RESUMEN
Purpose: Due to intrinsic defensive response, ferroptosis-activating targeted therapy fails to achieve satisfactory clinical benefits. Though p62-Keap1-Nrf2 axis is activated to form a negative feedback loop during ferroptosis induction, how p62 is activated remains largely unknown. Methods: MTS assay was applied to measure cell growth. Lipid ROS was detected with C11-BODIPY reagent by flow cytometer. Quantitative real-time PCR (qPCR) and western blotting were performed to determine mRNA and protein level. Immunofluorescence (IF) was performed to examine the distribution of proteins. Fluorescence recovery after photobleaching (FRAP) was adopted to evaluate p62 phase separation. Immunoprecipitation (IP), co-IP and Proximal ligation assay (PLA) were performed to detected protein posttranslational modifications and protein-protein interactions. Tumor xenograft model was employed to inspect in vivo growth of pancreatic cancer cells. Results: Upon ferroptosis induction, Nuclear Factor E2 Related Factor 2 (Nrf2) protein and its downstream genes such as HMOX1 and NQO1 were upregulated. Knockdown of p62 significantly reversed Nrf2 upregulation and Keap1 decrease after ferroptosis induction. Knockdown of either p62 or Nrf2 remarkably sensitized ferroptosis induction. Due to augmented p62 phase separation, formation of p62 bodies were increased to recruit Keap1 after ferroptosis induction. Protein arginine methyltransferase 6 (PRMT6) mediated asymmetric dimethylarginine (ADMA) of p62 to increase its oligomerization, promoting p62 phase separation and p62 body formation. Knockdown of p62 or PRMT6 notably sensitized pancreatic cancer cells to ferroptosis both in vitro and in vivo through suppressing Nrf2 signaling. Conclusion: During ferroptosis induction, PRMT6 mediated p62 ADMA to promote its phase separation, sequestering Keap1 to activate Nrf2 signaling and inhibit ferroptosis. Therefore, targeting PRMT6-mediated p62 ADMA could be a new option to sensitize ferroptosis for cancer treatment.
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Arginina , Ferroptosis , Proteína 1 Asociada A ECH Tipo Kelch , Factor 2 Relacionado con NF-E2 , Proteína-Arginina N-Metiltransferasas , Proteína-Arginina N-Metiltransferasas/metabolismo , Proteína-Arginina N-Metiltransferasas/genética , Humanos , Animales , Arginina/metabolismo , Arginina/análogos & derivados , Factor 2 Relacionado con NF-E2/metabolismo , Factor 2 Relacionado con NF-E2/genética , Ratones , Línea Celular Tumoral , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Proteína 1 Asociada A ECH Tipo Kelch/genética , Retroalimentación Fisiológica , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Neoplasias Pancreáticas/genética , Proteína Sequestosoma-1/metabolismo , Proteína Sequestosoma-1/genética , Ratones Desnudos , Transducción de Señal , Separación de Fases , Proteínas de Unión al ARNRESUMEN
BACKGROUND: Aberrant activation of macrophages is associated with pathogenesis of acute lung injury (ALI). However, the potential pathogenesis has not been explored. OBJECTIVES: We aimed to identify whether histone deacetylase (HDAC) 10 is involved in lipopolysaccharide (LPS)-exposed ALI and reveal the underlying pathogenesis by which it promotes lung inflammation in LPS-exposed ALI via modifying P62 with deacetylation. METHODS: We constructed an ALI mice model stimulated with LPS to determine the positive effect of Hdac10 deficiency. Moreover, we cultured murine alveolar macrophage cell line (MH-S cells) and primary bone marrow-derived macrophages (BMDMs) to explore the pro-inflammatory activity and mechanism of HDAC10 after LPS challenge. RESULTS: HDAC10 expression was increased both in mice lung tissues and macrophage cell lines and promoted inflammatory cytokines production exposed to LPS. Hdac10 deficiency inhibited autophagy and inflammatory response after LPS stimulation. In vivo, Hdac10fl/fl-LysMCre mice considerably attenuated lung inflammation and inflammatory cytokines release exposed to LPS. Mechanistically, HDAC10 interacts with P62 and mediates P62 deacetylation at lysine 165 (K165), by which it promotes P62 expression and increases inflammatory cytokines production. Importantly, we identified that Salvianolic acid B (SAB), an HDAC10 inhibitor, reduces lung inflammatory response in LPS-stimulated ALI. CONCLUSION: These results uncover a previously unknown role for HDAC10 in regulating P62 deacetylation and aggravating lung inflammation in LPS-induced ALI, implicating that targeting HDAC10 is an effective therapy for LPS-exposed ALI.
