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1.
Artigo em Chinês | MEDLINE | ID: mdl-32148224

RESUMO

OBJECTIVE: To investigate the molecule mechanism of nuclear translocation of hypoxia-inducible factor-1α (HIF-1α) in influenza A (H1N1) virus infected-alveolar epithelial cells. METHODS: Human lung adenocarcinoma epithelial cells (A549 cells) were cultured in vitro, and cells in logarithmic growth phase were selected for experiments. (1) Experiment 1: the A549 cell model with H1N1 virus infection was established by using H1N1 virus infected cells with multiplicity of infection (MOI) 1.0 for 24 hours (H1N1 virus infection group), and the blank control group was set up. Importin 4 and Importin 7 protein expressions were detected by Western Blot to investigate whether HIF-1α nuclear translocation depended on Importin 4 or Importin 7. (2) Experiment 2: the A549 cells were infected with H1N1 virus under different MOI (0, 0.1, 0.5, 1.0, 2.0, 4.0) for 24 hours. Then the A549 cells were infected with H1N1 virus (MOI 1.0) for different time (0, 3, 6, 12, 18, 24, 36 hours). The septin 9 isoform 1 (SEPT9_i1) mRNA expression was detected by real-time fluorescence quantitative reverse transcription-polymerase chain reaction (RT-PCR) to investigate the effect of different MOI and infection time on the expression of SEPT9_i1. (3) Experiment 3: a cell model with SEPT9_i1 silencing was established by transfection of small interfering RNA (siRNA) for 24 hours (siRNA-SEPT9_i1 group), and the blank control group and blank vector control group (siControl group) were set up. Then the cells in the three groups were infected with H1N1 virus (MOI 1.0) for 24 hours after 24-hour transfection, and the SEPT9_i1 mRNA expression was detected by real-time fluorescence quantitative RT-PCR to investigate the interference efficiency of siRNA-SEPT9_i1. (4) Experiment 4: the cells were divided into siControl group and siRNA-SEPT9_i1 group. The transfection methods of two groups was as the same as experiment 3,and then the cells were infected with H1N1 virus (MOI 1.0) after 24-hour transfection. The distribution of HIF-1α was detected by immunofluorescence at 24 hours after infection. The M gene expression of virus was detected by real-time fluorescence quantitative RT-PCR at 6, 12, 24, 36, 48 hours after infection. The effects of SEPT9_i1 on HIF-1α translocation and virus replication were explored. (5) Experiment 5: the cells were divided into blank control group (complete medium), SP600125 group [100 µmol/L c-Jun N-terminal kinase (JNK) signaling pathway inhibitor SP600125 for 2 hours], H1N1 virus infection group (H1N1 virus of MOI 1.0 for 24 hours), H1N1 virus+SP600125 group (pretreated with 100 µmol/L SP600125 for 2 hours before 24-hour H1N1 virus infection). Real-time fluorescence quantitative RT-PCR was used to detect the expressions of SEPT_i1 mRNA and viral M gene to investigate the effect of JNK signaling pathway on SEPT9_i1 expression and virus replication. RESULTS: (1) Experiment 1: compared with the blank control group, the protein expressions of Importin 4 and Importin 7 in the H1N1 virus infection group had no significant changes [Importin 4 protein (Importin 4/GAPDH): 1.08±0.03 vs. 1.05±0.03, Importin 7 protein (Importin 7/GAPDH): 0.87±0.11 vs. 0.78±0.03, both P > 0.05]. These indicated that the HIF-1α nuclear translocation in A549 cells might not be independent of Importin 4 and Importin 7 during H1N1 virus infection. (2) Experiment 2: the SEPT9_i1 mRNA expression in A549 cells was increased with the increase in MOI and infection time of H1N1 virus, and peaked at MOI 2.0 or 18 hours after infection, and the differences were statistically significant as compared with MOI 0 or 0 hour after infection (2-ΔΔCT: 1.39±0.05 vs. 1.00±0.00 at MOI 2.0, 1.47±0.04 vs. 1.00±0.00 at 18 hours, both P < 0.01). This indicated that the SEPT9_i1 expression in A549 cells was related to the MOI and the infection time during H1N1 virus infection. (3) Experiment 3: compared with the blank control group, the SEPT9_i1 mRNA expression in A549 cells was significantly decreased in the siRNA-SEPT9_i1 group (2-ΔΔCT: 0.38±0.11 vs. 1.00±0.00, P < 0.01), and there was no significant difference between the siControl group and blank control group (2-ΔΔCT: 1.03±0.16 vs. 1.00±0.00, P > 0.05). This indicated that SEPT9_i1 silence could inhibit the expression of SEPT9_i1 mRNA in H1N1 virus-infected A549 cells. (4) Experiment 4: HIF-1α nuclear translocation in the H1N1 virus-infected A549 cells in the siRNA-SEPT9_i1 group was significantly reduced as compared with the siControl group. The virus M gene expression after H1N1 virus infection in the siControl group was gradually increased, and peaked at 48 hours. The expression of virus M gene in A549 cells in the siRNA-SEPT9_i1 group was significantly down-regulated, and showed a statistically significant difference at 48 hours as compared with the siControl group (2-ΔΔCT: 3.47±0.66 vs. 8.17±0.38, P < 0.05). This indicated that HIF-1α nuclear translocation and virus replication in H1N1 virus-infected A549 cells were inhibited after silencing SEPT9_i1. (5) Experiment 5: the expressions of SEPT9_i1 mRNA and virus M gene in A549 cells in the H1N1 virus infection group were significantly higher than those in the blank control group. However, the expressions of SEPT9_i1 mRNA and viral M gene in A549 cells in the H1N1 virus+SP600125 group were significantly lower than those in the H1N1 virus infection group (2-ΔΔCT: SEPT9_i1 mRNA was 0.12±0.10 vs. 1.53±0.14, viral M gene was 2.13±0.10 vs. 4.66±0.14, both P < 0.05). There was no significant difference in above indicators between the SP600125 group and the blank control group. This indicated that the JNK signaling pathway could regulate the expression of SEPT9_i1 in A549 cells during H1N1 virus infection, and the JNK signaling pathway inhibition could down-regulate the expression of SEPT9_i1 and inhibit virus replication. CONCLUSIONS: The H1N1 virus regulates the expression of SEPT9_i1 by activating the JNK signaling pathway, thus increase HIF-1α transport efficiency and H1N1 replication.


