RESUMEN
Bone marrow-derived mesenchymal/stromal stem cells (MSCs) became a major focus of research since the anti-inflammatory features and the osteogenic commitment of these cells can prevent the inflamm-aging and various form of osteopenia in humans and animals. We previously showed that p62/SQSTM1 plasmid can prompt release of anti-inflammatory cytokines/chemokines by MSC when injected in adult mice. Furthermore, it can enhance osteoblastogenesis at the expense of adipogenesis and ameliorate bone density and bone remodeling. On the other hand, absence of p62 partially exhausted MSC pool caused expansion of fat cells within bone marrow and pro-inflammatory mediator's accumulation. Given the critical function of p62 as molecular hub of MSC dynamics, here, using MSCs from p62 knockout adult mice, we investigated the effect of this protein on MSC survival and bone-forming molecule cascades. We found that the main osteogenic routes are impaired in absence of p62. In particular, lack of p62 can suppress Smads activation, and Osterix and CREBs expression, thus significantly modifying the schedule of MSCs differentiation. MSCs obtained from p62-/- mice have also demonstrate an amplified NFκB/ Smad1/5/8 colocalization along with NFκB activation in the nucleus, which precludes Smads binding to target promoters. Considering the "teamwork" of TGFß, PTH and BMP2 on MSC homeostatic behavior, we consider that p62 exerts an essential role as a hub protein. Lastly, ex vivo pulsing p62-deficient MSCs, which then will be administered to a patient as a cell therapy, may be considered as a treatment for bone and bone marrow disorders.
Asunto(s)
Células Madre Mesenquimatosas , Osteogénesis , Proteína Sequestosoma-1 , Animales , Médula Ósea/metabolismo , Diferenciación Celular , Células Madre Mesenquimatosas/metabolismo , Ratones , Ratones Noqueados , Proteína Sequestosoma-1/genética , Proteína Sequestosoma-1/fisiologíaRESUMEN
Selective autophagy mediates the degradation of cytoplasmic cargos, such as damaged organelles, invading pathogens, and protein aggregates. However, whether it targets double-stranded RNA (dsRNA) of intracellular pathogens is still largely unknown. Here, we show that selective autophagy regulates the degradation of the infectious bursal disease virus (IBDV) dsRNA genome. The amount of dsRNA decreased greatly in cells that overexpressed the autophagy-required protein VPS34 or autophagy cargo receptor SQSTM1, while it increased significantly in SQSTM1 or VPS34 knockout cells or by treating wild-type cells with the autophagy inhibitor chloroquine or wortmannin. Confocal microscopy and structured illumination microscopy showed SQSTM1 colocalized with dsRNA during IBDV infection. A pull-down assay further confirmed the direct binding of SQSTM1 to dsRNA through amino acid sites R139 and K141. Overexpression of SQSTM1 inhibited the replication of IBDV, while knockout of SQSTM1 promoted IBDV replication. Therefore, our findings reveal the role of SQSTM1 in clearing viral dsRNA through selective autophagy, highlighting the antiviral role of autophagy in the removal of the viral genome.
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Autofagia/fisiología , Infecciones por Birnaviridae/prevención & control , Virus de la Enfermedad Infecciosa de la Bolsa/fisiología , ARN Bicatenario/metabolismo , ARN Viral/metabolismo , Proteína Sequestosoma-1/fisiología , Células HEK293 , Humanos , Virus de la Enfermedad Infecciosa de la Bolsa/genética , Replicación ViralRESUMEN
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 ubiquitinproteasome degradation pathway. Numerous studies have shown that SQSTM1/p62 functions at multiple levels, including involvement in genetic stability or modification, posttranscriptional 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 tumorrelated and tumor microenvironmentrelated signaling pathways, particularly positive feedback loops and epithelialmesenchymal transitionrelated pathways. Therefore, functioning as a protooncogene or tumor suppressor gene in various types of cancer and tumorassociated 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.
