Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 1.290
Filtrar
1.
J Med Chem ; 64(10): 6596-6607, 2021 05 27.
Artigo em Inglês | MEDLINE | ID: mdl-33974430

RESUMO

Nuclear export factor chromosome region maintenance 1 (CRM1) is an attractive anticancer and antiviral drug target that spurred several research efforts to develop its inhibitor. Noncovalent CRM1 inhibitors are desirable, but none is reported to date. Here, we present the crystal structure of yeast CRM1 in complex with S109, a substructure of CBS9106 (under clinical test). Superimposition with the LFS-829 (another covalent CRM1 inhibitor) complex inspired the design of a noncovalent CRM1 inhibitor. Among nine synthesized compounds, noncovalent CRM1 inhibitor 1 (NCI-1) showed a high affinity to human and yeast CRM1 in the absence or presence of GST-bound Ras-related nuclear protein (RanGTP). Unlike covalent inhibitors, the crystal structure showed that NCI-1 is bound in the "open" nuclear export signal (NES) groove of CRM1, simultaneously occupying two hydrophobic pockets. NCI-1 additionally inhibited the nuclear export and proliferation of cells harboring the human CRM1-C528S mutant. Our work opens up the avenue of noncovalent CRM1 inhibitor development toward a more potent, less toxic, and broad-spectrum anticancer/antiviral therapy.


Assuntos
Desenho de Fármacos , Proteínas Fúngicas/antagonistas & inibidores , Carioferinas/antagonistas & inibidores , Receptores Citoplasmáticos e Nucleares/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/química , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Antineoplásicos/química , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Sítios de Ligação , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cristalografia por Raios X , Ensaios de Seleção de Medicamentos Antitumorais , Proteínas Fúngicas/metabolismo , Humanos , Carioferinas/metabolismo , Simulação de Dinâmica Molecular , Piridinas/química , Piridinas/metabolismo , Piridinas/farmacologia , Receptores Citoplasmáticos e Nucleares/metabolismo , Saccharomyces cerevisiae/metabolismo , Bibliotecas de Moléculas Pequenas/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia
2.
Chimia (Aarau) ; 75(4): 319-322, 2021 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-33902802

RESUMO

Cellular homeostasis importantly relies on the correct nucleoplasmic distribution of a large number of RNA molecules and proteins, which are shuttled by specialized transport receptors. The nuclear import receptor importin-5, also called IPO5, RanBP5 or karyopherin ß3, mediates the translocation of proteins to the nucleus, and thus regulates critical signaling pathways and cellular functions. The normal function of IPO5 appears to be disrupted in cancer cells due to aberrant overexpression. IPO5 also demonstrated a pivotal role in viral replication. The constant increasing number of publications shows an interest within the scientific community as a therapeutic target due to its pivotal role in protein trafficking.


Assuntos
Proteínas Nucleares , beta Carioferinas , Transporte Ativo do Núcleo Celular , Núcleo Celular/metabolismo , Carioferinas/metabolismo , Proteínas Nucleares/metabolismo , beta Carioferinas/metabolismo
3.
Int J Nanomedicine ; 16: 2833-2847, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33883894

RESUMO

Introduction: Peptides can be rationally designed as non-covalent inhibitors for molecularly targeted therapy. However, it remains challenging to efficiently deliver the peptides into the targeted cells, which often severely affects their therapeutic efficiency. Methods: Herein, we created a novel non-covalent peptide inhibitor against nuclear export factor CRM1 by a structure-guided drug design method and targetedly delivered the peptide into cancer cells by a nanoparticle-mediated gene expression system for use as a cancer therapy. Results: The nuclear export signal (NES)-optimized CRM1 peptide inhibitor colocalized with CRM1 to the nuclear envelope and inhibited nuclear export in cancer cell lines in vitro. The crystal structures of the inhibitors complexed with CRM1 were solved. In contrast to the covalent inhibitors, the peptides were similarly effective against cells harboring the CRM1 C528S mutation. Moreover, a plasmid encoding the peptides was delivered by a iRGD-modified nanoparticle to efficiently target and transfect the cancer cells in vivo after intravenous administration. The peptides could be selectively expressed in the tumor, resulting in the efficient inhibition of subcutaneous melanoma xenografts without obvious systemic toxicity. Discussion: This work provides an effective strategy to design peptide-based molecularly targeted therapeutics, which could lead to the development of future targeted therapy.


