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1.
Elife ; 132024 Nov 13.
Artigo em Inglês | MEDLINE | ID: mdl-39535529

RESUMO

Mammalian sperm delve into the female reproductive tract to fertilize the female gamete. The available information about how sperm regulate their motility during the final journey to the fertilization site is extremely limited. In this work, we investigated the structural and functional changes in the sperm flagellum after acrosomal exocytosis (AE) and during the interaction with the eggs. The evidence demonstrates that the double helix actin network surrounding the mitochondrial sheath of the midpiece undergoes structural changes prior to the motility cessation. This structural modification is accompanied by a decrease in diameter of the midpiece and is driven by intracellular calcium changes that occur concomitant with a reorganization of the actin helicoidal cortex. Midpiece contraction occurs in a subset of cells that undergo AE, and live-cell imaging during in vitro fertilization showed that the midpiece contraction is required for motility cessation after fusion is initiated. These findings provide the first evidence of the F-actin network's role in regulating sperm motility, adapting its function to meet specific cellular requirements during fertilization, and highlighting the broader significance of understanding sperm motility.


Assuntos
Actinas , Fertilização , Motilidade dos Espermatozoides , Cauda do Espermatozoide , Espermatozoides , Masculino , Animais , Motilidade dos Espermatozoides/fisiologia , Fertilização/fisiologia , Cauda do Espermatozoide/fisiologia , Cauda do Espermatozoide/metabolismo , Actinas/metabolismo , Espermatozoides/fisiologia , Camundongos , Feminino , Exocitose/fisiologia , Cálcio/metabolismo
2.
J Neurochem ; 168(9): 3268-3283, 2024 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-39126680

RESUMO

Dynamins are large GTPases whose primary function is not only to catalyze membrane scission during endocytosis but also to modulate other cellular processes, such as actin polymerization and vesicle trafficking. Recently, we reported that centronuclear myopathy associated dynamin-2 mutations, p.A618T, and p.S619L, impair Ca2+-induced exocytosis of the glucose transporter GLUT4 containing vesicles in immortalized human myoblasts. As exocytosis and endocytosis occur within rapid timescales, here we applied high-temporal resolution techniques, such as patch-clamp capacitance measurements and carbon-fiber amperometry to assess the effects of these mutations on these two cellular processes, using bovine chromaffin cells as a study model. We found that the expression of any of these dynamin-2 mutants inhibits a dynamin and F-actin-dependent form of fast endocytosis triggered by single action potential stimulus, as well as inhibits a slow compensatory endocytosis induced by 500 ms square depolarization. Both dynamin-2 mutants further reduced the exocytosis induced by 500 ms depolarizations, and the frequency of release events and the recruitment of neuropeptide Y (NPY)-labeled vesicles to the cell cortex after stimulation of nicotinic acetylcholine receptors with 1,1-dimethyl-4-phenyl piperazine iodide (DMPP). They also provoked a significant decrease in the Ca2+-induced formation of new actin filaments in permeabilized chromaffin cells. In summary, our results indicate that the centronuclear myopathy (CNM)-linked p.A618T and p.S619L mutations in dynamin-2 affect exocytosis and endocytosis, being the disruption of F-actin dynamics a possible explanation for these results. These impaired cellular processes might underlie the pathogenic mechanisms associated with these mutations.


Assuntos
Células Cromafins , Dinamina II , Endocitose , Exocitose , Mutação , Miopatias Congênitas Estruturais , Células Cromafins/metabolismo , Endocitose/fisiologia , Endocitose/genética , Dinamina II/genética , Dinamina II/metabolismo , Animais , Exocitose/fisiologia , Miopatias Congênitas Estruturais/genética , Miopatias Congênitas Estruturais/patologia , Miopatias Congênitas Estruturais/metabolismo , Mutação/genética , Bovinos , Humanos , Actinas/metabolismo , Actinas/genética , Células Cultivadas , Técnicas de Patch-Clamp , Glândulas Suprarrenais/metabolismo , Glândulas Suprarrenais/patologia
3.
Int J Mol Sci ; 25(13)2024 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-39000353

RESUMO

Connexins (Cxs) are transmembrane proteins that assemble into gap junction channels (GJCs) and hemichannels (HCs). Previous researches support the involvement of Rho GTPases and actin microfilaments in the trafficking of Cxs, formation of GJCs plaques, and regulation of channel activity. Nonetheless, it remains uncertain whether distinct types of Cxs HCs and GJCs respond differently to Rho GTPases or changes in actin polymerization/depolymerization dynamics. Our investigation revealed that inhibiting RhoA, a small GTPase that controls actin polymerization, or disrupting actin microfilaments with cytochalasin B (Cyto-B), resulted in reduced GJCs plaque size at appositional membranes and increased transport of HCs to non-appositional plasma membrane regions. Notably, these effects were consistent across different Cx types, since Cx26 and Cx43 exhibited similar responses, despite having distinct trafficking routes to the plasma membrane. Functional assessments showed that RhoA inhibition and actin depolymerization decreased the activity of Cx43 GJCs while significantly increasing HC activity. However, the functional status of GJCs and HCs composed of Cx26 remained unaffected. These results support the hypothesis that RhoA, through its control of the actin cytoskeleton, facilitates the transport of HCs to appositional cell membranes for GJCs formation while simultaneously limiting the positioning of free HCs at non-appositional cell membranes, independently of Cx type. This dynamic regulation promotes intercellular communications and reduces non-selective plasma membrane permeability through a Cx-type dependent mechanism, whereby the activity of Cx43 HCs and GJCs are differentially affected but Cx26 channels remain unchanged.