Asunto(s)
Neoplasias/etiología , Proteína Sequestosoma-1/fisiología , Agresión , Transición Epitelial-Mesenquimal , Humanos , Neoplasias/patología , Proteína Sequestosoma-1/genética , Microambiente TumoralRESUMEN
Macroautophagy/autophagy plays a critical role in antiviral immunity through targeting viruses and initiating host immune responses. The receptor protein, SQSTM1/p62 (sequestosome 1), plays a vital role in selective autophagy. It serves as a receptor targeting ubiquitinated proteins or pathogens to phagophores for degradation. In this study, we explored the reciprocal regulation between selective autophagy receptor SQSTM1 and Seneca Valley virus (SVV). SVV infection induced autophagy. Autophagy promoted SVV infection in pig cells but played opposite functions in human cells. Overexpression of SQSTM1 decreased viral protein production and reduced viral titers. Further study showed that SQSTM1 interacted with SVV VP1 and VP3 independent of its UBA domain. SQSTM1 targeted SVV VP1 and VP3 to phagophores for degradation to inhibit viral replication. To counteract this, SVV evolved strategies to circumvent the host autophagic machinery to promote viral replication. SVV 3Cpro targeted the receptor SQSTM1 for cleavage at glutamic acid 355, glutamine 392, and glutamine 395 and abolished its capacity to mediate selective autophagy. At the same time, the 3Cpro-mediated SQSTM1 cleavage products lost the ability to inhibit viral propagation. Collectively, our results provide evidence for selective autophagy in host against viruses and reveal potential viral strategies to evade autophagic machinery for successful pathogenesis.Abbreviations: Baf.A1: bafilomycin A1; Co-IP: co-immunoprecipitation; hpi: h post-infection; LIR: LC3-interacting region; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; MOI: multiplicity of infection; PB1: N-terminal Phox/Bem1p; Rap.: rapamycin; Seneca Valley virus: SVV; SQSTM1/p62: sequestosome 1; SQSTM1-N355: residues 1 to 355 of SQSTM1; SQSTM1-C355: residues 355 to 478 of SQSTM1; SQSTM1-N392: residues 1 to 392 of SQSTM1; SQSTM1-C392: residues 392 to 478 of SQSTM1; SQSTM1-N388: residues 1 to 388 of SQSTM1; SQSTM1-N397: residues 1 to 397 of SQSTM1; UBA: ubiquitin association; Ubi: ubiquitin.
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Autofagia , Picornaviridae/fisiología , Proteína Sequestosoma-1/metabolismo , Proteínas Estructurales Virales/metabolismo , Replicación Viral , Animales , Línea Celular , Células HEK293 , Humanos , Proteína Sequestosoma-1/fisiología , Especificidad de la Especie , Porcinos , Proteínas Estructurales Virales/fisiologíaRESUMEN
Macroautophagy/autophagy plays an important role in the control of viral infections and viruses have evolved multiple strategies to interfere with autophagy to avoid destruction and promote their own replication and spread. Here we report that the deubiquitinase encoded in the N-terminal domain of the Epstein-Barr virus (EBV) large tegument protein, BPLF1, regulates selective autophagy. Mass spectrometry analysis identified several vesicular traffic and autophagy related proteins as BPLF1 interactors and potential substrates, suggesting that the viral protein targets this cellular defense during productive infection. Direct binding of BPLF1 to the autophagy receptor SQSTM1/p62 (sequestosome 1) was confirmed by co-immunoprecipitation of transfected BPLF1 and by in vitro affinity isolation of bacterially expressed proteins. Expression of the catalytically active BPLF1 was associated with decreased SQSTM1/p62 ubiquitination and failure to recruit LC3 to SQSTM1/p62-positive aggregates. Selective autophagy was inhibited as illustrated by the accumulation of large protein aggregates in BPLF1-positive cells co-transfected with an aggregate-prone HTT (huntingtin)-Q109 construct, and by a slower autophagy-dependent clearance of protein aggregates upon transfection of BPLF1 in cells expressing a tetracycline-regulated HTT-Q103. The inhibition of aggregate clearance was restored by overexpression of a SQSTM1/p62[E409A,K420R] mutant that does not require ubiquitination of Lys420 for cargo loading. These findings highlight a previously unrecognized role of the viral deubiquitinase in the regulation of selective autophagy, which may promote infection and the production of infectious virus.Abbreviations: BPLF1, BamH1 fragment left open reading frame-1; EBV, Epstein-Barr virus; GFP, green fluorescent protein; HTT, huntingtin; MAP1LC3/LC3, microtubule associated protein 1 light chain 3; PB1, Phox and Bem1 domain; PE, phosphatidylethanolamine; SQSTM1/p62, sequestosome 1; UBA, ubiquitin-associated domain.
