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1.
Vet Microbiol ; 239: 108461, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31767078

RESUMO

Corynebacterium pseudotuberculosis, a broad host-spectrum zoonotic pathogen, causes caseous lymphadenitis (CLA) in small ruminants and is responsible for considerable economic losses in the livestock industry worldwide. Macrophages play a pivotal role in the immunopathogenesis of CLA. However, the immunoregulatory mechanisms of macrophages against C. pseudotuberculosis remains poorly understood. In the present study, for the first time, the partial exoproteome of murine peritoneal macrophages infected with C. pseudotuberculosis was profiled and the differential expression of the identified proteins was analyzed. In macrophages, infection with C. pseudotuberculosis, rather than with heat-killed bacteria, induced release of diverse proteins. Three unconventional proteins: cofilin-1, peroxiredoxin-1, and galectin-3 were significantly expressed and released by infected macrophages into the culture supernatant. These proteins are involved in the host inflammatory response and may be responsible for the excessive inflammation of CLA. In C. pseudotuberculosis-infected macrophages, the release of cofilin-1 and peroxiredoxin-1 was predominant at later stages of infection, while the release of galectin-3 was independent of time. Taken together, the present work contributes to our understanding of the functional role of macrophage response to C. pseudotuberculosis infection.


Assuntos
Cofilina 1/imunologia , Infecções por Corynebacterium/imunologia , Corynebacterium pseudotuberculosis/imunologia , Galectina 3/imunologia , Macrófagos/imunologia , Peroxirredoxinas/imunologia , Cofilina 1/genética , Infecções por Corynebacterium/fisiopatologia , Galectina 3/genética , Regulação da Expressão Gênica/imunologia , Macrófagos/microbiologia , Peroxirredoxinas/genética
2.
Nat Cell Biol ; 21(11): 1370-1381, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31685997

RESUMO

Cell migration is hypothesized to involve a cycle of behaviours beginning with leading edge extension. However, recent evidence suggests that the leading edge may be dispensable for migration, raising the question of what actually controls cell directionality. Here, we exploit the embryonic migration of Drosophila macrophages to bridge the different temporal scales of the behaviours controlling motility. This approach reveals that edge fluctuations during random motility are not persistent and are weakly correlated with motion. In contrast, flow of the actin network behind the leading edge is highly persistent. Quantification of actin flow structure during migration reveals a stable organization and asymmetry in the cell-wide flowfield that strongly correlates with cell directionality. This organization is regulated by a gradient of actin network compression and destruction, which is controlled by myosin contraction and cofilin-mediated disassembly. It is this stable actin-flow polarity, which integrates rapid fluctuations of the leading edge, that controls inherent cellular persistence.


Assuntos
Actinas/genética , Movimento Celular/genética , Drosophila melanogaster/embriologia , Mecanotransdução Celular , Peixe-Zebra/embriologia , Actinas/metabolismo , Animais , Polaridade Celular , Rastreamento de Células , Cofilina 1/genética , Cofilina 1/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Embrião não Mamífero , Regulação da Expressão Gênica no Desenvolvimento , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Hemócitos/citologia , Hemócitos/metabolismo , Queratinócitos/citologia , Queratinócitos/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Macrófagos/citologia , Macrófagos/metabolismo , Miosinas/genética , Miosinas/metabolismo , Cultura Primária de Células , Peixe-Zebra/genética , Peixe-Zebra/metabolismo
3.
J Oral Pathol Med ; 48(10): 959-966, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31359510

RESUMO

BACKGROUND: The overexpression of metastasis-associated protein 2 (MTA2) contributes to human tumor progression and metastasis in various tumor cells. However, the role of MTA2 in human oral cancer progression remains unknown. MATERIALS AND METHODS: MTA2 expression in human oral tumor tissues and cell lines was measured by immunohistochemistry and Western blotting. Cell proliferation and cell cycle were analyzed using MTT assay and flow cytometry. The effects of MTA2 on oral cell migration and invasion were investigated using migration and invasion assays. The expression of MTA2, p-cofilin-1, and MTA2-induced LC3-II levels were measured using Western blotting and an immunofluorescence assay. RESULTS: Based on the human oral cancer tissue array and TCGA database, we found that MTA2 was increased in oral cancer tissues than in non-tumor oral tissues (P < .01). Moreover, MTA2 is significantly associated with tumor grade (P < .01) and the overall survival rate of patients with grade III tumor (P < .05). MTA2 expression in oral cancer cells was markedly higher than that in normal oral cells. Cell proliferation and cell cycle were not significantly changed in the cells inhibited by MTA2. MTA2 knockdown can inhibit cell migration and invasion of human oral cancer cells. Furthermore, we suggest that MTA2 inhibition enhances p-cofilin and LC3-II expression, and the knockdown of LC3-II expression in cells inhibited by MTA2 had the opposite effect. CONCLUSION: These results indicate that MTA2 may serve as a candidate prognostic biomarker and that targeting autophagy is a potential therapeutic strategy for treating human oral cancer.