Assuntos
Citoesqueleto de Actina , Conexina 26 , Conexina 43 , Junções Comunicantes , Proteína rhoA de Ligação ao GTP , Citoesqueleto de Actina/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Junções Comunicantes/metabolismo , Conexina 43/metabolismo , Conexina 26/metabolismo , Humanos , Animais , Membrana Celular/metabolismo , Actinas/metabolismo
4.
mBio ; 15(7): e0072624, 2024 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-38847540

RESUMO

The modulation of actin polymerization is a common theme among microbial pathogens. Even though microorganisms show a wide repertoire of strategies to subvert the activity of actin, most of them converge in the ones that activate nucleating factors, such as the Arp2/3 complex. Brucella spp. are intracellular pathogens capable of establishing chronic infections in their hosts. The ability to subvert the host cell response is dependent on the capacity of the bacterium to attach, invade, avoid degradation in the phagocytic compartment, replicate in an endoplasmic reticulum-derived compartment and egress. Even though a significant number of mechanisms deployed by Brucella in these different phases have been identified and characterized, none of them have been described to target actin as a cellular component. In this manuscript, we describe the identification of a novel virulence factor (NpeA) that promotes niche formation. NpeA harbors a short linear motif (SLiM) present within an amphipathic alpha helix that has been described to bind the GTPase-binding domain (GBD) of N-WASP and stabilizes the autoinhibited state. Our results show that NpeA is secreted in a Type IV secretion system-dependent manner and that deletion of the gene diminishes the intracellular replication capacity of the bacterium. In vitro and ex vivo experiments demonstrate that NpeA binds N-WASP and that the short linear motif is required for the biological activity of the protein.IMPORTANCEThe modulation of actin-binding effectors that regulate the activity of this fundamental cellular protein is a common theme among bacterial pathogens. The neural Wiskott-Aldrich syndrome protein (N-WASP) is a protein that several pathogens target to hijack actin dynamics. The highly adapted intracellular bacterium Brucella has evolved a wide repertoire of virulence factors that modulate many activities of the host cell to establish successful intracellular replication niches, but, to date, no effector proteins have been implicated in the modulation of actin dynamics. We present here the identification of a virulence factor that harbors a short linear motif (SLiM) present within an amphipathic alpha helix that has been described to bind the GTPase-binding domain (GBD) of N-WASP stabilizing its autoinhibited state. We demonstrate that this protein is a Type IV secretion effector that targets N-WASP-promoting intracellular survival and niche formation.


Assuntos
Proteínas de Bactérias , Fatores de Virulência , Proteína Neuronal da Síndrome de Wiskott-Aldrich , Fatores de Virulência/metabolismo , Fatores de Virulência/genética , Proteína Neuronal da Síndrome de Wiskott-Aldrich/metabolismo , Proteína Neuronal da Síndrome de Wiskott-Aldrich/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Humanos , Sistemas de Secreção Tipo IV/metabolismo , Sistemas de Secreção Tipo IV/genética , Animais , Camundongos , Ligação Proteica , Brucella/metabolismo , Brucella/genética , Brucella/patogenicidade , Motivos de Aminoácidos , Actinas/metabolismo , Brucelose/microbiologia , Macrófagos/microbiologia , Interações Hospedeiro-Patógeno
5.
Head Neck Pathol ; 18(1): 40, 2024 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-38727794

RESUMO

BACKGROUND: Odontogenic lesions constitute a heterogeneous group of lesions. CLIC4 protein regulates different cellular processes, including epithelial-mesenchymal transition and fibroblast-myofibroblast transdifferentiation. This study analyzed CLIC4, E-cadherin, Vimentin, and α-SMA immunoexpression in epithelial odontogenic lesions that exhibit different biological behavior. METHODS: It analyzed the immunoexpression of CLIC4, E-cadherin, and Vimentin in the epithelial cells, as well as CLIC4 and α-SMA in the mesenchymal cells, of ameloblastoma (AM) (n = 16), odontogenic keratocyst (OKC) (n = 20), and adenomatoid odontogenic tumor (AOT) (n = 8). Immunoexpressions were categorized as score 0 (0% positive cells), 1 (< 25%), 2 (≥ 25% - < 50%), 3 (≥ 50% - < 75%), or 4 (≥ 75%). RESULTS: Cytoplasmic CLIC4 immunoexpression was higher in AM and AOT (p < 0.001) epithelial cells. Nuclear-cytoplasmic CLIC4 was higher in OKC's epithelial lining (p < 0.001). Membrane (p = 0.012) and membrane-cytoplasmic (p < 0.001) E-cadherin immunoexpression were higher in OKC, while cytoplasmic E-cadherin expression was higher in AM and AOT (p < 0.001). Vimentin immunoexpression was higher in AM and AOT (p < 0.001). Stromal CLIC4 was higher in AM and OKC (p = 0.008). Similarly, α-SMA immunoexpression was higher in AM and OKC (p = 0.037). Correlations in these proteins' immunoexpression were observed in AM and OKC (p < 0.05). CONCLUSIONS: CLIC4 seems to regulate the epithelial-mesenchymal transition, modifying E-cadherin and Vimentin expression. In mesenchymal cells, CLIC4 may play a role in fibroblast-myofibroblast transdifferentiation. CLIC4 may be associated with epithelial odontogenic lesions with aggressive biological behavior.


