RESUMEN
Nuclear import receptors (NIRs) not only transport RNA-binding proteins (RBPs) but also modify phase transitions of RBPs by recognizing nuclear localization signals (NLSs). Toxic arginine-rich poly-dipeptides from C9orf72 interact with NIRs and cause nucleocytoplasmic transport deficit. However, the molecular basis for the toxicity of arginine-rich poly-dipeptides toward NIRs function as phase modifiers of RBPs remains unidentified. Here we show that arginine-rich poly-dipeptides impede the ability of NIRs to modify phase transitions of RBPs. Isothermal titration calorimetry and size-exclusion chromatography revealed that proline:arginine (PR) poly-dipeptides tightly bind karyopherin-ß2 (Kapß2) at 1:1 ratio. The nuclear magnetic resonances of Kapß2 perturbed by PR poly-dipeptides partially overlapped with those perturbed by the designed NLS peptide, suggesting that PR poly-dipeptides target the NLS binding site of Kapß2. The findings offer mechanistic insights into how phase transitions of RBPs are disabled in C9orf72-related neurodegeneration.
Asunto(s)
Transporte Activo de Núcleo Celular/genética , Proteína C9orf72/química , Péptidos/química , beta Carioferinas/química , Sitios de Unión , Proteína C9orf72/genética , Proteína C9orf72/metabolismo , Clonación Molecular , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Células HeLa , Humanos , Modelos Moleculares , Señales de Localización Nuclear/genética , Señales de Localización Nuclear/metabolismo , Péptidos/genética , Péptidos/metabolismo , Transición de Fase , Unión Proteica , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Proteína FUS de Unión a ARN/genética , Proteína FUS de Unión a ARN/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , beta Carioferinas/antagonistas & inhibidores , beta Carioferinas/genética , beta Carioferinas/metabolismoRESUMEN
The SARS-CoV-2 pandemic has accelerated the study of existing drugs. The mixture of homologs called ivermectin (avermectin-B1a [HB1a] + avermectin-B1b [HB1b]) has shown antiviral activity against SARS-CoV-2 in vitro. However, there are few reports on the behavior of each homolog. We investigated the interaction of each homolog with promising targets of interest associated with SARS-CoV-2 infection from a biophysical and computational-chemistry perspective using docking and molecular dynamics. We observed a differential behavior for each homolog, with an affinity of HB1b for viral structures, and of HB1a for host structures considered. The induced disturbances were differential and influenced by the hydrophobicity of each homolog and of the binding pockets. We present the first comparative analysis of the potential theoretical inhibitory effect of both avermectins on biomolecules associated with COVID-19, and suggest that ivermectin through its homologs, has a multiobjective behavior.
Asunto(s)
Antivirales/química , Proteasas 3C de Coronavirus/antagonistas & inhibidores , ADN Helicasas/antagonistas & inhibidores , Ivermectina/análogos & derivados , alfa Carioferinas/antagonistas & inhibidores , beta Carioferinas/antagonistas & inhibidores , Animales , Antivirales/farmacología , Sitios de Unión , COVID-19/virología , Proteasas 3C de Coronavirus/química , Proteasas 3C de Coronavirus/metabolismo , ADN Helicasas/química , ADN Helicasas/metabolismo , Humanos , Ivermectina/química , Ivermectina/farmacología , Cinética , Ratones , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Unión Proteica , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , SARS-CoV-2/química , SARS-CoV-2/efectos de los fármacos , SARS-CoV-2/enzimología , Termodinámica , alfa Carioferinas/química , alfa Carioferinas/metabolismo , beta Carioferinas/química , beta Carioferinas/metabolismo , Tratamiento Farmacológico de COVID-19RESUMEN
Programmed death-ligand 1 (PD-L1) is an immune checkpoint molecule that negatively regulates anti-tumor immunity. Recent reports indicate that anti-cancer treatments, such as radiation therapy, increase PD-L1 expression on the surface of tumor cells. We previously reported that the nuclear transport receptor karyopherin-ß1 (KPNB1) is involved in radiation-increased PD-L1 expression on head-and-neck squamous cell carcinoma cells. However, the mechanisms underlying KPNB1-mediated, radiation-increased PD-L1 expression remain unknown. Thus, the mechanisms of radiation-increased, KPNB1-mediated PD-L1 expression were investigated by focusing on the transcription factor interferon regulatory factor 1 (IRF1), which is reported to regulate PD-L1 expression. Western blot analysis showed that radiation increased IRF1 expression. In addition, flow cytometry showed that IRF1 knockdown decreased cell surface PD-L1 expression of irradiated cells but had a limited effect on non-irradiated cells. These findings suggest that the upregulation of IRF1 after irradiation is required for radiation-increased PD-L1 expression. Notably, immunofluorescence and western blot analyses revealed that KPNB1 inhibitor importazole not only diffused nuclear localization of IRF1 but also decreased IRF1 upregulation by irradiation, which attenuated radiation-increased PD-L1 expression. Taken together, these findings suggest that KPNB1 mediates radiation-increased cell surface PD-L1 expression through both upregulation and nuclear import of IRF1.
