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Hippo signaling controls organ size and tumor progression through a conserved pathway leading to nuclear translocation of the transcriptional effector Yki/Yap/Taz. Most of our understanding of Hippo signaling pertains to its cytoplasmic regulation, but how the pathway is controlled in the nucleus remains poorly understood. Here we uncover an evolutionarily conserved mechanism by which CDK7 promotes Yki/Yap/Taz stabilization in the nucleus to sustain Hippo pathway outputs. We found that a modular E3 ubiquitin ligase complex CRL4DCAF12 binds and targets Yki/Yap/Taz for ubiquitination and degradation, whereas CDK7 phosphorylates Yki/Yap/Taz at S169/S128/S90 to inhibit CRL4DCAF12 recruitment, leading to Yki/Yap/Taz stabilization. As a consequence, inactivation of CDK7 reduced organ size and inhibited tumor growth, which could be reversed by restoring Yki/Yap activity. Our study identifies an unanticipated layer of Hippo pathway regulation, defines a novel mechanism by which CDK7 regulates tissue growth, and implies CDK7 as a drug target for Yap/Taz-driven cancer.
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Carcinogênese/genética , Quinases Ciclina-Dependentes/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimologia , Drosophila melanogaster/metabolismo , Proteínas Nucleares/metabolismo , Transativadores/metabolismo , Animais , Antineoplásicos/farmacologia , Carcinogênese/efeitos dos fármacos , Linhagem Celular , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Quinases Ciclina-Dependentes/genética , Drosophila melanogaster/genética , Ativação Enzimática , Regulação Neoplásica da Expressão Gênica/genética , Técnicas de Silenciamento de Genes , Humanos , Neoplasias Hepáticas/enzimologia , Neoplasias Hepáticas/fisiopatologia , Camundongos , Tamanho do Órgão/genética , Fenilenodiaminas/farmacologia , Proteólise , Pirimidinas/farmacologia , Proteínas de Sinalização YAP , Quinase Ativadora de Quinase Dependente de CiclinaRESUMO
Acute lung injury (ALI) and its severe manifestation, acute respiratory distress syndrome (ARDS), represent critical clinical syndromes with multifactorial origins, notably stemming from sepsis within intensive care units (ICUs). Despite their high mortality rates, no selective cure is available beside ventilation support. Apoptosis plays a complex and pivotal role in the pathophysiology of acute lung injury. Excessive apoptosis of alveolar epithelial and microvascular endothelial cells can lead to disruption of lung epithelial barrier integrity, impairing the body's ability to exchange blood and gas. At the same time, apoptosis of damaged or dysfunctional cells, including endothelial and epithelial cells, can help maintain tissue integrity and accelerate recovery from organ pro-inflammatory stress. The balance between pro-survival and pro-apoptotic signals in lung injury determines patient outcomes, making the modulation of apoptosis an area of intense research in the quest for more effective therapies. Here we found that protein tyrosine phosphatase receptor type O (PTPRO), a poorly understood receptor-like protein tyrosine phosphatase, is consistently upregulated in multiple tissue types of mice under septic conditions and in the lung alveolar epithelial cells. PTPRO reduction by its selective short-interfering RNA (siRNA) leads to excessive apoptosis in lung alveolar epithelial cells without affecting cell proliferation. Consistently PTPRO overexpression by a DNA construct attenuates apoptotic signaling induced by LPS. These effects of PTPTO on cellular apoptosis are dependent on an ErbB2/PI3K/Akt/NFκB signaling pathway. Here we revealed a novel regulatory pathway of cellular apoptosis by PTPRO in lung alveolar epithelial cells during sepsis.
