RESUMEN
Cell size is a crucial physical property that significantly impacts cellular physiology and function. However, the influence of cell size on stem cell specification remains largely unknown. Here, we investigated the dynamic changes in cell size during the differentiation of human pluripotent stem cells into definitive endoderm (DE). Interestingly, cell size exhibited a gradual decrease as DE differentiation progressed with higher stiffness. Furthermore, the application of hypertonic pressure or chemical to accelerate the reduction in cell size significantly and specifically enhanced DE differentiation. By functionally intervening in mechanosensitive elements, we have identified actomyosin activity as a crucial mediator of both DE differentiation and cell size reduction. Mechanistically, the reduction in cell size induces actomyosin-dependent angiomotin (AMOT) nuclear translocation, which suppresses Yes-associated protein (YAP) activity and thus facilitates DE differentiation. Together, our study has established a novel connection between cell size diminution and DE differentiation, which is mediated by AMOT nuclear translocation. Additionally, our findings suggest that the application of osmotic pressure can effectively promote human endodermal lineage differentiation.
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Actomiosina , Angiomotinas , Diferenciación Celular , Tamaño de la Célula , Endodermo , Transducción de Señal , Factores de Transcripción , Proteínas Señalizadoras YAP , Humanos , Endodermo/citología , Endodermo/metabolismo , Actomiosina/metabolismo , Proteínas Señalizadoras YAP/metabolismo , Factores de Transcripción/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Presión Osmótica , Células Madre Pluripotentes/metabolismo , Células Madre Pluripotentes/citología , Núcleo Celular/metabolismoRESUMEN
Adriamycin (ADR) is widely used against breast cancer, but subsequent resistance always occurs. YAP, a downstream protein of angiomotin (AMOT), importantly contributes to ADR resistance, whereas the mechanism is largely unknown. MCF-7 cells and MDA-MB-231 cells were used to establish ADR-resistant cell. Then, mRNA and protein expressions of AMOT and YAP expressions were determined. After AMOT transfection alone or in combination with YAP, the sensitivity of the cells to ADR were evaluated in vitro by examining cell proliferation, apoptosis, and cell cycle, as well as in vivo by examining tumor growth. Additionally, the expressions of proteins in YAP pathway were determined in AMOT-overexpressing cells. In the ADR-resistant cells, the expression of AMOT was decreased while YAP was increased, respectively, and the nucleus localization of YAP was increased at the same time. After AMOT overexpression, these were inhibited, whereas the cell sensitivity to ADR was enhanced. However, the AMOT-induced changes were significantly suppressed by YAP knockdown. The consistent results in vivo showed that AMOT enhanced the inhibition of ADR on tumor growth, and inhibited YAP signaling, evidenced by decreased levels of YAP, CycD1, and p-ERK. Our data revealed that decreased AMOT contributed to ADR resistance in breast cancer cells, which was importantly negatively mediated YAP. These observations provide a potential therapy against breast cancer with ADR resistance.
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Angiomotinas , Apoptosis , Neoplasias de la Mama , Proliferación Celular , Doxorrubicina , Resistencia a Antineoplásicos , Ratones Desnudos , Proteínas Señalizadoras YAP , Humanos , Doxorrubicina/farmacología , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Resistencia a Antineoplásicos/efectos de los fármacos , Femenino , Proliferación Celular/efectos de los fármacos , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Células MCF-7 , Proteínas Señalizadoras YAP/metabolismo , Animales , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Ratones , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Ratones Endogámicos BALB C , Antibióticos Antineoplásicos/farmacología , Transducción de Señal/efectos de los fármacos , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Proteínas de Microfilamentos/metabolismo , Proteínas de Microfilamentos/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Ciclo Celular/efectos de los fármacosRESUMEN
BACKGROUND: Colorectal cancer (CRC) ranks as the third most common cancer and is second in terms of mortality worldwide. Circular RNAs are involved in the occurrence and development of malignant tumors by functioning either as oncogenes or tumor suppressors. METHOD: This study investigated the functions of hsa_circ_0001278 in CRC. We analyzed the expression of hsa_circ_0001278 in CRC tissues and adjacent normal tissues. In order to understand the roles of hsa_circ_0001278 in CRC in terms of cellular biological behavior, in vitro experiments were conducted. A mechanistic study was designed to investigate the regulatory effect of hsa_circ_0001278 on CRC. RESULTS: Hsa_circ_0001278 was found to be significantly upregulated in CRC specimens. The functional analysis indicated that hsa_circ_0001278 promotes aggressive phenotypes of CRC cells. Further mechanistic studies revealed that hsa_circ_0001278 sponges miR-338-5p to regulate angiomotin-like 1 (AMOTL1), thereby facilitating CRC progression. CONCLUSION: Our results demonstrate that hsa_circ_0001278 promotes malignant behaviors in CRC cells by sponging miR-338-5p to regulate AMOTL1 expression. This suggests that hsa_circ_0001278 may serve as a novel target for CRC treatment.
