RESUMO
Ribonucleoprotein (RNP) condensates partition RNA and protein into multiple liquid phases. The multiphasic feature of condensate-enriched components creates experimental challenges for distinguishing membraneless condensate functions from the surrounding dilute phase. We combined fluorescence lifetime imaging microscopy (FLIM) with phasor plot filtering and segmentation to resolve condensates from the dilute phase. Condensate-specific lifetimes were used to track protein-protein interactions by measuring FLIM-Förster resonance energy transfer (FRET). We used condensate FLIM-FRET to evaluate whether mRNA decapping complex subunits can form decapping-competent interactions within P-bodies. Condensate FLIM-FRET revealed the presence of core subunit interactions within P-bodies under basal conditions and the disruption of interactions between the decapping enzyme (Dcp2) and a critical cofactor (Dcp1A) during oxidative stress. Our results show a context-dependent plasticity of the P-body interaction network, which can be rewired within minutes in response to stimuli. Together, our FLIM-based approaches provide investigators with an automated and rigorous method to uncover and track essential protein-protein interaction dynamics within RNP condensates in live cells.
Assuntos
Transferência Ressonante de Energia de Fluorescência , Ribonucleoproteínas , Humanos , Ribonucleoproteínas/metabolismo , Microscopia de Fluorescência , Citoplasma/metabolismo , Condensados Biomoleculares/metabolismo , Condensados Biomoleculares/química , Células HeLa , Estresse Oxidativo , Endorribonucleases/metabolismo , Endorribonucleases/genética , Ligação ProteicaRESUMO
Objective. Carboxypeptidase E (CPE) plays an important role in the biosynthesis of neurotransmitters and peptide hormones including insulin. It also promotes cell proliferation, survival, and invasion of tumor cells. The endoplasmic reticulum stress, hypoxia, and nutrient supply are significant factors of malignant tumor growth including glioblastoma. There are data indicating that the knockdown of the endoplasmic reticulum to nucleus signaling 1 (ERN1) suppressed glioblastoma cell proliferation and increased invasiveness of these cells. The present study aims to investigate the regulation of the CPE gene in U87MG glioblastoma cells by ERN1 knockdown, hypoxia, and glucose or glutamine deprivations with the intent to reveal the role of ERN1 signaling in the regulation of this gene expression and function in tumorigenesis. Methods. Human glioblastoma cells U87MG (transfected by an empty vector; control) and ERN1 knockdown cells with inhibited ERN1 endoribonuclease and protein kinase (dnERN1) or only ERN1 endoribonuclease (dnrERN1) were used. Hypoxia was introduced by dimethyloxalylglycine; for glucose and glutamine deprivations, the cells were cultured in DMEM medium without glucose or glutamine for 16 h, respectively. The expression level of the CPE gene was studied by quantitative RT-PCR and normalized to ACTB. Results. It was found that inhibition of endoribonuclease and protein kinase activities of ERN1 led to a strong up-regulation of CPE gene expression in glioblastoma cells. The expression of this gene also increased in glioblastoma cells after silencing ERN1. At the same time, the expression of this gene did not significantly change in cells with inhibited ERN1 endoribonuclease only. The expression of the CPE gene was resistant to hypoxia in control U87MG cells, but increased in cells with ERN1 knockdown. The expression of this gene was up-regulated under glutamine deprivation in control glioblastoma cells, but decreased upon ERN1 knockdown. However, glucose deprivation decreased the expression of CPE gene in both types of used cells, but ERN1 inhibition enhanced this effect. Conclusion. The results of the present study demonstrate that inhibition of ERN1 strongly up-regulated the expression of pro-oncogenic CPE gene through protein kinase activity of ERN1 and that increased CPE gene expression possibly participates in ERN1 knockdown-mediated invasiveness of glioblastoma cells.