Assuntos
Ameloblastoma , Caderinas , Canais de Cloreto , Transição Epitelial-Mesenquimal , Tumores Odontogênicos , Vimentina , Humanos , Transição Epitelial-Mesenquimal/fisiologia , Canais de Cloreto/metabolismo , Canais de Cloreto/análise , Caderinas/metabolismo , Tumores Odontogênicos/patologia , Tumores Odontogênicos/metabolismo , Ameloblastoma/patologia , Ameloblastoma/metabolismo , Vimentina/metabolismo , Adulto , Feminino , Cistos Odontogênicos/patologia , Cistos Odontogênicos/metabolismo , Masculino , Actinas/metabolismo , Adulto Jovem , Pessoa de Meia-Idade , Antígenos CD/metabolismo , Adolescente
6.
Int J Mol Sci ; 25(5)2024 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-38473701

RESUMO

This work analyzes the role of the tight junction (TJ) protein ZO-2 on mechanosensation. We found that the lack of ZO-2 reduced apical membrane rigidity measured with atomic force microscopy, inhibited the association of γ-actin and JAM-A to the cell border, and instead facilitated p114RhoGEF and afadin accumulation at the junction, leading to an enhanced mechanical tension at the TJ measured by FRET, with a ZO-1 tension probe, and increased tricellular TJ tension. Simultaneously, adherens junction tension measured with an E-cadherin probe was unaltered. The stability of JAM-A and ZO-2 binding was assessed by a collaborative in silico study. The absence of ZO-2 also impacted the cell response to the substrate, as monolayers plated in 20 kPa hydrogels developed holes not seen in parental cultures and displayed a retarded elongation and formation of cell aggregates. The absence of ZO-2 was sufficient to induce YAP and Snail nuclear accumulation in cells cultured over glass, but when ZO-2 KD cells were plated in nanostructured ridge arrays, they displayed an increased abundance of nuclear Snail and conspicuous internalization of claudin-4. These results indicate that the absence of ZO-2 also impairs the response of cells to substrate stiffness and exacerbates transformation triggered by substrate topography.


Assuntos
Actinas , Junções Íntimas , Actinas/metabolismo , Junções Íntimas/metabolismo , Proteína da Zônula de Oclusão-1/metabolismo , Fosfoproteínas/metabolismo
7.
Clin Exp Pharmacol Physiol ; 51(4): e13851, 2024 04.
Artigo em Inglês | MEDLINE | ID: mdl-38452757

RESUMO

Benign prostatic hyperplasia (BPH) is characterised by increases in prostate volume and contraction. Downregulation of the nitric oxide (NO)-cyclic guanosine monophosphate (cGMP) signalling pathway contributes to prostate dysfunctions. Previous studies in cancer cells or vessels have shown that the epigenetic mechanisms control the gene and protein expression of the enzymes involved in the production of NO and cGMP. This study is aimed to evaluate the effect of a 2-week treatment of 5-azacytidine (5-AZA), a DNA-methyltransferase inhibitor, in the prostate function of mice fed with a high-fat diet. Functional, histological, biochemical and molecular assays were carried out. Obese mice presented greater prostate weight, α-actin expression and contractile response induced by the α-1adrenoceptors agonist. The relaxation induced by the NO-donor and the protein expression of endothelial nitric oxide synthase (eNOS) and soluble guanylate cyclase (sGC) were significantly decreased in the prostate of obese mice. The treatment with 5-AZA reverted the higher expression of α-actin, reduced the hypercontractility state of the prostate and increased the expression of eNOS and sGC and intraprostatic levels of cGMP. When prostates from obese mice treated with 5-AZA were incubated in vitro with inhibitors of the NOS or sGC, the inhibitory effect of 5-AZA was reverted, therefore, showing the involvement of NO and cGMP. In conclusion, our study paves the way to develop or repurpose therapies that recover the expression of eNOS and sGC and, hence, to improve prostate function in BPH.


Assuntos
Óxido Nítrico , Hiperplasia Prostática , Masculino , Humanos , Camundongos , Animais , Óxido Nítrico/metabolismo , Guanilato Ciclase/metabolismo , Próstata/metabolismo , Camundongos Obesos , Guanosina Monofosfato/metabolismo , Azacitidina/metabolismo , Hiperplasia Prostática/metabolismo , Actinas/metabolismo , GMP Cíclico/metabolismo
8.
J Econ Entomol ; 117(2): 629-637, 2024 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-38245820

RESUMO

Rhyzopertha dominica is a serious stored grain insect pest around the world. Real-time quantitative polymerase chain reaction (RT-qPCR) is a widely used experimental method in molecular biology for detecting the expression of target genes. As appropriate reference genes are essential for normalizing gene expression, the selection of suitable reference genes is the basis of RT-qPCR experiments. In this study, the expression profiles of 7 candidate reference genes of rps3, rps6, rps13, actin, gadph, tubulin, and 18S rRNA were analyzed under 4 different experimental conditions. The expression stability of candidate genes was evaluated using the ΔCt, GeNorm, BestKeeper, NormFinder, and RefFinder methods. The results revealed that different reference genes were suitable for various experiments. Specifically, rps3 and rps6 were appropriate for the developmental stages and all samples: 18S rRNA and rps13 for temperature-related experiments, actin and rps6 for sex-related experiments, and rps6 and gadph for starvation stress. Our results lay essential groundwork for the normalization of RT-qPCR analyses and contribute to genomic and gene functional research of R. dominica.


