RESUMEN
Introduction: Oxysterol-binding protein (OSBP) is known for its crucial role in lipid transport, facilitating cholesterol exchange between the Golgi apparatus and endoplasmic reticulum membranes. Despite its established function in cellular processes, its involvement in coronavirus replication remains unclear. Methods: In this study, we investigated the role of OSBP in coronavirus replication and explored the potential of a novel OSBP-binding compound, ZJ-1, as an antiviral agent against coronaviruses, including SARS-CoV-2. We utilized a combination of biochemical and cellular assays to elucidate the interactions between OSBP and SARS-CoV-2 non-structural proteins (Nsps) and other viral proteins. Results: Our findings demonstrate that OSBP positively regulates coronavirus replication. Moreover, treatment with ZJ-1 resulted in reduced OSBP levels and exhibited potent antiviral effects against multiple coronaviruses. Through our investigation, we identified specific interactions between OSBP and SARS-CoV-2 Nsps, particularly Nsp3, Nsp4, and Nsp6, which are involved in double-membrane vesicle formation-a crucial step in viral replication. Additionally, we observed that Nsp3 a.a.1-1363, Nsp4, and Nsp6 target vesicle-associated membrane protein (VAMP)-associated protein B (VAP-B), which anchors OSBP to the ER membrane. Interestingly, the interaction between OSBP and VAP-B is disrupted by Nsp3 a.a.1-1363 and partially impaired by Nsp6. Furthermore, we identified SARS-CoV-2 orf7a, orf7b, and orf3a as additional OSBP targets, with OSBP contributing to their stabilization. Conclusion: Our study highlights the significance of OSBP in coronavirus replication and identifies it as a promising target for the development of antiviral therapies against SARS-CoV-2 and other coronaviruses. These findings underscore the potential of OSBP-targeted interventions in combating coronavirus infections.
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Antivirales , Receptores de Esteroides , SARS-CoV-2 , Proteínas no Estructurales Virales , Replicación Viral , Replicación Viral/efectos de los fármacos , Humanos , SARS-CoV-2/efectos de los fármacos , SARS-CoV-2/fisiología , Antivirales/farmacología , Receptores de Esteroides/metabolismo , Proteínas no Estructurales Virales/metabolismo , COVID-19/virología , COVID-19/metabolismo , Chlorocebus aethiops , Células Vero , Proteínas Virales/metabolismo , Células HEK293 , Animales , Retículo Endoplásmico/metabolismo , Retículo Endoplásmico/virología , Proteínas Viroporinas/metabolismo , Proteasas Similares a la Papaína de Coronavirus/metabolismo , Unión ProteicaRESUMEN
Innate immune responses are linked to key metabolic pathways, yet the proximal signaling events that connect these systems remain poorly understood. Here we show that phosphofructokinase 1, liver type (PFKL), a rate-limiting enzyme of glycolysis, is phosphorylated at Ser775 in macrophages following several innate stimuli. This phosphorylation increases the catalytic activity of PFKL, as shown by biochemical assays and glycolysis monitoring in cells expressing phosphorylation-defective PFKL variants. Using a genetic mouse model in which PFKL Ser775 phosphorylation cannot take place, we observe that upon activation, glycolysis in macrophages is lower than in the same cell population of wild-type animals. Consistent with their higher glycolytic activity, wild-type cells have higher levels of HIF1α and IL-1ß than PfklS775A/S775A after LPS treatment. In an in vivo inflammation model, PfklS775A/S775A mice show reduced levels of MCP-1 and IL-1ß. Our study thus identifies a molecular link between innate immune activation and early induction of glycolysis.
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Glucólisis , Subunidad alfa del Factor 1 Inducible por Hipoxia , Inmunidad Innata , Interleucina-1beta , Macrófagos , Animales , Macrófagos/metabolismo , Macrófagos/inmunología , Ratones , Fosforilación , Interleucina-1beta/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Receptores de Reconocimiento de Patrones/metabolismo , Receptores de Reconocimiento de Patrones/genética , Fosfofructoquinasa-1/metabolismo , Fosfofructoquinasa-1/genética , Lipopolisacáridos/farmacología , Ratones Endogámicos C57BL , Humanos , Quimiocina CCL2/metabolismo , Quimiocina CCL2/genética , Inflamación/metabolismo , Masculino , Reprogramación MetabólicaRESUMEN
Fms-like tyrosine kinase 3 (FLT3) is often overexpressed or constitutively activated by internal tandem duplication (ITD) and tyrosine kinase domain (TKD) mutations in acute myeloid leukemia (AML). Despite the use of receptor tyrosine kinase inhibitors (TKI) in FLT3-ITD-positive AML, the prognosis of patients is still poor, and further improvement of therapy is required. Targeting FLT3 independent of mutations by antibody-drug conjugates (ADCs) is a promising strategy for AML therapy. Here, we report the development and preclinical characterization of a novel FLT3-targeting ADC, 20D9-ADC, which was generated by applying the innovative P5 conjugation technology. In vitro, 20D9-ADC mediated potent cytotoxicity to Ba/F3 cells expressing transgenic FLT3 or FLT3-ITD, to AML cell lines, and to FLT3-ITD-positive patient-derived xenograft AML cells. In vivo, 20D9-ADC treatment led to a significant tumor reduction and even durable complete remission in AML xenograft models. Furthermore, 20D9-ADC demonstrated no severe hematotoxicity in in vitro colony formation assays using concentrations that were cytotoxic in AML cell line treatment. The combination of 20D9-ADC with the TKI midostaurin showed strong synergy in vitro and in vivo, leading to reduction of aggressive AML cells below the detection limit. Our data indicate that targeting FLT3 with an advanced new-generation ADC is a promising and potent antileukemic strategy, especially when combined with FLT3-TKI in FLT3-ITD-positive AML.
