RESUMEN
Migration of endothelial and many other cells requires spatiotemporal regulation of protrusive and contractile cytoskeletal rearrangements that drive local cell shape changes. Unexpectedly, the small GTPase Rho, a crucial regulator of cell movement, has been reported to be active in both local cell protrusions and retractions, raising the question of how Rho activity can coordinate cell migration. Here, we show that Rho activity is absent in local protrusions and active during retractions. During retractions, Rho rapidly activated ezrin-radixin-moesin proteins (ERMs) to increase actin-membrane attachment, and, with a delay, nonmuscle myosin 2 (NM2). Rho activity was excitable, with NM2 acting as a slow negative feedback regulator. Strikingly, inhibition of SLK/LOK kinases, through which Rho activates ERMs, caused elongated cell morphologies, impaired Rho-induced cell contractions, and reverted Rho-induced blebbing. Together, our study demonstrates that Rho activity drives retractions by sequentially enhancing ERM-mediated actin-membrane attachment for force transmission and NM2-dependent contractility.
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Actomiosina , Movimiento Celular , Forma de la Célula , Proteínas del Citoesqueleto , Actomiosina/metabolismo , Humanos , Proteínas del Citoesqueleto/metabolismo , Proteínas del Citoesqueleto/genética , Proteínas de Unión al GTP rho/metabolismo , Proteínas de Microfilamentos/metabolismo , Proteínas de la Membrana/metabolismo , Actinas/metabolismo , Animales , Miosina Tipo II/metabolismoRESUMEN
BACKGROUND: The actin-binding protein anillin (ANLN) functions as an oncogene in various cancers but has not been fully studied in oral squamous cell carcinoma (OSCC). This study aimed to investigate the expression of ANLN in OSCC tissues and cell lines, to better understand its role in mediating proliferative, angiogenic, invasive, and metastatic capabilities in this type of cancer. METHODS: ANLN mRNA and protein levels were assessed using qPCR and western immunoblotting. The expression intensity of ANLN was evaluated using immunohistochemical (IHC) staining. Biological functional assays were employed to characterize the behavior of OSCC cells influenced by ANLN. Additionally, comprehensive bioinformatics analysis, including GO analysis and KEGG enrichment analysis, was performed on differentially expressed genes in ANLN-mediated pathways. RESULTS: OSCC tumors and cell lines exhibited higher ANLN expression. Silencing of ANLN significantly suppressed OSCC cell proliferation, as evidenced by a significant reduction in the Ki-67 index both in vitro and in vivo. The migration and invasive ability of OSCC cells were markedly diminished, coinciding with a decrease in epithelial-mesenchymal transition activity. ANLN was also found to promote angiogenic activity in OSCC cells, partly through synergistic effects mediated by vascular endothelial growth factor A (VEGFA). Downregulation of ANLN expression led to decreased VEGFA levels, resulting in reduced angiogenesis characterized by fewer vascular branches. CONCLUSIONS: Our findings highlight the promising role of ANLN as a biomarker for both diagnostic and prognostic in OSCC. Targeting ANLN with inhibitory strategies could impede the oncogenesis processes at the core of OSCC development, presenting significant opportunities for advancing therapeutic interventions.
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Movimiento Celular , Proliferación Celular , Proteínas de Microfilamentos , Neoplasias de la Boca , Invasividad Neoplásica , Neovascularización Patológica , Humanos , Neoplasias de la Boca/patología , Neoplasias de la Boca/genética , Neoplasias de la Boca/metabolismo , Proliferación Celular/genética , Neovascularización Patológica/genética , Neovascularización Patológica/patología , Neovascularización Patológica/metabolismo , Movimiento Celular/genética , Invasividad Neoplásica/genética , Proteínas de Microfilamentos/metabolismo , Proteínas de Microfilamentos/genética , Carcinoma de Células Escamosas/patología , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Animales , Carcinoma de Células Escamosas de Cabeza y Cuello/patología , Carcinoma de Células Escamosas de Cabeza y Cuello/genética , Carcinoma de Células Escamosas de Cabeza y Cuello/metabolismo , Silenciador del Gen , Ratones , Transición Epitelial-Mesenquimal/genética , Femenino , Masculino , AngiogénesisRESUMEN
miR-31 is a highly conserved microRNA that plays crucial roles in cell proliferation, migration and differentiation. We discovered that miR-31 and some of its validated targets are enriched on the mitotic spindle of the dividing sea urchin embryo and mammalian cells. Using the sea urchin embryo, we found that miR-31 inhibition led to developmental delay correlated with increased cytoskeletal and chromosomal defects. We identified miR-31 to directly suppress several actin remodeling transcripts, including ß-actin, Gelsolin, Rab35 and Fascin. De novo translation of Fascin occurs at the mitotic spindle of sea urchin embryos and mammalian cells. Importantly, miR-31 inhibition leads to a significant a increase of newly translated Fascin at the spindle of dividing sea urchin embryos. Forced ectopic localization of Fascin transcripts to the cell membrane and translation led to significant developmental and chromosomal segregation defects, highlighting the importance of the regulation of local translation by miR-31 at the mitotic spindle to ensure proper cell division. Furthermore, miR-31-mediated post-transcriptional regulation at the mitotic spindle may be an evolutionarily conserved regulatory paradigm of mitosis.