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Lesión Pulmonar Aguda , Histona Desacetilasas , Lipopolisacáridos , Lisina , Ratones Endogámicos C57BL , Animales , Lesión Pulmonar Aguda/inducido químicamente , Lesión Pulmonar Aguda/prevención & control , Lesión Pulmonar Aguda/metabolismo , Lesión Pulmonar Aguda/genética , Lesión Pulmonar Aguda/patología , Lipopolisacáridos/toxicidad , Ratones , Acetilación , Histona Desacetilasas/metabolismo , Histona Desacetilasas/genética , Histona Desacetilasas/deficiencia , Lisina/metabolismo , Ratones Noqueados , Masculino , Proteína Sequestosoma-1/metabolismo , Proteína Sequestosoma-1/genética , Células Mieloides/metabolismoRESUMEN
Thiram, a prevalent dithiocarbamate insecticide in agriculture, is widely employed as a crop insecticide and preservative. Chronic exposure to thiram has been linked to various irreversible damages, including tibial cartilage dysplasia, erythrocytotoxicity, renal issues, and immune system compromise. Limited research exists on its effects on reproductive organs. This study investigated the reproductive toxicology in mouse testes exposure to varying concentrations (0, 30, 60, and 120 mg/kg) of thiram. Our study uncovered a series of adverse effects in mice subjected to thiram exposure, including emaciation, stunted growth, decreased water intake, and postponed testicular maturation. Biochemical analysis in thiram-exposed mice showed elevated levels of LDH and AST, while ALP, TG, ALT, and urea were decreased. Histologically, thiram disrupted the testis' microarchitecture and compromised its barrier function by widening the gap between spermatogenic cells and promoting fibrosis. The expression of pro-apoptotic genes (Bax, APAF1, Cytc, and Caspase-3) was downregulated, whereas Bcl-2 expression increased in thiram-treated mice compared to controls. Conversely, the expression of Atg5 was upregulated, and mTOR and p62 expression decreased, with a trend towards lower LC3b levels. Thiram also disrupted the blood-testis barrier, significantly reducing the mRNA expression of zona occludens-1 (ZO-1) and occludin. In conclusion, chronic exposure to high thiram concentrations (120 mg/kg) caused testicular tissue damage, affecting the blood-testis barrier and modulating apoptosis and autophagy through the Bcl-2/Bax and mTOR/Atg5/p62 pathways. This study contributes to understanding the molecular basis of thiram-induced reproductive toxicity and underscores the need for further research and precautions for those chronically exposed to thiram and its environmental residuals.
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Apoptosis , Proteína 5 Relacionada con la Autofagia , Autofagia , Barrera Hematotesticular , Proteínas Proto-Oncogénicas c-bcl-2 , Serina-Treonina Quinasas TOR , Testículo , Tiram , Proteína X Asociada a bcl-2 , Animales , Masculino , Apoptosis/efectos de los fármacos , Ratones , Serina-Treonina Quinasas TOR/metabolismo , Barrera Hematotesticular/efectos de los fármacos , Testículo/efectos de los fármacos , Testículo/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteína 5 Relacionada con la Autofagia/metabolismo , Proteína 5 Relacionada con la Autofagia/genética , Autofagia/efectos de los fármacos , Tiram/toxicidad , Proteína X Asociada a bcl-2/metabolismo , Proteína X Asociada a bcl-2/genética , Proteína Sequestosoma-1/metabolismo , Proteína Sequestosoma-1/genética , Insecticidas/toxicidad , Transducción de Señal/efectos de los fármacosRESUMEN
Butachlor is widely used in agriculture around the world and therefore poses environmental and public health hazards due to persistent and poor biodegradability. Ferroptosis is a type of iron-mediated cell death controlled by glutathione (GSH) and GPX4 inhibition. P62 is an essential autophagy adaptor that regulates Keap1 to activate nuclear factor erythroid 2-related factor 2 (Nrf2), which effectively suppresses lipid peroxidation, thereby relieving ferroptosis. Here, we found that butachlor caused changes in splenic macrophage structure, especially impaired mitochondrial morphology with disordered structure, which is suggestive of the occurrence of ferroptosis. This was further confirmed by the detection of iron metabolism, the GSH system, and lipid peroxidation. Mechanistically, butachlor suppressed the protein level of p62 and promoted Keap1-mediated degradation of Nrf2, which results in decreased GPX4 expression and accelerated splenic macrophage ferroptosis. These findings suggest that targeting the p62-Nrf2-GPX4 signaling axis may be a promising strategy for treating inflammatory diseases.
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Ferroptosis , Macrófagos , Ratones Endogámicos C57BL , Factor 2 Relacionado con NF-E2 , Fosfolípido Hidroperóxido Glutatión Peroxidasa , Transducción de Señal , Bazo , Animales , Humanos , Masculino , Ratones , Ferroptosis/efectos de los fármacos , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Proteína 1 Asociada A ECH Tipo Kelch/genética , Peroxidación de Lípido/efectos de los fármacos , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Factor 2 Relacionado con NF-E2/genética , Fosfolípido Hidroperóxido Glutatión Peroxidasa/metabolismo , Fosfolípido Hidroperóxido Glutatión Peroxidasa/genética , Proteína Sequestosoma-1/metabolismo , Proteína Sequestosoma-1/genética , Transducción de Señal/efectos de los fármacos , Bazo/efectos de los fármacos , Bazo/citología , Bazo/metabolismoRESUMEN
Autophagy plays a pivotal role in diverse biological processes, including the maintenance and differentiation of neural stem cells (NSCs). Interestingly, while complete deletion of Fip200 severely impairs NSC maintenance and differentiation, inhibiting canonical autophagy via deletion of core genes, such as Atg5, Atg16l1, and Atg7, or blockade of canonical interactions between FIP200 and ATG13 (designated as FIP200-4A mutant or FIP200 KI) does not produce comparable detrimental effects. This highlights the likely critical involvement of the non-canonical functions of FIP200, the mechanisms of which have remained elusive. Here, utilizing genetic mouse models, we demonstrated that FIP200 mediates non-canonical autophagic degradation of p62/sequestome1, primarily via TAX1BP1 in NSCs. Conditional deletion of Tax1bp1 in fip200 hGFAP conditional knock-in (cKI) mice led to NSC deficiency, resembling the fip200 hGFAP conditional knockout (cKO) mouse phenotype. Notably, reintroducing wild-type TAX1BP1 not only restored the maintenance of NSCs derived from tax1bp1-knockout fip200 hGFAP cKI mice but also led to a marked reduction in p62 aggregate accumulation. Conversely, a TAX1BP1 mutant incapable of binding to FIP200 or NBR1/p62 failed to achieve this restoration. Furthermore, conditional deletion of Tax1bp1 in fip200 hGFAP cKO mice exacerbated NSC deficiency and p62 aggregate accumulation compared to fip200 hGFAP cKO mice. Collectively, these findings illustrate the essential role of the FIP200-TAX1BP1 axis in mediating the non-canonical autophagic degradation of p62 aggregates towards NSC maintenance and function, presenting novel therapeutic targets for neurodegenerative diseases.
