Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 375
Filtrar
1.
Nat Struct Mol Biol ; 27(2): 210-220, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32015554

RESUMO

Neddylation is the post-translational protein modification most closely related to ubiquitination. Whereas the ubiquitin-like protein NEDD8 is well studied for its role in activating cullin-RING E3 ubiquitin ligases, little is known about other substrates. We developed serial NEDD8-ubiquitin substrate profiling (sNUSP), a method that employs NEDD8 R74K knock-in HEK293 cells, allowing discrimination of endogenous NEDD8- and ubiquitin-modification sites by MS after Lys-C digestion and K-εGG-peptide enrichment. Using sNUSP, we identified 607 neddylation sites dynamically regulated by the neddylation inhibitor MLN4924 and the de-neddylating enzyme NEDP1, implying that many non-cullin proteins are neddylated. Among the candidates, we characterized lysine 112 of the actin regulator cofilin as a novel neddylation event. Global inhibition of neddylation in developing neurons leads to cytoskeletal defects, altered actin dynamics and neurite growth impairments, whereas site-specific neddylation of cofilin at K112 regulates neurite outgrowth, suggesting that cofilin neddylation contributes to the regulation of neuronal actin organization.


Assuntos
Actinas/metabolismo , Cofilina 1/metabolismo , Proteína NEDD8/metabolismo , Neurônios/metabolismo , Animais , Linhagem Celular , Células Cultivadas , Técnicas de Introdução de Genes , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Proteína NEDD8/genética , Neurônios/citologia , Mutação Puntual , Ratos , Ratos Sprague-Dawley , Ubiquitina/metabolismo , Ubiquitinação
2.
Artigo em Inglês | MEDLINE | ID: mdl-31676439

RESUMO

OBJECTIVE: Highly elevated plasma levels of interleukin-10 (IL-10) are causally associated with "Disappearing HDL Syndrome" and low plasma LDL-cholesterol, but the underlying mechanism is poorly understood. Fluid-phase endocytosis, a process highly dependent on actin dynamics, enables cells to internalize relatively high amounts of extracellular fluids and solutes. We sought to investigate whether IL-10 induces lipoprotein uptake by fluid-phase endocytosis in macrophages. METHODS AND RESULTS: Macrophages (RAW264.7, Kupffer and human) were incubated with vehicle (PBS) or IL-10 (20 ng/ml) for 7 days. Uptake of HDL, LDL, and/or fluid-phase endocytosis probes (albumin-Alexa680®, 70 kDa FITC-Dextran and Lucifer Yellow, LY) was evaluated by FACS. Intracellular cofilin and phosphorylated cofilin (p-cofilin) levels were determined by immunoblotting. Macrophage uptake of lipoproteins and probes was non-saturable and increased after IL-10 incubation (p < 0.0001). Furthermore, pre-incubation with fluid-phase endocytosis inhibitors (LY294002, Latrunculin A, and Amiloride) significantly reduced uptake (p < 0.05). IL-10 increased the cofilin/p-cofilin ratio (p = 0.021), signifying increased cofilin activation and hence filamentous actin. Consistently, phalloidin staining revealed increased filamentous actin in macrophages after IL-10 treatment (p = 0.0018). Finally, RNA-seq analysis demonstrated enrichment of gene sets related to actin filament dynamics, membrane ruffle formation and endocytosis in IL-10-treated macrophages (p < 0.05). IL-10 did not alter mRNA levels of Ldlr, Vldlr, Scarb1, Cd36 or Lrp1. In primary human monocyte-derived macrophages and murine Kupffer cells, IL-10 incubation also increased uptake of lipoproteins, albumin and LY (p < 0.01). CONCLUSIONS: Interleukin-10 induces the uptake of HDL and LDL by fluid-phase endocytosis by increasing actin-filament rearrangement in macrophages, thus providing a plausible mechanism contributing to "Disappearing HDL Syndrome".