Assuntos
Actinas , Besouros , Animais , Actinas/genética , Actinas/metabolismo , RNA Ribossômico 18S/genética , Besouros/genética , Besouros/metabolismo , Genes de Insetos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Padrões de Referência , Perfilação da Expressão Gênica/métodos
9.
Exp Eye Res ; 238: 109745, 2024 01.
Artigo em Inglês | MEDLINE | ID: mdl-38043763

RESUMO

The epiretinal membrane is a fibrocontractile tissue that forms on the inner surface of the retina, causing visual impairment ranging from mild to severe, and even retinal detachment. Müller glial cells actively participate in the formation of this membrane. Current research is constantly seeking for new therapeutic approaches that aim to prevent or treat cellular dysfunctions involved in the progression of this common fibrosis condition. The Rho GTPases signaling pathway regulates several processes associated with the epiretinal membrane, such as cell proliferation, migration, and contraction. Rho kinase (ROCK), an effector of the RhoA GTPase, is an interesting potential therapeutic target. This study aimed to evaluate the effects of a ROCK inhibitor (Y27632) on human Müller cells viability, growth, cytoskeletal organization, expression of extracellular matrix components, myofibroblast differentiation, migration, and contractility. Müller cells of the MIO-M1 lineage were cultured and treated for different periods with the inhibitor. Viability was evaluated by MTT assay and trypan blue exclusion method, and growth was evaluated by growth curve and BrdU incorporation assay. The actin cytoskeleton was stained with fluorescent phalloidin, intermediate filaments and microtubules were analyzed with immunofluorescence for vimentin and α-tubulin. Gene and protein expression of collagens I and V, laminin and fibronectin were evaluated by rt-PCR and immunofluorescence. Chemotactic and spontaneous cell migration were studied by transwell assay and time-lapse observation of live cells, respectively. Cell contractility was assessed by collagen gel contraction assay. The results showed that ROCK inhibition by Y27632 did not affect cell viability, but decreased cell growth and proliferation after 72 h. There was a change in cell morphology and organization of F-actin, with a reduction in the cell body, disappearance of stress fibers and formation of long, branched cell extensions. Microtubules and vimentin filaments were also affected, possibly because of F-actin alterations. The inhibitor also reduced gene expression and immunoreactivity of smooth muscle α-actin, a marker of myofibroblasts. The expression of extracellular matrix components was not affected by the inhibitor. Chemotactic cell migration showed no significant changes, while cell contractility was substantially reduced. No spontaneous migration of MIO-M1 cells was observed. In conclusion, pharmacological inhibition of ROCK in Müller cells could be a potentially promising approach to treat epiretinal membranes by preventing cell proliferation, contractility and transdifferentiation, without affecting cell viability.


Assuntos
Membrana Epirretiniana , Quinases Associadas a rho , Humanos , Actinas/metabolismo , Células Ependimogliais/metabolismo , Vimentina/metabolismo , Sobrevivência Celular , Membrana Epirretiniana/metabolismo , Células Cultivadas , Matriz Extracelular/metabolismo
10.
mBio ; 14(6): e0282223, 2023 Dec 19.
Artigo em Inglês | MEDLINE | ID: mdl-38014993

RESUMO

IMPORTANCE: Mitochondria constitute major sources of H2O2 and other reactive oxygen species in eukaryotic cells. The division of these organelles is crucial for multiple processes in cell biology and relies on highly regulated mechano-GTPases that are oligomerization dependent and belong to the dynamin-related protein family, like A. nidulans DnmA. Our previous work demonstrated that H2O2 induces mitochondrial constriction, division, and remodeling of the outer membrane. Here, we show that H2O2 also induces a DnmA aggregation consistent with higher-order oligomerization and its recruitment to mitochondria. The study of this response uncovered that H2O2 induces the depolymerization and reorganization of actin as well as the critical role that cysteines 450 and 776 play in DnmA function. Our results provide new insights into the mechanisms of reactive oxygen species cell signaling and how they can regulate the dynamics of the actin cytoskeleton and the division of mitochondria and peroxisomes.


