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1.
Artigo em Inglês | MEDLINE | ID: mdl-31913659

RESUMO

It has been reported that actin polymerization is regulated by protein tyrosine phosphorylation in smooth muscle upon contractile stimulation. The role of protein serine/threonine phosphorylation in modulating actin dynamics is under-investigated. Ste20-like kinase (SLK) is a serine/threonine protein kinase that plays a role in apoptosis, cell cycle, proliferation, and migration. The function of SLK in smooth muscle is mostly unknown. Here, SLK knockdown (KD) inhibited the acetylcholine (ACh)-induced actin polymerization and contraction without affecting myosin light chain phosphorylation at Ser-19 in human airway smooth muscle. Stimulation with ACh induced paxillin phosphorylation at Ser-272, which was reduced in SLK KD cells. However, SLK did not catalyze paxillin Ser-272 phosphorylation in vitro. But, SLK KD attenuated polo-like kinase 1 (Plk1) phosphorylation at Thr-210. Plk1 mediated paxillin phosphorylation at Ser-272 in vitro. Expression of the non-phosphorylatable paxillin mutant S272A (substitution of alanine at Ser-272) attenuated the agonist-enhanced F-actin/G-actin ratios without affecting myosin light chain phosphorylation. Because neuronal Wiskott-Aldrich Syndrome Protein (N-WASP) phosphorylation at Tyr-256 (an indication of its activation) promotes actin polymerization, we also assessed the role of paxillin phosphorylation in N-WASP activation. S272A paxillin inhibited the ACh-enhanced N-WASP phosphorylation at Tyr-256. Together, these results suggest that SLK regulates paxillin phosphorylation at Ser-272 via Plk1, which modulates N-WASP activation and actin polymerization in smooth muscle. SLK-mediated actin cytoskeletal reorganization may facilitate force transmission between the contractile units and the extracellular matrix.

2.
Front Oncol ; 9: 1319, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31828041

RESUMO

Background: ALK and ROS1 rearrangement accounts for 3-6% and 1-3% of non-small cell lung cancers, respectively, while coexistence of them in the same patient is extremely rare. Only three cases have ever been reported with concurrent ALK/ROS1 fusions in the same tumor indicating tumor heterogeneity. Therefore, comprehensive genetic profiling via next-generation sequencing (NGS) is needed to provide fully molecular diagnosis. Case Presentation: A 50-year old Chinese female with resectable stage IB bilateral lung adenocarcinomas (ADCs) harbored EML4 exon 6-ALK exon 19 and TPM3 exon 8-ROS1 exon 35 fusions in the right lower and the left upper tumors, respectively, identified by clinical NGS test targeting 425 cancer-relevant genes. The results were further confirmed at RNA level using RNA-seq. Genomic evolution analysis reveals that these bilateral tumors are synchronous multiple primary lung cancers with no shared somatic alterations for both genes and arm-level copy number variations (CNVs). No recurrence was observed during 12 months of post-surgery follow-up. Conclusions: Our case is the first report of concurrent ALK/ROS1 fusions as distinct driver events of synchronous multiple primary lung cancers, and highlights the importance of individual genetic testing for each of the multiple primary tumors for fully molecular diagnosis and precise treatment decision-making.

3.
Future Oncol ; 15(22): 2585-2593, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31339066

RESUMO

Aim: Crizotinib has been used to counter MET amplification in different human malignancies. However, transient responses were observed in some patients with rapid acquisition of resistant mutations in MET. Materials & methods: MET mutations stably expressed Ba/F3 cell lines were used for IC50 detection. Signaling pathway analysis was done using 293T cell line. Results: Four MET mutations conferred resistance to crizotinib with sustained activation of downstream signaling pathways of MET. On the other hand, the four MET mutations displayed different response to type II tyrosine kinase inhibitors with variable deterioration of the downstream signals. Conclusion: This study suggested that patients carrying MET V1092L, D1228G or Y1230H mutations could benefit from type II tyrosine kinase inhibitor treatment, but not patients with G1163R or D1228Y/N mutations.


