RESUMO
BACKGROUND: Tobacco smoking is the leading cause of preventable death and disease worldwide, with over 8 million annual deaths attributed to cigarette smoking. This study investigates the impact of cigarette smoke and heated tobacco products (HTPs) on microglial function, focusing on toxicological profiles, inflammatory responses, and oxidative stress using ISO standard and clinically relevant conditions of exposure. METHODS: We assessed cell viability, reactive oxygen species (ROS) production, lipid peroxidation, mitochondrial function, unfolded protein response, and inflammation in human microglial cells (HMC3) exposed to cigarette smoke, HTP aerosol or nicotine. RESULTS: Our findings show that cigarette smoke significantly reduces microglial viability, increases ROS formation, induces lipid peroxidation, and reduces intracellular glutathione levels. Cigarette smoke also alters the expression of genes involved in mitochondrial dynamics and biogenesis, leading to mitochondrial dysfunction. Additionally, cigarette smoke impairs the unfolded protein response, activates the NF-κB pathway, and induces a pro-inflammatory state characterized by increased TNF and IL-18 expression. Furthermore, cigarette smoke causes DNA damage and decreases the expression of the aging marker Klotho ß. In contrast, HTP, exhibited a lesser degree of microglial toxicity, with reduced ROS production, lipid peroxidation, and mitochondrial dysfunction compared to conventional cigarettes. CONCLUSION: These results highlight the differential toxicological profile of cigarette smoke and HTP on microglial cells, suggesting a potential harm reduction strategy for neurodegenerative disease for smokers unwilling or unable to quit.
Assuntos
Sobrevivência Celular , Inflamação , Peroxidação de Lipídeos , Microglia , Mitocôndrias , Estresse Oxidativo , Espécies Reativas de Oxigênio , Fumaça , Produtos do Tabaco , Resposta a Proteínas não Dobradas , Estresse Oxidativo/efeitos dos fármacos , Humanos , Espécies Reativas de Oxigênio/metabolismo , Inflamação/patologia , Microglia/efeitos dos fármacos , Microglia/metabolismo , Microglia/patologia , Produtos do Tabaco/efeitos adversos , Fumaça/efeitos adversos , Mitocôndrias/metabolismo , Mitocôndrias/efeitos dos fármacos , Peroxidação de Lipídeos/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Linhagem Celular , Temperatura Alta , NF-kappa B/metabolismo , Nicotiana/efeitos adversos , Dano ao DNARESUMO
Activating transcription factor 4 (ATF4) plays a central role in the integrated stress response (ISR) and one overlapping branch of the unfolded protein response (UPR). We recently reported that the splicing inhibitor isoginkgetin (IGG) induced ATF4 protein along with several known ATF4-regulated transcripts in a response that resembled the ISR and UPR. However, the contribution of ATF4-dependent and -independent transcriptional responses to IGG exposure was not known. Here we used RNA-sequencing in HCT116 colon cancer cells and an isogenic subline lacking ATF4 to investigate the contribution of ATF4 to IGG-induced changes in gene expression. Approximately 85% of the IGG-responsive DEGs in HCT116 cells were also differentially expressed in response to the ER stressor thapsigargin (Tg) and these were enriched for genes associated with the UPR and ISR. Most of these were positively regulated by IGG with impaired responses in the ATF4-deficient cells. Nonetheless, there were DEGs that responded similarly in both cell lines. The ATF4-independent IGG-induced DEGs included several metal responsive transcripts encoding metallothionines and a zinc transporter. Taken together, the predominant IGG response was ATF4-dependent in these cells and resembled the UPR and ISR while a second less prominent response involved the ATF4-independent regulation of metal responsive mRNAs.
Assuntos
Fator 4 Ativador da Transcrição , Biflavonoides , Humanos , Células HCT116 , Fator 4 Ativador da Transcrição/metabolismo , Fator 4 Ativador da Transcrição/genética , Biflavonoides/farmacologia , Tapsigargina/farmacologia , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacosRESUMO
Mitochondria are essential organelles that play critical roles in energy metabolism, apoptosis and various cellular processes. Accumulating evidence suggests that mitochondria are also involved in cancer development and progression. The mitochondrial unfolded protein response (UPRmt) is a complex cellular process that is activated when the protein-folding capacity of the mitochondria is overwhelmed. The core machinery of UPRmt includes upstream regulatory factors, mitochondrial chaperones and proteases. These components work together to eliminate misfolded proteins, increase protein-folding capacity, and restore mitochondrial function. Recent studies have shown that UPRmt is dysregulated in various cancers and contributes to tumor initiation, growth, metastasis, and therapeutic resistance. Considering the pivotal role of the UPRmt in oncogenesis, numerous compounds and synthetic drugs targeting UPRmt-related components induce cancer cell death and suppress tumor growth. In this review, we comprehensively summarize recent studies on the molecular mechanisms of UPRmt activation in C. elegans and mammals and elucidate the conceptual framework, functional aspects, and implications of the UPRmt for cancer therapy. In summary, we paint a developmental landscape of the UPRmt in different types of cancer and offer valuable insights for the development of novel cancer treatment strategies by targeting the UPRmt.