Assuntos
Carboxipeptidase H , Estresse do Retículo Endoplasmático , Endorribonucleases , Regulação Neoplásica da Expressão Gênica , Glioblastoma , Proteínas Serina-Treonina Quinases , Humanos , Glioblastoma/metabolismo , Glioblastoma/genética , Glioblastoma/patologia , Carboxipeptidase H/metabolismo , Carboxipeptidase H/genética , Linhagem Celular Tumoral , Estresse do Retículo Endoplasmático/fisiologia , Endorribonucleases/metabolismo , Endorribonucleases/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/metabolismo , Glucose/metabolismo , Técnicas de Silenciamento de Genes , Hipóxia Celular/fisiologia , Transdução de Sinais/fisiologiaRESUMO
Eukaryotic cells possess surveillance mechanisms that detect and degrade defective transcripts. Aberrant transcripts include mRNAs with a premature termination codon (PTC), targeted by the nonsense-mediated decay (NMD) pathway, and mRNAs lacking a termination codon, targeted by the nonstop decay (NSD) pathway. The eukaryotic exosome, a ribonucleolytic complex, plays a crucial role in mRNA processing and turnover through its catalytic subunits PM/Scl100 (Rrp6 in yeast), DIS3 (Rrp44 in yeast), and DIS3L1. Additionally, eukaryotic cells have other ribonucleases, such as SMG6 and XRN1, that participate in RNA surveillance. However, the specific pathways through which ribonucleases recognize and degrade mRNAs remain elusive. In this study, we characterized the involvement of human ribonucleases, both nuclear and cytoplasmic, in the mRNA surveillance mechanisms of NMD and NSD. We performed knockdowns of SMG6, PM/Scl100, XRN1, DIS3, and DIS3L1, analyzing the resulting changes in mRNA levels of selected natural NMD targets by RT-qPCR. Additionally, we examined the levels of different human ß-globin variants under the same conditions: wild-type, NMD-resistant, NMD-sensitive, and NSD-sensitive. Our results demonstrate that all the studied ribonucleases are involved in the decay of certain endogenous NMD targets. Furthermore, we observed that the ribonucleases SMG6 and DIS3 contribute to the degradation of all ß-globin variants, with an exception for ßNS in the former case. This is also the case for PM/Scl100, which affects all ß-globin variants except the NMD-sensitive variants. In contrast, DIS3L1 and XRN1 show specificity for ß-globin WT and NMD-resistant variants. These findings suggest that eukaryotic ribonucleases are target-specific rather than pathway-specific. In addition, our data suggest that ribonucleases play broader roles in mRNA surveillance and degradation mechanisms beyond just NMD and NSD.
Assuntos
Exorribonucleases , Complexo Multienzimático de Ribonucleases do Exossomo , Degradação do RNAm Mediada por Códon sem Sentido , RNA Mensageiro , Humanos , Exorribonucleases/metabolismo , Exorribonucleases/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Complexo Multienzimático de Ribonucleases do Exossomo/genética , Complexo Multienzimático de Ribonucleases do Exossomo/metabolismo , Códon sem Sentido/genética , Células HeLa , Estabilidade de RNA/genética , Ribonucleases/genética , Ribonucleases/metabolismo , Endorribonucleases , Proteínas Associadas aos Microtúbulos , Proteínas de Ligação ao GTPRESUMO
High-grade serious ovarian cancer (HGSOC) is an aggressive malignancy that remains refractory to current immunotherapies. While advanced stage disease has been extensively studied, the cellular and molecular mechanisms that promote early immune escape in HGSOC remain largely unexplored. Here, we report that primary HGSO tumors program neutrophils to inhibit T cell anti-tumor function by activating the endoplasmic reticulum (ER) stress sensor IRE1α. We found that intratumoral neutrophils exhibited overactivation of ER stress response markers compared with their counterparts at non-tumor sites. Selective deletion of IRE1α in neutrophils delayed primary ovarian tumor growth and extended the survival of mice with HGSOC by enabling early T cell-mediated tumor control. Notably, loss of IRE1α in neutrophils sensitized tumor-bearing mice to PD-1 blockade, inducing HGSOC regression and long-term survival in ~ 50% of the treated hosts. Hence, neutrophil-intrinsic IRE1α facilitates early adaptive immune escape in HGSOC and targeting this ER stress sensor might be used to unleash endogenous and immunotherapy-elicited immunity that controls metastatic disease.