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Células Epiteliais Alveolares , Apoptose , Lipopolissacarídeos , Proteínas Tirosina Fosfatases Classe 3 Semelhantes a Receptores , Animais , Humanos , Masculino , Camundongos , Lesão Pulmonar Aguda/metabolismo , Lesão Pulmonar Aguda/patologia , Células Epiteliais Alveolares/metabolismo , Células Epiteliais Alveolares/efeitos dos fármacos , Células Epiteliais Alveolares/patologia , Apoptose/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Camundongos Endogâmicos C57BL , Proteínas Tirosina Fosfatases Classe 3 Semelhantes a Receptores/metabolismo , Proteínas Tirosina Fosfatases Classe 3 Semelhantes a Receptores/genética , Sepse/metabolismo , Sepse/patologia , Transdução de Sinais/efeitos dos fármacosRESUMO
Here, a scheme for a controllable nonreciprocal phonon laser is proposed in a hybrid photonic molecule system consisting of a whispering-gallery mode (WGM) optomechanical resonator and a χ(2)-nonlinear WGM resonator, by directionally quantum squeezing one of two coupled resonator modes. The directional quantum squeezing results in a chiral photon interaction between the resonators and a frequency shift of the squeezed resonator mode with respect to the unsqueezed bare mode. We show that the directional quantum squeezing can modify the effective optomechanical coupling in the optomechanical resonator, and analyze the impacts of driving direction and squeezing extent on the phonon laser action in detail. Our analytical and numerical results indicate that the controllable nonreciprocal phonon laser action can be effectively realized in this system. The proposed scheme uses an all-optical and chip-compatible approach without spinning resonators, which may be more beneficial for integrating and packaging of the system on a chip. Our proposal may provide a new route to realize integratable phonon devices for on-chip nonreciprocal phonon manipulations, which may be used in chiral quantum acoustics, topological phononics, and acoustical information processing.
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A theoretical model for studying the temperature properties of singlemode-multimode-singlemode (SMS) fiber structure fabricated by absorptive multimode fiber (MMF) cladding is established. Moreover, an SMS-based temperature sensor is fabricated and experimentally demonstrated. Experimental results show that the dip wavelength of the transmission spectrum changes linearly with temperature, which is in good agreement with the simulated results obtained by using the model. Further, a comprehensive study of temperature characteristics affected by the thermo-optic effect, thermal expansion effect, and thermal effect of absorption characteristics is performed for SMS fiber optic structures with different refractive indexes, thermo-optic coefficients, and absorption properties of MMF cladding, MMF core diameters, and thermal expansion coefficients of packaging shell. According to the obtained rules, investigations are carried out into the thermal response of an SMS fiber structure resulting from combined thermal effects for temperature performance optimization. Excellent temperature stability with a temperature sensitivity of 0 pm/°C or good temperature sensitivity of -441.58 pm/°C is achieved accordingly.
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Photonic hyper-parallel quantum information processing (QIP) can simplify the quantum circuit and improve the information-processing speed, as well as reduce the quantum resource consumption and suppress the photonic dissipation noise. Here, utilizing the singly charged semiconductor quantum dot (QD) inside single-sided optical microcavity as the potentially experimental platform, we propose five schemes for heralded four-qubit hyper-controlled-not (hyper-CNOT) gates, covering all cases of four-qubit hyper-CNOT gates operated on both the polarization and spatial-mode degrees of freedom (DoFs) of a two-photon system. The novel heralding mechanism improves the fidelity of each hyper-CNOT gate to unity in principle without the strict restriction of strong coupling. The adaptability and scalability of the schemes make the hyper-CNOT gates more accessible under current experimental technologies. These heralded high-fidelity photonic hyper-CNOT gates can therefore have immense utilization potentials in high-capacity quantum communication and fast quantum computing, which are of far-reaching significance for QIP.