Assuntos
Mitocôndrias , Neoplasias , Resposta a Proteínas não Dobradas , Humanos , Neoplasias/metabolismo , Neoplasias/patologia , Mitocôndrias/metabolismo , Animais , Progressão da DoençaRESUMO
Cerebral dopamine neurotrophic factor (CDNF) is an unconventional neurotrophic factor that is a disease-modifying drug candidate for Parkinson's disease. CDNF has pleiotropic protective effects on stressed cells, but its mechanism of action remains incompletely understood. Here, we use state-of-the-art advanced structural techniques to resolve the structural basis of CDNF interaction with GRP78, the master regulator of the unfolded protein response (UPR) pathway. Subsequent binding studies confirm the obtained structural model of the complex, eventually revealing the interaction site of CDNF and GRP78. Finally, mutating the key residues of CDNF mediating its interaction with GRP78 not only results in impaired binding of CDNF but also abolishes the neuroprotective activity of CDNF-derived peptides in mesencephalic neuron cultures. These results suggest that the molecular interaction with GRP78 mediates the neuroprotective actions of CDNF and provide a structural basis for development of next generation CDNF-based therapeutic compounds against neurodegenerative diseases.
Assuntos
Chaperona BiP do Retículo Endoplasmático , Proteínas de Choque Térmico , Resposta a Proteínas não Dobradas , Chaperona BiP do Retículo Endoplasmático/metabolismo , Humanos , Proteínas de Choque Térmico/metabolismo , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/genética , Animais , Ligação Proteica , Fatores de Crescimento Neural/metabolismo , Fatores de Crescimento Neural/química , Fármacos Neuroprotetores/farmacologia , Fármacos Neuroprotetores/química , Fármacos Neuroprotetores/metabolismo , Neurônios/metabolismo , Modelos Moleculares , Sítios de LigaçãoRESUMO
BACKGROUND: Alpha-1 antitrypsin deficiency (AATD) is an inherited disease, the common variant caused by a Pi*Z mutation in the SERPINA1 gene. Pi*Z AAT increases the risk of pulmonary emphysema and liver disease. Berberine (BBR) is a nature dietary supplement and herbal remedy. Emerging evidence revealed that BBR has remarkable liver-protective properties against various liver diseases. In the present study, we investigated the therapeutic effects and toxicities of BBR in Pi*Z hepatocytes and Pi*Z transgenic mice. METHODS: Huh7.5 and Huh7.5Z (which carries the Pi*Z mutation) cells were treated with different concentrations of BBR for 48 hours. MTT was performed for cell viability assay. Intracellular AAT levels were evaluated by western blot. In vivo studies were carried out in wild type, native phenotype AAT (Pi*M), and Pi*Z AAT transgenic mice. Mice were treated with 50 mg/kg/day of BBR or solvent only by oral administration for 30 days. Western blot and liver histopathological examinations were performed to evaluate therapeutic benefits and liver toxicity of BBR. RESULTS: BBR reduced intracellular AAT levels in Huh7.5Z cells, meanwhile, no Pi*Z-specific toxicity was observed. However, BBR did not reduce liver AAT load but significantly potentiated liver inflammation and fibrosis accompanying the activation of unfolded protein response and mTOR in Pi*Z mice, but not in wild type and Pi*M mice. CONCLUSIONS: BBR exacerbated liver inflammation and fibrosis specifically in Pi*Z mice. This adverse effect may be associated with the activation of unfolded protein response and mTOR. This study implicates that BBR should be avoided by AATD patients.
