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1.
J Cell Biol ; 220(8)2021 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-34106209

RESUMO

The mechanisms regulating the disassembly of branched actin networks formed by the Arp2/3 complex still remain to be fully elucidated. In addition, the impact of Arp3 isoforms on the properties of Arp2/3 are also unexplored. We now demonstrate that Arp3 and Arp3B isocomplexes promote actin assembly equally efficiently but generate branched actin networks with different disassembly rates. Arp3B dissociates significantly faster than Arp3 from the network, and its depletion increases actin stability. This difference is due to the oxidation of Arp3B, but not Arp3, by the methionine monooxygenase MICAL2, which is recruited to the actin network by coronin 1C. Substitution of Arp3B Met293 by threonine, the corresponding residue in Arp3, increases actin network stability. Conversely, replacing Arp3 Thr293 with glutamine to mimic Met oxidation promotes disassembly. The ability of MICAL2 to enhance network disassembly also depends on cortactin. Our observations demonstrate that coronin 1C, cortactin, and MICAL2 act together to promote disassembly of branched actin networks by oxidizing Arp3B-containing Arp2/3 complexes.


Assuntos
Citoesqueleto de Actina/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Proteína 3 Relacionada a Actina/metabolismo , Proteínas dos Microfilamentos/metabolismo , Oxirredutases/metabolismo , Citoesqueleto de Actina/genética , Complexo 2-3 de Proteínas Relacionadas à Actina/genética , Proteína 3 Relacionada a Actina/genética , Cortactina/genética , Cortactina/metabolismo , Células HeLa , Humanos , Proteínas dos Microfilamentos/genética , Microscopia de Fluorescência , Oxirredução , Oxirredutases/genética , Vírus Vaccinia/genética , Vírus Vaccinia/metabolismo
2.
Elife ; 102021 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-33949307

RESUMO

Despite extensive studies on the actin regulators that direct microfilament dynamics, how these regulators are combinatorially utilized in organismal tissues to generate 3D structures is an unresolved question. Here, we present an in-depth characterization of cortical actin cap dynamics and their regulation in vivo. We identify rapid phases of initiation, expansion, duplication, and disassembly and examine the functions of seven different actin and/or nucleator regulators (ANRPs) in guiding these behaviors. We find ANRPs provide distinct activities in building actin cap morphologies - specifically, while DPod1 is a major regulator of actin intensities, Cortactin is required for continued cortical growth, while Coronin functions in both growth and intensity and is required for Cortactin localization to the cap periphery. Unexpectedly, cortical actin populations recover more rapidly after regulator disruption, suggestive of a deep competition for limited G-actin pools, and we measure in vivo Arp2/3 recruitment efficiencies through an ectopic relocalization strategy. Our results illustrate how the coordination of multiple actin regulators can orchestrate organized and dynamic actin structures in a developmental system.


Assuntos
Actinas/química , Actinas/fisiologia , Cortactina/genética , Regulação da Expressão Gênica , 4-Butirolactona/análogos & derivados , 4-Butirolactona/metabolismo , Actinas/genética , Animais , Linhagem Celular , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Cortactina/metabolismo , Drosophila , Feminino , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo
3.
Cells ; 10(4)2021 04 08.
Artigo em Inglês | MEDLINE | ID: mdl-33917849

RESUMO

It was reported that lumican inhibits the activity of metalloproteinase MMP-14 and melanoma cell migration in vitro and in vivo. Moreover, Snail triggers epithelial-to-mesenchymal transition and the metastatic potential of cancer cells. Therefore, the aim of this study was to examine the effect of lumican on Mock and Snail overexpressing melanoma B16F1 cells in vivo. Lung metastasis was analyzed after intravenous injections of Mock-B16F1 and Snail-B16F1 cells in Lum+/+ and Lum-/- mice. At day 14, mice were sacrificed, and lungs were collected. The number of lung metastatic nodules was significantly higher in mice injected with Snail-B16F1 cells as compared to mice injected with Mock-B16F1 cells confirming the pro-metastatic effect of Snail. This effect was stronger in Lum-/- mice as compared to Lum+/+, suggesting that endogenous lumican of wild-type mice significantly inhibits metastasis to lungs. Scanning electron and confocal microscopy investigations demonstrated that lumican inhibits the development of elongated cancer cell phenotypes which are known to develop invadopodia releasing MMPs. Moreover, lumican was shown to affect the expression of cyclin D1, cortactin, vinculin, hyaluronan synthase 2, heparanase, MMP-14 and the phosphorylation of FAK, AKT, p130 Cas and GSK3α/ß. Altogether, these data demonstrated that lumican significantly inhibits lung metastasis in vivo, as well as cell invasion in vitro, suggesting that a lumican-based strategy targeting Snail-induced metastasis could be useful for melanoma treatment.


Assuntos
Biomarcadores Tumorais/metabolismo , Lumicana/metabolismo , Melanoma/patologia , Podossomos/patologia , Neoplasias Cutâneas/patologia , Animais , Ciclo Celular , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Forma Celular , Cortactina/metabolismo , Ciclina D1/metabolismo , Matriz Extracelular/metabolismo , Adesões Focais/metabolismo , Humanos , Ácido Hialurônico/metabolismo , Neoplasias Pulmonares/secundário , Melanoma/metabolismo , Melanoma/ultraestrutura , Camundongos Endogâmicos C57BL , Metástase Neoplásica , Fosforilação , Transdução de Sinais , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/ultraestrutura , Fatores de Transcrição da Família Snail/metabolismo , Vinculina/metabolismo
4.
Exp Oncol ; 43(1): 73-76, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33785719

RESUMO

AIM: To study cellular localization of full-length breakpoint cluster region (BCR), Pleckstrin homology domain of BCR and cortactin and determine whether they can coexist in cell nucleus. MATERIALS AND METHODS: HEK293T cell line was transfected with pECFP-BCR, pEGFP-PH and pmTagRFP-N1-CTTN using polyethyleneimine. Live cells were imaged in cell culture dishes with glass coverslip attached to the bottom with Leica SP8 STED 3D confocal microscope in the environmental chamber. Obtained images were processed and analyzed with Fiji software. RESULTS: We identified colocalization of full-length BCR and cortactin in nucleus of cell undergoing terminal phase of cell division. We did not observe nuclear localization of cortactin in non-dividing cell. Both Pleckstrin homology domain and full-length BCR exhibited cytoplasmic as well as nuclear localization. CONCLUSIONS: Colocalization of BCR with cortactin in cell nucleus indicates their potential role in regulation of actin network allowing for the maintenance of nuclear architecture and DNA integrity.


Assuntos
Actinas/metabolismo , Núcleo Celular/metabolismo , Cortactina/metabolismo , Proteínas Proto-Oncogênicas c-bcr/metabolismo , Células HEK293 , Humanos
5.
J Neurosci ; 41(14): 3068-3081, 2021 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-33622779

RESUMO

Dendritic spines act as the receptive contacts at most excitatory synapses. Spines are enriched in a network of actin filaments comprised of two kinetically distinct pools. The majority of spine actin is highly dynamic and regulates spine size, structural plasticity, and postsynaptic density organization. The remainder of the spine actin network is more stable, but the function of this minor actin population is not well understood, as tools to study it have not been available. Previous work has shown that disruption of the Abl2/Arg nonreceptor tyrosine kinase in mice compromises spine stability and size. Here, using cultured hippocampal neurons pooled from both sexes of mice, we provide evidence that binding to cortactin tethers Abl2 in spines, where Abl2 and cortactin maintain the small pool of stable actin required for dendritic spine stability. Using fluorescence recovery after photobleaching of GFP-actin, we find that disruption of Abl2:cortactin interactions eliminates stable actin filaments in dendritic spines, significantly reducing spine density. A subset of spines remaining after Abl2 depletion retain their stable actin pool and undergo activity-dependent spine enlargement, associated with increased cortactin and GluN2B levels. Finally, tonic increases in synaptic activity rescue spine loss following Abl2 depletion by promoting cortactin enrichment in vulnerable spines. Together, our findings strongly suggest that Abl2:cortactin interactions promote spine stability by maintaining pools of stable actin filaments in spines.SIGNIFICANCE STATEMENT Dendritic spines contain two kinetically distinct pools of actin. The more abundant, highly dynamic pool regulates spine shape, size, and plasticity. The function of the smaller, stable actin network is not well understood, as tools to study it have not been available. We demonstrate here that Abl2 and its substrate and interaction partner, cortactin, are essential to maintain the stable pool in spines. Depletion of the stable actin pool via disruption of Abl2 or cortactin, or interactions between the proteins, significantly reduces spine stability. We also provide evidence that tonic increases in synaptic activity promote spine stability via enrichment of cortactin in spines, suggesting that synaptic activity acts on the stable actin pool to stabilize dendritic spines.


Assuntos
Citoesqueleto de Actina/metabolismo , Cortactina/metabolismo , Espinhas Dendríticas/metabolismo , Proteínas Tirosina Quinases/metabolismo , Citoesqueleto de Actina/genética , Actinas/genética , Actinas/metabolismo , Animais , Animais Recém-Nascidos , Cortactina/genética , Espinhas Dendríticas/genética , Feminino , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Ligação Proteica/fisiologia , Proteínas Tirosina Quinases/genética
6.
Sci Rep ; 11(1): 1375, 2021 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-33446758

RESUMO

Dendritic spines are the primary sites of excitatory transmission in the mammalian brain. Spines of cerebellar Purkinje Cells (PCs) are plastic, but they differ from forebrain spines in a number of important respects, and the mechanisms of spine plasticity differ between forebrain and cerebellum. Our previous studies indicate that in hippocampal spines cortactin-a protein that stabilizes actin branch points-resides in the spine core, avoiding the spine shell. To see whether the distribution of cortactin differs in PC spines, we examined its subcellular organization using quantitative preembedding immunoelectron microscopy. We found that cortactin was enriched in the spine shell, associated with the non-synaptic membrane, and was also situated within the postsynaptic density (PSD). This previously unrecognized distribution of cortactin within PC spines may underlie structural and functional differences in excitatory spine synapses between forebrain, and cerebellum.


Assuntos
Cortactina/metabolismo , Espinhas Dendríticas/metabolismo , Densidade Pós-Sináptica/metabolismo , Células de Purkinje/metabolismo , Animais , Espinhas Dendríticas/ultraestrutura , Masculino , Densidade Pós-Sináptica/ultraestrutura , Células de Purkinje/ultraestrutura , Ratos , Ratos Wistar
7.
J Cell Mol Med ; 25(1): 132-146, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33191645

RESUMO

It has been reported that chemokine CX3 CL1 can regulate various tumours by binding to its unique receptor CX3 CR1. However, the effect of CX3 CL1-CX3 CR1 on the lung adenocarcinoma and lung squamous cell carcinoma is still unclear. Here, we showed that CX3 CL1 can further invasion and migration of lung adenocarcinoma A549 and lung squamous cell carcinoma H520. In addition, Western blot and immunofluorescence test indicated CX3 CL1 up-regulated the phosphorylation level of cortactin, which is a marker of cell pseudopodium. Meanwhile, the phosphorylation levels of c-Src and c-Abl, which are closely related to the regulation of cortactin phosphorylation, are elevated. Nevertheless, the src/abl inhibitor bosutinib and mutations of cortactin phosphorylation site could inhibit the promotion effect of CX3 CL1 on invasion and migration of A549 and H520. Moreover, these results of MTT, Hoechst staining and Western blot suggested that CX3 CL1 had no effect on the proliferation and apoptosis of A549 and H520 in vitro. The effects of CX3 CL1 were also verified by the subcutaneous tumour formation in nude mice, which showed that it could promote proliferation and invasion of A549 in vivo. In summary, our results indicated that CX3 CL1 furthered invasion and migration in lung cancer cells partly via activating cortactin, and CX3 CL1 may be a potential molecule in regulating the migration and invasion of lung cancer.


Assuntos
Quimiocina CXCL1/metabolismo , Cortactina/metabolismo , Neoplasias Pulmonares/metabolismo , Fosfotirosina/metabolismo , Animais , Apoptose , Receptor 1 de Quimiocina CX3C/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Humanos , Neoplasias Pulmonares/patologia , Masculino , Camundongos Nus , Invasividade Neoplásica , Fosforilação , Proteínas Proto-Oncogênicas c-abl/metabolismo , Transdução de Sinais/efeitos dos fármacos , Quinases da Família src/metabolismo
8.
Int J Mol Sci ; 22(1)2020 Dec 25.
Artigo em Inglês | MEDLINE | ID: mdl-33375626

RESUMO

The fidelity of actin dynamics relies on protein quality control, but the underlying molecular mechanisms are poorly defined. During mitosis, the cochaperone BCL2-associated athanogene 3 (BAG3) modulates cell rounding, cortex stability, spindle orientation, and chromosome segregation. Mitotic BAG3 shows enhanced interactions with its preferred chaperone partner HSPB8, the autophagic adaptor p62/SQSTM1, and HDAC6, a deacetylase with cytoskeletal substrates. Here, we show that depletion of BAG3, HSPB8, or p62/SQSTM1 can recapitulate the same inhibition of mitotic cell rounding. Moreover, depletion of either of these proteins also interfered with the dynamic of the subcortical actin cloud that contributes to spindle positioning. These phenotypes were corrected by drugs that limit the Arp2/3 complex or HDAC6 activity, arguing for a role for BAG3 in tuning branched actin network assembly. Mechanistically, we found that cortactin acetylation/deacetylation is mitotically regulated and is correlated with a reduced association of cortactin with HDAC6 in situ. Remarkably, BAG3 depletion hindered the mitotic decrease in cortactin-HDAC6 association. Furthermore, expression of an acetyl-mimic cortactin mutant in BAG3-depleted cells normalized mitotic cell rounding and the subcortical actin cloud organization. Together, these results reinforce a BAG3's function for accurate mitotic actin remodeling, via tuning cortactin and HDAC6 spatial dynamics.


Assuntos
Actinas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Cortactina/metabolismo , Proteínas de Choque Térmico/metabolismo , Desacetilase 6 de Histona/metabolismo , Mitose , Chaperonas Moleculares/metabolismo , Acetilação , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Ativação Enzimática , Humanos , Ligação Proteica
9.
Sci Rep ; 10(1): 10667, 2020 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-32606387

RESUMO

Smooth muscle cell migration is essential for many diverse biological processes such as pulmonary/cardiovascular development and homeostasis. Abi1 (Abelson interactor 1) is an adapter protein that has been implicated in nonmuscle cell migration. However, the role and mechanism of Abi1 in smooth muscle migration are largely unknown. Here, Abi1 knockdown by shRNA reduced human airway smooth muscle cell migration, which was restored by Abi1 rescue. Abi1 localized at the tip of lamellipodia and its protrusion coordinated with F-actin at the leading cell edge of live cells. In addition, we identified profilin-1 (Pfn-1), a G-actin transporter, as a new partner for Abi1. Abi1 knockdown reduced the recruitment of Pfn-1 to the leading cell edge. Moreover, Abi1 knockdown reduced the localization of the actin-regulatory proteins c-Abl (Abelson tyrosine kinase) and N-WASP (neuronal Wiskott-Aldrich Syndrome Protein) at the cell edge without affecting other migration-related proteins including pVASP (phosphorylated vasodilator stimulated phosphoprotein), cortactin and vinculin. Furthermore, we found that c-Abl and integrin ß1 regulated the positioning of Abi1 at the leading edge. Taken together, the results suggest that Abi1 regulates cell migration by affecting Pfn-1 and N-WASP, but not pVASP, cortactin and focal adhesions. Integrin ß1 and c-Abl are important for the recruitment of Abi1 to the leading edge.


Assuntos
Actinas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Movimento Celular/fisiologia , Proteínas do Citoesqueleto/metabolismo , Músculo Liso/metabolismo , Miócitos de Músculo Liso/metabolismo , Adolescente , Células Cultivadas , Cortactina/metabolismo , Feminino , Adesões Focais/metabolismo , Humanos , Integrina beta1/metabolismo , Masculino , Profilinas/metabolismo , Pseudópodes/metabolismo , Vinculina/metabolismo , Proteína Neuronal da Síndrome de Wiskott-Aldrich/metabolismo
10.
Mol Brain ; 13(1): 105, 2020 07 25.
Artigo em Inglês | MEDLINE | ID: mdl-32711564

RESUMO

Proper dendrite morphogenesis and neuronal migration are crucial for cerebral cortex development and neural circuit formation. In this study, we sought to determine if the histone deacetylase HDAC6 plays a role in dendrite development and neuronal migration of pyramidal neurons during cerebral cortex development. It was observed that knockdown of HDAC6 leads to defective dendrite morphogenesis and abnormal Golgi polarization in vitro, and the expression of wild type cortactin or deacetyl-mimetic cortactin 9KR rescued the defective phenotypes of the HDAC6 knockdown neurons. This suggests that HDAC6 promotes dendritic growth and Golgi polarization through cortactin deacetylation in vitro. We also demonstrated that ectopic expression of SIRT2, a cytoplasmic NAD+ - dependent deacetylase, suppresses the defects of HDAC6 knockdown neurons. These results indicate that HDAC6 and SIRT2 may be functionally redundant during dendrite development. Neurons transfected with both HDAC6 and SIRT2 shRNA or acetyl-mimetic cortactin 9KQ showed slow radial migration compared to the control cells during cerebral cortex development. Furthermore, a large portion of cortactin 9KQ-expressing pyramidal neurons at layer II/III in the cerebral cortex failed to form an apical dendrite toward the pial surface and had an increased number of primary dendrites, and the percentage of neurons with dendritic Golgi decreased in cortactin 9KQ-expressing cells, compared to control neurons. Taken together, this study suggests that HDAC6 and SIRT2 regulate neuronal migration and dendrite development through cortactin deacetylation in vivo.


Assuntos
Movimento Celular , Córtex Cerebral/embriologia , Córtex Cerebral/metabolismo , Cortactina/metabolismo , Dendritos/metabolismo , Desacetilase 6 de Histona/metabolismo , Neurogênese , Sirtuína 2/metabolismo , Acetilação , Animais , Complexo de Golgi/metabolismo , Hipocampo/citologia , Camundongos Endogâmicos ICR , Ratos , Tubulina (Proteína)/metabolismo
11.
Cell Death Dis ; 11(7): 572, 2020 07 24.
Artigo em Inglês | MEDLINE | ID: mdl-32709847

RESUMO

The pRb-E2F pathway is a critical point of regulation in the cell cycle and loss of control of the pathway is a hallmark of cancer. E2F1 is the major target through which pRb exerts its effects and arginine methylation by PRMT5 plays a key role in dictating E2F1 activity. Here we have explored the functional role of the PRMT5-E2F1 axis and highlight its influence on different aspects of cancer cell biology including viability, migration, invasion and adherence. Through a genome-wide expression analysis, we identified a distinct set of genes under the control of PRMT5 and E2F1, including some highly regulated genes, which influence cell migration, invasio and adherence through a PRMT5-dependent mechanism. Most significantly, a coincidence was apparent between the expression of PRMT5 and E2F1 in human tumours, and elevated levels of PRMT5 and E2F1 correlated with poor prognosis disease. Our results suggest a causal relationship between PRMT5 and E2F1 in driving the malignant phenotype and thereby highlight an important pathway for therapeutic intervention.


Assuntos
Movimento Celular , Fator de Transcrição E2F1/metabolismo , Neoplasias/metabolismo , Neoplasias/patologia , Proteína-Arginina N-Metiltransferases/metabolismo , Transdução de Sinais , Linhagem Celular Tumoral , Movimento Celular/genética , Cortactina/genética , Cortactina/metabolismo , Regulação para Baixo/genética , Fator de Transcrição E2F1/genética , Adesões Focais/metabolismo , Regulação Neoplásica da Expressão Gênica , Genoma Humano , Humanos , Invasividade Neoplásica , Neoplasias/genética , Proteína-Arginina N-Metiltransferases/antagonistas & inibidores , Proteína-Arginina N-Metiltransferases/genética , Transdução de Sinais/genética
12.
Biomed Res Int ; 2020: 6590303, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32626753

RESUMO

Objective: Long noncoding RNA (LncRNA) SBF2-AS1 was reportedly to function as an oncogene in several types of cancers, such as hepatocellular carcinoma, nonsmall cell lung cancer, glioma, and colorectal cancer. However, the biological roles and regulatory mechanisms of SBF2-AS1 in gastric cancer (GC) are unknown. Methods: The expression of SBF2-AS1 and miR-545 were examined in GC tissues and cell lines via real-time quantitative PCR. The relationship of SBF2-AS1 with miR-545 was verified via dual-luciferase reporter gene assay and RNA immunoprecipitation. The influences of SBF2-AS1 on cell proliferation, migration, and invasion were determined using cell counting Kit-8 (CCK-8), wound healing, and transwell invasion assays, respectively. Results: LncRNA SBF2-AS1 expression was upregulated in GC tissues, especially in advanced clinical stage cases. Moreover, increased SBF2-AS1 indicated a poor survival rate. Functionally, the downregulation of SBF2-AS1 by siRNA in GC cells suppressed the proliferation, migration, and invasion. In terms of mechanism, SBF2-AS1 can directly bind to miR-545 and regulate its expression. Moreover, SBF2-AS1 knockdown significantly decreased the expression of EMS1, which was the direct target of miR-545. Importantly, inhibition of miR-545 or overexpression of EMS1 partially reversed SBF2-AS1-depletion-caused suppression on proliferation, migration, and invasion. Conclusion: These findings elucidated a crucial role of SBF2-AS1 as a miR-545 sponge in GC cells, suggesting that SBF2-AS1 might be a potential target for GC.


Assuntos
Cortactina , MicroRNAs , RNA Longo não Codificante , Neoplasias Gástricas , Linhagem Celular Tumoral , Cortactina/genética , Cortactina/metabolismo , Progressão da Doença , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Masculino , MicroRNAs/genética , MicroRNAs/metabolismo , Pessoa de Meia-Idade , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Estômago/química , Estômago/patologia , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/mortalidade , Neoplasias Gástricas/patologia
13.
J Virol ; 94(19)2020 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-32727880

RESUMO

Hepatitis C virus (HCV) exploits cellular proteins to facilitate viral propagation. To identify the cellular factors involved in the HCV life cycle, we previously performed protein microarray assays using either HCV nonstructural 5A (NS5A) protein or core protein as a probe. Interestingly, cellular cortactin strongly interacted with both NS5A and core. Cortactin is an actin-binding protein critically involved in tumor progression by regulating the migration and invasion of cancerous cells. Protein interaction between cortactin and NS5A or core was confirmed by coimmunoprecipitation and immunofluorescence assays. We showed that cortactin interacted with NS5A and core via the N-terminal acidic domain of cortactin. Cortactin expression levels were not altered by HCV infection. Small interfering RNA (siRNA)-mediated knockdown of cortactin dramatically decreased HCV protein expression and infectivity levels, whereas overexpression of cortactin increased viral propagation. Ectopic expression of the siRNA-resistant cortactin recovered the viral infectivity, suggesting that cortactin was specifically required for HCV propagation. We further showed that cortactin was involved in the assembly step without affecting viral entry, HCV internal ribosome entry site (IRES)-mediated translation, and the replication steps of the HCV life cycle. Of note, silencing of cortactin markedly reduced both NS5A and core protein levels on the lipid droplets (LDs), and this effect was reversed by the overexpression of cortactin. Importantly, NS5A and core promoted cell migration by activating the phosphorylation of cortactin at tyrosine residues 421 and 466. Taken together, these data suggest that cortactin is not only involved in HCV assembly but also plays an important role in the cell migration.IMPORTANCE Cortactin is a cytoskeletal protein that regulates cell migration in response to a number of extracellular stimuli. The functional involvement of cortactin in the virus life cycle is not yet fully understood. The most significant finding is that cortactin strongly interacted with both hepatitis C virus (HCV) core and NS5A. Cortactin is involved in HCV assembly by tethering core and NS5A on the lipid droplets (LDs) with no effect on LD biogenesis. It was noteworthy that HCV NS5A and core activated cortactin by phosphorylation at tyrosines 421 and 466 to regulate cell migration. Collectively, our study shows that cortactin is a novel host factor involved in viral production and HCV-associated pathogenesis.


Assuntos
Cortactina/metabolismo , Hepacivirus/fisiologia , Proteínas não Estruturais Virais/metabolismo , Vírion/fisiologia , Montagem de Vírus/fisiologia , Linhagem Celular , Proteínas do Citoesqueleto/metabolismo , Células HEK293 , Hepatite C/virologia , Antígenos da Hepatite C/metabolismo , Humanos , Imunoprecipitação , Fosforilação , RNA Interferente Pequeno/genética , Internalização do Vírus , Replicação Viral
14.
BMC Cancer ; 20(1): 281, 2020 Apr 06.
Artigo em Inglês | MEDLINE | ID: mdl-32252688

RESUMO

BACKGROUND: Esophageal squamous cell carcinoma (ESCC) is an aggressive cancer with one of the highest world incidences in the Eastern Cape region of South Africa. Several genome wide studies have been performed on ESCC cohorts from Asian countries, North America, Malawi and other parts of the world but none have been conducted on ESCC tumors from South Africa to date, where the molecular pathology and etiology of this disease remains unclear. We report here tumor associated copy number changes observed in 51 ESCC patients' samples from the Eastern Cape province of South Africa. METHODS: We extracted tumor DNA from 51 archived ESCC specimens and interrogated tumor associated DNA copy number changes using Affymetrix® 500 K SNP array technology. The Genomic Identification of Significant Targets in Cancer (GISTIC 2.0) algorithm was applied to identify significant focal regions of gains and losses. Gains of the top recurrent cancer genes were validated by fluorescence in situ hybridization and their protein expression assessed by immunohistochemistry. RESULTS: Twenty-three significant focal gains were identified across samples. Gains involving the CCND1, MYC, EGFR and JAG1 loci recapitulated those described in studies on Asian and Malawian cohorts. The two most significant gains involved the chromosomal sub-bands 3q28, encompassing the TPRG1 gene and 11q13.3 including the CTTN, PPFIA1and SHANK2 genes. There was no significant homozygous loss and the most recurrent hemizygous deletion involved the B3GAT1 gene on chromosome 11q25. Focal gains on 11q13.3 in 37% of cases (19/51), consistently involved CTTN and SHANK2 genes. Twelve of these cases (23,5%), had a broader region of gain that also included the CCND1, FGF19, FGF4 and FGF3 genes. SHANK2 and CTTN are co-amplified in several cancers, these proteins interact functionally together and are involved in cell motility. Immunohistochemistry confirmed both Shank2 (79%) and cortactin (69%) protein overexpression in samples with gains of these genes. In contrast, cyclin D1 (65%) was moderately expressed in samples with CCND1 DNA gain. CONCLUSIONS: This study reports copy number changes in a South African ESCC cohort and highlights similarities and differences with cohorts from Asia and Malawi. Our results strongly suggest a role for CTTN and SHANK2 in the pathogenesis of ESCC in South Africa.


Assuntos
Biomarcadores Tumorais/genética , Variações do Número de Cópias de DNA , Doenças Endêmicas/estatística & dados numéricos , Neoplasias Esofágicas/genética , Carcinoma de Células Escamosas do Esôfago/genética , Biomarcadores Tumorais/metabolismo , Hibridização Genômica Comparativa/métodos , Cortactina/genética , Cortactina/metabolismo , Neoplasias Esofágicas/epidemiologia , Neoplasias Esofágicas/patologia , Carcinoma de Células Escamosas do Esôfago/epidemiologia , Carcinoma de Células Escamosas do Esôfago/patologia , Humanos , Imuno-Histoquímica/métodos , Hibridização in Situ Fluorescente/métodos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , África do Sul/epidemiologia
15.
Cells ; 9(3)2020 02 27.
Artigo em Inglês | MEDLINE | ID: mdl-32120844

RESUMO

Epigenetic regulation plays an important role in the development and progression of nasopharyngeal carcinoma (NPC), but the epigenetic mechanisms underlying NPC metastasis remain poorly understood. Here, we demonstrate that hypermethylation of the UCHL1 promoter leads to its downregulation in NPC. Restoration of UCHL1 inhibited the migration and invasion of NPC cells in vitro and in vivo, and knockdown of UCHL1 promoted NPC cell migration and invasion in vitro and in vivo. Importantly, we found that UCHL1 interacts with CTTN, and may function as a ligase promoting CTTN degradation by increasing K48-linked ubiquitination of CTTN. Additionally, restoration of CTTN in NPC cells that overexpressed UCHL1 rescued UCHL1 suppressive effects on NPC cell migration and invasion, which indicated that CTTN is a functional target of UCHL1 in NPC. Our findings revealed that UCHL1 acts as a tumor suppressor gene in NPC and thus provided a novel therapeutic target for NPC treatment.


Assuntos
Cortactina/metabolismo , Metilação de DNA/genética , Carcinoma Nasofaríngeo/genética , Carcinoma Nasofaríngeo/patologia , Proteólise , Ubiquitina Tiolesterase/genética , Animais , Linhagem Celular Tumoral , Movimento Celular/genética , Regulação para Baixo/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Humanos , Lisina/metabolismo , Camundongos Endogâmicos BALB C , Camundongos Nus , Invasividade Neoplásica , Metástase Neoplásica , Regiões Promotoras Genéticas , Ligação Proteica , Ubiquitina Tiolesterase/metabolismo , Ubiquitinação
16.
J Ethnopharmacol ; 255: 112738, 2020 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-32147479

RESUMO

ETHNOPHARMACOLOGICAL RELEVANCE: Qian Yang Yu Yin Granule (QYYY) is a Chinese herbal formulation. It is used to treat hypertensive nephropathy for decades in China, but it is unknown that the exact mechanism of QYYY on hypertensive nephropathy. AIMS OF STUDY: The present study was to elucidate its epigenetic mechanism of QYYY on hypertensive nephropathy. MATERIALS AND METHODS: In the current study, HEK293T cells' proliferation induced by Ang II was chosen to observe epigenetic mechanisms of QYYY on renal damage. The cell proliferation was examined by MTT assays and ethynyldeoxyuridine analysis. Cell cycle analysis was performed. After treatment with QYYY, expression of Nicotinamide N-methyltransferase (NNMT), sirtuin1(SIRT1), S-adenosylhomocysteine(SAH), histone H3K4 methylation, and cortactin acetylation(acetyl-cortactin,ac-cortactin) were further investigated by western-blotting and real time PCR. DNA methylation was detected by ELISA. The study also observed the changes of SIRT1, SAH, H3K4 methylation, acetyl-cortactin when NNMT over-expressed by lentivirus transfection. Angiotensin II(Ang II) induced renal damage in spontaneously hypertensive rats(SHR). After eight weeks treatment of QYYY, blood pressure, serum and urine creatinine, and urinary microalbumin(mAlb) were assessed. The concentration of N1 -methylnicotinamide were detected by liquid chromatography with tandem mass spectrometry. The protein of NNMT, ac-cortactin, H3K3me3 were also assessed in vivo. RESULTS: QYYY inhibited HEK293T cells' proliferation, down-regulated the expression of NNMT, SAH, acetyl-cortactin and DNA methylation, up-regulated the expression of SIRT1, histone H3K4 trimethylation(H3K4me3). Over-expression of NNMT increased the expression of SAH and acetyl-cortactin, and reduced the expression of SIRT1 and H3K4me3. The study also demonstrated that QYYY promoted urinary creatinine excretion and reduced serum creatinine and urinary mAlb in SHR. QYYY decreased the concentration of N1 -methylnicotinamide in Ang II group. QYYY decreased the protein of NNMT, ac-cortactin and increased H3K4me3 in vivo. CONCLUSION: The results showed that QYYY alleviated renal impairment of SHR and inhibited HEK293T cells' proliferation induced by Ang II through the pathway of epigenetic mechanism linked to Nicotinamide N-Methyltransferase (NNMT) expression, including histone methylation, DNA methylation and acetyl-cortactin. This study unveiled a novel molecular mechanism by which QYYY controlled the progression of hypertensive nephropathy.


Assuntos
Medicamentos de Ervas Chinesas/farmacologia , Epigênese Genética/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Hipertensão/tratamento farmacológico , Nefropatias/prevenção & controle , Rim/efeitos dos fármacos , Nicotinamida N-Metiltransferase/metabolismo , Acetilação , Angiotensina II , Animais , Proliferação de Células/efeitos dos fármacos , Cortactina/metabolismo , Metilação de DNA/efeitos dos fármacos , Modelos Animais de Doenças , Células Epiteliais/enzimologia , Células Epiteliais/patologia , Células HEK293 , Histonas/metabolismo , Humanos , Hipertensão/complicações , Hipertensão/enzimologia , Hipertensão/genética , Rim/enzimologia , Rim/patologia , Nefropatias/induzido quimicamente , Nefropatias/enzimologia , Nefropatias/genética , Masculino , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY , S-Adenosil-Homocisteína/metabolismo , Sirtuína 1/metabolismo
17.
FEBS J ; 287(22): 4862-4880, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32160387

RESUMO

The small leucine-rich proteoglycan lumican regulates estrogen receptors (ERs)-associated functional properties of breast cancer cells, expression of matrix macromolecules, and epithelial-to-mesenchymal transition. However, it is not known whether the ER-dependent lumican effects on breast cancer cells are related to the expression of integrins and their intracellular signaling pathways. Here, we analyzed the effects of lumican in three breast cancer cell lines: the highly metastatic ERß-positive MDA-MB-231, cells with the respective ERß-suppressed (shERßMDA-MB-231), and lowly invasive ERα-positive MCF-7/c breast cancer cells. Scanning electron microscopy, confocal microscopy, real-time PCR, western blot, and cell adhesion assays were performed. Lumican effects on breast cancer cell morphology were also investigated in 3-dimensional collagen cultures. Lumican treatment induced cell-cell contacts and cell grouping and inhibited microvesicles and microvilli formation. The expression of the cell surface adhesion receptor CD44, its isoform and variants, hyaluronan (HA), and HA synthases was also investigated. Lumican inhibited the expression of CD44 and HA synthases, and its effect on cell adhesion revealed a major role of α1, α2, α3, αVß3, and αVß5 integrins in MDA-MB-231 cells, but not in MCF-7/c cells. Lumican upregulated the expression of α2 and ß1 integrin subunits both in MDA-MB-231 and in shERßMDA-MB-231 as compared to MCF-7/c cells. Downstream signaling pathways for integrins, such as FAK, ERK 1/2 MAPK 42/44, and Akt, were found to be downregulated by lumican. Our data shed light to the molecular mechanisms responsible for the anticancer activity of lumican in invasive breast cancer.


Assuntos
Neoplasias da Mama/metabolismo , Forma Celular/efeitos dos fármacos , Cortactina/metabolismo , Integrinas/metabolismo , Lumicana/farmacologia , Transdução de Sinais/efeitos dos fármacos , Neoplasias da Mama/patologia , Neoplasias da Mama/ultraestrutura , Adesão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Quinase 1 de Adesão Focal/metabolismo , Humanos , Receptores de Hialuronatos/metabolismo , Hialuronan Sintases/metabolismo , Células MCF-7 , Microscopia Eletrônica de Varredura , Fosforilação/efeitos dos fármacos
18.
Am J Pathol ; 190(5): 958-967, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32084363

RESUMO

Cortactin is an actin-binding protein expressed in virtually all cell types. It regulates several cell functions, including adhesion and migration. Cortactin overexpression is associated with increased metastasis formation and worse outcome in different types of solid tumors, thus highlighting a critical role of cortactin in cancer progression. Mechanistically, this is due to increased invadopodia formation and matrix metalloproteinase secretion. Cortactin has been until recently considered absent in hematopoietic cells because these cells express the cortactin homolog hematopoietic cell-specific lyn substrate-1. However, many recent reports describe functional expression of cortactin in different hematopoietic cells, such as macrophages, dendritic cells, and lymphocytes. Of note, cortactin is strongly overexpressed in leukemic cell lines and primary patient-derived leukemic cells. In B-cell chronic lymphocytic leukemia, this is associated with poor prognosis and increased chemotaxis; in B-cell acute lymphoblastic leukemia, high cortactin levels correlate with treatment failure and relapse. Moreover, cortactin has been proposed as a diagnostic marker for non-Hodgkin B-cell lymphomas. This review summarizes current knowledge on cortactin expression in hematopoietic cells and discusses the functional implications for different hematological malignancies.


Assuntos
Células Sanguíneas/metabolismo , Cortactina/metabolismo , Neoplasias Hematológicas/metabolismo , Animais , Adesão Celular/fisiologia , Movimento Celular/fisiologia , Humanos
19.
Viruses ; 12(1)2020 01 12.
Artigo em Inglês | MEDLINE | ID: mdl-31940955

RESUMO

Influenza A virus (IAV) exploits host factors to multiply and cause disease. An in-depth knowledge of this interaction of IAV with the host will aid the development of anti-IAV intervention strategies. Previously, we demonstrated that host cortactin, an actin filament-binding protein promotes IAV infection, but undergoes degradation via a lysosome-associated apoptotic pathway during the late stages of IAV infection. Next, we wanted to further understand the mechanisms and significance of this phenomenon. By using the RNA interference screens and site-directed mutagenesis followed by western blotting, we found that lysosome protease, cathepsin C is involved in cortactin degradation in human cells infected with IAV. Furthermore, executioner apoptotic caspase, caspase-3 not caspase-6 or caspase-7 is involved in cortactin degradation during IAV infection, and caspase-3 cleavage site is located in the first actin-binding repeat of cortactin polypeptide. Finally, when expressed ectopically, the cleavage-resistant cortactin mutants decreased the amount of IAV progeny released from infected cells that was enhanced by the cleavage-sensitive cortactin wild type. These data strengthen the hypothesis proposed earlier that host cortactin plays an inhibitory role during the late stages of IAV infection, and IAV is facilitating its degradation to undermine such function.


Assuntos
Actinas/metabolismo , Caspases/metabolismo , Cortactina/metabolismo , Interações Hospedeiro-Patógeno , Vírus da Influenza A/fisiologia , Células A549 , Caspase 3/metabolismo , Catepsina C/metabolismo , Humanos , Ligação Proteica , Replicação Viral
20.
Glia ; 68(5): 947-962, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-31743496

RESUMO

Bmal1 is an essential component of the molecular clockwork, which drives circadian rhythms in cell function. In Bmal1-deficient (Bmal1-/-) mice, chronodisruption is associated with cognitive deficits and progressive brain pathology including astrocytosis indicated by increased expression of glial fibrillary acidic protein (GFAP). However, relatively little is known about the impact of Bmal1-deficiency on astrocyte morphology prior to astrocytosis. Therefore, in this study we analysed astrocyte morphology in young (6-8 weeks old) adult Bmal1-/- mice. At this age, overall GFAP immunoreactivity was not increased in Bmal1-deficient mice. At the ultrastructural level, we found a decrease in the volume fraction of the fine astrocytic processes that cover the hippocampal mossy fiber synapse, suggesting an impairment of perisynaptic processes and their contribution to neurotransmission. For further analyses of actin cytoskeleton, which is essential for distal process formation, we used cultured Bmal1-/- astrocytes. Bmal1-/- astrocytes showed an impaired formation of actin stress fibers. Moreover, Bmal1-/- astrocytes showed reduced levels of the actin-binding protein cortactin (CTTN). Cttn promoter region contains an E-Box like element and chromatin immunoprecipitation revealed that Cttn is a potential Bmal1 target gene. In addition, the level of GTP-bound (active) Rho-GTPase (Rho-GTP) was reduced in Bmal1-/- astrocytes. In summary, our data demonstrate that Bmal1-deficiency affects morphology of the fine astrocyte processes prior to strong upregulation of GFAP, presumably because of impaired Cttn expression and reduced Rho-GTP activation. These morphological changes might result in altered synaptic function and, thereby, relate to cognitive deficits in chronodisruption.


Assuntos
Fatores de Transcrição ARNTL/metabolismo , Citoesqueleto de Actina/metabolismo , Astrócitos/metabolismo , Fibras Musgosas Hipocampais/metabolismo , Sinapses/metabolismo , Fatores de Transcrição ARNTL/genética , Animais , Cortactina/genética , Cortactina/metabolismo , Proteína Glial Fibrilar Ácida/metabolismo , Masculino , Camundongos , Camundongos Knockout , Transmissão Sináptica/fisiologia
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