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Proteínas Relacionadas con la Autofagia , Autofagia , Células-Madre Neurales , Animales , Células-Madre Neurales/fisiología , Células-Madre Neurales/metabolismo , Ratones , Autofagia/fisiología , Proteínas Relacionadas con la Autofagia/genética , Proteínas Relacionadas con la Autofagia/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Ratones Noqueados , Proteína Sequestosoma-1/metabolismo , Proteína Sequestosoma-1/genética , Regulación de la Expresión Génica , Proteínas de NeoplasiasRESUMEN
As the largest organ in the human body, skeletal muscle is essential for breathing support, movement initiation, and maintenance homeostasis. It has been shown that programmed cell death (PCD), which includes autophagy, apoptosis, and necrosis, is essential for the development of skeletal muscle. A novel form of PCD called ferroptosis is still poorly understood in relation to skeletal muscle. In this study, we observed that the activation of ferroptosis significantly impeded the differentiation of C2C12 myoblasts into myotubes and concurrently suppressed the expression of OTUB1, a crucial deubiquitinating enzyme. OTUB1-silenced C2C12 mouse myoblasts were used to investigate the function of OTUB1 in ferroptosis. The results show that OTUB1 knockdown in vitro significantly increased C2C12 ferroptosis and inhibited myogenesis. Interestingly, the induction of ferroptosis resulting from OTUB1 knockdown was concomitant with the activation of autophagy. Furthermore, OTUB1 interacted with the P62 protein and stabilized its expression by deubiquitinating it, thereby inhibiting autophagy-dependent ferroptosis and promoting myogenesis. All of these findings demonstrate the critical role that OTUB1 plays in controlling ferroptosis, and we suggest that focusing on the OTUB1-P62 axis may be a useful tactic in the treatment and prevention of disorders involving the skeletal muscle.
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Autofagia , Diferenciación Celular , Cisteína Endopeptidasas , Ferroptosis , Desarrollo de Músculos , Fibras Musculares Esqueléticas , Mioblastos , Animales , Ratones , Fibras Musculares Esqueléticas/metabolismo , Ferroptosis/genética , Cisteína Endopeptidasas/metabolismo , Cisteína Endopeptidasas/genética , Mioblastos/metabolismo , Mioblastos/citología , Línea Celular , Enzimas Desubicuitinizantes/metabolismo , Enzimas Desubicuitinizantes/genética , Ubiquitinación , Humanos , Proteína Sequestosoma-1/metabolismo , Proteína Sequestosoma-1/genéticaRESUMEN
Dihydropyrazines (DHPs) are formed by non-enzymatic glycation reactions in vivo and in food. We recently reported that 3-hydro-2,2,5,6-tetramethylpyrazine (DHP-3), which is a methyl-substituted DHP, caused severe oxidative stress and cytotoxicity. However, the molecular mechanisms underlying the cytotoxic pathways of the DHP response remain elusive. Because oxidative stress induces endoplasmic reticulum (ER) stress and autophagy, we investigated the ability of DHP-3 to modulate the ER stress and autophagy pathways. DHP-3 activated the ER stress pathway by increasing inositol-requiring enzyme 1 (IRE1) and PKR-like ER kinase (PERK) phosphorylation and transcription factor 6 (ATF6) expression. Moreover, DHP-3 increased the expression of activating transcription factor 4 (ATF4) and C/EBP homologous protein (CHOP), which are downstream targets of PERK. In addition, DHP-3 inhibited the autophagy pathway by increasing the accumulation of microtubule-associated protein 1 light chain 3 alpha-phosphatidylethanolamine conjugate (LC3-II) and p62/sequestosome 1 (p62), while decreasing autophagic flux. Taken together, these results indicate that DHP-3 activates the ER stress pathway and inhibits the autophagy pathway, suggesting that the resulting removal of damaged organelles is inadequate.
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Factor de Transcripción Activador 4 , Factor de Transcripción Activador 6 , Autofagia , Estrés del Retículo Endoplásmico , Proteínas Serina-Treonina Quinasas , Pirazinas , eIF-2 Quinasa , Humanos , Autofagia/efectos de los fármacos , Estrés del Retículo Endoplásmico/efectos de los fármacos , Pirazinas/farmacología , Células Hep G2 , Factor de Transcripción Activador 4/metabolismo , Factor de Transcripción Activador 4/genética , eIF-2 Quinasa/metabolismo , Factor de Transcripción Activador 6/metabolismo , Factor de Transcripción Activador 6/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Factor de Transcripción CHOP/metabolismo , Factor de Transcripción CHOP/genética , Endorribonucleasas/metabolismo , Endorribonucleasas/genética , Fosforilación , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Estrés Oxidativo/efectos de los fármacos , Proteína Sequestosoma-1/metabolismo , Proteína Sequestosoma-1/genética , Transducción de Señal/efectos de los fármacos , Proteínas Asociadas a Microtúbulos/metabolismoRESUMEN
Sequestosome 1 (SQSTM1/p62) is a selective autophagy adapter protein that participates in antiviral and bacterial immune responses and plays an important regulatory role in clearing the proteins to be degraded and maintaining intracellular protein homeostasis. In this study, two p62 genes were cloned from common carp (Cyprinus carpio), namely Ccp62-1 and Ccp62-2, and conducted bioinformatics analysis on them. The results showed that Ccp62s had the same structural domain (Phox and Bem1 domain, ZZ-type zinc finger domain, and ubiquitin-associated domain) as p62 from other species. Ccp62s were widely expressed in various tissues of fish, and highly expressed in immune organs such as gills, spleen, head kidney, etc. Subcellular localization study showed that they were mainly distributed in punctate aggregates in the cytoplasm. After stimulation with Aeromonas hydrophila and spring viraemia of carp virus (SVCV), the expression level of Ccp62s was generally up-regulated. Overexpression of Ccp62s in EPC cells could inhibit SVCV replication. Upon A. hydrophila challenge, the bacterial load in Ccp62s-overexpressing group was significantly reduced, the expression levels of pro-inflammatory cytokines and interferon factors were increased, and the survival rate of the fish was improved. These results indicated that Ccp62s were involved in the immune response of common carp to bacterial and viral infections.