Assuntos
Berberina , Cirrose Hepática , Camundongos Transgênicos , alfa 1-Antitripsina , Animais , Berberina/farmacologia , Camundongos , alfa 1-Antitripsina/genética , alfa 1-Antitripsina/metabolismo , Humanos , Cirrose Hepática/metabolismo , Cirrose Hepática/patologia , Cirrose Hepática/tratamento farmacológico , Cirrose Hepática/induzido quimicamente , Modelos Animais de Doenças , Serina-Treonina Quinases TOR/metabolismo , Fígado/efeitos dos fármacos , Fígado/patologia , Fígado/metabolismo , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Hepatócitos/patologia , Hepatite/patologia , Hepatite/metabolismo , Hepatite/tratamento farmacológico , Hepatite/etiologia , Resposta a Proteínas não Dobradas/efeitos dos fármacosRESUMO
Cancer cachexia manifests as whole body wasting, however, the precise mechanisms governing the alterations in skeletal muscle and cardiac anabolism have yet to be fully elucidated. In this study, we explored changes in anabolic processes in both skeletal and cardiac muscles in the Yoshida AH-130 ascites hepatoma model of cancer cachexia. AH-130 tumor-bearing rats experienced significant losses in body weight, skeletal muscle, and heart mass. Skeletal and cardiac muscle loss was associated with decreased ribosomal (r)RNA, and hypophosphorylation of the eukaryotic factor 4E binding protein 1. Endoplasmic reticulum stress was evident by higher activating transcription factor mRNA in skeletal muscle and growth arrest and DNA damage-inducible protein (GADD)34 mRNA in both skeletal and cardiac muscles. Tumors provoked an increase in tissue expression of interferon-γ in the heart, while an increase in interleukin-1ß mRNA was apparent in both skeletal and cardiac muscles. We conclude that compromised skeletal muscle and heart mass in the Yoshida AH-130 ascites hepatoma model involves a marked reduction translational capacity and efficiency. Furthermore, our observations suggest that endoplasmic reticulum stress and tissue production of pro-inflammatory factors may play a role in the development of skeletal and cardiac muscle wasting.
Assuntos
Caquexia , Músculo Esquelético , Miocárdio , Resposta a Proteínas não Dobradas , Animais , Caquexia/metabolismo , Caquexia/etiologia , Caquexia/patologia , Caquexia/genética , Masculino , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Ratos , Miocárdio/metabolismo , Miocárdio/patologia , Ratos Wistar , Estresse do Retículo Endoplasmático , Neoplasias Hepáticas Experimentais/metabolismo , Neoplasias Hepáticas Experimentais/patologia , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Fator de Transcrição CHOP/metabolismo , Fator de Transcrição CHOP/genéticaRESUMO
Our study investigates the impact of FGF23 overexpression on SaOS-2 cells to elucidate its role in cellular stress and morphology, contributing to the understanding of skeletal pathologies like X-linked hypophosphatemia (XLH). Using transmission electron microscopy and protein analysis (Western blot), we analyzed the rough endoplasmic reticulum (rER) and mitochondria in SaOS-2 cells with FGF23 overexpression compared to controls. We found significant morphological changes, including enlarged and elongated rER and mitochondria, with increased contact zones, suggesting enhanced interaction and adaptation to elevated protein synthesis and secretion demands. Additionally, we observed higher apoptosis rates of the cells after 24-72 h in vitro and upregulated proteins associated with ER stress and apoptosis, such as CHOP, XBP1 (spliced and unspliced), GRP94, eIF2α, and BAX. These findings indicate a robust activation of the unfolded protein response (UPR) and apoptotic pathways due to FGF23 overexpression. Our results highlight the critical role of ER and mitochondrial interactions in cellular stress responses and provide new insights into the mechanistic link between FGF23 signaling and cellular homeostasis. In conclusion, our study underscores the importance of analyzing UPR-related pathways in the development of therapeutic strategies for skeletal and systemic diseases and contributes to a broader understanding of diseases like XLH.
Assuntos
Apoptose , Estresse do Retículo Endoplasmático , Raquitismo Hipofosfatêmico Familiar , Fator de Crescimento de Fibroblastos 23 , Fatores de Crescimento de Fibroblastos , Mitocôndrias , Fator de Crescimento de Fibroblastos 23/metabolismo , Humanos , Fatores de Crescimento de Fibroblastos/metabolismo , Raquitismo Hipofosfatêmico Familiar/metabolismo , Raquitismo Hipofosfatêmico Familiar/patologia , Raquitismo Hipofosfatêmico Familiar/genética , Mitocôndrias/metabolismo , Resposta a Proteínas não Dobradas , Linhagem Celular Tumoral , Modelos Biológicos , Estresse FisiológicoRESUMO
BACKGROUND: Misfolded proteins accumulate in the liver due to endoplasmic reticulum stress (ERS) caused by high blood glucose levels in diabetes. This triggers the unfolded protein response (UPR), which if persistently activated, results in cellular dysfunction. Chronic ER stress increases inflammation, insulin resistance, and apoptosis. There is growing interest in using native plants and traditional medicine for diabetes treatment. The stevia plant has recently gained attention for its potential therapeutic effects. This study investigates the protective effects of aquatic stevia extract on liver damage, ER stress, and the UPR pathway in streptozotocin (STZ)-induced diabetic rats. METHODS: Rats were randomly divided into four groups: a control group that received 1 ml of water; a diabetic group induced by intraperitoneal injection of STZ (60 mg/kg); a diabetic group treated with metformin (500 mg/kg); and a diabetic group treated with aquatic extracts of stevia (400 mg/kg). After 28 days, various parameters were assessed, including inflammatory markers, oxidative stress indices, antioxidant levels, gene expression, stereology, and liver tissue pathology. RESULT: Compared to the diabetic control group, treatment with stevia significantly decreased serum glucose, liver enzymes, inflammatory markers, and oxidative stress while increasing body weight and antioxidant levels. Additionally, stevia extract manipulated UPR gene expression and reduced apoptosis pathway activation. Histological examination revealed improved liver tissue morphology in stevia-treated diabetic rats. CONCLUSION: These findings suggest that aquatic stevia extract mitigates ER stress in diabetic rats by modulating the IRE-1 arm of the UPR and apoptosis pathways, highlighting its potential therapeutic benefits for diabetes-related liver complications.