Assuntos
Cofilina 1/genética , Histona Desacetilases/genética , Proteínas Associadas aos Microtúbulos/genética , Neoplasias Bucais/patologia , Proteínas Repressoras/genética , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Técnicas de Silenciamento de Genes , Humanos , Metástase Neoplásica
4.
Bull Exp Biol Med ; 167(3): 393-395, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31346877

RESUMO

We studied the expression of mRNA and the level of CAP1 (adenylate cyclase-associated protein 1) and cofilin proteins in the tissues of patients with non-small cell lung cancer. The expression of mRNA and the level of CAP1 in tumor tissue increased during growth of the primary tumor and its metastasis. It was shown that with the growth of the primary tumor, the content of cofilin in the tumor tissue decreases against the background of increased expression of its mRNA; in regional metastasis, the content of cofilin and expression of the corresponding mRNA increased. It was found that increased content of the studied proteins in the tumor tissue increased the risk of metastasis.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Proteínas de Ciclo Celular/genética , Cofilina 1/genética , Proteínas do Citoesqueleto/genética , Neoplasias Pulmonares/genética , RNA Mensageiro/biossíntese , Carcinoma Pulmonar de Células não Pequenas/patologia , Proteínas de Ciclo Celular/metabolismo , Cofilina 1/metabolismo , Proteínas do Citoesqueleto/metabolismo , Feminino , Humanos , Neoplasias Pulmonares/patologia , Masculino , Metástase Neoplásica/patologia
5.
Health Phys ; 116(6): 749-759, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30913056

RESUMO

Gamma radiation causes cell injury and leads to an increased risk of cancer, so it is of practical significance to identify biomarkers for gamma radiation. We used proteomic analysis to identify differentially expressed proteins in liver tissues of C57BL/6J mice treated with gamma radiation from Cs for 360 d. We confirmed obvious pathological changes in mouse liver tissues after irradiation. Compared with the control group, 74 proteins showed a fold change of ≥1.5 in the irradiated groups. We selected 24 proteins for bioinformatics analysis and peptide mass fingerprinting and found that 20 of the identified proteins were meaningful. These proteins were associated with tumorigenesis, tumor suppression, catalysis, cell apoptosis, cytoskeleton, metabolism, gene transcription, T-cell response, and other pathways. We confirmed that both cofilin-1 and destrin were up regulated in the irradiated groups by western blot and real-time polymerase chain reaction. Our findings indicate that cofilin-1 and destrin are sensitive to gamma radiation and may be potential biomarkers for gamma radiation. Whether these proteins are involved in radiation-induced tumorigenesis requires further investigation.


Assuntos
Biomarcadores/metabolismo , Cofilina 1/metabolismo , Destrina/metabolismo , Fígado/metabolismo , Proteoma/análise , Animais , Biomarcadores/análise , Cofilina 1/genética , Destrina/genética , Raios gama , Fígado/efeitos da radiação , Masculino , Camundongos , Camundongos Endogâmicos C57BL
6.
IUBMB Life ; 71(3): 364-375, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30550624

RESUMO

Emerging evidence on efficient tumor growth regulation by endogenous lectins directs interest to determine on a proof-of-principle level the range of information on alterations provided by full-scale analysis using phosphoproteomics. In our pilot study, we tested galectin-4 (gal-4) that is a growth inhibitor for colon cancer cells (CRC), here working with the LS 180 line. In order to cover monitoring of short- and long-term effects stable isotope labeling by amino acids in cell culture-based quantitative phosphoproteomic analyses were conducted on LS 180 cell preparations collected 1 and 72 h after adding gal-4 to the culture medium. After short-term treatment, 981 phosphosites, all of them S/T based, were detected by phosphoproteomics. Changes higher than 1.5-fold were seen for eight sites in seven proteins. Most affected were the BET1 homolog (BET1), whose level of phosphorylation at S50 was about threefold reduced, and centromere protein F (CENPF), extent of phosphorylation at S3119 doubling in gal-4-treated cells. Phosphoproteome analysis after 72 h of treatment revealed marked changes at 33 S/T-based phosphosites from 29 proteins. Prominent increase of phosphorylation was observed for cofilin-1 at position S3. Extent of phosphorylation of the glutamine transporter SLC1A5 at position S503 was decreased by a factor of 3. Altered phosphorylation of BET1, CENPF, and cofilin-1 as well as a significant effect of gal-4 treatment on glutamine uptake by cells were substantiated by independent methods in the Vaco 432, Colo 205, CX 1, and HCT 116 cell lines. With the example of gal-4 which functions as a tumor suppressor in CRC cells, we were able to prove that cell surface binding of the lectin not only markedly influences the cell proteome, but also has a bearing on malignancy-associated intracellular protein phosphorylation. These results underscore the potential of this approach to give further work on elucidating the details of signaling underlying galectin-triggered growth inhibition a clear direction. © 2018 IUBMB Life, 71(3):364-375, 2019.


