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1.
Nat Commun ; 12(1): 4096, 2021 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-34215746

RESUMO

Non-centrosomal microtubule arrays serve crucial functions in cells, yet the mechanisms of their generation are poorly understood. During budding of the epithelial tubes of the salivary glands in the Drosophila embryo, we previously demonstrated that the activity of pulsatile apical-medial actomyosin depends on a longitudinal non-centrosomal microtubule array. Here we uncover that the exit from the last embryonic division cycle of the epidermal cells of the salivary gland placode leads to one centrosome in the cells losing all microtubule-nucleation capacity. This restriction of nucleation activity to the second, Centrobin-enriched, centrosome is key for proper morphogenesis. Furthermore, the microtubule-severing protein Katanin and the minus-end-binding protein Patronin accumulate in an apical-medial position only in placodal cells. Loss of either in the placode prevents formation of the longitudinal microtubule array and leads to loss of apical-medial actomyosin and impaired apical constriction. We thus propose a mechanism whereby Katanin-severing at the single active centrosome releases microtubule minus-ends that are then anchored by apical-medial Patronin to promote formation of the longitudinal microtubule array crucial for apical constriction and tube formation.


Assuntos
Divisão Celular/fisiologia , Centrossomo/metabolismo , Microtúbulos/metabolismo , Actinas , Actomiosina/metabolismo , Animais , Centrossomo/ultraestrutura , Proteínas do Citoesqueleto/metabolismo , Drosophila , Katanina , Masculino , Proteínas dos Microfilamentos/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Morfogênese , Glândulas Salivares , Tubulina (Proteína)/metabolismo
2.
Nat Commun ; 12(1): 4413, 2021 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-34285210

RESUMO

Enhanced neovasculogenesis, especially vasculogenic mimicry (VM), contributes to the development of triple-negative breast cancer (TNBC). Breast tumor-initiating cells (BTICs) are involved in forming VM; however, the specific VM-forming BTIC population and the regulatory mechanisms remain undefined. We find that tumor endothelial marker 8 (TEM8) is abundantly expressed in TNBC and serves as a marker for VM-forming BTICs. Mechanistically, TEM8 increases active RhoC level and induces ROCK1-mediated phosphorylation of SMAD5, in a cascade essential for promoting stemness and VM capacity of breast cancer cells. ASB10, an estrogen receptor ERα trans-activated E3 ligase, ubiquitylates TEM8 for degradation, and its deficiency in TNBC resulted in a high homeostatic level of TEM8. In this work, we identify TEM8 as a functional marker for VM-forming BTICs in TNBC, providing a target for the development of effective therapies against TNBC targeting both BTIC self-renewal and neovasculogenesis simultaneously.


Assuntos
Biomarcadores Tumorais/metabolismo , Proteínas dos Microfilamentos/metabolismo , Células-Tronco Neoplásicas/patologia , Neovascularização Patológica/patologia , Receptores de Superfície Celular/metabolismo , Neoplasias de Mama Triplo Negativas/patologia , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Biomarcadores Tumorais/antagonistas & inibidores , Mama/patologia , Mama/cirurgia , Carcinogênese/efeitos dos fármacos , Carcinogênese/patologia , Linhagem Celular Tumoral , Autorrenovação Celular/efeitos dos fármacos , Feminino , Humanos , Mastectomia , Camundongos , Proteínas dos Microfilamentos/antagonistas & inibidores , Pessoa de Meia-Idade , Células-Tronco Neoplásicas/efeitos dos fármacos , Neovascularização Patológica/tratamento farmacológico , Receptores de Superfície Celular/antagonistas & inibidores , Neoplasias de Mama Triplo Negativas/irrigação sanguínea , Neoplasias de Mama Triplo Negativas/terapia , Ensaios Antitumorais Modelo de Xenoenxerto
3.
Head Face Med ; 17(1): 18, 2021 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-34082790

RESUMO

BACKGROUND: Oral cancer is a malignant disease that threatenshuman life and greatly reducespatientquality of life. ANLN was reported to promote the progression of cancer. This study aims to investigate the role of ANLNin oral cancer and the underlying molecular mechanism. METHODS: ANLN expression was downregulated by RNAi technology. The effect of ANLN on cell behaviors, including proliferation, cell cycle progression, invasion, and apoptosis, was detected. Western blotting analysis was used to explore the mechanism by whichANLN functions in oral cancer. RESULTS: Data from TCGA database showed that ANLN was expressed at significantly higher levels in tumor tissues thanin normal control tissues. Patients with higher ANLN expression exhibitedshorter survivaltimes. ANLN was alsoabundantly expressedin the cancer cell lines CAL27 and HN30. When ANLN was knocked down in CAL27 and HN30 cells, cell proliferation and colony formation weredecreased. The cell invasion ability was also inhibited. However, the cell apoptosis rate was increased. In addition, the levels of critical members of the PI3K signaling pathway, includingPI3K, mTOR, Akt, and PDK-1, were significantlyreducedafter ANLN was knocked down in CAL27 cells. CONCLUSIONS: ANLN contributes to oral cancerprogressionand affects activation ofthe PI3K/mTOR signaling pathway. This study providesa new potential targetfor drug development and treatment in oral cancer.