Assuntos
Diabetes Mellitus Experimental , Estresse do Retículo Endoplasmático , Fígado , Estresse Oxidativo , Extratos Vegetais , Stevia , Animais , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Stevia/química , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/metabolismo , Extratos Vegetais/farmacologia , Ratos , Masculino , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Estresse Oxidativo/efeitos dos fármacos , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Glicemia/efeitos dos fármacos , Glicemia/metabolismo , Estreptozocina , Apoptose/efeitos dos fármacos , Antioxidantes/farmacologia , Hipoglicemiantes/farmacologiaRESUMO
Type 1 diabetes (T1D) is a T lymphocyte-mediated autoimmune disease caused by pancreatic ß|-cell destruction, which eventually leads to reduced insulin level and increased blood glucose level (Syed, 2022). As a multifactorial disease, T1D is characterized by a genetic predisposition associated with various environmental and cellular elements (Syed, 2022). Pancreatic ß cells have long been considered the "innocent victims" in T1D pathogenesis since the pancreas is attacked by the immune cells, resulting in a process known as insulitis, in which the immune cells infiltrate pancreatic islets and secrete pro-inflammatory cytokines. However, growing evidence suggests that various ß|-cell stresses, dysfunction, and death contribute to T1D pathogenesis, as it has been observed that ß|-cell dysfunction in autoantibody-positive (Aab+) individuals exists long before T1D diagnosis (Evans-Molina et al., 2018).
Assuntos
Senescência Celular , Diabetes Mellitus Tipo 1 , Células Secretoras de Insulina , Resposta a Proteínas não Dobradas , Diabetes Mellitus Tipo 1/imunologia , Células Secretoras de Insulina/imunologia , Células Secretoras de Insulina/metabolismo , Animais , Camundongos , Humanos , Autoanticorpos/imunologiaRESUMO
African swine fever (ASF), caused by African swine fever virus (ASFV), is a devastating infectious disease of domestic pigs and wild boar, which threatens the global pig industry. Endoplasmic reticulum (ER) is a multifunctional signaling organelle in eukaryotic cells that is involved in protein synthesis, processing, posttranslational modification and quality control. As intracellular parasitic organisms, viruses have evolved several strategies to modulate ER functions to favor their life cycles. We have previously demonstrated that the differentially expressed genes associated with unfolded protein response (UPR), which represents a response to ER stress, are significantly enriched upon ASFV infection. However, the correlation between the ER stress or UPR and ASFV replication has not been illuminated yet. Here, we demonstrated that ASFV infection induces ER stress both in target cells and in vivo, and subsequently activates the activating transcription factor 6 (ATF6) branch of the UPR to facilitate viral replication. Mechanistically, ASFV infection disrupts intracellular calcium (Ca2+) homeostasis, while the ATF6 pathway facilitates ASFV replication by increasing the cytoplasmic Ca2+ level. More specifically, we demonstrated that ASFV infection triggers ER-dependent Ca2+ release via the inositol triphosphate receptor (IP3R) channel. Notably, we showed that the ASFV B117L protein plays crucial roles in ER stress and the downstream activation of the ATF6 branch, as well as the disruption of Ca2+ homeostasis. Taken together, our findings reveal for the first time that ASFV modulates the ER stress-ATF6-Ca2+ axis to facilitate viral replication, which provides novel insights into the development of antiviral strategies for ASFV.
