RESUMO
Vault ribonucleoprotein particles are naturally designed nanocages, widely found in the eukaryotic kingdom. Vaults consist of 78 copies of the major vault protein (MVP) that are organized in 2 symmetrical cup-shaped halves, of an approximate size of 70x40x40 nm, leaving a huge internal cavity which accommodates the vault poly(ADP-ribose) polymerase (vPARP), the telomerase-associated protein-1 (TEP1) and some small untranslated RNAs. Diverse hypotheses have been developed on possible functions of vaults, based on their unique capsular structure, their rapid movements and the distinct subcellular localization of the particles, implicating transport of cargo, but they are all pending confirmation. Vault particles also possess many attributes that can be exploited in nanobiotechnology, particularly in the creation of vehicles for the delivery of multiple molecular cargoes. Here we review what is known about the structure and dynamics of the vault complex and discuss a possible mechanism for the vault opening process. The recent findings in the characterization of the vaults in cells and in its natural microenvironment will be also discussed.
Assuntos
Partículas de Ribonucleoproteínas em Forma de Abóbada , Partículas de Ribonucleoproteínas em Forma de Abóbada/metabolismo , Partículas de Ribonucleoproteínas em Forma de Abóbada/química , Partículas de Ribonucleoproteínas em Forma de Abóbada/genética , Humanos , Animais , Poli(ADP-Ribose) Polimerases/metabolismo , Poli(ADP-Ribose) Polimerases/químicaRESUMO
As we previously revealed, major vault protein (MVP) is a virus-induced host factor, and its expression is crucial for innate immune responses. Nevertheless, the function of MVP in adaptive immunity is poorly known. Here, we demonstrate that Mvp knockout mice had attenuated antibody responses and reduced survival after rechallenge with homologous influenza A virus (IAV) relative to wild-type mice. Analysis of B cell populations showed that MVP promoted germinal center (GC) responses to develop optimal antiviral humoral immunity. Although MVP-deficient T cells and dendritic cells (DCs) were not intrinsically damaged, MVP promoted activating effector T cells and T follicular helper responses and regulated specific DC subsets. These findings suggest that MVP directs an effective adaptive immune response against IAV by directly engaging in GC reactions or indirectly augmenting cellular immunity via innate immune pathways.
Assuntos
Imunidade Adaptativa , Células Dendríticas , Imunidade Inata , Vírus da Influenza A , Camundongos Knockout , Infecções por Orthomyxoviridae , Partículas de Ribonucleoproteínas em Forma de Abóbada , Animais , Vírus da Influenza A/imunologia , Partículas de Ribonucleoproteínas em Forma de Abóbada/genética , Partículas de Ribonucleoproteínas em Forma de Abóbada/imunologia , Partículas de Ribonucleoproteínas em Forma de Abóbada/metabolismo , Camundongos , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/virologia , Células Dendríticas/imunologia , Camundongos Endogâmicos C57BL , Centro Germinativo/imunologia , Linfócitos B/imunologiaRESUMO
Background: Pancreatic adenocarcinoma (PAAD) is a formidable challenge in oncology research, with a complex pathogenesis that requires to be explored. Major Vault Protein (MVP) is the principal structural component of the vault complex, and its expression level is remarkably upregulated in various cancers. Extensive investigations have been conducted to explore the role of MVP in specific cancer contexts, yet the potential molecular mechanisms and biological functions of MVP in PAAD still remain considerably elusive. This study aims to explore the role of MVP as a novel immune-related biomarker in the pathogenesis and clinical treatment of PAAD. Methods: Gene expression data and clinical information were collected from TCGA, GTEx and GEO databases. Survival, prognostic and functional enrichment analysis were employed with R software. Immunological correlation analysis was performed using TIMER2.0, TIDE scores, TISIDB and TISCH. Epigenetic analysis was implemented by MethSurv, CPTAC, UALCAN, and cBioPortal. Drug analysis was conducted using Enrichr and CellMiner. Moreover, cellular experiments, like RNA interference, qRT-PCR, Western blot, cell cycle analysis, cell apoptosis analysis, colony formation assay, transwell assay, and wound healing assay, were performed for verifying the functional properties of MVP in the PAAD progression. Results: We demonstrated an abnormally upregulated expression of MVP in PAAD tissues, which notably correlated with an adverse prognosis in PAAD patients. Functional analysis suggested the conceivable involvement of MVP in immune modulation, and immunotherapy. Additionally, we identified genetic alterations, reduced promoter methylation, and heightened phosphorylation in MVP. We also clarified Suloctidil and Tetradioxin as the most notable potential drugs targeting MVP in PAAD. Moreover, our experimental observations consistently highlighted the significant impact of MVP deficiency on impeding PAAD cell proliferation, inhibiting cell migration, and accelerating cell apoptosis. Interestingly, a potential link between MVP and ERK or AKT pathways was displayed, which opens new avenues for further exploration of the molecular mechanisms of MVP-targeted therapies in PAAD. Conclusions: This study systematically describes MVP as an immune-related biomarker with remarkable potential for predicting the prognosis, tumor progression and immunotherapeutic efficacy in PAAD.
