RESUMEN

Structural plasticity of dendritic spines in the nucleus accumbens (NAc) is crucial for learning from aversive experiences. Activation of NMDA receptors (NMDARs) stimulates Ca2+-dependent signaling that leads to changes in the actin cytoskeleton, mediated by the Rho family of GTPases, resulting in postsynaptic remodeling essential for learning. We investigated how phosphorylation events downstream of NMDAR activation drive the changes in synaptic morphology that underlie aversive learning. Large-scale phosphoproteomic analyses of protein kinase targets in mouse striatal/accumbal slices revealed that NMDAR activation resulted in the phosphorylation of 194 proteins, including RhoA regulators such as ARHGEF2 and ARHGAP21. Phosphorylation of ARHGEF2 by the Ca2+-dependent protein kinase CaMKII enhanced its RhoGEF activity, thereby activating RhoA and its downstream effector Rho-associated kinase (ROCK/Rho-kinase). Further phosphoproteomic analysis identified 221 ROCK targets, including the postsynaptic scaffolding protein SHANK3, which is crucial for its interaction with NMDARs and other postsynaptic scaffolding proteins. ROCK-mediated phosphorylation of SHANK3 in the NAc was essential for spine growth and aversive learning. These findings demonstrate that NMDAR activation initiates a phosphorylation cascade crucial for learning and memory.

Asunto(s)

Proteínas del Tejido Nervioso , Plasticidad Neuronal , Proteoma , Receptores de N-Metil-D-Aspartato , Animales , Receptores de N-Metil-D-Aspartato/metabolismo , Plasticidad Neuronal/fisiología , Ratones , Fosforilación , Proteoma/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas del Tejido Nervioso/genética , Masculino , Transducción de Señal , Quinasas Asociadas a rho/metabolismo , Quinasas Asociadas a rho/genética , Ratones Endogámicos C57BL , Fosfoproteínas/metabolismo , Fosfoproteínas/genética , Aprendizaje/fisiología , Reacción de Prevención/fisiología , Factores de Intercambio de Guanina Nucleótido Rho/metabolismo , Factores de Intercambio de Guanina Nucleótido Rho/genética , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Sinapsis/metabolismo , Proteína de Unión al GTP rhoA/metabolismo , Espinas Dendríticas/metabolismo

RESUMEN

A significant number of the genetic alterations observed in cancer patients lie within nonprotein-coding segments of the genome, including regions coding for long noncoding RNAs (lncRNAs). LncRNAs display aberrant expression in breast cancer (BrCa), but the functional implications of this altered expression remain to be elucidated. By performing transcriptome screen in a triple negative BrCa (TNBC) isogenic 2D and 3D spheroid model, we observed aberrant expression of >1000 lncRNAs during BrCa progression. The chromatin-associated lncRNA MANCR shows elevated expression in metastatic TNBC. MANCR is upregulated in response to cellular stress and modulates DNA repair and cell proliferation. MANCR promotes metastasis as MANCR-depleted cells show reduced cell migration, invasion, and wound healing in vitro, and reduced metastatic lung colonization in xenograft experiments in vivo. Transcriptome analyses reveal that MANCR modulates expression and pre-mRNA splicing of genes, controlling DNA repair and checkpoint response. MANCR promotes the transcription of NET1A, a Rho-GEF that regulates DNA damage checkpoint and metastatic processes in cis, by differential promoter usage. Experiments suggest that MANCR regulates the expression of cancer-associated genes by modulating the association of various transcription factors and RNA-binding proteins. Our results identified the metastasis-promoting activities of MANCR in TNBC by cis-regulation of gene expression.

Asunto(s)

Regulación Neoplásica de la Expresión Génica , Metástasis de la Neoplasia , ARN Largo no Codificante , Neoplasias de la Mama Triple Negativas , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Humanos , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/patología , Neoplasias de la Mama Triple Negativas/metabolismo , Línea Celular Tumoral , Animales , Femenino , Ratones , Ciclo Celular/genética , Proliferación Celular/genética , Factores de Intercambio de Guanina Nucleótido Rho/genética , Factores de Intercambio de Guanina Nucleótido Rho/metabolismo , Movimiento Celular/genética , Reparación del ADN/genética

RESUMEN

Glucocorticoid use may cause elevated intraocular pressure, leading to the development of glucocorticoid-induced glaucoma (GIG). However, the mechanism of GIG development remains incompletely understood. In this study, we subjected primary human trabecular meshwork cells (TMCs) and mice to dexamethasone treatment to mimic glucocorticoid exposure. The myofibroblast transdifferentiation of TMCs was observed in cellular and mouse models, as well as in human trabecular mesh specimens. This was demonstrated by the cytoskeletal reorganization, alterations in cell morphology, heightened transdifferentiation markers, increased extracellular matrix deposition, and cellular dysfunction. Knockdown of Rho guanine nucleotide exchange factor 26 (ARHGEF26) expression ameliorated dexamethasone-induced changes in cell morphology and upregulation of myofibroblast markers, reversed dysfunction and extracellular matrix deposition in TMCs, and prevented the development of dexamethasone-induced intraocular hypertension. And, this process may be related to the TGF-ß pathway. In conclusion, glucocorticoids induced the myofibroblast transdifferentiation in TMCs, which played a crucial role in the pathogenesis of GIG. Inhibition of ARHGEF26 expression protected TMCs by reversing myofibroblast transdifferentiation. This study demonstrated the potential of reversing the myofibroblast transdifferentiation of TMCs as a new target for treating GIG.