Asunto(s)
Antígeno B7-H1/antagonistas & inhibidores , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Factor 1 Regulador del Interferón/antagonistas & inhibidores , Neoplasias Pulmonares/metabolismo , Quinazolinas/farmacología , beta Carioferinas/antagonistas & inhibidores , Transporte Activo de Núcleo Celular , Línea Celular Tumoral , Humanos , Inmunoterapia/métodos , Factor 1 Regulador del Interferón/genética , Factor 1 Regulador del Interferón/metabolismo , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/radioterapia , Radiación IonizanteRESUMEN
Karyopherin beta 1 (Kpnß1) is a major nuclear import receptor that mediates the import of cellular cargoes into the nucleus. Recently it has been shown that Kpnß1 is highly expressed in several cancers, and its inhibition by siRNA induces apoptotic cancer cell death, while having little effect on non-cancer cells. This study investigated the effect of a novel small molecule, Inhibitor of Nuclear Import-60 (INI-60), on cancer cell biology, as well as nuclear import activities associated with Kpnß1, and cancer progression in vivo using cervical and oesophageal cancer cell lines. INI-60 treatment resulted in the inhibition of cancer cell proliferation, colony formation, migration and invasion, and induced a G1/S cell cycle arrest, followed by cancer cell death via apoptosis. Non-cancer cells were minimally affected by INI-60 at concentrations that inhibited cancer cells. INI-60 treatment altered the localisation of Kpnß1 and its cargoes, NFκB/p65, NFAT and AP-1, and the overexpression of Kpnß1 reduced INI-60 cytotoxicity. INI-60 also inhibited KYSE 30 oesophageal cancer cell line growth in vivo. Taken together, these results show that INI-60 inhibits the nuclear import of Kpnß1 cargoes and interferes with cancer cell biology. INI-60 presents as a potential therapeutic approach for cancers of different tissue origins and warrants further investigation as a novel anti-cancer agent.
Asunto(s)
Apoptosis/efectos de los fármacos , Puntos de Control del Ciclo Celular/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , beta Carioferinas/antagonistas & inhibidores , Transporte Activo de Núcleo Celular/efectos de los fármacos , Puntos de Control del Ciclo Celular/genética , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Femenino , Humanos , Neoplasias del Cuello Uterino/tratamiento farmacológico , Neoplasias del Cuello Uterino/genética , beta Carioferinas/genéticaRESUMEN
As of January 2021, SARS-CoV-2 has killed over 2 million individuals across the world. As such, there is an urgent need for vaccines and therapeutics to reduce the burden of COVID-19. Several vaccines, including mRNA, vector-based vaccines, and inactivated vaccines, have been approved for emergency use in various countries. However, the slow roll-out of vaccines and insufficient global supply remains a challenge to turn the tide of the pandemic. Moreover, vaccines are important tools for preventing the disease but therapeutic tools to treat patients are also needed. As such, since the beginning of the pandemic, repurposed FDA-approved drugs have been sought as potential therapeutic options for COVID-19 due to their known safety profiles and potential anti-viral effects. One of these drugs is ivermectin (IVM), an antiparasitic drug created in the 1970s. IVM later exerted antiviral activity against various viruses including SARS-CoV-2. In this review, we delineate the story of how this antiparasitic drug was eventually identified as a potential treatment option for COVID-19. We review SARS-CoV-2 lifecycle, the role of the nucleocapsid protein, the turning points in past research that provided initial 'hints' for IVM's antiviral activity and its molecular mechanism of action- and finally, we culminate with the current clinical findings.
Asunto(s)
Transporte Activo de Núcleo Celular/efectos de los fármacos , Antivirales/uso terapéutico , Tratamiento Farmacológico de COVID-19 , Ivermectina/uso terapéutico , SARS-CoV-2/efectos de los fármacos , Animales , Línea Celular , Chlorocebus aethiops , Proteínas de la Nucleocápside de Coronavirus/antagonistas & inhibidores , Proteínas de la Nucleocápside de Coronavirus/metabolismo , Reposicionamiento de Medicamentos , Humanos , Fosfoproteínas/antagonistas & inhibidores , Fosfoproteínas/metabolismo , Transporte de Proteínas/efectos de los fármacos , SARS-CoV-2/crecimiento & desarrollo , Células Vero , Replicación Viral/efectos de los fármacos , alfa Carioferinas/antagonistas & inhibidores , beta Carioferinas/antagonistas & inhibidoresRESUMEN
BACKGROUND: Inhibition of nuclear import via Karyopherin beta 1 (Kpnß1) shows potential as an anti-cancer approach. This study investigated the use of nuclear import inhibitor, INI-43, in combination with cisplatin. METHODS: Cervical cancer cells were pre-treated with INI-43 before treatment with cisplatin, and MTT cell viability and apoptosis assays performed. Activity and localisation of p53 and NFκB was determined after co-treatment of cells. RESULTS: Pre-treatment of cervical cancer cells with INI-43 at sublethal concentrations enhanced cisplatin sensitivity, evident through decreased cell viability and enhanced apoptosis. Kpnß1 knock-down cells similarly displayed increased sensitivity to cisplatin. Combination index determination using the Chou-Talalay method revealed that INI-43 and cisplatin engaged in synergistic interactions. p53 was found to be involved in the cell death response to combination treatment as its inhibition abolished the enhanced cell death observed. INI-43 pre-treatment resulted in moderately stabilized p53 and induced p53 reporter activity, which translated to increased p21 and decreased Mcl-1 upon cisplatin combination treatment. Furthermore, cisplatin treatment led to nuclear import of NFκB, which was diminished upon pre-treatment with INI-43. NFκB reporter activity and expression of NFκB transcriptional targets, cyclin D1, c-Myc and XIAP, showed decreased levels after combination treatment compared to single cisplatin treatment and this associated with enhanced DNA damage. CONCLUSIONS: Taken together, this study shows that INI-43 pre-treatment significantly enhances cisplatin sensitivity in cervical cancer cells, mediated through stabilization of p53 and decreased nuclear import of NFκB. Hence this study suggests the possible synergistic use of nuclear import inhibition and cisplatin to treat cervical cancer.