Assuntos
Adenocarcinoma , Biomarcadores Tumorais , Movimento Celular , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Neoplasias Pancreáticas , Partículas de Ribonucleoproteínas em Forma de Abóbada , Humanos , Neoplasias Pancreáticas/imunologia , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/metabolismo , Partículas de Ribonucleoproteínas em Forma de Abóbada/genética , Partículas de Ribonucleoproteínas em Forma de Abóbada/metabolismo , Adenocarcinoma/imunologia , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Linhagem Celular Tumoral , Prognóstico , ApoptoseRESUMO
Major Vault Protein (MVP) has emerged as a potential prognostic and immunological biomarker in various cancer types. This pan-cancer study aimed to investigate expression of MVP and its correlation with clinical outcomes and immune infiltration across diverse cancer types. We conducted an analysis of extensive transcriptomic and clinical data from publicly available databases. Our findings unveiled a significant association between MVP expression and cancer progression, with higher expression levels predicting poorer overall survival in multiple cancer types. Importantly, MVP expression demonstrated a close relationship with immune infiltration in the tumor microenvironment, showing that higher expression levels were associated with increased immune cell infiltration. We further validated expression of MVP and function in cancer cell lines A549 and AGS. These compelling results suggest that MVP holds promise as a valuable biomarker for prognostic assessment and the development of immunotherapeutic strategies across various cancer types. Consequently, targeting MVP may offer a compelling therapeutic approach in the treatment of human cancers.
Assuntos
Biomarcadores Tumorais , Neoplasias , Microambiente Tumoral , Partículas de Ribonucleoproteínas em Forma de Abóbada , Humanos , Neoplasias/imunologia , Neoplasias/mortalidade , Prognóstico , Microambiente Tumoral/imunologia , Biomarcadores Tumorais/metabolismo , Partículas de Ribonucleoproteínas em Forma de Abóbada/genética , Partículas de Ribonucleoproteínas em Forma de Abóbada/metabolismo , Regulação Neoplásica da Expressão Gênica , Linhagem Celular TumoralRESUMO
Cancer cells show significant dysregulation of genes expression, which may favor their survival in the tumor environment. In this study, the cellular vault's components MVP (major vault protein), TEP1 (telomerase-associated protein 1) and vPARP (vault poly(ADP-ribose) polymerase) were transiently or completely inhibited in U2OS cells (human bone osteosarcoma epithelial cells) to evaluate their impact on the cell proliferative and migratory capacity as well as on the development of their resistance to the drug vinorelbine. Comparative analysis of MVP protein expression level in normal colon tissue, primary colorectal tumor, and metastasis showed that the expression of this protein does not increase significantly in the primary tumor, but its expression increases in metastatic cells. Further comparative molecular analysis using the whole transcriptome microarrays for MVP-positive and MVP-negative cells showed that MVP is involved in regulating proliferation and migration of cancer cells. MVP may facilitate metastasis of colon cancer due to its impact on cell migration. Moreover, two vault proteins, MVP and TEP1, contribute the resistance to vinorelbine, while vPARP does not.
Assuntos
Neoplasias Colorretais/genética , Metástase Neoplásica/genética , Poli(ADP-Ribose) Polimerases/genética , Proteínas de Ligação a RNA/genética , Partículas de Ribonucleoproteínas em Forma de Abóbada/genética , Adulto , Idoso , Movimento Celular/genética , Proliferação de Células/genética , Neoplasias Colorretais/patologia , Intervalo Livre de Doença , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Masculino , Pessoa de Meia-Idade , Metástase Neoplásica/patologia , Transcriptoma/genéticaRESUMO
Porcine reproductive and respiratory syndrome (PRRS), a significant viral infectious disease that commonly occurs among farmed pigs, leads to considerable economic losses to the swine industry worldwide. Major vault protein (MVP) is a host factor that induces type â interferon (IFN) production. In this study, we evaluated the effect of MVP on PRRSV infection in CRL2843CD163 cell lines and porcine alveolar macrophages (PAMs). Our results showed that MVP expression was downregulated by PRRSV infection. Adenoviral overexpression of MVP inhibited PRRSV replication, whereas the siRNA knockdown of MVP promoted PRRSV replication. In addition, MVP knockdown has an adverse effect on the inhibitive role of MVP overexpression on PRRSV replication. Moreover, MVP could induce the expression of type â IFNs and IFN-stimulated gene 15 (ISG15) in PRRSV-infected PAMs. Based on these results, MVP may be a potential molecular target of drugs for the effective prevention and treatment of PRRSV infection.