Assuntos
Antineoplásicos/farmacologia , Galectina 4/farmacologia , Proteínas de Neoplasias/metabolismo , Fosfoproteínas/metabolismo , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteoma/metabolismo , Sistema ASC de Transporte de Aminoácidos/genética , Sistema ASC de Transporte de Aminoácidos/metabolismo , Transporte Biológico/efeitos dos fármacos , Isótopos de Carbono , Linhagem Celular Tumoral , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Cofilina 1/genética , Cofilina 1/metabolismo , Glutamina/metabolismo , Células HCT116 , Humanos , Marcação por Isótopo , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Antígenos de Histocompatibilidade Menor/genética , Antígenos de Histocompatibilidade Menor/metabolismo , Proteínas de Neoplasias/genética , Isótopos de Nitrogênio , Fosfoproteínas/genética , Fosforilação/efeitos dos fármacos , Proteoma/genética , Proteínas Qc-SNARE/genética , Proteínas Qc-SNARE/metabolismo , Proteínas Recombinantes/farmacologia
7.
Biochem Biophys Res Commun ; 509(2): 395-401, 2019 02 05.
Artigo em Inglês | MEDLINE | ID: mdl-30594393

RESUMO

Methamphetamine (METH) is a psychostimulant with severe neurotoxicity, which is related to an increase of blood-brain barrier (BBB) permeability. However, the exact mechanisms have not been fully illuminated. In the present study, male Sprague Dawley rats were treated with METH or saline with 8 injections (i.p.) at 12-h intervals and sacrificed 24 h after the last METH injection. To evaluate BBB permeability, 6 rats were administered with Evans blue (EB) by tail vein injection 1 h prior to sacrifice. EB levels significantly increased in both left and right frontal lobes in METH-treated rats, suggesting increase of BBB permeability, which was proved by the rearrangement of F-actin cytoskeleton and decreased expressions of tight junction (TJ) proteins in hippocampus. Over-expressions of RhoA, ROCK, myosin light chain (MLC), cofilin, phosphorylation (p)-MLC, p-cofilin and matrix metalloproteinase (MMP)-9 were observed, indicating activated RhoA/ROCK pathway. Rat brain microvascular endothelial cells (RBMECs) were isolated and treated with inhibitors of RhoA and ROCK followed by METH. Pretreatments of the inhibitors significantly decreased expressions of RhoA, ROCK, MLC, cofilin, p-MLC and p-cofilin, increased expressions of TJ proteins, suppressed F-actin cytoskeleton rearrangement and reduced the permeability of RBMECs. These results suggested that METH increased BBB permeability through activating the RhoA/ROCK pathway, which resulted in F-actin cytoskeleton rearrangement and down-regulation of TJ proteins.


Assuntos
Citoesqueleto de Actina/efeitos dos fármacos , Barreira Hematoencefálica/efeitos dos fármacos , Estimulantes do Sistema Nervoso Central/farmacologia , Hipocampo/efeitos dos fármacos , Metanfetamina/farmacologia , Junções Íntimas/efeitos dos fármacos , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/ultraestrutura , Actinas/genética , Actinas/metabolismo , Animais , Barreira Hematoencefálica/metabolismo , Cofilina 1/genética , Cofilina 1/metabolismo , Corantes/farmacocinética , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Azul Evans/farmacocinética , Regulação da Expressão Gênica , Hipocampo/citologia , Hipocampo/metabolismo , Masculino , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , Cadeias Leves de Miosina/genética , Cadeias Leves de Miosina/metabolismo , Permeabilidade/efeitos dos fármacos , Cultura Primária de Células , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Proteínas de Junções Íntimas/genética , Proteínas de Junções Íntimas/metabolismo , Junções Íntimas/metabolismo , Junções Íntimas/ultraestrutura , Proteínas rho de Ligação ao GTP/genética , Proteínas rho de Ligação ao GTP/metabolismo , Quinases Associadas a rho/genética , Quinases Associadas a rho/metabolismo
8.
J Cell Sci ; 132(2)2019 01 18.
Artigo em Inglês | MEDLINE | ID: mdl-30584064

RESUMO

A large number of neuronal proteins must show correct spatiotemporal localization in order to carry out their critical functions. The mRNA transcript for the somatodendritic protein activity-regulated cytoskeleton-associated protein (Arc; also known as Arg3.1) contains two conserved introns in the 3' untranslated region (UTR), and was proposed to be a natural target for nonsense-mediated mRNA decay (NMD). However, a well-known NMD component Upf1 has differential roles in transcriptional and translational regulation of Arc gene expression. Specifically, Upf1 suppresses Arc transcription by enhancing destabilization of mRNAs encoding various transcription factors, including Mef2a. Upf1 also binds to the Arc 3'UTR, resulting in suppression of translation. Surprisingly, the Arc transcript escapes from Upf1-mediated NMD by binding to Ago2 (also known as miRISC), which blocks NMD and further suppresses Arc mRNA translation. Upf1 knockdown triggered sustained Arc expression, which contributes to Cofilin (also known as Cfl1) hyperphosphorylation and abnormal neuronal outgrowth and branching. Collectively, these data reveal that multiple levels of Upf1-mediated inhibition of Arc gene expression may allow neurons to more effectively respond to changes in neuronal activity.