Asunto(s)

Transdiferenciación Celular , Dexametasona , Glaucoma , Miofibroblastos , Factores de Intercambio de Guanina Nucleótido Rho , Malla Trabecular , Dexametasona/farmacología , Malla Trabecular/efectos de los fármacos , Malla Trabecular/metabolismo , Malla Trabecular/citología , Transdiferenciación Celular/efectos de los fármacos , Animales , Humanos , Miofibroblastos/efectos de los fármacos , Miofibroblastos/metabolismo , Miofibroblastos/citología , Ratones , Factores de Intercambio de Guanina Nucleótido Rho/metabolismo , Factores de Intercambio de Guanina Nucleótido Rho/genética , Glaucoma/patología , Glaucoma/metabolismo , Células Cultivadas , Glucocorticoides/farmacología , Ratones Endogámicos C57BL , Masculino

RESUMEN

Background and Objective: GIT1 (G-protein-coupled receptor kinase interacting protein-1) has been found to be highly related with cancer cell invasion and metastasis in many cancer types. ß-Pix (p21-activated kinase-interacting exchange factor) is one of the proteins that interact with GIT1. Targeting GIT1/ß-Pix complex might be a potential therapeutic strategy for interfering cancer metastasis. However, at present, no well-recognized small-molecule inhibitor targeting GIT1/ß-Pix is available. Thus, we aim to discover novel GIT1/ß-Pix inhibitors with simple scaffold, high activity and low toxicity to develop new therapeutic strategies to restrain cancer metastasis. Methods: GIT1/ß-Pix inhibitors were identified from ChemBridge by virtual screening. Briefly, the modeling of GIT1 was performed and the establishment of GIT1/ß-Pix binding pocket enabled the virtual screening to identify the inhibitor. In addition, direct binding of the candidate molecules to GIT1 was detected by biolayer interferometry (BLI) to discover the hit compound. Furthermore, the inhibitory effect on invasion of stomach and colon cancer cells in vitro was carried out by the transwell assay and detection of epithelial-mesenchymal transition (EMT)-related proteins. Finally, the binding mode of hit compound to GIT1 was estimated by molecular dynamics simulation to analyze the key amino residues to guide further optimization. Results: We selected the top 50 compounds from the ChemBridge library by virtual screening. Then, by skeleton similarity analysis nine compounds were selected for further study. Furthermore, the direct interaction of nine compounds to GIT1 was detected by BLI to obtain the best affinitive compound. Finally, 17302836 was successfully identified (KD = 84.1±2.0 µM). In vitro tests on 17302836 showed significant anti-invasion effect on gastric cancer and colorectal cancer. Conclusion: We discovered a new GIT1/ß-Pix inhibitor (17302836) against gastrointestinal cancer invasion and metastasis. This study provides a promising candidate for developing new GIT1/ß-Pix inhibitors for tumor treatment.

Asunto(s)

Antineoplásicos , Proteínas de Ciclo Celular , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas de Ciclo Celular/metabolismo , Antineoplásicos/farmacología , Antineoplásicos/química , Antineoplásicos/síntesis química , Neoplasias Gastrointestinales/tratamiento farmacológico , Neoplasias Gastrointestinales/patología , Neoplasias Gastrointestinales/metabolismo , Bibliotecas de Moléculas Pequeñas/farmacología , Bibliotecas de Moléculas Pequeñas/química , Relación Estructura-Actividad , Descubrimiento de Drogas , Estructura Molecular , Proliferación Celular/efectos de los fármacos , Factores de Intercambio de Guanina Nucleótido Rho/antagonistas & inhibidores , Factores de Intercambio de Guanina Nucleótido Rho/metabolismo , Relación Dosis-Respuesta a Droga , Invasividad Neoplásica , Proteínas Adaptadoras Transductoras de Señales/antagonistas & inhibidores , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Evaluación Preclínica de Medicamentos , Movimiento Celular/efectos de los fármacos

RESUMEN

The giant cytoskeletal protein obscurin contains multiple cell signaling domains that influence cell migration. Here, we follow each of these pathways, examine how these pathways modulate epithelial cell migration, and discuss the cross-talk between these pathways. Specifically, obscurin uses its PH domain to inhibit phosphoinositide-3-kinase (PI3K)-dependent migration and its RhoGEF domain to activate RhoA and slow cell migration. While obscurin's effect on the PI3K pathway agrees with the literature, obscurin's effect on the RhoA pathway runs counter to most other RhoA effectors, whose activation tends to lead to enhanced motility. Obscurin also phosphorylates cadherins, and this may also influence cell motility. When taken together, obscurin's ability to modulate three independent cell migration pathways is likely why obscurin knockout cells experience enhanced epithelial to mesenchymal transition, and why obscurin is a frequently mutated gene in several types of cancer.