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Aeromonas hydrophila , Carpas , Enfermedades de los Peces , Proteínas de Peces , Infecciones por Bacterias Gramnegativas , Inmunidad Innata , Filogenia , Infecciones por Rhabdoviridae , Rhabdoviridae , Animales , Carpas/inmunología , Carpas/genética , Enfermedades de los Peces/inmunología , Proteínas de Peces/genética , Proteínas de Peces/inmunología , Aeromonas hydrophila/fisiología , Inmunidad Innata/genética , Rhabdoviridae/fisiología , Infecciones por Bacterias Gramnegativas/inmunología , Infecciones por Bacterias Gramnegativas/veterinaria , Infecciones por Rhabdoviridae/inmunología , Infecciones por Rhabdoviridae/veterinaria , Regulación de la Expresión Génica/inmunología , Proteína Sequestosoma-1/genética , Proteína Sequestosoma-1/inmunología , Perfilación de la Expresión Génica/veterinaria , Alineación de Secuencia/veterinaria , Secuencia de Aminoácidos , Autofagia/inmunologíaRESUMEN
In this study we have identified POLθ-S6K-p62 as a novel druggable regulator of radiation response in prostate cancer. Despite significant advances in delivery, radiotherapy continues to negatively affect treatment outcomes and quality of life due to resistance and late toxic effects to the surrounding normal tissues such as bladder and rectum. It is essential to develop new and effective strategies to achieve better control of tumor. We found that ribosomal protein S6K (RPS6KB1) is elevated in human prostate tumors, and contributes to resistance to radiation. As a downstream effector of mTOR signaling, S6K is known to be involved in growth regulation. However, the impact of S6K signaling on radiation response has not been fully explored. Here we show that loss of S6K led to formation of smaller tumors with less metastatic ability in mice. Mechanistically we found that S6K depletion reduced NFκB and SQSTM1 (p62) reporter activity and DNA polymerase θ (POLθ) that is involved in alternate end-joining repair. We further show that the natural compound berberine interacts with S6K in a in a hitherto unreported novel mode and that pharmacological inhibition of S6K with berberine reduces Polθ and downregulates p62 transcriptional activity via NFκB. Loss of S6K or pre-treatment with berberine improved response to radiation in prostate cancer cells and prevented radiation-mediated resurgence of PSA in animals implanted with prostate cancer cells. Notably, silencing POLQ in S6K overexpressing cells enhanced response to radiation suggesting S6K sensitizes prostate cancer cells to radiation via POLQ. Additionally, inhibition of autophagy with CQ potentiated growth inhibition induced by berberine plus radiation. These observations suggest that pharmacological inhibition of S6K with berberine not only downregulates NFκB/p62 signaling to disrupt autophagic flux but also decreases Polθ. Therefore, combination treatment with radiation and berberine inhibits autophagy and alternate end-joining DNA repair, two processes associated with radioresistance leading to increased radiation sensitivity.
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FN-kappa B , Neoplasias de la Próstata , Tolerancia a Radiación , Proteína Sequestosoma-1 , Transducción de Señal , Masculino , Animales , Humanos , Neoplasias de la Próstata/radioterapia , Neoplasias de la Próstata/patología , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/tratamiento farmacológico , Proteína Sequestosoma-1/metabolismo , Proteína Sequestosoma-1/genética , FN-kappa B/metabolismo , FN-kappa B/genética , Transducción de Señal/efectos de los fármacos , Ratones , Tolerancia a Radiación/efectos de los fármacos , Línea Celular Tumoral , ADN Polimerasa Dirigida por ADN/genética , ADN Polimerasa Dirigida por ADN/metabolismo , Proteínas Quinasas S6 Ribosómicas 70-kDa/metabolismo , Proteínas Quinasas S6 Ribosómicas 70-kDa/genéticaRESUMEN
BACKGROUND: Genetic abnormalities in the FGFR signalling occur in 40% of breast cancer (BCa) patients resistant to anti-ER therapy, which emphasizes the potential of FGFR-targeting strategies. Recent findings indicate that not only mutated FGFR is a driver of tumour progression but co-mutational landscapes and other markers should be also investigated. Autophagy has been recognized as one of the major mechanisms underlying the role of tumour microenvironment in promotion of cancer cell survival, and resistance to anti-ER drugs. The selective autophagy receptor p62/SQSTM1 promotes Nrf-2 activation by Keap1/Nrf-2 complex dissociation. Herein, we have analysed whether the negative effect of FGFR2 on BCa cell response to anti-ER treatment involves the autophagy process and/or p62/Keap1/Nrf-2 axis. METHODS: The activity of autophagy in ER-positive MCF7 and T47D BCa cell lines was determined by analysis of expression level of autophagy markers (p62 and LC3B) and monitoring of autophagosomes' maturation. Western blot, qPCR and proximity ligation assay were used to determine the Keap1/Nrf-2 interaction and Nrf-2 activation. Analysis of 3D cell growth in Matrigel® was used to assess BCa cell response to applied treatments. In silico gene expression analysis was performed to determine FGFR2/Nrf-2 prognostic value. RESULTS: We have found that FGFR2 signalling induced autophagy in AMPKα/ULK1-dependent manner. FGFR2 activity promoted dissociation of Keap1/Nrf-2 complex and activation of Nrf-2. Both, FGFR2-dependent autophagy and activation of Nrf-2 were found to counteract the effect of anti-ER drugs on BCa cell growth. Moreover, in silico analysis showed that high expression of NFE2L2 (gene encoding Nrf-2) combined with high FGFR2 expression was associated with poor relapse-free survival (RFS) of ER+ BCa patients. CONCLUSIONS: This study revealed the unknown role of FGFR2 signalling in activation of autophagy and regulation of the p62/Keap1/Nrf-2 interdependence, which has a negative impact on the response of ER+ BCa cells to anti-ER therapies. The data from in silico analyses suggest that expression of Nrf-2 could act as a marker indicating potential benefits of implementation of anti-FGFR therapy in patients with ER+ BCa, in particular, when used in combination with anti-ER drugs.