Assuntos
Espaço Intracelular/metabolismo , Carioferinas/antagonistas & inibidores , Melanoma Experimental/tratamento farmacológico , Nanopartículas/química , Peptídeos/farmacologia , Peptídeos/uso terapêutico , Receptores Citoplasmáticos e Nucleares/antagonistas & inibidores , Células A549 , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Sequência de Aminoácidos , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Técnicas de Transferência de Genes , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Carioferinas/química , Carioferinas/metabolismo , Melanoma Experimental/patologia , Proteínas Mutantes/metabolismo , Mutação/genética , Nanopartículas/ultraestrutura , Sinais de Exportação Nuclear , Peptídeos/química , Ligação Proteica/efeitos dos fármacos , Receptores Citoplasmáticos e Nucleares/química , Receptores Citoplasmáticos e Nucleares/metabolismo , Proteínas não Estruturais Virais/química
4.
J Cancer Res Clin Oncol ; 147(7): 2025-2033, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33856525

RESUMO

BACKGROUND: The nuclear pore complexes (NPCs) are built of about 30 different nucleoporins and act as key regulators of molecular traffic between the cytoplasm and the nucleus for sizeable proteins (> 40 kDa) which must enter the nucleus. Various nuclear transport receptors are involved in import and export processes of proteins through the nuclear pores. The most prominent nuclear export receptor is chromosome region maintenance 1 (CRM1), also known as exportin 1 (XPO1). One of its cargo proteins is the prolyl hydroxylase 2 (PHD2) which is involved in the initiation of the degradation of hypoxia-inducible factors (HIFs) under normoxia. HIFs are proteins that regulate the cellular adaptation under hypoxic conditions. They are involved in many aspects of cell viability and play an important role in the hypoxic microenvironment of cancer. In cancer, CRM1 is often overexpressed thus being a putative target for the development of new cancer therapies. The newly FDA-approved pharmaceutical Selinexor (KPT-330) selectively inhibits nuclear export via CRM1 and is currently tested in additional Phase-III clinical trials. In this study, we investigated the effect of CRM1 inhibition on the subcellular localization of HIF-1α and radiosensitivity. METHODS: Human hepatoma cells Hep3B and human osteosarcoma cells U2OS were treated with Selinexor. Intranuclear concentration of HIF-1α protein was measured using immunoblot analysis. Furthermore, cells were irradiated with 2-8 Gy after treatment with Selinexor compared to untreated controls. RESULTS: Selinexor significantly reduced the intranuclear level of HIF-1α protein in human hepatoma cells Hep3B and human osteosarcoma cells U2OS. Moreover, we demonstrated by clonogenic survival assays that Selinexor leads to dose-dependent radiosensitization in Hep3B-hepatoma and U2OS-osteosarcoma cells. CONCLUSION: Targeting the HIF pathway by Selinexor might be an attractive tool to overcome hypoxia-induced radioresistance.


Assuntos
Carcinoma Hepatocelular/radioterapia , Hidrazinas/farmacologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Carioferinas/antagonistas & inibidores , Osteossarcoma/radioterapia , Tolerância a Radiação/efeitos dos fármacos , Radiossensibilizantes/farmacologia , Receptores Citoplasmáticos e Nucleares/antagonistas & inibidores , Triazóis/farmacologia , Apoptose , Neoplasias Ósseas/tratamento farmacológico , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Neoplasias Ósseas/radioterapia , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Carioferinas/genética , Carioferinas/metabolismo , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/radioterapia , Osteossarcoma/tratamento farmacológico , Osteossarcoma/metabolismo , Osteossarcoma/patologia , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Células Tumorais Cultivadas
5.
Nat Commun ; 12(1): 1505, 2021 03 08.
Artigo em Inglês | MEDLINE | ID: mdl-33686072

RESUMO

Survivin's dual function as apoptosis inhibitor and regulator of cell proliferation is mediated via its interaction with the export receptor CRM1. This protein-protein interaction represents an attractive target in cancer research and therapy. Here, we report a sophisticated strategy addressing Survivin's nuclear export signal (NES), the binding site of CRM1, with advanced supramolecular tweezers for lysine and arginine. These were covalently connected to small peptides resembling the natural, self-complementary dimer interface which largely overlaps with the NES. Several biochemical methods demonstrated sequence-selective NES recognition and interference with the critical receptor interaction. These data were strongly supported by molecular dynamics simulations and multiscale computational studies. Rational design of lysine tweezers equipped with a peptidic recognition element thus allowed to address a previously unapproachable protein surface area. As an experimental proof-of-principle for specific transport signal interference, this concept should be transferable to any protein epitope with a flanking well-accessible lysine.