Assuntos
Estresse do Retículo Endoplasmático , Endorribonucleases , Neutrófilos , Neoplasias Ovarianas , Receptor de Morte Celular Programada 1 , Proteínas Serina-Treonina Quinases , Feminino , Animais , Neoplasias Ovarianas/imunologia , Neoplasias Ovarianas/patologia , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Neutrófilos/imunologia , Neutrófilos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Endorribonucleases/metabolismo , Endorribonucleases/genética , Camundongos , Humanos , Estresse do Retículo Endoplasmático/imunologia , Receptor de Morte Celular Programada 1/metabolismo , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Resistencia a Medicamentos Antineoplásicos , Inibidores de Checkpoint Imunológico/farmacologia , Inibidores de Checkpoint Imunológico/uso terapêutico , Cistadenocarcinoma Seroso/patologia , Cistadenocarcinoma Seroso/imunologia , Cistadenocarcinoma Seroso/metabolismo , Linhagem Celular Tumoral , Gradação de Tumores , Evasão Tumoral/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
Unfolded protein response (UPR) is a central stress response pathway that is hijacked by tumor cells for their survival. Here, we find that IRE1α signaling, one of the canonical UPR arms, is increased in prostate cancer (PCa) patient tumors. Genetic or small molecule inhibition of IRE1α in syngeneic mouse PCa models and an orthotopic model decreases tumor growth. IRE1α ablation in cancer cells potentiates interferon responses and activates immune system related pathways in the tumor microenvironment (TME). Single-cell RNA-sequencing analysis reveals that targeting IRE1α in cancer cells reduces tumor-associated macrophage abundance. Consistently, the small molecule IRE1α inhibitor MKC8866, currently in clinical trials, reprograms the TME and enhances anti-PD-1 therapy. Our findings show that IRE1α signaling not only promotes cancer cell growth and survival but also interferes with anti-tumor immunity in the TME. Thus, targeting IRE1α can be a promising approach for improving anti-PD-1 immunotherapy in PCa.
Assuntos
Endorribonucleases , Neoplasias da Próstata , Proteínas Serina-Treonina Quinases , Microambiente Tumoral , Resposta a Proteínas não Dobradas , Microambiente Tumoral/imunologia , Masculino , Animais , Neoplasias da Próstata/imunologia , Neoplasias da Próstata/patologia , Neoplasias da Próstata/genética , Neoplasias da Próstata/tratamento farmacológico , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Endorribonucleases/metabolismo , Endorribonucleases/genética , Humanos , Camundongos , Linhagem Celular Tumoral , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Resposta a Proteínas não Dobradas/imunologia , Transdução de Sinais , Imunoterapia/métodos , Receptor de Morte Celular Programada 1/metabolismo , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Camundongos Endogâmicos C57BL , Macrófagos Associados a Tumor/imunologia , Macrófagos Associados a Tumor/metabolismoRESUMO
Airway mucus hypersecretion, a crucial pathological feature of chronic obstructive pulmonary disease (COPD), contributes to the initiation, progression, and exacerbation of this disease. As a macromolecular mucin, the secretory behaviour of Mucin5AC (MUC5AC) is highly dependent on a series of modifying and folding processes that occur in the endoplasmic reticulum (ER). In this study, we focused on the ER quality control protein KDEL receptor (KDELR) and demonstrated that KDELR2 and MUC5AC were colocalized in the airway epithelium of COPD patients and COPD model rats. In addition, knockdown of KDELR2 markedly reduced the expression of MUC5AC both in vivo and in vitro and knockdown of ATF6 further decreased the levels of KDELR2. Furthermore, pretreatment with 4µ8C, an IRE1α inhibitor, led to a partial reduction in the expression of KDELR2 and MUC5AC both in vivo and in vitro, which indicated the involvement of IRE1α/XBP-1s in the upstream signalling cascade. Our study revealed that KDELR2 plays a crucial role in airway MUC5AC hypersecretion in COPD, which might be dependent on ATF6 and IRE1α/XBP-1s upstream signalling.