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MicroARNs , Biosíntesis de Proteínas , Huso Acromático , Animales , MicroARNs/metabolismo , MicroARNs/genética , Huso Acromático/metabolismo , Regulación del Desarrollo de la Expresión Génica , Humanos , Proteínas de Microfilamentos/metabolismo , Proteínas de Microfilamentos/genética , Mitosis/genética , Proteínas Portadoras/metabolismo , Proteínas Portadoras/genética , Desarrollo Embrionario/genética , Embrión no Mamífero/metabolismo , Segregación Cromosómica/genética , Actinas/metabolismo , Actinas/genética , Erizos de Mar/embriología , Erizos de Mar/genética , Erizos de Mar/metabolismoRESUMEN
Osteoarthritis (OA) is the most common form of arthritic disease, and phenotypic modification of chondrocytes is an important mechanism that contributes to the loss of cartilage homeostasis. This study identified that Fascin actin-bundling protein 1 (FSCN1) plays a pivotal role in regulating chondrocytes phenotype and maintaining cartilage homeostasis. Proteome-wide screening revealed markedly upregulated FSCN1 protein expression in human OA cartilage. FSCN1 accumulation was confirmed in the superficial layer of OA cartilage from humans and mice, primarily in dedifferentiated-like chondrocytes, associated with enhanced actin stress fiber formation and upregulated type I and III collagens. FSCN1-inducible knockout mice exhibited delayed cartilage degeneration following experimental OA surgery. Mechanistically, FSCN1 promoted actin polymerization and disrupted the inhibition of Decorin on TGF-ß1, leading to excessive TGF-ß1 production and ALK1/Smad1/5 signaling activation, thus, accelerated chondrocyte dedifferentiation. Intra-articular injection of FSCN1-overexpressing adeno-associated virus exacerbated OA progression in mice, which was mitigated by an ALK1 inhibitor. Moreover, FSCN1 inhibitor NP-G2-044 effectively reduced extracellular matrix degradation in OA mice, cultured human OA chondrocytes, and cartilage explants by suppressing ALK1/Smad1/5 signaling. These findings suggest that targeting FSCN1 represents a promising therapeutic approach for OA.
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Proteínas Portadoras , Condrocitos , Proteínas de Microfilamentos , Osteoartritis , Animales , Humanos , Masculino , Ratones , Proteínas Portadoras/metabolismo , Proteínas Portadoras/genética , Cartílago Articular/metabolismo , Cartílago Articular/patología , Condrocitos/metabolismo , Condrocitos/patología , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismo , Osteoartritis/patología , Osteoartritis/metabolismo , Osteoartritis/genética , Fenotipo , Receptores Odorantes , Transducción de SeñalRESUMEN
Plastin-3 (PLS3) encodes T-plastin, an actin-bundling protein mediating the formation of actin filaments by which numerous cellular processes are regulated. Loss-of-function genetic defects in PLS3 are reported to cause X-linked osteoporosis and childhood-onset fractures. However, the molecular etiology of PLS3 remains elusive. Functional compensation by actin-bundling proteins ACTN1, ACTN4, and FSCN1 was investigated in zebrafish following morpholino-mediated pls3 knockdown. Primary dermal fibroblasts from six patients with a PLS3 variant were also used to examine expression of these proteins during osteogenic differentiation. In addition, Pls3 knockdown in the murine MLO-Y4 cell line was employed to provide insights in global gene expression. Our results showed that ACTN1 and ACTN4 can rescue the skeletal deformities in zebrafish after pls3 knockdown, but this was inadequate for FSCN1. Patients' fibroblasts showed the same osteogenic transdifferentiation ability as healthy donors. RNA-seq results showed differential expression in Wnt1, Nos1ap, and Myh3 after Pls3 knockdown in MLO-Y4 cells, which were also associated with the Wnt and Th17 cell differentiation pathways. Moreover, WNT2 was significantly increased in patient osteoblast-like cells compared to healthy donors. Altogether, our findings in different bone cell types indicate that the mechanism of PLS3-related pathology extends beyond actin-bundling proteins, implicating broader pathways of bone metabolism.