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Hyperentangled-Bell-state analysis (HBSA) represents a key step in many quantum information processing schemes that utilize hyperentangled states. In this paper, we present a complete and faithful HBSA scheme for two-photon quantum systems hyperentangled in both the polarization and spatial-mode degrees of freedom, using a failure-heralded and fidelity-robust quantum swap gate for the polarization states of two photons (P-SWAP gate), constructed with a singly charged semiconductor quantum dot (QD) in a double-sided optical microcavity (double-sided QD-cavity system) and some linear-optical elements. Compared with the previously proposed complete HBSA schemes using different auxiliary tools such as parity-check quantum nondemonlition detectors or additional entangled states, our scheme significantly simplifies the analysis process and saves the quantum resource. Unlike the previous schemes based on the ideal optical giant circular birefringence induced by a single-electron spin in a double-sided QD-cavity system, our scheme guarantees the robust fidelity and relaxes the requirement on the QD-cavity parameters. These features indicate that our scheme may be more feasible and useful in practical applications based on the photonic hyperentanglement.
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Hedgehog (Hh) signaling in vertebrates depends on primary cilia. Upon stimulation, Hh pathway components, including Gli transcription factors, accumulate at primary cilia to transduce the Hh signal, but the mechanisms underlying their ciliary targeting remains largely unknown. Here, we show that the PY-type nuclear localization signal (PY-NLS)/karyopherinß2 (Kapß2) nuclear import system regulates Gli ciliary localization and Hh pathway activation. Mutating the PY-NLS in Gli or knockdown of Kapß2 diminished Gli ciliary localization. Kapß2 is required for the formation of Gli activator (GliA) in wild-type but not in Sufu mutant cells. Knockdown of Kapß2 affected Hh signaling in zebrafish embryos, as well as in vitro cultured cerebellum granule neuron progenitors (CGNPs) and SmoM2-driven medulloblastoma cells. Furthermore, Kapß2 depletion impaired the growth of cultured medulloblastoma cells, which was rescued by Gli overexpression. Interestingly, Kapß2 is a transcriptional target of the Hh pathway, thus forming a positive feedback loop for Gli activation. Our study unravels the molecular mechanism and cellular machinery regulating Gli ciliary localization and identifies Kapß2 as a critical regulator of the Hh pathway and a potential drug target for Hh-driven cancers.
Assuntos
Cílios/metabolismo , Proteínas Hedgehog/metabolismo , Sinais de Localização Nuclear , Proteína GLI1 em Dedos de Zinco/metabolismo , beta Carioferinas/metabolismo , Animais , Animais Geneticamente Modificados , Neoplasias Cerebelares/metabolismo , Embrião não Mamífero/metabolismo , Retroalimentação Fisiológica , Meduloblastoma/metabolismo , Camundongos , Células NIH 3T3 , Peixe-ZebraRESUMO
Hedgehog (Hh) signaling controls embryonic development and adult tissue homeostasis through the G protein coupled receptor (GPCR)-family protein Smoothened (Smo). Upon stimulation, Smo accumulates on the cell surface in Drosophila or primary cilia in vertebrates, which is thought to be essential for its activation and function, but the underlying mechanisms remain poorly understood. Here we show that Hh stimulates the binding of Smo to a plasma membrane-associated kinase Gilgamesh (Gish)/CK1γ and that Gish fine-tunes Hh pathway activity by phosphorylating a Ser/Thr cluster (CL-II) in the juxtamembrane region of Smo carboxyl-terminal intracellular tail (C-tail). We find that CL-II phosphorylation is promoted by protein kinase A (PKA)-mediated phosphorylation of Smo C-tail and depends on cell surface localization of both Gish and Smo. Consistent with CL-II being critical for high-threshold Hh target gene expression, its phosphorylation appears to require higher levels of Hh or longer exposure to the same level of Hh than PKA-site phosphorylation on Smo. Furthermore, we find that vertebrate CK1γ is localized at the primary cilium to promote Smo phosphorylation and Sonic hedgehog (Shh) pathway activation. Our study reveals a conserved mechanism whereby Hh induces a change in Smo subcellular localization to promote its association with and activation by a plasma membrane localized kinase, and provides new insight into how Hh morphogen progressively activates Smo.