Assuntos
Endorribonucleases , Mucina-5AC , Proteínas Serina-Treonina Quinases , Doença Pulmonar Obstrutiva Crônica , Idoso , Animais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Ratos , Fator 6 Ativador da Transcrição/metabolismo , Fator 6 Ativador da Transcrição/genética , Modelos Animais de Doenças , Retículo Endoplasmático/metabolismo , Estresse do Retículo Endoplasmático , Endorribonucleases/metabolismo , Endorribonucleases/genética , Mucina-5AC/metabolismo , Mucina-5AC/genética , Muco/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Doença Pulmonar Obstrutiva Crônica/metabolismo , Doença Pulmonar Obstrutiva Crônica/genética , Doença Pulmonar Obstrutiva Crônica/patologia , Ratos Sprague-Dawley , Transdução de Sinais , Proteína 1 de Ligação a X-Box/metabolismo , Proteína 1 de Ligação a X-Box/genéticaRESUMO
Cervical cancer is the fourth most common malignancy in women globally. Chemotherapies, targeted therapies, and immunotherapies in the treatment of cervical cancer are usually accompanied by effective and adverse effects. Therefore, finding other efficient and accurate molecular targets remains essential to improve the treatment benefits of cervical cancer patients. MCPIP1 (monocyte chemoattractant protein-induced protein 1) is a kind of endonuclease with a CCCH zinc finger domain and a PilT-N-terminal (PIN) domain, and its function in cervical cancer is unknown. We found that MCPIP1 inhibits cell proliferation and promotes cell apoptosis of cervical cancer. Additionally, MCPIP1 suppresses mRNA and protein expression of the apoptotic inhibitor XIAP by decreasing its mRNA stability. Mechanically, MCPIP1 binds to the XIAP mRNA via its CCCH zinc finger domain and degrades the XIAP mRNA via the endonuclease activity coming from its PIN domain. Our study clarifies that MCPIP1 promotes cervical cancer cell apoptosis by suppressing the expression of XIAP, thereby impeding cervical cancer progression. Moreover, targeted delivery of MCPIP1 with engineered Salmonella typhimurium leads to tumor growth retardation in the HeLa xenograft tumor model in mice. Therefore, our study may provide a theoretical basis for formulating clinical treatment strategies for cervical cancer.
Assuntos
Apoptose , Proliferação de Células , Estabilidade de RNA , Ribonucleases , Fatores de Transcrição , Neoplasias do Colo do Útero , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X , Humanos , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/terapia , Neoplasias do Colo do Útero/metabolismo , Neoplasias do Colo do Útero/patologia , Feminino , Ribonucleases/metabolismo , Ribonucleases/genética , Animais , Camundongos , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Células HeLa , Regulação Neoplásica da Expressão Gênica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Endorribonucleases/metabolismo , Endorribonucleases/genética , Camundongos Nus , Linhagem Celular Tumoral , Camundongos Endogâmicos BALB CRESUMO
Tuberculosis is a worldwide plague caused by the pathogen Mycobacterium tuberculosis (M. tb). Toxin-antitoxin (TA) systems are genetic elements abundantly present in prokaryotic organisms and regulate important cellular processes. MazEF is a TA system implicated in the formation of "persisters cells" of M. tb, which contain more than 10 such members. However, the exact function and inhibition mode of each MazF are not fully understood. Here we report crystal structures of MazF-mt3 in its apo form and in complex with the C-terminal half of MazE-mt3. Structural analysis suggested that two long but disordered ß1-ß2 loops would interfere with the binding of the cognate MazE-mt3 antitoxin. Similar loops are also present in the MazF-mt1 and -mt9 but are sustainably shortened in other M. tb MazF members, and these TA pairs behave distinctly in terms of their binding modes and their RNase activities. Systematic crystallographic and biochemical studies further revealed that the biochemical activities of M. tb toxins were combined results between the interferences from the characteristic loops and the electrostatic interactions between the cognate TA pairs. This study provides structural insight into the binding mode and the inhibition mechanism of the MazE/F TA pairs, which facilitate the structure-based peptide designs.
Assuntos
Proteínas de Bactérias , Endorribonucleases , Mycobacterium tuberculosis , Sistemas Toxina-Antitoxina , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Mycobacterium tuberculosis/metabolismo , Mycobacterium tuberculosis/genética , Sistemas Toxina-Antitoxina/genética , Endorribonucleases/química , Endorribonucleases/metabolismo , Endorribonucleases/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Toxinas Bacterianas/química , Toxinas Bacterianas/metabolismo , Toxinas Bacterianas/genética , Ligação Proteica , Cristalografia por Raios X , Modelos Moleculares , Antitoxinas/química , Antitoxinas/metabolismo , Antitoxinas/genética , Sequência de AminoácidosRESUMO
Microcystin-LR (MC-LR), a cyanobacterial toxin, is a potent carcinogen implicated in colorectal cancer (CRC) progression. However, its impact on the tumor microenvironment (TME) during CRC development remains poorly understood. This study investigates the interaction between tumor cells and macrophages mediated by MC-LR within the TME and its influence on CRC progression. CRC mice exposed to MC-LR demonstrated a significant transformation from adenoma to adenocarcinoma. The infiltration of macrophages increased, and the IRE1α/XBP1 pathway was activated in CRC cells after MC-LR exposure, influencing macrophage M2 polarization under co-culture conditions. Additionally, hexokinase 2 (HK2), a downstream target of the IRE1α/XBP1 pathway, was identified, regulating glycolysis and lactate production. The MC-LR-induced IRE1α/XBP1/HK2 axis enhanced lactate production in CRC cells, promoting M2 macrophage polarization. Furthermore, co-culturing MC-LR-exposed CRC cells with macrophages, along with the IRE1α/XBP1 pathway inhibitor 4µ8C and the hexokinase inhibitor 2-DG, suppressed M2 macrophage-induced CRC cell migration, clonogenicity, and M2 macrophage polarization. This study elucidates the mechanism by which MC-LR-mediated interactions through the IRE1α/XBP1 pathway promote CRC progression, highlighting potential therapeutic targets.