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Diferenciación Celular , Glicoproteínas de Membrana , Proteínas de Microfilamentos , Osteogénesis , Pez Cebra , Pez Cebra/metabolismo , Pez Cebra/genética , Animales , Osteogénesis/genética , Humanos , Proteínas de Microfilamentos/metabolismo , Proteínas de Microfilamentos/genética , Glicoproteínas de Membrana/metabolismo , Glicoproteínas de Membrana/genética , Ratones , Fibroblastos/metabolismo , Osteoblastos/metabolismo , Proteínas de Pez Cebra/metabolismo , Proteínas de Pez Cebra/genética , Técnicas de Silenciamiento del GenRESUMEN
Synaptopodin 2-like protein (SYNPO2L) is localized in the sarcomere of cardiomyocytes and is involved in heart morphogenesis. However, the molecular function of SYNPO2L in the heart is not fully understood. We investigated the interaction of SYNPO2L with sarcomeric α-actinin and actin filaments in cultured mouse cardiomyocytes. Immunofluorescence studies showed that SYNPO2L colocalized with α-actinin and actin filaments at the Z-discs of the sarcomere. Recombinant SYNPO2La or SYNPO2Lb caused a bundling of the actin filaments in the absence of α-actinin and enhanced the α-actinin-dependent formation of actin bundles. In addition, high-speed atomic force microscopy revealed that SYNPO2La directly bound to α-actinin via its globular ends. The interaction between α-actinin and SYNPO2La fixed the movements of the two proteins on the actin filaments. These results strongly suggest that SYNPO2L cooperates with α-actinin during actin bundle formation to facilitate sarcomere formation and maintenance.
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Actinina , Proteínas de Microfilamentos , Proteínas Musculares , Miocitos Cardíacos , Unión Proteica , Sarcómeros , Animales , Ratones , Citoesqueleto de Actina/metabolismo , Actinina/metabolismo , Actinas/metabolismo , Proteínas de Microfilamentos/metabolismo , Miocitos Cardíacos/metabolismo , Sarcómeros/metabolismo , Proteínas Musculares/metabolismoRESUMEN
In an experimental model of Alzheimer's disease in mice, oral administration of trehalose disaccharide reduces neuroinflammation assessed by the expression level of microglia activation marker Iba1 and affects the neutrophil degranulation activity. A potential anti-inflammatory effect of 4% trehalose solution associated with a decrease in the activity of leukocyte elastase in plasma was revealed.
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Enfermedad de Alzheimer , Modelos Animales de Enfermedad , Microglía , Trehalosa , Animales , Trehalosa/farmacología , Trehalosa/uso terapéutico , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Microglía/efectos de los fármacos , Microglía/metabolismo , Ratones , Proteínas de Microfilamentos/metabolismo , Inflamación/tratamiento farmacológico , Inflamación/metabolismo , Masculino , Proteínas de Unión al Calcio/metabolismo , Elastasa de Leucocito/metabolismo , Neutrófilos/efectos de los fármacos , Neutrófilos/metabolismo , Disacáridos/farmacología , Antiinflamatorios/farmacologíaRESUMEN
The Enabled/VASP homology 1 (EVH1) domain is a small module that interacts with proline-rich stretches in its ligands and is found in various signaling and scaffolding proteins. Mena, the mammalian homologue of Ena, is involved in diverse actin-associated events, such as membrane dynamics, bacterial motility, and tumor intravasation and extravasation. Two-dimensional (2D) 1H-15N HSQC NMR was used to study Mena EVH1 binding properties, defining the amino acids involved in ligand recognition for the physiological ligands ActA and PCARE, and a synthetic polyproline-inspired small molecule (hereafter inhibitor 6c). Chemical shift perturbations indicated that proline-rich segments bind in the conserved EVH1 hydrophobic cleft. The PCARE-derived peptide elicited more perturbations compared to the ActA-derived peptide, consistent with a previous report of a structural alteration in the solvent-exposed ß7-ß8 loop. Unexpectedly, EVH1 and the proline-rich segment of PTP1B did not exhibit NMR chemical shift perturbations; however, the high-resolution crystal structure implicated the conserved EVH1 hydrophobic cleft in ligand recognition. Intrinsic steady-state fluorescence and fluorescence polarization assays indicate that residues outside the proline-rich segment enhance the ligand affinity for EVH1 (Kd = 3-8 µM). Inhibitor 6c displayed tighter binding (Kd â¼ 0.3 µM) and occupies the same EVH1 cleft as physiological ligands. These studies revealed that the EVH1 domain enhances ligand affinity through recognition of residues flanking the proline-rich segments. Additionally, a synthetic inhibitor binds more tightly to the EVH1 domain than natural ligands, occupying the same hydrophobic cleft.