Assuntos
Interleucina-10/metabolismo , Lipoproteínas HDL/metabolismo , Lipoproteínas LDL/metabolismo , Macrófagos/metabolismo , Citoesqueleto de Actina/metabolismo , Animais , Aterosclerose/sangue , Aterosclerose/metabolismo , Células Cultivadas , Cofilina 1/metabolismo , Endocitose , Humanos , Camundongos , Cultura Primária de Células , Proteínas Recombinantes/metabolismo
3.
Nat Commun ; 10(1): 5320, 2019 11 22.
Artigo em Inglês | MEDLINE | ID: mdl-31757941

RESUMO

The ability of cells to generate forces through actin filament turnover was an early adaptation in evolution. While much is known about how actin filaments grow, mechanisms of their disassembly are incompletely understood. The best-characterized actin disassembly factors are the cofilin family proteins, which increase cytoskeletal dynamics by severing actin filaments. However, the mechanism by which severed actin filaments are recycled back to monomeric form has remained enigmatic. We report that cyclase-associated-protein (CAP) works in synergy with cofilin to accelerate actin filament depolymerization by nearly 100-fold. Structural work uncovers the molecular mechanism by which CAP interacts with actin filament pointed end to destabilize the interface between terminal actin subunits, and subsequently recycles the newly-depolymerized actin monomer for the next round of filament assembly. These findings establish CAP as a molecular machine promoting rapid actin filament depolymerization and monomer recycling, and explain why CAP is critical for actin-dependent processes in all eukaryotes.


Assuntos
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Proteínas de Transporte/metabolismo , Cofilina 1/metabolismo , Animais , Cristalografia por Raios X , Camundongos , Simulação de Dinâmica Molecular , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Coelhos
4.
Nat Commun ; 10(1): 5319, 2019 11 22.
Artigo em Inglês | MEDLINE | ID: mdl-31757952

RESUMO

Cellular actin networks can be rapidly disassembled and remodeled in a few seconds, yet in vitro actin filaments depolymerize slowly over minutes. The cellular mechanisms enabling actin to depolymerize this fast have so far remained obscure. Using microfluidics-assisted TIRF, we show that Cyclase-associated protein (CAP) and Cofilin synergize to processively depolymerize actin filament pointed ends at a rate 330-fold faster than spontaneous depolymerization. Single molecule imaging further reveals that hexameric CAP molecules interact with the pointed ends of Cofilin-decorated filaments for several seconds at a time, removing approximately 100 actin subunits per binding event. These findings establish a paradigm, in which a filament end-binding protein and a side-binding protein work in concert to control actin dynamics, and help explain how rapid actin network depolymerization is achieved in cells.


Assuntos
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Cofilina 1/metabolismo , Proteínas do Citoesqueleto/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Animais , Microfluídica , Microscopia de Fluorescência , Coelhos , Imagem Individual de Molécula
5.
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
6.
Pathol Res Pract ; 215(10): 152582, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31427165

RESUMO

Nowadays, histopathological criteria for melanocytic lesions are the mainstay prognostic factors for melanoma. However, there are cases in which these parameters fall short to predict melanoma spread. We recently demonstrated a correlation of cofilin-1 levels, a key protein for tumor invasion, with different histopathological parameters associated with melanoma malignancy as well as a negative correlation with survival. In order to broaden our previous findings, we aim to estimate the probability of a melanoma to metastasize as a function of both a conventional histopathological parameter (Breslow thickness, BT) and cofilin-1's immunohistochemical expression levels, which we propose as a potential marker for metastasis. We used a Bayesian approach to analyze clinical and cofilin-1 datasets formerly obtained from a patients' small cohort diagnosed with malignant melanocytic lesions since 2000 until 2008; classified at different tumor stages with or without detected metastasis and with at least 5 years of clinical follow-up. Low BT values exhibited wide variance to predict metastasis occurrence, while the differential diagnostic value of cofilin-1 confirmed BT diagnosis or resulted more precise to predict outcome. Particularly, the probability of metastasis estimation improved when cofilin-1 was combined with BT for specific cases, where BT displayed large uncertainties. Our analysis and the cofilin-1 determination provided statistically significant prognostic value in mid-low BT melanomas, which could complement further evaluation criteria to assist diagnosis and treatment decision-making. Moreover, the combined use of cofilin-1 with BT, if validated in follow-up studies, would be feasible to help patients' selection for treatment and optimize health resources.