Assuntos
Fator 6 Ativador da Transcrição , Vírus da Febre Suína Africana , Febre Suína Africana , Cálcio , Estresse do Retículo Endoplasmático , Resposta a Proteínas não Dobradas , Replicação Viral , Animais , Vírus da Febre Suína Africana/fisiologia , Vírus da Febre Suína Africana/genética , Fator 6 Ativador da Transcrição/metabolismo , Fator 6 Ativador da Transcrição/genética , Suínos , Febre Suína Africana/virologia , Febre Suína Africana/metabolismo , Cálcio/metabolismo , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/virologia , Células Vero , Chlorocebus aethiopsRESUMO
BACKGROUND: Currently, there is a lack of validated pharmacological interventions for non-alcoholic fatty liver disease (NAFLD), which is characterized by the accumulation of hepatic triglyceride. Zhimu-Huangbai (ZH) herb-pair is a traditional Chinese medicine that regulates glucose and lipid metabolism disorders. However, the precise mechanisms underlying the preventive effects of hepatic triglyceride induced by high-fat diet (HFD) remain elusive. PURPOSE: The study aimed to examine the impact of ZH herb-pair on NAFLD in mice and explore the underlying mechanisms, particularly its effects on endoplasmic reticulum (ER) stress and lipid metabolism. METHODS: NAFLD was induced in mice using HFD, and the treated mice were orally administered ZH, metformin (Glucophage) or lovastatin. The lipid metabolism factors, ER stress markers, and the unfolded protein response (UPR) branch factors were measured using immunohistochemistry, western blotting or qRT-PCR. Co-Immunoprecipitation (CoIP) was performed to reveal the connection between SCAP and SREBP-1c. Tunicamycin (TM) and plasmid delivery were used to induce acute ER stress or crease XBP1 gain function models. The main compounds in ZH binding to IRE1α protein were studied by molecular docking and cellular thermal shift assay (CETSA). RESULTS: Treatment with ZH significantly ameliorated hepatic steatosis and reduced lipid synthesis process mainly inhibiting the expression of mature active form of SREBP-1c through relieving ER stress. The expression of IRE1α and XBP1s was inhibited after treatment with ZH. In addition, ZH improved the fatty liver phenotype caused by XBP1 overexpression via decreasing srebp1c transcription. In vitro experimental results suggested that the main compounds in ZH decreased cellular TG contents. Mechanistically, ZH targeted IRE1α and inhibited XBP1s mRNA expression to relieve ER stress and inhibit SREBP-1c production. CONCLUSIONS: ZH herb-pair can protect against NAFLD by reducing the expression of SREBP-1c, in part, via regulating IRE1α/XBP1s pathway.
Assuntos
Medicamentos de Ervas Chinesas , Estresse do Retículo Endoplasmático , Endorribonucleases , Hepatopatia Gordurosa não Alcoólica , Proteínas Serina-Treonina Quinases , Animais , Humanos , Masculino , Camundongos , Dieta Hiperlipídica/efeitos adversos , Medicamentos de Ervas Chinesas/farmacologia , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Endorribonucleases/metabolismo , Metabolismo dos Lipídeos/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Lovastatina/farmacologia , Metformina/farmacologia , Camundongos Endogâmicos C57BL , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Triglicerídeos/metabolismo , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Proteína 1 de Ligação a X-Box/metabolismoRESUMO
Cisplatin is a commonly used chemotherapy agent with a nearly universal side effect of sensorineural hearing loss. The cellular mechanisms underlying cisplatin ototoxicity are poorly understood. Efforts in drug development to prevent or reverse cisplatin ototoxicity have largely focused on pathways of oxidative stress and apoptosis. An effective treatment for cisplatin ototoxicity, sodium thiosulfate (STS), while beneficial when used in standard risk hepatoblastoma, is associated with reduced survival in disseminated pediatric malignancy, highlighting the need for more specific drugs without potential tumor protective effects. The unfolded protein response (UPR) and endoplasmic reticulum (ER) stress pathways have been shown to be involved in the pathogenesis of noise-induced hearing loss and cochlear synaptopathy in vivo, and these pathways have been implicated broadly in cisplatin cytotoxicity. This study sought to determine whether the UPR can be targeted to prevent cisplatin ototoxicity. Neonatal cochlear cultures and HEK cells were exposed to cisplatin, and UPR marker gene expression and cell death measured. Treatment with ISRIB (Integrated Stress Response InhIBitor), a drug that activates eif2B and downregulates the pro-apoptotic PERK/CHOP pathway of the UPR, was tested for its ability to reduce apoptosis in HEK cells, hair-cell death in cochlear cultures, and hearing loss using an in vivo mouse model of cisplatin ototoxicity. Finally, to evaluate whether ISRIB might interfere with cisplatin chemoeffectiveness, we tested it in head and neck squamous cell carcinoma (HNSCC) cell-based assays of cisplatin cytotoxicity. Cisplatin exhibited a biphasic, non-linear dose-response of cell death and apoptosis that correlated with different patterns of UPR marker gene expression in HEK cells and cochlear cultures. ISRIB treatment protected against cisplatin-induced hearing loss and hair-cell death, but did not impact cisplatin's cytotoxic effects on HNSCC cell viability, unlike STS. These findings demonstrate that targeting the pro-apoptotic PERK/CHOP pathway with ISRIB can mitigate cisplatin ototoxicity without reducing anti-cancer cell effects, suggesting that this may be a viable strategy for drug development.