Asunto(s)

Movimiento Celular , Células Epiteliales , Proteínas Serina-Treonina Quinasas , Factores de Intercambio de Guanina Nucleótido Rho , Transducción de Señal , Proteína de Unión al GTP rhoA , Humanos , Células Epiteliales/metabolismo , Factores de Intercambio de Guanina Nucleótido Rho/metabolismo , Proteína de Unión al GTP rhoA/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Transición Epitelial-Mesenquimal , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Quinasas Asociadas a rho/metabolismo , Cadherinas/metabolismo

RESUMEN

RhoA and its effectors, the transcriptional coactivators myocardin-related transcription factor (MRTF) and serum response factor (SRF), control epithelial phenotype and are indispensable for profibrotic epithelial reprogramming during fibrogenesis. Context-dependent control of RhoA and fibrosis-associated changes in its regulators, however, remain incompletely characterized. We previously identified the guanine nucleotide exchange factor GEF-H1 as a central mediator of RhoA activation in renal tubular cells exposed to inflammatory or fibrotic stimuli. Here we found that GEF-H1 expression and phosphorylation were strongly elevated in two animal models of fibrosis. In the Unilateral Ureteral Obstruction mouse kidney fibrosis model, GEF-H1 was upregulated predominantly in the tubular compartment. GEF-H1 was also elevated and phosphorylated in a rat pulmonary artery banding (PAB) model of right ventricular fibrosis. Prolonged stimulation of LLC-PK1 tubular cells with tumor necrosis factor (TNF)-α or transforming growth factor (TGF)-ß1 increased GEF-H1 expression and activated a luciferase-coupled GEF-H1 promoter. Knockdown and overexpression studies revealed that these effects were mediated by RhoA, cytoskeleton remodeling, and MRTF, indicative of a positive feedback cycle. Indeed, silencing endogenous GEF-H1 attenuated activation of the GEF-H1 promoter. Of importance, inhibition of MRTF using CCG-1423 prevented GEF-H1 upregulation in both animal models. MRTF-dependent increase in GEF-H1 was prevented by inhibition of the transcription factor Sp1, and mutating putative Sp1 binding sites in the GEF-H1 promoter eliminated its MRTF-dependent activation. As the GEF-H1/RhoA axis is key for fibrogenesis, this novel MRTF/Sp1-dependent regulation of GEF-H1 abundance represents a potential target for reducing renal and cardiac fibrosis.NEW & NOTEWORTHY We show that expression of the RhoA regulator GEF-H1 is upregulated in tubular cells exposed to fibrogenic cytokines and in animal models of kidney and heart fibrosis. We identify a pathway wherein GEF-H1/RhoA-dependent MRTF activation through its noncanonical partner Sp1 upregulates GEF-H1. Our data reveal the existence of a positive feedback cycle that enhances Rho signaling through control of both GEF-H1 activation and expression. This feedback loop may play an important role in organ fibrosis.

Asunto(s)

Fibrosis , Factores de Intercambio de Guanina Nucleótido Rho , Factor de Transcripción Sp1 , Transactivadores , Proteína de Unión al GTP rhoA , Animales , Factor de Transcripción Sp1/metabolismo , Factor de Transcripción Sp1/genética , Proteína de Unión al GTP rhoA/metabolismo , Proteína de Unión al GTP rhoA/genética , Factores de Intercambio de Guanina Nucleótido Rho/metabolismo , Factores de Intercambio de Guanina Nucleótido Rho/genética , Transactivadores/metabolismo , Transactivadores/genética , Ratones , Ratas , Retroalimentación Fisiológica , Masculino , Ratones Endogámicos C57BL , Humanos , Transducción de Señal , Porcinos , Fosforilación , Modelos Animales de Enfermedad , Obstrucción Ureteral/metabolismo , Obstrucción Ureteral/patología , Obstrucción Ureteral/genética , Ratas Sprague-Dawley , Línea Celular , Factores de Transcripción