Assuntos
Cofilina 1/metabolismo , Melanoma/diagnóstico , Metástase Neoplásica/diagnóstico , Neoplasias Cutâneas/diagnóstico , Idoso , Idoso de 80 Anos ou mais , Argentina , Biomarcadores Tumorais/metabolismo , Feminino , Humanos , Masculino , Melanoma/metabolismo , Melanoma/patologia , Pessoa de Meia-Idade , Metástase Neoplásica/patologia , Estadiamento de Neoplasias , Prognóstico , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia
7.
Neuron ; 103(6): 1073-1085.e6, 2019 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-31400829

RESUMO

Injured axons fail to regenerate in the adult CNS, which contrasts with their vigorous growth during embryonic development. We explored the potential of re-initiating axon extension after injury by reactivating the molecular mechanisms that drive morphogenetic transformation of neurons during development. Genetic loss- and gain-of-function experiments followed by time-lapse microscopy, in vivo imaging, and whole-mount analysis show that axon regeneration is fueled by elevated actin turnover. Actin depolymerizing factor (ADF)/cofilin controls actin turnover to sustain axon regeneration after spinal cord injury through its actin-severing activity. This pinpoints ADF/cofilin as a key regulator of axon growth competence, irrespective of developmental stage. These findings reveal the central role of actin dynamics regulation in this process and elucidate a core mechanism underlying axon growth after CNS trauma. Thereby, neurons maintain the capacity to stimulate developmental programs during adult life, expanding their potential for plasticity. Thus, actin turnover is a key process for future regenerative interventions.


Assuntos
Actinas/metabolismo , Axônios/metabolismo , Cofilina 1/genética , Cofilina 2/genética , Destrina/genética , Cones de Crescimento/patologia , Regeneração Nervosa/genética , Traumatismos da Medula Espinal/genética , Animais , Axônios/patologia , Cofilina 1/metabolismo , Cofilina 2/metabolismo , Destrina/metabolismo , Cones de Crescimento/metabolismo , Microscopia Intravital , Camundongos , Microscopia Confocal , Neurônios/metabolismo , Neurônios/patologia , Ratos , Traumatismos da Medula Espinal/metabolismo , Traumatismos da Medula Espinal/patologia , Imagem com Lapso de Tempo
8.
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
9.
Oncol Rep ; 42(2): 805-816, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31233187

RESUMO

Metastasis is the primary cause of mortality in patients with non­small cell lung cancer (NSCLC). Actin cytoskeletal reorganization is usually accompanied by the epithelial­mesenchymal transition (EMT)­induced invasion and metastasis of cancer cells. In the present study, expression levels of the actin­associated protein cofilin­1 and of the pivotal EMT molecule Twist­1 were determined in NSCLC tissues. Using lung cancer tissue arrays, the identification of 67.4% of tissue spots that exhibited reciprocal levels of cofilin­1 and Twist­1 was achieved by immunohistochemical (IHC) staining. This reciprocal expression pattern was also detected in 21 out of 25 clinicopathological NSCLC tissue sections, and in 10 out of 15 NSCLC cell lines. In addition, high levels of cofilin­1 and low levels of Twist­1 accounted for 80 and 71.5% of the reciprocal expression pattern in tissue arrays and clinicopathological tissue samples, respectively. This pattern was also detected in normal lung tissues, stage I and II lung cancer tissues, and adenocarcinoma subtypes of NSCLC tissues. Although cofilin­1 and Twist­1 were expressed inversely, a positive correlation of these two proteins was present in normal lung tissues and lung tumor tissues. Furthermore, enforced expression of cofilin­1 suppressed the expression level of Twist­1 in NSCLC H1299 cells. An on­line Kaplan­Meier survival analytic tool allowed access to a public microarray dataset with a maximum of 1,926 NSCLC samples. The analysis revealed that high expression levels of both cofilin­1 (CFL1) and Twist­1 (TWIST1) genes were associated with decreased survival of NSCLC patients, notably with regard to the adenocarcinoma subtype. The analysis was conducted using the multivariate Cox regression model. Although the reciprocal association of the expression levels of cofilin­1 and Twist­1 with the survival rate of NSCLC patients requires additional information, it may be a significant indicator of the progression of NSCLC.