Assuntos
Actinas , Aspergillus nidulans , Peróxido de Hidrogênio , Mitocôndrias , Peroxissomos , Aspergillus nidulans/genética , Aspergillus nidulans/metabolismo , Peroxissomos/metabolismo , Mitocôndrias/metabolismo , Peróxido de Hidrogênio/metabolismo , Peróxido de Hidrogênio/farmacologia , Actinas/metabolismo , Cisteína/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/genética , Citoesqueleto de Actina/metabolismo , Espécies Reativas de Oxigênio/metabolismo
11.
Arch Oral Biol ; 155: 105793, 2023 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-37633029

RESUMO

OBJECTIVE: To evaluate the healing potential of Nile tilapia skin collagen using a rat model with experimentally induced traumatic oral ulcers. DESIGN: Male Wistar rats were segregated into three experimental groups (n = 8/group/euthanasia day). Ulcers were induced using a dermatological punch on the left buccal mucosa. The rats were then euthanized on days 1, 5, 10, 15, and 20 (ntotal=120 rats). Each group received topical treatment, 2x/day, with 1 % Nile tilapia skin collagen orabase (experimental group), only orabase (negative control), or Oncilom-A® orabase (positive control). Ulcer area, closure percentage, and body mass variation were measured. Slides were prepared for histological analysis, which included Picrosirius red staining (collagen analysis), and immunohistochemistry (platelet endothelial cell adhesion molecule, alpha-smooth muscle actin, and transforming growth factor-beta). RESULTS: On day 15, the experimental and positive control groups displayed smaller ulcer areas, a higher percentage of closure, complete re-epithelialization, superior histological repair scores, and a reduced count of polymorphonuclear cells in comparison to the negative control group (p < 0.05). Additionally, the experimental group exhibited an increased number of blood vessels, total collagen (types I and III) and expression of platelet endothelial cell adhesion molecule, alpha-smooth muscle actin, and transforming growth factor-beta relative to the negative and positive control groups (p < 0.05). By day 20, the experimental group showed a more significant weight gain compared to the other groups (p < 0.0001). CONCLUSIONS: Nile tilapia skin collagen orabase optimizes the healing of traumatic ulcers by stimulating re-epithelialization, angiogenesis, and collagenesis. Transforming growth factor-beta plays a significant role in this process.


Assuntos
Ciclídeos , Úlceras Orais , Ratos , Masculino , Animais , Cicatrização/fisiologia , Úlcera/metabolismo , Úlceras Orais/tratamento farmacológico , Ratos Wistar , Actinas/metabolismo , Pele , Colágeno/metabolismo , Fatores de Crescimento Transformadores/metabolismo , Moléculas de Adesão Celular/metabolismo
12.
Ann Hepatol ; 28(6): 101135, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37451514

RESUMO

INTRODUCTION AND OBJECTIVES: Congestive hepatopathy (CH) is a hepatic vascular disease that results in chronic liver congestion, which can lead to liver fibrosis. New uses of metformin have been discovered over the years. However, the function of metformin in congestive liver fibrosis is not yet fully understood. This study aimed to investigate the effect of metformin on liver fibrosis in a mouse model of CH. MATERIALS AND METHODS: Partial ligation of the inferior vena cava (pIVCL) was used to establish a mouse model of liver congestion. Metformin (0.1%) was added to the daily drinking water of the animals, and the effect of metformin on liver tissue was studied after 6 weeks. Hepatic stellate cells (HSCs) were also stimulated with CoCl2 to investigate the inhibitory impact of metformin on the mammalian target of rapamycin (mTOR)/hypoxia-inducible factor-1α (HIF-1α) pathway. RESULTS: Metformin attenuated liver congestion; decreased the expression of collagen, fibronectin, α-smooth muscle actin (α-SMA), and HIF-1α; and ameliorated liver fibrosis in pIVCL mice. The proliferation and migration of HSCs were inhibited by metformin in vitro, which prevented α-SMA expression and restrained HSC activation. The expression levels of phosphorylated-mTOR, HIF-1α, and vascular endothelial growth factor were also decreased. CONCLUSIONS: Metformin inhibits CH-induced liver fibrosis. Functionally, this beneficial effect may be the result of inhibition of HSC activation and of the mTOR/HIF-1α signaling pathway.


Assuntos
Proliferação de Células , Modelos Animais de Doenças , Células Estreladas do Fígado , Subunidade alfa do Fator 1 Induzível por Hipóxia , Cirrose Hepática , Metformina , Transdução de Sinais , Serina-Treonina Quinases TOR , Animais , Metformina/farmacologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Transdução de Sinais/efeitos dos fármacos , Células Estreladas do Fígado/efeitos dos fármacos , Células Estreladas do Fígado/metabolismo , Células Estreladas do Fígado/patologia , Cirrose Hepática/metabolismo , Cirrose Hepática/tratamento farmacológico , Cirrose Hepática/patologia , Proliferação de Células/efeitos dos fármacos , Masculino , Actinas/metabolismo , Movimento Celular/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fígado/efeitos dos fármacos , Fígado/patologia , Fígado/metabolismo , Fibronectinas/metabolismo , Colágeno/metabolismo , Antifibróticos/farmacologia , Veia Cava Inferior/patologia , Veia Cava Inferior/efeitos dos fármacos
13.
Redox Biol ; 64: 102784, 2023 08.
Artigo em Inglês | MEDLINE | ID: mdl-37356135