Assuntos
Proteínas dos Microfilamentos/metabolismo , Neoplasias Bucais , Fosfatidilinositol 3-Quinases , Transdução de Sinais , Carcinogênese/genética , Linhagem Celular Tumoral , Proliferação de Células , Humanos , Neoplasias Bucais/genética , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo
4.
Molecules ; 26(9)2021 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-34064330

RESUMO

Several genetic studies have identified a rare variant of triggering receptor expressed on myeloid cells 2 (TREM2) as a risk factor for Alzheimer's disease (AD). However, findings on the effects of TREM2 on Aß deposition are quite inconsistent in animal studies, requiring further investigation. In this study, we investigated whether elevation of TREM2 mitigates Aß pathology in TgCRND8 mice. We found that peripheral nerve injury resulted in a robust elevation of TREM2 exclusively in reactive microglia in the ipsilateral spinal cord of aged TgCRND8 mice at the age of 20 months. TREM2 expression appeared on day 1 post-injury and the upregulation was maintained for at least 28 days. Compared to the contralateral side, neither amyloid beta plaque load nor soluble Aß40 and Aß42 levels were attenuated upon TREM2 induction. We further showed direct evidence that TREM2 elevation in reactive microglia did not affect amyloid-ß pathology in plaque-bearing TgCRND8 mice by applying anti-TREM2 neutralizing antibody to selectively block TREM2. Our results question the ability of TREM2 to ameliorate established Aß pathology, discouraging future development of disease-modifying pharmacological treatments targeting TREM2 in the late stage of AD.


Assuntos
Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/metabolismo , Glicoproteínas de Membrana/metabolismo , Microglia/metabolismo , Microglia/patologia , Receptores Imunológicos/metabolismo , Envelhecimento/patologia , Animais , Plexo Braquial , Proteínas de Ligação ao Cálcio/metabolismo , Modelos Animais de Doenças , Masculino , Camundongos Transgênicos , Proteínas dos Microfilamentos/metabolismo , Nervos Periféricos/patologia , Medula Espinal/metabolismo , Medula Espinal/patologia , Corno Dorsal da Medula Espinal/patologia
5.
J Cell Sci ; 134(12)2021 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-34152390

RESUMO

Cytokinesis occurs at the end of mitosis/meiosis wherein the cytoplasms of daughter cells are separated. Before abscission, an intercellular bridge containing the remaining furrowing machinery, mitotic spindle and actin cytoskeleton connects the two daughter cells. To remove this actin and allow for the separation of daughter cells, Rab35 vesicles, loaded with the actin oxidizer MICAL1 and the inositol polyphosphate 5-phosphatase OCRL, are recruited to the midbody in a fine-tuned spatiotemporal manner. However, importantly, the means by which these vesicles are recruited is currently unclear. Here, we demonstrate that Rab11FIP1 is recruited to the midbody after Rab35 to scaffold it at the bridge and maintain Rab35 in this region. In the absence of Rab11FIP1, Rab35 dramatically drops from the midbody, inducing defects, such as cytokinetic delays and binucleation due to actin overaccumulation at the intercellular bridge, which can be rescued with Latrunculin A treatment. Importantly, we show that Rab11FIP1 is critical for Rab35 function in actin removal prior to cytokinesis. This article has an associated First Person interview with the first author of the paper.


Assuntos
Actinas , Citocinese , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Células HeLa , Humanos , Proteínas dos Microfilamentos/metabolismo , Mitose , Oxigenases de Função Mista , Fuso Acromático/metabolismo , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo
6.
Res Vet Sci ; 138: 39-48, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34091228

RESUMO

Methotrexate (MTX), an antifolate drug, is widely used in chemotherapeutic protocols for metastatic and primary brain tumors and some autoimmune diseases. Its efficacy for brain tumors is limited by the high incidence of central nervous system (CNS) complications. This investigation aimed to observe the morphological effects, including astroglial and microglial responses, following systemic short-term MTX administration in adult rats. Male Wistar rats received 5 or 10 mg/kg/day of MTX by intraperitoneal route for 4 consecutive days (respectively, MTX5 and MTX10 groups) or the same volume of 0.9% saline solution (control group). On the 5th day, brain samples were collected for hematoxylin-eosin and luxol fast blue staining techniques, as well as for immunohistochemical staining for glial fibrillary acidic protein (GFAP) expression in astrocytes and Iba1 (ionized calcium binding adaptor molecule 1) for microglia in the frontal cortex, hippocampus, hypothalamus and molecular/granular layers of the cerebellum. Morphometric analyses were performed using Image Pro-Plus software. Brain levels of the proinflammatory cytokines TNF-α and IL-1ß were determined by ELISA. No signs of neuronal loss or demyelination were observed in all groups. Increased GFAP and Iba1 expression was found in all areas from the MTX groups, although it was slightly higher in the MTX10 group compared to the MTX5. Both TNF-α and IL-1ß levels were decreased in the MTX5 group compared to controls. In the MTX10 group, TNF-α decreased, although IL-1ß was increased relative to controls. MTX administration induced microglial reaction and astrogliosis in several CNS areas. In the MTX5 group, it apparently occurred in the presence of decreased proinflammatory cytokines.


