RESUMO
About 20% of breast cancer patients are positive for HER2. The efficacy of current treatments is limited by primary and secondary resistance to trastuzumab. tRNA-derived fragments (tRFs) have shown crucial regulatory roles in various cancers. This study aimed to evaluate the role of tRF-27 in regulating the resistance of HER2-positive breast cancer against trastuzumab. tRF-27 was highly expressed in trastuzumab-resistant cells, and its expression level could predict the resistance to trastuzumab. High expression of tRF-27 promoted the growth and proliferation of trastuzumab-exposed cells. RNA-pulldown assay and mass spectrometry were performed to identify Ras GTPase-activating protein-binding proteins 1 and 2 (G3BPs) (two proteins targeted by tRF-27); RNA-immunoprecipitation (RIP) to confirm their bindings; co-immunoprecipitation (co-IP) and RNA-pulldown assay to determine the binding domains between G3BPs and tRF-27.tRF-27 bound to the nuclear transport factor 2 like domain(NTF2 domain) of G3BPs through a specific sequence. tRF-27 relied on G3BPs and NTF2 domain to increase trastuzumab tolerance. tRF-27 competed with lysosomal associated membrane protein 1(LAMP1) for NTF2 domain, thereby inhibiting lysosomal localization of G3BPs and tuberous sclerosis complex (TSC). Overexpression of tRF-27 inhibited phosphorylation of TSCs and promoted the activation of mechanistic target of rapamycin complex 1(MTORC1) to enhance cell proliferation and entice the resistance of HER2-positive breast cancer against trastuzumab.
Assuntos
Neoplasias da Mama , Alvo Mecanístico do Complexo 1 de Rapamicina , Trastuzumab , Humanos , Trastuzumab/farmacologia , Trastuzumab/uso terapêutico , Neoplasias da Mama/metabolismo , Neoplasias da Mama/tratamento farmacológico , Feminino , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos , Receptor ErbB-2/metabolismo , Animais , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , RNA de Transferência/metabolismo , Camundongos , RNA Helicases/metabolismo , Camundongos Nus , Proteínas com Motivo de Reconhecimento de RNA/metabolismoRESUMO
Coiled-coil domain-containing 124 protein is a multifunctional RNA-binding factor, and it was previously reported to interact with various biomolecular complexes localized at diverse subcellular locations, such as the ribosome, centrosome, midbody, and nucleoli. We aimed to better characterize the subcellular CCDC124 translocation by labelling this protein with a fluorescent tag, followed by laser scanning confocal microscopy methods. As traditional GFP-tagging of small proteins such as CCDC124 often faces limitations like potential structural perturbations of labeled proteins, and interference of the fluorescent-tag with their endogenous cellular functions, we aimed to label CCDC124 with the smallest possible split-GFP associated protein-tagging system (GFP11/GFP1-10) for better characterization of its subcellular localizations and its translocation dynamics. By recombinant DNA techniques we generated CCDC124-constructs labelled with either single of four tandem copies of GFP11 (GFP11 × 1::CCDC124, GFP11 × 4::CCDC124, or CCDC124::GFP11 × 4). We then cotransfected U2OS cells with these split-GFP constructs (GFP11 × 1(or X4)::CCDC124/GFP1-10) and analyzed subcellular localization of CCDC124 protein by laser scanning confocal microscopy. Tagging CCDC124 with four tandem copies of a 16-amino acid short GFP-derived peptide-tag (GFP11 × 4::CCDC124) allowed better characterization of the subcellular localization of CCDC124 protein in our model human bone osteosarcoma (U2OS) cells. Thus, by this novel methodology we successfully identified GFP11 × 4::CCDC124 molecules in G3BP1-overexpression induced stress-granules by live cell protein imaging for the first time. Our findings propose CCDC124 as a novel component of the stress granule which is a membraneless organelle involved in translational shut-down in response to cellular stress.
Assuntos
Grânulos Citoplasmáticos , Proteínas de Fluorescência Verde , Proteínas de Ligação a Poli-ADP-Ribose , Proteínas com Motivo de Reconhecimento de RNA , Humanos , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/genética , Proteínas com Motivo de Reconhecimento de RNA/química , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Fluorescência Verde/química , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/genética , Grânulos Citoplasmáticos/metabolismo , Grânulos Citoplasmáticos/química , Linhagem Celular Tumoral , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/química , Transporte Proteico , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/química , Microscopia Confocal/métodos , DNA Helicases , RNA HelicasesRESUMO
Stress triggers the formation of two distinct cytoplasmic biomolecular condensates: stress granules (SGs) and processing bodies (PBs), both of which may contribute to stress-responsive translation regulation. Though PBs can be present constitutively, stress can increase their number and size and lead to their interaction with stress-induced SGs. The mechanism of such interaction, however, is largely unknown. Formation of canonical SGs requires the RNA binding protein Ubiquitin-Associated Protein 2-Like (UBAP2L), which is a central SG node protein in the RNA-protein interaction network of SGs and PBs. UBAP2L binds to the essential SG and PB proteins G3BP and DDX6, respectively. Research on UBAP2L has mostly focused on its role in SGs, but not its connection to PBs. We find that UBAP2L is not solely an SG protein but also localizes to PBs in certain conditions, contributes to PB biogenesis and SG-PB interactions, and can nucleate hybrid granules containing SG and PB components in cells. These findings inform a new model for SG and PB formation in the context of UBAP2L's role.
