide strong evidence that keratin aggregation is the initial step in MDB formation in steatohepatitis-related mouse models. Interaction of p62 with keratin aggregates then leads to maturation i.e., enlargement and stabilization of the MDBs as well as recruitment of other MDB-associated proteins.

The cellular protein quality control system in association with aggresome formation contributes to protecting cells against aggregation-prone protein-induced toxicity. p62/Sequestosome 1 (p62) is a multifunctional protein which plays an important role in protein degradation and aggregation. Although poly-ubiquitination is usually required for p62-mediated protein degradation and aggresome formation, several p62 substrates are processed to form aggregate in an ubiquitination-independent manner. In this study we demonstrate that p62 directly interacts with pathogenic Machado Joseph Disease (MJD)-associated protein ataxin-3 with polyglutamine (polyQ) expansion. Moreover, p62 could regulate the aggresome formation of pathogenic ataxin-3 and protect cells against pathogenic ataxin-3-induced cell death.

Methylmercury (MeHg) is a hazardous environmental pollutant that causes serious toxicity in humans and animals, as well as proteotoxic stress. In our previous study, we found that MeHg induces the expression of p62/sequestosome 1 (p62) that selectively targets ubiquitinated proteins for degradation via autophagy, and that p62 might protect cells against MeHg toxicity. To further investigate the role of p62 in MeHg-induced stress responses, we evaluated the role of p62 in MeHg-induced endoplasmic reticulum (ER) stress in p62 knockout (p62KO) mouse embryonic fibroblasts (MEFs). Treatment of wild-type (WT) MEFs were treated with MeHg (1 μM) increased mRNA levels of Chop encoding C/EBP homologous protein, Trib3 encoding Tribbles homolog 3, and Dnajb9 encoding DnaJ heat-shock protein family (Hsp40) member B9 increased, suggesting that ER stress is elicited by MeHg stress. Additionally, p62KO MEFs treated with MeHg showed a higher mRNA expression of Chop and Trib3 relative to that in WT MEFs. Furthermore, knock-in of GFP-p62 to p62KO cells diminished the Chop and Trib3 induction responses to MeHg stress and resulted in a higher cell viability than that of p62KO MEFs. These results suggest that the protective role of p62 against MeHg toxicity is partly mediated by suppressing the ER stress response.

To investigate whether sequestosome 1/p62 (p62), a key cargo adaptor protein involved in both the ubiquitin-proteasome system and the autophagy-lysosome system, could directly regulate autophagy in vitro.

Sequestosome 1 (p62) is a multifunctional adapter protein involved in various physiological functions, such as selective autophagy and oxidative stress response. Hence, aberrant expression and defective regulation of p62 are thought to lead to the onset of various diseases, including cancer. The expression of p62 has been shown to be increased in breast cancer tissues, and is correlated with a poor prognosis. However, the role of p62 in the breast cancer pathophysiology is still unclear. Here, we aimed to analyze the effect of changes in p62 expression on breast cancer cell lines. DNA microarray analysis revealed that the expression of progesterone receptor (PR), which is one of the indices for the classification of breast cancer subtypes, was markedly suppressed by forced expression of p62. The protein expression of PR was also decreased by forced expression of p62, but increased by knockdown of p62. Moreover, we found that p62 knockdown induced the protein expression of argonaute 2 (AGO2). Luciferase reporter assay results showed that the gene expression of PR was promoted by AGO2. Furthermore, results revealed that overexpression of AGO2 partially rescued the decrease in PR expression induced by forced expression of p62. Collectively, our findings indicated that p62 accumulation suppressed the expression of AGO2, which in turn decreased the expression of PR, suggesting that p62 may serve as a marker of aggressive breast cancer and poor prognosis. Moreover, the p62-AGO2-PR axis was identified as a crucial signaling cascade in breast cancer progression.