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Unión Proteica , Humanos , Resonancia Magnética Nuclear Biomolecular , Dominios Proteicos , Ligandos , Modelos Moleculares , Péptidos/química , Péptidos/metabolismo , Prolina/metabolismo , Prolina/química , Secuencia de Aminoácidos , Sitios de Unión , Cristalografía por Rayos X , Proteínas de Microfilamentos/química , Proteínas de Microfilamentos/metabolismoRESUMEN
Endosomal membrane trafficking is mediated by specific protein coats and formation of actin-rich membrane domains. The Retromer complex coordinates with sorting nexin (SNX) cargo adaptors including SNX27, and the SNX27-Retromer assembly interacts with the Wiskott-Aldrich syndrome protein and SCAR homolog (WASH) complex which nucleates actin filaments establishing the endosomal recycling domain. Crystal structures, modeling, biochemical, and cellular validation reveal how the FAM21 subunit of WASH interacts with both Retromer and SNX27. FAM21 binds the FERM domain of SNX27 using acidic-Asp-Leu-Phe (aDLF) motifs similar to those found in the SNX1 and SNX2 subunits of the ESCPE-1 complex. Overlapping FAM21 repeats and a specific Pro-Leu containing motif bind three distinct sites on Retromer involving both the VPS35 and VPS29 subunits. Mutation of the major VPS35-binding site does not prevent cargo recycling; however, it partially reduces endosomal WASH association indicating that a network of redundant interactions promote endosomal activity of the WASH complex. These studies establish the molecular basis for how SNX27-Retromer is coupled to the WASH complex via overlapping and multiplexed motif-based interactions required for the dynamic assembly of endosomal membrane recycling domains.
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Endosomas , Nexinas de Clasificación , Proteínas de Transporte Vesicular , Humanos , Endosomas/metabolismo , Nexinas de Clasificación/metabolismo , Nexinas de Clasificación/genética , Nexinas de Clasificación/química , Proteínas de Transporte Vesicular/metabolismo , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/química , Proteínas de Microfilamentos/metabolismo , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/química , Unión Proteica , Cristalografía por Rayos X , Sitios de Unión , Modelos MolecularesRESUMEN
A large portion of neuropathic pain suffering patients may also concurrently experience neuropathic itch, with a negative impact on the quality of life. The limited understanding of neuropathic itch and the low efficacy of current anti-itch therapies dictate the urgent need of a better comprehension of molecular mechanisms involved and development of relevant animal models. This study was aimed to characterize the itching phenotype in a model of trauma-induced peripheral neuropathy, the spared nerve injury (SNI), and the molecular events underlying the overlap with the nociceptive behavior. SNI mice developed hyperknesis and spontaneous itch 7-14 days after surgery that was prevented by gabapentin treatment. Itch was associated with pain hypersensitivity, loss of intraepidermal nerve fiber (IENF) density and increased epidermal thickness. In coincidence with the peak of scratching behavior, SNI mice showed a spinal overexpression of IBA1 and GFAP, microglia and astrocyte markers respectively. An increase of the itch neuropeptide B-type natriuretic peptide (BNP) in NeuN+ cells, of its downstream effector interleukin 17 (IL17) along with increased pERK1/2 levels occurred in the spinal cord dorsal horn and DRG. A raise in BNP and IL17 was also detected at skin level. Stimulation of HaCat cells with conditioned medium from BV2-stimulated SH-SY5Y cells produced a dramatic reduction of HaCat cell viability. This study showed that SNI mice might represent a model for neuropathic itch and pain. Collectively, our finding suggest that neuropathic itch might initiate at spinal level, then affecting skin epidermis events, through a glia-mediated neuroinflammation-evoked BNP/IL17 mechanism.
