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1.
Nat Microbiol ; 4(11): 1840-1850, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31611641

RESUMO

The initial steps of HIV replication in host cells prime the virus for passage through the nuclear pore and drive the establishment of a productive and irreparable infection1,2. The timely release of the viral genome from the capsid-referred to as uncoating-is emerging as a critical parameter for nuclear import, but the triggers and mechanisms that orchestrate these steps are unknown. Here, we identify ß-karyopherin Transportin-1 (TRN-1) as a cellular co-factor of HIV-1 infection, which binds to incoming capsids, triggers their uncoating and promotes viral nuclear import. Depletion of TRN-1, which we characterized by mass spectrometry, significantly reduced the early steps of HIV-1 infection in target cells, including primary CD4+ T cells. TRN-1 bound directly to capsid nanotubes and induced dramatic structural damage, indicating that TRN-1 is necessary and sufficient for uncoating in vitro. Glycine 89 on the capsid protein, which is positioned within a nuclear localization signal in the cyclophilin A-binding loop, is critical for engaging the hydrophobic pocket of TRN-1 at position W730. In addition, TRN-1 promotes the efficient nuclear import of both viral DNA and capsid protein. Our study suggests that TRN-1 mediates the timely release of the HIV-1 genome from the capsid protein shell and efficient viral nuclear import.


Assuntos
Proteínas do Capsídeo/química , Proteínas do Capsídeo/metabolismo , Infecções por HIV/metabolismo , HIV-1/fisiologia , beta Carioferinas/química , beta Carioferinas/metabolismo , Transporte Ativo do Núcleo Celular , Sítios de Ligação , Linfócitos T CD4-Positivos/metabolismo , Capsídeo/química , Capsídeo/metabolismo , Deleção de Genes , Células HEK293 , Infecções por HIV/genética , Infecções por HIV/virologia , HIV-1/metabolismo , Células HeLa , Humanos , Espectrometria de Massas , Modelos Moleculares , Sinais de Localização Nuclear , Ligação Proteica , Conformação Proteica , RNA Viral/metabolismo , Desenvelopamento do Vírus , beta Carioferinas/genética
2.
Nat Commun ; 10(1): 4307, 2019 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-31541088

RESUMO

To facilitate proper mitotic cell partitioning, the Golgi disassembles by suppressing vesicle fusion. However, the underlying mechanism has not been characterized previously. Here, we report a Ran pathway-independent attenuation mechanism that allows Importin-α (a nuclear transport factor) to suppress the vesicle fusion mediated by p115 (a vesicular tethering factor) and is required for mitotic Golgi disassembly. We demonstrate that Importin-α directly competes with p115 for interaction with the Golgi protein GM130. This interaction, promoted by a phosphate moiety on GM130, is independent of Importin-ß and Ran. A GM130 K34A mutant, in which the Importin-α-GM130 interaction is specifically disrupted, exhibited abundant Golgi puncta during metaphase. Importantly, a mutant showing enhanced p115-GM130 interaction presented proliferative defects and G2/M arrest, demonstrating that Importin-α-GM130 binding modulates the Golgi disassembly that governs mitotic progression. Our findings illuminate that the Ran and kinase-phosphatase pathways regulate multiple aspects of mitosis coordinated by Importin-α (e.g. spindle assembly, Golgi disassembly).


Assuntos
Autoantígenos/metabolismo , Complexo de Golgi/metabolismo , Proteínas da Matriz do Complexo de Golgi/metabolismo , Proteínas de Membrana/metabolismo , Metáfase/fisiologia , Proteínas de Transporte Vesicular/metabolismo , alfa Carioferinas/metabolismo , Autoantígenos/genética , Cristalografia por Raios X , Pontos de Checagem da Fase G2 do Ciclo Celular , Células HEK293 , Humanos , Fusão de Membrana , Proteínas de Membrana/genética , Mitose/fisiologia , Fosforilação , Ligação Proteica , beta Carioferinas/metabolismo , Proteína ran de Ligação ao GTP/metabolismo
3.
Nucleic Acids Res ; 47(16): 8620-8631, 2019 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-31392978

RESUMO

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.