Assuntos
Antimetabólitos Antineoplásicos/administração & dosagem , Astrócitos/efeitos dos fármacos , Proteínas de Ligação ao Cálcio/metabolismo , Proteína Glial Fibrilar Ácida/metabolismo , Gliose/fisiopatologia , Metotrexato/administração & dosagem , Proteínas dos Microfilamentos/metabolismo , Microglia/efeitos dos fármacos , Animais , Astrócitos/metabolismo , Astrócitos/patologia , Relação Dose-Resposta a Droga , Gliose/induzido quimicamente , Gliose/patologia , Masculino , Microglia/metabolismo , Microglia/patologia , Ratos , Ratos Wistar
7.
Nat Commun ; 12(1): 2812, 2021 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-33990570

RESUMO

Trastuzumab is the backbone of HER2-directed gastric cancer therapy, but poor patient response due to insufficient cell sensitivity and drug resistance remains a clinical challenge. Here, we report that HER2 is involved in cell mitotic promotion for tumorigenesis by hyperactivating a crucial HER2-SHCBP1-PLK1 axis that drives trastuzumab sensitivity and is targeted therapeutically. SHCBP1 is an Shc1-binding protein but is detached from scaffold protein Shc1 following HER2 activation. Released SHCBP1 responds to HER2 cascade by translocating into the nucleus following Ser273 phosphorylation, and then contributing to cell mitosis regulation through binding with PLK1 to promote the phosphorylation of the mitotic interactor MISP. Meanwhile, Shc1 is recruited to HER2 for MAPK or PI3K pathways activation. Also, clinical evidence shows that increased SHCBP1 prognosticates a poor response of patients to trastuzumab therapy. Theaflavine-3, 3'-digallate (TFBG) is identified as an inhibitor of the SHCBP1-PLK1 interaction, which is a potential trastuzumab sensitizing agent and, in combination with trastuzumab, is highly efficacious in suppressing HER2-positive gastric cancer growth. These findings suggest an aberrant mitotic HER2-SHCBP1-PLK1 axis underlies trastuzumab sensitivity and offer a new strategy to combat gastric cancer.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Receptor ErbB-2/metabolismo , Proteínas Adaptadoras da Sinalização Shc/metabolismo , Neoplasias Gástricas/tratamento farmacológico , Neoplasias Gástricas/metabolismo , Trastuzumab/farmacologia , Animais , Antineoplásicos Imunológicos/farmacologia , Biflavonoides/farmacologia , Catequina/análogos & derivados , Catequina/farmacologia , Proteínas de Ciclo Celular/química , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Resistencia a Medicamentos Antineoplásicos/fisiologia , Feminino , Técnicas de Silenciamento de Genes , Humanos , Imuno-Histoquímica , Estimativa de Kaplan-Meier , Masculino , Camundongos , Proteínas dos Microfilamentos/metabolismo , Pessoa de Meia-Idade , Mitose/efeitos dos fármacos , Modelos Biológicos , Modelos Moleculares , Fosfoproteínas/metabolismo , Prognóstico , Domínios e Motivos de Interação entre Proteínas/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/química , Proteínas Proto-Oncogênicas/química , Receptor ErbB-2/antagonistas & inibidores , Proteínas Adaptadoras da Sinalização Shc/antagonistas & inibidores , Proteínas Adaptadoras da Sinalização Shc/química , Transdução de Sinais/efeitos dos fármacos , Neoplasias Gástricas/patologia , Ensaios Antitumorais Modelo de Xenoenxerto
8.
Nat Commun ; 12(1): 2739, 2021 05 20.
Artigo em Inglês | MEDLINE | ID: mdl-34016974

RESUMO

In addition to conspicuous large mesophyll chloroplasts, where most photosynthesis occurs, small epidermal chloroplasts have also been observed in plant leaves. However, the functional significance of this small organelle remains unclear. Here, we present evidence that Arabidopsis epidermal chloroplasts control the entry of fungal pathogens. In entry trials, specialized fungal cells called appressoria triggered dynamic movement of epidermal chloroplasts. This movement is controlled by common regulators of mesophyll chloroplast photorelocation movement, designated as the epidermal chloroplast response (ECR). The ECR occurs when the PEN2 myrosinase-related higher-layer antifungal system becomes ineffective, and blockage of the distinct steps of the ECR commonly decreases preinvasive nonhost resistance against fungi. Furthermore, immune components were preferentially localized to epidermal chloroplasts, contributing to antifungal nonhost resistance in the pen2 background. Our findings reveal that atypical small chloroplasts act as defense-related motile organelles by specifically positioning immune components in the plant epidermis, which is the first site of contact between the plant and pathogens. Thus, this work deepens our understanding of the functions of epidermal chloroplasts.