Assuntos
Carioferinas/química , Carioferinas/metabolismo , Domínios e Motivos de Interação entre Proteínas/efeitos dos fármacos , Receptores Citoplasmáticos e Nucleares/química , Receptores Citoplasmáticos e Nucleares/metabolismo , Survivina/química , Survivina/metabolismo , Sítios de Ligação , Proliferação de Células , Humanos , Proteínas Inibidoras de Apoptose/metabolismo , Modelos Moleculares , Sinais de Exportação Nuclear , Ligação Proteica , Conformação Proteica
6.
Biochem Biophys Res Commun ; 545: 105-111, 2021 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-33548622

RESUMO

Transforming growth factor ß1 (TGF-ß1) is one of the broad-spectrum growth-promoting factors that participate in tooth development. The influence of TGF-ß1 on the odontoblastic differentiation is still controvercy. Mouse primary dental papilla cells (mDPCs) as well as an immortalized mouse dental papilla cell line (mDPC6Ts) were treated with exogenous TGF-ß1 during odontoblastic differentiation. RT-qPCR, Western blot, alizarin red staining and ALP staining were carried out to investigate the influence of TGF-ß1 on odontoblastic differentiation. IPO7, important for SMAD complex translocation was also detected in mDPCs and mDPC6Ts in response to TGF-ß1. After silencing IPO7 by transfection, the translocation process of P-SMAD2 was investigated by nuclear and cytoplasmic extraction as well as co-immunoprecipitation assay. The odontogenic markers, mineralization and IPO7 expression were significantly up-regulated in TGF-ß1-treated mDPCs while down-regulated in mDPC6Ts. The total level of P-SMAD2 was not influenced by IPO7 in mDPCs, however, IPO7 could bind to P-SMAD2 and affect the nuclear-cytoplasm-shuttling of P-SMAD2. Our data demonstrated that TGF-ß1 plays opposite roles in odontoblast differentiation in mDPCs and immortalized mouse dental papilla cell line (mDPC6Ts), which is determined by IPO7.


Assuntos
Carioferinas/metabolismo , Odontoblastos/citologia , Odontoblastos/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Linhagem Celular , Células Cultivadas , Papila Dentária , Regulação para Baixo , Técnicas de Silenciamento de Genes , Carioferinas/antagonistas & inibidores , Carioferinas/genética , Camundongos , Odontogênese/genética , Odontogênese/fisiologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteína Smad2/metabolismo , Regulação para Cima
7.
Science ; 371(6536): 1350-1355, 2021 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-33632892

RESUMO

Mitogens trigger cell division in animals. In plants, cytokinins, a group of phytohormones derived from adenine, stimulate cell proliferation. Cytokinin signaling is initiated by membrane-associated histidine kinase receptors and transduced through a phosphorelay system. We show that in the Arabidopsis shoot apical meristem (SAM), cytokinin regulates cell division by promoting nuclear shuttling of Myb-domain protein 3R4 (MYB3R4), a transcription factor that activates mitotic gene expression. Newly synthesized MYB3R4 protein resides predominantly in the cytoplasm. At the G2-to-M transition, rapid nuclear accumulation of MYB3R4-consistent with an associated transient peak in cytokinin concentration-feeds a positive feedback loop involving importins and initiates a transcriptional cascade that drives mitosis and cytokinesis. An engineered nuclear-restricted MYB3R4 mimics the cytokinin effects of enhanced cell proliferation and meristem growth.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/citologia , Arabidopsis/metabolismo , Divisão Celular , Citocininas/metabolismo , Transativadores/metabolismo , Transporte Ativo do Núcleo Celular , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Pontos de Checagem do Ciclo Celular , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Regulação da Expressão Gênica de Plantas , Carioferinas/metabolismo , Meristema/metabolismo , Mitose/genética , Reguladores de Crescimento de Plantas/metabolismo , Transdução de Sinais , Transativadores/genética
8.
Ecotoxicol Environ Saf ; 208: 111668, 2021 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-33396178