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Modelos Animales de Enfermedad , Neuralgia , Enfermedades Neuroinflamatorias , Prurito , Animales , Prurito/metabolismo , Prurito/patología , Neuralgia/metabolismo , Neuralgia/etiología , Ratones , Masculino , Enfermedades Neuroinflamatorias/metabolismo , Humanos , Gabapentina/farmacología , Interleucina-17/metabolismo , Ratones Endogámicos C57BL , Ganglios Espinales/metabolismo , Ganglios Espinales/efectos de los fármacos , Ganglios Espinales/patología , Células HaCaT , Microglía/metabolismo , Microglía/efectos de los fármacos , Hiperalgesia/metabolismo , Proteínas de Microfilamentos/metabolismo , Asta Dorsal de la Médula Espinal/metabolismo , Asta Dorsal de la Médula Espinal/efectos de los fármacos , Proteínas de Unión al CalcioRESUMEN
Seizures induce hippocampal subregion dependent enhancements in microglia/macrophage phagocytosis and cytokine release that may contribute to the development of epilepsy. As a model of hyperactive mTOR induced epilepsy, neuronal subset specific phosphatase and tensin homolog (NS-Pten) knockout (KO) mice exhibit hyperactive mTOR signaling in the hippocampus, seizures that progress with age, and enhanced hippocampal microglia/macrophage activation. However, it is unknown where microglia/macrophages are most active within the hippocampus of NS-Pten KO mice. We quantified the density of IBA1 positive microglia/macrophages in the CA1, CA2/3, and dentate gyrus of NS-Pten KO and wildtype (WT) male and female mice at 4, 10, and 15 weeks of age. NS-Pten KO mice exhibited an overall increase in the number of IBA1 positive microglia/macrophages in each subregion and in the entire hippocampus. After accounting for differences in size, the whole hippocampus of NS-Pten KO mice still exhibited an increased density of IBA1 positive microglia/macrophages. Subregion analyses showed that this increase was restricted to the dentate gyrus of both male and female NS-Pten KO mice and to the CA1 of male NS-Pten KO mice. These data suggest enhanced microglia/macrophage activity may occur in the NS-Pten KO mice in a hippocampal subregion and sex-dependent manner. Future work should seek to determine whether these region-specific increases in microgliosis play a role in the progression of epilepsy in this model.
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Hipocampo , Macrófagos , Microglía , Fosfohidrolasa PTEN , Caracteres Sexuales , Animales , Femenino , Masculino , Ratones , Proteínas de Unión al Calcio/metabolismo , Recuento de Células , Hipocampo/metabolismo , Macrófagos/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas de Microfilamentos/metabolismo , Microglía/metabolismo , Fosfohidrolasa PTEN/genética , Fosfohidrolasa PTEN/deficiencia , Fosfohidrolasa PTEN/metabolismoRESUMEN
Nuclear migration is critical for the proper positioning of neurons in the developing brain. It is known that bidirectional microtubule motors are required for nuclear transport, yet the mechanism of the coordination of opposing motors is still under debate. Using mouse cerebellar granule cells, we demonstrate that Nesprin-2 serves as a nucleus-motor adaptor, coordinating the interplay of kinesin-1 and dynein. Nesprin-2 recruits dynein-dynactin-BicD2 independently of the nearby kinesin-binding LEWD motif. Both motor binding sites are required to rescue nuclear migration defects caused by the loss of function of Nesprin-2. In an intracellular cargo transport assay, the Nesprin-2 fragment encompassing the motor binding sites generates persistent movements toward both microtubule minus and plus ends. Nesprin-2 drives bidirectional cargo movements over a prolonged period along perinuclear microtubules, which advance during the migration of neurons. We propose that Nesprin-2 keeps the nucleus mobile by coordinating opposing motors, enabling continuous nuclear transport along advancing microtubules in migrating cells.