Assuntos
Biomarcadores Tumorais/metabolismo , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Cofilina 1/metabolismo , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/metabolismo , Proteínas Nucleares/metabolismo , Proteína 1 Relacionada a Twist/metabolismo , Adenocarcinoma de Pulmão/metabolismo , Adenocarcinoma de Pulmão/patologia , Adenocarcinoma de Pulmão/cirurgia , Adulto , Idoso , Idoso de 80 Anos ou mais , Apoptose , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma Pulmonar de Células não Pequenas/cirurgia , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Carcinoma de Células Escamosas/cirurgia , Estudos de Casos e Controles , Movimento Celular , Proliferação de Células , Transição Epitelial-Mesenquimal , Feminino , Seguimentos , Humanos , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/cirurgia , Masculino , Pessoa de Meia-Idade , Prognóstico , Taxa de Sobrevida , Células Tumorais Cultivadas
10.
Invest Ophthalmol Vis Sci ; 60(6): 2274-2285, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-31112612

RESUMO

Purpose: Retraction of the axon terminals of rod photoreceptors after retinal detachment breaks the first synapse in the visual pathway, resulting in visual impairment. Previous work showed that the mechanism of axonal retraction involves RhoA signaling and its downstream effector LIM Kinase (LIMK) activation. We examined the response of the downstream component cofilin, a direct binding protein of actin filaments, as well as the regulation by RhoA-LIMK-Cofilin signaling of actin assembly/disassembly, in the presynaptic ribbon terminal of injured rod cells. Methods: Injury was produced by retinal detachment or rod cell isolation. Detached porcine retina was probed for levels and localization of phosphorylated cofilin with Western blots and confocal microscopy, whereas rod cell cultures of dissociated salamander retina were examined for filamentous actin assembly/disassembly with a barbed end assay and phalloidin staining. Results: A detachment increased phosphorylation of cofilin in retinal explants; phosphorylation occurred in rod terminals in sections of detached retina. Isolation of rod cells resulted in axon retraction accompanied by an increase in actin barbed ends and a decrease in net filament labeling. All changes were significantly reduced by either Rho kinase (ROCK) or LIMK inhibition, using Y27632 or BMS-5, respectively. Cytochalasin D also reduced retraction and stabilized filaments in isolated rod cells. Conclusions: These results indicate that actin depolymerization via activation of RhoA downstream kinases and cofilin contributes to axon retraction. Preventing depolymerization, in addition to actomyosin contraction, may stabilize ribbon synapses after trauma.


Assuntos
Actinas/metabolismo , Cofilina 1/metabolismo , Retina/lesões , Descolamento Retiniano/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/fisiologia , Transdução de Sinais/fisiologia , Proteína rhoA de Ligação ao GTP/metabolismo , Amidas/farmacologia , Animais , Axônios/metabolismo , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , Quinases Lim/antagonistas & inibidores , Plasticidade Neuronal/fisiologia , Piridinas/farmacologia , Suínos , Quinases Associadas a rho/farmacologia , Proteína rhoA de Ligação ao GTP/antagonistas & inibidores
11.
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
12.
Chin J Nat Med ; 17(3): 198-208, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30910056