Assuntos
RNA Helicases , Proteínas com Motivo de Reconhecimento de RNA , Grânulos de Estresse , Humanos , Grânulos de Estresse/metabolismo , Grânulos de Estresse/genética , RNA Helicases/metabolismo , RNA Helicases/genética , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , Corpos de Processamento/metabolismo , Corpos de Processamento/genética , RNA Helicases DEAD-box/metabolismo , RNA Helicases DEAD-box/genética , Grânulos Citoplasmáticos/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/genética , Células HeLa , DNA Helicases/metabolismo , DNA Helicases/genética , Células HEK293 , Ligação Proteica , Proteínas de Transporte/metabolismo , Proteínas de Transporte/genética , Proteínas Proto-OncogênicasRESUMO
Stress granules (SGs) are induced by various environmental stressors, resulting in their compositional and functional heterogeneity. SGs play a crucial role in the antiviral process, owing to their potent translational repressive effects and ability to trigger signal transduction; however, it is poorly understood how these antiviral SGs differ from SGs induced by other environmental stressors. Here we identify that TRIM25, a known driver of the ubiquitination-dependent antiviral innate immune response, is a potent and critical marker of the antiviral SGs. TRIM25 undergoes liquid-liquid phase separation (LLPS) and co-condenses with the SG core protein G3BP1 in a dsRNA-dependent manner. The co-condensation of TRIM25 and G3BP1 results in a significant enhancement of TRIM25's ubiquitination activity towards multiple antiviral proteins, which are mainly located in SGs. This co-condensation is critical in activating the RIG-I signaling pathway, thus restraining RNA virus infection. Our studies provide a conceptual framework for better understanding the heterogeneity of stress granule components and their response to distinct environmental stressors.
Assuntos
Infecções por Vírus de RNA , Grânulos de Estresse , Proteínas com Motivo Tripartido , Ubiquitina-Proteína Ligases , Humanos , Grânulos Citoplasmáticos/metabolismo , Proteína DEAD-box 58/metabolismo , DNA Helicases/metabolismo , Células HEK293 , Células HeLa , Imunidade Inata , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/genética , Receptores Imunológicos/metabolismo , RNA Helicases/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/genética , Infecções por Vírus de RNA/virologia , Infecções por Vírus de RNA/metabolismo , Infecções por Vírus de RNA/imunologia , RNA de Cadeia Dupla/metabolismo , Transdução de Sinais , Grânulos de Estresse/metabolismo , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Proteínas com Motivo Tripartido/metabolismo , Proteínas com Motivo Tripartido/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , UbiquitinaçãoRESUMO
Acute stress caused by short-term exposure to deleterious chemicals can induce the aggregation of RNA-binding proteins (RBPs) in the cytosol and the formation of stress granules (SGs). The cytoplasmic RBP, Ras GTPase-activating protein-binding protein 1 (G3BP1) is a critical organizer of SG, and its aggregation is considered a hallmark of cellular stress. However, assembly of SG is a highly dynamic process that involves RBPs; hence, existing methods based on fixation processes or overexpression of RBPs exhibit limited efficacy in detecting the assembly of SG under stress conditions. In this study, we established a G3BP1- Green fluorescent protein (GFP) reporter protein in a human neuroblastoma cell line to overcome these limitations. GFP was introduced into the G3BP1 genomic sequence via homologous recombination to generate a G3BP1-GFP fusion protein and further analyze the aggregation processes. We validated the assembly of SG under stress conditions using the G3BP1-GFP reporter system. Additionally, this system supported the evaluation of bisphenol A-induced SG response in the established human neuroblastoma cell line. In conclusion, the established G3BP1-GFP reporter system enables us to monitor the assembly of the SG complex in a human neuroblastoma cell line in real time and can serve as an efficient tool for assessing potential neurotoxicity associated with short-term exposure to chemicals.
Assuntos
DNA Helicases , Proteínas de Fluorescência Verde , Neuroblastoma , Proteínas de Ligação a Poli-ADP-Ribose , RNA Helicases , Proteínas com Motivo de Reconhecimento de RNA , Humanos , Proteínas com Motivo de Reconhecimento de RNA/genética , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/genética , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Linhagem Celular Tumoral , RNA Helicases/genética , RNA Helicases/metabolismo , Neuroblastoma/patologia , DNA Helicases/metabolismo , Grânulos de Estresse , Estresse Fisiológico , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genéticaRESUMO
Evidence suggests associations between COVID-19 patients or vaccines and glycometabolic dysfunction and an even higher risk of the occurrence of diabetes. Herein, we retrospectively analyzed pancreatic lesions in autopsy tissues from 67 SARS-CoV-2 infected non-human primates (NHPs) models and 121 vaccinated and infected NHPs from 2020 to 2023 and COVID-19 patients. Multi-label immunofluorescence revealed direct infection of both exocrine and endocrine pancreatic cells by the virus in NHPs and humans. Minor and limited phenotypic and histopathological changes were observed in adult models. Systemic proteomics and metabolomics results indicated metabolic disorders, mainly enriched in insulin resistance pathways, in infected adult NHPs, along with elevated fasting C-peptide and C-peptide/glucose ratio levels. Furthermore, in elder COVID-19 NHPs, SARS-CoV-2 infection causes loss of beta (ß) cells and lower expressed-insulin in situ characterized by islet amyloidosis and necrosis, activation of α-SMA and aggravated fibrosis consisting of lower collagen in serum, an increase of pancreatic inflammation and stress markers, ICAM-1 and G3BP1, along with more severe glycometabolic dysfunction. In contrast, vaccination maintained glucose homeostasis by activating insulin receptor α and insulin receptor ß. Overall, the cumulative risk of diabetes post-COVID-19 is closely tied to age, suggesting more attention should be paid to blood sugar management in elderly COVID-19 patients.