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Autofagia/fisiología , Enzimas Desubicuitinizantes/fisiología , Herpesvirus Humano 4/fisiología , Proteína Sequestosoma-1/fisiología , Proteínas Reguladoras y Accesorias Virales/fisiología , Autofagia/genética , Enzimas Desubicuitinizantes/genética , Infecciones por Virus de Epstein-Barr/patología , Infecciones por Virus de Epstein-Barr/virología , Células HeLa , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/patogenicidad , Interacciones Microbiota-Huesped/genética , Interacciones Microbiota-Huesped/fisiología , Humanos , Proteína Huntingtina/genética , Proteína Huntingtina/metabolismo , Macroautofagia/genética , Macroautofagia/fisiología , Proteínas Asociadas a Microtúbulos/metabolismo , Mutación , Agregado de Proteínas/genética , Agregado de Proteínas/fisiología , Proteína Sequestosoma-1/genética , Transfección , Ubiquitinación , Proteínas Reguladoras y Accesorias Virales/genéticaRESUMEN
Irisin has gained attention because of its potential applications in the treatment of metabolic diseases. Accumulating evidence indicates that irisin attenuates obesity via the browning of white adipose tissue; however, the underlying mechanisms are unclear. Here, we evaluated the effects of irisin on adipocyte browning and the underlying mechanisms. The western blotting and immunofluorescence analyses demonstrated that irisin significantly induced the up-regulation of brown fat-specific proteins (PGC1α, PRDM16, and UCP-1) and HO-1 in 3T3-L1 adipocytes. Moreover, irisin significantly increased the levels of cytosolic p62 and nuclear Nrf2. These effects of irisin in the adipocytes were attenuated by treatment with SnPP or p62 siRNA. In addition, the browning effect of irisin was observed in BAT-WT-1 cells. These findings suggest that irisin induced browning effect via the p62/Nrf2/HO-1 signalling pathway and that it may be a potential candidate for preventing or treating obesity.
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Adipocitos Marrones/efectos de los fármacos , Adipocitos/efectos de los fármacos , Fibronectinas/farmacología , Hemo-Oxigenasa 1/fisiología , Proteínas de la Membrana/fisiología , Proteína Sequestosoma-1/fisiología , Células 3T3-L1 , Adipocitos/fisiología , Adipocitos Marrones/fisiología , Animales , Diferenciación Celular/efectos de los fármacos , Transdiferenciación Celular/efectos de los fármacos , Transdiferenciación Celular/genética , Células Cultivadas , Fibronectinas/fisiología , Metabolismo de los Lípidos/efectos de los fármacos , Metabolismo de los Lípidos/genética , Ratones , Factor 2 Relacionado con NF-E2/fisiología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiologíaRESUMEN
Vascular calcification is a complication of diseases and conditions such as chronic kidney disease, diabetes, and aging. Previous studies have demonstrated that high concentrations of inorganic phosphate (Pi) can induce oxidative stress and vascular smooth muscle cell calcification. KEAP1 (Kelch-like ECH-associated protein 1)/NF-E2-related factor 2 (NRF2) signaling has been shown to play important roles in protecting cells from oxidative stress. The current study aims to investigate the possible involvement of the KEAP1/NRF2/P62 -mediated antioxidant pathway in vascular calcification induced by high Pi levels. Exposure of vascular smooth muscle cells (VSMCs) to high Pi concentrations promoted the accumulation of reactive oxygen species (ROS) and the nuclear translocation of NRF2, along with an increase in P62 levels and a decrease in KEAP1 levels. A classic NRF2 activator, tert-butylhydroquinone (tBHQ), significantly decreased ROS levels and calcium deposition in VSMCs by promoting the nuclear translocation of NRF2 and upregulating P62 and KEAP1 expression. In contrast, silencing NRF2 and P62 with siRNAs increased the levels of ROS and calcium deposition in VSMCs. In conclusion, VSMC calcification can be alleviated by the activation of the KEAP1/NRF2/P62 antioxidative pathway, which could have a protective role when it is exogenously activated by tBHQ.