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Autofagia , Neoplasias de la Mama , Proteína 1 Asociada A ECH Tipo Kelch , Factor 2 Relacionado con NF-E2 , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos , Femenino , Humanos , Autofagia/efectos de los fármacos , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Línea Celular Tumoral , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Proteína 1 Asociada A ECH Tipo Kelch/genética , Células MCF-7 , Factor 2 Relacionado con NF-E2/metabolismo , Factor 2 Relacionado con NF-E2/genética , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/metabolismo , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/genética , Proteína Sequestosoma-1/metabolismo , Proteína Sequestosoma-1/genética , Transducción de Señal/efectos de los fármacos , Antagonistas de Estrógenos/farmacología , Moduladores de los Receptores de Estrógeno/farmacologíaRESUMEN
p62, also known as SQSTM1, has been shown to be closely related to the coronavirus. However, it remains unclear on the relationship between p62 and NIBV infection. Moreover, there are no available antibodies against the chicken p62 protein. Thus, this study aimed to prepare p62 polyclonal antibody and investigate the correlation between the p62 protein and NIBV infection. Here, PET-32a-p62 prokaryotic fusion expression vector was constructed for prokaryotic protein expression, and then p62 polyclonal antibody was prepared by immunizing rabbits. Lastly, these antibodies were then utilized in Western blotting (WB), immunohistochemistry (IHC), and immunofluorescence (IF) assays. The results showed that we successfully prepared chicken p62 polyclonal antibody. Meanwhile, WB and IF demonstrated that the expression of p62 showed a trend of first increase and then decrease after NIBV infection. IHC showed that the expression of p62 in the spleen, lung, kidney, bursa of Fabricius and trachea of chickens infected with NIBV in 11 dpi was significantly higher than that of normal chickens. Taken together, this study successfully prepared a polyclonal antibody for chicken p62 protein and confirmed its application and expression in chickens, as well as the expression of p62 in tissues after NIBV infection.
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Pollos , Infecciones por Coronavirus , Virus de la Bronquitis Infecciosa , Animales , Virus de la Bronquitis Infecciosa/inmunología , Infecciones por Coronavirus/inmunología , Infecciones por Coronavirus/virología , Enfermedades de las Aves de Corral/inmunología , Enfermedades de las Aves de Corral/virología , Proteína Sequestosoma-1/metabolismo , Proteína Sequestosoma-1/inmunología , Proteína Sequestosoma-1/genética , Anticuerpos/inmunología , Conejos , Anticuerpos Antivirales/inmunologíaRESUMEN
Three-dimensional cell cultures have improved the evaluation of drugs for cancer therapy, due to their high similarity to solid tumors. In melanoma, autophagy appears to show a dual role depending on the progression of the disease. p62 protein has been proposed for the evaluation of autophagic flux since its expression is an indicator of the state of autophagy. Pentoxifylline (PTX) and Norcantharidin (NCTD) are drugs that have been shown to possess anticancer effects. In this work, we used B16F1 mouse melanoma cells in two-dimensional (2D) monolayer cultures and three-dimensional (3D) spheroids to test the effect of PTX and NCTD over the p62 expression. We analyzed the effect on p62 expression through Western blot and immunofluorescence assays. Our results indicate that PTX decreases p62 expression in both cell culture models, while Norcantharidin increases its expression in 3D cultures at 24 h. Therefore, these drugs could have a potential therapeutic use for the regulation of autophagy in melanoma, depending on the state of evolution of the disease.