Assuntos
Proteínas de Transporte Nucleocitoplasmático/genética , Proteínas de Protozoários/genética , RNA Mensageiro/genética , RNA de Protozoário/genética , RNA de Transferência/genética , Trypanosoma brucei brucei/genética , beta Carioferinas/genética , Transporte Biológico , Núcleo Celular/genética , Núcleo Celular/metabolismo , Citoplasma/genética , Citoplasma/metabolismo , Regulação da Expressão Gênica , Conformação de Ácido Nucleico , Proteínas de Transporte Nucleocitoplasmático/antagonistas & inibidores , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Nucleosídeo Q/química , Nucleosídeo Q/metabolismo , Ligação Proteica , Biossíntese de Proteínas , Proteínas de Protozoários/antagonistas & inibidores , Proteínas de Protozoários/metabolismo , RNA Mensageiro/metabolismo , RNA de Protozoário/química , RNA de Protozoário/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , RNA de Transferência/química , RNA de Transferência/metabolismo , Transdução de Sinais , Trypanosoma brucei brucei/metabolismo , beta Carioferinas/antagonistas & inibidores , beta Carioferinas/metabolismo
4.
Cells ; 8(8)2019 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-31426451

RESUMO

Various growth factors and full-length cell surface receptors such as EGFR are translocated from the cell surface to the nucleoplasm, baffling cell biologists to the mechanisms and functions of this process. Elevated levels of nuclear EGFR correlate with poor prognosis in various cancers. In recent years, nuclear EGFR has been implicated in regulating gene transcription, cell proliferation and DNA damage repair. Different models have been proposed to explain how the receptors are transported into the nucleus. However, a clear consensus has yet to be reached. Recently, we described the nuclear envelope associated endosomes (NAE) pathway, which delivers EGFR from the cell surface to the nucleus. This pathway involves transport, docking and fusion of NAEs with the outer membrane of the nuclear envelope. EGFR is then presumed to be transported through the nuclear pore complex, extracted from membranes and solubilised. The SUN1/2 nuclear envelope proteins, Importin-beta, nuclear pore complex proteins and the Sec61 translocon have been implicated in the process. While this framework can explain the cell surface to nucleus traffic of EGFR and other cell surface receptors, it raises several questions that we consider in this review, together with implications for health and disease.


Assuntos
Endossomos/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Membrana Nuclear/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas Nucleares/metabolismo , Canais de Translocação SEC/metabolismo , beta Carioferinas/metabolismo , Linhagem Celular , Endocitose , Receptores ErbB/metabolismo , Humanos
5.
J Exp Clin Cancer Res ; 38(1): 296, 2019 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-31288861

RESUMO

BACKGROUND: Karyopherin nuclear transport receptors play important roles in tumour development and drug resistance and have been reported as potential biomarkers and therapeutic targets for tumour treatment. However, IPO5, one of the karyopherin nuclear transport receptor family members, remains largely uncharacterized in tumour progression. METHODS: The TCGA data, quantitative reverse transcription-PCR (qRT-PCR), western blotting, and IHC analyses were used to detect IPO5 expression in CRC tissues. A series of in vivo and in vitro experiments was utilized to demonstrate the function of IPO5 in CRC tissues. Mass spectrometry (MS), CO-IP technology, subcellular fractionation, and immunofluorescence were utilized to investigate the possible mechanisms of CRC. RESULTS: IPO5 was highly expressed and positively correlated with the clinicopathological characteristics of colorectal cancer tissues. Functional experiments indicated that IPO5 could promote the development of CRC. Mechanistically, we screened RASAL2, one cargo of IPO5, and further confirmed that IPO5 bound to the NLS sequence of RASAL2, mediating RASAL2 nuclear translocation and inducing RAS signal activation, thereby promoting the progression of CRC. CONCLUSIONS: Together, our results indicate that IPO5 is overexpressed in colorectal cancer cells. By transporting RASAL2, IPO5 may play a crucial role in CRC.


Assuntos
Proteínas de Transporte/metabolismo , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , beta Carioferinas/genética , beta Carioferinas/metabolismo , Transporte Ativo do Núcleo Celular , Adulto , Idoso , Animais , Proteínas de Transporte/química , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Neoplasias Colorretais/patologia , Modelos Animais de Doenças , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Fluoruracila/farmacologia , Xenoenxertos , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Modelos Biológicos , Gradação de Tumores , Metástase Neoplásica , Estadiamento de Neoplasias , Sinais de Localização Nuclear , Ligação Proteica , Carga Tumoral
6.
PLoS One ; 14(6): e0217897, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31170242

RESUMO

Protein-protein interactions are central to biological processes. In vitro methods to examine protein-protein interactions are generally categorized into two classes: in-solution and surface-based methods. Here, using the multivalent interactions between nucleocytoplasmic transport factors and intrinsically disordered FG repeat containing nuclear pore complex proteins as a model system, we examined the utility of three surface-based methods: atomic force microscopy, quartz crystal microbalance with dissipation, and surface plasmon resonance. Although results were comparable to those of previous reports, the apparent effect of mass transport limitations was demonstrated. Additional experiments with a loss-of-interaction FG repeat mutant variant demonstrated that the binding events that take place on surfaces can be unexpectedly complex, suggesting particular care must be exercised in interpretation of such data.