RESUMEN

Transmembrane signaling is a critical process by which changes in the extracellular environment are relayed to intracellular systems that induce changes in homeostasis. One family of intracellular systems are the guanine nucleotide exchange factors (GEFs), which catalyze the exchange of GTP for GDP bound to inactive guanine nucleotide binding proteins (G proteins). The resulting active G proteins then interact with downstream targets that control cell proliferation, growth, shape, migration, adhesion, and transcription. Dysregulation of any of these processes is a hallmark of cancer. The Dbl family of GEFs activates Rho family G proteins, which, in turn, alter the actin cytoskeleton and promote gene transcription. Although they have a common catalytic mechanism exercised by their highly conserved Dbl homology (DH) domains, Dbl GEFs are regulated in diverse ways, often involving the release of autoinhibition imposed by accessory domains. Among these domains, the pleckstrin homology (PH) domain is the most commonly observed and found immediately C-terminal to the DH domain. The domain has been associated with both positive and negative regulation. Recently, some atomic structures of Dbl GEFs have been determined that reemphasize the complex and central role that the PH domain can play in orchestrating regulation of the DH domain. Here, we discuss these newer structures, put them into context by cataloging the various ways that PH domains are known to contribute to signaling across the Dbl family, and discuss how the PH domain might be exploited to achieve selective inhibition of Dbl family RhoGEFs by small-molecule therapeutics. SIGNIFICANCE STATEMENT: Dysregulation via overexpression or mutation of Dbl family Rho guanine nucleotide exchange factors (GEFs) contributes to cancer and neurodegeneration. Targeting the Dbl homology catalytic domain by small-molecule therapeutics has been challenging due to its high conservation and the lack of a discrete binding pocket. By evaluating some new autoinhibitory mechanisms in the Dbl family, we demonstrate the great diversity of roles played by the regulatory domains, in particular the PH domain, and how this holds tremendous potential for the development of selective therapeutics that modulate GEF activity.

Asunto(s)

Carcinogénesis , Factores de Intercambio de Guanina Nucleótido , Factores de Intercambio de Guanina Nucleótido Rho , Humanos , Animales , Factores de Intercambio de Guanina Nucleótido/metabolismo , Factores de Intercambio de Guanina Nucleótido/química , Factores de Intercambio de Guanina Nucleótido/genética , Carcinogénesis/metabolismo , Carcinogénesis/genética , Factores de Intercambio de Guanina Nucleótido Rho/metabolismo , Factores de Intercambio de Guanina Nucleótido Rho/genética , Neoplasias/metabolismo , Neoplasias/genética , Neoplasias/patología , Transducción de Señal

RESUMEN

Extracellular vesicles are essential for intercellular communication and are involved in tumor progression. Inhibiting the direct release of extracellular vesicles seems to be an effective strategy in inhibiting tumor progression, but lacks of investigation. Here, we report a natural flavonoid compound, apigenin, could significantly inhibit the growth of hepatocellular carcinoma by preventing microvesicle secretion. Mechanistically, apigenin primarily targets the guanine nucleotide exchange factor ARHGEF1, inhibiting the activity of small G protein Cdc42, which is essential in regulating the release of microvesicles from tumor cells. In turn, this inhibits tumor angiogenesis related to VEGF90K transported on microvesicles, ultimately impeding tumor progression. Collectively, these findings highlight the therapeutic potential of apigenin and shed light on its anticancer mechanisms through inhibiting microvesicle biogenesis, providing a solid foundation for the refinement and practical application of apigenin.

Asunto(s)

Apigenina , Carcinoma Hepatocelular , Micropartículas Derivadas de Células , Neoplasias Hepáticas , Neovascularización Patológica , Factores de Intercambio de Guanina Nucleótido Rho , Humanos , Neovascularización Patológica/tratamiento farmacológico , Neovascularización Patológica/metabolismo , Animales , Apigenina/farmacología , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/metabolismo , Factores de Intercambio de Guanina Nucleótido Rho/metabolismo , Factores de Intercambio de Guanina Nucleótido Rho/genética , Micropartículas Derivadas de Células/metabolismo , Micropartículas Derivadas de Células/efectos de los fármacos , Carcinoma Hepatocelular/tratamiento farmacológico , Carcinoma Hepatocelular/patología , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/irrigación sanguínea , Ratones , Línea Celular Tumoral , Proteína de Unión al GTP cdc42/metabolismo , Proliferación Celular/efectos de los fármacos , Ensayos Antitumor por Modelo de Xenoinjerto , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Hep G2 , Ratones Desnudos , Angiogénesis

RESUMEN

BACKGROUND: Rostral ventrolateral medulla (RVLM) neuron hyperactivity raises sympathetic outflow, causing hypertension. MicroRNAs (miRNAs) contribute to diverse biological processes, but their influence on RVLM neuronal excitability and blood pressure (BP) remains widely unexplored. METHODS AND RESULTS: The RVLM miRNA profiles in spontaneously hypertensive rats were unveiled using RNA sequencing. Potential effects of these miRNAs in reducing neuronal excitability and BP and underlying mechanisms were investigated through various experiments. Six hundred thirty-seven miRNAs were identified, and reduced levels of miR-193b-3p and miR-346 were observed in the RVLM of spontaneously hypertensive rats. Increased miR-193b-3p and miR-346 expression in RVLM lowered neuronal excitability, sympathetic outflow, and BP in spontaneously hypertensive rats. In contrast, suppressing miR-193b-3p and miR-346 expression in RVLM increased neuronal excitability, sympathetic outflow, and BP in Wistar Kyoto and Sprague-Dawley rats. Cdc42 guanine nucleotide exchange factor (Arhgef9) was recognized as a target of miR-193b-3p. Overexpressing miR-193b-3p caused an evident decrease in Arhgef9 expression, resulting in the inhibition of neuronal apoptosis. By contrast, its downregulation produced the opposite effects. Importantly, the decrease in neuronal excitability, sympathetic outflow, and BP observed in spontaneously hypertensive rats due to miR-193b-3p overexpression was greatly counteracted by Arhgef9 upregulation. CONCLUSIONS: miR-193b-3p and miR-346 are newly identified factors in RVLM that hinder hypertension progression, and the miR-193b-3p/Arhgef9/apoptosis pathway presents a potential mechanism, highlighting the potential of targeting miRNAs for hypertension prevention.