Assuntos
COVID-19 , Diabetes Mellitus , Adulto , Animais , Humanos , Idoso , SARS-CoV-2 , Receptor de Insulina , Peptídeo C , DNA Helicases , Estudos Retrospectivos , Proteínas de Ligação a Poli-ADP-Ribose , RNA Helicases , Proteínas com Motivo de Reconhecimento de RNA , GlucoseRESUMO
Aberrant expression of forkhead box transcription factor 1 (FOXM1) plays critical roles in a variety of human malignancies and predicts poor prognosis. However, little is known about the crosstalk between FOXM1 and long noncoding RNAs (lncRNAs) in tumorigenesis. The present study identifies a previously uncharacterized lncRNA XLOC_008672 in gastric cancer (GC), which is regulated by FOXM1 and possesses multiple copies of tandem repetitive sequences. LncRNA microarrays are used to screen differentially expressed lncRNAs in FOXM1 knockdown GC cells, and then the highest fold downregulation lncRNA XLOC_008672 is screened out. Sequence analysis reveals that the new lncRNA contains 62 copies of 37-bp tandem repeats. It is transcriptionally activated by FOXM1 and functions as a downstream effector of FOXM1 in GC cells through in vitro and in vivo functional assays. Elevated expression of XLOC_008672 is found in GC tissues and indicates worse prognosis. Mechanistically, XLOC_008672 can bind to small nuclear ribonucleoprotein polypeptide A (SNRPA), thereby enhancing mRNA stability of Ras-GTPase-activating protein SH3 domain-binding protein 1 (G3BP1) and, consequently, facilitating GC cell proliferation and migration. Our study discovers a new uncharacterized lncRNA XLOC_008672 involved in GC carcinogenesis and progression. Targeting FOXM1/XLOC_008672/SNRPA/G3BP1 signaling axis might be a promising therapeutic strategy for GC.
Assuntos
Carcinogênese , Proliferação de Células , Proteína Forkhead Box M1 , Regulação Neoplásica da Expressão Gênica , RNA Longo não Codificante , Neoplasias Gástricas , Animais , Feminino , Humanos , Masculino , Camundongos , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , DNA Helicases , Proteína Forkhead Box M1/genética , Proteína Forkhead Box M1/metabolismo , Camundongos Nus , Proteínas de Ligação a Poli-ADP-Ribose/genética , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Prognóstico , RNA Helicases , Proteínas com Motivo de Reconhecimento de RNA/genética , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologia , Neoplasias Gástricas/metabolismo , Sequências de Repetição em Tandem/genéticaRESUMO
Patients with concurrent intrahepatic cholangiocarcinoma (ICC) and hepatolithiasis generally have poor prognoses. Hepatolithiasis is once considered the primary cause of ICC, although recent insights indicate that bacteria in the occurrence of hepatolithiasis can promote the progression of ICC. By constructing in vitro and in vivo ICC models and patient-derived organoids (PDOs), it is shown that Escherichia coli induces the production of a novel RNA, circGLIS3 (cGLIS3), which promotes tumor growth. cGLIS3 binds to hnRNPA1 and G3BP1, resulting in the assembly of stress granules (SGs) and suppression of hnRNPA1 and G3BP1 ubiquitination. Consequently, the IKKα mRNA is blocked in SGs, decreasing the production of IKKα and activating the NF-κB pathway, which finally results in chemoresistance and produces metastatic phenotypes of ICC. This study shows that a combination of Icaritin (ICA) and gemcitabine plus cisplatin (GP) chemotherapy can be a promising treatment strategy for ICC.