Asunto(s)
Transporte Activo de Núcleo Celular/efectos de los fármacos , Antineoplásicos/farmacología , Biomarcadores de Tumor/metabolismo , Cisplatino/farmacología , Sinergismo Farmacológico , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Pirroles/farmacología , Quinoxalinas/farmacología , Neoplasias del Cuello Uterino/tratamiento farmacológico , beta Carioferinas/antagonistas & inhibidores , Antineoplásicos/uso terapéutico , Apoptosis , Biomarcadores de Tumor/genética , Movimiento Celular , Proliferación Celular , Cisplatino/uso terapéutico , Quimioterapia Combinada , Femenino , Humanos , Pirroles/uso terapéutico , Quinoxalinas/uso terapéutico , Células Tumorales Cultivadas , Neoplasias del Cuello Uterino/metabolismo , Neoplasias del Cuello Uterino/patologíaRESUMEN
Karyopherins mediate the macromolecular transport between the cytoplasm and the nucleus and participate in cancer progression. However, the role and mechanism of importin-11 (IPO11), a member of the karyopherin family, in glioma progression remain undefined. Effects of IPO11 on glioma progression were detected using CCK-8, colony formation assay, flow cytometry analysis, caspase-3 activity assay, and Transwell invasion assay. Western blot analysis was used to detect the expression of active caspase-3, active caspase-7, active caspase-9, N-cadherin, Vimentin, E-cadherin, ß-catenin, and c-Myc. The activity of Wnt/ß-catenin pathway was evaluated by the T-cell factor/lymphoid enhancer factor (TCF/LEF) transcription factor reporter assay. Results showed that IPO11 knockdown inhibited proliferation and reduced colony number in glioma cells. IPO11 silencing promoted the apoptotic rate, increased expression levels of active caspase-3, caspase-7, and caspase-9, and enhanced caspase-3 activity. Moreover, IPO11 silencing inhibited glioma cell invasion by suppressing epithelial-to-mesenchymal transition (EMT). Mechanistically, IPO11 knockdown inactivated the Wnt/ß-catenin pathway. ß-Catenin overexpression abolished the effects of IPO11 silencing on the proliferation, apoptosis, and invasion in glioma cells. Furthermore, IPO11 silencing blocked the malignant phenotypes and repressed the Wnt/ß-catenin pathway in vivo. In conclusion, IPO11 knockdown suppressed the malignant phenotypes of glioma cells by inactivating the Wnt/ß-catenin pathway.
Asunto(s)
Neoplasias Encefálicas/metabolismo , Carcinógenos/metabolismo , Glioma/metabolismo , beta Carioferinas/metabolismo , Animales , Apoptosis/genética , Apoptosis/fisiología , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Carcinógenos/antagonistas & inhibidores , Línea Celular Tumoral , Proliferación Celular/genética , Proliferación Celular/fisiología , Femenino , Técnicas de Silenciamiento del Gen , Glioma/genética , Glioma/patología , Xenoinjertos , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Invasividad Neoplásica/genética , Invasividad Neoplásica/patología , Invasividad Neoplásica/fisiopatología , Fenotipo , Transcriptoma , Regulación hacia Arriba , Vía de Señalización Wnt , beta Catenina/genética , beta Catenina/metabolismo , beta Carioferinas/antagonistas & inhibidores , beta Carioferinas/genéticaRESUMEN
There is no cure for adult T cell leukemia/lymphoma (ATLL) associated with human T cell leukemia virus type 1 (HTLV-1), and novel targeted strategies are needed. NF-κB and AP-1 are crucial for ATLL, and both are transported to the nucleus by an importin (IPO)α/ß heterodimeric complex to activate target genes. In this study, we aimed to elucidate the function of IPOß1 in ATLL. The expression of IPOß1 was analyzed by western blotting and RT-PCR. Cell growth, viability, cell cycle, apoptosis and intracellular signaling cascades were examined by the water-soluble tetrazolium-8 assay, flow cytometry and western blotting. Xenograft tumors in severe combined immune deficient mice were used to evaluate the growth of ATLL cells in vivo. IPOß1 was upregulated in HTLV-1-infected T cell lines. Further, IPOß1 knockdown or the IPOß1 inhibitor importazole and the IPOα/ß1 inhibitor ivermectin reduced HTLV-1-infected T cell proliferation. However, the effect of inhibitors on uninfected T cells was less pronounced. Further, in HTLV-1-infected T cell lines, inhibitors suppressed NF-κB and AP-1 nuclear transport and DNA binding, induced apoptosis and poly (ADP-ribose) polymerase cleavage, and activated caspase-3, caspase-8 and caspase-9. Inhibitors also mediated G1 cell cycle arrest. Moreover, the expression of NF-κB- and AP-1-target proteins involved in cell cycle and apoptosis was reduced. In vivo, the IPOα/ß1 inhibitor ivermectin decreased ATLL tumor burden without side effects. IPOß1 mediated NF-κB and AP-1 translocation into ATLL cell nuclei, thereby regulating cell growth and survival, which provides new insights for targeted ATLL therapies. Thus, ivermectin, an anti-strongyloidiasis medication, could be a potent anti-ATLL agent.