RESUMO

Resistance to cisplatin (DDP)-based chemotherapy is a major cause of treatment failure in human gastric cancer (GC). It is necessary to identify the drugs to re-sensitize GC cells to DDP. In our previous research, Zuo Jin Wan Formula (ZJW) has been proved could increase the mitochondrial apoptosis via cofilin-1 in a immortalized cell line, SGC-7901/DDP. Due to the immortalized cells may still difficult highly recapitulate the important molecular events in vivo, primary GC cells model derived from clinical patient was constructed in the present study to further evaluate the effect of ZJW and the underlying molecular mechanism. Immunofluorescent staining was used to indentify primary cultured human GC cells. Western blotting was carried out to detect the protein expression. Cell Counting Kit-8 (CCK-8) was used to evaluate cell proliferation. Flow cytometry analysis was performed to assess cell apoptosis. ZJW inhibited proliferation and induced apoptosis in primary DDP-resistant GC cells. Notably, the apoptosis in GC cells was mediated by inducing cofilin-1 mitochondrial translocation, down-regulating Bcl-2 and up-regulating Bax expression. Surprisingly, the level of p-AKT protein was higher in DDP-resistant GC cells than that of the DDP-sensitive GC cells, and the activation of AKT could attenuate ZJW-induced sensitivity to DDP. These data revealed that ZJW can increase the chemosensitivity in DDP-resistant primary GC cells by inducing mitochondrial apoptosis and AKT inactivation. The combining chemotherapy with ZJW may be an effective therapeutic strategy for GC chemoresistance patients.


Assuntos
Cisplatino/uso terapêutico , Cofilina 1/metabolismo , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Medicamentos de Ervas Chinesas/farmacologia , Mitocôndrias/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Neoplasias Gástricas/tratamento farmacológico , Adulto , Idoso , Idoso de 80 Anos ou mais , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Cisplatino/farmacologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/patologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologia , Células Tumorais Cultivadas
13.
Med Hypotheses ; 125: 8-9, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30902157

RESUMO

To date, there is no clear evidence for memory formation. In this article, we provide a framework to understand how memory is formed. The information collected by sensory organs is converted to a digital current that enters the presynaptic neuron through axonal conductance. Digital waves are converted to analog waves in the synapses. The analog current of information flows into the postsynapse. The degree of Ca2+ influx in the postsynapse is proportional to the voltage of each wave of analog current. The activation (via dephosphorylation) of the phosphorylated phosphatase, Slingshot, is regulated by Ca2+ concentration in the spine. After dephosphorylation by Slingshot, activated cofilin binds the parallel actin bundle. The wide helical twist angle of an actin filament that has been decorated with cofilin confers high electric potential to the filament. Phosphorylation results in the deactivation of the actin filament bound to cofilin, which in turn results in the cleavage of cofilin and actin filament, followed by a decrease in the twist angle of the actin filament. Next, the electric potential energy is discharged by the actin filament as it returns to its non-cofilin bound state, resulting in the formation of additional analog waves in the postsynapse.


Assuntos
Hipocampo/fisiopatologia , Memória , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Animais , Axônios , Cálcio/metabolismo , Calmodulina/metabolismo , Cofilina 1/metabolismo , Hipocampo/fisiologia , Humanos , Memória de Longo Prazo , Modelos Neurológicos , Neurônios/fisiologia , Fosfoproteínas Fosfatases/metabolismo , Fosforilação , Eletricidade Estática
14.
Reprod Sci ; 26(11): 1499-1505, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-30808273

RESUMO

Endometriosis is an estrogen-dependent gynecological disease; however, the mechanism by which estradiol promotes the development of endometriosis, including invasion and proliferation, remains unclear. Estradiol is involved in cell invasion and proliferation by regulating the cytoskeleton. The abnormally high expression of cytoskeletal regulators (LIM kinase 1 [LIMK1] and cofilin1) is closely related to increased invasiveness and proliferation of eutopic endometrial stromal cells from endometriosis patients compared to normal eutopic endometrial cells. The aim of this study was to analyze the role of estradiol during invasion and proliferation through the LIMK1/cofilin1 pathway in the endometrium of women with endometriosis. To address this, primary eutopic endometrial stromal cells were isolated from the uteri of patients with endometriosis and cultured without estradiol. The phosphorylation of cofilin1 was analyzed by western blotting. Cell invasiveness and proliferation were evaluated following LIMK1 knockdown by RNA interference technology. We found that, before LIMK1silencing, the phosphorylation levels of cofilin1 and LIMK1 of eutopic endometrial stromal cells from endometriosis patients treated with estradiol were higher than cells not treated with estradiol (P < .05 and P < .01, respectively). The total levels of cofilin1 and LIMK1 protein did not change (P > .05 and P > .05, respectively). After LIMK1 silencing, the phosphorylation of cofilin1 by estradiol was significantly reduced, and invasiveness and proliferation were clearly and concurrently decreased (P < .05 and P < .05, respectively). Thus, the phosphorylation of cofilin1 by estradiol is mediated by LIMK1, and estradiol is involved in regulating cell invasion and proliferation in endometriotic patients through the LIMK1/cofilin1 pathway.