Asunto(s)
Proteína 1 Asociada A ECH Tipo Kelch/fisiología , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Factor 2 Relacionado con NF-E2/fisiología , Fosfatos/farmacología , Especies Reactivas de Oxígeno/metabolismo , Proteína Sequestosoma-1/fisiología , Transducción de Señal/fisiología , Calcificación Vascular/prevención & control , Línea Celular , Fluoresceínas/metabolismo , Humanos , Hidroquinonas/farmacología , Proteína 1 Asociada A ECH Tipo Kelch/biosíntesis , Proteína 1 Asociada A ECH Tipo Kelch/genética , Músculo Liso Vascular/efectos de los fármacos , Miocitos del Músculo Liso/efectos de los fármacos , Oxidación-Reducción , Estrés Oxidativo/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Interferencia de ARN , ARN Interferente Pequeño/genética , Proteína Sequestosoma-1/biosíntesis , Proteína Sequestosoma-1/genética , Regulación hacia Arriba , Calcificación Vascular/metabolismoRESUMEN
Accumulation of damaged mitochondria is implicated in a number of neurodegenerative disorders, including Parkinson's disease. Therefore, the machinery for mitochondrial quality control is important for the prevention of such diseases. It has been reported that Parkin- and p62/sequestosome 1 (SQSTM1)-mediated clustering and subsequent elimination of damaged mitochondria (termed mitophagy) are critical for maintaining the quality of mitochondria under stress induced by uncoupling agents such as carbonyl cyanide m-chlorophenyl hydrazone. However, the molecular mechanisms underlying mitochondrial translocation to the perinuclear region during mitophagy have not been adequately addressed to date. In this study, we found that BCL2-associated athanogene 6 (BAG6; also known as BAT3 or Scythe) is required for this process. Indeed, RNA interference-mediated depletion of endogenous BAG6 prevented Parkin-dependent relocalization of mitochondrial clusters to the perinuclear cytoplasmic region, whereas BAG6 knockdown did not affect the translocation of Parkin and p62/SQSTM1 to the depolarized mitochondria and subsequent aggregation. These results suggest that BAG6 is essential for cytoplasmic redistribution, but not for clustering, of damaged mitochondria.
Asunto(s)
Mitocondrias/metabolismo , Mitofagia/fisiología , Chaperonas Moleculares/genética , Proteínas Adaptadoras Transductoras de Señales/genética , Autofagia/efectos de los fármacos , Citosol/metabolismo , Células HeLa , Humanos , Chaperonas Moleculares/metabolismo , Proteína Sequestosoma-1/metabolismo , Proteína Sequestosoma-1/fisiología , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación/efectos de los fármacosRESUMEN
Increased muscle contractile activity, as observed with regular exercise, prevents oxidative stress-induced muscle wasting, at least partially, by improving the antioxidant defense system. Phosphorylated p62/sequestosome1 competitively binds to the Kelch-like ECH-associated protein 1, activating nuclear factor erythroid 2-related factor 2 (Nrf2), which stimulates transcription of antioxidant/electrophile responsive elements. However, it remains to be determined if this process is activated by regular exercise in skeletal muscle. Here, we demonstrate that muscle contractile activity increases antioxidants, Nrf2 translocation into nuclei, and Nrf2 DNA-binding activity in association with increased p62 phosphorylation (Ser351) in mouse oxidative skeletal muscle. Skeletal muscle-specific loss of Nrf2 [i.e., Nrf2 muscle-specific knockout (mKO) mice] abolished the expression of the Nrf2 target antioxidant gene NAD(P)H-quinone oxidoreductase 1 (NQO1) in both glycolytic and oxidative muscles but reduced exercise-mediated increases of antioxidants (i.e., copper/zinc superoxide dismutase (SOD) and extracellular SOD only in oxidative muscle. Interestingly, skeletal muscle-specific loss of p62 (i.e., p62 mKO mice) also abolished the expression of NQO1 and reduced exercise-mediated increases of the same antioxidants in soleus muscle. Collectively, these findings indicate that p62 and Nrf2 cooperatively regulate the exercise-mediated increase of antioxidants in oxidative muscle.-Yamada, M., Iwata, M., Warabi, E., Oishi, H., Lira, V. A., Okutsu, M. p62/SQSTM1 and Nrf2 are essential for exercise-mediated enhancement of antioxidant protein expression in oxidative muscle.