Assuntos
Macrófagos Alveolares/virologia , Vírus da Síndrome Respiratória e Reprodutiva Suína/fisiologia , Partículas de Ribonucleoproteínas em Forma de Abóbada/metabolismo , Animais , Linhagem Celular , Interferon Tipo I/genética , Interferon Tipo I/metabolismo , Macrófagos Alveolares/metabolismo , Suínos , Partículas de Ribonucleoproteínas em Forma de Abóbada/genética , Replicação ViralRESUMO
Rationale: Bone homeostasis is maintained by a balanced interplay of osteoblasts and osteoclasts. Osteoclasts are derived from monocyte/macrophage lineage. Major vault protein (MVP) is known to promote apoptosis and prevent metabolic diseases in macrophage. However, whether MVP is involved in osteoclastogenesis is unknown. Here, we identified an important function of MVP as a negative regulator of osteoclastogenesis and its therapeutic potential in preventing bone loss. Methods: Expression of MVP in osteoclasts was investigated in human tumor tissues with immunohistochemical staining. Next, we generated total body (Mvp-/- ) and monocyte-specific (Mvpf/fLyz2-Cre) MVP gene knockout mice to observe bone phenotype and osteoclastogenesis using micro-CT and bone histomorphometry. Moreover, we examined the effects of MVP on osteoclast differentiation, bone resorption, NFATc1 activation and calcium oscillations in vitro. Finally, we explored the clinical potential of targeting MVP in two osteoporosis mouse models and used an adeno-associated virus (AAV) gene to overexpress MVP locally in mice. Results: We found that Mvp-/- and Mvpf/fLyz2-Cre mice both exhibited osteoporosis-like phenotypes. MVP-deficiency also enhanced calcineurin-NFATc1 signaling and promoted NFATc1 activity, which led to enhanced osteoclastogenesis and bone resorption. Calcineurin inhibition using the small molecule inhibitor FK506 corrected the enhanced osteoclastogenesis in Mvpf/fLyz2-Cre group. Additionally, MVP reexpression in Mvpf/fLyz2-Cre group rescued calcineurin expression. MVP overexpression in wild-type mice prevented pathologic bone loss in mouse models of ovariectomized (OVX) and calvaria-adjacent lipopolysaccharide (LPS)-injected. Conclusions: Our data suggested that MVP negatively regulates osteoclast differentiation and bone resorption via inhibition of calcineurin-NFATc1 signaling. In osteoclast-related bone diseases such as osteoporosis, manipulation of MVP activity may be an attractive therapeutic target.
Assuntos
Calcineurina/metabolismo , Diferenciação Celular , Fatores de Transcrição NFATC/metabolismo , Osteoclastos/metabolismo , Transdução de Sinais , Partículas de Ribonucleoproteínas em Forma de Abóbada/metabolismo , Animais , Reabsorção Óssea/genética , Reabsorção Óssea/metabolismo , Reabsorção Óssea/patologia , Calcineurina/genética , Humanos , Camundongos , Camundongos Knockout , Fatores de Transcrição NFATC/genética , Partículas de Ribonucleoproteínas em Forma de Abóbada/genéticaRESUMO
Hedgehog signaling plays a pivotal role in embryonic pattern formation and diverse aspects of the postnatal biological process. Perturbation of the hedgehog pathway and overexpression of GLI1, a downstream transcription factor in the hedgehog pathway, are highly relevant to several malignancies including chondrosarcoma (CS). We previously found that knocking down expression of GLI1 attenuates the disrupted Indian hedgehog (IHH) signal pathway and suppresses cell survival in human CS cells. However, the underlying mechanisms regulating the expression of GLI1 are still unknown. Here, we demonstrated the implication of GLI1 in SMO-independent pathways in CS cells. A GLI1 binding protein, major vault protein (MVP), was identified using the affinity purification method. MVP promoted the nuclear transport and stabilization of GLI1 by compromising the binding affinity of GLI1 with suppressor of fused homolog (SUFU) and increased GLI1 expression via mTOR/S6K1 signaling cascade. Functionally, knockdown of MVP suppressed cell growth and induced apoptosis. Simultaneous inhibition of MVP and GLI1 strongly inhibits the growth of CS in vitro and in vivo. Moreover, IHC results showed that MVP, GLI1, and P-p70S6K1 were highly expressed and positively correlated with each other in 71 human CS tissues. Overall, our findings revealed a novel regulating mechanism for HH-independent GLI1 expression and provide a rationale for combination therapy in patients with advanced CS.