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Núcleo Celular , Dineínas , Cinesinas , Proteínas Asociadas a Microtúbulos , Microtúbulos , Proteínas del Tejido Nervioso , Neuronas , Animales , Microtúbulos/metabolismo , Neuronas/metabolismo , Cinesinas/metabolismo , Cinesinas/genética , Proteínas del Tejido Nervioso/metabolismo , Proteínas del Tejido Nervioso/genética , Dineínas/metabolismo , Núcleo Celular/metabolismo , Ratones , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Asociadas a Microtúbulos/genética , Transporte Activo de Núcleo Celular , Complejo Dinactina/metabolismo , Complejo Dinactina/genética , Movimiento Celular , Proteínas de Microfilamentos/metabolismo , Proteínas de Microfilamentos/genética , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Cerebelo/metabolismo , Cerebelo/citología , Sitios de Unión , HumanosRESUMEN
BACKGROUND: LARC patients commonly receive adjuvant therapy, however, hidden micrometastases still limit the improvement of OS. This study aims to investigate the impact of VASN in rectal cancer with pulmonary metastasis and understand the underlying molecular mechanisms to guide adjuvant chemotherapy selection. METHODS: Sequencing data from rectal cancer patients with pulmonary metastasis from Sun Yat-sen University Cancer Center (SYSUCC) and publicly available data were meticulously analyzed. The functional role of VASN in pulmonary metastasis was validated in vivo and in vitro. Coimmunoprecipitation (co-IP), immunofluorescence, and rescue experiments were conducted to unravel potential molecular mechanisms of VASN. Moreover, VASN expression levels in tumor samples were examined and analyzed for their correlations with pulmonary metastasis status, tumor stage, adjuvant chemotherapy benefit, and survival outcome. RESULTS: Our study revealed a significant association between high VASN expression and pulmonary metastasis in LARC patients. Experiments in vitro and in vivo demonstrated that VASN could promote the cell proliferation, metastasis, and drug resistance of colorectal cancer. Mechanistically, VASN interacts with the NOTCH1 protein, leading to concurrent activation of the NOTCH and MAPK pathways. Clinically, pulmonary metastasis and advanced tumor stage were observed in 90% of VASN-positive patients and 53.5% of VASN-high patients, respectively, and VASN-high patients had a lower five-year survival rate than VASN-low patients (26.7% vs. 83.7%). Moreover, the Cox analysis and OS analysis indicated that VASN was an independent prognostic factor for OS (HR = 7.4, P value < 0.001) and a predictor of adjuvant therapy efficacy in rectal cancer. CONCLUSIONS: Our study highlights the role of VASN in decreasing drug sensitivity and activating the NOTCH and MAPK pathways, which leads to tumorigenesis and pulmonary metastasis. Both experimental and clinical data support that rectal cancer patients with VASN overexpression detected in biopsies have a higher risk of pulmonary metastasis and adjuvant chemotherapy resistance.
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Resistencia a Antineoplásicos , Neoplasias Pulmonares , Neoplasias del Recto , Humanos , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/secundario , Femenino , Masculino , Neoplasias del Recto/patología , Neoplasias del Recto/metabolismo , Neoplasias del Recto/genética , Neoplasias del Recto/tratamiento farmacológico , Quimioterapia Adyuvante , Resistencia a Antineoplásicos/genética , Línea Celular Tumoral , Persona de Mediana Edad , Animales , Regulación Neoplásica de la Expresión Génica , Ratones Desnudos , Proliferación Celular/efectos de los fármacos , Receptor Notch1/metabolismo , Receptor Notch1/genética , Proteínas de Microfilamentos/metabolismo , Proteínas de Microfilamentos/genética , Sistema de Señalización de MAP Quinasas/efectos de los fármacosRESUMEN
The morphology of dendritic spines, the postsynaptic compartment of most excitatory synapses, decisively modulates the function of neuronal circuits as also evident from human brain disorders associated with altered spine density or morphology. Actin filaments (F-actin) form the backbone of spines, and a number of actin-binding proteins (ABP) have been implicated in shaping the cytoskeleton in mature spines. Instead, only little is known about the mechanisms that control the reorganization from unbranched F-actin of immature spines to the complex, highly branched cytoskeleton of mature spines. Here, we demonstrate impaired spine maturation in hippocampal neurons upon genetic inactivation of cyclase-associated protein 1 (CAP1) and CAP2, but not of CAP1 or CAP2 alone. We found a similar spine maturation defect upon overactivation of inverted formin 2 (INF2), a nucleator of unbranched F-actin with hitherto unknown synaptic function. While INF2 overactivation failed in altering spine density or morphology in CAP-deficient neurons, INF2 inactivation largely rescued their spine defects. From our data we conclude that CAPs inhibit INF2 to induce spine maturation. Since we previously showed that CAPs promote cofilin1-mediated cytoskeletal remodeling in mature spines, we identified them as a molecular switch that control transition from filopodia-like to mature spines.