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Autofagia , Compuestos Bicíclicos Heterocíclicos con Puentes , Pentoxifilina , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Animales , Ratones , Pentoxifilina/farmacología , Autofagia/efectos de los fármacos , Línea Celular Tumoral , Melanoma Experimental/metabolismo , Melanoma Experimental/tratamiento farmacológico , Melanoma Experimental/patología , Técnicas de Cultivo de Célula , Proteína Sequestosoma-1/metabolismo , Proteína Sequestosoma-1/genética , Antineoplásicos/farmacología , Esferoides Celulares/efectos de los fármacos , Esferoides Celulares/metabolismoRESUMEN
Extracellular secretion is an essential mechanism for α-synuclein (α-syn) proteostasis. Although it has been reported that neuronal activity affects α-syn secretion, the underlying mechanisms remain unclear. Here, we investigated the autophagic processes that regulate the physiological release of α-syn in mouse primary cortical neurons and SH-SY5Y cells. Stimulating neuronal activity with glutamate or depolarization with high KCl enhanced α-syn secretion. This glutamate-induced α-syn secretion was blocked by a mixture of NMDA receptor antagonist AP5 and AMPA receptor antagonist NBQX, as well as by cytosolic Ca2+ chelator BAPTA-AM. Additionally, mTOR inhibitor rapamycin increased α-syn and p62/SQSTM1 (p62) secretion, and this effect of rapamycin was reduced in primary cortical neurons deficient in the autophagy regulator beclin 1 (derived from BECN1+/- mice). Glutamate-induced α-syn and p62 secretion was suppressed by the knockdown of ATG5, which is required for autophagosome formation. Glutamate increased LC3-II generation and decreased intracellular p62 levels, and the increase in LC3-II levels was blocked by BAPTA-AM. Moreover, glutamate promoted co-localization of α-syn with LC3-positive puncta, but not with LAMP1-positive structures in the neuronal somas. Glutamate-induced α-syn and p62 secretion were also reduced by the knockdown of RAB8A, which is required for autophagosome fusion with the plasma membrane. Collectively, these findings suggest that stimulating neuronal activity mediates autophagic α-syn secretion in a cytosolic Ca2+-dependent manner, and autophagosomes may participate in autophagic secretion by functioning as α-syn carriers.
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Autofagia , Neuronas , Proteína Sequestosoma-1 , alfa-Sinucleína , Animales , alfa-Sinucleína/metabolismo , alfa-Sinucleína/genética , Neuronas/metabolismo , Ratones , Humanos , Proteína Sequestosoma-1/metabolismo , Proteína Sequestosoma-1/genética , Proteína 5 Relacionada con la Autofagia/metabolismo , Proteína 5 Relacionada con la Autofagia/genética , Ácido Glutámico/metabolismo , Beclina-1/metabolismo , Beclina-1/genética , Calcio/metabolismo , Línea Celular Tumoral , Ácido Egtácico/análogos & derivados , Ácido Egtácico/farmacología , Sirolimus/farmacologíaRESUMEN
Autophagy is an essential degradation program required for cell homeostasis. Among its functions is the engulfment and destruction of cytosolic pathogens, termed xenophagy. Not surprisingly, many pathogens use various strategies to circumvent or co-opt autophagic degradation. For poxviruses, it is known that infection activates autophagy, which however is not required for successful replication. Even though these complex viruses replicate exclusively in the cytoplasm, autophagy-mediated control of poxvirus infection has not been extensively explored. Using the prototypic poxvirus, vaccinia virus (VACV), we show that overexpression of the xenophagy receptors p62, NDP52, and Tax1Bp1 restricts poxvirus infection. While NDP52 and Tax1Bp1 were degraded, p62 initially targeted cytoplasmic virions before being shunted to the nucleus. Nuclear translocation of p62 was dependent upon p62 NLS2 and correlated with VACV kinase mediated phosphorylation of p62 T269/S272. This suggests that VACV targets p62 during the early stages of infection to avoid destruction and further implies that poxviruses exhibit multi-layered control of autophagy to facilitate cytoplasmic replication.
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Autofagia , Núcleo Celular , Proteína Sequestosoma-1 , Virus Vaccinia , Humanos , Transporte Activo de Núcleo Celular , Núcleo Celular/metabolismo , Núcleo Celular/virología , Células HEK293 , Células HeLa , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Fosforilación , Proteína Sequestosoma-1/metabolismo , Proteína Sequestosoma-1/genética , Vaccinia/metabolismo , Vaccinia/virología , Vaccinia/genética , Virus Vaccinia/metabolismo , Virus Vaccinia/genética , Replicación ViralRESUMEN
In females, the pathophysiological mechanism of poor ovarian response (POR) is not fully understood. Considering the expression level of p62 was significantly reduced in the granulosa cells (GCs) of POR patients, this study focused on identifying the role of the selective autophagy receptor p62 in conducting the effect of follicle-stimulating hormone (FSH) on antral follicles (AFs) formation in female mice. The results showed that p62 in GCs was FSH responsive and that its level increased to a peak and then decreased time-dependently either in ovaries or in GCs after gonadotropin induction in vivo. GC-specific deletion of p62 resulted in subfertility, a significantly reduced number of AFs and irregular estrous cycles, which were same as pathophysiological symptom of POR. By conducting mass spectrum analysis, we found the ubiquitination of proteins was decreased, and autophagic flux was blocked in GCs. Specifically, the level of nonubiquitinated Wilms tumor 1 homolog (WT1), a transcription factor and negative controller of GC differentiation, increased steadily. Co-IP results showed that p62 deletion increased the level of ubiquitin-specific peptidase 5 (USP5), which blocked the ubiquitination of WT1. Furthermore, a joint analysis of RNA-seq and the spatial transcriptome sequencing data showed the expression of steroid metabolic genes and FSH receptors pivotal for GCs differentiation decreased unanimously. Accordingly, the accumulation of WT1 in GCs deficient of p62 decreased steroid hormone levels and reduced FSH responsiveness, while the availability of p62 in GCs simultaneously ensured the degradation of WT1 through the ubiquitinâproteasome system and autophagolysosomal system. Therefore, p62 in GCs participates in GC differentiation and AF formation in FSH induction by dynamically controlling the degradation of WT1. The findings of the study contributes to further study the pathology of POR.