Assuntos
Células Epiteliais Alveolares/metabolismo , Células Epiteliais Alveolares/virologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Vírus da Influenza A Subtipo H1N1 , Células A549 , Transporte Ativo do Núcleo Celular , Humanos , Influenza Humana , Carioferinas/metabolismo , Sistema de Sinalização das MAP Quinases , Proteínas de Membrana Transportadoras/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Septinas/metabolismo
2.
Nat Commun ; 11(1): 270, 2020 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-31937751

RESUMO

Phase separation of substrates and effectors is proposed to enhance biological reaction rates and efficiency. Targeting protein for Xklp2 (TPX2) is an effector of branching microtubule nucleation in spindles and functions with the substrate tubulin by an unknown mechanism. Here we show that TPX2 phase separates into a co-condensate with tubulin, which mediates microtubule nucleation in vitro and in isolated cytosol. TPX2-tubulin co-condensation preferentially occurs on pre-existing microtubules, the site of branching microtubule nucleation, at the endogenous and physiologically relevant concentration of TPX2. Truncation and chimera versions of TPX2 suggest that TPX2-tubulin co-condensation enhances the efficiency of TPX2-mediated branching microtubule nucleation. Finally, the known inhibitor of TPX2, the importin-α/ß heterodimer, regulates TPX2 condensation in vitro and, consequently, branching microtubule nucleation activity in isolated cytosol. Our study demonstrates how regulated phase separation can simultaneously enhance reaction efficiency and spatially coordinate microtubule nucleation, which may facilitate rapid and accurate spindle formation.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Tubulina (Proteína)/metabolismo , Proteínas de Xenopus/metabolismo , Animais , Proteínas de Ciclo Celular/química , Citosol/metabolismo , Carioferinas/metabolismo , Meiose , Proteínas Associadas aos Microtúbulos/química , Centro Organizador dos Microtúbulos/metabolismo , Óvulo/citologia , Óvulo/metabolismo , Fuso Acromático/metabolismo , Proteínas de Xenopus/química , Xenopus laevis
3.
Gynecol Oncol ; 156(2): 308-314, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31822399

RESUMO

BACKGROUND: Selinexor is an oral inhibitor of the nuclear export protein Exportin 1 (XPO1) with demonstrated antitumor activity in solid and hematological malignancies. We evaluated the efficacy and safety of selinexor in heavily pretreated, recurrent gynecological malignancies. METHODS: In this phase 2 trial, patients received selinexor (35 or 50 mg/m2 twice-weekly [BIW] or 50 mg/m2 once-weekly [QW]) in 4-week cycles. Primary endpoint was disease control rate (DCR) including complete response (CR), partial response (PR) or stable disease (SD) ≥12 weeks. Secondary endpoints were progression-free survival (PFS), overall survival (OS) and safety. RESULTS: 114 patients with ovarian (N = 66), endometrial (N = 23) or cervical (N = 25) cancer were enrolled. Median number of prior regimens for ovarian, endometrial and cervical cancer was 6 (1-11), 2 (1-5), and 3 (1-6) respectively. DCR was 30% (ovarian 30%; endometrial 35%; cervical 24%), which included confirmed PRs in 8%, 9%, and 4% of patients with ovarian, endometrial, and cervical cancer respectively. Median PFS and OS for patients with ovarian, endometrial and cervical cancer were 2.6, 2.8 and 1.4 months, and 7.3, 7.0, and 5.0 months, respectively. Common Grade 3/4 adverse events (AEs) were thrombocytopenia (17%), fatigue (14%), anemia (10%), nausea (9%) and hyponatremia (9%). Patients with ovarian cancer receiving 50 mg/m2 QW had fewer high-grade AEs with similar efficacy as BIW treatment. CONCLUSIONS: Selinexor demonstrated single-agent activity and disease control in patients with heavily pretreated ovarian and endometrial cancers. Side effects were a function of dose level and treatment frequency, similar to previous reports, reversible and mitigated with supportive care.