Assuntos
Proliferação de Células/fisiologia , Cofilina 1/metabolismo , Endometriose/metabolismo , Endométrio/metabolismo , Estradiol/farmacologia , Quinases Lim/metabolismo , Adulto , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Endometriose/patologia , Endométrio/efeitos dos fármacos , Endométrio/patologia , Feminino , Humanos , Fosforilação/efeitos dos fármacos , Fosforilação/fisiologia , Células Estromais/efeitos dos fármacos , Células Estromais/metabolismo , Células Estromais/patologia
15.
Cell Death Dis ; 10(2): 126, 2019 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-30755597

RESUMO

Factors mediating mobilization of osteoblastic stem and progenitor cells from their bone marrow niche to be recruited to bone formation sites during bone remodeling are poorly known. We have studied secreted factors present in the bone marrow microenvironment and identified KIAA1199 (also known as CEMIP, cell migration inducing hyaluronan binding protein) in human bone biopsies as highly expressed in osteoprogenitor reversal cells (Rv.C) recruited to the eroded surfaces (ES), which are the future bone formation sites. In vitro, KIAA1199 did not affect the proliferation of human osteoblastic stem cells (also known as human bone marrow skeletal or stromal stem cells, hMSCs); but it enhanced cell migration as determined by scratch assay and trans-well migration assay. KIAA1199 deficient hMSCs (KIAA1199down) exhibited significant changes in cell size, cell length, ratio of cell width to length and cell roundness, together with reduction of polymerization actin (F-actin) and changes in phos-CFL1 (cofflin1), phos-LIMK1 (LIM domain kinase 1) and DSTN (destrin), key factors regulating actin cytoskeletal dynamics and cell motility. Moreover, KIAA1199down hMSC exhibited impaired Wnt signaling in TCF-reporter assay and decreased expression of Wnt target genes and these effects were rescued by KIAA1199 treatment. Finally, KIAA1199 regulated the activation of P38 kinase and its associated changes in Wnt-signaling. Thus, KIAA1199 is a mobilizing factor that interacts with P38 and Wnt signaling, and induces changes in actin cytoskeleton, as a mechanism mediating recruitment of hMSC to bone formation sites.


Assuntos
Movimento Celular , Hialuronoglucosaminidase/metabolismo , Células-Tronco Mesenquimais/metabolismo , Osteogênese/fisiologia , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Proliferação de Células , Tamanho Celular , Cofilina 1/metabolismo , Destrina/metabolismo , Células HEK293 , Humanos , Hialuronoglucosaminidase/genética , Quinases Lim/metabolismo , Sistema de Sinalização das MAP Quinases , Transfecção , Via de Sinalização Wnt
16.
Proc Natl Acad Sci U S A ; 116(7): 2595-2602, 2019 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-30692249

RESUMO

Proteins of the actin depolymerizing factor (ADF)/cofilin family are the central regulators of actin filament disassembly. A key function of ADF/cofilin is to sever actin filaments. However, how it does so in a physiological context, where filaments are interconnected and under mechanical stress, remains unclear. Here, we monitor and quantify the action of ADF/cofilin in different mechanical situations by using single-molecule, single-filament, and filament network techniques, coupled to microfluidics. We find that local curvature favors severing, while tension surprisingly has no effect on cofilin binding and weakly enhances severing. Remarkably, we observe that filament segments that are held between two anchoring points, thereby constraining their twist, experience a mechanical torque upon cofilin binding. We find that this ADF/cofilin-induced torque does not hinder ADF/cofilin binding, but dramatically enhances severing. A simple model, which faithfully recapitulates our experimental observations, indicates that the ADF/cofilin-induced torque increases the severing rate constant 100-fold. A consequence of this mechanism, which we verify experimentally, is that cross-linked filament networks are severed by cofilin far more efficiently than nonconnected filaments. We propose that this mechanochemical mechanism is critical to boost ADF/cofilin's ability to sever highly connected filament networks in cells.