Assuntos
Neoplasias Colorretais , Endorribonucleases , Macrófagos , Microcistinas , Transdução de Sinais , Animais , Humanos , Camundongos , Linhagem Celular Tumoral , Neoplasias Colorretais/patologia , Neoplasias Colorretais/metabolismo , Progressão da Doença , Endorribonucleases/metabolismo , Hexoquinase/metabolismo , Macrófagos/metabolismo , Macrófagos/efeitos dos fármacos , Toxinas Marinhas , Microcistinas/farmacologia , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Microambiente Tumoral/efeitos dos fármacos , Proteína 1 de Ligação a X-Box/metabolismoRESUMO
Eukaryotic gene expression is regulated at the transcriptional and post-transcriptional levels, with disruption of regulation contributing significantly to human diseases. The 5' m7G mRNA cap is a central node in post-transcriptional regulation, participating in both mRNA stabilization and translation efficiency. In mammals, DCP1a and DCP1b are paralogous cofactor proteins of the mRNA cap hydrolase DCP2. As lower eukaryotes have a single DCP1 cofactor, the functional advantages gained by this evolutionary divergence remain unclear. We report the first functional dissection of DCP1a and DCP1b, demonstrating that they are non-redundant cofactors of DCP2 with unique roles in decapping complex integrity and specificity. DCP1a is essential for decapping complex assembly and interactions between the decapping complex and mRNA cap-binding proteins. DCP1b is essential for decapping complex interactions with protein degradation and translational machinery. DCP1a and DCP1b impact the turnover of distinct mRNAs. The observation that different ontological groups of mRNA molecules are regulated by DCP1a and DCP1b, along with their non-redundant roles in decapping complex integrity, provides the first evidence that these paralogs have qualitatively distinct functions.
Assuntos
Endorribonucleases , Capuzes de RNA , Estabilidade de RNA , RNA Mensageiro , Humanos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Endorribonucleases/metabolismo , Endorribonucleases/genética , Capuzes de RNA/metabolismo , Capuzes de RNA/genética , Estabilidade de RNA/genética , Proteínas de Ligação ao Cap de RNA/metabolismo , Proteínas de Ligação ao Cap de RNA/genética , Células HEK293 , Biossíntese de Proteínas , Ligação Proteica , Regulação da Expressão Gênica , TransativadoresRESUMO
BACKGROUND: Patients with glioblastoma (GBM) have a poor prognosis and limited treatment options. The mRNA decapping enzyme scavenger (DCPS) is a cap-hydrolyzing enzyme. The DCPS inhibitor RG3039 exhibited excellent central nervous system bioavailability in vivo and was safe and well tolerated in healthy volunteers in a phase 1 clinical trial. In this study, we investigated the expression of DCPS in GBM and the anti-tumor activity of RG3039 in various preclinical models of GBM. METHODS: DCPS expression was examined in human GBM and paired peritumoral tissues. Its prognostic role was evaluated together with clinicopathological characteristics of patients. The anti-GBM effect of RG3039 was determined using GBM cell lines, patient-derived organoids, and orthotopic mouse models. The therapeutic mechanisms of DCPS inhibition were explored. RESULTS: DCPS is overexpressed in GBM and is associated with poor survival of patients with GBM. The DCPS inhibitor RG3039 exhibited robust anti-GBM activities in GBM cell lines, patient-derived organoids and orthotopic mouse models, with drug exposure achievable in humans. Mechanistically, RG3039 downregulated STAT5B expression, thereby suppressing proliferation, survival and colony formation of GBM cells. CONCLUSIONS: DCPS is a promising target for GBM. Inhibition of DCPS with RG3039 at doses achievable in humans downregulates STAT5B expression and reduces proliferation, survival and colony formation of GBM cells. Given the excellent anti-cancer activity and central nervous system bioavailability in vivo and good tolerance in humans, RG3039 warrants further study as a potential GBM therapy.