Assuntos
Proteínas do Citoesqueleto/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neuritos/metabolismo , Degradação do RNAm Mediada por Códon sem Sentido , Transativadores/metabolismo , Transcrição Genética , Animais , Linhagem Celular , Cofilina 1/genética , Cofilina 1/metabolismo , Proteínas do Citoesqueleto/genética , Camundongos , Proteínas do Tecido Nervoso/genética , Transativadores/genética
9.
Bull Exp Biol Med ; 166(2): 250-252, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30488206

RESUMO

The model of head and neck squamous cell carcinoma (HNSCC) was used to study the expression of genes encoding actin-binding proteins depending on the type of cell motility. The expression of SNAIL1 and CAPN2 mRNA in HNSCC tissue was higher than in specimens of dysplastic epithelium of the larynx and hypopharynx, which can be explained by activation of mesenchymal and amoeboid types of cell motility. In biopsy material of HNSCC patients with T1-2N0M0, expression of genes responsible for actin-binding proteins differed from that of patients with pretumor pathology of the larynx and hypopharynx: expression of FSCN was lower, while expressions of EZR and CAP1 were higher. The data attest that progression of HNSCC is associated with activation of both types of cell motility and with the changes in the expression of mRNA encoding cell motility proteins.


Assuntos
Calpaína/genética , Movimento Celular/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias de Cabeça e Pescoço/genética , Fatores de Transcrição da Família Snail/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Calpaína/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Cofilina 1/genética , Cofilina 1/metabolismo , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Progressão da Doença , Feminino , Neoplasias de Cabeça e Pescoço/metabolismo , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Hipofaringe/metabolismo , Hipofaringe/patologia , Laringe/metabolismo , Laringe/patologia , Masculino , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Pessoa de Meia-Idade , Profilinas/genética , Profilinas/metabolismo , Transdução de Sinais , Fatores de Transcrição da Família Snail/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Vimentina/genética , Vimentina/metabolismo , Proteínas rho de Ligação ao GTP/genética , Proteínas rho de Ligação ao GTP/metabolismo
10.
Respir Res ; 19(1): 229, 2018 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-30463566

RESUMO

BACKGROUND: Hyperoxia is a frequently employed therapy for prematurely born infants, induces lung injury and contributes to development of bronchopulmonary dysplasia (BPD). BPD is characterized by decreased cellular proliferation, cellular migration, and failure of injury repair systems. Actin binding proteins (ABPs) such as VASP, cofilin1, and profilin1 regulate cell proliferation and migration via modulation of actin dynamics. Lung mesenchymal stem cells (L-MSCs) initiate repair processes by proliferating, migrating, and localizing to sites of injury. These processes have not been extensively explored in hyperoxia induced lung injury and repair. METHODS: ABPs and CD146+ L-MSCs were analyzed by immunofluorescence in human lung autopsy tissues from infants with and without BPD and by western blot in lung tissue homogenates obtained from our murine model of newborn hyperoxic lung injury. RESULTS: Decreased F-actin content, ratio of VASPpS157/VASPpS239, and profilin 1 expression were observed in human lung tissues but this same pattern was not observed in lungs from hyperoxia-exposed newborn mice. Increases in cofilin1 expression were observed in both human and mouse tissues at 7d indicating a dysregulation in actin dynamics which may be related to altered growth. CD146 levels were elevated in human and newborn mice tissues (7d). CONCLUSION: Altered phosphorylation of VASP and expression of profilin 1 and cofilin 1 in human tissues indicate that the pathophysiology of BPD involves dysregulation of actin binding proteins. Lack of similar changes in a mouse model of hyperoxia exposure imply that disruption in actin binding protein expression may be linked to interventions or morbidities other than hyperoxia alone.


Assuntos
Displasia Broncopulmonar/metabolismo , Moléculas de Adesão Celular/metabolismo , Cofilina 1/biossíntese , Hiperóxia/metabolismo , Lesão Pulmonar/metabolismo , Proteínas dos Microfilamentos/metabolismo , Fosfoproteínas/metabolismo , Profilinas/biossíntese , Animais , Animais Recém-Nascidos , Displasia Broncopulmonar/patologia , Cofilina 1/genética , Feminino , Expressão Gênica , Humanos , Hiperóxia/patologia , Recém-Nascido , Lesão Pulmonar/patologia , Camundongos , Camundongos Endogâmicos C3H , Fosforilação/fisiologia , Gravidez , Profilinas/genética , Distribuição Aleatória
11.
Oncol Rep ; 40(4): 2298-2306, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30066939