Assuntos
Cisplatino , Estresse do Retículo Endoplasmático , Ototoxicidade , Resposta a Proteínas não Dobradas , Cisplatino/efeitos adversos , Cisplatino/toxicidade , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Animais , Ototoxicidade/prevenção & controle , Ototoxicidade/metabolismo , Ototoxicidade/etiologia , Humanos , Camundongos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Células HEK293 , Antineoplásicos/efeitos adversos , Antineoplásicos/toxicidade , Apoptose/efeitos dos fármacos , Cóclea/efeitos dos fármacos , Cóclea/metabolismo , Cóclea/patologia , Células Ciliadas Auditivas/efeitos dos fármacos , Células Ciliadas Auditivas/metabolismo , eIF-2 Quinase/metabolismoRESUMO
The complex structure of monoclonal antibodies (mAbs) expressed in Chinese hamster ovary (CHO) cells may result in the accumulation of unfolded proteins, triggering endoplasmic reticulum (ER) stress and an unfolded protein response (UPR). If the protein folding ability cannot maintain ER homeostasis, the cell will shut down protein translation and ultimately induce apoptosis. We co-overexpressed HsQSOX1b and survivin proteins in the antibody-producing cell line CHO-PAb to obtain a new cell line, CHO-PAb-QS. Compared with CHO-PAb cells, the survival time of CHO-PAb-QS cells in batch culture was extended by 2 days, and the antibody accumulation and productivity were increased by 52% and 45%, respectively. The proportion of (HC-LC)2 was approximately doubled in the CHO-PAb-QS cells, which adapted to the accelerated disulfide bond folding capacity by upregulating the UPR's strength and increasing the ER content. The results of the apoptosis assays indicated that the CHO-PAb-QS cell line exhibited more excellent resistance to apoptosis induced by ER stress. Finally, CHO-PAb-QS cells exhibited mild oxidative stress but did not significantly alter the redox status. This study demonstrated that strategies based on HsQSOX1b and survivin co-overexpression could facilitate protein disulfide bond folding and anti-apoptosis ability, enhancing antibody production efficiency in CHO cell lines.
Assuntos
Apoptose , Cricetulus , Dissulfetos , Dobramento de Proteína , Células CHO , Animais , Dissulfetos/metabolismo , Dissulfetos/química , Estresse do Retículo Endoplasmático , Resposta a Proteínas não Dobradas , Formação de Anticorpos , Anticorpos Monoclonais , Cricetinae , Survivina/metabolismo , Humanos , Retículo Endoplasmático/metabolismo , Estresse OxidativoRESUMO
ER-phagy is a specialized form of autophagy, defined by the lysosomal degradation of ER subdomains. ER-phagy has been implicated in relieving the ER from misfolded proteins during ER stress upon activation of the unfolded protein response (UPR). Here, we identified an essential role for the ER chaperone calnexin in regulating ER-phagy and the UPR in neurons. We showed that chemical induction of ER stress triggers ER-phagy in the somata and axons of primary cultured motoneurons. Under basal conditions, the depletion of calnexin leads to an enhanced ER-phagy in axons. However, upon ER stress induction, ER-phagy did not further increase in calnexin-deficient motoneurons. In addition to increased ER-phagy under basal conditions, we also detected an elevated proteasomal turnover of insoluble proteins, suggesting enhanced protein degradation by default. Surprisingly, we detected a diminished UPR in calnexin-deficient early cortical neurons under ER stress conditions. In summary, our data suggest a central role for calnexin in orchestrating both ER-phagy and the UPR to maintain protein homeostasis within the ER.
Assuntos
Calnexina , Estresse do Retículo Endoplasmático , Retículo Endoplasmático , Resposta a Proteínas não Dobradas , Calnexina/metabolismo , Animais , Retículo Endoplasmático/metabolismo , Camundongos , Autofagia , Neurônios Motores/metabolismo , Axônios/metabolismo , Células Cultivadas , Lisossomos/metabolismo , Neurônios/metabolismoRESUMO
Charcot-Marie-Tooth type 1B (CMT1B) is a peripheral neuropathy caused by mutations in the gene encoding myelin protein zero (MPZ), a key component of the myelin sheath in Schwann cells. Mutations in the MPZ gene can lead to protein misfolding, unfolded protein response (UPR), endoplasmic reticulum (ER) stress, or protein mistrafficking. Despite significant progress in understanding the disease mechanisms, there is currently no effective treatment for CMT1B, with therapeutic strategies primarily focused on supportive care. Gene therapy represents a promising therapeutic approach for treating CMT1B. To develop a treatment and better design preclinical studies, an in-depth understanding of the pathophysiological mechanisms and animal models is essential. In this review, we present a comprehensive overview of the disease mechanisms, preclinical models, and recent advancements in therapeutic research for CMT1B, while also addressing the existing challenges in the field. This review aims to deepen the understanding of CMT1B and to encourage further research towards the development of effective treatments for CMT1B patients.