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Caseína Quinase I/metabolismo , Membrana Celular/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas Hedgehog/metabolismo , Transdução de Sinais , Receptor Smoothened/metabolismo , Animais , Animais Geneticamente Modificados , Sítios de Ligação/genética , Western Blotting , Caseína Quinase I/genética , Linhagem Celular , Cílios/genética , Cílios/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/citologia , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Transferência Ressonante de Energia de Fluorescência , Proteínas Hedgehog/genética , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microscopia Confocal , Mutação , Fosforilação , Interferência de RNA , Receptor Smoothened/genética , Asas de Animais/metabolismoRESUMO
The tumor suppressor protein Suppressor of fused (Sufu) plays a conserved role in the Hedgehog (Hh) signaling pathway by inhibiting Cubitus interruptus (Ci)/Glioma-associated oncogene homolog (Gli) transcription factors, but the molecular mechanism by which Sufu inhibits Ci/Gli activity remains poorly understood. Here we show that Sufu can bind Ci/Gli through a C-terminal Sufu-interacting site (SIC) in addition to a previously identified N-terminal site (SIN), and that both SIC and SIN are required for optimal inhibition of Ci/Gli by Sufu. We show that Sufu can sequester Ci/Gli in the cytoplasm through binding to SIN while inhibiting Ci/Gli activity in the nucleus depending on SIC. We also find that binding of Sufu to SIC and the middle region of Ci can impede recruitment of the transcriptional coactivator CBP by masking its binding site in the C-terminal region of Ci. Indeed, moving the CBP-binding site to an "exposed" location can render Ci resistant to Sufu-mediated inhibition in the nucleus. Hence, our study identifies a previously unidentified and conserved Sufu-binding motif in the C-terminal region of Ci/Gli and provides mechanistic insight into how Sufu inhibits Ci/Gli activity in the nucleus.
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Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila/fisiologia , Proteínas Hedgehog/metabolismo , Proteínas Repressoras/metabolismo , Transdução de Sinais/fisiologia , Fatores de Transcrição/metabolismo , Animais , Sítios de Ligação , Western Blotting , Fracionamento Celular , Proteínas de Ligação a DNA/genética , Drosophila/metabolismo , Proteínas de Drosophila/genética , Imunoprecipitação , Luciferases , Proteínas Oncogênicas/genética , Proteínas Oncogênicas/metabolismo , Transativadores/genética , Transativadores/metabolismo , Fatores de Transcrição/genética , Proteína GLI1 em Dedos de ZincoRESUMO
The biological underpinnings and the pathological lesions of psychiatric disorders are centuries-old questions that have yet to be understood. Recent studies suggest that schizophrenia and related disorders likely have their origins in perturbed neurodevelopment and can result from a large number of common genetic variants or multiple, individually rare genetic alterations. It is thus conceivable that key neurodevelopmental pathways underline the various genetic changes and the still unknown pathological lesions in schizophrenia. Here, we report that mice defective of the nicastrin subunit of γ-secretase in oligodendrocytes have hypomyelination in the central nervous system. These mice have altered dopamine signaling and display profound abnormal phenotypes reminiscent of schizophrenia. In addition, we identify an association of the nicastrin gene with a human schizophrenia cohort. These observations implicate γ-secretase and its mediated neurodevelopmental pathways in schizophrenia and provide support for the "myelination hypothesis" of the disease. Moreover, by showing that schizophrenia and obsessive-compulsive symptoms could be modeled in animals wherein a single genetic factor is altered, our work provides a biological basis that schizophrenia with obsessive-compulsive disorder is a distinct subtype of schizophrenia.