Assuntos
Crizotinibe/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-met/genética , Neoplasias Gástricas/tratamento farmacológico , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Crizotinibe/efeitos adversos , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Mutação , Fosforilação/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologia
4.
Sci Rep ; 9(1): 7555, 2019 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-31101859

RESUMO

Allergic asthma is characterized by airway smooth muscle layer thickening, which is largely attributed to cell division that requires the formation of centrosomes. Centrosomes play a pivotal role in regulating bipolar spindle formation and cell division. Before mitosis, centrosomes undergo maturation characterized by expansion of pericentriolar material proteins, which facilitates spindle formation and mitotic efficiency of many cell types. Although polo-like kinase 1 (Plk1) has been implicated in centrosome maturation, the mechanisms by which Plk1 regulates the cellular process are incompletely elucidated. Here, we identified paxillin as a new Plk1-interacting protein in human airway smooth muscle cells. We unexpectedly found that phosphorylated paxillin (Ser-272) was localized in centrosomes of human smooth muscle cells, which regulated centrosome maturation and spindle assembly. Plk1 knockdown inhibited paxillin Ser-272 phosphorylation, centrosome maturation, and cell division. Furthermore, exposure to allergens enhanced airway smooth muscle layer and paxillin phosphorylation at this residue in mice, which was reduced by smooth muscle conditional knockout of Plk1. These findings suggest that Plk1 regulates centrosome maturation and cell division in part by modulating paxillin phosphorylation on Ser-272. Furthermore, Plk1 contributes to the pathogenesis of allergen-induced thickening of the airway smooth muscle layer by affecting paxillin phosphorylation at this position.

5.
Am J Respir Cell Mol Biol ; 61(2): 219-231, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30811945

RESUMO

Airway smooth muscle cells require coordinated protrusion and focal adhesion dynamics to migrate properly. However, the signaling cascades that connect these two processes remain incompletely understood. Glia maturation factor (GMF)-γ has been implicated in inducing actin debranching and inhibiting nucleation. In this study, we discovered that GMFγ phosphorylation at Y104 regulates human airway smooth muscle cell migration. Using high-resolution microscopy coupled with three-dimensional object-based quantitative image analysis software, Imaris 9.2.0, phosphomimetic mutant, Y104D-GMFγ, was enriched at nascent adhesions along the leading edge where it recruited activated neural Wiskott-Aldrich syndrome protein (N-WASP; pY256) to promote actin-branch formation, which enhanced lamellipodial dynamics and limited the growth of focal adhesions. Unexpectedly, we found that nonphosphorylated mutant, Y104F-GMFγ, was enriched in growing adhesions where it promoted a linear branch organization and focal adhesion clustering, and recruited zyxin to increase maturation, thus inhibiting lamellipodial dynamics and cell migration. The localization of GMFγ between the leading edge and focal adhesions was dependent upon myosin activity. Furthermore, c-Abl tyrosine kinase regulated the GMFγ phosphorylation-dependent processes. Together, these results unveil the importance of GMFγ phosphorylation in coordinating lamellipodial and focal adhesion dynamics to regulate cell migration.

6.
Sci Rep ; 8(1): 12635, 2018 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-30135525

RESUMO

Polo-like kinase 1 (Plk1) has been implicated in mitosis, cytokinesis, and proliferation. The mechanisms that regulate Plk1 expression remain to be elucidated. It is reported that miR-100 targets Plk1 in certain cancer cells. Here, treatment with miR-100 did not affect Plk1 protein expression in human airway smooth muscle cells. In contrast, treatment with miR-509 inhibited the expression of Plk1 in airway smooth muscle cells. Exposure to miR-509 inhibitor enhanced Plk1 expression in cells. Introduction of miR-509 reduced luciferase activity of a Plk1 3'UTR reporter. Mutation of miR-509 targeting sequence in Plk1 3'UTR resisted the reduction of the luciferase activity. Furthermore, miR-509 inhibited the PDGF-induced phosphorylation of MEK1/2 and ERK1/2, and cell proliferation without affecting the expression of c-Abl, a tyrosine kinase implicated in cell proliferation. Moreover, we unexpectedly found that vimentin filaments contacted paxillin-positive focal adhesions. miR-509 exposure inhibited vimentin phosphorylation at Ser-56, vimentin network reorganization, focal adhesion formation, and cell migration. The effects of miR-509 on ERK1/2 and vimentin were diminished in RNAi-resistant Plk1 expressing cells treated with miR-509. Taken together, these findings unveil previously unknown mechanisms that miR-509 regulates ERK1/2 and proliferation by targeting Plk1. miR-509 controls vimentin cytoskeleton reorganization, focal adhesion assembly, and cell migration through Plk1.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Adesões Focais/fisiologia , Sistema de Sinalização das MAP Quinases , MicroRNAs/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Vimentina/metabolismo , Regiões 3' não Traduzidas , Proteínas de Ciclo Celular/genética , Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Adesões Focais/genética , Adesões Focais/metabolismo , Humanos , MicroRNAs/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/fisiologia , Fosforilação , Cultura Primária de Células , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Transdução de Sinais , Vimentina/genética
7.
Respir Res ; 19(1): 4, 2018 01 05.
Artigo em Inglês | MEDLINE | ID: mdl-29304860

RESUMO

BACKGROUND: Airway smooth muscle contraction is critical for maintenance of appropriate airway tone, and has been implicated in asthma pathogenesis. Smooth muscle contraction requires an "engine" (myosin activation) and a "transmission system" (actin cytoskeletal remodeling). However, the mechanisms that control actin remodeling in smooth muscle are not fully elucidated. The adapter protein Crk-associated substrate (CAS) regulates actin dynamics and the contraction in smooth muscle. In addition, profilin-1 (Pfn-1) and Abelson tyrosine kinase (c-Abl) are also involved in smooth muscle contraction. The interplays among CAS, Pfn-1 and c-Abl in smooth muscle have not been previously investigated. METHODS: The association of CAS with Pfn-1 in mouse tracheal rings was evaluated by co-immunoprecipitation. Tracheal rings from c-Abl conditional knockout mice were used to assess the roles of c-Abl in the protein-protein interaction and smooth muscle contraction. Decoy peptides were utilized to evaluate the importance of CAS/Pfn-1 coupling in smooth muscle contraction. RESULTS: Stimulation with acetylcholine (ACh) increased the interaction of CAS with Pfn-1 in smooth muscle, which was regulated by CAS tyrosine phosphorylation and c-Abl. The CAS/Pfn-1 coupling was also modified by the phosphorylation of cortactin (a protein implicated in Pfn-1 activation). In addition, ACh activation promoted the spatial redistribution of CAS and Pfn-1 in smooth muscle cells, which was reduced by c-Abl knockdown. Inhibition of CAS/Pfn-1 interaction by a decoy peptide attenuated the ACh-induced actin polymerization and contraction without affecting myosin light chain phosphorylation. Furthermore, treatment with the Src inhibitor PP2 and the actin polymerization inhibitor latrunculin A attenuated the ACh-induced c-Abl tyrosine phosphorylation (an indication of c-Abl activation). CONCLUSIONS: Our results suggest a novel activation loop in airway smooth muscle: c-Abl promotes the CAS/Pfn-1 coupling and actin polymerization, which conversely facilitates c-Abl activation. The positive feedback may render c-Abl in active state after contractile stimulation.


Assuntos
Proteína Substrato Associada a Crk/metabolismo , Contração Muscular/fisiologia , Miócitos de Músculo Liso/fisiologia , Profilinas/metabolismo , Proteínas Proto-Oncogênicas c-abl/metabolismo , Sequência de Aminoácidos , Animais , Células Cultivadas , Feminino , Técnicas de Inativação de Genes , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Traqueia/citologia , Traqueia/fisiologia
8.
Microb Cell Fact ; 16(1): 202, 2017 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-29137648

RESUMO

BACKGROUND: Heat-stable antifungal factor (HSAF) is a polycyclic tetramate macrolactam secondary metabolite that exhibits broad-spectrum inhibitory activities against filamentous fungal pathogens. The native yield of this chemical is low. It is also a great challenge to synthesize HSAF artificially, due to its complex structure. Understanding the regulatory mechanism underlying HSAF biosynthesis could provide genetic basis for engineering high HSAF-producing strain. The transcription factor Clp is a global regulator that controls bacterial pathogenicity and the expression of one hundred related genes in the phytopathogenic bacterium Xanthomonas campestris pv. campestris (Xcc). Diffusible signal factor (DSF) chemical signaling is the only well-characterized upstream regulatory pathway that involves downstream Clp regulation in Xcc. Such a regulatory hierarchy between DSF signaling and Clp is also conserved in the Gram-negative biological control agent Lysobacter enzymogenes, where the DSF signaling system controls antifungal antibiotic HSAF biosynthesis via Clp. RESULTS: Here, using LLysobacter enzymogenes OH11 as a working organism, we examined a novel upstream regulator, LesR, a LuxR solo that controls Clp expression to modulate HSAF biosynthesis as well as cell aggregation. We found that the overexpression of lesR in strain OH11 almost entirely shut down HSAF production and accelerated cell aggregation. These changed phenotypes could be rescued by the introduction of plasmid-borne clp in the lesR overexpression background. Consistent with findings, we further found that overexpression of lesR led to a decrease in the Clp level. CONCLUSIONS: These results collectively have shown that LesR could exert its function, i.e., HSAF biosynthesis, via downstream Clp. These findings were subsequently validated by a comparative transcriptome analysis, where the regulatory action of LesR was found to largely overlap with that of Clp. Therefore, in addition to the well-known DSF signaling system, the present study reveals that LesR functions as a new upstream regulatory factor of Clp in L. enzymogenes. The key factor was important for the production of HSAF. The strains with high HSAF yield can presumably be constructed by deletion of the negative regulators or overexpression of the positive regulators by genetic engineering.