Assuntos
Núcleo Celular/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/química , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Sequências Repetitivas de Ácido Nucleico , Transporte Ativo do Núcleo Celular , Sequência de Aminoácidos , Mutação/genética , Ligação Proteica , Técnicas de Microbalança de Cristal de Quartzo , beta Carioferinas/metabolismo
7.
J Biol Chem ; 294(24): 9631-9641, 2019 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-31064840

RESUMO

Serine-arginine (SR) proteins are essential splicing factors that promote numerous steps associated with mRNA processing and whose biological function is tightly regulated through multi-site phosphorylation. In the nucleus, the cdc2-like kinases (CLKs) phosphorylate SR proteins on their intrinsically disordered Arg-Ser (RS) domains, mobilizing them from storage speckles to the splicing machinery. The CLKs have disordered N termini that bind tightly to RS domains, enhancing SR protein phosphorylation. The N termini also promote nuclear localization of CLKs, but their transport mechanism is presently unknown. To explore cytoplasmic-nuclear transitions, several classical nuclear localization sequences in the N terminus of the CLK1 isoform were identified, but their mutation had no effect on subcellular localization. Rather, we found that CLK1 amplifies its presence in the nucleus by forming a stable complex with the SR protein substrate and appropriating its NLS for transport. These findings indicate that, along with their well-established roles in mRNA splicing, SR proteins use disordered protein-protein interactions to carry their kinase regulator from the cytoplasm to the nucleus.


Assuntos
Arginina/metabolismo , Núcleo Celular/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Serina/metabolismo , Sequência de Aminoácidos , Células HeLa , Humanos , Fosforilação , Conformação Proteica , Proteínas Serina-Treonina Quinases/química , Proteínas Tirosina Quinases/química , Homologia de Sequência , Fatores de Processamento de Serina-Arginina/metabolismo , Especificidade por Substrato , beta Carioferinas/metabolismo
8.
Int J Oncol ; 54(6): 2039-2053, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30942445

RESUMO

Glioblastoma harbors frequent alterations in receptor tyrosine kinases, phosphatidylinositol­3 kinase (PI3K) and phosphatase and tensin homolog (PTEN) that dysregulate phospholipid signaling driven tumor proliferation and therapeutic resistance. Myristoylated alanine­rich C­kinase substrate (MARCKS) is a 32 kDa intrinsically unstructured protein containing a polybasic (+13) effector domain (ED), which regulates its electrostatic sequestration of phospholipid phosphatidylinositol (4,5)­bisphosphate (PIP2), and its binding to phosphatidylserine, calcium/calmodulin, filamentous actin, while also serving as a nuclear localization sequence. MARCKS ED is phosphorylated by protein kinase C (PKC) and Rho­associated protein kinase (ROCK) kinases; however, the impact of MARCKS on glioblastoma growth and radiation sensitivity remains undetermined. In the present study, using a tetracycline­inducible system in PTEN­null U87 cells, we demonstrate that MARCKS overexpression suppresses growth and enhances radiation sensitivity in vivo. A new image cytometer, Xcyto10, was utilized to quantify differences in MARCKS ED phosphorylation on localization and its association with filamentous actin. The overexpression of the non­phosphorylatable ED mutant exerted growth­suppressive and radiation­sensitizing effects, while the pseudo­phosphorylated ED mutant exhibited an enhanced colony formation and clonogenic survival ability. The identification of MARCKS protein­protein interactions using co­immunoprecipitation coupled with tandem mass spectrometry revealed novel MARCKS­associated proteins, including importin­ß and ku70. On the whole, the findings of this study suggest that the determination of the MARCKS ED phosphorylation status is essential to understanding the impact of MARCKS on cancer progression.


Assuntos
Neoplasias Encefálicas/patologia , Glioblastoma/patologia , Substrato Quinase C Rico em Alanina Miristoilada/metabolismo , Domínios Proteicos , Tolerância a Radiação , Animais , Neoplasias Encefálicas/mortalidade , Neoplasias Encefálicas/radioterapia , Linhagem Celular Tumoral , Proliferação de Células , Progressão da Doença , Feminino , Glioblastoma/mortalidade , Glioblastoma/radioterapia , Humanos , Autoantígeno Ku/metabolismo , Camundongos , Camundongos Nus , Fosforilação , Mapeamento de Interação de Proteínas , Análise de Sobrevida , Resultado do Tratamento , Ensaios Antitumorais Modelo de Xenoenxerto , beta Carioferinas/metabolismo
9.
Kaohsiung J Med Sci ; 35(3): 139-145, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30887716

RESUMO

Accumulating evidences indicates that chronic neuropathic pain is a kind of neuro-immune disorder with enhanced activation of the immune system. Although the prevalence is very high, neuropathic pain remains extremely difficult to cure. miRNAs are a group of short nonprotein coding RNAs, regulating target genes expression via targeting 3'-untranslated region. More and more research indicates that altered miRNAs expression profile relates to the pathogenesis of neuropathic pain. In this study, we firstly detected the expression of six candidate miRNAs in the plasma samples from 23 patients with neuropathic pain and 10 healthy controls. Subsequently, the level of miR-132 and miR-101 was detected in the sural nerve biopsies. We found miR-101 level was significantly repressed in both the plasma samples and sural nerve biopsies from neuropathic pain patients. Predicted by bioinformatics tools and confirmed by dual luciferase assay and immunoblotting, we identified that KPNB1 is a direct target of miR-101. The negative correlation between miR-101 and KPNB1 was also confirmed in the sural nerve biopsies, and miR-101 reduction relates to the activation of NF-κB signaling in vivo and in vitro which contributes to the pathogenesis of neuropathic pain.


Assuntos
Dor Crônica/genética , MicroRNAs/metabolismo , NF-kappa B/metabolismo , Neuralgia/genética , Transdução de Sinais , beta Carioferinas/metabolismo , Regiões 3' não Traduzidas/genética , Adulto , Idoso , Sequência de Bases , Estudos de Casos e Controles , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Dor Crônica/sangue , Feminino , Regulação da Expressão Gênica , Células HEK293 , Humanos , Interleucina-1beta/metabolismo , Masculino , MicroRNAs/sangue , MicroRNAs/genética , Pessoa de Meia-Idade , Neuralgia/sangue , Fator de Transcrição RelA/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , beta Carioferinas/genética
10.
Cell Death Dis ; 10(2): 118, 2019 02 11.
Artigo em Inglês | MEDLINE | ID: mdl-30742128

RESUMO

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.


Assuntos
Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , beta Carioferinas/metabolismo , Fator 4 Ativador da Transcrição/metabolismo , Animais , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Caspase 8/metabolismo , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Feminino , Glioblastoma/tratamento farmacológico , Glioblastoma/metabolismo , Glioblastoma/patologia , Humanos , Camundongos , Camundongos Nus , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/uso terapêutico , beta Carioferinas/antagonistas & inibidores , beta Carioferinas/genética
11.
EMBO J ; 38(6)2019 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-30745319

RESUMO

DSCAM and DSCAML1 are immunoglobulin and cell adhesion-type receptors serving important neurodevelopmental functions including control of axon growth, branching, neurite self-avoidance, and neuronal cell death. The signal transduction mechanisms or effectors of DSCAM receptors, however, remain poorly characterized. We used a human ORFeome library to perform a high-throughput screen in mammalian cells and identified novel cytoplasmic signaling effector candidates including the Down syndrome kinase Dyrk1a, STAT3, USP21, and SH2D2A. Unexpectedly, we also found that the intracellular domains (ICDs) of DSCAM and DSCAML1 specifically and directly interact with IPO5, a nuclear import protein of the importin beta family, via a conserved nuclear localization signal. The DSCAM ICD is released by γ-secretase-dependent cleavage, and both the DSCAM and DSCAML1 ICDs efficiently translocate to the nucleus. Furthermore, RNA sequencing confirms that expression of the DSCAM as well as the DSCAML1 ICDs alone can profoundly alter the expression of genes associated with neuronal differentiation and apoptosis, as well as synapse formation and function. Gain-of-function experiments using primary cortical neurons show that increasing the levels of either the DSCAM or the DSCAML1 ICD leads to an impairment of neurite growth. Strikingly, increased expression of either full-length DSCAM or the DSCAM ICD, but not the DSCAML1 ICD, significantly decreases synapse numbers in primary hippocampal neurons. Taken together, we identified a novel membrane-to-nucleus signaling mechanism by which DSCAM receptors can alter the expression of regulators of neuronal differentiation and synapse formation and function. Considering that chromosomal duplications lead to increased DSCAM expression in trisomy 21, our findings may help uncover novel mechanisms contributing to intellectual disability in Down syndrome.