Asunto(s)

Presión Sanguínea , Hipertensión , Bulbo Raquídeo , MicroARNs , Animales , Masculino , Ratas , Apoptosis , Presión Sanguínea/efectos de los fármacos , Presión Sanguínea/genética , Modelos Animales de Enfermedad , Hipertensión/fisiopatología , Hipertensión/genética , Hipertensión/metabolismo , Bulbo Raquídeo/metabolismo , Bulbo Raquídeo/fisiopatología , Bulbo Raquídeo/efectos de los fármacos , MicroARNs/genética , MicroARNs/metabolismo , Neuronas/metabolismo , Ratas Endogámicas SHR , Ratas Endogámicas WKY , Ratas Sprague-Dawley , Factores de Intercambio de Guanina Nucleótido Rho/genética , Factores de Intercambio de Guanina Nucleótido Rho/metabolismo , Sistema Nervioso Simpático/fisiopatología , Sistema Nervioso Simpático/metabolismo

RESUMEN

Synapses containing γ-aminobutyric acid (GABA) constitute the primary centers for inhibitory neurotransmission in our nervous system. It is unclear how these synaptic structures form and align their postsynaptic machineries with presynaptic terminals. Here, we monitored the cellular distribution of several GABAergic postsynaptic proteins in a purely glutamatergic neuronal culture derived from human stem cells, which virtually lacks any vesicular GABA release. We found that several GABAA receptor (GABAAR) subunits, postsynaptic scaffolds, and major cell-adhesion molecules can reliably coaggregate and colocalize at even GABA-deficient subsynaptic domains, but remain physically segregated from glutamatergic counterparts. Genetic deletions of both Gephyrin and a Gephyrin-associated guanosine di- or triphosphate (GDP/GTP) exchange factor Collybistin severely disrupted the coassembly of these postsynaptic compositions and their proper apposition with presynaptic inputs. Gephyrin-GABAAR clusters, developed in the absence of GABA transmission, could be subsequently activated and even potentiated by delayed supply of vesicular GABA. Thus, molecular organization of GABAergic postsynapses can initiate via a GABA-independent but Gephyrin-dependent intrinsic mechanism.

Asunto(s)

Proteínas Portadoras , Proteínas de la Membrana , Terminales Presinápticos , Receptores de GABA-A , Sinapsis , Ácido gamma-Aminobutírico , Humanos , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Ácido gamma-Aminobutírico/metabolismo , Receptores de GABA-A/metabolismo , Receptores de GABA-A/genética , Proteínas Portadoras/metabolismo , Proteínas Portadoras/genética , Terminales Presinápticos/metabolismo , Sinapsis/metabolismo , Transmisión Sináptica/fisiología , Factores de Intercambio de Guanina Nucleótido Rho/metabolismo , Factores de Intercambio de Guanina Nucleótido Rho/genética

RESUMEN

Benzene, a widely used industrial chemical, has been clarified to cause hematotoxicity. Our previous study suggested that miR-451a may play a role in benzene-induced impairment of erythroid differentiation. However, the mechanism underlying remains unclear. In this study, we explored the role of miR-451a and its underlying mechanisms in hydroquinone (HQ)-induced suppression of erythroid differentiation in K562 cells. 0, 1.0, 2.5, 5.0, 10.0, and 50 µM HQ treatment of K562 cells resulted in a dose-dependent inhibition of erythroid differentiation, as well as the expression of miR-451a. Bioinformatics analysis was conducted to predict potential target genes of miR-451a and dual-luciferase reporter assays confirmed that miR-451a can directly bind to the 3'-UTR regions of BATF, SETD5, and ARHGEF3 mRNAs. We further demonstrated that over-expression or down-regulation of miR-451a altered the expression of BATF, SETD5, and ARHGEF3, and also modified erythroid differentiation. In addition, BATF, SETD5, and ARHGEF3 were verified to play a role in HQ-induced inhibition of erythroid differentiation in this study. Knockdown of SETD5 and ARHGEF3 reversed HQ-induced suppression of erythroid differentiation while knockdown of BATF had the opposite effect. On the other hand, we also identified c-Jun as a potential transcriptional regulator of miR-451a. Forced expression of c-Jun increased miR-451a expression and reversed the inhibition of erythroid differentiation induced by HQ, whereas knockdown of c-Jun had the opposite effect. And the binding site of c-Jun and miR-451a was verified by dual-luciferase reporter assay. Collectively, our findings indicate that miR-451a and its downstream targets BATF, SETD5, and ARHGEF3 are involved in HQ-induced erythroid differentiation disorder, and c-Jun regulates miR-451a as a transcriptional regulator in this process.