Assuntos
Neoplasias dos Ductos Biliares , Colangiocarcinoma , Progressão da Doença , Escherichia coli , NF-kappa B , Grânulos de Estresse , Animais , Humanos , Camundongos , Neoplasias dos Ductos Biliares/metabolismo , Neoplasias dos Ductos Biliares/genética , Neoplasias dos Ductos Biliares/patologia , Colangiocarcinoma/metabolismo , Colangiocarcinoma/genética , Colangiocarcinoma/patologia , Modelos Animais de Doenças , DNA Helicases , Escherichia coli/genética , Escherichia coli/metabolismo , Gencitabina , NF-kappa B/metabolismo , NF-kappa B/genética , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/genética , RNA Helicases , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/genética , Transdução de Sinais/genética , Grânulos de Estresse/metabolismo , Grânulos de Estresse/genéticaRESUMO
Proper regulation of cellular response to environmental stress is crucial for maintaining biological homeostasis and is achieved by the balance between cell death processes, such as the formation of the pyroptosis-inducing NLRP3 inflammasome, and pro-survival processes, such as stress granule (SG) assembly. However, the functional interplay between these two stress-responsive organelles remains elusive. Here, we identified DHX33, a viral RNA sensor for the NLRP3 inflammasome, as a SG component, and the SG-nucleating protein G3BP as an NLRP3 inflammasome component. We also found that a decrease in intracellular potassium (K+) concentration, a key 'common' step in NLRP3 inflammasome activation, markedly inhibited SG assembly. Therefore, when macrophages are exposed to stress stimuli with the potential to induce both SGs and the NLRP3 inflammasome, such as cytoplasmic poly(I:C) stimulation, they preferentially form the NLRP3 inflammasome but avoid SG assembly by sequestering G3BP into the inflammasome and by inducing a reduction in intracellular K+ levels. Thus, under such conditions, DHX33 is primarily utilized as a viral RNA sensor for the inflammasome. Our data reveal the functional crosstalk between NLRP3 inflammasome-mediated pyroptosis and SG-mediated cell survival pathways and delineate a molecular mechanism that regulates cell-fate decisions and anti-viral innate immunity under stress.
Assuntos
Inflamassomos , Proteína 3 que Contém Domínio de Pirina da Família NLR , Grânulos de Estresse , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Inflamassomos/metabolismo , Humanos , Grânulos de Estresse/metabolismo , Camundongos , Animais , Potássio/metabolismo , RNA Helicases DEAD-box/metabolismo , RNA Helicases DEAD-box/genética , Piroptose , RNA Helicases/metabolismo , Macrófagos/metabolismo , Macrófagos/virologia , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Poli I-C/farmacologia , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/genética , DNA HelicasesRESUMO
Ras-GTPase-activating protein (GAP)-binding protein 1 (G3BP1) is an RNA-binding protein implicated in various malignancies. However, its role in nasopharyngeal carcinoma (NPC) remains elusive. This study elucidates the potential regulation mechanisms of G3BP1 and its significance in NPC advancement. Through knockdown and overexpression approaches, we validate G3BP1's oncogenic role by promoting proliferation, migration, and invasion in vitro and in vivo. Moreover, G3BP1 emerges as a key regulator of the JAK2/STAT3 signaling pathway, augmenting JAK2 expression via mRNA binding. Notably, epigallocatechin gallate (EGCG), a green tea-derived antioxidant, counteracts G3BP1-mediated pathway activation. Clinical analysis reveals heightened G3BP1, JAK2, and p-STAT3 as powerful prognostic markers, with G3BP1's expression standing as an independent indicator of poorer outcomes for NPC patients. In conclusion, the study unveils the oncogenic prowess of G3BP1, its orchestration of the JAK2/STAT3 signaling pathway, and its pivotal role in NPC progression.
Assuntos
DNA Helicases , Neoplasias Nasofaríngeas , Humanos , Carcinoma Nasofaríngeo/genética , DNA Helicases/metabolismo , RNA Helicases/genética , RNA Helicases/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Linhagem Celular Tumoral , Proteínas com Motivo de Reconhecimento de RNA/genética , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Transdução de Sinais/genética , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Neoplasias Nasofaríngeas/genética , Neoplasias Nasofaríngeas/patologia , Proliferação de Células/genética , Janus Quinase 2/genética , Janus Quinase 2/metabolismoRESUMO
BACKGROUND: Most patients with ovarian cancer (OC) treated with platinum-based chemotherapy have a dismal prognosis owing to drug resistance. However, the regulatory mechanisms of circular RNA (circRNA) and p53 ubiquitination are unknown in platinum-resistant OC. We aimed to identify circRNAs associated with platinum-resistant OC to develop a novel treatment strategy. METHODS: Platinum-resistant circRNAs were screened through circRNA sequencing and validated using quantitative reverse-transcription PCR in OC cells and tissues. The characteristics of circNUP50 were analysed using Sanger sequencing, oligo (dT) primers, ribonuclease R and fluorescence in situ hybridisation assays. Functional experimental studies were performed in vitro and in vivo. The mechanism underlying circNUP50-mediated P53 ubiquitination was investigated through circRNA pull-down analysis and mass spectrometry, luciferase reporters, RNA binding protein immunoprecipitation, immunofluorescence assays, cycloheximide chase assays, and ubiquitination experiments. Finally, a platinum and si-circNUP50 co-delivery nanosystem (Psc@DPP) was constructed to treat platinum-resistant OC in an orthotopic animal model. RESULTS: We found that circNUP50 contributes to platinum-resistant conditions in OC by promoting cell proliferation, affecting the cell cycle, and reducing apoptosis. The si-circNUP50 mRNA sequencing and circRNA pull-down analysis showed that circNUP50 mediates platinum resistance in OC by binding p53 and UBE2T, accelerating p53 ubiquitination. By contrast, miRNA sequencing and circRNA pull-down experiments indicated that circNUP50 could serve as a sponge for miR-197-3p, thereby upregulating G3BP1 to mediate p53 ubiquitination, promoting OC platinum resistance. Psc@DPP effectively overcame platinum resistance in an OC tumour model and provided a novel idea for treating platinum-resistant OC using si-circNUP50. CONCLUSIONS: This study reveals a novel molecular mechanism by which circNUP50 mediates platinum resistance in OC by modulating p53 ubiquitination and provides new insights for developing effective therapeutic strategies for platinum resistance in OC.