Asunto(s)
Supervivencia Celular/efectos de los fármacos , Leucemia-Linfoma de Células T del Adulto/patología , FN-kappa B/efectos de los fármacos , Quinazolinas/farmacología , beta Carioferinas/antagonistas & inhibidores , Animales , Apoptosis/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Femenino , Virus Linfotrópico T Tipo 1 Humano , Humanos , Ratones , Ratones SCID , Transducción de Señal/efectos de los fármacos , Linfocitos T/efectos de los fármacos , Factor de Transcripción AP-1/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
In addition to the role of programmed cell death ligand 1 (PD-L1) in facilitating tumour cells escape from immune surveillance, it is considered as a crucial effector in transducing intrinsic signals to promote tumour development. Our previous study has pointed out that PD-L1 promotes non-small cell lung cancer (NSCLC) cell proliferation, but the mechanism remains elusive. Here we first demonstrated that PD-L1 expression levels were positively correlated with p-MerTK levels in patient samples and NSCLC cell lines. In addition, PD-L1 knockdown led to the reduced phosphorylation level of MerTK in vitro. We next showed that PD-L1 regulated NSCLC cell proliferation via Gas6/MerTK signaling pathway in vitro and in vivo. To investigate the underlying mechanism, we unexpectedly found that PD-L1 translocated into the nucleus of cancer cells which was facilitated through the binding of Karyopherin ß1 (KPNB1). Nuclear PD-L1 (nPD-L1), coupled with transcription factor Sp1, regulated the synthesis of Gas6 mRNA and promoted Gas6 secretion to activate MerTK signaling pathway. Taken together, our results shed light on the novel role of nPD-L1 in NSCLC cell proliferation and reveal a new molecular mechanism underlying nPD-L1-mediated Gas6/MerTK signaling activation. All above findings provide the possible combinational implications for PD-L1 targeted immunotherapy in the clinic.
Asunto(s)
Antígeno B7-H1/antagonistas & inhibidores , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Neoplasias Pulmonares/metabolismo , beta Carioferinas/antagonistas & inhibidores , Tirosina Quinasa c-Mer/metabolismo , Adulto , Anciano , Animales , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/patología , Línea Celular Tumoral , Proliferación Celular , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Inmunoterapia/métodos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/patología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Persona de Mediana Edad , Fosforilación , Transducción de Señal , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
TNF-related apoptosis-inducing ligand (TRAIL) and an agonistic antibody against the death-inducing TRAIL receptor 5, DR5, are thought to selectively induce tumor cell death and therefore, have gained attention as potential therapeutics currently under investigation in several clinical trials. However, some tumor cells are resistant to TRAIL/DR5-induced cell death, even though they express DR5. Previously, we reported that DR5 is transported into the nucleus by importin ß1, and knockdown of importin ß1 upregulates cell surface expression of DR5 resulting in increased TRAIL sensitivity in vitro Here, we examined the impact of importin ß1 knockdown on agonistic anti-human DR5 (hDR5) antibody therapy. Drug-inducible importin ß1 knockdown sensitizes HeLa cells to TRAIL-induced cell death in vitro, and exerts an antitumor effect when combined with agonistic anti-hDR5 antibody administration in vivo Therapeutic importin ß1 knockdown, administered via the atelocollagen delivery system, as well as treatment with the importin ß inhibitor, importazole, induced regression and/or eradication of two human TRAIL-resistant tumor cells when combined with agonistic anti-hDR5 antibody treatment. Thus, these findings suggest that the inhibition of importin ß1 would be useful to improve the therapeutic effects of agonistic anti-hDR5 antibody against TRAIL-resistant cancers.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Carcinoma Hepatocelular/tratamiento farmacológico , Neoplasias Hepáticas/tratamiento farmacológico , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/antagonistas & inhibidores , Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , beta Carioferinas/antagonistas & inhibidores , Animales , Apoptosis , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Proliferación Celular , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Ratones , Ratones Endogámicos BALB C , Ligando Inductor de Apoptosis Relacionado con TNF/genética , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Infection by RNA viruses such as human immunodeficiency virus (HIV)-1, influenza, and dengue virus (DENV) represent a major burden for human health worldwide. Although RNA viruses replicate in the infected host cell cytoplasm, the nucleus is central to key stages of the infectious cycle of HIV-1 and influenza, and an important target of DENV nonstructural protein 5 (NS5) in limiting the host antiviral response. We previously identified the small molecule ivermectin as an inhibitor of HIV-1 integrase nuclear entry, subsequently showing ivermectin could inhibit DENV NS5 nuclear import, as well as limit infection by viruses such as HIV-1 and DENV. We show here that ivermectin's broad spectrum antiviral activity relates to its ability to target the host importin (IMP) α/ß1 nuclear transport proteins responsible for nuclear entry of cargoes such as integrase and NS5. We establish for the first time that ivermectin can dissociate the preformed IMPα/ß1 heterodimer, as well as prevent its formation, through binding to the IMPα armadillo (ARM) repeat domain to impact IMPα thermal stability and α-helicity. We show that ivermectin inhibits NS5-IMPα interaction in a cell context using quantitative bimolecular fluorescence complementation. Finally, we show for the first time that ivermectin can limit infection by the DENV-related West Nile virus at low (µM) concentrations. Since it is FDA approved for parasitic indications, ivermectin merits closer consideration as a broad spectrum antiviral of interest.