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Proteínas del Citoesqueleto , Espinas Dendríticas , Forminas , Hipocampo , Proteínas de Microfilamentos , Espinas Dendríticas/metabolismo , Animales , Ratones , Forminas/metabolismo , Forminas/genética , Proteínas de Microfilamentos/metabolismo , Proteínas de Microfilamentos/genética , Proteínas del Citoesqueleto/metabolismo , Proteínas del Citoesqueleto/genética , Hipocampo/metabolismo , Hipocampo/citología , Células Cultivadas , Neuronas/metabolismo , Actinas/metabolismo , Citoesqueleto de Actina/metabolismo , Ratones Noqueados , Humanos , Proteínas PortadorasRESUMEN
Recently, various cancer types have been identified to express a distinct subset of Interferon-stimulated genes (ISGs) that mediate therapy resistance. The mechanism through which cancer cells maintain prolonged Interferon stimulation effects to coordinate resistance remains unclear. Our research demonstrated that aberrant upregulation of TAGLN2 is associated with gastric cancer progression, and inhibiting its expression renders gastric cancer cells more susceptible to chemotherapy and radiation. We uncovered a novel role for TAGLN2 in the upregulation of resistance signature ISGs by enhancing YBX1-associated ssDNA aggregation and cGAS-STING pathway activation. TAGLN2 modulates YBX1 by recruiting c-Myc and SOX9 to YBX1 promoter region and directly interacting with AKT-YBX1, thereby enhancing YBX1 phosphorylation and nuclear translocation. Significantly, targeted downregulation of key proteins, inhibition of the TAGLN2-YBX1-AKT interaction (using Fisetin or MK2206) or disruption of the cGAS-STING pathway substantially reduced ssDNA accumulation, subsequent ISGs upregulation, and therapy resistance. The combination of Cisplatin with MK2206 displayed a synergistic effect in the higher TAGLN2-expressing xenograft tumors. Clinical analysis indicated that a derived nine-gene set effectively predicts therapeutic sensitivity and long-term prognosis in gastric cancer patients. These findings suggest that TAGLN2, YBX1 and induced ISGs are novel predictive markers for clinical outcomes, and targeting this axis is an attractive therapeutic sensitization strategy.
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Daño del ADN , Proteínas Proto-Oncogénicas c-akt , Transducción de Señal , Neoplasias Gástricas , Proteína 1 de Unión a la Caja Y , Humanos , Neoplasias Gástricas/genética , Neoplasias Gástricas/patología , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/tratamiento farmacológico , Proteína 1 de Unión a la Caja Y/metabolismo , Proteína 1 de Unión a la Caja Y/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Animales , Línea Celular Tumoral , Ratones , Resistencia a Antineoplásicos/genética , Compuestos Heterocíclicos con 3 Anillos/farmacología , Ratones Desnudos , Interferones/metabolismo , Regulación Neoplásica de la Expresión Génica , Cisplatino/farmacología , Cisplatino/uso terapéutico , Proteínas de Microfilamentos/metabolismo , Proteínas de Microfilamentos/genética , Nucleotidiltransferasas/metabolismo , Nucleotidiltransferasas/genética , Femenino , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Ratones Endogámicos BALB C , MasculinoRESUMEN
MICAL proteins represent a unique family of actin regulators crucial for synapse development, membrane trafficking, and cytokinesis. Unlike classical actin regulators, MICALs catalyze the oxidation of specific residues within actin filaments to induce robust filament disassembly. The potent activity of MICALs requires tight control to prevent extensive damage to actin cytoskeleton. However, the molecular mechanism governing MICALs' activity regulation remains elusive. Here, we report the cryo-EM structure of MICAL1 in the autoinhibited state, unveiling a head-to-tail interaction that allosterically blocks enzymatic activity. The structure also reveals the assembly of C-terminal domains via a tripartite interdomain interaction, stabilizing the inhibitory conformation of the RBD. Our structural, biochemical, and cellular analyses elucidate a multi-step mechanism to relieve MICAL1 autoinhibition in response to the dual-binding of two Rab effectors, revealing its intricate activity regulation mechanisms. Furthermore, our mutagenesis study of MICAL3 suggests the conserved autoinhibition and relief mechanisms among MICALs.