RESUMO

Neutrophil extracellular traps (NETs) are web-like structures of DNA coated with cytotoxic proteins and histones released by activated neutrophils through a process called NETosis. NETs release occurs through a sequence of highly organized events leading to chromatin expansion and rupture of nuclear and cellular membranes. In calcium ionophore-induced NETosis, the enzyme peptidylargine deiminase 4 (PAD4) mediates chromatin decondensation through histone citrullination, but the biochemical pathways involved in this process are not fully understood. Here we use live-imaging microscopy and proteomic studies of the neutrophil cellular fractions to investigate the early events in ionomycin-triggered NETosis. We found that before ionomycin-stimulated neutrophils release NETs, profound biochemical changes occur in and around their nucleus, such as, cytoskeleton reorganization, nuclear redistribution of actin-remodeling related proteins, and citrullination of actin-ligand and nuclear structural proteins. Ionomycin-stimulated neutrophils rapidly lose their characteristic polymorphic nucleus, and these changes are promptly communicated to the extracellular environment through the secretion of proteins related to immune response. Therefore, our findings revealed key biochemical mediators in the early process that subsequently culminates with nuclear and cell membranes rupture, and extracellular DNA release.


Assuntos
Citrulinação , Armadilhas Extracelulares , Actinas/metabolismo , Ionomicina/farmacologia , Ionomicina/metabolismo , Proteínas Nucleares/metabolismo , Ligantes , Proteômica , Neutrófilos/metabolismo , Armadilhas Extracelulares/metabolismo , Cromatina/metabolismo , DNA/metabolismo , Citoesqueleto/metabolismo
14.
Chem Biol Interact ; 382: 110593, 2023 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-37270087

RESUMO

The non-cholinergic molecular targets of organophosphate (OP) compounds have recently been investigated to explain their role in the generation of non-neurological diseases, such as immunotoxicity and cancer. Here, we evaluated the effects of malathion and its dialkylphosphate (DAP) metabolites on the cytoskeleton components and organization of RAW264.7 murine macrophages as non-cholinergic targets of OP and DAPs toxicity. All OP compounds affected actin and tubulin polymerization. Malathion, dimethyldithiophosphate (DMDTP) dimethylthiophosphate (DMTP), and dimethylphosphate (DMP) induced elongated morphologies and the formation of pseudopods rich in microtubule structures, and increased filopodia formation and general actin disorganization in RAW264.7 cells and slightly reduced stress fibers in the human fibroblasts GM03440, without significantly disrupting the tubulin or vimentin cytoskeleton. Exposure to DMTP and DMP increased cell migration in the wound healing assay but did not affect phagocytosis, indicating a very specific modification in the organization of the cytoskeleton. The induction of actin cytoskeleton rearrangement and cell migration suggested the activation of cytoskeletal regulators such as small GTPases. We found that DMP slightly reduced Ras homolog family member A activity but increased the activities of Ras-related C3 botulinum toxin substrate 1 (Rac1) and cell division control protein 42 (Cdc42) from 5 min to 2 h of exposure. Chemical inhibition of Rac1 with NSC23766 reduced cell polarization and treatment with DMP enhanced cell migration, but Cdc42 inhibition by ML-141 completely inhibited the effects of DMP. These results suggest that methylated OP compounds, especially DMP, can modify macrophage cytoskeleton function and configuration via activation of Cdc42, which may represent a potential non-cholinergic molecular target for OP compounds.


Assuntos
Inseticidas , Malation , Camundongos , Humanos , Animais , Malation/toxicidade , Malation/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Actinas/metabolismo , Tubulina (Proteína)/metabolismo , Citoesqueleto de Actina/metabolismo , Inseticidas/toxicidade , Inseticidas/metabolismo , Movimento Celular , Compostos Organofosforados/metabolismo , Organofosfatos/metabolismo
15.
Ann Hepatol ; 28(5): 101124, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37286166

RESUMO

INTRODUCTION AND OBJECTIVES: The development of hepatocellular carcinoma (HCC) is a multi-step process that accumulates genetic and epigenetic alterations, including changes in circular RNA (circRNA). This study aimed to understand the alterations in circRNA expression in HCC development and metastasis and to explore the biological functions of circRNA. MATERIALS AND METHODS: Ten pairs of adjacent chronic hepatitis tissues and HCC tissues from patients without venous metastases, and ten HCC tissues from patients with venous metastases were analyzed using human circRNA microarrays. Differentially expressed circRNAs were then validated by quantitative real-time PCR. In vitro and in vivo assays were performed to assess the roles of the circRNA in HCC progression. RNA pull-down assay, mass spectrometry analysis, and RNA-binding protein immunoprecipitation were conducted to explore the protein partners of the circRNA. RESULTS: CircRNA microarrays revealed that the expression patterns of circRNAs across the three groups were significantly different. Among these, hsa_circ_0098181 was validated to be lowly expressed and associated with poor prognosis in HCC patients. Ectopic expression of hsa_circ_0098181 delayed HCC metastasis in vitro and in vivo. Mechanistically, hsa_circ_0098181 sequestered eukaryotic translation elongation factor 2 (eEF2) and dissociated eEF2 from filamentous actin (F-actin) to prevent F-actin formation, which blocked activation of the Hippo signaling pathway. In addition, the RNA binding protein Quaking-5 bound directly to hsa_circ_0098181 and induced its biogenesis. CONCLUSIONS: Our study reveals changes in circRNA expression from chronic hepatitis, primary HCC, to metastatic HCC. Further, the QKI5-hsa_circ_0098181-eEF2-Hippo signaling pathway exerts a regulatory role in HCC.


Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , MicroRNAs , Humanos , Carcinoma Hepatocelular/patologia , RNA Circular/genética , Neoplasias Hepáticas/patologia , Fator 2 de Elongação de Peptídeos/genética , Fator 2 de Elongação de Peptídeos/metabolismo , Via de Sinalização Hippo , Actinas/metabolismo , Hepatite Crônica , MicroRNAs/genética , Regulação Neoplásica da Expressão Gênica
16.
Cells ; 12(3)2023 02 02.
Artigo em Inglês | MEDLINE | ID: mdl-36766835

RESUMO

Caveolae-associated signaling toward mitochondria contributes to the cardioprotective mechanisms against ischemia-reperfusion (I/R) injury induced by ischemic postconditioning. In this work, we evaluated the role that the actin-cytoskeleton network exerts on caveolae-mitochondria communication during postconditioning. Isolated rat hearts subjected to I/R and to postconditioning were treated with latrunculin A, a cytoskeleton disruptor. Cardiac function was compared between these hearts and those exposed only to I/R and to the cardioprotective maneuver. Caveolae and mitochondria structures were determined by electron microscopy and maintenance of the actin-cytoskeleton was evaluated by phalloidin staining. Caveolin-3 and other putative caveolae-conforming proteins were detected by immunoblot analysis. Co-expression of caveolin-3 and actin was evaluated both in lipid raft fractions and in heart tissue from the different groups. Mitochondrial function was assessed by respirometry and correlated with cholesterol levels. Treatment with latrunculin A abolishes the cardioprotective postconditioning effect, inducing morphological and structural changes in cardiac tissue, reducing F-actin staining and diminishing caveolae formation. Latrunculin A administration to post-conditioned hearts decreases the interaction between caveolae-forming proteins, the co-localization of caveolin with actin and inhibits oxygen consumption rates in both subsarcolemmal and interfibrillar mitochondria. We conclude that actin-cytoskeleton drives caveolae signaling to mitochondria during postconditioning, supporting their functional integrity and contributing to cardiac adaption against reperfusion injury.


Assuntos
Cavéolas , Traumatismo por Reperfusão , Ratos , Animais , Cavéolas/metabolismo , Actinas/metabolismo , Caveolina 3/metabolismo , Citoesqueleto/metabolismo , Caveolina 1/metabolismo , Traumatismo por Reperfusão/metabolismo , Mitocôndrias/metabolismo
17.
Exp Eye Res ; 226: 109336, 2023 01.
Artigo em Inglês | MEDLINE | ID: mdl-36455675

RESUMO

Aging increases the risks for developing fibrocontractile membranes on the retina, which causes significant macular distortion, as in the idiopathic epiretinal membrane (iERM). Retinal Müller glial cells are components of these membranes and may play a key role in the iERM pathogenesis. The transforming growth factor-ß (TGF-ß) induces Müller cell transdifferentiation into myofibroblast, reducing glial cell markers (glutamine synthetase, GS, and glial fibrillary acidic protein, GFAP) and increasing α-smooth muscle actin (α-SMA). Our aim was to investigate the effect of the TGF-ß inhibitor galunisertib (LY2157299) on the glial-mesenchymal transition and contraction of Müller cells. MIO-M1 human Müller cells were treated with TGF-ß1 (10 ng/mL), galunisertib (5, 10 and 20 µM) and TGF-ß1+galunisertib for 24h and 48h. Galunisertib cytotoxicity was analyzed by MTT and trypan blue, and TGF-ß1 blockade by phospho-SMAD3 immunofluorescence. Caspase-3 (cell death indicator), GS, GFAP and α-SMA expression was examined by immunofluorescence, Western blotting, and qPCR analysis. Cell contractility was determined by collagen gel contraction assay with Müller cells incorporated. Galunisertib did not show cytotoxicity at the concentrations evaluated and maintained the Müller cells phenotype, ensuring the GS expression. Galunisertib inhibited the TGF-ß1 pathway by decreasing phospho-SMAD3 immunoreactivity, attenuated the α-SMA expression, and prevented the contraction of Müller cells in collagen gel. Although more studies are needed, in vitro assays suggest that galunisertib may be a potential candidate to attenuate the formation of fibrocontractile membranes and prevent retinal detachment and consequent loss of vision.


Assuntos
Células Ependimogliais , Membrana Epirretiniana , Humanos , Células Ependimogliais/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Fator de Crescimento Transformador beta1/farmacologia , Fator de Crescimento Transformador beta1/metabolismo , Neuroglia/metabolismo , Actinas/metabolismo , Colágeno/metabolismo , Membrana Epirretiniana/metabolismo
18.
PLoS Negl Trop Dis ; 16(10): e0010788, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-36190932