Assuntos
Neoplasias Ósseas/metabolismo , Condrossarcoma/metabolismo , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Partículas de Ribonucleoproteínas em Forma de Abóbada/metabolismo , Proteína GLI1 em Dedos de Zinco/metabolismo , Animais , Neoplasias Ósseas/genética , Neoplasias Ósseas/patologia , Linhagem Celular Tumoral , Proliferação de Células , Condrossarcoma/genética , Condrossarcoma/patologia , Feminino , Regulação Neoplásica da Expressão Gênica , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Partículas de Ribonucleoproteínas em Forma de Abóbada/genética , Proteína GLI1 em Dedos de Zinco/genéticaRESUMO
The demographic shift toward an older population will increase the number of prostate cancer cases. A challenge in the treatment of prostate cancer is to avoid undertreatment of patients at high risk of progression following curative treatment. These men can benefit from early salvage treatment. An explorative cohort consisting of tissue from 16 patients who underwent radical prostatectomy, and were either alive or had died from prostate cancer within 10 years postsurgery, was analyzed by mass spectrometry analysis. Following proteomic and bioinformatic analyses, major vault protein (MVP) was identified as a putative prognostic biomarker. A publicly available tissue proteomics dataset and a retrospective cohort of 368 prostate cancer patients were used for validation. The prognostic value of the MVP was verified by scoring immunohistochemical staining of a tissue microarray. High level of MVP was associated with more than 4-fold higher risk for death from prostate cancer (hazard ratio = 4.41, 95% confidence interval: 1.45-13.38; P = 0.009) in a Cox proportional hazard models, adjusted for Cancer of the Prostate Risk Assessments Post-surgical (CAPRA-S) score and perineural invasion. Decision curve analyses suggested an improved standardized net benefit, ranging from 0.06 to 0.18, of adding MVP onto CAPRA-S score. This observation was confirmed by receiver operator characteristics curve analyses for the CAPRA-S score versus CAPRA-S and MVP score (area under the curve: 0.58 versus 0.73). From these analyses, one can infer that MVP levels in combination with CAPRA-S score might add onto established risk parameters to identify patients with lethal prostate cancer.
Assuntos
Neoplasias da Próstata/genética , Proteômica , Partículas de Ribonucleoproteínas em Forma de Abóbada/genética , Biomarcadores Tumorais/genética , Evolução Fatal , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Masculino , Neoplasias da Próstata/diagnóstico , Neoplasias da Próstata/mortalidade , Neoplasias da Próstata/patologiaRESUMO
Lipid rafts, also known as microdomains, are important components of cell membranes and are enriched in cholesterol, glycophospholipids and receptors. They are involved in various essential cellular processes, including endocytosis, exocytosis and cellular signaling. Receptors are concentrated at lipid rafts, through which cellular signaling can be transmitted. Pathogens exploit these signaling mechanisms to enter cells, proliferate and egress. However, lipid rafts also play an important role in initiating antimicrobial responses by sensing pathogens via clustered pathogen-sensing receptors and triggering downstream signaling events such as programmed cell death or cytokine production for pathogen clearance. In this review, we discuss how both host and pathogens use lipid rafts and associated proteins in an arms race to survive. Special attention is given to the involvement of the major vault protein, the main constituent of a ribonucleoprotein complex, which is enriched in lipid rafts upon infection with vaccinia virus.