Sequestosome 1 (SQSTM1)/p62 is an adapter protein mainly involved in the transportation, degradation and destruction of various proteins that cooperates with components of autophagy and the ubiquitin‑proteasome degradation pathway. Numerous studies have shown that SQSTM1/p62 functions at multiple levels, including involvement in genetic stability or modification, post‑transcriptional regulation and protein function. As a result, SQSTM1/p62 is a versatile protein that is a critical core regulator of tumor cell genetic stability, autophagy, apoptosis and other forms of cell death, malignant growth, proliferation, migration, invasion, metastasis and chemoradiotherapeutic response, and an indicator of patient prognosis. SQSTM1/p62 regulates these processes via its distinct molecular structure, through which it participates in a variety of activating or inactivating tumor‑related and tumor microenvironment‑related signaling pathways, particularly positive feedback loops and epithelial‑mesenchymal transition‑related pathways. Therefore, functioning as a proto‑oncogene or tumor suppressor gene in various types of cancer and tumor‑associated microenvironments, SQSTM1/p62 is capable of promoting or retarding malignant tumor aggression, giving rise to immeasurable effects on tumor occurrence and development, and on patient treatment and prognosis.

Diffuse myofiber necrosis in the context of inflammatory myopathy is the hallmark of immune-mediated necrotizing myopathy (IMNM). We have previously shown that skeletal muscle fibers of IMNM patients may display nonrimmed vacuoles and sarcoplasmic irregularities. The dysfunctional chaperone activity has been linked to the defective assembly of skeletal muscle proteins and their degradation via lysosomes, autophagy and the proteasomal machinery. This study was undertaken to highlight a chaperone-assisted selective autophagy (CASA) pathway, functionally involved in protein homeostasis, cell stress and the immune response in skeletal muscle of IMNM patients. Skeletal muscle biopsies from 54 IMNM patients were analyzed by immunostaining, as well as by qPCR. Eight biopsies of sIBM patients served as pathological controls, and eight biopsies of nondisease control subjects were included. Alteration of autophagy was detectable in all IMNM biopsy samples highlighted via a diffuse sarcoplasmic staining pattern by p62 and LC3 independent of vacuoles. This pattern was at variance with the coarse focal staining pattern mostly confined to rimmed vacuoles in sIBM. Colocalization of p62 with the chaperone proteins HSP70 and αB-crystalline points to the specific targeting of misfolded proteins to the CASA machinery. Bcl2-associated athanogene 3 (BAG3) positivity of these fibers emphasizes the selectivity of autophagy processes and these fibers also express MHC class I sarcolemma. Expression of genes involved in autophagy and endoplasmic reticulum (ER) stress pathways studied here is significantly upregulated in IMNM. We highlight that vacuoles without sarcolemmal features may arise in IMNM muscle biopsies, and they must not be confounded with sIBM-specific vacuoles. Further, we show the activation of selective autophagy and emphasize the role of chaperones in this context. CASA occurs in IMNM muscle, and specific molecular pathways of autophagy differ from the ones in sIBM, with p62 as a unique identifier of this process.

Nrf2 regulates the expression of numerous cytoprotective genes in mammalian cells. The activity of Nrf2 is regulated by the Cul3 adaptor Keap1, yet little is known regarding mechanisms of regulation of Keap1 itself. Here, we have used immunopurification of Keap1 and mass spectrometry, in addition to immunoblotting, to identify sequestosome 1 (SQSTM1) as a cellular binding partner of Keap1. SQSTM1 serves as a scaffold in various signaling pathways and shuttles polyubiquitinated proteins to the proteasomal and lysosomal degradation machineries. Ectopic expression of SQSTM1 led to a decrease in the basal protein level of Keap1 in a panel of cells. Furthermore, RNA interference (RNAi) depletion of SQSTM1 resulted in an increase in the protein level of Keap1 and a concomitant decrease in the protein level of Nrf2 in the absence of changes in Keap1 or Nrf2 mRNA levels. The increased protein level of Keap1 in cells depleted of SQSTM1 by RNAi was linked to a decrease in its rate of degradation; the half-life of Keap1 was almost doubled by RNAi depletion of SQSTM1. The decreased level of Nrf2 in cells depleted of SQSTM1 by RNAi was associated with decreases in the mRNA levels, protein levels, and function of several Nrf2-regulated cell defense genes. SQSTM1 was dispensable for the induction of the Keap1-Nrf2 pathway, as Nrf2 activation by tert-butylhydroquinone or iodoacetamide was not affected by RNAi depletion of SQSTM1. These findings demonstrate a physical and functional interaction between Keap1 and SQSTM1 and reveal an additional layer of regulation in the Keap1-Nrf2 pathway.