Assuntos
Doença de Charcot-Marie-Tooth , Modelos Animais de Doenças , Terapia Genética , Doença de Charcot-Marie-Tooth/terapia , Doença de Charcot-Marie-Tooth/genética , Doença de Charcot-Marie-Tooth/metabolismo , Humanos , Animais , Terapia Genética/métodos , Mutação , Proteína P0 da Mielina/genética , Proteína P0 da Mielina/metabolismo , Resposta a Proteínas não Dobradas/genética , Estresse do Retículo Endoplasmático/genéticaRESUMO
This study was designed to examine the association between myocardial concentrations of the trace elements Cu, Fe, Mn, Mo, and Zn and the expression of mitochondrial unfolded protein response (UPRmt) elements and the age of patients who received heart transplantation or a left-ventricular assist device (ageHTx/LVAD). Inductively coupled plasma mass spectrometry was used to determine the concentration of Cu, Fe, Mn, Mo, and Zn in the myocardium of control subjects and patients undergoing heart transplantation or left-ventricular assist device (LVAD) implantation. We used ELISA to quantify the expression of UPRmt proteins and 4-Hydroxynonenal (4-HNE), which served as a marker of oxidative-stress-induced lipid peroxidation. Concentrations of Cu, Mn, Mo, and Zn were similar in the control and heart failure (HF) myocardium, while Fe showed a significant decrease in the HF group compared to the control. A higher cumulative concentration of Fe and Zn in the myocardium was associated with reduced ageHTx/LVAD, which was not observed for other combinations of trace elements or their individual effects. The trace elements Cu, Mn, and Zn showed positive correlations with several UPRmt proteins, while Fe had a negative correlation with UPRmt effector protease YME1L. None of the trace elements correlated with 4-HNE in the myocardium. The concentrations of the trace elements Mn and Zn were significantly higher in the myocardium of patients with dilated cardiomyopathy than in patients with ischemic cardiomyopathy. A higher cumulative concentration of Fe and Zn in the myocardium was associated with a younger age at which patients received heart transplantation or LVAD, potentially suggesting an acceleration of HF. A positive correlation between myocardial Cu, Mn, and Zn and the expression of UPRmt proteins and a negative correlation between myocardial Fe and YME1L expression suggest that these trace elements exerted their actions on the human heart by interacting with the UPRmt. An altered generation of oxidative stress was not an underlying mechanism of the observed changes.
Assuntos
Ferro , Resposta a Proteínas não Dobradas , Zinco , Humanos , Zinco/metabolismo , Zinco/análise , Masculino , Ferro/metabolismo , Pessoa de Meia-Idade , Feminino , Adulto , Cardiotoxicidade/etiologia , Cardiotoxicidade/metabolismo , Estresse Oxidativo , Insuficiência Cardíaca/metabolismo , Miocárdio/metabolismo , Idoso , Transplante de Coração , Coração Auxiliar/efeitos adversos , Aldeídos/metabolismoRESUMO
During homeostasis, the endoplasmic reticulum (ER) maintains productive transmembrane and secretory protein folding that is vital for proper cellular function. The ER-resident HSP70 chaperone, binding immunoglobulin protein (BiP), plays a pivotal role in sensing ER stress to activate the unfolded protein response (UPR). BiP function is regulated by the bifunctional enzyme filamentation induced by cyclic-AMP domain protein (FicD) that mediates AMPylation and deAMPylation of BiP in response to changes in ER stress. AMPylated BiP acts as a molecular rheostat to regulate UPR signaling, yet little is known about the molecular consequences of FicD loss. In this study, we investigate the role of FicD in mouse embryonic fibroblast (MEF) response to pharmacologically and metabolically induced ER stress. We find differential BiP AMPylation signatures when comparing robust chemical ER stress inducers to physiological glucose starvation stress and recovery. Wildtype MEFs respond to pharmacological ER stress by down-regulating BiP AMPylation. Conversely, BiP AMPylation in wildtype MEFs increases upon metabolic stress induced by glucose starvation. Deletion of FicD results in widespread gene expression changes under baseline growth conditions. In addition, FicD null MEFs exhibit dampened UPR signaling, altered cell stress recovery response, and unconstrained protein secretion. Taken together, our findings indicate that FicD is important for tampering UPR signaling, stress recovery, and the maintenance of secretory protein homeostasis.