Assuntos
Glioblastoma , Humanos , Glioblastoma/tratamento farmacológico , Glioblastoma/patologia , Glioblastoma/genética , Glioblastoma/metabolismo , Animais , Linhagem Celular Tumoral , Ensaios Antitumorais Modelo de Xenoenxerto , Endorribonucleases/metabolismo , Endorribonucleases/antagonistas & inibidores , Proliferação de Células/efeitos dos fármacos , Feminino , Masculino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Camundongos Nus , Camundongos , Organoides/efeitos dos fármacos , Organoides/metabolismo , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , Pessoa de Meia-IdadeRESUMO
BACKGROUND: Ischemia-induced cellular damage and stress responses significantly impact cellular viability and function. Icariin (ICA), known for its protective effects, has been studied to understand its role in mitigating oxygen-glucose deprivation/reperfusion (OGD/R)-induced endoplasmic reticulum (ER) stress and ferroptosis in H9C2 cardiomyoblast cells. METHODS: We employed an in vitro OGD/R model using H9C2 cells. ICA's effects were analyzed across multiple concentrations. Key indicators of ER stress, autophagy, and ferroptosis-including markers like Bip, PERK, IRE1, ATF6, P62, FTH1, LC3II/LC3I, and NCOA4-were assessed using Western blotting, electron microscopy, and biochemical assays. Additionally, the role of the IRE1/JNK pathway in mitochondrial dynamics and its influence on mitochondrial dynamics protein was explored through specific inhibition and activation experiments. RESULTS: ICA significantly reduced the activation of UPR pathways, decreased autophagic vacuole formation, and maintained cell viability in response to OGD/R and Erastin-induced ferroptosis. These protective effects were associated with modulated autophagic processes, reduced lipid peroxidation, and decreased ferrous ion accumulation. Inhibition of the IRE1/JNK pathway and subsequent Drp1 activity demonstrated reduced mitochondrial recruitment and mitophagy, correlating with decreased ferroptosis markers and improved cell survival. CONCLUSION: Our findings highlight ICA's potential in modulating IRE1/JNK pathway, autophagy, providing a therapeutic avenue for mitigating ferroptosis in myocardial ischemia-reperfusion injury (MIRI).
Assuntos
Estresse do Retículo Endoplasmático , Ferroptose , Flavonoides , Traumatismo por Reperfusão Miocárdica , Proteínas Serina-Treonina Quinases , Ferroptose/efeitos dos fármacos , Flavonoides/farmacologia , Animais , Ratos , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Traumatismo por Reperfusão Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/patologia , Traumatismo por Reperfusão Miocárdica/tratamento farmacológico , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Linhagem Celular , Autofagia/efeitos dos fármacos , Endorribonucleases/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Glucose/metabolismo , Glucose/deficiência , Complexos MultienzimáticosRESUMO
The cytokine tumor necrosis factor (TNF) plays important roles in limiting infection but is also linked to sepsis. The mechanisms underlying these paradoxical roles are unclear. Here, we show that TNF limits the antimicrobial activity of Paneth cells (PCs), causing bacterial translocation from the gut to various organs. This TNF-induced lethality does not occur in mice with a PC-specific deletion in the TNF receptor, P55. In PCs, TNF stimulates the IFN pathway and ablates the steady-state unfolded protein response (UPR), effects not observed in mice lacking P55 or IFNAR1. TNF triggers the transcriptional downregulation of IRE1 key genes Ern1 and Ern2, which are key mediators of the UPR. This UPR deficiency causes a significant reduction in antimicrobial peptide production and PC antimicrobial activity, causing bacterial translocation to organs and subsequent polymicrobial sepsis, organ failure, and death. This study highlights the roles of PCs in bacterial control and therapeutic targets for sepsis.