Assuntos
Arabidopsis/imunologia , Cloroplastos/imunologia , Resistência à Doença/imunologia , Doenças das Plantas/imunologia , Epiderme Vegetal/imunologia , Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/microbiologia , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Auxilinas/genética , Auxilinas/metabolismo , Proteínas de Cloroplastos/genética , Proteínas de Cloroplastos/metabolismo , Cloroplastos/metabolismo , Colletotrichum/imunologia , Colletotrichum/patogenicidade , Interações Hospedeiro-Patógeno/imunologia , Magnaporthe/imunologia , Magnaporthe/patogenicidade , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Mutação , N-Glicosil Hidrolases/genética , N-Glicosil Hidrolases/metabolismo , Doenças das Plantas/microbiologia , Epiderme Vegetal/citologia , Epiderme Vegetal/metabolismo , Epiderme Vegetal/microbiologia , Folhas de Planta/citologia , Folhas de Planta/imunologia , Folhas de Planta/metabolismo , Folhas de Planta/microbiologia , Plantas Geneticamente Modificadas , Pseudomonas syringae/imunologia , Pseudomonas syringae/patogenicidade
9.
Anticancer Res ; 41(5): 2403-2410, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33952465

RESUMO

BACKGROUND/AIM: Fascin, an actin-bundling protein, plays an essential role in cancer metastasis. The Hippo pathway is critical for carcinogenesis and cancer stem cell self-renewal. Mammalian STE20-like kinase (MST) is a core component of the Hippo pathway. However, whether fascin and MST2 affect melanoma remain largely unknown. This study aimed to investigate the role of fascin and MST2 in melanoma development. MATERIALS AND METHODS: Surgically excised skin melanomas and the adjacent non-tumorous skin tissue from 30 cases were analyzed using immunohistochemistry for fascin and MST2. The melanoma cell line WM793 was employed for fascin and MST2 knock-down followed by western blotting, and melanoma xenografting in BALB/c mice. RESULTS: Immunohistochemistry revealed increased expression of fascin and decreased expression of MST2 in melanoma. The reverse correlation of fascin and MST2 was statistically significant. Fascin siRNA upregulated MST2 expression; however, MST2 siRNA did not significantly affect fascin expression in the WM793. WM793 xenografting followed by fascin knock-down inhibited tumor growth significantly in the animal study. CONCLUSION: Fascin is a regulator of the Hippo pathway and plays an important role in melanoma development. Therefore, fascin could be a potential therapeutic target for melanoma.


Assuntos
Proteínas de Transporte/metabolismo , Melanoma/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Neoplasias Cutâneas/metabolismo , Animais , Proteínas de Transporte/genética , Linhagem Celular Tumoral , Feminino , Humanos , Imuno-Histoquímica , Melanoma/genética , Melanoma/terapia , Camundongos Endogâmicos BALB C , Proteínas dos Microfilamentos/genética , Proteínas Serina-Treonina Quinases/genética , Interferência de RNA , Terapêutica com RNAi/métodos , Transdução de Sinais/genética , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/terapia , Ensaios Antitumorais Modelo de Xenoenxerto/métodos
10.
Nat Commun ; 12(1): 2610, 2021 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-33972531

RESUMO

Angiogenic sprouting relies on collective migration and coordinated rearrangements of endothelial leader and follower cells. VE-cadherin-based adherens junctions have emerged as key cell-cell contacts that transmit forces between cells and trigger signals during collective cell migration in angiogenesis. However, the underlying molecular mechanisms that govern these processes and their functional importance for vascular development still remain unknown. We previously showed that the F-BAR protein PACSIN2 is recruited to tensile asymmetric adherens junctions between leader and follower cells. Here we report that PACSIN2 mediates the formation of endothelial sprouts during angiogenesis by coordinating collective migration. We show that PACSIN2 recruits the trafficking regulators EHD4 and MICAL-L1 to the rear end of asymmetric adherens junctions to form a recycling endosome-like tubular structure. The junctional PACSIN2/EHD4/MICAL-L1 complex controls local VE-cadherin trafficking and thereby coordinates polarized endothelial migration and angiogenesis. Our findings reveal a molecular event at force-dependent asymmetric adherens junctions that occurs during the tug-of-war between endothelial leader and follower cells, and allows for junction-based guidance during collective migration in angiogenesis.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Antígenos CD/metabolismo , Caderinas/metabolismo , Proteínas de Ligação a DNA/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Proteínas dos Microfilamentos/metabolismo , Oxigenases de Função Mista/metabolismo , Neovascularização Patológica/metabolismo , Proteínas Nucleares/metabolismo , Junções Aderentes/genética , Junções Aderentes/metabolismo , Animais , Cateninas/metabolismo , Movimento Celular/genética , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neovascularização Patológica/genética , Transdução de Sinais/genética , Esferoides Celulares/metabolismo
11.
J Med Chem ; 64(11): 7453-7467, 2021 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-34032427

RESUMO

There has been significant attention concerning the biased agonism of G protein-coupled receptors (GPCRs), and it has resulted in various pharmacological benefits. 5-HT7R belongs to a GPCR, and it is a promising pharmaceutical target for the treatment of neurodevelopmental and neuropsychiatric disorders. Based on our previous research, we synthesized a series of 6-chloro-2'-methoxy biphenyl derivatives 1, 2, and 3 with a variety of amine scaffolds. These compounds were evaluated for their binding affinities to 5-HTR subtypes and their functional selectivity toward the Gs protein and the ß-arrestin signaling pathways of 5-HT7R. Among them, 2-(6-chloro-2'-methoxy-[1,1'-biphenyl]-3-yl)-N-ethylethan-1-amine, 2b, was found to be a G-protein-biased ligand of 5-HT7R. In an in vivo study with Shank3 transgenic mice, the self-grooming behavior test was performed with 2b, which increased the duration of self-grooming. The experiments further suggested that 5-HT7R is associated with autism spectrum disorders (ASDs) and could be a therapeutic target for the treatment of stereotypy in ASDs.