RESUMO

Lycorine, a natural alkaloid extracted from the Amaryllidaceae plant family, has been reported to exhibit anti­cancer effects in various types of cancer cells. However, the molecular mechanisms through which lycorine exhibits anti­hepatoblastoma activity are unclear. In the present study, the inhibitory effects of lycorine on the proliferation and migration of HepG2 hepatoblastoma cells were investigated. Lycorine inhibited the proliferation of HepG2 cells in a dose­dependent manner by inducing cell cycle arrest at the G2/M phase, via downregulation of cyclin A, cyclin B1 and cyclin dependent kinase 1. Additionally, wound healing and Transwell assays revealed that treatment with lycorine resulted in a decrease in the migratory ability of HepG2 cells. Also, treatment with lycorine decreased the expression levels of matrix metalloproteinase (MMP)­9 and MMP­2. Furthermore, lycorine induced the cleavage/activation of Rho associated coiled­coil containing protein kinase 1 (ROCK1) and the downregulation of cofilin, accompanied by an increase in polymerized filamentous actin and a loss of depolymerized globular actin. Furthermore, pre­incubation of cells with Y­27632, a specific ROCK1 inhibitor, markedly attenuated lycorine­induced anti­proliferative and anti­migration effects. Taken together, the results demonstrated that lycorine inhibited the proliferation and migration of HepG2 cells by suppressing ROCK1/cofilin­induced actin dynamics, which suggests that lycorine has the potential to be developed into a novel drug for hepatoblastoma treatment.


Assuntos
Actinas/metabolismo , Alcaloides de Amaryllidaceae/farmacologia , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Cofilina 1/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Hepatoblastoma/patologia , Fenantridinas/farmacologia , Quinases Associadas a rho/metabolismo , Apoptose/efeitos dos fármacos , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Cofilina 1/genética , Hepatoblastoma/tratamento farmacológico , Hepatoblastoma/metabolismo , Humanos , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Células Tumorais Cultivadas , Quinases Associadas a rho/genética
12.
PLoS One ; 13(8): e0201553, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30110355

RESUMO

Despite the prevalence and high heritability of Attention-Deficit/Hyperactivity Disorder (ADHD), genetic etiology remains elusive. Clinical evidence points in part to reduced function of the striatum, but which specific genes are differentially expressed and how they sculpt striatal physiology to predispose ADHD are not well understood. As an exploratory tool, a polygenic mouse model of ADHD was recently developed through selective breeding for high home cage activity. Relative to the Control line, the High-Active line displays hyperactivity and motor impulsivity which are ameliorated with amphetamine. This study compared gene expression in the striatum between Control and High-Active mice to develop a coherent hypothesis for how genes might affect striatal physiology and predispose ADHD-like symptoms. To this end, striatal transcriptomes of High-Active and Control mice were analyzed after mice were treated with saline or amphetamines. The pseudogene Gm6180 for n-cofilin (Cfl1) displayed 20-fold higher expression in High-Active mice corresponding with reduced Cfl1 expression suggesting synaptic actin dysregulation. Latrophilin 3 (Lphn3), which is associated with ADHD in human populations and is involved in synapse structure, and its ligand fibronectin leucine rich transmembrane protein 3 (Flrt3), were downregulated in High-Active mice. Multiple genes were altered in High-Active mice in a manner predicted to downregulate the canonical Wnt pathway. A smaller and different set of genes including glyoxalase (Glo1) were differentially regulated in High-Active as compared to Control in response to amphetamine. Together, results suggest genes involved in excitatory synapse regulation and maintenance are downregulated in ADHD-like mice. Consistent with the molecular prediction, stereological analysis of the striatum from a separate set of mice processed for imunohistochemical detection of synaptophysin revealed approximately a 46% reduction in synaptophysin immunoreactivity in High-Active relative to Control. Results provide a new set of molecular targets related to synapse maintenance for the next generation of ADHD medicines.


Assuntos
Anfetaminas/efeitos adversos , Transtorno do Deficit de Atenção com Hiperatividade/genética , Perfilação da Expressão Gênica/métodos , Análise de Sequência de RNA/métodos , Córtex Visual/química , Animais , Transtorno do Deficit de Atenção com Hiperatividade/induzido quimicamente , Cofilina 1/genética , Modelos Animais de Doenças , Potenciais Pós-Sinápticos Excitadores , Regulação da Expressão Gênica , Humanos , Lactoilglutationa Liase/genética , Masculino , Glicoproteínas de Membrana/genética , Camundongos , Pseudogenes , Receptores Acoplados a Proteínas-G/genética , Receptores de Peptídeos/genética , Via de Sinalização Wnt
13.
Exp Cell Res ; 370(2): 663-670, 2018 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-30036538

RESUMO

The matrix metalloproteinases (MMPs) are implicated in tumor invasion and metastasis. Given their multiple tumor promoting roles, MMPs are promising targets for the treatment of metastatic cancer. Using a siRNA library screen of 140 membrane trafficking genes, we identified 41 genes in HEC-1B and 36 in Ishikawa cancer cells that decreased metalloproteinases activity. The 16 genes common in both cancer cell lines that decreased MMPs activity are involved in cargo sorting, vesicle formation and vesicle recycling. The top two genes clathrin-B and cofilin-1 were chosen for post hoc functional studies. Higher expression of both genes was confirmed in cancer cells and knockdown with respective siRNAs inhibited their invasive potential and matrix metalloproteinases activity. Membrane Type 1- Matrix Metalloproteinase (MT1-MMP) is a master switch proteinase and regulator of invasion and metastasis. A marked decrease in MT1-MMP expression and activity was seen in clathrin-B and cofilin-1 knockdown cancer cells which was associated with a marked decreased expression of invadopodia formation proteins. Our results suggest that the decreased expression of clathrin-B and cofilin-1 decreases the expression of MT1-MMP and results in attenuation of MT1-MMP at the cell surface, thus inhibiting tumor cell invasion and metastasis.