Assuntos
Translocação Bacteriana , Celulas de Paneth , Sepse , Transdução de Sinais , Fator de Necrose Tumoral alfa , Animais , Celulas de Paneth/metabolismo , Sepse/microbiologia , Camundongos , Fator de Necrose Tumoral alfa/metabolismo , Resposta a Proteínas não Dobradas , Camundongos Endogâmicos C57BL , Camundongos Knockout , Endorribonucleases/metabolismo , Endorribonucleases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Peptídeos Antimicrobianos/metabolismoRESUMO
BACKGROUND: Inositol-requiring enzyme 1 (IRE1) is an endoplasmic reticulum (ER)-resident transmembrane protein that senses ER stress and mediates an essential arm of the unfolded protein response (UPR). IRE1 reduces ER stress by upregulating the expression of multiple ER chaperones through activation of X-box-binding protein 1 (XBP1). Emerging lines of evidence have revealed that IRE1-XBP1 axis serves as a multipurpose signal transducer during oncogenic transformation and cancer development. In this study, we explore how IRE1-XBP1 signaling promotes chemoresistance in lung cancer. METHODS: The expression patterns of UPR components and MRP1 were examined by Western blot. qRT-PCR was employed to determine RNA expression. The promoter activity was determined by luciferase reporter assay. Chemoresistant cancer cells were analyzed by viability, apoptosis. CUT & Tag (Cleavage under targets and tagmentation)-qPCR analysis was used for analysis of DNA-protein interaction. RESULTS: Here we show that activation of IRE1α-XBP1 pathway leads to an increase in MDR-related protein 1 (MRP1) expression, which facilitates drug extrusion and confers resistance to cytotoxic chemotherapy. At the molecular level, XBP1-induced c-Myc is necessary for SREBP1 expression, and SREBP1 binds to the MRP1 promoter to directly regulate its transcription. CONCLUSIONS: We conclude that IRE1α-XBP1 had important role in chemoresistance and appears to be a novel prognostic marker for lung cancer.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Resistencia a Medicamentos Antineoplásicos , Endorribonucleases , Neoplasias Pulmonares , Proteínas Serina-Treonina Quinases , Proteína 1 de Ligação a X-Box , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Endorribonucleases/metabolismo , Endorribonucleases/genética , Proteína 1 de Ligação a X-Box/metabolismo , Proteína 1 de Ligação a X-Box/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 1/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Regulação Neoplásica da Expressão Gênica , Linhagem Celular Tumoral , ApoptoseRESUMO
Over the past five decades, DNA restriction enzymes have revolutionized biotechnology. While these enzymes are widely used in DNA research and DNA engineering, the emerging field of RNA and mRNA therapeutics requires sequence-specific RNA endoribonucleases. Here, we describe EcoToxN1, a member of the type III toxin-antitoxin family of sequence-specific RNA endoribonucleases, and its use in RNA and mRNA analysis. This enzyme recognizes a specific pentamer in a single-stranded RNA and cleaves the RNA within this sequence. The enzyme is neither dependent on annealing of guide RNA or DNA oligos to the template nor does it require magnesium. Furthermore, it performs over a wide range of temperatures. With its unique functions and characteristics, EcoToxN1 can be classified as an RNA restriction enzyme. EcoToxN1 enables new workflows in RNA analysis and biomanufacturing, meeting the demand for faster, cheaper, and more robust analysis methods.
Assuntos
Endorribonucleases , RNA , RNA/metabolismo , RNA/química , Endorribonucleases/metabolismo , Endorribonucleases/química , Toxinas Bacterianas/metabolismo , Toxinas Bacterianas/genética , Toxinas Bacterianas/química , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , Análise de Sequência de RNARESUMO
The nonsense-mediated mRNA decay (NMD) pathway clears eukaryotic cells of mRNAs containing premature termination codons (PTCs) or normal stop codons located in specific contexts. It therefore plays an important role in gene expression regulation. The precise molecular mechanism of the NMD pathway has long been considered to differ substantially from yeast to metazoa, despite the involvement of universally conserved factors such as the central ATP-dependent RNA-helicase Upf1. Here, we describe the crystal structure of the yeast Upf1 bound to its recently identified but yet uncharacterized partner Nmd4, show that Nmd4 stimulates Upf1 ATPase activity and that this interaction contributes to the elimination of NMD substrates. We also demonstrate that a region of Nmd4 critical for the interaction with Upf1 in yeast is conserved in the metazoan SMG6 protein, another major NMD factor. We show that this conserved region is involved in the interaction of SMG6 with UPF1 and that mutations in this region affect the levels of endogenous human NMD substrates. Our results support the universal conservation of the NMD mechanism in eukaryotes.