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Secretases da Proteína Precursora do Amiloide/metabolismo , Comportamento Compulsivo , Glicoproteínas de Membrana/metabolismo , Bainha de Mielina/metabolismo , Oligodendroglia/metabolismo , Esquizofrenia/metabolismo , Secretases da Proteína Precursora do Amiloide/genética , Animais , Feminino , Humanos , Masculino , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Esquizofrenia/genéticaRESUMO
The Hedgehog (Hh) family of secreted proteins governs many key developmental processes by regulating Ci/Gli transcription factors at multiple levels including nuclear-cytoplasmic shuttling. Here, we investigate the mechanism underlying the regulation of Ci/Gli subcellular localization by identifying and characterizing a novel nuclear localization sequence (NLS) in the N-terminal conserved domain of Ci/Gli that matches the PY-NLS consensus. We demonstrate that the PY-NLS functions in parallel with a previously identified bipartite NLS to promote nuclear localization and activity of full-length Ci. We find that Transportin (Trn), the Drosophila homolog of Kapß2, is responsible for PY-NLS-mediated nuclear localization of Ci. Furthermore, we show that the tumor suppressor and conserved Hh pathway component Suppressor of fused (Sufu) opposes Trn-mediated Ci nuclear import by masking its PY-NLS. Finally, we provide evidence that Gli proteins also contain a functional PY-NLS and that mammal Sufu uses a similar mechanism to regulate nuclear translocation of Gli. Our study not only provides a mechanistic insight into how Sufu regulates Hh signaling and the subcellular localization of Ci/Gli, but also reveals a role for Trn/Kapß2 in developmental regulation.
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Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/fisiologia , Drosophila melanogaster/metabolismo , Carioferinas/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Oncogênicas/metabolismo , Proteínas Repressoras/fisiologia , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Transporte Ativo do Núcleo Celular , Sequência de Aminoácidos , Animais , Linhagem Celular , Sequência Conservada , Proteínas de Ligação a DNA/química , Proteínas de Drosophila/química , Dados de Sequência Molecular , Sinais de Localização Nuclear , Ligação Proteica , Transdução de Sinais , Fatores de Transcrição/química , Proteína GLI1 em Dedos de ZincoRESUMO
The plasmas generated by dielectric barrier discharge in atmospheric pressure air have wide application prospect in industry. In order to study generation condition and mechanism, the dielectric barrier uniform discharge in atmospheric pressure air has been studied experimentally with a micro-gap discharge device. Results of electrical characteristics indicate that it exist several current pulses with short width in half period of the applied voltage at a low voltage, a large number of micro-discharge filaments are observed. The discharge power increases with increasing peak value of applied voltage, the micro-discharge filaments increase meanwhile. When the peak of applied voltage reaches to 9.2 kV, only a discharge hump with a width of about 5.5 µs appears in a half period of the applied voltage, micro-discharge filaments cannot be discerned. The uniform discharge has been obtained finally as the micro-discharge filaments extend and superimpose randomly. The emission spectrum of dielectric barrier discharge scanning from 330 to 420 nm is collected. It is found that the intensity of 337.1 nm is stronger than that of 391.4 nm. If the intensity of 337.1 nm is considered as the datum reference, the intensity of 391.4 nm shows the magnitude of electron average energy. The molecule internal energy is evaluated by vibration temperature. The electron average energy and molecule internal energy have been investigated by optical emission spectra. It is found that both of them decrease with increasing the applied voltage. Results indicate that it is not easy to form filamentary discharge when the electrical energy is lower. The average electron energy of uniform discharge is lower than that of the filamentary discharge. These results are of great significance to the application of dielectric barrier uniform discharge obtained in air at atmospheric pressure.
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OBJECTIVE: The aim of this study was to investigate the expression and clinical significance of HIF-1α and DcR3 in endometriosis by analysing clinical case data. Tissue samples were collected for tissue chip analysis and staining, and human endometrial stromal cells were isolated and cultured for cell experiments. Additionally, experiments were conducted on collected peritoneal fluid to explore the association and role of HIF-1α and DcR3 in endometriosis. STUDY DESIGN: Patients who visited the Department of Obstetrics and Gynaecology at Central Hospital in Fengxian District, Shanghai, from January 2018 to December 2021 were recruited for this controlled study. Clinical data and tissue chip staining results were collected for multiple regression analysis on the clinical significance of HIF-1α and DcR3. Endometrial tissue, ovarian cysts, and pelvic fluid were collected, and human endometrial stromal cells were cultured. The impact of HIF-1α on DcR3 in different oxygen environments and its role in endometriosis were investigated through PCR, Western blotting, enzyme-linked immunosorbent assay, as well as adhesion and migration assays. RESULTS: In patients with endometriosis, the expression of DcR3 and HIF-1α was found to be upregulated and correlated in ectopic endometrium. The expression of DcR3 served as an indicator of the severity of endometriosis. Hypoxia induced the expression of DcR3, which was regulated by HIF-1α and promoted migration and adhesion. CONCLUSION: DcR3 can be used as a clinical indicator to assess the severity of endometriosis. The hypoxic environment in endometriosis enhances disease progression by regulating DcR3 through HIF-1α.