RESUMO

Cadmium is an environmental metal pollutant that has been a focus of research in recent years, which is reported to cause bone disease; however, its skeletal toxicity and the mechanism involved are not yet fully known. Therefore, this study used MC3T3-E1 subclone 14 cells to determine the mechanism of cadmium toxicity on bone. Cadmium chloride (Cd) significantly reduced cell viability in a concentration-dependent manner. Exposure to Cd inhibited osteoblast-related proteins (Runx2, Col-1, STC2) and decreased alkaline phosphatase (ALP) activity. Cd caused Exportin-1 accumulation and induced DNA damage. Cd significantly down-regulated caspase 9 and induced cleaved-PARP, cleaved-caspase 3 protein level. Treatment with JNK inhibitor, SP600125, suppressed cadmium-induced elevation in the ratio of phosphorylation of JNK to JNK. Inhibition of caspase with pan-caspase inhibitor, Z-VAD-FMK, prevented MC3T3-E1 subclone 14 cells from cadmium-induced reduction of Runx2, STC2, caspase 9, and accumulation of cleaved PARP and cleaved caspase 3. Cd-induced cell survival enhanced by SP600125 but rescued by Z-VAD-FMK or KPT-335. These results suggest that cadmium cytotoxicity on bone involved exportin 1 accumulation, phosphorylation of JNK, induction of DNA damage and pro-apoptosis, which was induced by activation of caspase-dependent pathways.


Assuntos
Apoptose/efeitos dos fármacos , Cádmio/toxicidade , Dano ao DNA/efeitos dos fármacos , Carioferinas/metabolismo , MAP Quinase Quinase 4/metabolismo , Osteoblastos/efeitos dos fármacos , Receptores Citoplasmáticos e Nucleares/metabolismo , Animais , Caspases/metabolismo , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Camundongos , Osteoblastos/metabolismo , Osteoblastos/patologia , Fosforilação/efeitos dos fármacos
9.
Nat Commun ; 11(1): 6349, 2020 12 11.
Artigo em Inglês | MEDLINE | ID: mdl-33311513

RESUMO

Human heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) serves as a key regulating protein in RNA metabolism. Malfunction of hnRNPA1 in nucleo-cytoplasmic transport or dynamic phase separation leads to abnormal amyloid aggregation and neurodegeneration. The low complexity (LC) domain of hnRNPA1 drives both dynamic phase separation and amyloid aggregation. Here, we use cryo-electron microscopy to determine the amyloid fibril structure formed by hnRNPA1 LC domain. Remarkably, the structure reveals that the nuclear localization sequence of hnRNPA1 (termed PY-NLS), which is initially known to mediate the nucleo-cytoplamic transport of hnRNPA1 through binding with karyopherin-ß2 (Kapß2), represents the major component of the fibril core. The residues that contribute to the binding of PY-NLS with Kapß2 also exert key molecular interactions to stabilize the fibril structure. Notably, hnRNPA1 mutations found in familial amyotrophic lateral sclerosis (ALS) and multisystem proteinopathoy (MSP) are all involved in the fibril core and contribute to fibril stability. Our work illuminates structural understandings of the pathological amyloid aggregation of hnRNPA1 and the amyloid disaggregase activity of Kapß2, and highlights the multiple roles of PY-NLS in hnRNPA1 homeostasis.


Assuntos
Amiloide/metabolismo , Núcleo Celular/metabolismo , Ribonucleoproteína Nuclear Heterogênea A1/química , Ribonucleoproteína Nuclear Heterogênea A1/genética , Ribonucleoproteína Nuclear Heterogênea A1/metabolismo , Transporte Ativo do Núcleo Celular , Esclerose Amiotrófica Lateral , Microscopia Crioeletrônica , Células HEK293 , Humanos , Carioferinas/metabolismo , Modelos Moleculares , Mutação , Conformação Proteica , Domínios Proteicos
10.
Leukemia ; 34(11): 2875-2886, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32624581

RESUMO

The transport of proteins across the nuclear membrane is a highly regulated process, essential for the cell function. This transport is actively mediated by members of the karyopherin family, termed importins, or exportins, depending on the direction of transport. These proteins play an active part in tumorigenesis, through aberrant localization of their cargoes, which include oncogenes, tumor-suppressor genes and mediators of key signal transduction pathways. Overexpression of importins and exportins is reported in many malignancies, with implications in cell growth and viability, differentiation, drug resistance, and tumor microenvironment. Given their broad significance across tumors and pathways, much effort is being put to develop specific inhibitors as a novel anticancer therapeutics. Already, selinexor, a specific inhibitor of exportin-1 (XPO1), is approved for clinical use. This review will focus on the role of importins and exportins in hematological malignancies. We will discuss current preclinical and clinical data on importins and exportins, and demonstrate how our growing understanding of their functions has identified new therapeutic targets.