Assuntos
Neoplasias dos Genitais Femininos/tratamento farmacológico , Hidrazinas/administração & dosagem , Carioferinas/antagonistas & inibidores , Recidiva Local de Neoplasia/tratamento farmacológico , Receptores Citoplasmáticos e Nucleares/antagonistas & inibidores , Triazóis/administração & dosagem , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Coortes , Feminino , Neoplasias dos Genitais Femininos/metabolismo , Neoplasias dos Genitais Femininos/patologia , Humanos , Hidrazinas/efeitos adversos , Carioferinas/metabolismo , Pessoa de Meia-Idade , Gradação de Tumores , Recidiva Local de Neoplasia/patologia , Intervalo Livre de Progressão , Receptores Citoplasmáticos e Nucleares/metabolismo , Triazóis/efeitos adversos
4.
Biosci Biotechnol Biochem ; 84(2): 290-296, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31619134

RESUMO

Chromosome region maintenance 1 (CRM1) plays a critical role in tumorigenesis and progression through modulating nuclear export of several proteins. However, the precise effects of CRM1 inhibitor on gastric carcinoma have not yet been illustrated. Here, we investigated the potential anti-cancer activities of leptomycin B, the most potent CRM1 antagonist, on cultured gastric carcinoma cells. Our findings demonstrate that CRM1 was highly expressed in four gastric carcinoma cell lines. Leptomycin B inhibited the viability of HGC-27 and AGS cells in a dose- and time-dependent pattern. Leptomycin B at the dose of 10 nM or 100 nM suppressed the migration and invasion of HGC-27 and AGS cells. Leptomycin B elevated the expressions of autophagy-related protein LC3-II and autophagy substrate p62. Moreover, leptomycin B enhanced the LC3-positive puncta formation in cells. Our data suggest that leptomycin B may exert an anti-cancer activity possibly through interfering autophagy function in gastric carcinoma cells.


Assuntos
Antibióticos Antineoplásicos/farmacologia , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Invasividade Neoplásica/prevenção & controle , Neoplasias Gástricas/patologia , Autofagia/efeitos dos fármacos , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Ácidos Graxos Insaturados/farmacologia , Humanos , Carioferinas/antagonistas & inibidores , Carioferinas/metabolismo , Receptores Citoplasmáticos e Nucleares/antagonistas & inibidores , Receptores Citoplasmáticos e Nucleares/metabolismo
5.
Asian Pac J Cancer Prev ; 20(12): 3747-3754, 2019 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-31870117

RESUMO

OBJECTIVES: Exportin 1(XPO1), a nuclear exporter protein, has been gaining recognition in cancer progression and treatment. This study aimed to evaluate the association between the overexpression of XPO1 with NF-κB, Ki67 and clinicopathological characteristics in colorectal cancer (CRC) tissue samples and to explore the anti-proliferative effect of KPT-330, as XPO1 inhibitor, in colorectal cancer cell line. METHODS: Forty CRC tissue samples were analyzed by immunostaining for the expressions of XPO1, NF-κB and Ki67 and then the anti-proliferative effect of the KPT-330 was also evaluated in HT29 colorectal cancer cell line. RESULTS: XPO1 overexpression was observed in 52.5% of CRC and significantly apparent with strong intensity in tumor cells compared to the normal adjacent epithelium (P<0.001). Regarding to the histopathological characteristics, the XPO1 overexpression significantly associated with advanced tumor stages (P=0.049) and has great tendency towards moderate/poorly differentiated tumors. Although the XPO1 overexpression was strongly associated with high Ki67 expression (P=0.001), only Ki67 expression showed significant association with tumor size (P=0.012). No significant association was detected between the XPO1 overexpression and NF-κB, while the NF-κB positive expression was significantly associated with lymph node metastasis and Ki67 expression at P=0.027 and P= 0.007, respectively. The in vitro experiments showed a great impact of KPT-330, as XPO1 inhibitor, to inhibit cancer growth in dose and time dependent manner and significantly diminished the colony formation (P<0.001) of HT29 cells- associated with the expression of Ki67 (P<0.001). CONCLUSION: XPO1 overexpression and NF-κB expression may serve as potential biomarker associated with CRC pathogenesis and proliferation, while the KPT-330 is effectively inhibited-colon cancer growth in vitro. Further studies considering the prognostication role of XPO1 overexpression in CRC are required.
.


Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias Colorretais/patologia , Carioferinas/metabolismo , Antígeno Ki-67/metabolismo , NF-kappa B/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/metabolismo , Feminino , Seguimentos , Células HT29 , Humanos , Hidrazinas/farmacologia , Carioferinas/antagonistas & inibidores , Metástase Linfática , Masculino , Pessoa de Meia-Idade , Prognóstico , Receptores Citoplasmáticos e Nucleares/antagonistas & inibidores , Triazóis/farmacologia , Adulto Jovem
6.
Proc Natl Acad Sci U S A ; 116(50): 25137-25146, 2019 12 10.
Artigo em Inglês | MEDLINE | ID: mdl-31767767

RESUMO

Stochastic neuronal cell fate choice involving notch-independent mechanisms is a poorly understood biological process. The Caenorhabditis elegans AWC olfactory neuron pair asymmetrically differentiates into the default AWCOFF and induced AWCON subtypes in a stochastic manner. Stochastic choice of the AWCON subtype is established using gap junctions and SLO BK potassium channels to repress a calcium-activated protein kinase pathway. However, it is unknown how the potassium channel-repressed calcium signaling is translated into the induction of the AWCON subtype. Here, we identify a detailed working mechanism of how the homeodomain-like transcription factor NSY-7, previously described as a repressor in the maintenance of AWC asymmetry, couples SLO BK potassium channels to transactivation of sox-2 expression for the induction of the AWCON subtype through the identification of a unique imb-2 (transportin 1) allele. imb-2 loss-of-function mutants are not viable; however, we identify a viable imb-2 allele from an unbiased forward genetic screen that reveals a specific role of imb-2 in AWC olfactory neuron asymmetry. IMB-2 specifically drives nuclear import of NSY-7 within AWC neurons to transactivate the expression of the high mobility group (HMG)-box transcription factor SOX-2 for the specification of the AWCON subtype. This study provides mechanistic insight into how NSY-7 couples SLO BK potassium channels to transactivation of sox-2 expression for the induction of the AWCON subtype. Our findings also provide structure-function insight into a conserved amino acid residue of transportins in brain development and suggest its dysfunction may lead to human neurological disorders.