Asunto(s)
Transporte Activo de Núcleo Celular/efectos de los fármacos , Ivermectina/farmacología , alfa Carioferinas/antagonistas & inhibidores , beta Carioferinas/antagonistas & inhibidores , Animales , Línea Celular , Chlorocebus aethiops , Cricetinae , Infecciones por Flavivirus/tratamiento farmacológico , Riñón/citología , Unión Proteica , Células Vero , alfa Carioferinas/metabolismo , beta Carioferinas/metabolismoRESUMEN
Transfer RNAs (tRNAs) are central players in protein synthesis, which in Eukarya need to be delivered from the nucleus to the cytoplasm by specific transport receptors, most of which belong to the evolutionarily conserved beta-importin family. Based on the available literature, we identified two candidates, Xpo-t and Xpo-5 for tRNA export in Trypanosoma brucei. However, down-regulation of expression of these genes did not disrupt the export of tRNAs to the cytoplasm. In search of alternative pathways, we tested the mRNA export complex Mex67-Mtr2, for a role in tRNA nuclear export, as described previously in yeast. Down-regulation of either exporter affected the subcellular distribution of tRNAs. However, contrary to yeast, TbMex67 and TbMtr2 accumulated different subsets of tRNAs in the nucleus. While TbMtr2 perturbed the export of all the tRNAs tested, silencing of TbMex67, led to the nuclear accumulation of tRNAs that are typically modified with queuosine. In turn, inhibition of tRNA nuclear export also affected the levels of queuosine modification in tRNAs. Taken together, the results presented demonstrate the dynamic nature of tRNA trafficking in T. brucei and its potential impact not only on the availability of tRNAs for protein synthesis but also on their modification status.
Asunto(s)
Proteínas de Transporte Nucleocitoplasmático/genética , Proteínas Protozoarias/genética , ARN Mensajero/genética , ARN Protozoario/genética , ARN de Transferencia/genética , Trypanosoma brucei brucei/genética , beta Carioferinas/genética , Transporte Biológico , Núcleo Celular/genética , Núcleo Celular/metabolismo , Citoplasma/genética , Citoplasma/metabolismo , Regulación de la Expresión Génica , Conformación de Ácido Nucleico , Proteínas de Transporte Nucleocitoplasmático/antagonistas & inhibidores , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Nucleósido Q/química , Nucleósido Q/metabolismo , Unión Proteica , Biosíntesis de Proteínas , Proteínas Protozoarias/antagonistas & inhibidores , Proteínas Protozoarias/metabolismo , ARN Mensajero/metabolismo , ARN Protozoario/química , ARN Protozoario/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , ARN de Transferencia/química , ARN de Transferencia/metabolismo , Transducción de Señal , Trypanosoma brucei brucei/metabolismo , beta Carioferinas/antagonistas & inhibidores , beta Carioferinas/metabolismoRESUMEN
Prostate cancer (PCa) initiation and progression requires activation of numerous oncogenic signaling pathways. Nuclear-cytoplasmic transport of oncogenic factors is mediated by Karyopherin proteins during cell transformation. However, the role of nuclear transporter proteins in PCa progression has not been well defined. Here, we report that the KPNB1, a key member of Karyopherin beta subunits, is highly expressed in advanced prostate cancers. Further study showed that targeting KPNB1 suppressed the proliferation of prostate cancer cells. The knockdown of KPNB1 reduced nuclear translocation of c-Myc, the expression of downstream cell cycle modulators, and phosphorylation of regulator of chromatin condensation 1 (RCC1), a key protein for spindle assembly during mitosis. Meanwhile, CHIP assay demonstrated the binding of c-Myc to KPNB1 promoter region, which indicated a positive feedback regulation of KPNB1 expression mediated by the c-Myc. In addition, NF-κB subunit p50 translocation to nuclei was blocked by KPNB1 inhibition, which led to an increase in apoptosis and a decrease in tumor sphere formation of PCa cells. Furthermore, subcutaneous xenograft tumor models with a stable knockdown of KPNB1 in C42B PCa cells validated that the inhibition of KPNB1 could suppress the growth of prostate tumor in vivo. Moreover, the intravenously administration of importazole, a specific inhibitor for KPNB1, effectively reduced PCa tumor size and weight in mice inoculated with PC3 PCa cells. In summary, our data established the functional link between KPNB1 and PCa prone c-Myc, NF-kB, and cell cycle modulators. More importantly, inhibition of KPNB1 could be a new therapeutic target for PCa treatment.