Assuntos
Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Neoplasias Hematológicas/etiologia , Neoplasias Hematológicas/metabolismo , Terapia de Alvo Molecular , Transdução de Sinais/efeitos dos fármacos , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Biomarcadores Tumorais , Transformação Celular Neoplásica/efeitos dos fármacos , Transformação Celular Neoplásica/metabolismo , Ensaios Clínicos como Assunto , Desenvolvimento de Medicamentos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Neoplasias Hematológicas/tratamento farmacológico , Neoplasias Hematológicas/patologia , Humanos , Carioferinas/antagonistas & inibidores , Carioferinas/metabolismo
11.
J Virol ; 94(17)2020 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-32581109

RESUMO

Retroviral Gag polyproteins orchestrate the assembly and release of nascent virus particles from the plasma membranes of infected cells. Although it was traditionally thought that Gag proteins trafficked directly from the cytosol to the plasma membrane, we discovered that the oncogenic avian alpharetrovirus Rous sarcoma virus (RSV) Gag protein undergoes transient nucleocytoplasmic transport as an intrinsic step in virus assembly. Using a genetic approach in yeast, we identified three karyopherins that engage the two independent nuclear localization signals (NLSs) in Gag. The primary NLS is in the nucleocapsid (NC) domain of Gag and binds directly to importin-α, which recruits importin-ß to mediate nuclear entry. The second NLS (TNPO3), which resides in the matrix (MA) domain, is dependent on importin-11 and transportin-3 (TNPO3), which are known as MTR10p and Kap120p in yeast, although it is not clear whether these import factors are independent or additive. The functions of importin-α/importin-ß and importin-11 have been verified in avian cells, whereas the role of TNPO3 has not been studied. In this report, we demonstrate that TNPO3 directly binds to Gag and mediates its nuclear entry. To our surprise, this interaction did not require the cargo-binding domain (CBD) of TNPO3, which typically mediates nuclear entry for other binding partners of TNPO3, including SR domain-containing splicing factors and tRNAs that reenter the nucleus. These results suggest that RSV hijacks this host nuclear import pathway using a unique mechanism, potentially allowing other cargo to simultaneously bind TNPO3.IMPORTANCE RSV Gag nuclear entry is facilitated using three distinct host import factors that interact with nuclear localization signals in the Gag MA and NC domains. Here, we show that the MA region is required for nuclear import of Gag through the TNPO3 pathway. Gag nuclear entry does not require the CBD of TNPO3. Understanding the molecular basis for TNPO3-mediated nuclear trafficking of the RSV Gag protein may lead to a deeper appreciation for whether different import factors play distinct roles in retrovirus replication.


Assuntos
Produtos do Gene gag/metabolismo , Domínios Proteicos , Vírus do Sarcoma de Rous/fisiologia , Internalização do Vírus , beta Carioferinas/metabolismo , Transporte Ativo do Núcleo Celular , Núcleo Celular , Produtos do Gene gag/genética , Carioferinas/metabolismo , Sinais de Localização Nuclear/metabolismo , Nucleocapsídeo/metabolismo , Transporte Proteico , Saccharomyces cerevisiae , Montagem de Vírus , alfa Carioferinas/metabolismo , beta Carioferinas/genética
12.
Virology ; 546: 25-37, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32452415

RESUMO

Bovine adenovirus-3 (BAdV-3) is a non enveloped, icosahedral DNA virus containing a genome of 34446 bps. The intermediate region of BAdV-3 encodes pIX and IVa2 proteins. Here, we report the characterization of BAdV-3 IVa2. Anti-IVa2 serum detected a 50 kDa protein at 24-48 h post infection in BAdV-3 infected cells. The IVa2 localizes to nucleus and nucleolus of BAdV-3 infected cells. Analysis of mutant IVa2 demonstrated that amino acids 1-25 and 373-448 are required for nuclear and nucleolar localization of IVa2, respectively. The nuclear import of IVa2 utilize importin α -1 of importin nuclear import pathway. Although deletion/substitution of amino acids 4-18 is sufficient to abrogate the nuclear localization of IVa2, amino acids 1-25 are required for nuclear localization of a cytoplasmic protein. Furthermore, we demonstrate that amino acids 1-25 and 120-140 of IVa2 interact with importin α-1 and pV proteins, respectively in BAdV-3 infected cells.