Assuntos
Suscetibilidade a Doenças , Interações Hospedeiro-Patógeno , Microdomínios da Membrana/metabolismo , Animais , Apoptose , Citocinas/biossíntese , Endocitose , Regulação da Expressão Gênica , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Humanos , Imunomodulação , Microdomínios da Membrana/efeitos dos fármacos , Oxirredução , Ligação Proteica , Receptores de Reconhecimento de Padrão/metabolismo , Transdução de Sinais , Partículas de Ribonucleoproteínas em Forma de Abóbada/genética , Partículas de Ribonucleoproteínas em Forma de Abóbada/metabolismo , Internalização do VírusAssuntos
Antineoplásicos/uso terapêutico , Transtorno Autístico/genética , Transtornos Cromossômicos/genética , Histona-Lisina N-Metiltransferase/genética , Deficiência Intelectual/genética , Leucemia Mieloide Aguda/tratamento farmacológico , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína de Leucina Linfoide-Mieloide/genética , Partículas de Ribonucleoproteínas em Forma de Abóbada/genética , Criança , Deleção Cromossômica , Cromossomos Humanos Par 16/genética , Citarabina/uso terapêutico , Daunorrubicina/uso terapêutico , Etoposídeo/uso terapêutico , Feminino , Transplante de Células-Tronco Hematopoéticas , Humanos , Leucemia Mieloide Aguda/genéticaRESUMO
Streptomyces Sp FJS31-2 is a strain isolated from special habitat soils in the early stage of our laboratory for producing a new type of halogenated type II polyketide antibiotic with good anti-MRSA activity. In this experiment, a variety of chromatographic and spectroscopic methods was used to isolate and identify a milbemycin compound VM48130 from the ethyl acetate extract of the fermentation products. To investigate its bioactivity, Cell Counting Kit-8 (CCK-8) assay was used to test the cytotoxic activity of the compound against a variety of cancer cells (human liver cancer cell line MHCC97H and SK-Hep1, human nasopharyngeal carcinoma cell line CNE1, mouse melanoma cell line B16, human colon cancer cell line LOVO, human lung adenocarcinoma cell line A549) and normal cells (human bronchial epithelial cell line 16HBE, human normal liver cell line L02, human nasopharyngeal epithelial cell line NP69). The results showed that the compound had significant cytotoxic activity against the above cancer cells, and the IC50 values were 21.96 ± 1.45, 22.18 ± 0.55, 19.42 ± 0.71, 18.61 ± 1.68, 18.62 ± 0.67, 18.52 ± 0.64 µM, respectively. Furthermore, the CCK-8 method was used to evaluate the compound's reversal of cisplatin resistance in multidrug resistant cisplatin-resistant human lung adenocarcinoma (A549/DDP) cells. The results indicated that when the compound concentration was 0.5 µM, the reversal fold (RF) reached 6.25 and showed a dose-dependent effect. At 5 µM, the RF reached 8.35, which was approximately equivalent to the reversal effect of the positive drug verapamil at the same concentration. The expression of MDR1, MRP1, LRP, MAST1 resistance genes and the corresponding proteins were analyzed by quantitative RT-PCR and Western blot assay, and found that the compound could significantly down-regulate the expression of these genes and proteins. These results indicated that VM48130 had the potential of being a lead compound for the treatment or adjuvant treatment of cancer.
Assuntos
Adenocarcinoma de Pulmão/tratamento farmacológico , Antineoplásicos/farmacologia , Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Neoplasias Pulmonares/tratamento farmacológico , Macrolídeos/farmacologia , Streptomyces , Células A549 , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/metabolismo , Adenocarcinoma de Pulmão/patologia , Antineoplásicos/isolamento & purificação , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Concentração Inibidora 50 , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Macrolídeos/isolamento & purificação , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Streptomyces/química , Partículas de Ribonucleoproteínas em Forma de Abóbada/genética , Partículas de Ribonucleoproteínas em Forma de Abóbada/metabolismoRESUMO
The vault ribonucleoprotein (RNP), comprising vault RNA (vtRNA) and telomerase-associated protein 1 (TEP1), is found in many eukaryotes. However, previous studies of vtRNAs, for example in mammalian cells, have failed to reach a definitive conclusion about their function. vtRNAs are related to Y RNAs, which are complexed with Ro protein and influence Ro's function in noncoding RNA (ncRNA) quality control and processing. In Trypanosoma brucei, the small noncoding TBsRNA-10 was first described in a survey of the ncRNA repertoire in this organism. Here, we report that TBsRNA-10 in T. brucei is a vtRNA, based on its association with TEP1 and sequence similarity to those of other known and predicted vtRNAs. We observed that like vtRNAs in other species, TBsRNA-10 is transcribed by RNA polymerase III, which in trypanosomes also generates the spliceosomal U-rich small nuclear RNAs. In T. brucei, spliced leader (SL)-mediated trans-splicing of pre-mRNAs is an obligatory step in gene expression, and we found here that T. brucei's vtRNA is highly enriched in a non-nucleolar locus in the cell nucleus implicated in SL RNP biogenesis. Using a newly developed permeabilized cell system for the bloodstream form of T. brucei, we show that down-regulated vtRNA levels impair trans-spliced mRNA production, consistent with a role of vtRNA in trypanosome mRNA metabolism. Our results suggest a common theme for the functions of vtRNAs and Y RNAs. We conclude that by complexing with their protein-binding partners TEP1 and Ro, respectively, these two RNA species modulate the metabolism of various RNA classes.