Asunto(s)
Proliferación Celular/efectos de los fármacos , Neoplasias de la Próstata/patología , Quinazolinas/farmacología , ARN Interferente Pequeño/farmacología , beta Carioferinas/antagonistas & inhibidores , beta Carioferinas/genética , Animales , Línea Celular Tumoral , Proliferación Celular/genética , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Masculino , Ratones , Ratones Desnudos , Células PC-3 , Neoplasias de la Próstata/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a cytokine with potential anticancer effect, but innate and adaptive TRAIL resistance in majority of cancers limit its clinical application. Karyopherin ß1 (KPNB1) inhibition in cancer cells has been reported to abrogate the nuclear import of TRAIL receptor DR5 and facilitate its localization on the cell surface ready for TRAIL stimulation. However, our study reveals a more complicated mechanism. Genetic or pharmacological inhibition of KPNB1 potentiated TRAIL-induced apoptosis selectively in glioblastoma cells mainly by unfolded protein response (UPR). First, it augmented ATF4-mediated DR5 expression and promoted the assembly of death-inducing signaling complex (DISC). Second, it freed Bax and Bak from Mcl-1. Third, it downregulated FLIPL and FLIPS, inhibitors of caspase-8 cleavage, partly through upregulating ATF4-induced 4E-BP1 expression and disrupting the cap-dependent translation initiation. Meanwhile, KPNB1 inhibition-induced undesirable autophagy and accelerated cleaved caspase-8 clearance. Inhibition of autophagic flux maintained cleaved caspase-8 and aggravated apoptosis induced by KPNB1 inhibitor plus TRAIL, which were abolished by caspase-8 inhibitor. These results unveil new molecular mechanism for optimizing TRAIL-directed therapeutic efficacy against cancer.
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Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Ligando Inductor de Apoptosis Relacionado con TNF/farmacología , beta Carioferinas/metabolismo , Factor de Transcripción Activador 4/metabolismo , Animales , Apoptosis/efectos de los fármacos , Autofagia/efectos de los fármacos , Caspasa 8/metabolismo , Línea Celular Tumoral , Resistencia a Antineoplásicos , Femenino , Glioblastoma/tratamiento farmacológico , Glioblastoma/metabolismo , Glioblastoma/patología , Humanos , Ratones , Ratones Desnudos , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Ligando Inductor de Apoptosis Relacionado con TNF/uso terapéutico , beta Carioferinas/antagonistas & inhibidores , beta Carioferinas/genéticaRESUMEN
Defects in nucleocytoplasmic transport have been identified as a key pathogenic event in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) mediated by a GGGGCC hexanucleotide repeat expansion in C9ORF72, the most common genetic cause of ALS/FTD. Furthermore, nucleocytoplasmic transport disruption has also been implicated in other neurodegenerative diseases with protein aggregation, suggesting a shared mechanism by which protein stress disrupts nucleocytoplasmic transport. Here, we show that cellular stress disrupts nucleocytoplasmic transport by localizing critical nucleocytoplasmic transport factors into stress granules, RNA/protein complexes that play a crucial role in ALS pathogenesis. Importantly, inhibiting stress granule assembly, such as by knocking down Ataxin-2, suppresses nucleocytoplasmic transport defects as well as neurodegeneration in C9ORF72-mediated ALS/FTD. Our findings identify a link between stress granule assembly and nucleocytoplasmic transport, two fundamental cellular processes implicated in the pathogenesis of C9ORF72-mediated ALS/FTD and other neurodegenerative diseases.