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Antineoplásicos , Leucemia Mieloide Aguda , Humanos , Tirosina Quinasa 3 Similar a fms/genética , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patología , MutaciónRESUMEN
Cilia are protrusions of the cell surface and composed of hundreds of proteins many of which are evolutionary and functionally well conserved. In cells assembling motile cilia the expression of numerous ciliary components is under the control of the transcription factor FOXJ1. Here, we analyse the evolutionary conserved FOXJ1 target CFAP161 in Xenopus and mouse. In both species Cfap161 expression correlates with the presence of motile cilia and depends on FOXJ1. Tagged CFAP161 localises to the basal bodies of multiciliated cells of the Xenopus larval epidermis, and in mice CFAP161 protein localises to the axoneme. Surprisingly, disruption of the Cfap161 gene in both species did not lead to motile cilia-related phenotypes, which contrasts with the conserved expression in cells carrying motile cilia and high sequence conservation. In mice mutation of Cfap161 stabilised the mutant mRNA making genetic compensation triggered by mRNA decay unlikely. However, genes related to microtubules and cilia, microtubule motor activity and inner dyneins were dysregulated, which might buffer the Cfap161 mutation.
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Cilios/metabolismo , Factores de Transcripción Forkhead/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/metabolismo , Animales , Axonema/metabolismo , Cuerpos Basales/metabolismo , Células Epidérmicas/metabolismo , Epidermis/metabolismo , Femenino , Masculino , Ratones , Microtúbulos/metabolismoRESUMEN
The p14ARF protein is a well-known regulator of p53-dependent and p53-independent tumor-suppressive activities. In unstressed cells, p14ARF is predominantly sequestered in the nucleoli, bound to its nucleolar interaction partner NPM. Upon genotoxic stress, p14ARF undergoes an immediate redistribution to the nucleo- and cytoplasm, where it promotes activation of cell cycle arrest and apoptosis. Here, we identify p14ARF as a novel interaction partner and substrate of PRMT1 (protein arginine methyltransferase 1). PRMT1 methylates several arginine residues in the C-terminal nuclear/nucleolar localization sequence (NLS/NoLS) of p14ARF . In the absence of cellular stress, these arginines are crucial for nucleolar localization of p14ARF . Genotoxic stress causes augmented interaction between PRMT1 and p14ARF , accompanied by arginine methylation of p14ARF . PRMT1-dependent NLS/NoLS methylation promotes the release of p14ARF from NPM and nucleolar sequestration, subsequently leading to p53-independent apoptosis. This PRMT1-p14ARF cooperation is cancer-relevant and indicative for PDAC (pancreatic ductal adenocarcinoma) prognosis and chemotherapy response of pancreatic tumor cells. Our data reveal that PRMT1-mediated arginine methylation is an important trigger for p14ARF 's stress-induced tumor-suppressive function.
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Neoplasias Pancreáticas/metabolismo , Proteína-Arginina N-Metiltransferasas/metabolismo , Proteínas Represoras/metabolismo , Proteína p14ARF Supresora de Tumor/metabolismo , Animales , Apoptosis/fisiología , Ciclo Celular/fisiología , Línea Celular , Línea Celular Tumoral , Nucléolo Celular/metabolismo , Núcleo Celular/metabolismo , Células HEK293 , Células HeLa , Humanos , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Neoplasias Pancreáticas/patología , Pronóstico , Células Sf9 , Proteína p53 Supresora de Tumor/metabolismo , Neoplasias PancreáticasRESUMEN
Eukaryotic DNA replication initiates during S phase from origins that have been licensed in the preceding G1 phase. Here, we compare ChIP-seq profiles of the licensing factors Orc2, Orc3, Mcm3, and Mcm7 with gene expression, replication timing, and fork directionality profiles obtained by RNA-seq, Repli-seq, and OK-seq. Both, the origin recognition complex (ORC) and the minichromosome maintenance complex (MCM) are significantly and homogeneously depleted from transcribed genes, enriched at gene promoters, and more abundant in early- than in late-replicating domains. Surprisingly, after controlling these variables, no difference in ORC/MCM density is detected between initiation zones, termination zones, unidirectionally replicating regions, and randomly replicating regions. Therefore, ORC/MCM density correlates with replication timing but does not solely regulate the probability of replication initiation. Interestingly, H4K20me3, a histone modification proposed to facilitate late origin licensing, was enriched in late-replicating initiation zones and gene deserts of stochastic replication fork direction. We discuss potential mechanisms specifying when and where replication initiates in human cells.