Assuntos
Chaperona BiP do Retículo Endoplasmático , Estresse do Retículo Endoplasmático , Fibroblastos , Glucose , Resposta a Proteínas não Dobradas , Animais , Camundongos , Embrião de Mamíferos/metabolismo , Embrião de Mamíferos/citologia , Retículo Endoplasmático/metabolismo , Chaperona BiP do Retículo Endoplasmático/metabolismo , Fibroblastos/metabolismo , Glucose/metabolismo , Proteínas de Choque Térmico/metabolismo , Proteínas de Choque Térmico/genética , Camundongos Knockout , Nucleotidiltransferases/metabolismo , Nucleotidiltransferases/genética , Transdução de SinaisRESUMO
BACKGROUND: Myocardial infarction (MI) significantly contributes to the global mortality rate, often leading to heart failure (HF) due to left ventricular remodeling. Key factors in the pathomechanism of HF include nitrosative/oxidative stress, inflammation, and endoplasmic reticulum (ER) stress. Furthermore, while a high-fat diet (HFD) is known to exacerbate post-MI cardiac remodeling, its impact on these critical factors in the context of HF is not as well understood. AIMS: This study aimed to assess the impact of post-MI HF and HFD on inflammation, nitro-oxidative stress, ER stress, and unfolded protein response (UPR). METHODS: The study was performed on fragments of the left ventricle harvested from 30 male adult Sprague Dawley rats, which were divided into four groups based on diet (normal-fat vs. high-fat) and surgical procedure (sham operation vs. coronary artery ligation to induce MI). We assessed body weight, NT-proBNP levels, protein levels related to nitrosative/oxidative stress, ER stress, UPR, apoptosis, and nitric oxide synthases, through Western Blot and ELISA. RESULTS: HFD and MI significantly influenced body weight and NT-proBNP concentrations. HFD elevated 3-nitrotyrosine and myeloperoxidase levels and altered nitric oxide synthase levels. HFD and MI significantly affected ER stress markers and activated or inhibited UPR pathways. CONCLUSIONS: The study demonstrates significant impacts of post-MI HF and dietary fat content on cardiac function and stress markers in a rat model. The interaction between HFD and MI on UPR activation suggests the importance of dietary management in post-MI recovery and HF prevention.
Assuntos
Dieta Hiperlipídica , Estresse do Retículo Endoplasmático , Insuficiência Cardíaca , Infarto do Miocárdio , Estresse Oxidativo , Ratos Sprague-Dawley , Resposta a Proteínas não Dobradas , Animais , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Masculino , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/etiologia , Insuficiência Cardíaca/patologia , Dieta Hiperlipídica/efeitos adversos , Ratos , Modelos Animais de Doenças , Apoptose , Peptídeo Natriurético Encefálico/metabolismo , Fragmentos de Peptídeos/metabolismo , Miocárdio/metabolismo , Miocárdio/patologiaRESUMO
In recent years, the world has seen an alarming increase in obesity and is closely associated with insulin resistance, which is a state of low-grade inflammation, the latter characterized by elevated levels of proinflammatory cytokines in blood and tissues. A shift in energy balance alters systemic metabolic regulation and the important role that chronic inflammation, endoplasmic reticulum (ER) dysfunction, and activation of the unfolded protein response (UPR) plays in this process.Why obesity is so closely associated with insulin resistance and inflammation is not understood well. This suggests that there are probably many causes for obesity-related insulin resistance and inflammation. One of the faulty mechanisms is protein homeostasis, protein quality control system included protein folding, chaperone activity, and ER-associated degradation leading to endoplasmic reticulum (ER) stress.The ER is a vast membranous network responsible for the trafficking of a wide range of proteins and plays a central role in integrating multiple metabolic signals critical in cellular homeostasis. Conditions that may trigger unfolded protein response activation include increased protein synthesis, the presence of mutant or misfolded proteins, inhibition of protein glycosylation, imbalance of ER calcium levels, glucose and energy deprivation, hypoxia, pathogens, or pathogen-associated components and toxins. Thus, characterizing the mechanisms contributing to obesity and identifying potential targets for its prevention and treatment will have a great impact on the control of associated conditions, particularly T2D.
Assuntos
Estresse do Retículo Endoplasmático , Obesidade , Resposta a Proteínas não Dobradas , Humanos , Obesidade/metabolismo , Animais , Retículo Endoplasmático/metabolismo , Resistência à Insulina , Inflamação/metabolismoRESUMO
T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy that commonly affects children and adolescents with a poor prognosis. The terminal unfolded protein response (UPR) is an emerging anti-cancer approach, although its role in pediatric T-ALL remains unclear. In our pediatric T-ALL cohort from different centers, a lower QRICH1 expression was found associated with a worse prognosis of pediatric T-ALL. Overexpression of QRICH1 significantly inhibited cell proliferation and stimulated apoptosis of T-ALL both in vitro and in vivo. Upregulation of QRICH1 significantly downregulated 78 KDa glucose-regulated protein (GRP78) and upregulated CHOP, thus activating the terminal UPR. Co-overexpression of GRP78 in T-ALL cells overexpressing QRICH1 partially reverted the inhibited proliferation and stimulated apoptosis. QRICH1 bound to the residues Asp212 and Glu155 of the nucleotide-binding domain (NBD) of GRP78, thereby inhibiting its ATP hydrolysis activity. In addition, QRICH1 was associated with endoplasmic reticulum (ER) stress in T-ALL, and overexpression of QRICH1 reversed drug resistance. Overall, low QRICH1 expression is an independent risk factor for a poor prognosis of pediatric T-ALL. By inhibiting GRP78, QRICH1 suppresses pediatric T-ALL.