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Transporte Activo de Núcleo Celular/fisiología , Esclerosis Amiotrófica Lateral/patología , Ataxina-2/metabolismo , Proteína C9orf72/genética , Demencia Frontotemporal/patología , Transporte Activo de Núcleo Celular/efectos de los fármacos , Anciano , Esclerosis Amiotrófica Lateral/metabolismo , Arsenitos/toxicidad , Ataxina-2/antagonistas & inhibidores , Ataxina-2/genética , Proteína C9orf72/metabolismo , Expansión de las Repeticiones de ADN/genética , Femenino , Demencia Frontotemporal/metabolismo , Células HEK293 , Humanos , Masculino , Glicoproteínas de Membrana/metabolismo , Persona de Mediana Edad , Proteínas de Complejo Poro Nuclear/metabolismo , Estrés Oxidativo/efectos de los fármacos , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Compuestos de Sodio/toxicidad , alfa Carioferinas/antagonistas & inhibidores , alfa Carioferinas/genética , alfa Carioferinas/metabolismo , beta Carioferinas/antagonistas & inhibidores , beta Carioferinas/genética , beta Carioferinas/metabolismo , Proteína de Unión al GTP ran/antagonistas & inhibidores , Proteína de Unión al GTP ran/genética , Proteína de Unión al GTP ran/metabolismoRESUMEN
BACKGROUND/AIMS: Transforming growth factor-ß3 (TGF-ß3) has been proved to perturb the blood-testis barrier (BTB) by accelerating junction protein endocytosis in Sertoli cells (SCs) to accommodate the traversing of preleptotene spermatocytes across the BTB around stage VIII in rat. Yet the molecular network underlying the impairment of TGF-ß3 on BTB integrity is not fully elucidated. Our study herein was designed to investigate the participation of microRNA-142-3p (miR-142-3p), which has been reported to affect TGF-ß3 signaling via different pathways, during BTB dynamics and the corresponding mechanisms. METHODS: MiRNA mimic or agomiRNA was co-administered with or without TGF-ß3 in the cultured SCs or in the rat testis. The SC permeability barrier function was reflected by measuring the transepithelial resistance (TER) and the permeability of the sodium fluorescein (Na-F). The BTB integrity was detected by the permeation of biotin. A luciferase reporter assay was used to testify the potential target of miR-142-3p, lethal giant larvae 2 (Lgl2). Laser capture microdissection (LCM) was applied to acquire cell components of different stages of seminiferious tubules, followed by detection of the expression levels of miR-142-3p, TGF-ß3, and Lgl2 by qPCR. The SC barrier function was also detected as above in the presence of TGF-ß3 after Lgl2 knockdown. RESULTS: We revealed a reversion of TGF-ß3-induced BTB impairment after miR-142-3p treatment both in vitro and in vivo. Meanwhile, the activation of Cdc42 and reduction in occludin aroused by TGF-ß3 were also reversed by miR-142-3p. The predicted binding of miR-142-3p with 3'-untranslated region (3'-UTR) of Lgl2, was verified by the luciferase assay. Moreover, an increased Lgl2 level in TGF-ß3-treated SCs was found and correlated stage-specific expressions of TGF-ß3, miR-142-3p, and Lgl2 were revealed. Knockdown of Lgl2 in SCs was shown to partially antagonize the BTB disruption mediated by TGF-ß3. CONCLUSIONS: Collectively, our results suggest a resistance of miR-142-3p on the BTB impairment caused by TGF-ß3 during the seminiferous epithelial cycle by targeting Lgl2.
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Barrera Hematotesticular/efectos de los fármacos , MicroARNs/metabolismo , Factor de Crecimiento Transformador beta/farmacología , beta Carioferinas/metabolismo , Regiones no Traducidas 3' , Animales , Antagomirs/metabolismo , Secuencia de Bases , Células Cultivadas , Células HEK293 , Humanos , Inmunoprecipitación , Masculino , MicroARNs/antagonistas & inhibidores , MicroARNs/genética , Ocludina/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Ratas , Ratas Sprague-Dawley , Alineación de Secuencia , Células de Sertoli/citología , Células de Sertoli/efectos de los fármacos , Células de Sertoli/metabolismo , beta Carioferinas/antagonistas & inhibidores , beta Carioferinas/genética , Proteína de Unión al GTP cdc42/metabolismoRESUMEN
In addition to the traditional epidermal growth factor receptor (EGFR) signaling pathways, nuclear EGFR has been shown to control multiple cellular functions, including cell proliferation and invasion. It has been reported that EGFR is transported into the nucleus after forming a complex with KPNA/KPNB1 or KPNB1. Herein, it is shown that EGFR can interact with both KP and KPNA, but EGF-activated EGFR mostly binds with KPNB1 through the pull-down assay. Also, a small organic molecule (1), an effective binder of KPNB1, inhibits the interaction between EGFR and KPNB1 in the nonclassical transport pathway, but not KPNA. Furthermore, treatment of cancer cells with 1 noticeably blocks the nuclear entry of EGFR, which results in significant suppression of invasion by lung cancer H1299 cells. These findings show that 1 is an effective inhibitor of EGFR/KPNB1 interactions in vitro, it may be used in cellular studies as a tool to determine the role of nuclear EGFR, and it is a drug candidate.
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Núcleo Celular/efectos de los fármacos , Neoplasias Pulmonares/metabolismo , Bibliotecas de Moléculas Pequeñas/farmacología , Tiazoles/farmacología , beta Carioferinas/antagonistas & inhibidores , Línea Celular Tumoral , Núcleo Celular/química , Núcleo Celular/metabolismo , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/química , Receptores ErbB/metabolismo , Humanos , Neoplasias Pulmonares/patología , Estructura Molecular , Bibliotecas de Moléculas Pequeñas/química , Tiazoles/química , beta Carioferinas/química , beta Carioferinas/metabolismoRESUMEN
Therapeutics are currently unavailable for Venezuelan equine encephalitis virus (VEEV), which elicits flu-like symptoms and encephalitis in humans, with an estimated 14% of cases resulting in neurological disease. Here we identify anti-VEEV agents using in silico structure-based-drug-design (SBDD) for the first time, characterising inhibitors that block recognition of VEEV capsid protein (C) by the host importin (IMP) α/ß1 nuclear transport proteins. From an initial screen of 1.5 million compounds, followed by in silico refinement and screening for biological activity in vitro, we identified 21 hit compounds which inhibited IMPα/ß1:C binding with IC50s as low as 5 µM. Four compounds were found to inhibit nuclear import of C in transfected cells, with one able to reduce VEEV replication at µM concentration, concomitant with reduced C nuclear accumulation in infected cells. Further, this compound was inactive against a mutant VEEV that lacks high affinity IMPα/ß1:C interaction, supporting the mode of its antiviral action to be through inhibiting C nuclear localization. This successful application of SBDD paves the way for lead optimization for VEEV antivirals, and is an exciting prospect to identify inhibitors for the many other viral pathogens of significance that require IMPα/ß1 in their infectious cycle.