Assuntos
Infecções por Adenoviridae/veterinária , Doenças dos Bovinos/virologia , Nucléolo Celular/virologia , Mastadenovirus/metabolismo , Proteínas Virais/química , Proteínas Virais/metabolismo , Transporte Ativo do Núcleo Celular , Infecções por Adenoviridae/genética , Infecções por Adenoviridae/metabolismo , Infecções por Adenoviridae/virologia , Motivos de Aminoácidos , Animais , Bovinos , Doenças dos Bovinos/genética , Doenças dos Bovinos/metabolismo , Nucléolo Celular/genética , Nucléolo Celular/metabolismo , Núcleo Celular/genética , Núcleo Celular/metabolismo , Núcleo Celular/virologia , Genoma Viral , Carioferinas/genética , Carioferinas/metabolismo , Mastadenovirus/química , Mastadenovirus/genética , Ligação Proteica , Transporte Proteico , Proteínas Virais/genética
13.
J Med Chem ; 63(8): 3881-3895, 2020 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-32223194

RESUMO

Exportin-1 (also named as CRM1) plays a prominent role in autoimmune disorders and has emerged as a potential therapeutic target for colitis. Here we report on the rational structure-based discovery of a small-molecule antagonist of exportin-1, LFS-829, with low-range nanomolar activities. The co-crystallographic structure, surface plasmon resonance binding assay, and cell-based phenotypic nuclear export functional assay validated that exportin-1 is a key target of LFS-829. Moreover, we demonstrated that the C528S mutation or the knockdown on exportin-1 can abolish the cellular activities of LFS-829. Strikingly, oral administration of LFS-829 can significantly reverse the pathological features of colitis model mice. We revealed that LFS-829 can attenuate dual NF-κB signaling and the Nrf2 cytoprotection pathway via targeting exportin-1 in colitis mice. Moreover, LFS-829 has a very low risk of cardiotoxicity and acute toxicity. Therefore, LFS-829 holds great promise for the treatment of colitis and may warrant translation for use in clinical trials.


Assuntos
Colite/tratamento farmacológico , Sistemas de Liberação de Medicamentos/métodos , Descoberta de Drogas/métodos , Hidrazinas/administração & dosagem , Carioferinas/antagonistas & inibidores , Receptores Citoplasmáticos e Nucleares/antagonistas & inibidores , Triazóis/administração & dosagem , Sequência de Aminoácidos , Animais , Colite/metabolismo , Colite/patologia , Relação Dose-Resposta a Droga , Células HEK293 , Humanos , Hidrazinas/química , Carioferinas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Receptores Citoplasmáticos e Nucleares/metabolismo , Triazóis/química
14.
PLoS One ; 15(4): e0232036, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32343715

RESUMO

The NUP98 and NUP214 nucleoporins (NUPs) are recurrently fused to heterologous proteins in leukemia. The resulting chimeric oncoproteins retain the phenylalanine-glycine (FG) repeat motifs of the NUP moiety that mediate interaction with the nuclear export receptor Crm1. NUP fusion leukemias are characterized by HOXA gene upregulation; however, their molecular pathogenesis remains poorly understood. To investigate the role of Crm1 in mediating the leukemogenic properties of NUP chimeric proteins, we took advantage of the Sequestosome-1 (SQSTM1)-NUP214 fusion. SQSTM1-NUP214 retains only a short C-terminal portion of NUP214 which contains FG motifs that mediate interaction with Crm1. We introduced point mutations targeting these FG motifs and found that the ability of the resulting SQSTM1-NUP214FGmut protein to interact with Crm1 was reduced by more than 50% compared with SQSTM1-NUP214. Mutation of FG motifs affected transforming potential: while SQSTM1-NUP214 impaired myeloid maturation and conferred robust colony formation to transduced hematopoietic progenitors in a serial replating assay, the effect of SQSTM1-NUP214FGmut was considerably diminished. Moreover, SQSTM1-NUP214 caused myeloid leukemia in all transplanted mice, whereas none of the SQSTM1-NUP214FGmut reconstituted mice developed leukemia. These oncogenic effects coincided with the ability of SQSTM1-NUP214 and SQSTM1-NUP214FGmut to upregulate the expression of Hoxa and Meis1 genes in hematopoietic progenitors. Indeed, chromatin immunoprecipitation assays demonstrated that impaired SQSTM1-NUP214 interaction with Crm1 correlated with impaired binding of the fusion protein to Hoxa and Meis1 genes. These findings highlight the importance of Crm1 in mediating the leukemogenic properties of SQSTM1-NUP214, and suggest a conserved role of Crm1 in recruiting oncoproteins to their effector genes.


Assuntos
Proteínas de Homeodomínio/genética , Carioferinas/metabolismo , Leucemia/metabolismo , Proteína Meis1/genética , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Proteínas de Fusão Oncogênica/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Proteína Sequestossoma-1/genética , Motivos de Aminoácidos , Animais , Linhagem Celular , Regulação Neoplásica da Expressão Gênica , Leucemia/genética , Leucemia/patologia , Camundongos , Mutagênese Sítio-Dirigida , Transplante de Neoplasias , Complexo de Proteínas Formadoras de Poros Nucleares/química , Proteínas de Fusão Oncogênica/química , Proteínas de Fusão Oncogênica/genética , Mutação Puntual , Regulação para Cima
15.
Nat Commun ; 11(1): 1845, 2020 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-32296071

RESUMO

XPO5 mediates nuclear export of miRNA precursors in a RanGTP-dependent manner. However, XPO5-associated RNA species have not been determined globally and it is unclear whether XPO5 has any additional functions other than nuclear export. Here we show XPO5 pervasively binds to double-stranded RNA regions found in some clustered primary miRNA precursors and many cellular RNAs. Surprisingly, the binding of XPO5 to pri-miRNAs such as mir-17~92 and mir-15b~16-2 and highly structured RNAs such as vault RNAs is RanGTP-independent. Importantly, XPO5 enhances the processing efficiency of pri-mir-19a and mir-15b~16-2 by the DROSHA/DGCR8 microprocessor. Genetic deletion of XPO5 compromises the biogenesis of most miRNAs and leads to severe defects during mouse embryonic development and skin morphogenesis. This study reveals an unexpected function of XPO5 for recognizing and facilitating the nuclear cleavage of clustered pri-miRNAs, identifies numerous cellular RNAs bound by XPO5, and demonstrates physiological functions of XPO5 in mouse development.


Assuntos
Guanosina Trifosfato/metabolismo , Carioferinas/metabolismo , MicroRNAs/metabolismo , Animais , Ensaio de Desvio de Mobilidade Eletroforética , Células HEK293 , Humanos , Carioferinas/genética , Camundongos , Plasmídeos/genética , Reação em Cadeia da Polimerase , RNA Mensageiro/metabolismo
16.
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
17.
Sci Rep ; 10(1): 2034, 2020 02 10.
Artigo em Inglês | MEDLINE | ID: mdl-32042025

RESUMO

The NAD-dependent deacetylase Sirtuin-2 (SIRT2) functions in diverse cellular processes including the cell cycle, metabolism, and has important roles in tumorigenesis and bacterial infection. SIRT2 predominantly resides in the cytoplasm but can also function in the nucleus. Consequently, SIRT2 localisation and its interacting partners may greatly impact its function and need to be defined more clearly. In this study we used mass spectrometry to determine the interactomes of SIRT2 in whole cells and in specific cellular fractions; cytoplasm, nucleus and chromatin. Using this approach, we identified novel interacting partners of SIRT2. These included a number of proteins that function in nuclear import. We show that multiple importins interact with and contribute to the basal nuclear shuttling of SIRT2 and that one of these, IPO7 is required for SIRT2 mediated H3K18 deacetylation in response to bacterial infection. Furthermore, we reveal that the unstructured C-terminus of SIRT2 negatively regulates importin-binding and nuclear transport. This study demonstrates that SIRT2 is actively transported into the nucleus via a process regulated by its C-terminus and provides a resource of SIRT2 interacting partners.