Assuntos
MicroRNAs , Neoplasias Ovarianas , Enzimas de Conjugação de Ubiquitina , Animais , Humanos , Feminino , Cisplatino/farmacologia , Cisplatino/uso terapêutico , MicroRNAs/metabolismo , RNA Circular/genética , RNA Circular/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , DNA Helicases/genética , DNA Helicases/metabolismo , Linhagem Celular Tumoral , Proteínas de Ligação a Poli-ADP-Ribose/genética , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , RNA Helicases/genética , RNA Helicases/metabolismo , RNA Helicases/uso terapêutico , Proteínas com Motivo de Reconhecimento de RNA/genética , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Ubiquitinação , Proliferação de Células , Resistencia a Medicamentos AntineoplásicosRESUMO
Stress granule formation is triggered by the release of mRNAs from polysomes and is promoted by the action of the RNA-binding proteins G3BP1/2. Stress granules have been implicated in several disease states, including cancer and neurodegeneration. Consequently, compounds that limit stress granule formation or promote their dissolution have potential as both experimental tools and novel therapeutics. Herein, we describe two small molecules, G3BP inhibitor a and b (G3Ia and G3Ib), designed to bind to a specific pocket in G3BP1/2 that is targeted by viral inhibitors of G3BP1/2 function. In addition to disrupting the co-condensation of RNA, G3BP1, and caprin 1 in vitro, these compounds inhibit stress granule formation in cells treated prior to or concurrent with stress and dissolve pre-existing stress granules. These effects are consistent across multiple cell types and a variety of initiating stressors. Thus, these compounds represent powerful tools to probe the biology of stress granules and hold promise for therapeutic interventions designed to modulate stress granule formation.
Assuntos
DNA Helicases , RNA Helicases , Grânulos de Estresse , DNA Helicases/genética , Proteínas de Ligação a Poli-ADP-Ribose/genética , RNA Helicases/genética , Proteínas com Motivo de Reconhecimento de RNA/genéticaRESUMO
Immune checkpoint inhibitors (ICIs) show promise as second-line treatment for advanced bladder cancer (BLCA); however, their responsiveness is limited by the immune evasion mechanisms in tumor cells. This study conduct a Cox regression analysis to screen mRNA-binding proteins and reveals an association between Ras GTPase-activating protein-binding protein 1 (G3BP1) and diminished effectiveness of ICI therapy in patients with advanced BLCA. Subsequent investigation demonstrates that G3BP1 enhances immune evasion in BLCA cells by downregulating major histocompatibility complex class I (MHC-I) through phosphoinositide 3-kinase (PI3K)/Akt signaling activation. Mechanistically, G3BP1 interacts with splicing factor synergistic lethal with U5 snRNA 7 (SLU7) to form a complex with poly(A)-binding protein cytoplasmic 1 and eukaryotic translation initiation factor 4 gamma 1. This complex stabilizes the closed-loop structure of the mRNAs of class IA PI3Ks and consequently facilitates their translation and stabilization, thereby activating PI3K/Akt signaling to downregulate MHC-I. Consistently, targeting G3BP1 with epigallocatechin gallate (EGCG) impedes immune evasion and sensitizes BLCA cells to anti-programmed cell death (PD)-1 antibodies in mice. Thus, G3BP1 and SLU7 collaboratively contribute to immune evasion in BLCA, indicating that EGCG is a precision therapeutic agent to enhance the effectiveness of anti-PD-1 therapy.
Assuntos
DNA Helicases , Neoplasias da Bexiga Urinária , Humanos , Animais , Camundongos , DNA Helicases/genética , DNA Helicases/metabolismo , RNA Helicases/genética , RNA Helicases/metabolismo , Fosfatidilinositol 3-Quinases , Proteínas de Ligação a Poli-ADP-Ribose/genética , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fosfatidilinositol 3-Quinase/metabolismo , Evasão da Resposta Imune , Proteínas com Motivo de Reconhecimento de RNA/genética , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Proteínas de Transporte/genética , Neoplasias da Bexiga Urinária/tratamento farmacológico , Fatores de Processamento de RNARESUMO