Assuntos
Antibacterianos/metabolismo , Proteínas de Bactérias/biossíntese , Endopeptidase Clp/genética , Regulação Bacteriana da Expressão Gênica , Lysobacter/genética , Antifúngicos/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/farmacologia , Lysobacter/fisiologia , Metabolismo Secundário , Transdução de Sinais
9.
AMB Express ; 7(1): 123, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-28618714

RESUMO

Ax21 family proteins have been shown to play regulatory roles in plant- and animal-pathogenic species in the bacterial family Xanthomonadaceae, but the protein have not been investigated previously in the non-pathogenic members of this bacterial family. Lysobacter enzymogenes, is a non-pathogenic species known for its capacity as a biocontrol agent of plant pathogens. It is also noted for the production of antimicrobial secondary metabolites, heat stable antifungal factor (HSAF) and WAP-8294A2, that have potential for agricultural and pharmaceutical applications. The species also displays type IV pili-dependent twitching motility and the production of multiple extracellular lytic enzymes as additional biocontrol-related traits. Here, we show that L. enzymogenes strain OH11 possesses three genes widely separated in the OH11 genome that code for unique Ax21-like proteins (Lsp). By comparing the wildtype OH11 with mutant strains having a single lsp gene or a combination of lsp genes deleted, we found that each Lsp protein individually is involved in positive regulation of HSAF and WAP-8294A2 biosynthesis, but the proteins collectively do not exert additive effects in this regulation. None of the Lsp proteins were found to influence twitching motility or the production of three extracellular lytic enzymes. This study is the first to provide evidence linking Ax21-family proteins to antibiotic biosynthesis and, hence, adds new insights into the diversity of regulatory functions of Ax21 family proteins in bacteria.

10.
J Biol Chem ; 291(45): 23693-23703, 2016 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-27662907

RESUMO

Polo-like kinase 1 (Plk1) is a serine/threonine-protein kinase that has been implicated in mitosis, cytokinesis, and smooth muscle cell proliferation. The role of Plk1 in smooth muscle contraction has not been investigated. Here, stimulation with acetylcholine induced Plk1 phosphorylation at Thr-210 (an indication of Plk1 activation) in smooth muscle. Contractile stimulation also activated Plk1 in live smooth muscle cells as evidenced by changes in fluorescence resonance energy transfer signal of a Plk1 sensor. Moreover, knockdown of Plk1 in smooth muscle attenuated force development. Smooth muscle conditional knock-out of Plk1 also diminished contraction of mouse tracheal rings. Plk1 knockdown inhibited acetylcholine-induced vimentin phosphorylation at Ser-56 without affecting myosin light chain phosphorylation. Expression of T210A Plk1 inhibited the agonist-induced vimentin phosphorylation at Ser-56 and contraction in smooth muscle. However, myosin light chain phosphorylation was not affected by T210A Plk1. Ste20-like kinase (SLK) is a serine/threonine-protein kinase that has been implicated in spindle orientation and microtubule organization during mitosis. In this study knockdown of SLK inhibited Plk1 phosphorylation at Thr-210 and activation. Finally, asthma is characterized by airway hyperresponsiveness, which largely stems from airway smooth muscle hyperreactivity. Here, smooth muscle conditional knock-out of Plk1 attenuated airway resistance and airway smooth muscle hyperreactivity in a murine model of asthma. Taken together, these findings suggest that Plk1 regulates smooth muscle contraction by modulating vimentin phosphorylation at Ser-56. Plk1 activation is regulated by SLK during contractile activation. Plk1 contributes to the pathogenesis of asthma.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Contração Muscular , Músculo Liso/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Vimentina/metabolismo , Animais , Proteínas de Ciclo Celular/genética , Linhagem Celular , Técnicas de Silenciamento de Genes , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Traqueia/fisiologia
11.
Respir Res ; 17(1): 91, 2016 07 25.
Artigo em Inglês | MEDLINE | ID: mdl-27457922

RESUMO

BACKGROUND: The intermediate filament protein vimentin undergoes reversible phosphorylation and dephosphorylation at Ser-56, which plays an important role in regulating the contraction-relaxation cycles of smooth muscle. The protein phosphatases that mediate vimentin dephosphorylation in smooth muscle have not been previously investigated. METHODS: The associations of protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A) with vimentin in mouse tracheal rings was evaluated by co-immunoprecipitation. Lentivirus-mediated shRNA against PP1 was used to assess the role of PP1 in vimentin dephosphorylation and the vimentin-associated process in smooth muscle. RESULTS: Co-immunoprecipitation analysis showed that vimentin interacted with PP1, but barely with PP2A, in airway smooth muscle. Knockdown of PP1 by lentivirus-mediated shRNA increased the acetylcholine-induced vimentin phosphorylation and smooth muscle contraction. Because vimentin phosphorylation is able to modulate p130 Crk-associated substrate (p130CAS) and actin polymerization, we also evaluated the role of PP1 in the biological processes. Silencing of PP1 also enhanced the agonist-induced the dissociation of p130CAS from vimentin and F/G-actin ratios (an index of actin polymerization). However, PP1 knockdown did not affect c-Abl tyrosine phosphorylation, an important molecule that controls actin dynamics. CONCLUSIONS: Taken together, these findings suggest that PP1 is a key protein serine/threonine phosphatase that controls vimentin Ser-56 dephosphorylation in smooth muscle. PP1 regulates actin polymerization by modulating the dissociation of p130CAS from vimentin, but not by affecting c-Abl tyrosine kinase.


Assuntos
Músculo Liso/enzimologia , Proteína Fosfatase 1/metabolismo , Processamento de Proteína Pós-Traducional , Traqueia/enzimologia , Vimentina/metabolismo , Actinas/metabolismo , Animais , Proteína Substrato Associada a Crk/metabolismo , Relação Dose-Resposta a Droga , Feminino , Masculino , Camundongos Endogâmicos C57BL , Contração Muscular , Músculo Liso/efeitos dos fármacos , Cadeias Leves de Miosina/metabolismo , Ácido Okadáico/farmacologia , Fosforilação , Ligação Proteica , Proteína Fosfatase 1/genética , Proteínas Proto-Oncogênicas c-abl/metabolismo , Interferência de RNA , Serina , Fatores de Tempo , Técnicas de Cultura de Tecidos , Traqueia/efeitos dos fármacos , Transfecção
12.
Sci Rep ; 6: 26881, 2016 05 31.
Artigo em Inglês | MEDLINE | ID: mdl-27241275

RESUMO

Lysobacter species are Gram-negative bacteria that are emerging as new sources of antibiotics, including HSAF (Heat Stable Antifungal Factor), which was identified from L. enzymogenes with a new mode of action. LesR, a LuxR solo, was recently shown to regulate the HSAF biosynthesis via an unidentified mechanism in L. enzymogenes OH11. Here, we used a comparative proteomic approach to identify the LesR targets and found that LesR influenced the expression of 33 proteins belonging to 10 functional groups, with 9 proteins belonging to the TBDR (TonB-Dependent Receptor) family. The fundamental role of bacterial TBDR in nutrient uptake motivates us to explore their potential regulation on HSAF biosynthesis which is also modulated by nutrient condition. Six out of 9 TBDR coding genes were individually in-frame deleted. Phenotypic and gene-expression assays showed that TBDR7, whose level was lower in a strain overexpressing lesR, was involved in regulating HSAF yield. TBDR7 was not involved in the growth, but played a vital role in transcribing the key HSAF biosynthetic gene. Taken together, the current lesR-based proteomic study provides the first report that TBDR7 plays a key role in regulating antibiotic (HSAF) biosynthesis, a function which has never been found for TBDRs in bacteria.


Assuntos
Antibacterianos/biossíntese , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Lysobacter/genética , Proteínas de Membrana/genética , Receptores de Superfície Celular/genética , Proteínas Repressoras/genética , Transativadores/genética , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Deleção de Genes , Ontologia Genética , Lysobacter/metabolismo , Proteínas de Membrana/metabolismo , Anotação de Sequência Molecular , Proteômica , Receptores de Superfície Celular/metabolismo , Proteínas Repressoras/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Transativadores/metabolismo , Transcrição Genética
13.
Bioorg Med Chem Lett ; 26(8): 2084-7, 2016 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-26965857

RESUMO

Four new norterpene cyclic peroxides (1-4), together with three known norterpene cyclic peroxides were isolated from the Xisha Islands Sponge Diacarnus megaspinorhabdosa. Their structures were elucidated on the basis of spectroscopic analyses and comparison with the related model compounds. The compounds (1-7) were evaluated for the inhibitory activity against the malaria parasite Plasmodium falciparum, all of them showed significant antimalarial activity with IC50 values in the range of 1.6-8.6 µM.


Assuntos
Antimaláricos/farmacologia , Peróxidos/farmacologia , Plasmodium falciparum/efeitos dos fármacos , Poríferos/química , Animais , Antimaláricos/síntese química , Antimaláricos/química , Antimaláricos/isolamento & purificação , Relação Dose-Resposta a Droga , Estrutura Molecular , Testes de Sensibilidade Parasitária , Peróxidos/síntese química , Peróxidos/química , Peróxidos/isolamento & purificação , Relação Estrutura-Atividade
14.
J Biol Chem ; 290(14): 8913-24, 2015 Apr 03.
Artigo em Inglês | MEDLINE | ID: mdl-25713069

RESUMO

ß-Catenin is a key component that connects transmembrane cadherin with the actin cytoskeleton at the cell-cell interface. However, the role of the ß-catenin/cadherin interaction in smooth muscle has not been well characterized. Here stimulation with acetylcholine promoted the recruitment of ß-catenin to N-cadherin in smooth muscle cells/tissues. Knockdown of ß-catenin by lentivirus-mediated shRNA attenuated smooth muscle contraction. Nevertheless, myosin light chain phosphorylation at Ser-19 and actin polymerization in response to contractile activation were not reduced by ß-catenin knockdown. In addition, the expression of the ß-catenin armadillo domain disrupted the recruitment of ß-catenin to N-cadherin. Force development, but not myosin light chain phosphorylation and actin polymerization, was reduced by the expression of the ß-catenin armadillo domain. Furthermore, actin polymerization and microtubules have been implicated in intracellular trafficking. In this study, the treatment with the inhibitor latrunculin A diminished the interaction of ß-catenin with N-cadherin in smooth muscle. In contrast, the exposure of smooth muscle to the microtubule depolymerizer nocodazole did not affect the protein-protein interaction. Together, these findings suggest that smooth muscle contraction is mediated by the recruitment of ß-catenin to N-cadherin, which may facilitate intercellular mechanotransduction. The association of ß-catenin with N-cadherin is regulated by actin polymerization during contractile activation.


Assuntos
Caderinas/metabolismo , Músculo Liso/fisiologia , beta Catenina/metabolismo , Actinas/metabolismo , Células Cultivadas , Humanos , Mecanotransdução Celular , Microtúbulos/metabolismo , Contração Muscular , Músculo Liso/citologia , Músculo Liso/metabolismo , Polimerização
16.
Mar Drugs ; 12(8): 4399-416, 2014 Aug 08.
Artigo em Inglês | MEDLINE | ID: mdl-25110917

RESUMO

Five new norditerpene endoperoxides, named diacarperoxides H-L (1-5), and a new norditerpene diol, called diacardiol B (6), were isolated from the South China Sea sponge, Diacarnus megaspinorhabdosa. Their structures, including conformations and absolute configurations, were determined by using spectroscopic analyses, computational approaches and chemical degradation. Diacarperoxides H-J (1-3) showed some interesting stereochemical issues, as well as antimalarial activity.


Assuntos
Antimaláricos/química , Diterpenos/química , Peróxidos/química , Poríferos/química , Animais , Antimaláricos/farmacologia , Linhagem Celular Tumoral , China , Diterpenos/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Humanos , Estrutura Molecular , Oceanos e Mares , Peróxidos/farmacologia , Plasmodium falciparum/efeitos dos fármacos , Estereoisomerismo
17.
Am J Physiol Cell Physiol ; 307(3): C288-95, 2014 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-24920679

RESUMO

Histone deacetylases (HDACs) are a family of enzymes that mediate nucleosomal histone deacetylation and gene expression. Some members of the HDAC family have also been implicated in nonhistone protein deacetylation, which modulates cell-cycle control, differentiation, and cell migration. However, the role of HDACs in smooth muscle contraction is largely unknown. Here, HDAC8 was localized both in the cytoplasm and the nucleus of mouse and human smooth muscle cells. Knockdown of HDAC8 by lentivirus-encoding HDAC8 shRNA inhibited force development in response to acetylcholine. Treatment of smooth muscle tissues with HDAC8 inhibitor XXIV (OSU-HDAC-44) induced relaxation of precontracted smooth muscle tissues. In addition, cortactin is an actin-regulatory protein that undergoes deacetylation during migration of NIH 3T3 cells. In this study, acetylcholine stimulation induced cortactin deacetylation in mouse and human smooth muscle tissues, as evidenced by immunoblot analysis using antibody against acetylated lysine. Knockdown of HDAC8 by RNAi or treatment with the inhibitor attenuated cortactin deacetylation and actin polymerization without affecting myosin activation. Furthermore, expression of a charge-neutralizing cortactin mutant inhibited contraction and actin dynamics during contractile activation. These results suggest a novel mechanism for the regulation of smooth muscle contraction. In response to contractile stimulation, HDAC8 may mediate cortactin deacetylation, which subsequently promotes actin filament polymerization and smooth muscle contraction.


Assuntos
Cortactina/metabolismo , Histona Desacetilases/genética , Contração Muscular/fisiologia , Músculo Liso/fisiologia , Miócitos de Músculo Liso/fisiologia , Proteínas Repressoras/genética , Acetilação , Citoesqueleto de Actina/fisiologia , Animais , Benzamidas/farmacologia , Diferenciação Celular , Movimento Celular , Células Cultivadas , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/farmacocinética , Humanos , Ácidos Hidroxâmicos/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Contração Muscular/efeitos dos fármacos , Contração Muscular/genética , Mutação , Miosinas/metabolismo , Fosforilação , Interferência de RNA , RNA Interferente Pequeno , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/farmacocinética
18.
Am J Respir Cell Mol Biol ; 51(5): 652-9, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-24818551

RESUMO

Actin dynamics plays an essential role in regulating airway smooth muscle contraction. The mechanisms that regulate actin dynamics in smooth muscle are not completely understood. Glia maturation factor (GMF) is a protein that has been reported to inhibit actin nucleation and to induce actin network debranching in vitro. The role of GMF in human smooth muscle cells and tissues has not been investigated. In this study, knockdown of GMF-γ by RNA interference enhanced actin polymerization and contraction in human airway smooth muscle (HASM) cells and tissues without affecting myosin phosphorylation (another important biochemical change during contractile activation). Activation of HASM cells and tissues with acetylcholine induced dissociation of GMF-γ from Arp2 of the Arp2/3 complex. Acetylcholine stimulation also increased GMF-γ phosphorylation at Tyr-104. GMF-γ phosphorylation at this residue was mediated by c-Abl tyrosine kinase. The GMF-γ mutant Y104F (phenylalanine substitution at Tyr-104) had higher association with Arp2 in HASM cells upon contractile activation. Furthermore, expression of mutant Y104F GMF-γ attenuated actin polymerization and contraction in smooth muscle. Thus, we propose a novel mechanism for the regulation of actin dynamics and smooth muscle contraction. In unstimulated smooth muscle, GMF-γ binds to the Arp2/3 complex, which induces actin disassembly and retains lower levels of F-actin. Upon contractile stimulation, phosphorylation at Tyr-104 mediated by c-Abl tyrosine kinase leads to the dissociation of GMF-γ from Arp2/3, by which GMF-γ no longer induces actin disassembly. Reduced actin disassembly renders F-actin in higher level, which facilitates smooth muscle contraction.


Assuntos
Actinas/metabolismo , Fator de Maturação da Glia/metabolismo , Contração Muscular/fisiologia , Miócitos de Músculo Liso/metabolismo , Transdução de Sinais/fisiologia , Acetilcolina/farmacologia , Proteína 2 Relacionada a Actina/metabolismo , Células Cultivadas , Agonistas Colinérgicos/farmacologia , Humanos , Músculo Liso/citologia , Músculo Liso/fisiologia , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Fosforilação/fisiologia , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-abl/metabolismo , Sistema Respiratório/citologia , Transdução de Sinais/efeitos dos fármacos , Tirosina/metabolismo
19.
J Biol Chem ; 289(20): 14157-69, 2014 May 16.
Artigo em Inglês | MEDLINE | ID: mdl-24700464

RESUMO

Profilin-1 (Pfn-1) is an actin-regulatory protein that has a role in modulating smooth muscle contraction. However, the mechanisms that regulate Pfn-1 in smooth muscle are not fully understood. Here, stimulation with acetylcholine induced an increase in the association of the adapter protein cortactin with Pfn-1 in smooth muscle cells/tissues. Furthermore, disruption of the protein/protein interaction by a cell-permeable peptide (CTTN-I peptide) attenuated actin polymerization and smooth muscle contraction without affecting myosin light chain phosphorylation at Ser-19. Knockdown of cortactin by lentivirus-mediated RNAi also diminished actin polymerization and smooth muscle force development. However, cortactin knockdown did not affect myosin activation. In addition, cortactin phosphorylation has been implicated in nonmuscle cell migration. In this study, acetylcholine stimulation induced cortactin phosphorylation at Tyr-421 in smooth muscle cells. Phenylalanine substitution at this position impaired cortactin/Pfn-1 interaction in response to contractile activation. c-Abl is a tyrosine kinase that is necessary for actin dynamics and contraction in smooth muscle. Here, c-Abl silencing inhibited the agonist-induced cortactin phosphorylation and the association of cortactin with Pfn-1. Finally, treatment with CTTN-I peptide reduced airway resistance and smooth muscle hyperreactivity in a murine model of asthma. These results suggest that the interaction of cortactin with Pfn-1 plays a pivotal role in regulating actin dynamics, smooth muscle contraction, and airway hyperresponsiveness in asthma. The association of cortactin with Pfn-1 is regulated by c-Abl-mediated cortactin phosphorylation.


Assuntos
Cortactina/metabolismo , Contração Muscular , Músculo Liso/fisiologia , Profilinas/metabolismo , Acetilcolina/farmacologia , Actinas/química , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Proteínas Adaptadoras de Transdução de Sinal/genética , Sequência de Aminoácidos , Animais , Brônquios/citologia , Brônquios/efeitos dos fármacos , Brônquios/fisiologia , Cortactina/química , Proteínas do Citoesqueleto/deficiência , Proteínas do Citoesqueleto/genética , Técnicas de Silenciamento de Genes , Humanos , Técnicas In Vitro , Camundongos , Dados de Sequência Molecular , Contração Muscular/efeitos dos fármacos , Músculo Liso/citologia , Músculo Liso/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/farmacologia , Fosforilação/efeitos dos fármacos , Ligação Proteica , Multimerização Proteica/efeitos dos fármacos , Estrutura Quaternária de Proteína , Proteínas Proto-Oncogênicas c-abl/deficiência , Proteínas Proto-Oncogênicas c-abl/genética , Proteínas Proto-Oncogênicas c-abl/metabolismo , Traqueia/citologia , Traqueia/efeitos dos fármacos , Traqueia/fisiologia , Tirosina/metabolismo
20.
Am J Physiol Cell Physiol ; 306(8): C753-61, 2014 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-24477238

RESUMO

c-Abl is a nonreceptor protein tyrosine kinase that has a role in regulating smooth muscle cell proliferation and contraction. The role of c-Abl in smooth muscle cell migration has not been investigated. In the present study, c-Abl was found in the leading edge of smooth muscle cells. Knockdown of c-Abl by RNA interference attenuated smooth muscle cell motility as evidenced by time-lapse microscopy. Furthermore, the actin-associated proteins cortactin and profilin-1 (Pfn-1) have been implicated in cell migration. In this study, cell adhesion induced cortactin phosphorylation at Tyr-421, an indication of cortactin activation. Phospho-cortactin and Pfn-1 were also found in the cell edge. Pfn-1 directly interacted with cortactin in vitro. Silencing of c-Abl attenuated adhesion-induced cortactin phosphorylation and Pfn-1 localization in the cell edge. To assess the role of cortactin/Pfn-1 coupling, we developed a cell-permeable peptide. Treatment with the peptide inhibited the interaction of cortactin with Pfn-1 without affecting cortactin phosphorylation. Moreover, treatment with the peptide impaired the recruitment of Pfn-1 to the leading edge and cell migration. Finally, ß1-integrin was required for the recruitment of c-Abl to the cell edge. Inhibition of actin dynamics impaired the spatial distribution of c-Abl. These results suggest that ß1-integrin may recruit c-Abl to the leading cell edge, which may regulate cortactin phosphorylation in response to cell adhesion. Phosphorylated cortactin may facilitate the recruitment of Pfn-1 to the cell edge, which promotes localized actin polymerization, leading edge formation, and cell movement. Conversely, actin dynamics may strengthen the recruitment of c-Abl to the leading edge.


Assuntos
Movimento Celular/fisiologia , Miócitos de Músculo Liso/enzimologia , Miócitos de Músculo Liso/fisiologia , Proteínas Proto-Oncogênicas c-abl/metabolismo , Animais , Far-Western Blotting , Adesão Celular , Células Cultivadas , Cortactina/genética , Cortactina/metabolismo , Regulação Enzimológica da Expressão Gênica , Humanos , Integrina beta1/genética , Integrina beta1/metabolismo , Fosforilação , Profilinas/genética , Profilinas/metabolismo , Proteínas Proto-Oncogênicas c-abl/genética , Interferência de RNA , Transdução Genética
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