Assuntos
Proteínas de Protozoários/genética , RNA de Protozoário/genética , Trans-Splicing/genética , Trypanosoma brucei brucei/genética , Partículas de Ribonucleoproteínas em Forma de Abóbada/genética , Pareamento de Bases/genética , Sequência de Bases , Nucléolo Celular/metabolismo , Sequência Conservada/genética , DNA Polimerase III/metabolismo , Proteínas de Protozoários/metabolismo , Interferência de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA de Protozoário/química , Transcrição GênicaRESUMO
The presence and absence of RNA modifications regulates RNA metabolism by modulating the binding of writer, reader, and eraser proteins. For 5-methylcytosine (m5C) however, it is largely unknown how it recruits or repels RNA-binding proteins. Here, we decipher the consequences of m5C deposition into the abundant non-coding vault RNA VTRNA1.1. Methylation of cytosine 69 in VTRNA1.1 occurs frequently in human cells, is exclusively mediated by NSUN2, and determines the processing of VTRNA1.1 into small-vault RNAs (svRNAs). We identify the serine/arginine rich splicing factor 2 (SRSF2) as a novel VTRNA1.1-binding protein that counteracts VTRNA1.1 processing by binding the non-methylated form with higher affinity. Both NSUN2 and SRSF2 orchestrate the production of distinct svRNAs. Finally, we discover a functional role of svRNAs in regulating the epidermal differentiation programme. Thus, our data reveal a direct role for m5C in the processing of VTRNA1.1 that involves SRSF2 and is crucial for efficient cellular differentiation.
Assuntos
5-Metilcitosina/metabolismo , Metilação de DNA , Células Epidérmicas/citologia , Metiltransferases/metabolismo , RNA/metabolismo , Partículas de Ribonucleoproteínas em Forma de Abóbada/genética , Diferenciação Celular , Linhagem Celular , Citosina/metabolismo , Células Epidérmicas/metabolismo , Células HEK293 , Células HeLa , Células-Tronco Embrionárias Humanas/citologia , Humanos , Metiltransferases/genética , RNA/genética , Partículas de Ribonucleoproteínas em Forma de Abóbada/metabolismoRESUMO
BACKGROUND: Major vault protein (MVP) is the major component of vault, a eukaryotic organelle involved in multiple cellular processes, and is important in multiple cellular processes and diseases including the drug resistance in cancer chemotherapies. However, the role of MVP in lung cancer remains unclear. METHODS: We examined MVP expression in 120 non-small cell lung cancer (NSCLC) tumors and matched normal tissues by immunohistochemistry. Its relationship with NSCLC prognosis was determined by investigating the patient cohort and analyzing the data from a published dataset consisting with more than 1900 lung cancer patients. We further performed shRNA-introduced knockdown of MVP in Lewis lung carcinoma (LLC) cells and examined its effects on the tumor formation in a xenograft mouse model and the tumor cell proliferation, apoptosis, and signal transduction in vitro. RESULTS: We found that MVP was up-regulated significantly in tumor tissues compared with the matched tumor-adjacent normal tissues. The increased expression of MVP in lung adenocarcinoma was associated with a better prognosis. Knockdown of MVP in LLC cells promoted xenografted lung cancer formation in mice, which was accompanied with accelerated tumor cell proliferation and suppressed cell apoptosis in vitro. Knockdown of MVP stimulated STAT3 phosphorylation, nuclear localization, and activation of JAK2 and RAF/MEK/ERK pathways in LLC cells. Administration of STAT3 inhibitor WP1066 could prevent MVP knockdown induced tumorigenesis. CONCLUSIONS: Our findings demonstrate that MVP may act as a lung tumor suppressor via inhibiting STAT3 pathway. MVP would be a potential target for novel therapies of lung adenocarcinoma.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/patologia , Neoplasias Pulmonares/patologia , Fator de Transcrição STAT3/metabolismo , Regulação para Cima , Partículas de Ribonucleoproteínas em Forma de Abóbada/metabolismo , Idoso , Animais , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Proliferação de Células , Sobrevivência Celular , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Masculino , Camundongos , Pessoa de Meia-Idade , Transplante de Neoplasias , Fosforilação , Prognóstico , Transdução de Sinais , Análise de Sobrevida , Partículas de Ribonucleoproteínas em Forma de Abóbada/genéticaRESUMO
Macrophage-orchestrated, low-grade chronic inflammation plays a pivotal role in obesity and atherogenesis. However, the underlying regulatory mechanisms remain incompletely understood. Here, we identify major vault protein (MVP), the main component of unique cellular ribonucleoprotein particles, as a suppressor for NF-κB signaling in macrophages. Both global and myeloid-specific MVP gene knockout aggravates high-fat diet induced obesity, insulin resistance, hepatic steatosis and atherosclerosis in mice. The exacerbated metabolic disorders caused by MVP deficiency are accompanied with increased macrophage infiltration and heightened inflammatory responses in the microenvironments. In vitro studies reveal that MVP interacts with TRAF6 preventing its recruitment to IRAK1 and subsequent oligomerization and ubiquitination. Overexpression of MVP and its α-helical domain inhibits the activity of TRAF6 and suppresses macrophage inflammation. Our results demonstrate that macrophage MVP constitutes a key constraint of NF-κB signaling thereby suppressing metabolic diseases.