Assuntos
Compostos de Bifenilo/química , Ligantes , Receptores de Serotonina/metabolismo , Animais , Comportamento Animal/efeitos dos fármacos , Compostos de Bifenilo/metabolismo , Compostos de Bifenilo/farmacologia , Avaliação Pré-Clínica de Medicamentos , Estabilidade de Medicamentos , Meia-Vida , Humanos , Masculino , Camundongos , Camundongos Endogâmicos ICR , Camundongos Transgênicos , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Microssomos/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Serotonina/química , Relação Estrutura-Atividade
12.
Life Sci ; 278: 119577, 2021 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-33961858

RESUMO

OBJECTIVES: This study aims to investigate the role of demethylase ALKBH5 mediated demethylation of TAGLN mRNA in the occurrence of Hirschsprung's disease (HSCR), and to clarify how ALKBH5 reduces the m6A level of TAGLN mRNA and inhibits its degradation, thereby inhibiting the proliferation and migration of neural crest cells, and potentially contributing to the occurrence of HSCR. MATERIAL AND METHODS: Quantitative real-time PCR (qRT-PCR) and Western-Blot (WB) were conducted to test the expression level of ALKBH5 and TAGLN genes. Cell function assays were adopted to detect cell phenotypes. The qRT-PCR and methylated RNA immunoprecipitation (MeRIP-qPCR) were used to test the regulation of TAGLN by ALKBH5. RESULTS: 1. Compared with control intestinal tissue, the expression level of TAGLN and ALKBH5 in the aganglionic intestinal tissue of HSCR is increased. 2. The MeRIP-PCR and dualluciferase report confirmed that ALKBH5 could bind to m6A sites of TAGLN mRNA and reduce the m6A level of TAGLN mRNA. 3. In vitro cell experiments confirmed that overexpression of ALKBH5 can inhibit the degradation of TAGLN mRNA, increase the expression of TAGLN, thereby inhibiting cell proliferation and migration. 4. A zebrafish model of ALKBH5 overexpression was constructed. Studies have shown that ALKBH5 could inhibit the proliferation and migration of zebrafish enteric neurons. CONCLUSIONS: ALKBH5 could demethylate TAGLN mRNA and up-regulate TAGLN expression, leading to the inhibition of proliferation and migration of enteric neural crest cells and contributing to the occurrence of HSCR.


Assuntos
Homólogo AlkB 5 da RNA Desmetilase/metabolismo , Doença de Hirschsprung/metabolismo , Intestinos/patologia , Proteínas dos Microfilamentos/metabolismo , Proteínas Musculares/metabolismo , Crista Neural/metabolismo , Peixe-Zebra/genética , Animais , Sítios de Ligação , Estudos de Casos e Controles , Movimento Celular , Proliferação de Células , Feminino , Perfilação da Expressão Gênica , Células HEK293 , Doença de Hirschsprung/genética , Humanos , Lactente , Masculino , Metiltransferases/metabolismo
13.
ACS Chem Biol ; 16(6): 1003-1010, 2021 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-34009928

RESUMO

Imaging RNA-protein interaction in the cellular space with single molecule sensitivity is attractive for studying gene expression and regulation, but remains a challenge. In this study, we reported a photoactivatable trimolecular fluorescence complementation (TriFC) system based on fluorescent protein, mIrisFP, to identify and visualize RNA-protein interactions in living mammalian cells. We also combined this TriFC system with photoactivated localization microscopy (PALM), named the TriFC-PALM technique, which allowed us to image the RNA-protein interactions with single molecule sensitivity. Using this TriFC-PALM technique, we identified the actin-bundling protein, FSCN1, specifically interacting with the HOX Transcript Antisense RNA (HOTAIR). The TriFC-PALM imaging acquired a higher resolution compared with the traditional method of total internal reflection (TIRF) imaging. The TriFC-PALM thus provides a useful tool for imaging and identifying the RNA-protein interactions inside cells at the nanometer scale.