Assuntos
Degradação do RNAm Mediada por Códon sem Sentido , RNA Helicases , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , RNA Helicases/metabolismo , RNA Helicases/genética , Humanos , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , Transativadores/metabolismo , Transativadores/genética , Adenosina Trifosfatases/metabolismo , Adenosina Trifosfatases/genética , Ligação Proteica , Cristalografia por Raios X , EndorribonucleasesRESUMO
The catalytic domain of the calcium-dependent endoribonuclease EndoU from Homo sapiens was expressed in E. coli with 13C and 15N labeling. A nearly complete assignment of backbone 1H, 15N, and 13C resonances was obtained, as well as a secondary structure prediction based on the assigned chemical shifts. The predicted secondary structures were almost identical to the published crystal structure of calcium-activated EndoU. This is the first NMR study of an eukaryotic member of the EndoU-like superfamily of ribonucleases.
Assuntos
Cálcio , Endorribonucleases , Ressonância Magnética Nuclear Biomolecular , Endorribonucleases/química , Endorribonucleases/metabolismo , Humanos , Cálcio/metabolismo , Isótopos de Nitrogênio , Estrutura Secundária de Proteína , Sequência de AminoácidosRESUMO
The global health crisis caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) urges the development of new antiviral agents with broad coronavirus coverage. Due to its key role in viral evasion from the host innate immune response, the coronavirus Nsp15 uridine-specific endoribonuclease (EndoU) is of high interest as a drug target. Considering that the isatin scaffold is well-known for its versatile pharmacological properties, we synthesized and evaluated a series of compounds carrying an isatin core. The initial compounds were selected on the basis of in silico predictions. After biochemical assays showed moderate inhibition of SARS-CoV-2 EndoU-mediated RNA cleavage, structural analogues were rationally designed to enhance the interaction with the target. This included the incorporation of a nitrile group since this dipole can improve ADME and facilitate polar interactions with proteins and can operate as hydroxy or carboxy surrogate. A straightforward solvent free and green, microwave-assisted synthetic process was established to achieve the development of the different target compounds. The best compound exhibited inhibitory activity in enzymatic EndoU assays, and reduced the SARS-CoV-2 viral RNA load by almost 68,000-fold in the low micromolar range similarly to the established antiviral agent GS-441524.
Assuntos
Antivirais , Endorribonucleases , Isatina , SARS-CoV-2 , Proteínas não Estruturais Virais , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/enzimologia , Antivirais/farmacologia , Antivirais/química , Antivirais/síntese química , Isatina/farmacologia , Isatina/química , Isatina/análogos & derivados , Endorribonucleases/antagonistas & inibidores , Endorribonucleases/metabolismo , Humanos , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/metabolismo , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/síntese química , Relação Estrutura-Atividade , Tratamento Farmacológico da COVID-19 , Estrutura Molecular , Simulação de Acoplamento MolecularRESUMO
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
Pseudomonas aeruginosa is a widespread nosocomial pathogen with a significant to cause both severe planktonic acute and biofilm-related chronic infections. Small RNAs (sRNAs) are noncoding regulatory molecules that are stabilized by the RNA chaperone Hfq to trigger various virulence-related signaling pathways. Here, we identified an Hfq-binding sRNA in P. aeruginosa PAO1, PqsS, which promotes bacterial pathogenicity and pseudomonas quinolone signal quorum sensing (pqs QS) system. Specifically, PqsS enhanced acute bacterial infections by inducing host cell death and promoting rhamnolipid-regulated swarming motility. Meanwhile, PqsS reduced chronic infection traits including biofilm formation and antibiotic resistance. Moreover, PqsS repressed pqsL transcript, increasing PQS levels for pqs QS. A PQS-rich environment promoted PqsS expression, thus forming a positive feedback loop. Furthermore, we demonstrated that the PqsS interacts and destabilizes the pqsL mRNA by recruiting RNase E to drive degradation. These findings provide insights for future research on P. aeruginosa pathogenesis and targeted treatment.