Assuntos
Aterosclerose/imunologia , Fígado Gorduroso/imunologia , Inflamação/imunologia , Macrófagos/imunologia , Obesidade/imunologia , Partículas de Ribonucleoproteínas em Forma de Abóbada/metabolismo , Tecido Adiposo/patologia , Animais , Aterosclerose/etiologia , Aterosclerose/metabolismo , Biópsia , Células da Medula Óssea , Dieta Hiperlipídica/efeitos adversos , Modelos Animais de Doenças , Fígado Gorduroso/etiologia , Fígado Gorduroso/metabolismo , Feminino , Técnicas de Inativação de Genes , Humanos , Quinase I-kappa B/metabolismo , Inflamação/etiologia , Quinases Associadas a Receptores de Interleucina-1/metabolismo , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout para ApoE , NF-kappa B/metabolismo , Obesidade/etiologia , Obesidade/metabolismo , Obesidade/patologia , Cultura Primária de Células , Transdução de Sinais/imunologia , Fator 6 Associado a Receptor de TNF/metabolismo , Ubiquitinação , Partículas de Ribonucleoproteínas em Forma de Abóbada/genética , Partículas de Ribonucleoproteínas em Forma de Abóbada/imunologiaRESUMO
Vault RNAs (vtRNA) are small non-coding RNAs transcribed by RNA polymerase III found in many eukaryotes. Although they have been linked to drug resistance, apoptosis, and viral replication, their molecular functions remain unclear. Here, we show that vault RNAs directly bind the autophagy receptor sequestosome-1/p62 in human and murine cells. Overexpression of human vtRNA1-1 inhibits, while its antisense LNA-mediated knockdown enhances p62-dependent autophagy. Starvation of cells reduces the steady-state and p62-bound levels of vault RNA1-1 and induces autophagy. Mechanistically, p62 mutants that fail to bind vtRNAs display increased p62 homo-oligomerization and augmented interaction with autophagic effectors. Thus, vtRNA1-1 directly regulates selective autophagy by binding p62 and interference with oligomerization, a critical step of p62 function. Our data uncover a striking example of the potential of RNA to control protein functions directly, as previously recognized for protein-protein interactions and post-translational modifications.
Assuntos
Autofagia/genética , Partículas de Ribonucleoproteínas em Forma de Abóbada/genética , Partículas de Ribonucleoproteínas em Forma de Abóbada/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Linhagem Celular , Células HeLa , Humanos , Camundongos , Células RAW 264.7 , RNA/metabolismo , RNA não Traduzido/metabolismo , RNA não Traduzido/fisiologia , Proteína Sequestossoma-1/genética , Proteína Sequestossoma-1/metabolismoRESUMO
INTRODUCTION: Clinical studies suggest that obesity, in addition to promoting breast cancer aggressiveness, is associated with a decrease in chemotherapy efficacy, although the mechanisms involved remain elusive. As chemotherapy is one of the main treatments for aggressive or metastatic breast cancer, we investigated whether adipocytes can mediate resistance to doxorubicin (DOX), one of the main drugs used to treat breast cancer, and the mechanisms associated. METHODS: We used a coculture system to grow breast cancer cells with in vitro differentiated adipocytes as well as primary mammary adipocytes isolated from lean and obese patients. Drug cellular accumulation, distribution, and efflux were studied by immunofluorescence, flow cytometry, and analysis of extracellular vesicles. Results were validated by immunohistochemistry in a series of lean and obese patients with cancer. RESULTS: Adipocytes differentiated in vitro promote DOX resistance (with cross-resistance to paclitaxel and 5-fluorouracil) in a large panel of human and murine breast cancer cell lines independently of their subtype. Subcellular distribution of DOX was altered in cocultivated cells with decreased nuclear accumulation of the drug associated with a localized accumulation in cytoplasmic vesicles, which then are expelled into the extracellular medium. The transport-associated major vault protein (MVP), whose expression was upregulated by adipocytes, mediated both processes. Coculture with human mammary adipocytes also induced chemoresistance in breast cancer cells (as well as the related MVP-induced DOX efflux) and their effect was amplified by obesity. Finally, in a series of human breast tumors, we observed a gradient of MVP expression, which was higher at the invasive front, where tumor cells are at close proximity to adipocytes, than in the tumor center, highlighting the clinical relevance of our results. High expression of MVP in these tumor cells is of particular interest since they are more likely to disseminate to give rise to chemoresistant metastases. CONCLUSIONS: Collectively, our study shows that adipocytes induce an MVP-related multidrug-resistant phenotype in breast cancer cells, which could contribute to obesity-related chemoresistance.