Assuntos
Citoesqueleto de Actina/fisiologia , Cofilina 1/fisiologia , Destrina/fisiologia , Citoesqueleto de Actina/metabolismo , Animais , Fenômenos Biomecânicos , Cofilina 1/metabolismo , Destrina/metabolismo , Humanos , Cinética , Ligação Proteica , Coelhos , Proteínas Recombinantes/metabolismo
17.
FASEB J ; 33(1): 668-682, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30024789

RESUMO

Nogo-A is a key inhibitory molecule of axon regeneration in oligodendrocytes. However, little is known about its role in adult neurons. In this study, we showed an important function of Nogo-A on regulation of inflammatory pain in dorsal root ganglion (DRG) neurons. In adult rats with complete Freund's adjuvant (CFA) hind paw inflammation, DRG neurons showed a significant increase in Nogo-A expression. Disruption of Nogo-A signaling with Nogo-66 receptor antagonist peptide, Nogo-A blocking antibody, Nogo-A short hairpin RNA, or Nogo-A gene knockout attenuated CFA-induced inflammatory heat hyperalgesia. Moreover, disruption of Nogo-A signaling suppressed the function and expression in DRG neurons of the transient receptor potential vanilloid subfamily member (TRPV)-1 channel, which is known to be the endogenous transducer of noxious heat during inflammation. These effects were accompanied with a reduction in LIM domain kinase (LIMK)/cofilin phosphorylation and actin polymerization. Similar disruption of actin filament architecture by direct action of Latrunculin A reduced the TRPV-1 activity and up-regulation of TRPV-1 protein caused by CFA. We conclude that Nogo-A plays an essential role in the development of inflammatory heat hyperalgesia, partly through maintaining TRPV-1 function via activation of the LIMK/cofilin pathway, which regulates actin filament dynamics. These findings support a therapeutic potential of modulating Nogo-A signaling in pain management.-Hu, F., Liu, H.-C., Su, D.-Q., Chen, H.-J., Chan, S.-O., Wang, Y., Wang, J. Nogo-A promotes inflammatory heat hyperalgesia by maintaining TRPV-1 function in the rat dorsal root ganglion neuron.


Assuntos
Gânglios Espinais/patologia , Temperatura Alta/efeitos adversos , Hiperalgesia/etiologia , Inflamação Neurogênica/complicações , Neurônios/patologia , Proteínas Nogo/metabolismo , Canais de Cátion TRPV/metabolismo , Animais , Cofilina 1/metabolismo , Gânglios Espinais/imunologia , Gânglios Espinais/metabolismo , Hiperalgesia/metabolismo , Hiperalgesia/patologia , Quinases Lim/metabolismo , Masculino , Inflamação Neurogênica/metabolismo , Inflamação Neurogênica/patologia , Neurônios/imunologia , Neurônios/metabolismo , Ratos , Ratos Sprague-Dawley
18.
Biochemistry ; 58(1): 40-47, 2019 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-30499293