Assuntos
Proteínas Luminescentes/metabolismo , Proteínas/metabolismo , RNA/metabolismo , Proteínas de Transporte/análise , Proteínas de Transporte/metabolismo , Células HEK293 , Células HeLa , Humanos , Proteínas Luminescentes/análise , Proteínas dos Microfilamentos/análise , Proteínas dos Microfilamentos/metabolismo , Microscopia de Fluorescência/métodos , Imagem Óptica/métodos , Proteínas/análise , RNA/análise , RNA Longo não Codificante/análise , RNA Longo não Codificante/metabolismo
14.
Aging (Albany NY) ; 13(10): 14198-14218, 2021 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-34016787

RESUMO

We investigated the role of long non-coding RNA (lncRNA) LOC146880 in esophageal squamous cell carcinoma (ESCC). LOC146880 was significantly upregulated in ESCC tissues (n = 21) and cell lines compared to the corresponding controls. Higher LOC146880 expression correlated with poorer overall survival (OS) of ESCC patients. Moreover, CREB-binding protein (CBP) and H3K27 acetylation levels were significantly higher in the LOC146880 promoter in ESCC cell lines than in the controls. LOC146880 silencing inhibited in vitro proliferation, invasion, migration, and epithelial-mesenchymal transition of ESCC cells. LOC146880 silencing also induced G1-phase cell cycle arrest and apoptosis in ESCC cells. Bioinformatics analysis, dual luciferase reporter assays, and RNA immunoprecipitation assays showed that LOC146880 regulates FSCN1 expression in ESCC cells by sponging miR-328-5p. Moreover, FSCN1 expression correlated with activation of the MAPK signaling pathway in ESCC cells and tissues. In vivo xenograft tumor volume and liver metastasis were significantly reduced in nude mice injected with LOC146880-silenced ESCC cells as compared to those injected with control shRNA-transfected ESCC cells. These findings show that the LOC146880/miR-328-5p/FSCN1/MAPK axis regulates ESCC progression in vitro and in vivo. LOC146880 is thus a promising prognostic biomarker and potential therapeutic target in ESCC.


Assuntos
Proteínas de Transporte/metabolismo , Progressão da Doença , Neoplasias Esofágicas/genética , Carcinoma de Células Escamosas do Esôfago/genética , Carcinoma de Células Escamosas do Esôfago/patologia , Sistema de Sinalização das MAP Quinases/genética , MicroRNAs/metabolismo , Proteínas dos Microfilamentos/metabolismo , RNA Longo não Codificante/metabolismo , Acetilação , Sequência de Bases , Proteínas de Transporte/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Sobrevivência Celular/genética , Transição Epitelial-Mesenquimal/genética , Neoplasias Esofágicas/patologia , Regulação Neoplásica da Expressão Gênica , Histonas/metabolismo , Humanos , Lisina/metabolismo , Proteínas dos Microfilamentos/genética , Fragmentos de Peptídeos/metabolismo , RNA Longo não Codificante/genética , Sialoglicoproteínas/metabolismo , Transcrição Genética
15.
Cell Mol Life Sci ; 78(13): 5275-5301, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34023917

RESUMO

For a long time, PLS3 (plastin 3, also known as T-plastin or fimbrin) has been considered a rather inconspicuous protein, involved in F-actin-binding and -bundling. However, in recent years, a plethora of discoveries have turned PLS3 into a highly interesting protein involved in many cellular processes, signaling pathways, and diseases. PLS3 is localized on the X-chromosome, but shows sex-specific, inter-individual and tissue-specific expression variability pointing towards skewed X-inactivation. PLS3 is expressed in all solid tissues but usually not in hematopoietic cells. When escaping X-inactivation, PLS3 triggers a plethora of different types of cancers. Elevated PLS3 levels are considered a prognostic biomarker for cancer and refractory response to therapies. When it is knocked out or mutated in humans and mice, it causes osteoporosis with bone fractures; it is the only protein involved in actin dynamics responsible for osteoporosis. Instead, when PLS3 is upregulated, it acts as a highly protective SMN-independent modifier in spinal muscular atrophy (SMA). Here, it seems to counteract reduced F-actin levels by restoring impaired endocytosis and disturbed calcium homeostasis caused by reduced SMN levels. In contrast, an upregulation of PLS3 on wild-type level might cause osteoarthritis. This emphasizes that the amount of PLS3 in our cells must be precisely balanced; both too much and too little can be detrimental. Actin-dynamics, regulated by PLS3 among others, are crucial in a lot of cellular processes including endocytosis, cell migration, axonal growth, neurotransmission, translation, and others. Also, PLS3 levels influence the infection with different bacteria, mycosis, and other pathogens.


Assuntos
Glicoproteínas de Membrana/metabolismo , Proteínas dos Microfilamentos/metabolismo , Neurônios Motores/fisiologia , Atrofia Muscular Espinal/fisiopatologia , Osteoclastos/fisiologia , Osteoporose/fisiopatologia , Animais , Humanos , Glicoproteínas de Membrana/genética , Proteínas dos Microfilamentos/genética , Osteoclastos/citologia
16.
Nat Commun ; 12(1): 3232, 2021 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-34050140

RESUMO

Arrays of regularly spaced nucleosomes dominate chromatin and are often phased by alignment to reference sites like active promoters. How the distances between nucleosomes (spacing), and between phasing sites and nucleosomes are determined remains unclear, and specifically, how ATP-dependent chromatin remodelers impact these features. Here, we used genome-wide reconstitution to probe how Saccharomyces cerevisiae ATP-dependent remodelers generate phased arrays of regularly spaced nucleosomes. We find that remodelers bear a functional element named the 'ruler' that determines spacing and phasing in a remodeler-specific way. We use structure-based mutagenesis to identify and tune the ruler element residing in the Nhp10 and Arp8 modules of the INO80 remodeler complex. Generally, we propose that a remodeler ruler regulates nucleosome sliding direction bias in response to (epi)genetic information. This finally conceptualizes how remodeler-mediated nucleosome dynamics determine stable steady-state nucleosome positioning relative to other nucleosomes, DNA bound factors, DNA ends and DNA sequence elements.