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Músculo Esquelético/metabolismo , Factor 2 Relacionado con NF-E2/fisiología , Condicionamiento Físico Animal , Proteína Sequestosoma-1/fisiología , Superóxido Dismutasa/biosíntesis , Animales , Núcleo Celular/enzimología , Células Cultivadas , Citoplasma/enzimología , Glucólisis , Fuerza de la Mano , Proteína 1 Asociada A ECH Tipo Kelch/biosíntesis , Proteína 1 Asociada A ECH Tipo Kelch/genética , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Contracción Muscular/fisiología , Fibras Musculares Esqueléticas/metabolismo , Factor 2 Relacionado con NF-E2/deficiencia , Factor 2 Relacionado con NF-E2/genética , Oxidación-Reducción , Transporte de Proteínas , Músculo Cuádriceps/metabolismo , Carrera , Proteína Sequestosoma-1/deficiencia , Proteína Sequestosoma-1/genética , Superóxido Dismutasa/genéticaRESUMEN
Autophagy maintains hematopoietic stem cell integrity and prevents malignant transformation. In addition to bulk degradation, selective autophagy serves as an intracellular quality control mechanism and requires autophagy receptors, such as p62 (SQSTM1), to specifically bridge the ubiquitinated cargos into autophagosomes. Here, we investigated the function of p62 in acute myeloid leukemia (AML) in vitro and in murine in vivo models of AML. Loss of p62 impaired expansion and colony-forming ability of leukemia cells and prolonged latency of leukemia development in mice. High p62 expression was associated with poor prognosis in human AML. Using quantitative mass spectrometry, we identified enrichment of mitochondrial proteins upon immunoprecipitation of p62. Loss of p62 significantly delayed removal of dysfunctional mitochondria, increased mitochondrial superoxide levels, and impaired mitochondrial respiration. Moreover, we demonstrated that the autophagy-dependent function of p62 is essential for cell growth and effective mitochondrial degradation by mitophagy. Our results highlight the prominent role of selective autophagy in leukemia progression, and specifically, the importance of mitophagy to maintain mitochondrial integrity.
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Autofagia , Leucemia Experimental/patología , Leucemia Mieloide Aguda/patología , Mitofagia , Proteína Sequestosoma-1/metabolismo , Proteína Sequestosoma-1/fisiología , Animales , Estudios de Seguimiento , Humanos , Leucemia Experimental/genética , Leucemia Experimental/metabolismo , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Ratones , Ratones Noqueados , Pronóstico , Tasa de Supervivencia , Células Tumorales CultivadasRESUMEN
In hepatocellular carcinoma (HCC), dysregulated expression of DDX5 (DEAD box protein 5) and impaired autophagy have been reported separately. However, the relationship between them has not been explored. Here we present evidence to show that, by interacting with autophagic receptor p62, DDX5 promotes autophagy and suppresses tumorigenesis. DDX5 inversely correlated with p62/sequestosome 1 (SQSTM1) expression in hepatitis B virus (HBV)-associated and non-HBV-associated HCCs. Patients with low DDX5 expression showed poor prognosis after tumor resection. We found that DDX5 overexpression induced, while DDX5 knockdown attenuated, autophagic flux in HepG2 and Huh7 cells. DDX5 promoted p62 degradation and markedly reduced the half-life of p62. Moreover, DDX5 overexpression dramatically reduced, while DDX5 knockdown promoted, cancer cell growth and tumorigenesis in vitro and in vivo. We found that DDX5 bound to p62 and interfered with p62/TRAF6 (tumor necrosis factor receptor-associated factor 6) interaction. Further findings revealed that the N-terminal domain of DDX5, involved in the interaction with p62, was sufficient to induce autophagy independent of its RNA binding and helicase activity. DDX5 overexpression decreased p62/TRAF6-mediated lysine 63-linked ubiquitination of mammalian target of rapamycin (mTOR) and subsequently inhibited the mTOR signaling pathway. Knockdown of TRAF6 blocked DDX5-induced autophagy. Furthermore, we showed that miR-17-5p downregulated DDX5 and impaired autophagy. Inhibition of miR-17-5p promoted autophagic flux and suppressed tumor growth in HCC xenograft models. Conclusion: Our findings define a noncanonical pathway that links miR-17-5p, DDX5, p62/TRAF6, autophagy, and HCC. These findings open an avenue for the treatment of HCC.
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Autofagia/fisiología , Carcinogénesis , ARN Helicasas DEAD-box/fisiología , Proteína Sequestosoma-1/fisiología , Humanos , Neoplasias Hepáticas , Células Tumorales CultivadasRESUMEN
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.