Asunto(s)

Factores de Transcripción con Cremalleras de Leucina de Carácter Básico , Diferenciación Celular , MicroARNs , Factores de Intercambio de Guanina Nucleótido Rho , Humanos , MicroARNs/genética , MicroARNs/metabolismo , Diferenciación Celular/efectos de los fármacos , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Células K562 , Factores de Intercambio de Guanina Nucleótido Rho/genética , Factores de Intercambio de Guanina Nucleótido Rho/metabolismo , Células Eritroides/efectos de los fármacos , Células Eritroides/metabolismo , Proteínas Proto-Oncogénicas c-jun/metabolismo , Proteínas Proto-Oncogénicas c-jun/genética , Metiltransferasas/genética , Metiltransferasas/metabolismo

RESUMEN

Microtubules regulate cell polarity and migration via local activation of focal adhesion turnover, but the mechanism of this process is insufficiently understood. Molecular complexes containing KANK family proteins connect microtubules with talin, the major component of focal adhesions. Here, local optogenetic activation of KANK1-mediated microtubule/talin linkage promoted microtubule targeting to an individual focal adhesion and subsequent withdrawal, resulting in focal adhesion centripetal sliding and rapid disassembly. This sliding is preceded by a local increase of traction force due to accumulation of myosin-II and actin in the proximity of the focal adhesion. Knockdown of the Rho activator GEF-H1 prevented development of traction force and abolished sliding and disassembly of focal adhesions upon KANK1 activation. Other players participating in microtubule-driven, KANK-dependent focal adhesion disassembly include kinases ROCK, PAK, and FAK, as well as microtubules/focal adhesion-associated proteins kinesin-1, APC, and αTAT. Based on these data, we develop a mathematical model for a microtubule-driven focal adhesion disruption involving local GEF-H1/RhoA/ROCK-dependent activation of contractility, which is consistent with experimental data.

Asunto(s)

Adhesiones Focales , Cinesinas , Microtúbulos , Factores de Intercambio de Guanina Nucleótido Rho , Adhesiones Focales/metabolismo , Microtúbulos/metabolismo , Humanos , Factores de Intercambio de Guanina Nucleótido Rho/metabolismo , Factores de Intercambio de Guanina Nucleótido Rho/genética , Cinesinas/metabolismo , Cinesinas/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas del Citoesqueleto/metabolismo , Proteínas del Citoesqueleto/genética , Miosina Tipo II/metabolismo , Talina/metabolismo , Talina/genética , Animales

RESUMEN

Actin cortex patterning and dynamics are critical for cell shape changes. These dynamics undergo transitions during development, often accompanying changes in collective cell behavior. Although mechanisms have been established for individual cells' dynamic behaviors, the mechanisms and specific molecules that result in developmental transitions in vivo are still poorly understood. Here, we took advantage of two developmental systems in Drosophila melanogaster to identify conditions that altered cortical patterning and dynamics. We identified a Rho guanine nucleotide exchange factor (RhoGEF) and Rho GTPase activating protein (RhoGAP) pair required for actomyosin waves in egg chambers. Specifically, depletion of the RhoGEF, Ect2, or the RhoGAP, RhoGAP15B, disrupted actomyosin wave induction, and both proteins relocalized from the nucleus to the cortex preceding wave formation. Furthermore, we found that overexpression of a different RhoGEF and RhoGAP pair, RhoGEF2 and Cumberland GAP (C-GAP), resulted in actomyosin waves in the early embryo, during which RhoA activation precedes actomyosin assembly by ∼4 s. We found that C-GAP was recruited to actomyosin waves, and disrupting F-actin polymerization altered the spatial organization of both RhoA signaling and the cytoskeleton in waves. In addition, disrupting F-actin dynamics increased wave period and width, consistent with a possible role for F-actin in promoting delayed negative feedback. Overall, we showed a mechanism involved in inducing actomyosin waves that is essential for oocyte development and is general to other cell types, such as epithelial and syncytial cells.

Asunto(s)

Proteínas de Drosophila , Drosophila melanogaster , Proteínas Activadoras de GTPasa , Animales , Proteínas Activadoras de GTPasa/metabolismo , Proteínas Activadoras de GTPasa/genética , Drosophila melanogaster/metabolismo , Drosophila melanogaster/crecimiento & desarrollo , Drosophila melanogaster/genética , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Actomiosina/metabolismo , Factores de Intercambio de Guanina Nucleótido Rho/metabolismo , Factores de Intercambio de Guanina Nucleótido Rho/genética , Femenino , Factores de Intercambio de Guanina Nucleótido/metabolismo , Factores de Intercambio de Guanina Nucleótido/genética , Embrión no Mamífero/metabolismo , Tipificación del Cuerpo

RESUMEN

How cancer cells determine their shape in response to three-dimensional (3D) geometric and mechanical cues is unclear. We develop an approach to quantify the 3D cell shape of over 60,000 melanoma cells in collagen hydrogels using high-throughput stage-scanning oblique plane microscopy (ssOPM). We identify stereotypic and environmentally dependent changes in shape and protrusivity depending on whether a cell is proximal to a flat and rigid surface or is embedded in a soft environment. Environmental sensitivity metrics calculated for small molecules and gene knockdowns identify interactions between the environment and cellular factors that are important for morphogenesis. We show that the Rho guanine nucleotide exchange factor (RhoGEF) TIAM2 contributes to shape determination in environmentally independent ways but that non-muscle myosin II, microtubules, and the RhoGEF FARP1 regulate shape in ways dependent on the microenvironment. Thus, changes in cancer cell shape in response to 3D geometric and mechanical cues are modulated in both an environmentally dependent and independent fashion.