Assuntos
Transporte Ativo do Núcleo Celular/fisiologia , Núcleo Celular/metabolismo , Carioferinas/metabolismo , Sirtuína 2/metabolismo , Acetilação , Citoplasma/metabolismo , Células HeLa , Humanos
18.
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
19.
J Virol ; 94(4)2020 01 31.
Artigo em Inglês | MEDLINE | ID: mdl-31748386

RESUMO

Parvoviruses are an important platform for gene and cancer therapy. Their cell entry and the following steps, including nuclear import, are inefficient, limiting their use in therapeutic applications. Two models exist on parvoviral nuclear entry: the classical import of the viral capsid using nuclear transport receptors of the importin (karyopherin) family or the direct attachment of the capsid to the nuclear pore complex leading to the local disintegration of the nuclear envelope. Here, by laser scanning confocal microscopy and in situ proximity ligation analyses combined with coimmunoprecipitation, we show that infection requires importin ß-mediated access to the nuclear pore complex and nucleoporin 153-mediated interactions on the nuclear side. The importin ß-capsid interaction continued within the nucleoplasm, which suggests a mixed model of nuclear entry in which the classical nuclear import across the nuclear pore complex is accompanied by transient ruptures of the nuclear envelope, also allowing the passive entry of importin ß-capsid complexes into the nucleus.IMPORTANCE Parvoviruses are small DNA viruses that deliver their DNA into the postmitotic nuclei, which is an important step for parvoviral gene and cancer therapies. Limitations in virus-receptor interactions or endocytic entry do not fully explain the low transduction/infection efficiency, indicating a bottleneck after virus entry into the cytoplasm. We thus investigated the transfer of parvovirus capsids from the cytoplasm to the nucleus, showing that the nuclear import of the parvovirus capsid follows a unique strategy, which differs from classical nuclear import and those of other viruses.


Assuntos
Infecções por Parvoviridae/metabolismo , Parvovirus/metabolismo , beta Carioferinas/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Capsídeo/metabolismo , Proteínas do Capsídeo/genética , Linhagem Celular , Núcleo Celular/virologia , Citoplasma/metabolismo , Citosol/metabolismo , Carioferinas/metabolismo , Membrana Nuclear/metabolismo , Poro Nuclear/metabolismo , Parvovirus/imunologia , Internalização do Vírus , Replicação Viral , alfa Carioferinas/metabolismo
20.
J Cardiovasc Transl Res ; 13(4): 593-600, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-31768947

RESUMO

Transcriptomic signature of XPO1 was highly expressed and inversely related to left ventricular function in ischemic cardiomyopathy patients. We hypothesized that treatment with AAV9-shXPO1 attenuates left ventricular dysfunction and remodeling in a myocardial infarction rat model. We induced myocardial infarction by coronary ligation in Sprague-Dawley rats (n = 10), which received AAV9-shXPO1 (n = 5) or placebo AAV9-scramble (n = 5) treatment. Serial echocardiographic assessment was performed throughout the study. After myocardial infarction, AAV9-shXPO1-treated rats showed partial recovery of left ventricular fractional shortening (16.8 ± 2.8 vs 24.6 ± 4.1%, P < 0.05) and a maintained left ventricular dimension (6.17 ± 0.95 vs 4.70 ± 0.93 mm, P < 0.05), which was not observed in non-treated rats. Furthermore, lower levels of EXP-1 (P < 0.05) and lower collagen fibers and fibrosis in cardiac tissue were observed. However, no differences were found in the IL-6 or TNFR1 plasma levels of the myocardium of AAV9-shXPO1 rats. AAV9-shXPO1 administration attenuates cardiac dysfunction and remodeling in rats after myocardial infarction, producing the gene silencing of XPO1.


Assuntos
Carioferinas/metabolismo , Contração Miocárdica , Infarto do Miocárdio/terapia , Miocárdio/metabolismo , RNA Interferente Pequeno/administração & dosagem , Terapêutica com RNAi , Receptores Citoplasmáticos e Nucleares/metabolismo , Função Ventricular Esquerda , Remodelação Ventricular , Animais , Modelos Animais de Doenças , Colágenos Fibrilares/metabolismo , Fibrose , Carioferinas/genética , Masculino , Infarto do Miocárdio/genética , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/fisiopatologia , Miocárdio/patologia , Interferência de RNA , Ratos Sprague-Dawley , Receptores Citoplasmáticos e Nucleares/genética
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...