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Endometriose , Subunidade alfa do Fator 1 Induzível por Hipóxia , Membro 6b de Receptores do Fator de Necrose Tumoral , Feminino , Humanos , Endometriose/metabolismo , Endométrio/metabolismo , Hipóxia/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Células Estromais/metabolismo , Membro 6b de Receptores do Fator de Necrose Tumoral/metabolismoRESUMO
Septic encephalopathy (SE) represents a severe inflammatory syndrome linked to elevated septic mortality rates, lacking specific therapeutic interventions, and often resulting in enduring neurological sequelae. The present investigation endeavors to elucidate the involvement of C-X-C Motif Chemokine Receptor 2 (CXCR2) in the pathogenesis of SE and to explore the potential of CXCR2 modulation as a therapeutic avenue for SE. Employing a murine SE model induced by lipopolysaccharide (LPS) administration, CXCR2 knockout mice and the CXCR2 inhibitor SB225002 were utilized to assess neutrophil recruitment, endothelial integrity, and transendothelial migration. Our findings substantiate that either CXCR2 deficiency or its inhibition curtails neutrophil recruitment without impacting their adhesion to cerebral endothelial cells. This phenomenon is contingent upon endothelial CXCR2 expression rather than CXCR2's presence on neutrophils. Furthermore, the CXCR2 blockade preserves the integrity of tight junction protein ZO-1 and mitigates F-actin stress fiber formation in cerebral endothelial cells following septic challenge. Mechanistically, CXCL1-mediated CXCR2 activation triggers cerebral endothelial actin contraction via Rho signaling, thereby facilitating neutrophil transmigration in SE. These observations advocate for the potential therapeutic efficacy of CXCR2 inhibition in managing SE.
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The Hedgehog (Hh) family of secreted proteins governs embryonic development and adult tissue homeostasis through the Gli family of transcription factors. Gli is thought to be activated at the tip of primary cilium, but the underlying mechanism has remained poorly understood. Here, we show that Unc-51-like kinase 4 (Ulk4), a pseudokinase and a member of the Ulk kinase family, acts in conjunction with another Ulk family member Stk36 to promote Gli2 phosphorylation and Hh pathway activation. Ulk4 interacts with Stk36 through its N-terminal region containing the pseudokinase domain and with Gli2 via its regulatory domain to bridge the kinase and substrate. Although dispensable for Hh-induced Stk36 kinase activation, Ulk4 is essential for Stk36 ciliary tip localization, Gli2 phosphorylation, and activation. In response to Hh, both Ulk4 and Stk36 colocalize with Gli2 at ciliary tip, and Ulk4 and Stk36 depend on each other for their ciliary tip accumulation. We further show that ciliary localization of Ulk4 depends on Stk36 kinase activity and phosphorylation of Ulk4 on Thr1023, and that ciliary tip accumulation of Ulk4 is essential for its function in the Hh pathway. Taken together, our results suggest that Ulk4 regulates Hh signaling by promoting Stk36-mediated Gli2 phosphorylation and activation at ciliary tip.