OBJECTIVE: Dynamic changes in ischemic pathology after stroke suggested a "critical window" of enhanced neuroplasticity immediately after stroke onset. Although physical exercise has long been considered a promising strategy of stroke rehabilitation, very early physical exercise may exacerbate brain injury. Since remote ischemic conditioning (RIC) promotes neuroprotection and neuroplasticity, the present study combined RIC with sequential exercise to establish a new rehabilitation strategy for a better rehabilitative outcome. METHODS: A total of 120 adult male Sprague-Dawley rats were used and divided into five groups: (1) sham, (2) stroke, (3) stroke with exercise, (4) stroke with RIC, and (5) stroke with RIC followed by exercise. Brain damage was evaluated by infarct volume, neurological deficit, cell death, and lactate dehydrogenase (LDH) activity. Long-term functional outcomes were determined by grid walk tests, rotarod tests, beam balance tests, forelimb placing tests, and the Morris water maze. Neuroplasticity was evaluated through measurements of both mRNA and protein levels of synaptogenesis (synaptophysin [SYN], post-synaptic density protein-95 [PSD-95], and brain-derived neurotrophic factor [BDNF]) and angiogenesis (vascular endothelial growth factor [VEGF], angiopoietin-1 [Ang-1], and angiopoietin-2 [Ang-2]). Inflammasome activation was measured by concentrations of interleukin-18 (IL-18) and IL-1ß detected by enzyme-linked immunosorbent assay (ELISA) kits, mRNA expressions of NLR pyrin domain containing 3 (NLRP3), apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain (ASC), IL-18 and IL-1ß, and protein quantities of NLRP3, ASC, cleaved-caspase-1, gasdermin D-N (GSDMD-N), and IL-18 and IL-1ß. Stress granules (SGs), including GTPase-activating protein-binding protein 1 (G3BP1), T cell-restricted intracellular antigen-1 (TIA1), and DEAD-box RNA helicase 3X (DDX3X) were evaluated at mRNA and protein levels. The interactions between DDX3X with NLRP3 or G3BP1 were determined by immunofluorescence and co-immunoprecipitation. RESULTS: Early RIC decreased infarct volumes, neurological deficits, cell death, and LDH activity at post-stroke Day 3 (p < 0.05). All treatment groups showed significant improvement in functional outcomes, including sensory, motor, and cognitive functions. RIC and exercise, as compared to RIC or physical exercise alone, had improved functional outcomes after stroke (p < 0.05), as well as synaptogenesis and angiogenesis (p < 0.05). RIC significantly reduced mRNA and protein expressions of NLRP3 (p < 0.05). SGs formation peaked at 0 h after ischemia, then progressively decreased until 24 h postreperfusion, which was reversed by RIC (p < 0.05). The assembly of SGs consumed DDX3X and then inhibited NLRP3 inflammasome activation. CONCLUSIONS: RIC followed by exercise induced a better rehabilitation in ischemic rats, while early RIC alleviated ischemia-reperfusion injury via stress-granule-mediated inhibition of NLRP3 inflammasome.
Assuntos
Lesões Encefálicas , Reabilitação do Acidente Vascular Cerebral , Acidente Vascular Cerebral , Humanos , Ratos , Masculino , Animais , Inflamassomos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Interleucina-18/metabolismo , Ratos Sprague-Dawley , DNA Helicases/metabolismo , Grânulos de Estresse , Fator A de Crescimento do Endotélio Vascular/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose , RNA Helicases/metabolismo , Proteínas com Motivo de Reconhecimento de RNA , Lesões Encefálicas/patologia , Infarto , RNA MensageiroRESUMO
CONTEXT.: Genetic profiling data of prostatic adenocarcinoma are derived from predominantly White patients. In African Americans, prostatic adenocarcinoma has a poorer prognosis, raising the possibility of distinct genetic alterations. OBJECTIVE.: To investigate the genomic alterations of prostatic adenocarcinoma metastatic to regional lymph nodes in African American patients, with an emphasis on SPOP mutation. DESIGN.: We retrospectively reviewed African American patients with pN1 prostatic adenocarcinoma managed with radical prostatectomy and lymph node dissection. Comprehensive molecular profiling was performed, and androgen receptor signaling scores were calculated. RESULTS.: Nineteen patients were included. The most frequent genetic alteration was SPOP mutations (5 of 17; 29.4% [95% CI: 10.3-56.0]). While most alterations were associated with a high androgen receptor signaling score, mutant SPOP was exclusively associated with a low median and interquartile range (IQR) androgen receptor signaling score (0.788 [IQR 0.765-0.791] versus 0.835 [IQR 0.828-0.842], P = .003). In mutant SPOP, mRNA expression of SPOP inhibitor G3BP1 and SPOP substrates showed a significantly decreased expression of AR (33.40 [IQR 28.45-36.30] versus 59.53 [IQR 53.10-72.83], P = .01), TRIM24 (3.95 [IQR 3.28-5.03] versus 9.80 [IQR 7.39-11.70], P = .008), and NCOA3 (15.19 [IQR 10.59-15.93] versus 21.88 [IQR 18.41-28.33], P = .046). CONCLUSIONS.: African American patients with metastatic prostate adenocarcinoma might have a higher prevalence of mutant SPOP (30%), compared to â¼10% in unselected cohorts with lower expressions of SPOP substrates. In our study, in patients with mutant SPOP, the mutation was associated with decreased SPOP substrate expression and androgen receptor signaling, raising concern for suboptimal efficacy of androgen deprivation therapy in this subset of patients.