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BACKGROUND: Although exosomal microRNAs (exo-miRNAs) regulate angiogenesis, they are not sufficient for the development of anti-vascular drugs for tongue squamous cell carcinoma (TSCC). miR-205-5p is an exo-miRNA that is highly expressed in the saliva of patients with oral SCC. OBJECTIVE: We aimed to clarify the role and molecular mechanism of exosomal miR-205-5p in regulating TSCC angiogenesis. METHODS: Effect of exosomes derived from TSCC cells on human umbilical vein endothelial cell (HUVEC) function was determined using the CCK-8, Transwell, Transwell-Matrigel, and Matrigel-based tube formation assays. Protein levels were detected by western blot. The binding between miR-205-5p and the 3'UTR of AMOT was verified using a luciferase reporter assay. RESULTS: Exosomal miR-205-5p (exo-miR-205-5p) promoted the proliferation, migration, and invasion of HUVECs, increased the number of tubes formed by HUVECs, and increased the vascular endothelial growth factor receptor 2 levels in HUVECs. Exo-miR-205-5p downregulated the AMOT level in HUVECs. Results of the luciferase reporter assay showed that miR-205-5p could bind to the 3'UTR of AMOT. AMOT overexpression blocked the effect of exo-miR-205-5p on HUVEC functions. CONCLUSION: Exo-miR-205-5p derived from TSCC regulates the angiogenic activity of HUVECs by targeting AMOT and might be a new molecular target for the development of anti-vascular drugs for TSCC.
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The scaffolding protein angiomotin (AMOT) is indispensable for vertebrate embryonic angiogenesis. Here, we report that AMOT undergoes cleavage in the presence of lysophosphatidic acid (LPA), a lipid growth factor also involved in angiogenesis. AMOT cleavage is mediated by aspartic protease DNA damage-inducible 1 homolog 2 (DDI2), and the process is tightly regulated by a signaling axis including neurofibromin 2 (NF2), tankyrase 1/2 (TNKS1/2), and RING finger protein 146 (RNF146), which induce AMOT membrane localization, poly ADP ribosylation, and ubiquitination, respectively. In both zebrafish and mice, the genetic inactivation of AMOT cleavage regulators leads to defective angiogenesis, and the phenotype is rescued by the overexpression of AMOT-CT, a C-terminal AMOT cleavage product. In either physiological or pathological angiogenesis, AMOT-CT is required for vascular expansion, whereas uncleavable AMOT represses this process. Thus, our work uncovers a signaling pathway that regulates angiogenesis by modulating a cleavage-dependent activation of AMOT.
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Angiomotinas , Pez Cebra , Animales , Ratones , Pez Cebra/metabolismo , Proteínas de Microfilamentos/metabolismo , Péptido Hidrolasas , Péptidos y Proteínas de Señalización Intercelular/genéticaRESUMEN
The function of biomolecular condensates is often restricted by condensate dissolution. Whether condensates can be suppressed without condensate dissolution is unclear. Here, we show that upstream regulators of the Hippo signaling pathway form functionally antagonizing condensates, and their coalescence into a common phase provides a mode of counteracting the function of biomolecular condensates without condensate dissolution. Specifically, the negative regulator SLMAP forms Hippo-inactivating condensates to facilitate pathway inhibition by the STRIPAK complex. In response to cell-cell contact or osmotic stress, the positive regulators AMOT and KIBRA form Hippo-activating condensates to facilitate pathway activation. The functionally antagonizing SLMAP and AMOT/KIBRA condensates further coalesce into a common phase to inhibit STRIPAK function. These findings provide a paradigm for restricting the activity of biomolecular condensates without condensate dissolution, shed light on the molecular principles of multiphase organization, and offer a conceptual framework for understanding upstream regulation of the Hippo signaling pathway.