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Proteínas Hedgehog , Fatores de Transcrição Kruppel-Like , Feminino , Gravidez , Humanos , Fosforilação , Proteínas Hedgehog/metabolismo , Proteína Gli2 com Dedos de Zinco/metabolismo , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Fatores de Transcrição/metabolismo , Proteína GLI1 em Dedos de Zinco/genética , Proteína GLI1 em Dedos de Zinco/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismoRESUMO
Objective To investigate the effects of C-X-C motif chemokine ligand 1 (CXCL1) and its receptor CXCR2 on the cerebral endothelial cytoskeleton rearrangement and permeability in the inflammation of septic encephalopathy. Methods The murine model of septic encephalopathy was established by intraperitoneal injection of LPS (10 mg/kg). The levels of TNF-α and CXCL1 in the whole brain tissue were detected by ELISA. The expression of CXCR2 was detected by Western blot analysis after bEND.3 cells were stimulated with 500 ng/mL LPS and 200 ng/mL TNF-α. After treated with CXCL1(150 ng/mL), the changes of endothelial filamentous actin (F-actin) rearrangement in bEND.3 cells were observed by immuno-fluorescence staining. In the cerebral endothelial permeability test, bEND.3 cells were randomly divided into PBS control group, CXCL1 group, and CXCL1 combined with CXCR2 antagonist SB225002 group. Then endothelial transwell permeability assay kit was used to detect the endothelial permeability changes. After stimulated with CXCL1 in bEND.3 cells, Western blot analysis was used to detect the expression of protein kinase B (AKT) and phosphorylated-AKT (p-AKT). Results Intraperitoneal injection of LPS significantly increased the levels of TNF-α and CXCL1 in the whole brain. LPS and TNF-α both upregulated the expression of CXCR2 protein in bEND.3 cells. CXCL1 stimulation induced the endothelial cytoskeleton contraction, increased paracellular gap formation and elevated endothelial permeability in bEND.3 cells, which was inhibited by the pretreatment with SB225002(CXCR2 antagonist). Furthermore, CXCL1 stimulation also enhanced the phosphorylation of AKT in bEND.3 cells. Conclusion CXCL1 induces the cytoskeleton contraction and increased permeability through AKT phosphorylation in bEND.3 cells, which can be effectively inhibited by CXCR2 antagonist SB225002.
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Encefalopatias , Células Endoteliais , Animais , Camundongos , Proteínas Proto-Oncogênicas c-akt , Fosforilação , Lipopolissacarídeos , Fator de Necrose Tumoral alfa , Citoesqueleto , EndotélioRESUMO
TAR DNA-binding protein 43 (TDP-43) is associated with a spectrum of neurodegenerative diseases. Although TDP-43 resembles heterogeneous nuclear ribonucleoproteins, its RNA targets and physiological protein partners remain unknown. Here we identify RNA targets of TDP-43 from cortical neurons by RNA immunoprecipitation followed by deep sequencing (RIP-seq). The canonical TDP-43 binding site (TG)(n) is 55.1-fold enriched, and moreover, a variant with adenine in the middle, (TG)(n)TA(TG)(m), is highly abundant among reads in our TDP-43 RIP-seq library. TDP-43 RNA targets can be divided into three different groups: those primarily binding in introns, in exons, and across both introns and exons. TDP-43 RNA targets are particularly enriched for Gene Ontology terms related to synaptic function, RNA metabolism, and neuronal development. Furthermore, TDP-43 binds to a number of RNAs encoding for proteins implicated in neurodegeneration, including TDP-43 itself, FUS/TLS, progranulin, Tau, and ataxin 1 and -2. We also identify 25 proteins that co-purify with TDP-43 from rodent brain nuclear extracts. Prominent among them are nuclear proteins involved in pre-mRNA splicing and RNA stability and transport. Also notable are two neuron-enriched proteins, methyl CpG-binding protein 2 and polypyrimidine tract-binding protein 2 (PTBP2). A PTBP2 consensus RNA binding motif is enriched in the TDP-43 RIP-seq library, suggesting that PTBP2 may co-regulate TDP-43 RNA targets. This work thus reveals the protein and RNA components of the TDP-43-containing ribonucleoprotein complexes and provides a framework for understanding how dysregulation of TDP-43 in RNA metabolism contributes to neurodegeneration.