Assuntos
Adenocarcinoma , Proteínas de Transporte , Neoplasias da Próstata , Humanos , Masculino , Adenocarcinoma/genética , Adenocarcinoma/patologia , Antagonistas de Androgênios , Negro ou Afro-Americano/genética , DNA Helicases , Linfonodos/patologia , Proteínas Nucleares/genética , Projetos Piloto , Proteínas de Ligação a Poli-ADP-Ribose , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Receptores Androgênicos/genética , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Estudos Retrospectivos , RNA Helicases/metabolismo , Proteínas com Motivo de Reconhecimento de RNARESUMO
BACKGROUND: Oral squamous cell carcinoma (OSCC), exhibiting high morbidity and malignancy, is the most common type of oral cancer. The abnormal expression of RNA-binding proteins (RBPs) plays important roles in the occurrence and progression of cancer. The objective of the present study was to establish a prognostic assessment model of RBPs and to evaluate the prognosis of OSCC patients. METHODS: Gene expression data in The Cancer Genome Atlas (TCGA) were analyzed by univariate Cox regression analysis model that established a novel nine RBPs, which were used to build a prognostic risk model. A multivariate Cox proportional regression model and the survival analysis were used to evaluate the prognostic risk model. Moreover, the receive operator curve (ROC) analysis was tested further the efficiency of prognostic risk model based on data from TCGA database and Gene Expression Omnibus (GEO). RESULTS: Nine RBPs' signatures (ACO1, G3BP1, NMD3, RNGTT, ZNF385A, SARS, CARS2, YARS and SMAD6) with prognostic value were identified in OSCC patients. Subsequently, the patients were further categorized into high-risk group and low-risk in the overall survival (OS) and disease-free survival (DFS), and external validation dataset. ROC analysis was significant for both the TCGA and GEO. Moreover, GSEA revealed that patients in the high-risk group significantly enriched in many critical pathways correlated with tumorigenesis than the low, including cell cycle, adheres junctions, oocyte meiosis, spliceosome, ERBB signaling pathway and ubiquitin-mediated proteolysis. CONCLUSIONS: Collectively, we developed and validated a novel robust nine RBPs for OSCC prognosis prediction. The nine RBPs could serve as an independent and reliable prognostic biomarker and guiding clinical therapy for OSCC patients.
Assuntos
Carcinoma de Células Escamosas , Neoplasias de Cabeça e Pescoço , Neoplasias Bucais , Humanos , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço , Neoplasias Bucais/genética , Prognóstico , DNA Helicases , Proteínas de Ligação a Poli-ADP-Ribose , RNA Helicases , Proteínas com Motivo de Reconhecimento de RNA , Proteínas de Ligação a RNA/genéticaRESUMO
BACKGROUND: Non-small cell lung cancer (NSCLC) is a prevalent and aggressive malignancy with limited therapeutic options. Despite advances in treatment, NSCLC remains a major cause of cancer-related death worldwide. Tumor heterogeneity and therapy resistance present challenges in achieving remission. Research is needed to provide molecular insights, identify new targets, and develop personalized therapies to improve outcomes. METHODS: The protein expression level and prognostic value of DHX38 in NSCLC were explored in public databases and NSCLC tissue microarrays. DHX38 knockdown and overexpression cell lines were established to evaluate the role of DHX38 in NSCLC. In vitro and in vivo functional experiments were conducted to assess proliferation and metastasis. To determine the underlying molecular mechanism of DHX38 in human NSCLC, proteins that interact with DHX38 were isolated by IP and identified by LC-MS. KEGG analysis of DHX38-interacting proteins revealed the molecular pathway of DHX38 in human NSCLC. Abnormal pathway activation was verified by Western blot analysis and immunohistochemical (IHC) staining. A molecule-specific inhibitor was further used to explore potential therapeutic targets for NSCLC. The pathway-related target that interacted with DHX38 was verified by co-immunoprecipitation(co-IP) experiments. In cell lines with stable DHX38 overexpression, the target protein was knocked down to explore its complementary effect on DHX38 overexpression-induced tumor promotion. RESULTS: The protein expression of DHX38 was increased in NSCLC, and patients with high DHX38 expression levels had a poor prognosis. In vitro and in vivo experiments showed that DHX38 promoted the proliferation, migration and invasion of human NSCLC cells. DHX38 overexpression caused abnormal activation of the MAPK pathway and promoted epithelial-mesenchymal transition (EMT) in tumours. SCH772984, a novel specific ERK1/2 inhibitor, significantly reduced the increases in cell proliferation, migration and invasion caused by DHX38 overexpression. The co-IP experiments confirmed that DHX38 interacted with the Ras GTPase-activating protein-binding protein G3BP1. DHX38 regulated the expression of G3BP1. Knocking down G3BP1 in cells with stable DHX38 overexpression prevented DHX38-induced tumor cell proliferation, migration and invasion. Silencing G3BP1 reversed the MAPK pathway activation and EMT induced by DHX38 overexpression. CONCLUSION: In NSCLC, DHX38 functions as a tumor promoter. DHX38 modulates G3BP1 expression, leading to the activation of the MAPK signaling pathway, thus promoting tumor cell proliferation, metastasis, and the progression of epithelial-mesenchymal transition (EMT) in non-small cell lung cancer.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Humanos , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Neoplasias Pulmonares/metabolismo , DNA Helicases/metabolismo , Transição Epitelial-Mesenquimal , Linhagem Celular Tumoral , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , RNA Helicases/metabolismo , Proliferação de Células , Movimento Celular , Regulação Neoplásica da Expressão Gênica , Fatores de Processamento de RNA/metabolismo , RNA Helicases DEAD-box/metabolismoAssuntos