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Vía de Señalización Hippo , Proteínas Serina-Treonina Quinasas , Transducción de SeñalRESUMEN
Ebola virus (EBOV) and Marburg virus (MARV) continue to emerge and cause severe hemorrhagic disease in humans. A comprehensive understanding of the filovirus-host interplay will be crucial for identifying and developing antiviral strategies. The filoviral VP40 matrix protein drives virion assembly and egress, in part by recruiting specific WW domain-containing host interactors via its conserved PPxY late (L) domain motif to positively regulate virus egress and spread. In contrast to these positive regulators of virus budding, a growing list of WW domain-containing interactors that negatively regulate virus egress and spread have been identified, including BAG3, YAP/TAZ, and WWOX. In addition to host WW domain regulators of virus budding, host PPxY-containing proteins also contribute to regulating this late stage of filovirus replication. For example, angiomotin (AMOT) is a multi-PPxY-containing host protein that functionally interacts with many of the same WW domain-containing proteins that regulate virus egress and spread. In this report, we demonstrate that host WWOX, which negatively regulates egress of VP40 virus-like particles (VLPs) and recombinant vesicular stomatitis virus (VSV) M40 virus, interacts with and suppresses the expression of AMOT. We found that WWOX disrupts AMOT's scaffold-like tubular distribution and reduces AMOT localization at the plasma membrane via lysosomal degradation. In sum, our findings reveal an indirect and novel mechanism by which modular PPxY-WW domain interactions between AMOT and WWOX regulate PPxY-mediated egress of filovirus VP40 VLPs. A better understanding of this modular network and competitive nature of protein-protein interactions will help to identify new antiviral targets and therapeutic strategies. IMPORTANCE Filoviruses (Ebola virus [EBOV] and Marburg virus [MARV]) are zoonotic, emerging pathogens that cause outbreaks of severe hemorrhagic fever in humans. A fundamental understanding of the virus-host interface is critical for understanding the biology of these viruses and for developing future strategies for therapeutic intervention. Here, we reveal a novel mechanism by which host proteins WWOX and AMOTp130 interact with each other and with the filovirus matrix protein VP40 to regulate VP40-mediated egress of virus-like particles (VLPs). Our results highlight the biological impact of competitive interplay of modular virus-host interactions on both the virus life cycle and the host cell.
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Ebolavirus , Marburgvirus , Oxidorreductasa que Contiene Dominios WW , Angiomotinas/metabolismo , Ebolavirus/fisiología , Humanos , Marburgvirus/metabolismo , Proteínas de la Matriz Viral/metabolismo , Liberación del Virus/fisiología , Oxidorreductasa que Contiene Dominios WW/metabolismoRESUMEN
The modulation of protein-protein interactions (PPIs) has developed into a well-established field of drug discovery. Despite the advances achieved in the field, many PPIs are still deemed as 'undruggable' targets and the design of PPIs stabilizers remains a significant challenge. The application of fragment-based methods for the identification of drug leads and to evaluate the 'tractability' of the desired protein target has seen a remarkable development in recent years. In this study, we explore the molecular characteristics of the 14-3-3/Amot-p130 PPI and the conceptual possibility of targeting this interface using X-ray crystallography fragment-based screening. We report the first structural elucidation of the 14-3-3 binding motif of Amot-p130 and the characterization of the binding mode and affinities involved. We made use of fragments to probe the 'ligandability' of the 14-3-3/Amot-p130 composite binding pocket. Here we disclose initial hits with promising stabilizing activity and an early-stage selectivity toward the Amot-p130 motifs over other representatives 14-3-3 partners. Our findings highlight the potential of using fragments to characterize and explore proteins' surfaces and might provide a starting point toward the development of small molecules capable of acting as molecular glues.
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Like most enveloped viruses, HIV must acquire a lipid membrane as it assembles and buds through the plasma membrane of infected cells to spread infection. Several sets of host cell machinery facilitate this process, including proteins of the endosomal sorting complexes required for transport pathway, which mediates the membrane fission reaction required to complete viral budding, as well as angiomotin (AMOT) and NEDD4L, which bind one another and promote virion membrane envelopment. AMOT and NEDD4L interact through the four NEDD4L WW domains and three different AMOT Pro-Pro-x (any amino acid)-Tyr (PPxY) motifs, but these interactions are not yet well defined. Here, we report that individual AMOT PPxY and NEDD4L WW domains interact with the following general affinity hierarchies: AMOT PPxY1>PPxY2>PPxY3 and NEDD4L WW3>WW2>WW1â¼WW4. The unusually high-affinity of the AMOT PPxY1-NEDD4L WW3 interaction accounts for most of the AMOT-NEDD4L binding and is critical for stimulating HIV-1 release. Comparative structural, binding, and virological analyses reveal that complementary ionic and hydrophobic contacts on both sides of the WW-PPxY core interaction account for the unusually high affinity of the AMOT PPxY1-NEDD4L WW3 interaction. Taken together, our studies reveal how the first AMOT PPxY1 motif binds the third NEDD4L WW domain to stimulate HIV-1 viral envelopment and promote infectivity.
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Angiomotinas/metabolismo , Infecciones por VIH/metabolismo , VIH-1/metabolismo , Ubiquitina-Proteína Ligasas Nedd4/metabolismo , Ensamble de Virus , Secuencias de Aminoácidos , Línea Celular , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Infecciones por VIH/patología , Infecciones por VIH/transmisión , Infecciones por VIH/virología , VIH-1/aislamiento & purificación , VIH-1/patogenicidad , Humanos , Dominios ProteicosRESUMEN
Purpose To explore the regulatory relationship between Chloride intracellular channel 1 (CLIC1) and Angiomotin (AMOT)-p130, and reveal the role of AMOT-p130 in gastric cancer (GC). Methods Immunohistochemistry was performed to analyze the expression of CLIC1 and AMOT-p130 in GC tissues and adjacent tissues. The expression of AMOT-p130 upon CLIC1 silencing was analyzed using RT-PCR, western blot, and immunofluorescence in GC cells. Transwell and wound-healing assays were performed to detect migration and invasion in GC cells. The changes in EMT-related proteins were detected using western blot. Results Our study found that high CLIC1 expression was significantly associated with low AMOT-p130 expression in GC tissues. Silencing CLIC1 expression in MGC-803 cells (MGC-803 CLIC1 KO) and AGS cells (AGS CLIC1 KO) decreased the invasive and migratory abilities of tumor cells, which were induced by the upregulation of AMOT-p130. Subsequently, we demonstrated that AMOT-p130 inhibits the invasive and migratory abilities of GC cells by inhibiting epithelialmesenchymal transition. Conclusions Our study suggests that AMOT-p130 could inhibit epithelialmesenchymal transition in GC cells. CLIC1 may participate in the metastatic progression of GC by downregulating the expression of AMOT-p130 (AU)
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Humanos , Masculino , Femenino , Persona de Mediana Edad , Canales de Cloruro/metabolismo , Transición Epitelial-Mesenquimal , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteínas de Microfilamentos/metabolismo , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patología , Inmunohistoquímica , Línea Celular Tumoral , Invasividad Neoplásica , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The underlying molecular mechanisms of intervertebral disc degeneration (IDD) remain unclear. This study aimed to identify the crucial molecules and explore the function of noncoding RNAs and related pathways in IDD. We randomly selected three samples each from an IDD and a spinal cord injury group (control) for RNA-sequencing. We identified 463 differentially-expressed long noncoding RNAs (lncRNAs), 47 differentially-expressed microRNAs (miRNAs), and 1,334 differentially-expressed mRNAs in IDD. Three hundred fifty-eight lncRNAs as cis-regulators could potentially target 865 genes. Protein-protein interaction (PPI) network analysis confirmed that IL-6, VEGFA, IGF1, MMP9, CXCL8, FGF2, IL1B, CCND1, ITGAM, PTPRC, FOS and PTGS2 were hub genes. We built a competing endogenous RNA (ceRNA) network and identified lncRNA XIST-hsa-miR-4775-PLA2G7 and lncRNA XIST-hsa-miR-424-5p-AMOT/TGFBR3 ceRNA axes. Quantitative real-time PCR (qRT-PCR) was implemented in 15 IDD samples and 15 controls to validate differentially-expressed genes in ceRNA axes. From the ceRNA network, gene ontology (GO) enrichment analysis indicated that noncoding RNAs were associated with several biological processes, including extracellular matrix organization, extracellular structure organization, leukocyte migration, and mesenchyme development. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that noncoding RNAs were associated with several pathways including the AGE-RAGE signaling pathway, PI3K-Akt signaling pathway, axon guidance, and osteoclast differentiation. These results indicate that some specific noncoding RNAs and ceRNA axes may be vital during the development of IDD, and may have potential as alternative diagnostic biomarkers as well as novel therapeutic strategies for IDD.
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Perfilación de la Expresión Génica , Degeneración del Disco Intervertebral/genética , MicroARNs/genética , ARN Largo no Codificante/genética , ARN Mensajero/genética , Análisis de Secuencia de ARN , Adulto , Anciano , Estudios de Casos y Controles , Regulación hacia Abajo/genética , Femenino , Ontología de Genes , Humanos , Masculino , MicroARNs/metabolismo , Persona de Mediana Edad , Mapas de Interacción de Proteínas/genética , ARN Largo no Codificante/metabolismo , ARN Mensajero/metabolismo , Transcriptoma/genética , Regulación hacia Arriba/genéticaRESUMEN
The Hippo signaling pathway is known to regulate cell differentiation, proliferation and apoptosis. Whereas activation of the Hippo signaling pathway leads to phosphorylation and cytoplasmic retention of the transcriptional coactivator YAP, decreased Hippo signaling results in nuclear import of YAP and subsequent transcription of pro-proliferative genes. Hence, a dynamic and precise regulation of the Hippo signaling pathway is crucial for organ size control and the prevention of tumor formation. The transcriptional activity of YAP is controlled by a growing number of upstream regulators including the family of WWC proteins. WWC1, WWC2 and WWC3 represent cytosolic scaffolding proteins involved in intracellular transport processes and different signal transduction pathways. Earlier in vitro experiments demonstrated that WWC proteins positively regulate the Hippo pathway via the activation of large tumor suppressor kinases 1/2 (LATS1/2) kinases and the subsequent cytoplasmic accumulation of phosphorylated YAP. Later, reduced WWC expression and subsequent high YAP activity were shown to correlate with the progression of human cancer in different organs. Although the function of WWC proteins as upstream regulators of Hippo signaling was confirmed in various studies, their important role as tumor modulators is often overlooked. This review has been designed to provide an update on the published data linking WWC1, WWC2 and WWC3 to cancer, with a focus on Hippo pathway-dependent mechanisms.
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PURPOSE: To explore the regulatory relationship between Chloride intracellular channel 1 (CLIC1) and Angiomotin (AMOT)-p130, and reveal the role of AMOT-p130 in gastric cancer (GC). METHODS: Immunohistochemistry was performed to analyze the expression of CLIC1 and AMOT-p130 in GC tissues and adjacent tissues. The expression of AMOT-p130 upon CLIC1 silencing was analyzed using RT-PCR, western blot, and immunofluorescence in GC cells. Transwell and wound-healing assays were performed to detect migration and invasion in GC cells. The changes in EMT-related proteins were detected using western blot. RESULTS: Our study found that high CLIC1 expression was significantly associated with low AMOT-p130 expression in GC tissues. Silencing CLIC1 expression in MGC-803 cells (MGC-803 CLIC1 KO) and AGS cells (AGS CLIC1 KO) decreased the invasive and migratory abilities of tumor cells, which were induced by the upregulation of AMOT-p130. Subsequently, we demonstrated that AMOT-p130 inhibits the invasive and migratory abilities of GC cells by inhibiting epithelial-mesenchymal transition. CONCLUSIONS: Our study suggests that AMOT-p130 could inhibit epithelial-mesenchymal transition in GC cells. CLIC1 may participate in the metastatic progression of GC by downregulating the expression of AMOT-p130.
Asunto(s)
Canales de Cloruro/metabolismo , Transición Epitelial-Mesenquimal , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteínas de Microfilamentos/metabolismo , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patología , Angiomotinas , Línea Celular Tumoral , Movimiento Celular , Canales de Cloruro/genética , Femenino , Silenciador del Gen , Humanos , Masculino , Persona de Mediana Edad , Invasividad Neoplásica , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Estadificación de Neoplasias , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Regulación hacia Arriba , Cicatrización de HeridasRESUMEN
The Ebola virus (EBOV) VP40 matrix protein (eVP40) orchestrates assembly and budding of virions in part by hijacking select WW-domain-bearing host proteins via its PPxY late (L)-domain motif. Angiomotin (Amot) is a multifunctional PPxY-containing adaptor protein that regulates angiogenesis, actin dynamics, and cell migration/motility. Amot also regulates the Hippo signaling pathway via interactions with the WW-domain-containing Hippo effector protein Yes-associated protein (YAP). In this report, we demonstrate that endogenous Amot is crucial for positively regulating egress of eVP40 virus-like particles (VLPs) and for egress and spread of authentic EBOV. Mechanistically, we show that ectopic YAP expression inhibits eVP40 VLP egress and that Amot co-expression rescues budding of eVP40 VLPs in a dose-dependent and PPxY-dependent manner. Moreover, results obtained with confocal and total internal reflection fluorescence microscopy suggested that Amot's role in actin organization and dynamics also contributes to promoting eVP40-mediated egress. In summary, these findings reveal a functional and competitive interplay between virus and host proteins involving the multifunctional PPxY-containing adaptor Amot, which regulates both the Hippo pathway and actin dynamics. We propose that our results have wide-ranging implications for understanding the biology and pathology of EBOV infections.
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Ebolavirus/fisiología , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteínas de Microfilamentos/metabolismo , Citoesqueleto de Actina/metabolismo , Secuencias de Aminoácidos , Angiomotinas , Proteínas de Ciclo Celular/metabolismo , Células HEK293 , Fiebre Hemorrágica Ebola/patología , Fiebre Hemorrágica Ebola/transmisión , Fiebre Hemorrágica Ebola/virología , Vía de Señalización Hippo , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Proteínas de Microfilamentos/antagonistas & inhibidores , Proteínas de Microfilamentos/genética , Microscopía Confocal , Nucleoproteínas/química , Nucleoproteínas/genética , Nucleoproteínas/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Transducción de Señal , Factores de Transcripción/metabolismo , Proteínas del Núcleo Viral/química , Proteínas del Núcleo Viral/genética , Proteínas del Núcleo Viral/metabolismo , Virión/fisiología , Liberación del VirusRESUMEN
Micro/nano-topography (MNT) is an important variable affecting osseointegration of bone biomaterials, but the underlying mechanisms are not fully understood. We probed the role of a AMOT130/YAP pathway in osteoblastic differentiation of bone marrow mesenchymal stems cultured on titanium (Ti) carrying MNTs. Ti surfaces with two well-defined MNTs (TiO2 nanotubes of different diameters and wall thicknesses) were prepared by anodization. Rat BMSCs were cultured on flat Ti and Ti surfaces carrying MNTs, and cell behaviors (i.e., morphology, F-actin development, osteoblastic differentiation, YAP localization) were studied. Ti surfaces carrying MNTs increased F-actin formation, osteoblastic gene expression, and protein AMOT130 production in BMSCs (all vs. flat Ti), and the surface carrying larger nantubes was more effective, confirming osteoblastic differentiation induced by MNTs. Elevation of the AMOT130 level (by inhibiting its degradation) increased the osteoblastic gene expression, F-actin formation, and nuclear localization of YAP. These show that, AMOT130/YAP is an important pathway mediating the translation of MNT signals to BMSC osteoblastic commitment, likely via the cascade: AMOT130 promotion of F-actin formation, increased YAP nuclear import, and activation of osteoblastic gene expression.
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Proteínas Reguladoras de la Apoptosis/genética , Péptidos y Proteínas de Señalización Intercelular/genética , Mecanotransducción Celular/genética , Proteínas de la Membrana/genética , Células Madre Mesenquimatosas/efectos de los fármacos , Osteogénesis/efectos de los fármacos , Titanio/farmacología , Actinas/genética , Actinas/metabolismo , Fosfatasa Alcalina/genética , Fosfatasa Alcalina/metabolismo , Angiomotinas , Animales , Proteínas Reguladoras de la Apoptosis/metabolismo , Técnicas de Cultivo de Célula , Diferenciación Celular/efectos de los fármacos , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Regulación de la Expresión Génica , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteínas de la Membrana/metabolismo , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Nanotubos/química , Osteoblastos/citología , Osteoblastos/efectos de los fármacos , Osteoblastos/metabolismo , Osteocalcina/genética , Osteocalcina/metabolismo , Osteogénesis/genética , Osteopontina/genética , Osteopontina/metabolismo , Cultivo Primario de Células , Ratas , Ratas Sprague-Dawley , Propiedades de Superficie , Titanio/química , Proteínas Señalizadoras YAPRESUMEN
Ammonium (NH4+) toxicity inhibits shoot growth in Arabidopsis, but the underlying mechanisms remain poorly characterized. Here, we show that a novel Arabidopsis mutant, ammonium tolerance 1 (amot1), exhibits enhanced shoot growth tolerance to NH4+. Molecular cloning revealed that amot1 is a new allele of EIN3, a key regulator of ethylene responses. The amot1 mutant and the allelic ein3-1 mutants show greater NH4+ tolerance than the wild type. Moreover, transgenic plants overexpressing EIN3 (EIN3ox) are more sensitive to NH4+ toxicity The ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) increases shoot sensitivity to NH4+, whereas the ethylene perception inhibitor Ag+ decreases sensitivity. NH4+ induces ACC and ethylene accumulation. Furthermore, ethylene-insensitive mutants such as etr1-3 and ein3eil1 display enhanced NH4+ tolerance. In contrast, the ethylene overproduction mutant eto1-1 exhibits decreased ammonium tolerance. AMOT1/EIN3 positively regulates shoot ROS accumulation, leading to oxidative stress under NH4+ stress, a trait that may be related to increased expression of peroxidase-encoding genes. These findings demonstrate the role of AMOT1/EIN3 in NH4+ tolerance and confirm the strong link between NH4+ toxicity symptoms and the accumulation of hydrogen peroxide.
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Compuestos de Amonio/toxicidad , Proteínas de Arabidopsis/genética , Arabidopsis/genética , Proteínas de Unión al ADN/genética , Factores de Transcripción/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica de las Plantas , Mutación , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismo , Factores de Transcripción/metabolismoRESUMEN
The Hippo pathway controls cell proliferation, differentiation, and survival by regulating the Yes-associated protein (YAP) transcriptional coactivator in response to various stimuli, including the mechanical environment. The major YAP regulators are the LATS1/2 kinases, which phosphorylate and inhibit YAP. LATS1/2 are activated by phosphorylation on a hydrophobic motif (HM) outside of the kinase domain by MST1/2 and other kinases. Phosphorylation of the HM motif then triggers autophosphorylation of the kinase in the activation loop to fully activate the kinase, a process facilitated by MOB1. The angiomotin family of proteins (AMOT, AMOTL1, and AMOTL2) bind LATS1/2 and promote its kinase activity and YAP phosphorylation through an unknown mechanism. Here we show that angiomotins increase Hippo signaling through multiple mechanisms. We found that, by binding LATS1/2, SAV1, and YAP, angiomotins function as a scaffold that connects LATS1/2 to both its activator SAV1-MST1 and its target YAP. Deletion of all three angiomotins reduced the association of LATS1 with SAV1-MST1 and decreased MST1/2-mediated LATS1/2-HM phosphorylation. Angiomotin deletion also reduced LATS1/2's ability to associate with and phosphorylate YAP. In addition, we found that angiomotins have an unexpected function along with MOB1 to promote autophosphorylation of LATS1/2 on the activation loop motif independent of HM phosphorylation. These results indicate that angiomotins enhance Hippo signaling by stimulating LATS1/2 autophosphorylation and by connecting LATS1/2 with both its activator SAV1-MST1/2 and its substrate YAP.
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Proteínas Portadoras/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Secuencias de Aminoácidos , Angiomotinas , Proteínas Portadoras/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Vía de Señalización Hippo , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Proteínas de la Membrana/genética , Proteínas de Microfilamentos , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosforilación , Unión Proteica , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/genética , Transducción de Señal , Factores de Transcripción , Proteínas Supresoras de Tumor/química , Proteínas Supresoras de Tumor/genética , Proteínas Señalizadoras YAPRESUMEN
Aberrant vascular smooth muscle cell (VSMC) migration has been implicated in a variety of vascular disorders, while the signal pathways governing this process remain unclear. Here, we investigated whether miRNAs, which are strong post-transcriptional regulators of gene expression, could alter VSMC migration. We detected the expression of miR-4463 in the plasma of patients with atherosclerosis and in human aortic smooth muscle cells under hypoxia-ischemia condition, and investigated the migration effect and its downstream pathways. The results have shown that whether in clinical AS patients or hypoxic cells, the expression of miR-4463 was lower than that of normal group, then the number of migrating cells in the miR-4463 mimic intervention group was significantly decreased compared with the normal group and miR-4463 inhibitor instead. Furthermore, the expression of angiomotin (AMOT) in gastrocnemius muscle and femoral artery of patients was significantly higher than that of the control group. The protein level of AMOT in miR-4463 mimic intervention group was significantly decreased, and its level was reversed by inhibiting miR-4463. In summary, these results indicate that miR-4463 is a novel modulator of VSMC migration by targetting AMOT expression. Regulating miR-4463 or its specific downstream target genes in VSMCs may represent an attractive approach for the treatment of vascular diseases.