Assuntos
Cofilina 1/genética , Neoplasias do Endométrio/genética , Interferência de RNA/fisiologia , Sesquiterpenos/metabolismo , Detecção Precoce de Câncer/métodos , Neoplasias do Endométrio/diagnóstico , Matriz Extracelular/metabolismo , Feminino , Humanos , Metaloproteinase 14 da Matriz/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinases da Matriz/metabolismo , Metaloendopeptidases , Invasividade Neoplásica/genética
14.
Hum Mol Genet ; 27(17): 3060-3078, 2018 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-29878125

RESUMO

Hyper-activation of extracellular signal-regulated kinase (ERK) 1/2 contributes to heart dysfunction in cardiomyopathy caused by mutations in the lamin A/C gene (LMNA cardiomyopathy). The mechanism of how this affects cardiac function is unknown. We show that active phosphorylated ERK1/2 directly binds to and catalyzes the phosphorylation of the actin depolymerizing factor cofilin-1 on Thr25. Cofilin-1 becomes active and disassembles actin filaments in a large array of cellular and animal models of LMNA cardiomyopathy. In vivo expression of cofilin-1, phosphorylated on Thr25 by endogenous ERK1/2 signaling, leads to alterations in left ventricular function and cardiac actin. These results demonstrate a novel role for cofilin-1 on actin dynamics in cardiac muscle and provide a rationale on how increased ERK1/2 signaling leads to LMNA cardiomyopathy.


Assuntos
Actinas/metabolismo , Cardiomiopatia Dilatada/patologia , Cofilina 1/metabolismo , Lamina Tipo A/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Mutação , Actinas/genética , Adolescente , Adulto , Animais , Cardiomiopatia Dilatada/genética , Cardiomiopatia Dilatada/metabolismo , Estudos de Casos e Controles , Cofilina 1/genética , Feminino , Coração/fisiologia , Humanos , Lamina Tipo A/metabolismo , Masculino , Camundongos , Pessoa de Meia-Idade , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/genética , Fosforilação , Transdução de Sinais , Adulto Jovem
15.
Clin Cancer Res ; 24(12): 2951-2962, 2018 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-29563135

RESUMO

Purpose: Successful immunotherapies for IDHmut gliomas require better knowledge of T-cell target antigens. Here, we elucidated their antigen repertoire recognized by spontaneous T-cell responses using an unbiased proteomic approach.Experimental Design: Protein fractionations of tissue lysates from IDHmut gliomas (n = 4) were performed. Fractions were tested by IFNγ ELISpot assay for recognition through patients' T cells. Proteins of immunogenic fractions were identified by mass spectrometry and validated by in silico-predicted synthetic long peptides in patients of origin, additional IDHmut glioma patients (n = 16), and healthy donors (n = 13). mRNA and protein expression of immunogenic antigens was analyzed in tumor tissues and IDHmut glioma stem-like cells (GSC). HLA-A*02-restricted T-cell epitopes were functionally determined by short peptides and numbers of antigen-specific T cells by HLA-peptide tetramer analysis.Results: A total of 2,897 proteins were identified in immunogenic tumor fractions. Based on a thorough filter process, 79 proteins were selected as potential T-cell antigens. Twenty-six of these were recognized by the patients' T cells, and five of them (CRKII, CFL1, CNTN1, NME2, and TKT) in up to 56% unrelated IDHmut glioma patients. Most immunogenic tumor-associated antigens (TAA) were expressed in IDHmut gliomas and GSCs, while being almost absent in normal brain tissues. Finally, we identified HLA-A*02-restricted epitopes for CRKII, NME2, and TKT that were recognized by up to 2.82% of antigen-specific peripheral cytotoxic T cells in IDHmut glioma patients.Conclusions: By analyzing the repertoire of T-cell target antigens in IDHmut glioma patients, we identified five novel immunogenic TAAs and confirmed their expression on IDHmut tumors and GSCs. Clin Cancer Res; 24(12); 2951-62. ©2018 AACR.