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Actinas , Microscopía por Crioelectrón , Oxigenasas de Función Mixta , Humanos , Actinas/metabolismo , Oxigenasas de Función Mixta/metabolismo , Oxigenasas de Función Mixta/química , Proteínas de Microfilamentos/metabolismo , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/química , Unión Proteica , Citoesqueleto de Actina/metabolismo , Modelos Moleculares , Proteínas de Unión al GTP rab/metabolismo , Proteínas de Unión al GTP rab/genética , Proteínas del Citoesqueleto/metabolismo , Proteínas del Citoesqueleto/química , Proteínas del Citoesqueleto/genética , Dominios Proteicos , CalponinasRESUMEN
Nuclear actin-based movements support DNA double-strand break (DSB) repair. However, molecular determinants that promote filamentous actin (F-actin) formation on the damaged chromatin remain undefined. Here we describe the DYRK1A kinase as a nuclear activity that promotes local F-actin assembly to support DSB mobility and repair, accomplished in part by its targeting of actin nucleator spire homolog 1 (Spir1). Indeed, perturbing DYRK1A-dependent phosphorylation of S482 mis-regulated Spir1 accumulation at damaged-modified chromatin, and led to compromised DSB-associated actin polymerization and attenuated DNA repair. Our findings uncover a role of the DYRK1A-Spir1 axis in nuclear actin dynamics during early DSB responses, and highlight the intricate details of nuclear cytoskeletal network in DSB repair and genome stability maintenance.
Asunto(s)
Actinas , Núcleo Celular , Cromatina , Roturas del ADN de Doble Cadena , Quinasas DyrK , Proteínas de Microfilamentos , Proteínas Nucleares , Proteínas Serina-Treonina Quinasas , Proteínas Tirosina Quinasas , Humanos , Actinas/metabolismo , Núcleo Celular/metabolismo , Cromatina/metabolismo , Reparación del ADN , Quinasas DyrK/genética , Quinasas DyrK/metabolismo , Células HeLa , Proteínas de Microfilamentos/metabolismo , Proteínas de Microfilamentos/genética , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Proteínas Tirosina Quinasas/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismoRESUMEN
Human cortical neurons (hCNs) exhibit high dendritic complexity and synaptic density, and the maturation process is greatly protracted. However, the molecular mechanism governing these specific features remains unclear. Here, we report that the hominoid-specific gene TBC1D3 promotes dendritic arborization and protracts the pace of synaptogenesis. Ablation of TBC1D3 in induced hCNs causes reduction of dendritic growth and precocious synaptic maturation. Forced expression of TBC1D3 in the mouse cortex protracts synaptic maturation while increasing dendritic growth. Mechanistically, TBC1D3 functions via interaction with MICAL1, a monooxygenase that mediates oxidation of actin filament. At the early stage of differentiation, the TBC1D3/MICAL1 interaction in the cytosol promotes dendritic growth via F-actin oxidation and enhanced actin dynamics. At late stages, TBC1D3 escorts MICAL1 into the nucleus and downregulates the expression of genes related with synaptic maturation through interaction with the chromatin remodeling factor ATRX. Thus, this study delineates the molecular mechanisms underlying human neuron development.
Asunto(s)
Proteínas de Microfilamentos , Transducción de Señal , Sinapsis , Humanos , Animales , Sinapsis/metabolismo , Ratones , Proteínas de Microfilamentos/metabolismo , Proteínas de Microfilamentos/genética , Proteínas Activadoras de GTPasa/metabolismo , Proteínas Activadoras de GTPasa/genética , Actinas/metabolismo , Neuronas/metabolismo , Dendritas/metabolismo , ADN Helicasas/metabolismo , Neurogénesis , Oxigenasas de Función Mixta/metabolismo , Oxigenasas de Función Mixta/genética , Diferenciación Celular , CalponinasRESUMEN
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.
Asunto(s)
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
It has been shown that the formation of filopodia is a key step in tumor cell metastasis, but there is limited research regarding its mechanism. In this study, we demonstrated that fatty acid synthase (FASN) promoted filopodia formation in liver cancer cells by regulating fascin actin-bundling protein 1 (FSCN1), a marker protein for filopodia. Mechanistically, on the one hand, the accumulation of FASN is caused by the enhanced deubiquitination of FASN mediated by UCHL5 (ubiquitin c-terminal hydrolase L5). In this pathway, low expression of SIAH1 (Seven in absentia homolog 1) can decrease the ubiquitination and degradation of ADRM1 (adhesion regulating molecule 1) thereby increasing its protein level, which will recruit and activate the deubiquitination enzyme UCHL5, leading to FASN undergo deubiquitination and escape from proteasomal degradation. On the other hand, the accumulation of FASN is related to its weakened ubiquitination, where SIAH1 directly acts as a ubiquitin ligase toward FASN, and low expression of SIAH1 reduces the ubiquitination and degradation of FASN. Both the two pathways are involved in the regulation of FASN in liver cancer. Our results reveal a novel mechanism for FASN accumulation due to the low expression of SIAH1 in human liver cancer and suggest an important role of FASN in filopodia formation in liver cancer cells.