RESUMO

Host cell invasion is a critical step for infection by Trypanosoma cruzi, the agent of Chagas disease. In natural infection, T. cruzi metacyclic trypomastigote (MT) forms establish the first interaction with host cells. The gp35/50 mucin molecules expressed in MT have been implicated in cell invasion process, but the mechanisms involved are not well understood. We performed a series of experiments to elucidate the mode of gp35/50-mediated MT internalization. Comparing two parasite strains from genetically divergent groups, G strain (TcI) and CL strain (TcVI), expressing variant forms of mucins, we demonstrated that G strain mucins participate in MT invasion. Only G strain-derived mucins bound to HeLa cells in a receptor-dependent manner and significantly inhibited G strain MT invasion. CL strain MT internalization was not affected by mucins from either strain. HeLa cell invasion by G strain MT was associated with actin recruitment and did not rely on lysosome mobilization. To examine the involvement of annexin A2, which plays a role in actin dynamic, annexin A2-depleted HeLa cells were generated. Annexin A2-deficient cell lines were significantly more resistant than wild type controls to G strain MT invasion. In a co-immunoprecipitation assay, to check whether annexin A2 might be the receptor for mucins, protein A/G magnetic beads crosslinked with monoclonal antibody to G strain mucins were incubated with detergent extracts of MT and HeLa cells. Binding of gp35/50 mucins to annexin A2 was detected. Both G strain MT and purified mucins induced focal adhesion kinase activation in HeLa cells. By confocal immunofluorescence microscopy, colocalization of invading G strain MT with clathrin was visualized. Inhibition of clathrin-coated vesicle formation reduced parasite internalization. Taken together, our data indicate that gp35/50-mediated MT invasion is accomplished through interaction with host cell annexin A2 and clathrin-dependent endocytosis.


Assuntos
Anexina A2 , Doença de Chagas , Trypanosoma cruzi , Actinas/metabolismo , Anexina A2/metabolismo , Anticorpos Monoclonais , Doença de Chagas/parasitologia , Clatrina , Detergentes/metabolismo , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Células HeLa , Humanos , Mucinas/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Trypanosoma cruzi/fisiologia
19.
Plant Foods Hum Nutr ; 77(4): 521-528, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36048356

RESUMO

High-fructose diet is associated with an increased risk of dyslipidemia, metabolic syndrome, and the development of non-alcoholic fatty liver disease (NAFLD) through chronic inflammation. The present study aimed to elucidate the potential benefit of daily consumption of Smallanthus sonchifolius (yacon) roots, rich in fructooligosaccharides (FOS), on the progression to liver fibrosis, in a rat model of NAFLD induced by a high-fructose diet. Male Wistar rats were fed a standard diet (CD, n = 6) or a standard diet plus 10% fructose solution (FD; n = 18). After 20 weeks, FD rats were randomly separated into the following groups (n = 6, each): FD; FD treated with yacon flour (340 mg FOS/body weight; FD + Y) and FD treated with fenofibrate (30 mg/kg body weight; FD + F), for 16 weeks. Daily intake of yacon flour significantly reduced body weight gain, plasma lipid levels, transaminase activities, and improved systemic insulin response in FD rats. In the liver, yacon treatment decreased fructose-induced steatosis and inflammation, and reduced total collagen deposition (64%). Also, yacon decreased TGF-ß1 mRNA expression (78%), followed by decreased nuclear localization of p-Smad2/3 in liver tissue. Yacon significantly reduced the expression of α-smooth muscle actin (α-SMA), Col1α1, and Col3α1 mRNAs (85, 44, and 47%, respectively), inhibiting the activation of resident hepatic stellate cells (HSCs). These results suggested that yacon roots have the potential to ameliorate liver damage caused by long-term consumption of a high-fructose diet, being a promising nutritional strategy in NAFLD management.


Assuntos
Asteraceae , Fenofibrato , Hepatopatia Gordurosa não Alcoólica , Animais , Ratos , Actinas/metabolismo , Dieta , Fenofibrato/metabolismo , Frutose/efeitos adversos , Inflamação , Insulina/metabolismo , Lipídeos , Fígado/metabolismo , Hepatopatia Gordurosa não Alcoólica/induzido quimicamente , Hepatopatia Gordurosa não Alcoólica/prevenção & controle , Ratos Wistar , RNA Mensageiro , Transaminases/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Aumento de Peso
20.
Cells ; 11(18)2022 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-36139464

RESUMO

This review will briefly outline the major signaling pathways in PMA-activated neutrophils. PMA is widely used to understand neutrophil pathways and formation of NETs. PMA activates PKC; however, we highlight some isoforms that contribute to specific functions. PKC α, ß and δ contribute to ROS production while PKC ßII and PKC ζ are involved in cytoskeleton remodeling. Actin polymerization is important for the chemotaxis of neutrophils and its remodeling is connected to ROS balance. We suggest that, although ROS and production of NETs are usually observed together in PMA-activated neutrophils, there might be a regulatory mechanism balancing both. Interestingly, we suggest that serine proteases might determine the PAD4 action. PAD4 could be responsible for the activation of the NF-κB pathway that leads to IL-1ß release, triggering the cleavage of gasdermin D by serine proteases such as elastase, leading to pore formation contributing to release of NETs. On the other hand, when serine proteases are inhibited, NETs are formed by citrullination through the PAD4 pathway. This review puts together results from the last 31 years of research on the effects of PMA on the neutrophil and proposes new insights on their interpretation.


Assuntos
Armadilhas Extracelulares , Neutrófilos , Actinas/metabolismo , Armadilhas Extracelulares/metabolismo , NF-kappa B/metabolismo , Neutrófilos/metabolismo , Elastase Pancreática/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Serina Proteases/metabolismo , Acetato de Tetradecanoilforbol/farmacologia
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