Assuntos
Adipócitos/metabolismo , Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Obesidade/complicações , Partículas de Ribonucleoproteínas em Forma de Abóbada/metabolismo , Células 3T3 , Tecido Adiposo/citologia , Adulto , Idoso , Animais , Antineoplásicos/uso terapêutico , Mama/citologia , Mama/patologia , Mama/cirurgia , Neoplasias da Mama/patologia , Neoplasias da Mama/cirurgia , Linhagem Celular Tumoral , Técnicas de Cocultura , Doxorrubicina/farmacologia , Doxorrubicina/uso terapêutico , Resistência a Múltiplos Medicamentos , Resistencia a Medicamentos Antineoplásicos , Feminino , Fluoruracila/farmacologia , Fluoruracila/uso terapêutico , Humanos , Mastectomia , Camundongos , Pessoa de Meia-Idade , Paclitaxel/farmacologia , Paclitaxel/uso terapêutico , Cultura Primária de Células , RNA Interferente Pequeno/metabolismo , Partículas de Ribonucleoproteínas em Forma de Abóbada/genéticaRESUMO
IFN production is crucial for hosts to defend against viral infection, yet it must be tightly controlled to maintain immune homeostasis. TANK-binding kinase 1 (TBK1) is a pivotal kinase in the IFN induction signaling pathway, but it is negatively regulated by multiple molecules to avoid the excessive expression of IFN in mammals. However, the identified TBK1 suppressors and the mechanisms are rare in fish. In this study, we show that zebrafish major vault protein (MVP) recruits and degrades TBK1 in a lysosome-dependent manner to inhibit IFN production. Through viral infection, polyinosinic:polycytidylic acid and RIG-I-like receptor factor stimulation upregulated IFN expression, but overexpression of MVP significantly subverted these inductions. On the protein level, MVP interacted with TBK1, and interestingly, MVP recruited TBK1 from a uniformly distributed state in the cytoplasm to an aggregated state. Finally, MVP mediated the lysosome-dependent degradation of TBK1 and decreased the IFN response and IFN-stimulated genes expression. Our findings reveal that zebrafish MVP is a negative regulator of IFN production by restricting the activation of TBK1, supplying evidence of the balanced mechanisms of IFN expression in lower vertebrates.
Assuntos
Proteínas de Peixes/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Partículas de Ribonucleoproteínas em Forma de Abóbada/metabolismo , Viroses/imunologia , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/imunologia , Animais , Animais Geneticamente Modificados , Proteínas de Peixes/genética , Regulação da Expressão Gênica , Células HEK293 , Humanos , Interferons/genética , Lisossomos/metabolismo , Poli I-C/imunologia , Agregação Patológica de Proteínas , Ligação Proteica , Proteólise , Transdução de Sinais , Partículas de Ribonucleoproteínas em Forma de Abóbada/genéticaRESUMO
Resistance to chemotherapy remains a significant problem in the treatment of breast cancer, especially for triple-negative breast cancer (TNBC), in which standard systemic therapy is currently limited to chemotherapeutic agents. Our study aimed to better understand the molecular mechanisms that lead to failure of chemotherapy in TNBC. Herein, we observed elevated expression of Notch1 and major vault protein (MVP) in MDA-MB-231DDPR cells compared to their parental counterparts. We demonstrated that Notch1 could positively regulate the expression of MVP. Also, Notch1 intracellular domain (ICD) was capable of binding to CBF-1 on the promoter of MVP to drive its transcription, resulting in activation of AKT pathway and promoting the progress of epithelial to mesenchymal transition (EMT). Conversely, silencing of Notch1 and MVP suppressed AKT pathway, reduced EMT and enhanced the sensitivity of TNBC cells to cisplatin and doxorubicin. Survival analysis indicated that the MVP was closely related to shorter recurrence-free survival (RFS) in patients with TNBC. Collectively, this study provides evidence that Notch1 activates AKT pathway and promotes EMT partly through direct activation of MVP. Targeting Notch1/MVP pathway appears to have potential in overcoming chemoresistance in TNBC.