Assuntos
Núcleo Celular/metabolismo , Carioferinas/metabolismo , Neurônios Receptores Olfatórios/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/fisiologia , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Sinalização do Cálcio/fisiologia , Junções Comunicantes/metabolismo , Carioferinas/genética , Fatores de Transcrição SOXB1/genética , Processos Estocásticos
7.
Elife ; 82019 11 27.
Artigo em Inglês | MEDLINE | ID: mdl-31774393

RESUMO

Intracellular Wolbachia bacteria manipulate arthropod reproduction to promote their own inheritance. The most prevalent mechanism, cytoplasmic incompatibility (CI), traces to a Wolbachia deubiquitylase, CidB, and CidA. CidB has properties of a toxin, while CidA binds CidB and rescues embryonic viability. CidB is also toxic to yeast where we identified both host effects and high-copy suppressors of toxicity. The strongest suppressor was karyopherin-α, a nuclear-import receptor; this required nuclear localization-signal binding. A protein-interaction screen of Drosophila extracts using a substrate-trapping catalytic mutant, CidB*, also identified karyopherin-α; the P32 protamine-histone exchange factor bound as well. When CidB* bound CidA, these host protein interactions disappeared. These associations would place CidB at the zygotic male pronucleus where CI defects first manifest. Overexpression of karyopherin-α, P32, or CidA in female flies suppressed CI. We propose that CidB targets nuclear-protein import and protamine-histone exchange and that CidA rescues embryos by restricting CidB access to its targets.


Assuntos
Proteína Centromérica A/metabolismo , Citoplasma/metabolismo , Proteínas de Drosophila/metabolismo , Histonas/metabolismo , Protaminas/metabolismo , Wolbachia/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Animais Geneticamente Modificados , Proteína Centromérica A/genética , Cruzamentos Genéticos , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Drosophila melanogaster/microbiologia , Escherichia coli/metabolismo , Feminino , Regulação da Expressão Gênica , Carioferinas/genética , Carioferinas/metabolismo , Masculino , Proteínas Recombinantes , Reprodução , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , alfa Carioferinas/isolamento & purificação , alfa Carioferinas/metabolismo
8.
Int J Mol Sci ; 20(23)2019 Nov 26.
Artigo em Inglês | MEDLINE | ID: mdl-31779129

RESUMO

Ribosomal proteins are highly expressed, and the quality of ribosomal proteins must be rigorously controlled to build up a functional ribosome. Rpl43, ribosomal protein large subunit 43, is located nearby the E-site of ribosomes. In our previous study, we found that Puf6, Loc1, and Rpl43 form a trimeric complex in Saccharomyces cerevisiae. Rpl43 protein levels are under-accumulated in the absence of PUF6 or LOC1. However, why the loss of Puf6 or Loc1 decreased the protein levels of Rpl43 remained unclear. In the present study, we further dissected the connections among these three proteins and found that the processing defects of pre-ribosomal RNA in puf6Δ and loc1Δ are similar to those of the mutant with depletion of Rpl43. The stability of newly synthesized Rpl43 protein decreased slightly in puf6Δ and significantly in loc1Δ. We also found that Puf6 and Loc1 could interact with nascent Rpl43 co-translationally via the N-terminus of Rpl43. While the association and dissociation of Rpl43 with karyopherins did not depend on Puf6 and Loc1, Puf6 and Loc1 interacted with nascent Rpl43 in collaboration. While the N-terminus of Puf6 contained nuclear localization signals for transport, the PUF (Pumilio) domain was essential to interaction with Loc1, Rpl43, and 60S subunits. The C-terminus of Loc1 is more important for interaction with Puf6 and Rpl43. In this study, we found that Puf6 and Loc1 are the dedicated chaperones of ribosomal protein Rpl43 and also analyzed the potential interaction domains among the three proteins. Correct formation of the Puf6, Loc1, and Rpl43 ternary complex is required to properly proceed to the next step in 60S biogenesis.


Assuntos
Proteínas Nucleares/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas Ribossômicas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Sítios de Ligação , Regulação Fúngica da Expressão Gênica , Carioferinas/metabolismo , Modelos Moleculares , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Mutação , Proteínas Nucleares/química , Proteínas Nucleares/genética , Ligação Proteica , Conformação Proteica , Estabilidade Proteica , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Proteínas Ribossômicas/química , Proteínas Ribossômicas/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética
9.
Int J Mol Sci ; 20(19)2019 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-31569391