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Proteínas de Unión al ARN/fisiología , Proteína Sequestosoma-1/fisiología , Animales , Humanos , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Proteína Sequestosoma-1/genética , Proteína Sequestosoma-1/metabolismo , Transducción de SeñalRESUMEN
Glucocorticoids are widely used to treat inflammatory disorders; however, prolonged use of glucocorticoids results in side effects including osteoporosis, diabetes and obesity. Compound A (CpdA), identified as a selective NR3C1/glucocorticoid receptor (nuclear receptor subfamily 3, group C, member 1) modulator, exhibits an inflammation-suppressive effect, largely in the absence of detrimental side effects. To understand the mechanistic differences between the classic glucocorticoid dexamethasone (DEX) and CpdA, we looked for proteins oppositely regulated in bone marrow-derived macrophages using an unbiased proteomics approach. We found that the autophagy receptor SQSTM1 but not NR3C1 mediates the anti-inflammatory action of CpdA. CpdA drives SQSTM1 upregulation by recruiting the NFE2L2 transcription factor to its promoter. In contrast, the classic NR3C1 ligand dexamethasone recruits NR3C1 to the Sqstm1 promoter and other NFE2L2-controlled gene promoters, resulting in gene downregulation. Both DEX and CpdA induce autophagy, with marked different autophagy characteristics and morphology. Suppression of LPS-induced Il6 and Ccl2 genes by CpdA in macrophages is hampered upon Sqstm1 silencing, confirming that SQSTM1 is essential for the anti-inflammatory capacity of CpdA, at least in this cell type. Together, these results demonstrate how off-target mechanisms of selective NR3C1 ligands may contribute to a more efficient anti-inflammatory therapy.
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Acetatos/farmacología , Antiinflamatorios/farmacología , Inflamación/genética , Inflamación/prevención & control , Macrófagos/efectos de los fármacos , Receptores de Glucocorticoides/agonistas , Proteína Sequestosoma-1/fisiología , Tiramina/análogos & derivados , Animales , Células Cultivadas , Dexametasona/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Macrófagos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Receptores de Glucocorticoides/metabolismo , Proteína Sequestosoma-1/genética , Activación Transcripcional/efectos de los fármacos , Tiramina/farmacologíaRESUMEN
In the process of recent hit-to-lead studies, not only in industry but also in academia, early evaluation of metabolic properties has been one of the key aspects supporting a higher probability of success in drug discovery. In this review, we introduce the development of chemical seeds targeting the Kelch-like ECH-associated protein-1 (Keap1) as an example of an academic hit-to-lead study considering metabolic stability. Keap1 regulates the function of nuclear factor erythroid 2-related factor 2 (Nrf2), which induces various antioxidative or detoxification proteins. An inhibitor of protein-protein interaction (PPI) between Keap1 and Nrf2 to activate Nrf2 is expected to be a novel target for drug discovery. However, Nrf2 is also activated in several cancers, such as human hepatocellular carcinoma, and causes chemoresistance, which is mediated by phosphorylated p62/Sqstm1 (p-p62), an autophagy-related protein that also undergoes a PPI with Keap1. In this case, an Nrf2 suppressor could be used to attenuate drug resistance. We discovered inhibitors against the Nrf2-Keap1 PPI and p-p62-Keap1 PPI using high-throughput screening and established the synthetic routes for the hit compounds and their derivatives. Furthermore, we assessed the metabolic stability of both of the PPI inhibitors in human liver microsomes and identified the metabolic sites.
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1-Naftilamina/análogos & derivados , Dimetilfumarato , Descubrimiento de Drogas , Isotiocianatos , Proteína 1 Asociada A ECH Tipo Kelch , Factor 2 Relacionado con NF-E2 , Ácido Oleanólico/análogos & derivados , Mapas de Interacción de Proteínas/efectos de los fármacos , Sulfonamidas , 1-Naftilamina/química , 1-Naftilamina/farmacología , Autofagia , Dimetilfumarato/química , Dimetilfumarato/farmacología , Ensayos Analíticos de Alto Rendimiento , Humanos , Isotiocianatos/química , Isotiocianatos/farmacología , Proteína 1 Asociada A ECH Tipo Kelch/fisiología , Microsomas Hepáticos/metabolismo , Factor 2 Relacionado con NF-E2/fisiología , Ácido Oleanólico/química , Ácido Oleanólico/farmacología , Fosforilación , Proteína Sequestosoma-1/fisiología , Sulfonamidas/química , Sulfonamidas/farmacología , SulfóxidosRESUMEN