Assuntos
Transporte Ativo do Núcleo Celular , Moléculas de Adesão Celular/metabolismo , Núcleo Celular/metabolismo , Neuritos/fisiologia , Sinapses/fisiologia , Secretases da Proteína Precursora do Amiloide/genética , Secretases da Proteína Precursora do Amiloide/metabolismo , Animais , Adesão Celular , Moléculas de Adesão Celular/genética , Núcleo Celular/genética , Células HEK293 , Hipocampo/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Neurogênese , Neurônios/metabolismo , Domínios Proteicos , Domínios e Motivos de Interação entre Proteínas , beta Carioferinas/genética , beta Carioferinas/metabolismo
12.
Int J Mol Sci ; 20(2)2019 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-30650545

RESUMO

LRRC59 (leucine-rich repeat-containing protein 59) is a tail-anchored protein with a single transmembrane domain close to its C-terminal end that localizes to the endoplasmic reticulum (ER) and the nuclear envelope. Here, we investigate the mechanisms of membrane integration of LRRC59 and its targeting to the inner nuclear membrane (INM). Using purified microsomes, we show that LRRC59 can be post-translationally inserted into ER-derived membranes. The TRC-pathway, a major route for post-translational membrane insertion, is not required for LRRC59. Like emerin, another tail-anchored protein, LRRC59 reaches the INM, as demonstrated by rapamycin-dependent dimerization assays. Using different approaches to inhibit importin α/ß-dependent nuclear import of soluble proteins, we show that the classic nuclear transport machinery does not play a major role in INM-targeting of LRRC59. Instead, the size of the cytoplasmic domain of LRRC59 is an important feature, suggesting that targeting is governed by passive diffusion.


Assuntos
Retículo Endoplasmático/metabolismo , Proteínas de Membrana/metabolismo , Membrana Nuclear/metabolismo , Células HeLa , Humanos , Proteínas de Membrana/química , Microssomos/metabolismo , Modelos Biológicos , Domínios Proteicos , Processamento de Proteína Pós-Traducional , Transporte Proteico , Relação Estrutura-Atividade , beta Carioferinas/metabolismo
13.
Nat Microbiol ; 4(4): 578-586, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30692667

RESUMO

Influenza A virus is a pathogen of great medical impact. To develop novel antiviral strategies, it is essential to understand the molecular aspects of virus-host cell interactions in detail. During entry, the viral ribonucleoproteins (vRNPs) that carry the RNA genome must be released from the incoming particle before they can enter the nucleus for replication. The uncoating process is facilitated by histone deacetylase 6 (ref.1). However, the precise mechanism of shell opening and vRNP debundling is unknown. Here, we show that transportin 1, a member of the importin-ß family proteins, binds to a PY-NLS2 sequence motif close to the amino terminus of matrix protein (M1) exposed during acid priming of the viral core. It promotes the removal of M1 and induces disassembly of vRNP bundles. Next, the vRNPs interact with importin-α/ß and enter the nucleus. Thus, influenza A virus uses dual importin-ßs for distinct steps in host cell entry.


Assuntos
Vírus da Influenza A/fisiologia , Influenza Humana/metabolismo , Ribonucleoproteínas/metabolismo , Proteínas Virais/metabolismo , Internalização do Vírus , beta Carioferinas/metabolismo , Núcleo Celular/genética , Núcleo Celular/metabolismo , Núcleo Celular/virologia , Humanos , Vírus da Influenza A/genética , Influenza Humana/genética , Influenza Humana/virologia , Ribonucleoproteínas/genética , Proteínas da Matriz Viral/genética , Proteínas da Matriz Viral/metabolismo , Proteínas Virais/genética , Replicação Viral
14.
BMC Cancer ; 18(1): 1123, 2018 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-30445944