Assuntos
Biomarcadores Tumorais , Glioma/genética , Glioma/metabolismo , Isocitrato Desidrogenase/genética , Mutação , Linfócitos T/metabolismo , Antígenos de Neoplasias/imunologia , Linhagem Celular Tumoral , Cromatografia Líquida , Cofilina 1/genética , Cofilina 1/metabolismo , Contactina 1/genética , Contactina 1/metabolismo , Citocinas/metabolismo , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Mapeamento de Epitopos , Glioma/imunologia , Humanos , Imuno-Histoquímica , Isocitrato Desidrogenase/metabolismo , Nucleosídeo NM23 Difosfato Quinases/genética , Nucleosídeo NM23 Difosfato Quinases/metabolismo , Proteoma , Proteômica/métodos , Proteínas Proto-Oncogênicas c-crk/genética , Proteínas Proto-Oncogênicas c-crk/metabolismo , Linfócitos T/imunologia , Espectrometria de Massas em Tandem
16.
EMBO J ; 37(5)2018 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-29440227

RESUMO

In neuronal development, dynamic rearrangement of actin promotes axonal growth cone extension, and spatiotemporal translation of local mRNAs in response to guidance cues directs axonal growth cone steering, where cofilin plays a critical role. While regulation of cofilin activity is well studied, regulatory mechanism for cofilin mRNA translation in neurons is unknown. In eukaryotic cells, proteins can be synthesized by cap-dependent or cap-independent mechanism via internal ribosome entry site (IRES)-mediated translation. IRES-mediated translation has been reported in various pathophysiological conditions, but its role in normal physiological environment is poorly understood. Here, we report that 5'UTR of cofilin mRNA contains an IRES element, and cofilin is predominantly translated by IRES-mediated mechanism in neurons. Furthermore, we show that IRES-mediated translation of cofilin is required for both axon extension and axonal growth cone steering. Our results provide new insights into the function of IRES-mediated translation in neuronal development.


Assuntos
Axônios/fisiologia , Cofilina 1/genética , Cones de Crescimento/fisiologia , Sítios Internos de Entrada Ribossomal/genética , Neurogênese/genética , Regiões 5' não Traduzidas/genética , Animais , Encéfalo/embriologia , Sistemas CRISPR-Cas , Linhagem Celular , Proliferação de Células/genética , Cofilina 1/metabolismo , Camundongos , Biossíntese de Proteínas/genética , RNA Mensageiro/genética
17.
Am J Physiol Lung Cell Mol Physiol ; 314(5): L799-L807, 2018 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-29345194

RESUMO

With every deep inspiration (DI) or sigh, the airway wall stretches, as do the airway smooth muscle cells in the airway wall. In response, the airway smooth muscle cell undergoes rapid stretch-induced cytoskeletal fluidization. As a molecular mechanism underlying the cytoskeletal fluidization response, we demonstrate a key role for the actin-severing protein cofilin. Using primary human airway smooth muscle cells, we simulated a DI by imposing a transient stretch of physiological magnitude and duration. We used traction microscopy to measure the resulting changes in contractile forces. After a transient stretch, cofilin-knockdown cells exhibited a 29 ± 5% decrease in contractile force compared with prestretch conditions. By contrast, control cells exhibited a 67 ± 6% decrease ( P < 0.05, knockdown vs. control). Consistent with these contractile force changes with transient stretch, actin filaments in cofilin-knockdown cells remained largely intact, whereas actin filaments in control cells were rapidly disrupted. Furthermore, in cofilin-knockdown cells, contractile force at baseline was higher and rate of remodeling poststretch was slower than in control cells. Additionally, the severing action of cofilin was restricted to the release phase of the transient stretch. We conclude that the actin-severing activity of cofilin is an important factor in stretch-induced cytoskeletal fluidization and may account for an appreciable part of the bronchodilatory effects of a DI.


Assuntos
Citoesqueleto de Actina/fisiologia , Cofilina 1/metabolismo , Citoesqueleto/fisiologia , Contração Muscular/fisiologia , Miócitos de Músculo Liso/fisiologia , Sistema Respiratório/metabolismo , Células Cultivadas , Cofilina 1/antagonistas & inibidores , Cofilina 1/genética , Humanos , Mecanotransdução Celular , Miócitos de Músculo Liso/citologia , RNA Interferente Pequeno/genética , Sistema Respiratório/citologia , Reologia
18.
J Cardiovasc Electrophysiol ; 29(3): 412-420, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29377394

RESUMO

INTRODUCTION: Reticulated platelet (RP) content is increased in nonvalvular atrial fibrillation (NVAF). The purpose of this study was to determine if platelet content, morphology, and RP proportion are modulated by platelet genes. METHODS AND RESULTS: Expression of six platelet-predominate genes impacting platelet formation and release, platelet count, and RP content was assessed in NVAF patients before and 3-4 months after pulmonary veins isolation (PVI) and compared to normal sinus rhythm (NSR) controls. RNA from isolated platelets was reverse-transcribed assayed against selected genes utilizing real-time qPCR, and expressed as mean cycle threshold (ΔCt) using beta-2-microglobulin as endogenous control. RP content was assessed by flow cytometry. A fourfold lower expression of CFL1 gene coding for nonmuscle cofilin (7.8 ± 0.9 vs. 5.7 ± 1.6, P < 0.001) and twofold lower expression of four other genes were associated with similar platelet counts but fourfold higher (28.7+7.0 vs. 6.7+5.4, P < 0.001) RP content (%) in 97 NVAF cases compared to 51 NSR controls. Three to 4 months after PVI, RP decreased by 28%, while CFL1 gene expression increased over twofold but TUBA4A gene expression decreased almost twofold; NFE2 and MYL6 gene expression remained unchanged. CONCLUSIONS: NVAF is associated with notable downregulation of genes directing platelet production and size but increased RP content. PVI impacts the expression of many of these genes, implying a direct relationship between atrial fibrillation and platelet biogenesis.