BACKGROUND: Obesity is closely related to the abnormal differentiation of adipocytes, which are subjected to high plasma levels of free fatty acids (FFAs). As the most abundant FFA in the bloodstream, oleic acid (OA) has the ability to induce adipogenic differentiation in human adipose-derived stromal cells (hADSCs). Recently, p62, an autophagy mediator, has been shown to play a role in obesity and adipose tissue metabolism. Therefore, the aim of this study was to investigate the roles of autophagy and mitochondrial function at different stages of OA (in combination with insulin and dexamethasone)-induced adipogenesis in hADSCs. METHODS: The hADSCs were incubated with OA, insulin, and dexamethasone after pretreatment with autophagy inhibitors or knockdown of p62 with shRNA. The adiposeness level was then analyzed by oil red O staining in the cells. The related proteins or mRNA levels were detected by western blot analysis or quantitative real-time polymerase chain reaction (PCR). RESULTS: Treatment with 80 µM OA (substituted for isobutylmethylxantine; IBMX) for 10 days successfully induced hADSCs to adipocytes. During OA-induced adipogenesis, autophagy was induced, with an increased LC3II/I ratio on day 3 and a decreased protein level of p62 on and after day 3. Inhibition of autophagy with 3-methyladenine (3MA) at the early stage (day 0 to day 3) of differentiation, but not at the middle or late stage, significantly decreased OA-induced adipogenesis; while knockdown of p62 with shRNA significantly promoted adipogenesis in hADSCs. Moreover, the copy number of mtDNA (the ND1 gene) and the protein level of TOM20, a mitochondrial membrane protein, were increased following OA treatment, which was related to the stability of mitochondria. Interestingly, knockdown of p62 increased the mito-LC3II/I and cyto-LC3II/I ratios by 110.1% and 73.3%, respectively. The increase in the ratio of mito-LC3II/I was higher than that of cyto-LC3II/I. Furthermore, p62 knockdown-enhanced adipogenesis in hADSCs was abolished by inhibiting mitophagy with cyclosporine A. CONCLUSIONS: These results suggested that p62 plays a protective role in adipogenesis of hADSCs through regulating mitophagy.
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Adipogénesis , Tejido Adiposo/citología , Mitofagia , Proteína Sequestosoma-1/fisiología , Adulto , Autofagia , Femenino , Humanos , Ácido Oléico/farmacología , Células del Estroma/efectos de los fármacos , Células del Estroma/metabolismo , Células del Estroma/fisiologíaRESUMEN
Negative regulation of immune pathways is essential to achieve resolution of immune responses and to avoid excess inflammation. DNA stimulates type I IFN expression through the DNA sensor cGAS, the second messenger cGAMP, and the adaptor molecule STING Here, we report that STING degradation following activation of the pathway occurs through autophagy and is mediated by p62/SQSTM1, which is phosphorylated by TBK1 to direct ubiquitinated STING to autophagosomes. Degradation of STING was impaired in p62-deficient cells, which responded with elevated IFN production to foreign DNA and DNA pathogens. In the absence of p62, STING failed to traffic to autophagy-associated vesicles. Thus, DNA sensing induces the cGAS-STING pathway to activate TBK1, which phosphorylates IRF3 to induce IFN expression, but also phosphorylates p62 to stimulate STING degradation and attenuation of the response.
Asunto(s)
Nucleotidiltransferasas/fisiología , Proteínas Serina-Treonina Quinasas/fisiología , Proteína Sequestosoma-1/fisiología , Animales , Autofagia , Línea Celular , ADN/metabolismo , Humanos , Ratones Endogámicos C57BL , Ratones Noqueados , Transducción de SeñalRESUMEN
BACKGROUND AND PURPOSE: SQSTM1/p62 is a multifunctional, stress-induced, scaffold protein involved in multiple cellular processes including autophagic clearance, regulation of inflammatory responses and redox homeostasis. Its altered function has been associated with different human pathologies, such as neurodegenerative, metabolic and bone diseases (down-regulation), and cancerogenesis (up-regulation). However, its role in the off-target effects of clinically used drugs is still not understood. EXPERIMENTAL APPROACH: We evaluated the expression of p62 in cultured Hep3B cells and their derived ρ° cells (lacking mitochondria), along with markers of autophagy and mitochondrial dysfunction. The effects of efavirenz were compared with those of known pharmacological stressors, rotenone, thapsigargin and CCCP, and we also used transient silencing with siRNA and p62 overexpression. Western blotting, quantRT-PCR and fluorescence microscopy were used to assay these effects and their underlying mechanisms. KEY RESULTS: In Hep3B cells, efavirenz augmented p62 protein content, an effect not observed in the corresponding ρ° cells. p62 up-regulation followed enhanced SQSTM1 expression mediated through the transcription factor CHOP/DDIT3, while other well-known regulators (NF-kB and Nrf2) were not involved. Inhibition of autophagy with 3MA or with transient silencing of Atg5 did not affect SQSTM1 expression in efavirenz-treated cells while p62 overexpression ameliorated the deleterious effect of efavirenz on cell viability. CONCLUSION AND IMPLICATIONS: In our model, p62 exerted a specific, autophagy-independent role and protected against efavirenz-induced mitochondrial ROS generation and activation of the NLRP3 inflammasome. These findings add to the multifunctional nature of p62 and may help to understand the off-target effects of clinically useful drugs.