Assuntos
Montagem e Desmontagem da Cromatina , Nucleossomos/metabolismo , Sequências Reguladoras de Ácido Nucleico/genética , Animais , Proteínas de Drosophila/genética , Proteínas de Drosophila/isolamento & purificação , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Epigênese Genética , Genoma Fúngico/genética , Proteínas de Grupo de Alta Mobilidade/genética , Proteínas de Grupo de Alta Mobilidade/isolamento & purificação , Proteínas de Grupo de Alta Mobilidade/metabolismo , Histonas/genética , Histonas/metabolismo , Larva/genética , Larva/metabolismo , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/isolamento & purificação , Proteínas dos Microfilamentos/metabolismo , Mutagênese , Nucleossomos/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/isolamento & purificação , Proteínas de Saccharomyces cerevisiae/metabolismo , Sequenciamento Completo do Genoma
17.
Int J Mol Sci ; 22(6)2021 Mar 17.
Artigo em Inglês | MEDLINE | ID: mdl-33802838

RESUMO

Osteoarthritis (OA) is a multifactorial disease which is characterized by a change in the homeostasis of the extracellular matrix (ECM). The ECM is essential for the function of the articular cartilage and plays an important role in cartilage mechanotransduction. To provide a better understanding of the interaction between the ECM and the actin cytoskeleton, we investigated the localization and expression of the Ca2+-dependent proteins cartilage oligomeric matrix protein (COMP), thrombospondin-1 (TSP-1), plastin 3 (PLS3) and stromal interaction molecule 1 (STIM1). We investigated 16 patients who suffered from varus knee OA and performed a topographical analysis of the cartilage from the medial and lateral compartment of the proximal tibial plateau. In a varus knee, OA is more pronounced in the medial compared to the lateral compartment as a result of an overloading due to the malalignment. We detected a location-dependent staining of PLS3 and STIM1 in the articular cartilage tissue. The staining intensity for both proteins correlated with the degree of cartilage degeneration. The staining intensity of TSP-1 was clearly reduced in the cartilage of the more affected medial compartment, an observation that was confirmed in cartilage extracts by immunoblotting. The total amount of COMP was unchanged; however, slight changes were detected in the localization of the protein. Our results provide novel information on alterations in OA cartilage suggesting that Ca2+-dependent mechanotransduction between the ECM and the actin cytoskeleton might play an essential role in the pathomechanism of OA.


Assuntos
Cartilagem Articular/metabolismo , Articulação do Joelho/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas dos Microfilamentos/metabolismo , Osteoartrite do Joelho/metabolismo , Molécula 1 de Interação Estromal/metabolismo , Trombospondinas/metabolismo , Idoso , Idoso de 80 Anos ou mais , Proteína de Matriz Oligomérica de Cartilagem/metabolismo , Condrócitos/metabolismo , Feminino , Humanos , Articulação do Joelho/patologia , Masculino , Pessoa de Meia-Idade , Osteoartrite do Joelho/patologia , Transporte Proteico
18.
Int J Mol Sci ; 22(9)2021 Apr 27.
Artigo em Inglês | MEDLINE | ID: mdl-33925417

RESUMO

Craniofacial neuropathic pain affects millions of people worldwide and is often difficult to treat. Two key mechanisms underlying this condition are a loss of the negative control exerted by inhibitory interneurons and an early microglial reaction. Basic features of these mechanisms, however, are still poorly understood. Using the chronic constriction injury of the infraorbital nerve (CCI-IoN) model of neuropathic pain in mice, we have examined the changes in the expression of GAD, the synthetic enzyme of GABA, and GlyT2, the membrane transporter of glycine, as well as the microgliosis that occur at early (5 days) and late (21 days) stages post-CCI in the medullary and upper spinal dorsal horn. Our results show that CCI-IoN induces a down-regulation of GAD at both postinjury survival times, uniformly across the superficial laminae. The expression of GlyT2 showed a more discrete and heterogeneous reduction due to the basal presence in lamina III of 'patches' of higher expression, interspersed within a less immunoreactive 'matrix', which showed a more substantial reduction in the expression of GlyT2. These patches coincided with foci lacking any perceptible microglial reaction, which stood out against a more diffuse area of strong microgliosis. These findings may provide clues to better understand the neural mechanisms underlying allodynia in neuropathic pain syndromes.