RESUMO

Actin depolymerizing factor (ADF)/cofilin is the main protein family promoting the disassembly of actin filaments, which is essential for numerous cellular functions. ADF/cofilin proteins disassemble actin filaments through different reactions, as they bind to their sides, sever them, and promote the depolymerization of the resulting ADF/cofilin-saturated filaments. Moreover, the efficiency of ADF/cofilin is known to be very sensitive to pH. ADF/cofilin thus illustrates two challenges in actin biochemistry: separating the different regulatory actions of a single protein and characterizing them as a function of specific biochemical conditions. Here, we investigate the different reactions of ADF/cofilin on actin filaments, at four different pH values ranging from 6.6 to 7.8, using single-filament microfluidics techniques. We show that decreasing the pH decreases the effective filament severing rate by increasing the rate at which filaments become saturated by ADF/cofilin, thereby reducing the number of ADF/cofilin domain boundaries, where severing can occur. The severing rate per domain boundary, however, remains unchanged at different pH values. The ADF/cofilin-decorated filaments ("cofilactin" filaments) depolymerize from both ends. We show here that, at physiological pH (7.0-7.4), the pointed end depolymerization of cofilactin filaments is barely faster than that of bare filaments. In contrast, cofilactin barbed ends undergo an "unstoppable" depolymerization (depolymerizing for minutes despite the presence of free actin monomers and capping protein in solution), throughout our pH range. We thus show that, at physiological pH, the main contribution of ADF/cofilin to filament depolymerization is at the barbed end.


Assuntos
Citoesqueleto de Actina/metabolismo , Fatores de Despolimerização de Actina/metabolismo , Actinas/metabolismo , Cofilina 1/metabolismo , Citoesqueleto de Actina/química , Fatores de Despolimerização de Actina/química , Actinas/química , Animais , Cofilina 1/química , Humanos , Concentração de Íons de Hidrogênio , Camundongos , Coelhos
19.
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
20.
Biomed Pharmacother ; 109: 751-761, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30551528

RESUMO

INTRODUCTION: Esophageal squamous cell carcinoma (ESCC) represents an aggressive malignancy often accompanied with a poor prognosis. Owing to the poor mortality and morbidity rates associated with this malignancy, a deeper understanding of the finer molecular changes that occur in ESCC is required in order to identify novel potential targets for early detection and therapy. At present the mechanism by which ESCC functions on a molecular level is not fully understood. Hence, the aim of the present study was to ascertain as to whether microRNA-384 (miR-384) influences the progression of ESCC. MATERIAL AND METHODS: Bioinformatics analysis was initially conducted to identify ESCC-related differentially expressed genes and predict regulatory miRs. After the target relationship between miR-384 and LIMK1 had been verified, the expression of miR-384 and LIMK1 in the EC9706 cell line was altered in an attempt to investigate the regulatory roles of miR-384 in the expression of the LIMK1/cofilin signaling pathway-related genes, cell proliferation, invasion, cell cycle distribution and apoptosis, in addition to lymph node metastasis (LNM) and tumor growth in nude mice. RESULTS: Microarray-based gene expression profiling indicated that miR-384 affected the progression of ESCC through the LIMK1-mediated LIMK1/cofilin signaling pathway. Furthermore, miR-384 and Bax were observed to be poorly expressed, while LIMK1, cofilin and Bcl-2 were highly expressed in ESCC. The obtained evidences indicating that miR-384 targeted and negatively regulated LIMK1. Upregulation of miR-384 or LIMK1 inhibition was determined to block the LIMK1/cofilin signaling pathway, repress cell proliferation, invasion, cell cycle, LNM and tumor growth, while promote cell apoptosis in ESCC. CONCLUSION: Collectively, based on the key findings of the study, miR-384 could sequester LIMK1, which acts to suppress activation of the LIMK1/cofilin signaling pathway, thus ultimately inhibiting the development and progression of ESCC.


Assuntos
Cofilina 1/metabolismo , Progressão da Doença , Neoplasias Esofágicas/metabolismo , Carcinoma de Células Escamosas do Esôfago/metabolismo , Quinases Lim/metabolismo , MicroRNAs/biossíntese , Adulto , Idoso , Animais , Linhagem Celular Tumoral , Cofilina 1/antagonistas & inibidores , Neoplasias Esofágicas/patologia , Neoplasias Esofágicas/prevenção & controle , Carcinoma de Células Escamosas do Esôfago/patologia , Carcinoma de Células Escamosas do Esôfago/prevenção & controle , Feminino , Humanos , Quinases Lim/antagonistas & inibidores , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Pessoa de Meia-Idade , Ligação Proteica/fisiologia , Ratos , Ratos Nus , Transdução de Sinais/fisiologia
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA