p62/SQSTM1 - steering the cell through health and disease.

SQSTM1 (also known as p62) is a multifunctional stress-inducible scaffold protein involved in diverse cellular processes. Its functions are tightly regulated through an extensive pattern of post-translational modifications, and include the isolation of cargos degraded by autophagy, induction of the antioxidant response by the Keap1-Nrf2 system, as well as the regulation of endosomal trafficking, apoptosis and inflammation. Accordingly, malfunction of SQSTM1 is associated with a wide range of diseases, including bone and muscle disorders, neurodegenerative and metabolic diseases, and multiple forms of cancer. In this Review, we summarize current knowledge regarding regulation, post-translational modifications and functions of SQSTM1, as well as how they are dysregulated in various pathogenic contexts.

p62 bodies: cytosolic zoning by phase separation.

Cellular zoning or partitioning is critical in preventing macromolecules from random diffusion and orchestrating the spatiotemporal dynamics of biochemical reactions. Along with membranous organelles, membraneless organelles contribute to the precise regulation of biochemical reactions inside cells. In response to environmental cues, membraneless organelles rapidly form through liquid-liquid phase separation, sequester certain proteins and RNAs, mediate specific reactions and dissociate. Among membraneless organelles, ubiquitin-positive condensates, namely, p62 bodies, maintain cellular homeostasis through selective autophagy of themselves to contribute to intracellular quality control. p62 bodies also activate the anti-oxidative stress response regulated by the KEAP1-NRF2 system. In this review, we present an overview of recent advancements in cellular and molecular biology related to p62 bodies, highlighting their dynamic nature and functions.

The Role of KEAP1-NRF2 System in Atopic Dermatitis and Psoriasis.

The Kelch-like erythroid cell-derived protein with cap'n'collar homology-associated protein 1 (KEAP1)-nuclear factor erythroid-2-related factor 2 (NRF2) system, a thiol-based sensor-effector apparatus, exerts antioxidative and anti-inflammatory effects and maintains skin homeostasis. Thus, NRF2 activation appears to be a promising treatment option for various skin diseases. However, NRF2-mediated defense responses may deteriorate skin inflammation in a context-dependent manner. Atopic dermatitis (AD) and psoriasis are two common chronic inflammatory skin diseases caused by a defective skin barrier, dysregulated immune responses, genetic predispositions, and environmental factors. This review focuses on the role of the KEAP1-NRF2 system in the pathophysiology of AD and psoriasis and the therapeutic approaches that utilize this system.

NRF2 pathway activation attenuates ageing-related renal phenotypes due to α-klotho deficiency.

Oxidative stress is one of the major causes of the age-related functional decline in cells and tissues. The KEAP1-NRF2 system plays a central role in the regulation of redox balance, and NRF2 activation exerts antiageing effects by controlling oxidative stress in aged tissues. α-Klotho was identified as an ageing suppressor protein based on the premature ageing phenotypes of its mutant mice, and its expression is known to gradually decrease during ageing. Because α-klotho has been shown to possess antioxidant function, ageing-related phenotypes of α-klotho mutant mice seem to be attributable to increased oxidative stress at least in part. To examine whether NRF2 activation antagonizes ageing-related phenotypes caused by α-klotho deficiency, we crossed α-klotho-deficient (Kl-/-) mice with a Keap1-knockdown background, in which the NRF2 pathway is constitutively activated in the whole body. NRF2 pathway activation in Kl-/- mice extended the lifespan and dramatically improved ageing-related renal phenotypes. With elevated expression of antioxidant genes accompanied by an oxidative stress decrease, the antioxidant effects of NRF2 seem to make a major contribution to the attenuation of ageing-related renal phenotypes of Kl-/- mice. Thus, NRF2 is expected to exert an antiageing function by partly compensating for the functional decline of α-Klotho during physiological ageing.

Structural insights into the multiple binding modes of Dimethyl Fumarate (DMF) and its analogs to the Kelch domain of Keap1.

The activation of the nuclear factor erythroid 2-related factor 2 (Nrf2) transcription function has been implicated in the protection of neurodegenerative diseases. The cytoplasmic protein, Kelch-like ECH-associated protein 1 (Keap1), negatively regulates Nrf2. The Keap1-Nrf2 pathway is a potential therapeutic target for tackling free-radical damage. Dimethyl fumarate (DMF) is currently an approved drug for the treatment of relapsing multiple sclerosis. Recent studies showed that DMF modifies the reactive cysteines in the BTB domain of Keap1 and thus activates Nrf2 transcription function. Intriguingly, our crystal structure studies revealed that DMF also binds to the ß-propeller domain (Keap1-DC) of Keap1. The crystal structure of the complex, refined to 1.54 Å resolution, revealed unexpected features: DMF binds (a) to the Nrf2-binding site (bottom region of Keap1-DC, site 1) with moderate interaction, and (b) to the top region of Keap1-DC, near to the blade II (site 2). The specificity of the binding 'site 2' was found to be unique to blade II of the ß-propeller domain. The newly identified 'site 2' region in Keap1-DC may have a different functional role to regulate Nrf2. Moreover, the crystal structures of Keap1-DC in complex with the DMF analogs, including monoethyl fumarate, fumarate, and itaconate, also exhibited similar binding modes with Keap1-DC. Binding studies confirmed that DMF binds, in a nanomolar range, to the Keap1-DC region as well as the BTB domain of Keap1. Furthermore, the competitive binding assay in the presence of the Nrf2 peptide affirmed the direct binding of DMF at the Nrf2-binding region of Keap1-DC. Overall, our studies suggest that the drug molecule, DMF, binds at multiple sites of Keap1 and thus potentially activates Nrf2 function through covalent as well as the noncovalent mode of action, to combat oxidative stress. DATABASE: Structural data are available in RCSB-protein data bank database(s) under the accession numbers 6LRZ, 7C60, and 7C5E.

Skeletal muscle-specific Keap1 disruption modulates fatty acid utilization and enhances exercise capacity in female mice.

Skeletal muscle health is important for the prevention of various age-related diseases. The loss of skeletal muscle mass, which is known as sarcopenia, underlies physical disability, poor quality of life and chronic diseases in elderly people. The transcription factor NRF2 plays important roles in the regulation of the cellular defense against oxidative stress, as well as the metabolism and mitochondrial activity. To determine the contribution of skeletal muscle NRF2 to exercise capacity, we conducted skeletal muscle-specific inhibition of KEAP1, which is a negative regulator of NRF2, and examined the cell-autonomous and non-cell-autonomous effects of NRF2 pathway activation in skeletal muscles. We found that NRF2 activation in skeletal muscles increased slow oxidative muscle fiber type and improved exercise endurance capacity in female mice. We also observed that female mice with NRF2 pathway activation in their skeletal muscles exhibited enhanced exercise-induced mobilization and ß-oxidation of fatty acids. These results indicate that NRF2 activation in skeletal muscles promotes communication with adipose tissues via humoral and/or neuronal signaling and facilitates the utilization of fatty acids as an energy source, resulting in increased mitochondrial activity and efficient energy production during exercise, which leads to improved exercise endurance.

The KEAP1-NRF2 System in Healthy Aging and Longevity.

Aging is inevitable, but the inherently and genetically programmed aging process is markedly influenced by environmental factors. All organisms are constantly exposed to various stresses, either exogenous or endogenous, throughout their lives, and the quality and quantity of the stresses generate diverse impacts on the organismal aging process. In the current oxygenic atmosphere on earth, oxidative stress caused by reactive oxygen species is one of the most common and critical environmental factors for life. The Kelch-like ECH-associated protein 1-NFE2-related factor 2 (KEAP1-NRF2) system is a critical defense mechanism of cells and organisms in response to redox perturbations. In the presence of oxidative and electrophilic insults, the thiol moieties of cysteine in KEAP1 are modified, and consequently NRF2 activates its target genes for detoxification and cytoprotection. A number of studies have clarified the contributions of the KEAP1-NRF2 system to the prevention and attenuation of physiological aging and aging-related diseases. Accumulating knowledge to control stress-induced damage may provide a clue for extending healthspan and treating aging-related diseases. In this review, we focus on the relationships between oxidative stress and aging-related alterations in the sensory, glandular, muscular, and central nervous systems and the roles of the KEAP1-NRF2 system in aging processes.

Nrf-2 as a therapeutic target in acute kidney injury.

Multifaceted cellular pathways exhibit a crucial role in the preservation of homeostasis at the molecular, cellular, and organism levels. One of the most important of these protective cascades is Nuclear factor E2-related factor (Nrf-2) that regulates the expression of several genes responsible for cellular detoxification, antioxidant function, anti-inflammation, drug/xenobiotic transportation, and stress-related factors. A growing body of evidence provides information regarding the protective role of Nrf-2 against a number of kidney diseases. Acute kidney injury (AKI) is a substantial clinical problem that causes a huge social burden. In the kidneys, Nrf-2 exerts a dynamic role in improving the injury triggered by inflammation and oxidative stress. Understanding of the exact molecular mechanisms underlying AKI is vital in order to determine the equilibrium between renal adaptation and malfunction and thus reduce disease progression. This review highlights the role of Nrf-2 targeting against AKI and provides evidence that targeting Nrf-2 to prevail oxidative damage and its consequences might exhibit protective effects in kidney diseases.

Ellagic acid ameliorates oxidative stress and insulin resistance in high glucose-treated HepG2 cells via miR-223/keap1-Nrf2 pathway.

As a promising new target, miR-233 may regulate oxidative stress by targeting keap1-Nrf2 system to affect the pathological process of liver injury in T2DM. Ellagic acid (EA) is versatile for protecting oxidative stress damage and metabolic disorders. In the present study, we investigated the effect of EA on oxidative stress and insulin resistance in high glucose-induced T2DM HepG2 cells and examined the role of miR-223/keap1-Nrf2 pathway in system. HepG2 cells were incubated in 30 mM of glucose, with or without EA (15 and 30 µM) or metformin (Met, 150 µM) for 12 h. Glucose consumption, phosphorylation of IRS1, Akt and ERK under insulin stimulation, ROS and O2- production, MDA level, SOD activity and miR-223 expression, as well as protein levels of keap1, Nrf2, HO-1, SOD1 and SOD2 were analyzed. Furthermore, dual luciferase reporter assay, miR-223 mimic and inhibitor were implemented in cellular studies to explore the possible mechanism. EA upregulated glucose consumption, IRS1, Akt and ERK phosphorylation under insulin stimulation, reduced ROS and O2- production and MDA level, and increased SOD activity in high glucose-exposed HepG2 cells. In addition, EA elevated miR-223 expression level, downregulated mRNA and protein levels of keap1, and upregulated Nrf2, HO-1, SOD1 and SOD2 protein levels in this cell model. What's more, dual luciferase reporter assay, miR-223 mimic and inhibitor transfection confirmed that EA activated keap1-Nrf2 system via elevating miR-223. The miR-223, a negative regulator of keap1, represents an attractive therapeutic target in hepatic injury in T2DM. EA ameliorates oxidative stress and insulin resistance via miR-223-mediated keap1-Nrf2 activation in high glucose-induced T2DM HepG2 cells.

Trehalose protects against oxidative stress by regulating the Keap1-Nrf2 and autophagy pathways.

Dysfunction of autophagy, which regulates cellular homeostasis by degrading organelles and proteins, is associated with pathogenesis of various diseases such as cancer, neurodegeneration and metabolic disease. Trehalose, a naturally occurring nontoxic disaccharide found in plants, insects, microorganisms and invertebrates, but not in mammals, was reported to function as a mechanistic target of the rapamycin (mTOR)-independent inducer of autophagy. In addition, trehalose functions as an antioxidant though its underlying molecular mechanisms remain unclear. In this study, we showed that trehalose not only promoted autophagy, but also increased p62 protein expression, in an autophagy-independent manner. In addition, trehalose increased nuclear translocation of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) in a p62-dependent manner and enhance expression of its downstream antioxidant factors, heme oxygenase-1 (Ho-1) and nicotinamide adenine dinucleotide phosphate quinone dehydrogenase 1 (Nqo1). Moreover, treatment with trehalose significantly reduced amount of reactive oxygen species. Collectively, these results suggested that trehalose can function as a novel activator of the p62-Keap1/Nrf2 pathway, in addition to inducing autophagy. Therefore, trehalose may be useful to treat many chronic diseases involving oxidative stress and dysfunction of autophagy.

Multiple regulations of Keap1/Nrf2 system by dietary phytochemicals.

Keap1/Nrf2 system plays a critical role on cellular protection by regulating many antioxidant and detoxification enzyme genes through the antioxidant response element (ARE). Thus, it must work constantly to prevent the accumulation of reactive oxygen species (ROS) because excess ROS are associated with many diseases such as cancer, cardiovascular complications, inflammation, and neurodegeneration. Dietary phytochemicals widely distributing in fruits and vegetables have been considered to possess cancer chemopreventive potential through the induction of Keap1/Nrf2 system-mediated antioxidant and detoxification enzymes in a variety of manners. The data are extensive and are not well classified on the molecular mechanisms. In this review, we first briefly introduce the current knowledge on Keap1/Nrf2 system regulation including Keap1-dependent and Keap1-independent cascades, and epigenetic pathway. Then, we summarize the molecular targets of Keap1/Nrf2 system by dietary phytochemicals, and finally review the crosstalk between Keap1/Nrf2 system and other cellular signaling pathways to regulate diverse chronic diseases by dietary phytochemicals. These comprehensive data will help us to understand the potential effects of dietary phytochemicals on the prevention of chronic diseases and maintenance of human health.