Assuntos
Microglia/metabolismo , Neuralgia/etiologia , Corno Dorsal da Medula Espinal/metabolismo , Animais , Comportamento Animal , Proteínas de Ligação ao Cálcio/metabolismo , Densitometria , Modelos Animais de Doenças , Proteínas da Membrana Plasmática de Transporte de Glicina/metabolismo , Hiperalgesia/etiologia , Masculino , Nervo Maxilar/lesões , Camundongos Endogâmicos C57BL , Proteínas dos Microfilamentos/metabolismo , Microglia/patologia , Corno Dorsal da Medula Espinal/patologia , Núcleo Inferior Caudal do Nervo Trigêmeo/metabolismo , Núcleo Inferior Caudal do Nervo Trigêmeo/patologia
19.
Nutrients ; 13(5)2021 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-33922475

RESUMO

Among therapeutic approaches that have been investigated, targeting of receptors implicated in managing neuroinflammation has been described. One such family of receptors comprises the formyl peptide receptors (FPRs) whose ligands could play a role in host defense. The murine FPR gene family includes at least six members while in humans there are only three. The two most important members are the Fpr1 and Fpr2. Fpr1encodes murine FPR1, which is considered the murine orthologue of human FPR. Resveratrol, a non-flavonoid polyphenol rich in red wine and grapes, apart from its beneficial health effects and anti-inflammatory properties, has been reported to reduce neuroinflammation in different neurodegenerative disease models. Resveratrol anti-inflammatory responses involve the activation of the protein deacetylase sirtuin 1 (SIRT1) gene. In this work we have investigated in an LPS-based murine model of neuroinflammation the role of FPR1, examining not only if this receptor undergoes a reduction of its expression during neuroinflammation, but also whether treatment with resveratrol was able to modulate its expression leading to an amelioration of neuroinflammatory picture in a murine model of neuroinflammation. Results of this work showed that FPR1 together with SIRT1 resulted upregulated by resveratrol treatment and that this increase is associated with an amelioration of the neuroinflammatory picture, as demonstrated by the induction of IL-10 and IL1-RA expression and the downregulation of proinflammatory mediators, such as TNF-α and IL-1ß. The expression and the modulation of FPR1 by resveratrol may be evaluated in order to propose a novel anti-inflammatory and pro-resolving therapeutic approach for the reduction of the detrimental effects associated with neuro-inflammation based neurodegenerative diseases and also as a promising strategy to promote human health by a diet rich in antioxidative bioactive compounds.


Assuntos
Astrócitos/patologia , Inflamação/metabolismo , Inflamação/patologia , Microglia/patologia , Receptores de Formil Peptídeo/metabolismo , Resveratrol/farmacologia , Animais , Anti-Inflamatórios/farmacologia , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Modelos Animais de Doenças , Proteína Glial Fibrilar Ácida/metabolismo , Inflamação/induzido quimicamente , Mediadores da Inflamação/metabolismo , Proteína Antagonista do Receptor de Interleucina 1/metabolismo , Lipopolissacarídeos , Masculino , Camundongos , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Microglia/efeitos dos fármacos , Microglia/metabolismo , Modelos Biológicos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais , Sirtuína 1/genética , Sirtuína 1/metabolismo , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo
20.
Oxid Med Cell Longev ; 2021: 5564884, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33859778

RESUMO

Vascular smooth muscle cell (VSMC) apoptosis is a major defining feature of abdominal aortic aneurysm (AAA) and mainly caused by inflammatory cell infiltration. Smooth muscle (SM) 22α prevents AAA formation through suppressing NF-κB activation. However, the role of SM22α in VSMC apoptosis is controversial. Here, we identified that SM22α loss contributed to apoptosis of VSMCs via activation of macrophages. Firstly, deficiency of SM22α enhanced the interaction of VSMCs with macrophages. Macrophages were retained and activated by Sm22α -/- VSMCs via upregulating VCAM-1 expression. The ratio of apoptosis was increased by 1.62-fold in VSMCs treated with the conditional media (CM) from activated RAW264.7 cells, compared to that of the control CM (P < 0.01), and apoptosis of Sm22α -/- VSMCs was higher than that of WT VSMCs (P < 0.001). Next, circRasGEF1B from activated macrophages was delivered into VSMCs promoting ZFP36 expression via stabilization of ZFP36 mRNA. Importantly, circRasGEF1B, as a scaffold, guided ZFP36 to preferentially bind to and decay Bcl-2 mRNA in a sequence-specific manner and triggered apoptosis of VSMCs, especially in Sm22α -/- VSMCs. These findings reveal a novel mechanism by which the circRasGEF1B-ZFP36 axis mediates macrophage-induced VSMC apoptosis via decay of Bcl-2 mRNA, whereas Sm22α -/- VSMCs have a higher sensitivity to apoptosis.


Assuntos
Macrófagos/citologia , Macrófagos/metabolismo , Proteínas dos Microfilamentos/deficiência , Proteínas Musculares/deficiência , Músculo Liso Vascular/citologia , Músculo Liso Vascular/metabolismo , RNA Circular/metabolismo , Animais , Apoptose/fisiologia , Comunicação Celular/fisiologia , Técnicas de Reprogramação Celular , Humanos , Masculino , Camundongos , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Células RAW 264.7 , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais , Tristetraprolina/biossíntese , Tristetraprolina/genética , Tristetraprolina/metabolismo
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