Asunto(s)

Forma de la Célula , Factores de Intercambio de Guanina Nucleótido , Humanos , Factores de Intercambio de Guanina Nucleótido/metabolismo , Factores de Intercambio de Guanina Nucleótido/genética , Línea Celular Tumoral , Microtúbulos/metabolismo , Miosina Tipo II/metabolismo , Factores de Intercambio de Guanina Nucleótido Rho/metabolismo , Factores de Intercambio de Guanina Nucleótido Rho/genética , Melanoma/patología , Melanoma/metabolismo

RESUMEN

Recent findings indicate that Solo, a RhoGEF, is involved in cellular mechanical stress responses. However, the mechanism of actin cytoskeletal remodeling via Solo remains unclear. Therefore, this study aimed to identify Solo-interacting proteins using the BioID, a proximal-dependent labeling method, and elucidate the molecular mechanisms of function of Solo. We identified PDZ-RhoGEF (PRG) as a Solo-interacting protein. PRG colocalized with Solo in the basal area of cells, depending on Solo localization, and enhanced actin polymerization at the Solo accumulation sites. Additionally, Solo and PRG interaction was necessary for actin cytoskeletal remodeling. Furthermore, the purified Solo itself had little or negligible GEF activity, even its GEF-inactive mutant directly activated the GEF activity of PRG through interaction. Moreover, overexpression of the Solo and PRG binding domains, respectively, had a dominant-negative effect on actin polymerization and actin stress fiber formation in response to substrate stiffness. Therefore, Solo restricts the localization of PRG and regulates actin cytoskeletal remodeling in synergy with PRG in response to the surrounding mechanical environment.

Asunto(s)

Citoesqueleto de Actina , Actinas , Factores de Intercambio de Guanina Nucleótido Rho , Humanos , Citoesqueleto de Actina/metabolismo , Factores de Intercambio de Guanina Nucleótido Rho/metabolismo , Actinas/metabolismo , Dominios PDZ , Unión Proteica , Citoesqueleto/metabolismo , Animales , Células HEK293

RESUMEN

Small Ras homologous guanosine triphosphatase (Rho GTPase) family proteins are highly associated with tumorigenesis and development. As intrinsic exchange activity regulators of Rho GTPases, Rho guanine nucleotide exchange factors (RhoGEFs) have been demonstrated to be closely involved in tumor development and received increasing attention. They mainly contain two families: the diffuse B-cell lymphoma (Dbl) family and the dedicator of cytokinesis (Dock) family. More and more emphasis has been paid to the Dbl family members for their abnormally high expression in various cancers and their correlation to poor prognosis. In this review, the common and distinctive structures of Dbl family members are discussed, and their roles in cancer are summarized with a focus on Ect2, Tiam1/2, P-Rex1/2, Vav1/2/3, Trio, KALRN, and LARG. Significantly, the strategies targeting Dbl family RhoGEFs are highlighted as novel therapeutic opportunities for cancer.

Asunto(s)

Linfoma de Células B , Neoplasias , Humanos , Factores de Intercambio de Guanina Nucleótido Rho/metabolismo , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Proteínas de Unión al GTP rho/metabolismo , Carcinogénesis

RESUMEN

Guanine nucleotide exchange factor H1 (GEF-H1) is a unique protein modulated by the GDP/GTP exchange. As a regulator of the Rho-GTPase family, GEF-H1 can be activated through a microtubule-depended mechanism and phosphorylation regulation, enabling it to perform various pivotal biological functions across multiple cellular activities. These include the regulation of Rho-GTPase, cytoskeleton formation, cellular barrier, cell cycle, mitosis, cell differentiation, and vesicle trafficking. Recent studies have revealed its crucial effect on the tumor microenvironment (TME) components, promoting tumor initiation and progress. Consequently, an in-depth exploration of GEF-H1's biological roles and association with tumors holds promise for its potential as a valuable molecular target in tumor treatment.

Asunto(s)

Neoplasias , Proteína de Unión al GTP rhoA , Humanos , Factores de Intercambio de Guanina Nucleótido Rho/metabolismo , Proteína de Unión al GTP rhoA/metabolismo , Microtúbulos/metabolismo , Proteínas , Neoplasias/metabolismo , Microambiente Tumoral

RESUMEN

AIMS: Tau is a key player in Alzheimer's disease (AD) and other Tauopathies. Tau pathology in the brain directly correlates with neurodegeneration in AD. The recent identification of a rapid variant of AD demands an urgent need to uncover underlying mechanisms leading to differential progression in AD. Accordingly, we aimed to dissect the underlying differential mechanisms of toxicity associated with the Tau protein in AD subtypes and to find out subtype-dependent biomarkers and therapeutic targets. METHODS: To identify and characterise subtype-specific Tau-associated mechanisms of pathology, we performed comparative interactome mapping of Tau protein in classical AD (cAD) and rapidly progressive AD (rpAD) cases using co-immunoprecipitation coupled with quantitative mass spectrometry. The mass spectrometry data were extensively analysed using several bioinformatics approaches. RESULTS: The comparative interactome mapping of Tau protein revealed distinct and unique interactors (DPYSL4, ARHGEF2, TUBA4A and UQCRC2) in subtypes of AD. Interestingly, an analysis of the Tau-interacting proteins indicated enrichment of mitochondrial organisation processes, including negative regulation of mitochondrion organisation, mitochondrial outer membrane permeabilisation involved in programmed cell death, regulation of autophagy of mitochondrion and necroptotic processes, specifically in the rpAD interactome. While, in cAD, the top enriched processes were related to oxidation-reduction process, transport and monocarboxylic acid metabolism. CONCLUSIONS: Overall, our results provide a comprehensive map of Tau-interacting protein networks in a subtype-dependent manner and shed light on differential functions/pathways in AD subtypes. This comprehensive map of the Tau-interactome has provided subsets of disease-related proteins that can serve as novel biomarkers/biomarker panels and new drug targets.

Asunto(s)

Enfermedad de Alzheimer , Tauopatías , Humanos , Proteínas tau/metabolismo , Enfermedad de Alzheimer/patología , Tauopatías/patología , Encéfalo/patología , Biomarcadores , Factores de Intercambio de Guanina Nucleótido Rho/metabolismo

RESUMEN

Obscurin, encoded by the OBSCN gene, is a muscle protein consisting of three main splice isoforms, obscurin-A, obscurin-B, and obscurin kinase-only protein (also known as KIAA1639 or Obsc-kin). Obscurin is located at the M-band and Z-disks and interacts with titin and myomesin. It plays an important role in the stability and maintenance of the A- and M-bands and the subsarcolemmal organization of the microtubule network. Furthermore, obscurin is involved in Ca2+ regulation and sarcoplasmic reticulum function and is connected to several other muscle proteins. OBSCN gene variants have been reported to be relatively common in inherited cardiomyopathies. Here we reported two young patients with a history of cramps, myalgia, exercise intolerance, rhabdomyolysis, and myoglobinuria without any evidence of concomitant cardiomyopathy in association with novel OBSCN variants (c.24822C>A and c.2653+1G>C). Obscurin-deficient muscle fibers seem to have increased susceptibility to damage triggered by exercise that may lead to rhabdomyolysis. More studies are needed to clarify the diverse clinical phenotypes and the pathophysiology of OBSCN gene variants.

Asunto(s)

Proteínas Musculares , Rabdomiólisis , Humanos , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Sarcómeros , Retículo Sarcoplasmático/metabolismo , Rabdomiólisis/genética , Rabdomiólisis/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Factores de Intercambio de Guanina Nucleótido Rho/genética , Factores de Intercambio de Guanina Nucleótido Rho/metabolismo

RESUMEN

Directed cell migration requires a local fine-tuning of Rho GTPase activity to control protrusion formation, cell-cell contraction, and turnover of cellular adhesions. The Rho guanine nucleotide exchange factor (GEF) TRIO is ideally suited to control RhoGTPase activity because it combines two distinct catalytic domains to control Rac1 and RhoA activity in one molecule. However, at the cellular level, this molecular feature also requires a tight spatiotemporal control of TRIO activity. Here, we analyze the dynamic localization of Trio in Xenopus cranial neural crest (NC) cells, where we have recently shown that Trio is required for protrusion formation and migration. Using live cell imaging, we find that the GEF2 domain, but not the GEF1 domain of Trio, dynamically colocalizes with EB3 at microtubule plus-ends. Microtubule-mediated transport of Trio appears to be relevant for its function in NC migration, as a mutant GEF2 construct lacking the SxIP motif responsible for microtubule plus-end localization was significantly impaired in its ability to rescue the Trio loss-of-function phenotype compared to wild-type GEF2. Furthermore, by analyzing microtubule dynamics in migrating NC cells, we observed that loss of Trio function stabilized microtubules at cell-cell contact sites compared to controls, whereas they were destabilized at the leading edge of NC cells. Our data suggest that Trio is transported by microtubules to distinct subcellular locations where it has different functions in controlling microtubule stability, cell morphology, and cell-cell interaction during directed NC migration.

Asunto(s)

Microtúbulos , Cresta Neural , Animales , Factores de Intercambio de Guanina Nucleótido Rho/genética , Factores de Intercambio de Guanina Nucleótido Rho/metabolismo , Cresta Neural/metabolismo , Microtúbulos/metabolismo , Xenopus laevis , Movimiento Celular/genética