RESUMO

BACKGROUND: Karyopherin ß1 (Kpnß1) is the main nuclear import protein involved in the transport of cargoes from the cytoplasm into the cell nucleus. Previous research has found Kpnß1 to be significantly overexpressed in cervical cancer and other cancer tissues, and further studies showed that inhibition of Kpnß1 expression by siRNA resulted in cancer cell death, while non-cancer cells were minimally affected. These results suggest that Kpnß1 has potential as an anticancer therapeutic target, thus warranting further research into the association between Kpnß1 expression and cancer progression. Here, the biological effects associated with Kpnß1 overexpression were investigated in order to further elucidate the relationship between Kpnß1 and the cancer phenotype. METHODS: To evaluate the effect of Kpnß1 overexpression on cell biology, cell proliferation, cell cycle, cell morphology and cell adhesion assays were performed. To determine whether Kpnß1 overexpression influences cell sensitivity to chemotherapeutic agents like Cisplatin, cell viability assays were performed. Expression levels of key proteins were analysed by Western blot analysis. RESULTS: Our data revealed that Kpnß1 overexpression, above that which was already detected in cancer cells, resulted in reduced proliferation of cervical cancer cells. Likewise, normal epithelial cells showed reduced proliferation after Kpnß1 overxpression. Reduced cancer cell proliferation was associated with a delay in cell cycle progression, as well as changes in the morphology and adhesion properties of cells. Additionally, Kpnß1 overexpressing HeLa cells exhibited increased sensitivity to cisplatin, as shown by decreased cell viability and increased apoptosis, where p53 and p21 inhibition reduced and enhanced cell sensitivity to Cisplatin, respectively. CONCLUSIONS: Overall, our results suggest that a tight balance of Kpnß1 expression is required for cellular function, and that perturbation of this balance results in negative effects associated with a variety of biological processes.


Assuntos
Neoplasias do Colo do Útero/metabolismo , beta Carioferinas/metabolismo , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Adesão Celular/efeitos dos fármacos , Adesão Celular/fisiologia , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/fisiologia , Linhagem Celular Tumoral , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/fisiologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Cisplatino/farmacologia , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Citoplasma/efeitos dos fármacos , Citoplasma/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/fisiologia , Células HeLa , Humanos , RNA Interferente Pequeno/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Neoplasias do Colo do Útero/tratamento farmacológico
15.
PLoS Pathog ; 14(11): e1007408, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30496303

RESUMO

Human myxovirus resistance 2 (MX2/MXB) is an interferon-induced post-entry inhibitor of human immunodeficiency virus type-1 (HIV-1) infection. While the precise mechanism of viral inhibition remains unclear, MX2 is localized to the nuclear envelope, and blocks the nuclear import of viral cDNAs. The amino-terminus of MX2 (N-MX2) is essential for anti-viral function, and mutation of a triple arginine motif at residues 11 to 13 abrogates anti-HIV-1 activity. In this study, we sought to investigate the role of N-MX2 in anti-viral activity by identifying functionally relevant host-encoded interaction partners through yeast-two-hybrid screening. Remarkably, five out of seven primary candidate interactors were nucleoporins or nucleoporin-like proteins, though none of these candidates were identified when screening with a mutant RRR11-13A N-MX2 fragment. Interactions were confirmed by co-immunoprecipitation, and RNA silencing experiments in cell lines and primary CD4+ T cells demonstrated that multiple components of the nuclear pore complex and nuclear import machinery can impact MX2 anti-viral activity. In particular, the phenylalanine-glycine (FG) repeat containing cytoplasmic filament nucleoporin NUP214, and transport receptor transportin-1 (TNPO1) were consistently required for full MX2, and interferon-mediated, anti-viral function. Both proteins were shown to interact with the triple arginine motif, and confocal fluorescence microscopy revealed that their simultaneous depletion resulted in diminished MX2 accumulation at the nuclear envelope. We therefore propose a model whereby multiple components of the nuclear import machinery and nuclear pore complex help position MX2 at the nuclear envelope to promote MX2-mediated restriction of HIV-1.