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Hormona Folículo Estimulante , Células de la Granulosa , Folículo Ovárico , Proteína Sequestosoma-1 , Ubiquitinación , Proteínas WT1 , Animales , Hormona Folículo Estimulante/metabolismo , Hormona Folículo Estimulante/farmacología , Femenino , Proteínas WT1/metabolismo , Proteínas WT1/genética , Ratones , Folículo Ovárico/metabolismo , Folículo Ovárico/efectos de los fármacos , Células de la Granulosa/metabolismo , Células de la Granulosa/efectos de los fármacos , Proteína Sequestosoma-1/metabolismo , Proteína Sequestosoma-1/genética , Ratones Endogámicos C57BL , Autofagia/efectos de los fármacos , Proteolisis/efectos de los fármacos , Humanos , Ratones NoqueadosRESUMEN
Influenza A virus (IAV) continues to pose serious threats to the global animal industry and public health security. Identification of critical host factors engaged in the life cycle of IAV and elucidation of the underlying mechanisms of their action are particularly important for the discovery of potential new targets for the development of anti-influenza drugs. Herein, we identified Hydroxyacyl-CoA Dehydratase 3 (HACD3) as a new host factor that supports the replication of IAV. Downregulating the expression of HACD3 reduced the level of viral PB1 protein in IAV-infected cells and in cells that were transiently transfected to express PB1. Silencing HACD3 expression had no effect on the level of PB1 mRNA but could promote the lysosome-mediated autophagic degradation of PB1 protein. Further investigation revealed that HACD3 interacted with PB1 and selective autophagic receptor SQSTM1/p62, and HACD3 competed with SQSTM1/p62 for the interaction with PB1, which prevented PB1 from SQSTM1/p62-mediated autophagic degradation. Collectively, these findings establish that HACD3 plays a positive regulatory role in IAV replication by stabilizing the viral PB1 protein.
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Autofagia , Virus de la Influenza A , Gripe Humana , Proteínas Virales , Replicación Viral , Animales , Perros , Humanos , Células A549 , Células HEK293 , Interacciones Huésped-Patógeno/genética , Virus de la Influenza A/fisiología , Gripe Humana/metabolismo , Gripe Humana/virología , Proteolisis , Proteína Sequestosoma-1/metabolismo , Proteína Sequestosoma-1/genética , Proteínas Virales/metabolismo , Replicación Viral/genéticaRESUMEN
Aggregation of proteins containing expanded polyglutamine (polyQ) repeats is the cytopathologic hallmark of a group of dominantly inherited neurodegenerative diseases, including Huntington's disease (HD). Huntingtin (Htt), the disease protein of HD, forms amyloid-like fibrils by liquid-to-solid phase transition. Macroautophagy has been proposed to clear polyQ aggregates, but the efficiency of aggrephagy is limited. Here, we used cryo-electron tomography to visualize the interactions of autophagosomes with polyQ aggregates in cultured cells in situ. We found that an amorphous aggregate phase exists next to the radially organized polyQ fibrils. Autophagosomes preferentially engulfed this amorphous material, mediated by interactions between the autophagy receptor p62/SQSTM1 and the non-fibrillar aggregate surface. In contrast, amyloid fibrils excluded p62 and evaded clearance, resulting in trapping of autophagic structures. These results suggest that the limited efficiency of autophagy in clearing polyQ aggregates is due to the inability of autophagosomes to interact productively with the non-deformable, fibrillar disease aggregates.
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Amiloide , Autofagosomas , Autofagia , Proteína Huntingtina , Enfermedad de Huntington , Péptidos , Agregado de Proteínas , Proteína Sequestosoma-1 , Péptidos/metabolismo , Péptidos/química , Péptidos/genética , Humanos , Proteína Huntingtina/metabolismo , Proteína Huntingtina/genética , Proteína Huntingtina/química , Autofagosomas/metabolismo , Autofagosomas/ultraestructura , Proteína Sequestosoma-1/metabolismo , Proteína Sequestosoma-1/genética , Amiloide/metabolismo , Amiloide/química , Amiloide/genética , Enfermedad de Huntington/metabolismo , Enfermedad de Huntington/genética , Enfermedad de Huntington/patología , Microscopía por Crioelectrón , Animales , Agregación Patológica de Proteínas/metabolismo , Agregación Patológica de Proteínas/genéticaRESUMEN
Diabetes-associated cognitive dysfunction (DCD) is a severe complication of diabetes mellitus (DM), threatening the life quality of the diabetic population. However, there is still a lack of effective approaches for its intervention. Eicosapentaenoic acid (EPA) is an omega-3 polyunsaturated fatty acid that was not previously investigated for its effect on DCD. In this study, EPA was found to improve DCD in a mouse model of type 2 DM (T2DM) induced by streptozotocin and a high-fat diet, exhibiting profound protective effects on cognitive dysfunction, neuronal loss, and cerebral oxidative stress and inflammation. While EPA did not attenuate advanced glycation end product-induced neuron injury, we hypothesized that EPA might protect neurons by regulating microglia polarization, the effect of which was confirmed by the co-culture of neurons and lipopolysaccharide-stimulated microglia. RNA sequencing identified nuclear factor-erythroid-2-related factor 2 (NRF2) antioxidant signaling as a major target of EPA in microglia. Mechanistically, EPA increased sequestosome-1 (SQSTM1 or P62) levels that might structurally inhibit Kelch-like ECH associated protein 1 (KEAP1), leading to nuclear translocation of NRF2. P62 and NRF2 predominantly mediated EPA's effect since the knockdown of P62 or NRF2 abolished EPA's protective effect on microglial oxidative stress and inflammation and sequential neuron injuries. Moreover, the regulation of P62/KEPA1/NRF2 axes by EPA was confirmed in the hippocampi of diabetic mice. The present work presents EPA as an effective nutritional approach and microglial P62/KEAP1/NRF2 as molecular targets for the intervention of DCD.