Eosinofilia , Transtornos Mieloproliferativos , Neoplasias , Humanos , Receptor beta de Fator de Crescimento Derivado de Plaquetas/genética , DNA Helicases , Proteínas de Ligação a Poli-ADP-Ribose , RNA Helicases/uso terapêutico , Proteínas com Motivo de Reconhecimento de RNA , Transtornos Mieloproliferativos/diagnóstico , Transtornos Mieloproliferativos/tratamento farmacológico , Transtornos Mieloproliferativos/genética , Eosinofilia/diagnóstico , Eosinofilia/genética , Eosinofilia/tratamento farmacológico , Proteínas de Fusão Oncogênica/genéticaRESUMO
OBJECTIVE: Cells can produce stress granules (SGs) to protect itself from damage under stress. The cGAS-STING pathway is one of the important pattern recognition pathways in the natural immune system. This study was investigated whether human mesenchymal stem cells (hMSCs) could protect the liver by inducing M2 macrophages to produce SGs during acute drug induced liver injury (DILI) induced by acetaminophen (APAP). METHODS: After intragastric administration of APAP in vivo to induce DILI mice model, hMSCs were injected into the tail vein. The co-culture system of hMSCs and M2 macrophages was established in vitro. It was further use SGs inhibitor anisomicin to intervene M2 macrophages. The liver histopathology, liver function, reactive oxygen species (ROS) level, apoptosis pathway, endoplasmic reticulum stress (ERS) level, SGs markers (G3BP1/TIA-1), cGAS-STING pathway, TNF-α, IL-6, IL-1ß mRNA levels in liver tissue and M2 macrophages were observed. RESULTS: In vivo experiments, it showed that hMSCs could alleviate liver injury, inhibite the level of ROS, apoptosis and ERS, protect liver function in DILI mice. The mount of M2 was increased in the liver. hMSCs could also induce the production of SGs, inhibit the cGAS-STING pathway and reduce TNF-α, IL-6, IL-1ß mRNA expression. The results in vitro showed that hMSCs could induce the production of SGs in macrophages, inhibit the cGAS-STING pathway, promote the secretion of IL-4 and IL-13, and reduce TNF-α, IL-6, IL-1ß mRNA level in cells. In the process of IL-4 inducing M2 macrophage activation, anisomycin could inhibit the production of SGs, activate the cGAS-STING pathway, and promote the inflammatory factor TNF-α, IL-6, IL-1ß mRNA expression in cells. CONCLUSIONS: HMSCs had a protective effect on acute DILI in mice induced by APAP. Its mechanism might involve in activating M2 type macrophages, promoting the production of SGs, inhibiting the cGAS-STING pathway, and reducing the expression of pro-inflammatory factors in macrophages, to reduce hepatocytes damage.
Assuntos
Doença Hepática Induzida por Substâncias e Drogas , Células-Tronco Mesenquimais , Humanos , Camundongos , Animais , Fator de Necrose Tumoral alfa/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Acetaminofen/toxicidade , Acetaminofen/metabolismo , Interleucina-6/metabolismo , DNA Helicases/metabolismo , Interleucina-4/metabolismo , Grânulos de Estresse , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , RNA Helicases/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Macrófagos/metabolismo , Nucleotidiltransferases/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , RNA Mensageiro/metabolismo , Células-Tronco Mesenquimais/metabolismoRESUMO
BACKGROUND: Ras-GTPase-activating protein binding protein 1 (G3BP1) is an oncogenic factor, which highly expressed in a variety of cancers. In recent years, G3BP1 has been reported to promote the development of prostate cancer by inhibiting the degradation of AR through inhibiting SPOP. However, whether G3BP1 contributes in a similar manner to the abnormal accumulation of ERα, which is also an important target for hormone therapy, remains unknown. This article addresses this issue and explores potential mechanisms. METHODS: Bioinformatics tools were used for G3BP1 expression analysis, survival analysis, and clinical association analysis. Immunohistochemical staining was used to examine the correlation between G3BP1 and ERα in EC patients. In addition, western blot and co-immunoprecipitation were used to detect the half-life of G3BP1 and mutant, and the effect of G3BP1 and mutant on the ubiquitination and degradation of ERα mediated by SPOP. Then, the oncogenic functions of G3BP1 dependent on the SPOP/ERα axis were determined by CCK8 cell proliferation assay, colony formation assay and cell migration assay. Finally, we established the EC cells treated or untreated with fulvestrant, exploring the possibility of fulvestrant combined with the reduction of G3BP1 to improve the efficacy of fulvestrant. RESULTS: G3BP1 is abnormally high expressed and characterized by high-frequency mutation in EC. In addition, there is a positive correlation between G3BP1 protein and ERα protein. Mechanistically, both G3BP1 and mutant, the latter is displaying the longer half-life, competitively bind SPOP with ERα, thereby inhibiting SPOP-mediated ubiquitination and degradation of ERα. Functionally, G3BP1 and mutant promote the proliferation and migration of EC cells by regulating the G3BP1/SPOP/ERα axis. However, fulvestrant can reverse the cancer-promoting effects of G3BP1 and mutant. CONCLUSIONS: G3BP1 and its mutant positively regulate ERα signaling pathway by inhibiting SPOP-mediated ubiquitination and degradation of ERα, indicating the promising effect of fulvestrant on the suppression the occurrence and development of EC with high expressed G3BP1 and G3BP1 mutants. Video Abstract.