Assuntos
Fibrilação Atrial/cirurgia , Plaquetas/metabolismo , Ablação por Cateter , Veias Pulmonares/cirurgia , Potenciais de Ação , Idoso , Fibrilação Atrial/sangue , Fibrilação Atrial/diagnóstico , Fibrilação Atrial/fisiopatologia , Plaquetas/patologia , Estudos de Casos e Controles , Ablação por Cateter/efeitos adversos , Cofilina 1/sangue , Cofilina 1/genética , Feminino , Regulação da Expressão Gênica , Frequência Cardíaca , Humanos , Masculino , Proteínas de Membrana/sangue , Proteínas de Membrana/genética , Pessoa de Meia-Idade , Cadeias Leves de Miosina/sangue , Cadeias Leves de Miosina/genética , Subunidade p45 do Fator de Transcrição NF-E2/sangue , Subunidade p45 do Fator de Transcrição NF-E2/genética , Contagem de Plaquetas , Veias Pulmonares/fisiopatologia , Receptores de Progesterona/sangue , Receptores de Progesterona/genética , Fatores de Tempo , Resultado do Tratamento , Tubulina (Proteína)/sangue , Tubulina (Proteína)/genética
19.
Biochem Biophys Res Commun ; 506(2): 372-377, 2018 11 25.
Artigo em Inglês | MEDLINE | ID: mdl-29102633

RESUMO

For the acquisition of the ability to fertilize the egg, mammalian spermatozoa should undergo a series of biochemical transformations in the female reproductive tract, collectively called capacitation. The capacitated sperm can undergo the acrosomal exocytosis process near or on the oocyte, which allows the spermatozoon to penetrate and fertilize it. One of the main processes in capacitation involves dynamic cytoskeletal remodeling particularly of actin. Actin polymerization occurs during sperm capacitation and the produced F-actin should be depolymerized prior to the acrosomal exocytosis. In the present review, we describe the mechanisms that regulate F-actin formation during sperm capacitation and the F-actin dispersion prior to the acrosomal exocytosis. During sperm capacitation, the actin severing proteins gelsolin and cofilin are inactive and they undergo activation prior to the acrosomal exocytosis.


Assuntos
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Cálcio/metabolismo , Cofilina 1/metabolismo , Gelsolina/metabolismo , Espermatozoides/metabolismo , Reação Acrossômica , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/ultraestrutura , Actinas/química , Actinas/genética , Animais , Cofilina 1/genética , Exocitose , Feminino , Gelsolina/genética , Regulação da Expressão Gênica , Humanos , Masculino , Oócitos/citologia , Oócitos/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Transdução de Sinais , Capacitação Espermática , Espermatozoides/citologia
20.
Nat Cell Biol ; 19(12): 1389-1399, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-29131140

RESUMO

Re-establishment of nuclear structure and chromatin organization after cell division is integral for genome regulation or development and is frequently altered during cancer progression. The mechanisms underlying chromatin expansion in daughter cells remain largely unclear. Here, we describe the transient formation of nuclear actin filaments (F-actin) during mitotic exit. These nuclear F-actin structures assemble in daughter cell nuclei and undergo dynamic reorganization to promote nuclear protrusions and volume expansion throughout early G1 of the cell cycle. Specific inhibition of this nuclear F-actin assembly impaired nuclear expansion and chromatin decondensation after mitosis and during early mouse embryonic development. Biochemical screening for mitotic nuclear F-actin interactors identified the actin-disassembling factor cofilin-1. Optogenetic regulation of cofilin-1 revealed its critical role for controlling timing, turnover and dynamics of F-actin assembly inside daughter cell nuclei. Our findings identify a cell-cycle-specific and spatiotemporally controlled form of nuclear F-actin that reorganizes the mammalian nucleus after mitosis.


Assuntos
Actinas/metabolismo , Cromatina/metabolismo , Citoesqueleto de Actina/química , Citoesqueleto de Actina/metabolismo , Actinas/química , Animais , Blastocisto/metabolismo , Núcleo Celular/metabolismo , Tamanho do Núcleo Celular , Montagem e Desmontagem da Cromatina/fisiologia , Cofilina 1/genética , Cofilina 1/metabolismo , Fase G1/fisiologia , Camundongos , Mitose/fisiologia , Modelos Biológicos , Células NIH 3T3 , Optogenética , Multimerização Proteica
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