Roles of Nrf2 in cell proliferation and differentiation.

The Keap1-Nrf2 system plays pivotal roles in defense mechanisms by regulating cellular redox homeostasis. Nrf2 is an inducible transcription factor that activates a battery of genes encoding antioxidant proteins and phase II enzymes in response to oxidative stress and electrophilic xenobiotics. The activity of Nrf2 is regulated by Keap1, which promotes the ubiquitination and subsequent degradation of Nrf2 under normal conditions and releases the inhibited Nrf2 activity upon exposure to the stresses. Though an impressive contribution of the Keap1-Nrf2 system to the protection from exogenous and endogenous electrophilic insults has been well established, a line of evidence has suggested that the Keap1-Nrf2 system has various novel functions, particularly in cell proliferation and differentiation. Because the proliferation and differentiation of diverse cell types are often influenced and modulated by the cellular redox balance, Nrf2 has been considered to control these cellular processes by regulating the cellular levels of reactive oxygen species (ROS). In addition, analyses of the genome-wide distribution of Nrf2 have identified new sets of Nrf2 target genes whose products are involved in cell proliferation and differentiation but not necessarily in the regulation of oxidative stress. Considering the most characteristic features of Nrf2 as an inducible transcription factor, a newly emerged concept proposes that the Keap1-Nrf2 system translates environmental stresses into regulatory network signals in cell fate determination. In this review, we introduce the contribution of Nrf2 to lineage-specific differentiation, maintenance and differentiation of stem cells, and proliferation of normal and cancer cells, and we discuss how the response to fluctuating environments modulates cell behavior through the Keap1-Nrf2 system.

p62/SQSTM1 functions as a signaling hub and an autophagy adaptor.

p62/SQSTM1 is a stress-inducible cellular protein that is conserved among metazoans but not in plants and fungi. p62/SQSTM1 has multiple domains that mediate its interactions with various binding partners and it serves as a signaling hub for diverse cellular events such as amino acid sensing and the oxidative stress response. In addition, p62/SQSTM1 functions as a selective autophagy receptor for degradation of ubiqutinated substrates. In the present review, we describe the current knowledge about p62 with regard to mammalian target of rapamycin complex 1 activation, the Keap1-Nrf2 pathway and selective autophagy.

Activation of the NRF2 pathway and its impact on the prognosis of anaplastic glioma patients.

BACKGROUND: Nuclear factor erythroid 2-related factor 2 (NRF2) plays pivotal roles in cytoprotection. We aimed at clarifying the contribution of the NRF2 pathway to malignant glioma pathology. METHODS: NRF2 target gene expression and its association with prognosis were examined in 95 anaplastic gliomas with or without isocitrate dehydrogenase (IDH) 1/2 gene mutations and 52 glioblastomas. To explore mechanisms for the altered activity of the NRF2 pathway, we examined somatic mutations and expressions of the NRF2 gene and those encoding NRF2 regulators, Kelch-like ECH-associated protein 1 (KEAP1) and p62/SQSTSM. To clarify the functional interaction between IDH1 mutations and the NRF2 pathway, we introduced a mutant IDH1 to T98 glioblastoma-derived cells and examined the NRF2 activity in these cells. RESULTS: NRF2 target genes were elevated in 13.7% and 32.7% of anaplastic gliomas and glioblastomas, respectively. Upregulation of NRF2 target genes correlated with poor prognosis in anaplastic gliomas but not in glioblastomas. Neither somatic mutations of NRF2/KEAP1 nor dysregulated expression of KEAP1/p62 explained the increased expression of NRF2 target genes. In most cases of anaplastic glioma with mutated IDH1/2, NRF2 and its target genes were downregulated. This was reproducible in IDH1 R132H-expressing T98 cells. In minor cases of IDH1/2-mutant anaplastic gliomas with increased expression of NRF2 target genes, the clinical outcomes were significantly poor. CONCLUSIONS: The NRF2 activity is increased in a significant proportion of malignant gliomas in general but decreased in the majority of IDH1/2-mutant anaplastic gliomas. It is plausible that the NRF2 pathway plays an important role in tumor progression of anaplastic gliomas with IDH1/2 mutations.