Paget disease of bone (PDB) is a common disorder characterized by focal and disorganized increases of bone turnover. Genetic factors are important in the pathogenesis of PDB. We and others recently mapped the third locus associated with the disorder, PDB3, at 5q35-qter. In the present study, by use of 24 French Canadian families and 112 unrelated subjects with PDB, the PDB3 locus was confined to approximately 300 kb. Within this interval, two disease-related haplotype signatures were observed in 11 families and 18 unrelated patients. This region encoded the ubiquitin-binding protein sequestosome 1 (SQSTM1/p62), which is a candidate gene for PDB because of its association with the NF-kappaB pathway. Screening SQSTM1/p62 for mutations led to the identification of a recurrent nonconservative change (P392L) flanking the ubiquitin-associated domain (UBA) (position 394-440) of the protein that was not present in 291 control individuals. Our data demonstrate that two independent mutational events at the same position in SQSTM1/p62 caused PDB in a high proportion of French Canadian patients.

Transforming growth factor beta (TGFβ) receptor trafficking regulates many TGFβ-dependent cellular outcomes including epithelial to mesenchymal transition (EMT). EMT in A549 non-small cell lung cancer (NSCLC) cells has recently been linked to the regulation of cellular autophagy. Here, we investigated the role of the autophagy cargo receptor, p62/sequestosome 1 (SQSTM1), in regulating TGFβ receptor trafficking, TGFβ1-dependent Smad2 phosphorylation and EMT in A549 NSCLC cells. Using immunofluorescence microscopy, p62/SQSTM1 was observed to co-localize with TGFβ receptors in the late endosome. Small interfering RNA (SiRNA)-mediated silencing of p62/SQSTM1 resulted in an attenuated time-course of Smad2 phosphorylation but did not alter Smad2 nuclear translocation. However, p62/SQSTM1 silencing promoted TGFβ1-dependent EMT marker expression, actin stress fiber formation and A549 cell migration. We further observed that Smad4-independent TGFβ1 signaling decreased p62/SQSTM1 protein levels via a proteasome-dependent mechanism. Although p62/SQSTM1 silencing did not impede TGFβ-dependent autophagy, our results suggest that p62/SQSTM1 may aid in maintaining A549 cells in an epithelial state and TGFβ1 decreases p62/SQSTM1 prior to inducing EMT and autophagy.

p62/sequestosome 1 (p62) is a multi-domain protein that functions as a receptor for ubiquitinated targets in the selective autophagy and serves as a scaffold in various signaling cascades. p62 have been reported to be up-regulated in several human malignancies, but the biological roles and significance of p62 are still poorly understood in colorectal carcinoma. We immunohistochemically evaluated p62 in 118 colorectal adenocarcinoma and 28 colorectal adenoma cases. We used four colon carcinoma cells (HCT8, HT29, COLO320, and SW480) in the in vitro studies. p62 immunoreactivity was detected in 11% of colorectal adenoma cases and 31% of adenocarcinoma cases, while it was negligible in the normal epithelium. The immunohistochemical p62 status was significantly associated with synchronous liver metastasis, and it turned out to be an independent adverse prognostic factor in colorectal cancer patients. Following in vitro studies revealed that HCT8 and HT29 cells transfected with p62-specific siRNA showed significantly decreased cell proliferation activity, whereas COLO320 and SW480 cells transfected with p62 expression plasmid showed significantly increased cell proliferation activity. The p62-mediated cell proliferation was not associated with the autophagy activity. These findings suggest that p62 promotes the cell proliferation mainly as a scaffold protein, and that the p62 status is a potent prognostic factor in colorectal carcinoma patients.