Assuntos
Infecções por HIV/metabolismo , HIV-1/fisiologia , Proteínas de Resistência a Myxovirus/metabolismo , Transporte Ativo do Núcleo Celular , Antivirais/metabolismo , Células HEK293 , Infecções por HIV/virologia , Células HeLa , Interações Hospedeiro-Patógeno , Humanos , Interferons/metabolismo , Proteínas de Resistência a Myxovirus/genética , Membrana Nuclear/metabolismo , Poro Nuclear/metabolismo , Poro Nuclear/fisiologia , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Replicação Viral , beta Carioferinas/metabolismo
16.
J Am Chem Soc ; 140(49): 17234-17240, 2018 12 12.
Artigo em Inglês | MEDLINE | ID: mdl-30398334

RESUMO

The efficient delivery of proteins into cells is needed to fully realize the potential of protein-based therapeutics. Current protein delivery strategies generally suffer from poor endosomal escape and low tolerance for serum. Here, the genetic fusion of a supercharged polypeptide, called SCP, to a protein provides a generic method for intracellular protein delivery. It allows efficient protein endocytosis and endosomal escape and is capable of potently delivering various proteins with a range of charges, sizes, and bioactivities into the nucleus of living cells. SCP is discovered to bind directly to the nuclear import protein importin ß1 and gains access to the nucleus. Furthermore, SCP shows minimal hemolytic activity and stability in serum and lacks toxicity and immunogenicity in vivo. Effective gene editing can be achieved by SCP-mediated delivery of Cas9 protein and guide RNA. This study may provide an efficient and useful tool for the design and development of cell-nuclear-targeted drug delivery.


Assuntos
Peptídeos Penetradores de Células/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Animais , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Peptídeos Penetradores de Células/sangue , Peptídeos Penetradores de Células/genética , Peptídeos Penetradores de Células/toxicidade , Endocitose/fisiologia , Escherichia coli/genética , Feminino , Humanos , Camundongos Endogâmicos BALB C , Estabilidade Proteica , Proteínas Recombinantes de Fusão/sangue , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/toxicidade , beta Carioferinas/metabolismo
17.
Biochem Biophys Res Commun ; 507(1-4): 67-73, 2018 12 09.
Artigo em Inglês | MEDLINE | ID: mdl-30396568

RESUMO

Mutations in the gene encoding BCL-6 corepressor (BCOR) are responsible for oculofaciocardiodental (OFCD) syndrome, which is a rare X-linked dominant disorder characterized by radiculomegaly of permanent teeth as the most typical symptom. To function as a transcriptional corepressor, BCOR needs to enter the nucleus; however, the molecular pathway for its nuclear translocation during dental root formation remains unclear. The purpose of this study was to determine the mechanism underlying BCOR transport into the nucleus. Our results showed that human periodontal ligament (PDL) cells expressed karyopherin α (KPNA)2, KPNA4, and KPNA6 belonging to a family of nuclear import proteins, which interacted with BCOR in the immunoprecipitation assay. Site-directed mutagenesis targeting the two nuclear localization signals (NLSs) within BCOR reduced its nuclear translocation; however, co-expression of KPNA2, KPNA4, or KPNA6 with BCOR carrying a previously described mutation which eliminated one of the two NLSs significantly increased nuclear accumulation of the mutant BCOR, indicating participation of KPNA in BCOR nuclear translocation. Comparative expression profiling of PDL cells isolated from normal and OFCD patients revealed significant downregulation of SMAD4, GLI1, and nuclear factor 1-C (NFIC) mRNA expression, suggesting that BCOR mutations cause hyperactive root formation in OFCD syndrome by inhibiting SMAD4-Hedgehog-NFIC signaling implicated in dental root development. Our study contributes to understanding of the mechanisms providing nuclear import of BCOR during root formation.


Assuntos
Núcleo Celular/metabolismo , Ligamento Periodontal/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Repressoras/metabolismo , alfa Carioferinas/metabolismo , Transporte Ativo do Núcleo Celular , Adolescente , Adulto , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Células COS , Criança , Regulação para Baixo/genética , Humanos , Masculino , Proteínas Mutantes/metabolismo , Mutação/genética , Sinais de Localização Nuclear/metabolismo , Ligação Proteica , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas/genética , Proteínas Repressoras/química , Proteínas Repressoras/genética , Frações Subcelulares/metabolismo , Transcrição Genética , beta Carioferinas/metabolismo
18.
Exp Cell Res ; 371(2): 372-378, 2018 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-30153455

RESUMO

The neural cell adhesion molecule (NCAM) is important for neural development and for plasticity in adult brain. Previous studies demonstrated a calmodulin-dependent import of a transmembrane fragment of NCAM into the nucleus that regulates gene expression. In a protein macroarray we identified importin-ß1 as a potential interaction partner of NCAM's cytoplasmic tail. The interaction was verified and an importin-ß1-dependent import of NCAM into the nucleus could be demonstrated using quantitative immunofluorescence analysis. Generation of NCAM deletion mutants revealed that the last amino acids of the cytoplasmic region of NCAM are dispensable whereas other parts of NCAM's cytoplasmic tail take part in its nuclear translocation. With this study we propose an alternative nuclear route for NCAM via the classical importin-mediated import.