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Proteínas de la Cápside/efectos de los fármacos , Descubrimiento de Drogas/métodos , Virus de la Encefalitis Equina Venezolana/efectos de los fármacos , Transporte Activo de Núcleo Celular/efectos de los fármacos , Animales , Antivirales/farmacología , Cápside , Proteínas de la Cápside/metabolismo , Núcleo Celular/metabolismo , Chlorocebus aethiops , Simulación por Computador , Diseño de Fármacos , Virus de la Encefalitis Equina Venezolana/patogenicidad , Humanos , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Células Vero , Replicación Viral/efectos de los fármacos , alfa Carioferinas/antagonistas & inhibidores , alfa Carioferinas/metabolismo , beta Carioferinas/antagonistas & inhibidores , beta Carioferinas/metabolismoRESUMEN
Repetitive elements, including LINE-1 (L1), comprise approximately half of the human genome. These elements can potentially destabilize the genome by initiating their own replication and reintegration into new sites (retrotransposition). In somatic cells, transcription of L1 elements is repressed by distinct molecular mechanisms, including DNA methylation and histone modifications, to repress transcription. Under conditions of hypomethylation (e.g. in tumor cells), a window of opportunity for L1 derepression arises, and additional restriction mechanisms become crucial. We recently demonstrated that the microRNA miR-128 represses L1 activity by directly binding to L1 ORF2 RNA. In this study, we tested whether miR-128 can also control L1 activity by repressing cellular proteins important for L1 retrotransposition. We found that miR-128 targets the 3' UTR of nuclear import factor transportin 1 (TNPO1) mRNA. Manipulation of miR-128 and TNPO1 levels demonstrated that induction or depletion of TNPO1 affects L1 retrotransposition and nuclear import of an L1-ribonucleoprotein complex (using L1-encoded ORF1p as a proxy for L1-ribonucleoprotein complexes). Moreover, TNPO1 overexpression partially reversed the repressive effect of miR-128 on L1 retrotransposition. Our study represents the first description of a protein factor involved in nuclear import of the L1 element and demonstrates that miR-128 controls L1 activity in somatic cells through two independent mechanisms: direct binding to L1 RNA and regulation of a cellular factor necessary for L1 nuclear import and retrotransposition.
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Regiones no Traducidas 3' , Regulación hacia Abajo , Regulación de la Expresión Génica , Elementos de Nucleótido Esparcido Largo , MicroARNs/metabolismo , ARN Mensajero/antagonistas & inhibidores , beta Carioferinas/antagonistas & inhibidores , Sustitución de Aminoácidos , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Transporte Biológico , Biología Computacional , Genes Reporteros , Células HeLa , Humanos , Inmunoprecipitación , Mutagénesis Sitio-Dirigida , Mutación , Interferencia de ARN , ARN Mensajero/metabolismo , ARN Interferente Pequeño/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , beta Carioferinas/genética , beta Carioferinas/metabolismoRESUMEN
Insulin-like growth factor-binding protein (IGFBP)-5 is a secreted protein that binds to IGFs and modulates IGF actions, as well as regulates cell proliferation, migration, and apoptosis independent of IGF. Proper cellular localization is critical for the effective function of most signaling molecules. In previous studies, we have shown that the nuclear IGFBP-5 comes from ER-cytosol retro-translocation. In this study, we further investigated the pathway mediating IGFBP-5 nuclear import after it retro-translocation. Importin-α5 was identified as an IGFBP-5-interacting protein with a yeast two-hybrid system, and its interaction with IGFBP-5 was further confirmed by GST pull down and co-immunoprecipitation. Binding affinity of IGFBP-5 and importins were determined by surface plasmon resonance (IGFBP-5/importin-ß: KD=2.44e-7, IGFBP-5/importin-α5: KD=3.4e-7). Blocking the importin-α5/importin-ß nuclear import pathway using SiRNA or dominant negative impotin-ß dramatically inhibited IGFBP-5-EGFP nuclear import, though importin-α5 overexpress does not affect IGFBP-5 nuclear import. Furthermore, nuclear IGFBP-5 was quantified using luciferase report assay. When deleted the IGFBP-5 nuclear localization sequence (NLS), IGFBP-5ΔNLS loss the ability to translocate into the nucleus and accumulation of IGFBP-5ΔNLS was visualized in the cytosol. Altogether, our findings provide a substantially evidence showed that the IGFBP-5 nuclear import is mediated by importin-α/importin-ß complex, and NLS is critical domain in IGFBP-5 nuclear translocation.