RESUMO

Gastric cancer remains an unmet clinical problem in urgent need of newer and effective treatments. Here we show that the nuclear export protein, Exportin 1 (XPO1, chromosome region maintenance 1 or CRM1), is a promising molecular target in gastric cancer. We demonstrate significant overexpression of XPO1 in a cohort of histologically diverse gastric cancer patients with primary and metastatic disease. XPO1 RNA interference suppressed gastric cancer cell growth. Anti-tumor activity was observed with specific inhibitor of nuclear export (SINE) compounds (selinexor/XPOVIO), second-generation compound KPT-8602/eltanexor, KPT-185 and +ve control Leptomycin B in three distinct gastric cancer cell lines. SINE compounds inhibited gastric cancer cell proliferation, disrupted spheroid formation, induced apoptosis and halted cell cycle progression at the G1/S phase. Anti-tumor activity was concurrent with nuclear retention of tumor suppressor proteins and inhibition of colony formation. In combination studies, SINE compounds enhanced the efficacy of nab-paclitaxel in vitro and in vivo. More significantly, using non-coding RNA sequencing studies, we demonstrate for the first time that SINE compounds can alter the expression of non-coding RNAs (microRNAs and piwiRNAs). SINE treatment caused statistically significant downregulation of oncogenic miR-33b-3p in two distinct cell lines. These studies demonstrate the therapeutic significance of XPO1 in gastric cancer that warrants further clinical investigation.


Assuntos
Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Carioferinas/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Neoplasias Gástricas/metabolismo , Antineoplásicos/química , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Sobrevivência Celular , Humanos , Paclitaxel/química , Paclitaxel/farmacologia
10.
EMBO Rep ; 20(11): e48150, 2019 11 05.
Artigo em Inglês | MEDLINE | ID: mdl-31544310

RESUMO

STK38 (also known as NDR1) is a Hippo pathway serine/threonine protein kinase with multifarious functions in normal and cancer cells. Using a context-dependent proximity-labeling assay, we identify more than 250 partners of STK38 and find that STK38 modulates its partnership depending on the cellular context by increasing its association with cytoplasmic proteins upon nutrient starvation-induced autophagy and with nuclear ones during ECM detachment. We show that STK38 shuttles between the nucleus and the cytoplasm and that its nuclear exit depends on both XPO1 (aka exportin-1, CRM1) and STK38 kinase activity. We further uncover that STK38 modulates XPO1 export activity by phosphorylating XPO1 on serine 1055, thus regulating its own nuclear exit. We expand our model to other cellular contexts by discovering that XPO1 phosphorylation by STK38 regulates also the nuclear exit of Beclin1 and YAP1, key regulator of autophagy and transcriptional effector, respectively. Collectively, our results reveal STK38 as an activator of XPO1, behaving as a gatekeeper of nuclear export. These observations establish a novel mechanism of XPO1-dependent cargo export regulation by phosphorylation of XPO1's C-terminal auto-inhibitory domain.


Assuntos
Autofagia , Núcleo Celular/metabolismo , Carioferinas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Proteínas de Transporte/metabolismo , Cromatografia Líquida , Biologia Computacional/métodos , Humanos , Fosforilação , Ligação Proteica , Mapeamento de Interação de Proteínas , Transporte Proteico , Transdução de Sinais , Espectrometria de Massas em Tandem
11.
EMBO J ; 38(16): e101168, 2019 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-31414556

RESUMO

Nuclear actin participates in many essential cellular processes including gene transcription, chromatin remodelling and mRNA processing. Actin shuttles into and out the nucleus through the action of dedicated transport receptors importin-9 and exportin-6, but how this transport is regulated remains unclear. Here, we show that RASSF1A is a novel regulator of actin nucleocytoplasmic trafficking and is required for the active maintenance of nuclear actin levels through supporting binding of exportin-6 (XPO6) to RAN GTPase. RASSF1A (Ras association domain family 1 isoform A) is a tumour suppressor gene frequently silenced by promoter hypermethylation in all major solid cancers. Specifically, we demonstrate that endogenous RASSF1A localises to the nuclear envelope (NE) and is required for nucleocytoplasmic actin transport and the concomitant regulation of myocardin-related transcription factor A (MRTF-A), a co-activator of the transcription factor serum response factor (SRF). The RASSF1A/RAN/XPO6/nuclear actin pathway is aberrant in cancer cells where RASSF1A expression is lost and correlates with reduced MRTF-A/SRF activity leading to cell adhesion defects. Taken together, we have identified a previously unknown mechanism by which the nuclear actin pool is regulated and uncovered a previously unknown link of RASSF1A and MRTF-A/SRF in tumour suppression.


Assuntos
Actinas/metabolismo , Neoplasias da Mama/genética , Neoplasias Hepáticas/genética , Membrana Nuclear/metabolismo , Fator de Resposta Sérica/genética , Proteínas Supressoras de Tumor/metabolismo , Transporte Biológico , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Citoplasma/metabolismo , Metilação de DNA , Regulação para Baixo , Feminino , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Células HeLa , Humanos , Carioferinas/metabolismo , Neoplasias Hepáticas/metabolismo , Prognóstico , Fator de Resposta Sérica/metabolismo , Transativadores/metabolismo , Proteínas Supressoras de Tumor/genética
12.
Theriogenology ; 139: 178-190, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31421412