Assuntos
Moléculas de Adesão Celular Neuronais/metabolismo , Núcleo Celular/metabolismo , Citosol/metabolismo , Neurônios/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , beta Carioferinas/metabolismo , Transporte Ativo do Núcleo Celular/genética , Animais , Células COS , Moléculas de Adesão Celular Neuronais/genética , Linhagem Celular Tumoral , Núcleo Celular/ultraestrutura , Citosol/ultraestrutura , Expressão Gênica , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Humanos , Neurônios/ultraestrutura , Análise Serial de Proteínas , Ligação Proteica , Transporte Proteico , Ratos , Proteínas Recombinantes de Fusão/genética , beta Carioferinas/genética
19.
Biochem Biophys Res Commun ; 503(3): 1792-1797, 2018 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-30064907

RESUMO

Importin α is nuclear transport receptor that recognises nuclear localisation sequences (NLS). The protein has two domains: armadillo (ARM) repeats containing NLS-binding sites and the importin ß-binding (IBB) domain. The IBB domain mimics an NLS and can bind to the ARM repeats, preventing NLS binding. This phenomenon, called auto-inhibition, is a key regulatory feature for binding and release of NLS-containing cargo by importin α and mutants that lack auto-inhibition show impaired viability in Saccharomyces cerevisiae. The genome of the human malaria parasite, Plasmodium falciparum, contains a single gene for importin α and here we show that the native protein expressed by this gene lacks auto-inhibition, suggesting that P. falciparum parasites possess unconventional mechanisms for regulation of cargo binding and release. Mutation of a single SKR motif (conserved in Plasmodium species) to KRR in P. falciparum importin α restores auto-inhibition. This is the first report of a single-celled eukaryote that has evolved with a single importin α isoform lacking auto-inhibition.


Assuntos
Plasmodium falciparum/metabolismo , beta Carioferinas/metabolismo , Clonagem Molecular , Sinais de Localização Nuclear/metabolismo , Plasmodium falciparum/genética , beta Carioferinas/genética
20.
Exp Cell Res ; 370(2): 454-460, 2018 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-29981748

RESUMO

YB-1 nuclear translocation/accumulation caused by anticancer agents leads to malignant transformation. Nuclear import of YB-1 requires a nuclear localization signal (YB-NLS). Previously, we identified five nucleocytoplasmic-shuttling proteins as YB-NLS binding proteins, and showed that they co-accumulate in the nucleus with YB-1 in response to treatment with actinomycin D. In addition, another group reported that transportin-1 is the molecule responsible for YB-1 nuclear translocation, binding to a region (PY-NLS) consistent with the YB-NLS. Recently, we found that indirubin 3'-oxime inhibits the nuclear localization of YB-1 in HepG2 cells and increases their sensitivity to actinomycin D. Here, we found that YB-1 nuclear translocation is dependent on the cellular mRNA level and that indirubin 3'-oxime inhibits the interaction between YB-1 and transportin-1. Interestingly, in cells showing inhibition of actinomycin D-induced YB-1 nuclear translocation by the compound, the YB-NLS-binding proteins as well as transportin-1 and its cargos were imported to the nucleus. Furthermore, the compound inhibited nuclear localization of the GFP-conjugated full-length YB-1 but not that of GFP-conjugated YB-NLS. These results indicate that indirubin 3'-oxime is a specific inhibitor of anticancer agent-induced YB-1 nuclear translocation, interacting with YB-1 itself in a region other than the YB-NLS/PY-NLS. This compound would increase the efficacy of cancer therapy.


Assuntos
Carcinoma Hepatocelular/tratamento farmacológico , Núcleo Celular/efeitos dos fármacos , Indóis/farmacologia , Sinais de Localização Nuclear/efeitos dos fármacos , Oximas/farmacologia , Antineoplásicos/farmacologia , Carcinoma Hepatocelular/metabolismo , Núcleo Celular/metabolismo , Células Hep G2/efeitos dos fármacos , Humanos , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/metabolismo , Proteínas de Transporte Nucleocitoplasmático/metabolismo , beta Carioferinas/efeitos dos fármacos , beta Carioferinas/metabolismo
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