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Proteínas de Ligação a DNA/metabolismo , Neurônios/fisiologia , Estabilidade de RNA/fisiologia , RNA Mensageiro/metabolismo , Proteinopatias TDP-43/metabolismo , Animais , Córtex Cerebral/citologia , Proteínas de Ligação a DNA/química , Biblioteca Gênica , Genômica , Células HeLa , Humanos , Camundongos , Peso Molecular , Neurônios/citologia , Proteômica , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , RatosRESUMO
The Hedgehog (Hh) family of secreted proteins governs embryonic development and adult tissue homeostasis by regulating the abundance, localization, and activity of the GPCR family protein Smoothened (Smo). Smo trafficking and subcellular accumulation are controlled by multiple posttranslational modifications (PTMs) including phosphorylation, ubiquitination, and sumoylation, which appears to be conserved from Drosophila to mammals. Smo ubiquitination is dynamically regulated by E3 ubiquitin ligases and deubiquitinases (dubs) and is opposed by Hh signaling. By contrast, Smo sumoylation is stimulated by Hh, which counteracts Smo ubiquitination by recruiting the dub USP8. We describe cell-base assays for Smo ubiquitination and its regulation by Hh and the E3 ligases in Drosophila. We also describe assays for Smo sumoylation in both Drosophila and mammalian cultured cells.
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Sumoilação , Ubiquitinação , Animais , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Proteínas Hedgehog/metabolismo , Mamíferos/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptor Smoothened/genética , Receptor Smoothened/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Hedgehog (Hh) signaling culminates in the conversion of the latent transcription factor Cubitus interruptus (Ci)/Gli from a repressor form (CiR/GliR) into an activator form (CiA/GliA). While sequential phosphorylation of Ci/Gli by protein kinase A(PKA), glycogen synthase kinase 3 (GSK3), and casein kinase 1 (CK1) is essential for its proteolytic processing that generates CiR/GliR, sequential phosphorylation of Ci/Gli by the Fused (Fu)/Unc-51 like kinase (Ulk) family kinases Fu/Ulk3/Stk36 and CK1 contributes to the formation of CiA/GliA. Fu/Ulk3/Stk36-mediated phosphorylation of Ci/Gli is stimulated by Hh, leading to altered interaction between Ci/Gli and the Hh pathway repressor Sufu. Here we describe both in vitro and in vivo assays that determine Ci/Gli phosphorylation by the Fu/Ulk family kinases and its regulation by Hh.
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Fosforilação , Caseína Quinase I/genética , Proteínas Quinases Dependentes de AMP Cíclico , Proteínas de Ligação a DNA , Quinase 3 da Glicogênio Sintase , Proteínas Hedgehog/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismo , Proteína GLI1 em Dedos de ZincoRESUMO
The GPCR-family protein Smoothened (Smo) is an obligatory signal transducer of the Hedgehog (Hh) signaling pathway. Binding of Hh to its receptor Patched (Ptc) alleviates Ptc-mediated inhibition of Smo, allowing Smo to activate the Cubitus interruptus (Ci)/Gli family of zinc finger transcription factors. The activation of Smo is an early and crucial event in Hh signal transduction. Studies have shown that Hh induces cell surface/ciliary accumulation and phosphorylation of Smo by multiple kinases, including protein kinase A (PKA), casein kinase 1 (CK1), casein kinase 2 (CK2), G protein-coupled receptor kinase 2 (Gprk2/GRK2), and atypical PKC (aPKC). Here, we describe the assays used to examine the phosphorylation and activity of Smo, including in vitro kinase assay, phospho-specific antibodies, luciferase reporter assay, cell surface accumulation, and ciliary localization assays. These assays provide powerful tools to study Smo phosphorylation and activation, leading to mechanistic insight into Smo regulation.