Asunto(s)
Autofagia/efectos de los fármacos , Autofagia/fisiología , Benzoxazinas/toxicidad , Proteína Sequestosoma-1/fisiología , Alquinos , Línea Celular Tumoral , Ciclopropanos , Relación Dosis-Respuesta a Droga , Hepatocitos/efectos de los fármacos , Hepatocitos/fisiología , Humanos , Especies Reactivas de Oxígeno/metabolismo , Inhibidores de la Transcriptasa Inversa/toxicidadRESUMEN
P62, also called sequestosome1 (SQSTM1), is the selective cargo receptor for autophagy to degenerate misfolded proteins. It has also been found to assist and connect parkin in pink1/parkin mitophagy pathway. Previous studies showed that p62 was in association with neurodegenerative diseases, and one of the diseases pathogenesis is P62 induced autophagy and mitophagy dysfunction. Autophagy is an important process to eliminate misfolded proteins. Intracellular aggregation including α-synuclein, Huntingtin, tau protein and ß-amyloid (Aß) protein are the misfolded proteins found in PD, HD and AD, respectively. P62 induced autophagy failure significantly accelerates misfolded protein aggregation. Mitophagy is the special autophagy, functions as the selective scavenger towards the impaired mitochondria. Mitochondrial dysfunction was confirmed greatly contribute to the occurrence of neurodegenerative diseases. Through assistance and connection with parkin, P62 is vital for regulating mitophagy, thus, aberrant P62 could influence the balance of mitophagy, and further disturb mitochondrial quality control. Therefore, accumulation of misfolded proteins leads to the aberrant P62 expression, aberrant P62 influence the balance of mitophagy, forming a vicious circle afterwards. In this review, we summarize the observations on the function of P62 relevant to autophagy and mitophagy in neurodegenerative diseases, hoping to give some clear and objective opinions to further study.
Asunto(s)
Autofagia , Mitofagia , Enfermedades Neurodegenerativas/patología , Proteína Sequestosoma-1/fisiología , Animales , Humanos , Deficiencias en la ProteostasisRESUMEN
p62, a protein capable of binding both ubiquitin and autophagy substrates, is well established as a key regulator in cancer and neurodegenerative diseases. Recently, there has been accumulating evidence that p62 is also a pivotal regulator in metabolic diseases, such as obesity, T2DM, NAFLD, metabolic bone disease, gout and thyroid disease. This review summarizes the emerging role of p62 on these diseases by considering its functional domains, phenotypes in genetically modified animals, clinically observed alterations, and its effects on downstream metabolic signaling pathways. At the same time, we highlight the need to explore the roles played by p62 in the gastrointestinal environment and immune system, and the extent to which its elevated expression may confer protection against metabolic disorders.
Asunto(s)
Enfermedades Metabólicas/genética , Enfermedades Metabólicas/metabolismo , Proteína Sequestosoma-1/fisiología , Animales , Autofagia/genética , Humanos , Ratones , Ratones Transgénicos , Transducción de SeñalRESUMEN
Many studies reveal that BAG3 plays a critical role in the regulation of protein degradation via macroautophagy. However, it remains unknown whether BAG3 affects the quality control of α-synuclein (SNCA), a Parkinson's disease-related protein. In this study, we demonstrated the increases of BAG3 expression in the ventral midbrain of SNCAA53T transgenic mice and also in MG132-treated PC12 cells overexpressing wild-type SNCA (SNCAWT-PC12). Moreover, we showed that BAG3 overexpression was sufficient to enhance the autophagy activity while knockdown of Bag3 reduced it in SNCAWT-PC12 cells. Immunoprecipitation revealed that BAG3 interacted with heat shock protein 70 and sequestosome 1. The immunostaining also showed the perinuclear accumulation and colocalization of BAG3 with these 2 proteins, as well as with LC3 dots in tyrosine hydroxylase-positive neurons in the midbrain of SNCAA53T mice. BAG3 overexpression was able to modulate SNCA degradation via macroautophagy which was prevented by Atg5 knockdown. Taken together, these results indicate that BAG3 plays a relevant role in regulating SNCA clearance via macroautophagy, and the heat shock protein 70-BAG3-sequestosome 1 complex may be involved in this process.