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Disfunción Cognitiva , Diabetes Mellitus Tipo 2 , Ácido Eicosapentaenoico , Proteína 1 Asociada A ECH Tipo Kelch , Factor 2 Relacionado con NF-E2 , Transducción de Señal , Animales , Masculino , Ratones , Disfunción Cognitiva/etiología , Disfunción Cognitiva/prevención & control , Diabetes Mellitus Experimental/complicaciones , Diabetes Mellitus Tipo 2/complicaciones , Diabetes Mellitus Tipo 2/metabolismo , Ácido Eicosapentaenoico/farmacología , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Proteína 1 Asociada A ECH Tipo Kelch/genética , Ratones Endogámicos C57BL , Microglía/efectos de los fármacos , Microglía/metabolismo , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Factor 2 Relacionado con NF-E2/genética , Estrés Oxidativo/efectos de los fármacos , Proteína Sequestosoma-1/metabolismo , Proteína Sequestosoma-1/genética , Transducción de Señal/efectos de los fármacosRESUMEN
Myocardial ischemia/reperfusion injury (MIRI) is an irreversible adverse event during the management of coronary heart disease that lacks effective controls. The underlying mechanism of MIRI still requires further investigation. Recent studies have suggested that overexpression of ATF3 protects against MIRI by regulating inflammatory responses, ferroptosis, and autophagy. The downstream target of ATF3, EGR1, also showed cardioprotective properties against MIRI by promoting autophagy. Therefore, further investigating the effect of ATF3/EGR1 pathway on MIRI-induced inflammation and autophagy is needed. Cardiomyocyte MIRI model was established by challenging H9C2 cells with hypoxia/reoxygenation (H/R). The ATF3 overexpression-H/R cell model by transfecting ATF3 plasmid into the H9C2 cell line. The transcription levels of ATF3 and EGR1 were determined using RT-qPCR, the levels of TNF-α and IL-6 were determined using ELISA kits, the protein expression of LC3 I, LC3 II, and P62 was determined via WB, and microstructure of H9C2 cell was observed by transmission electron microscopy (TEM). Overexpression of ATF3 significantly downregulated Egr1 levels, indicating that EGR1 might be the target of ATF3. By upregulating ATF3 levels, the extracellular levels of the inflammatory cytokines TNF-α and IL-6 significantly decreased, and the protein expression of the autophagy markers LC3 I, LC3 II, and P62 significantly increased. TEM results revealed that the cell line in the H/R-ATF3 group exhibited a higher abundance of autophagosome enclosures of mitochondria. The results indicated that ATF3/EGR1 may alleviate inflammation and improve autophagy in an H/R-induced MIRI model of cardiomyocytes.
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Factor de Transcripción Activador 3 , Autofagia , Proteína 1 de la Respuesta de Crecimiento Precoz , Inflamación , Daño por Reperfusión Miocárdica , Miocitos Cardíacos , Factor de Necrosis Tumoral alfa , Factor de Transcripción Activador 3/metabolismo , Factor de Transcripción Activador 3/genética , Autofagia/genética , Proteína 1 de la Respuesta de Crecimiento Precoz/metabolismo , Proteína 1 de la Respuesta de Crecimiento Precoz/genética , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Animales , Inflamación/metabolismo , Inflamación/patología , Inflamación/genética , Ratas , Línea Celular , Daño por Reperfusión Miocárdica/metabolismo , Daño por Reperfusión Miocárdica/patología , Daño por Reperfusión Miocárdica/genética , Factor de Necrosis Tumoral alfa/metabolismo , Factor de Necrosis Tumoral alfa/genética , Interleucina-6/metabolismo , Interleucina-6/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Asociadas a Microtúbulos/genética , Transducción de Señal , Proteína Sequestosoma-1/metabolismo , Proteína Sequestosoma-1/genéticaRESUMEN
Triple-negative breast cancer (TNBC) is characterized by the complex tumor microenvironment (TME) consisting of an abundance of mesenchymal stem cells (MSCs), which is known to facilitate epithelial-to-mesenchymal transition (EMT). The development of single-cell genomics is a powerful method for defining the intricate genetic landscapes of malignancies. In this study, we have employed single-cell RNA sequencing (scRNA-seq) to dissect the intra-tumoral heterogeneity and analyze the single-cell transcriptomic landscape to detect rare consequential cell subpopulations of significance. The scRNA-seq analysis of TNBC and Normal patient derived samples revealed that EMT markers and transcription factors were most upregulated in MSC population. Further, exploration of gene expression analysis among TNBC and Normal patient-derived MSCs ascertained the role of SQSTM1/P62 and Wnt/ß-catenin in TNBC progression. Wnt/ß-catenin and Wnt/PCP signaling pathways are prominent contributors of EMT, stemness, and cancer stem cell (CSC) properties of TNBC. SQSTM1/P62 cooperates with the components of the Wnt/PCP signaling pathway and is critically involved at the interface of autophagy and EMT. Moreover, siRNA targeting SQSTM1/P62 and inhibitor of Wnt/ß-catenin (FH535) in conjunction was used to explore molecular modification of EMT and stemness markers. Although SQSTM1/P62 is not crucial for cell survival, cytotoxicity assay revealed synergistic interaction between the siRNA/inhibitor. Modulation of these important pathways helped in reduction of expression of genes and proteins contributing to CSC properties. Gene and protein expression analysis revealed the induction of EMT to MET. Moreover, co-treatment resulted in inactivation of non-canonical Wnt VANGL2-JNK signaling axis. The synergistic impact of inhibition of SQSTM1/P62 and Wnt/ß-catenin signaling facilitates the development of a potential therapeutic regimen for TNBC.