Asunto(s)
Aorta/patología , Arteriosclerosis Obliterante/patología , Péptidos y Proteínas de Señalización Intercelular/genética , Proteínas de la Membrana/genética , MicroARNs/metabolismo , Miocitos del Músculo Liso/citología , Anciano , Anciano de 80 o más Años , Angiomotinas , Antígenos CD/genética , Antígenos CD/metabolismo , Aorta/citología , Arteriosclerosis Obliterante/genética , Cadherinas/genética , Cadherinas/metabolismo , Estudios de Casos y Controles , Hipoxia de la Célula/genética , Movimiento Celular/genética , Células Cultivadas , Femenino , Regulación de la Expresión Génica , Humanos , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Masculino , Proteínas de la Membrana/metabolismo , MicroARNs/antagonistas & inhibidores , MicroARNs/genética , Proteínas de Microfilamentos , Persona de Mediana Edad , Músculo Liso Vascular/citología , Miocitos del Músculo Liso/fisiología , Regulación hacia ArribaRESUMEN
Members in Motin family, or Angiomotins (AMOTs), are adaptor proteins that localize in the membranous, cytoplasmic or nuclear fraction in a cell context-dependent manner. They control the bioprocesses such as migration, tight junction formation, cell polarity, and angiogenesis. Emerging evidences have demonstrated that AMOTs participate in cancer initiation and progression. Many of the previous studies have focused on the involvement of AMOTs in Hippo-YAP1 pathway. However, it has been controversial for years that AMOTs serve as either positive or negative growth regulators in different cancer types because of the various cellular origins. The molecular mechanisms of these opposite roles of AMOTs remain elusive. This review comprehensively summarized how AMOTs function physiologically and how their dysregulation promotes or inhibits tumorigenesis. Better understanding the functional roles of AMOTs in cancers may lead to an improvement of clinical interventions as well as development of novel therapeutic strategies for cancer patients.
Asunto(s)
Carcinogénesis/metabolismo , Carcinogénesis/patología , Proteínas de la Membrana/metabolismo , Animales , Desarrollo Embrionario , Humanos , Proteínas de la Membrana/química , Modelos Biológicos , Neovascularización Fisiológica , Transducción de SeñalRESUMEN
In placental mammalian development, the first cell differentiation produces two distinct lineages that emerge according to their position within the embryo: the trophectoderm (TE, placenta precursor) differentiates in the surface, while the inner cell mass (ICM, fetal body precursor) forms inside. Here, we discuss how such position-dependent lineage specifications are regulated by the RHOA subfamily of small GTPases and RHO-associated coiled-coil kinases (ROCK). Recent studies in mouse show that activities of RHO/ROCK are required to promote TE differentiation and to concomitantly suppress ICM formation. RHO/ROCK operate through the HIPPO signaling pathway, whose cell position-specific modulation is central to establishing unique gene expression profiles that confer cell fate. In particular, activities of RHO/ROCK are essential in outside cells to promote nuclear localization of transcriptional co-activators YAP/TAZ, the downstream effectors of HIPPO signaling. Nuclear localization of YAP/TAZ depends on the formation of apicobasal polarity in outside cells, which requires activities of RHO/ROCK. We propose models of how RHO/ROCK regulate lineage specification and lay out challenges for future investigations to deepen our understanding of the roles of RHO/ROCK in preimplantation development. Finally, as RHO/ROCK may be inhibited by certain pharmacological agents, we discuss their potential impact on human preimplantation development in relation to fertility preservation in women.