RESUMO

The function of estrogen-related receptor (ERR) in testicular cells is at the beginning of exploration. Our previous findings showed that expression pattern of estrogen-related receptor (ERR) in mouse Leydig cell depends on physiological status of the cell. Exogenous hormones/hormonally active chemicals affect ERR expression. In Leydig cells in vitro, ERRα and ERRγ show opposing regulatory properties. The aim of this study was to examine the role of ERR in epigenetic processes in cells with altered level of secreted estrogens; mouse tumor Leydig cells and bank vole Leydig cells, respectively. In Leydig cells, ERRα and ERRγ were silenced via siRNA. mRNA and protein expression and protein localization of molecules required for miRNA biogenesis and function (Exportin 5, Dicer, Drosha and Argonaute 2; Ago2) were studied with the use of qRT-PCR, Western blotting, and immunohistochemistry. Global DNA methylation and histone deacetylation status together with estradiol secretion were determined with fluorometric, and immunoenzymatic assays. Regardless of ERR type knockdown in tumor Leydig cells, downregulation (P < 0.05; P < 0.01; P < 0.001) of Exportin5, Dicer, Drosha but not Ago2 was revealed while at protein level only Drosha was downregulated (P < 0.01) by both ERRα and ERRγ. Oppositely, Exportin5, Dicer and Ago2 showed ERR type-dependent regulation (downregulation; P < 0.01 by ERRα and upregulation; P < 0.01; P < 0.001 by ERRγ). In ERR-silenced vole Leydig cells, expression of Exportin5, endonucleases and Ago2 was not changed. Immunolocalization of Dicer and Ago2 was independent of the cell origin in contrast to localization of Exportin5 and Drosha which was dependent on the cell origin and ERR type knockdown. Absence of ERR effected on cell methylation status (ERRα increased it; P < 0.01 while ERRγ decreased it; P < 0.01, P < 0.001) but it not changed histone deacetylates activity. ERRα and ERRγ silencing decreased (P < 0.01, P < 0.001) estradiol secretion in both tumor and vole Leydig cells. In mouse and bank vole Leydig cell, Exportin5, Dicer, Drosha and Ago2 expression as well as methylation status are regulated by ERR in a manner related to receptor type, molecule type, cell origin and level of secreted estrogen.


Assuntos
Arvicolinae/metabolismo , Metilação de DNA , Células Intersticiais do Testículo/metabolismo , Receptores Estrogênicos/fisiologia , Acetilação , Animais , Proteínas Argonauta/genética , Proteínas Argonauta/metabolismo , Células Cultivadas , Regulação da Expressão Gênica , Carioferinas/genética , Carioferinas/metabolismo , Masculino , Camundongos , MicroRNAs/metabolismo , Modelos Biológicos , Interferência de RNA , Receptores Estrogênicos/antagonistas & inibidores , Ribonuclease III/genética , Ribonuclease III/metabolismo
13.
Mol Syst Biol ; 15(8): e8939, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31464369

RESUMO

Cells respond to environmental fluctuations by regulating multiple transcriptional programs. This response can be studied by measuring the effect of environmental changes on the transcriptome or the proteome of the cell at the end of the response. However, the dynamics of the response reflect the working of the regulatory mechanisms in action. Here, we utilized a fluorescent stress reporter gene to track the dynamics of protein production in yeast responding to environmental stress. The response is modulated by changes in both the duration and rate of transcription. We probed the underlying molecular pathways controlling these two dimensions using a library of ~1,600 single- and double-mutant strains. Dissection of the effects of these mutants and the interactions between them identified distinct modulators of response duration and response rate. Using a combination of mRNA-seq and live-cell microscopy, we uncover mechanisms by which Msn2/4, Mck1, Msn5, and the cAMP/PKA pathway modulate the response of a large module of stress-induced genes in two discrete regulatory phases. Our results and analysis show that transcriptional stress response is regulated by multiple mechanisms that overlap in time and cellular location.


Assuntos
Proteínas de Ligação a DNA/genética , Regulação Fúngica da Expressão Gênica , Quinase 3 da Glicogênio Sintase/genética , RNA Mensageiro/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Fatores de Transcrição/genética , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas de Ligação a DNA/metabolismo , Perfilação da Expressão Gênica , Interação Gene-Ambiente , Genes Reporter , Quinase 3 da Glicogênio Sintase/deficiência , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Carioferinas/genética , Carioferinas/metabolismo , Mutação , Cloreto de Potássio/farmacologia , RNA Mensageiro/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transdução de Sinais , Estresse Fisiológico , Fatores de Transcrição/metabolismo , Transcrição Genética
14.
Cell ; 178(4): 964-979.e20, 2019 08 08.
Artigo em Inglês | MEDLINE | ID: mdl-31398345

RESUMO

PIWI-interacting RNAs (piRNAs) guide transposon silencing in animals. The 22-30 nt piRNAs are processed in the cytoplasm from long non-coding RNAs that often lack RNA processing hallmarks of export-competent transcripts. By studying how these transcripts achieve nuclear export, we uncover an RNA export pathway specific for piRNA precursors in the Drosophila germline. This pathway requires Nxf3-Nxt1, a variant of the hetero-dimeric mRNA export receptor Nxf1-Nxt1. Nxf3 interacts with UAP56, a nuclear RNA helicase essential for mRNA export, and CG13741/Bootlegger, which recruits Nxf3-Nxt1 and UAP56 to heterochromatic piRNA source loci. Upon RNA cargo binding, Nxf3 achieves nuclear export via the exportin Crm1 and accumulates together with Bootlegger in peri-nuclear nuage, suggesting that after export, Nxf3-Bootlegger delivers precursor transcripts to the piRNA processing sites. These findings indicate that the piRNA pathway bypasses nuclear RNA surveillance systems to export unprocessed transcripts to the cytoplasm, a strategy also exploited by retroviruses.


Assuntos
Transporte Ativo do Núcleo Celular/fisiologia , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Heterocromatina/metabolismo , Proteínas de Transporte Nucleocitoplasmático/metabolismo , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Animais Geneticamente Modificados , Proteínas Argonauta/metabolismo , Linhagem Celular , Núcleo Celular/metabolismo , Citoplasma/metabolismo , RNA Helicases DEAD-box/metabolismo , Elementos de DNA Transponíveis , Inativação Gênica , Células Germinativas/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Carioferinas/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Transcrição Genética
15.
Cell Physiol Biochem ; 53(2): 366-387, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31385665

RESUMO

BACKGROUND/AIMS: The extracellular signal-regulated kinases (ERK) 1 and 2 (ERK1/2) are members of the mitogen-activated protein kinase (MAPK) family. Upon stimulation, these kinases translocate from the cytoplasm to the nucleus, where they induce physiological processes such as proliferation and differentiation. The mechanism of translocation of this kinase involves phosphorylation of two Ser residues within a nuclear translocation signal (NTS), which allows binding to importin7 and a subsequent penetration via nuclear pores. However, the regulation of this process and the protein kinases involved are not yet clear. METHODS: To answer this point we developed specific anti phospho-SPS antibody, used this and other antibodies in Western blots and crystalized the phospho-mimetic mutated ERK. RESULTS: Here we show that the phosphorylation of both Ser residues is mediated mainly by casein kinase 2 (CK2) and that active ERK may assist in the phosphorylation of the N-terminal Ser. We also demonstrate that the phosphorylation is dependent on the release of ERK from cytoplasmic anchoring proteins. Crystal structure of the phosphomimetic ERK revealed that the NTS phosphorylation creates an acidic patch in ERK. Our model is that in resting cells ERK is bound to cytoplasmic anchors, which prevent its NTS phosphorylation. Upon stimulation, phosphorylation of the ERK TEY domain releases ERK and allows phosphorylation of its NTS by CK2 and active ERK to generate a negatively charged patch in ERK, binding to importin 7 and nuclear translocation. CONCLUSION: These results provide an important role of CK2 in regulating nuclear ERK activities.


Assuntos
Núcleo Celular/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Transporte Ativo do Núcleo Celular , Caseína Quinase II/metabolismo , Linhagem Celular , Humanos , Carioferinas/metabolismo , Fosforilação , Ligação Proteica , Receptores Citoplasmáticos e Nucleares/metabolismo
16.
Eur J Med Chem ; 180: 224-237, 2019 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-31306909

RESUMO

Cytotoxic effects of (R)-4'-methylklavuzon were investigated on hepatocellular carcinoma cells (HuH-7 and HepG2) and HuH-7 EpCAM+/CD133+ cancer stem cells. IC50 of (R)-4'-methylklavuzon was found as 1.25 µM for HuH-7 parental cells while it was found as 2.50 µM for HuH-7 EpCAM+/CD133+ cancer stem cells. (R)-4'-methylklavuzon tended to show more efficient in vitro cytotoxicity with its lower IC50 values on hepatocellular carcinoma cell lines compared to its lead molecule, goniothalamin and FDA-approved drugs, sorafenib and regorafenib. Cell-based Sirtuin/HDAC enzyme activity measurements revealed that endogenous Sirtuin/HDAC enzymes were reduced by 40% compared to control. SIRT1 protein levels were upregulated indicating triggered DNA repair mechanism. p53 was overexpressed in HepG2 cells. (R)-4'-methylklavuzon inhibited CRM1 protein providing increased retention of p53 and RIOK2 protein in the nucleus. HuH-7 parental and EpCAM+/CD133+ cancer stem cell spheroids lost intact morphology. 3D HepG2 spheroid viabilities were decreased in a correlation with upregulation in p53 protein levels.


Assuntos
Antígeno AC133/antagonistas & inibidores , Antineoplásicos/farmacologia , Carcinoma Hepatocelular/tratamento farmacológico , Molécula de Adesão da Célula Epitelial/antagonistas & inibidores , Carioferinas/antagonistas & inibidores , Neoplasias Hepáticas/tratamento farmacológico , Naftalenos/farmacologia , Receptores Citoplasmáticos e Nucleares/antagonistas & inibidores , Sirtuína 1/antagonistas & inibidores , Antígeno AC133/metabolismo , Antineoplásicos/síntese química , Antineoplásicos/química , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Molécula de Adesão da Célula Epitelial/metabolismo , Células Hep G2 , Humanos , Carioferinas/metabolismo , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Estrutura Molecular , Naftalenos/síntese química , Naftalenos/química , Receptores Citoplasmáticos e Nucleares/metabolismo , Sirtuína 1/metabolismo , Relação Estrutura-Atividade , Células Tumorais Cultivadas
17.
Nat Cell Biol ; 21(7): 856-866, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31235936

RESUMO

Tumour-derived microvesicles (TMVs) comprise a class of extracellular vesicles released from tumour cells that are now understood to facilitate communication between the tumour and the surrounding microenvironment. Despite their significance, the regulatory mechanisms governing the trafficking of bioactive cargos to TMVs at the cell surface remain poorly defined. Here we describe a molecular pathway for the delivery of microRNA (miRNA) cargo to nascent TMVs involving the dissociation of a pre-miRNA/Exportin-5 complex from Ran-GTP following nuclear export and its subsequent transfer to a cytoplasmic shuttle comprised of ARF6-GTP and GRP1. As such, ARF6 activation increases the pre-miRNA cargo contained within TMVs through a process that requires the casein kinase 2-mediated phosphorylation of RanGAP1. Furthermore, TMVs were found to contain pre-miRNA processing machinery including Dicer and Argonaute-2, which allow for cell-free pre-miRNA processing within shed vesicles. These findings offer cellular targets to block the loading and processing of pre-miRNAs within TMVs.


Assuntos
Fatores de Ribosilação do ADP/metabolismo , Carioferinas/metabolismo , MicroRNAs/genética , Microambiente Tumoral/fisiologia , Fatores de Ribosilação do ADP/genética , Transporte Ativo do Núcleo Celular/fisiologia , Micropartículas Derivadas de Células/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Humanos , Carioferinas/genética , Neoplasias/metabolismo , Fosforilação , Interferência de RNA/fisiologia
18.
Cell Oncol (Dordr) ; 42(5): 645-661, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31147963

RESUMO

PURPOSE: Anastasis is newly discovered process by which cells recover from late-stage apoptosis upon removal of a death stimulus. Recent reports suggest that cells may recover, even after the initiation of mitochondrial outer-membrane permeabilization (MOMP) and caspase activation. Here, we specifically studied the reversibility of late-stage apoptosis in cervical (HeLa) and breast (MDA-MB-231) cancer cells in relation to the extent of MOMP (limited or widespread). In addition, we explored the molecular factors involved in the anastatic process. METHODS: The extent of MOMP was assessed using time lapse confocal microscopic imaging, considering mitochondrial cytochrome c-GFP release as a marker for MOMP. Anastatic cells were generated by specifically recovering late-stage apoptotic (annexin V/PI positive) cervical and breast cancer cells. Molecular signaling events involved in death reversal were assessed using LC-MS/MS and qRT-PCR. Targeted chemical inhibition and shRNA-based gene silencing studies were employed to explore the role of the nuclear export pathway in anastasis and increased oncogenicity. RESULTS: Time-lapse imaging of drug-treated Cyt-c-GFP expressing cancer cells revealed cell recovery despite widespread MOMP. A few recovered anastatic cells were noted and these were found to proliferate through a selection-type of survival. They showed increased drug-resistance, migration and invasive potential compared to non-anastatic cancer cells. Network analysis using 49 proteins uniquely expressed in anastatic cells indicated upregulation of nuclear export/import, redox and Ras signaling pathways in both HeLa and MDA-MB-231 anastatic cells, indicating common molecular mechanisms in different cell types. Inhibition of XPO1 significantly reduced the recovery of apoptotic cells and abrogated acquired oncogenic transformation in the anastatic cancer cells. CONCLUSIONS: Our study indicates that cancer cells can revert from apoptosis even after the induction of widespread MOMP. We noted a significant role of the nuclear-export pathway in the anastatic process of cancer cells. Inhibition of anastasis through the nuclear export pathway may be a potential therapeutic strategy for targeting drug-resistance, metastasis and recurrence problems during cancer treatment.


Assuntos
Núcleo Celular/metabolismo , Carioferinas/antagonistas & inibidores , Membranas Mitocondriais/metabolismo , Neoplasias/metabolismo , Receptores Citoplasmáticos e Nucleares/antagonistas & inibidores , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Caspases/metabolismo , Movimento Celular , Sobrevivência Celular/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HeLa , Humanos , Carioferinas/metabolismo , Mitocôndrias/metabolismo , Invasividade Neoplásica , Proteômica , Receptores Citoplasmáticos e Nucleares/metabolismo , Transdução de Sinais/efeitos dos fármacos
19.
Cells ; 8(6)2019 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-31181773

RESUMO

Protein nuclear transport is an integral process to many cellular pathways and often plays a critical role during viral infection. To overcome the barrier presented by the nuclear membrane and gain access to the nucleus, virally encoded proteins have evolved ways to appropriate components of the nuclear transport machinery. By binding karyopherins, or the nuclear pore complex, viral proteins influence their own transport as well as the transport of key cellular regulatory proteins. This review covers how viral proteins can interact with different components of the nuclear import machinery and how this influences viral replicative cycles. We also highlight the effects that viral perturbation of nuclear transport has on the infected host and how we can exploit viruses as tools to study novel mechanisms of protein nuclear import. Finally, we discuss the possibility that drugs targeting these transport pathways could be repurposed for treating viral infections.


Assuntos
Transporte Ativo do Núcleo Celular/fisiologia , Poro Nuclear/metabolismo , Vírus/patogenicidade , Interações Hospedeiro-Patógeno , Humanos , Carioferinas/metabolismo , Transporte Proteico , Replicação Viral , Vírus/metabolismo
20.
Cell Mol Life Sci ; 76(18): 3543-3551, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31129857

RESUMO

Maintenance of genomic stability during cell division is one of the most important cellular tasks, and it critically depends on the faithful replication of the genetic material and its equal partitioning into daughter cells, gametes, or spores in the case of yeasts. Defective mitotic spindle assembly and disassembly both result in changes in cellular ploidy that ultimately impinge proliferation fitness and might increase tumor malignancy. Although a great progress has been made in understanding how spindles are assembled to orchestrate chromosome segregation, much less is known about how they are disassembled once completed their function. Here, we review two recently uncovered mechanisms of spindle disassembly that operate at different stages of the fission yeast life cycle.


Assuntos
Schizosaccharomyces/metabolismo , Fuso Acromático/fisiologia , Transporte Ativo do Núcleo Celular , Instabilidade Genômica , Carioferinas/metabolismo , Meiose , Mitose , Membrana Nuclear/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo
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