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Replicación del ADN/genética , Proteínas de Mantenimiento de Minicromosoma/genética , Modelos Genéticos , Complejo de Reconocimiento del Origen/genética , Línea Celular Tumoral , Humanos , Proteínas de Mantenimiento de Minicromosoma/metabolismo , Complejo de Reconocimiento del Origen/metabolismoRESUMEN
BACKGROUND & AIMS: RING finger protein 43 (RNF43) is a tumor suppressor that frequently is mutated in gastric tumors. The link between RNF43 and modulation of Wingless-related integration site (WNT) signaling has not been shown clearly in the stomach. Because mutations in RNF43 are highly enriched in microsatellite-unstable gastric tumors, which show defects in DNA damage response (DDR), we investigated whether RNF43 is involved in DDR in the stomach. METHODS: DDR activation and cell viability upon γ-radiation was analyzed in gastric cells where expression of RNF43 was depleted. Response to chemotherapeutic agents 5-fluorouracil and cisplatin was analyzed in gastric cancer cell lines and xenograft tumors. In addition, involvement of RNF43 in DDR activation was analyzed upon Helicobacter pylori infection in wild-type and Rnf43ΔEx8 mice. Furthermore, a cohort of human gastric biopsy specimens was analyzed for RNF43 expression and mutation status as well as for activation of DDR. RESULTS: RNF43 depletion conferred resistance to γ-radiation and chemotherapy by dampening the activation of DDR, thereby preventing apoptosis in gastric cells. Upon Helicobacter pylori infection, RNF43 loss of function reduced activation of DDR and apoptosis. Furthermore, RNF43 expression correlated with DDR activation in human gastric biopsy specimens, and RNF43 mutations found in gastric tumors conferred resistance to DNA damage. When exploring the molecular mechanisms behind these findings, a direct interaction between RNF43 and phosphorylated H2A histone family member X (γH2AX) was observed. CONCLUSIONS: We identified a novel function for RNF43 in the stomach as a regulator of DDR. Loss of RNF43 function in gastric cells confers resistance to DNA damage-inducing radiotherapy and chemotherapy, suggesting RNF43 as a possible biomarker for therapy selection.
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Carcinogénesis/patología , Daño del ADN , Gastritis/patología , Infecciones por Helicobacter/complicaciones , Neoplasias Gástricas/patología , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/fisiología , Adulto , Anciano , Anciano de 80 o más Años , Animales , Apoptosis , Carcinogénesis/metabolismo , Estudios de Casos y Controles , Proliferación Celular , Femenino , Gastritis/etiología , Gastritis/metabolismo , Infecciones por Helicobacter/microbiología , Helicobacter pylori/aislamiento & purificación , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Persona de Mediana Edad , Neoplasias Gástricas/etiología , Neoplasias Gástricas/metabolismo , Células Tumorales Cultivadas , Ubiquitina-Proteína Ligasas/genética , Vía de Señalización Wnt , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Cilia are complex cellular protrusions consisting of hundreds of proteins. Defects in ciliary structure and function, many of which have not been characterised molecularly, cause ciliopathies: a heterogeneous group of human syndromes. Here, we report on the FOXJ1 target gene Cfap206, orthologues of which so far have only been studied in Chlamydomonas and Tetrahymena In mouse and Xenopus, Cfap206 was co-expressed with and dependent on Foxj1 CFAP206 protein localised to the basal body and to the axoneme of motile cilia. In Xenopus crispant larvae, the ciliary beat frequency of skin multiciliated cells was enhanced and bead transport across the epidermal mucociliary epithelium was reduced. Likewise, Cfap206 knockout mice revealed ciliary phenotypes. Electron tomography of immotile knockout mouse sperm flagella indicated a role in radial spoke formation reminiscent of FAP206 function in Tetrahymena Male infertility, hydrocephalus and impaired mucociliary clearance of the airways in the absence of laterality defects in Cfap206 mutant mice suggests that Cfap206 may represent a candidate for the subgroup of human primary ciliary dyskinesias caused by radial spoke defects.
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Encéfalo/embriología , Encéfalo/metabolismo , Proteínas del Citoesqueleto/metabolismo , Factores de Transcripción Forkhead/metabolismo , Pulmón/metabolismo , Depuración Mucociliar , Motilidad Espermática , Animales , Axonema/metabolismo , Cuerpos Basales/metabolismo , Cilios/metabolismo , Proteínas del Citoesqueleto/química , Desarrollo Embrionario , Células Epiteliales/metabolismo , Fluorescencia , Hidrocefalia/patología , Infertilidad Masculina/patología , Masculino , Ratones Noqueados , Moco/metabolismo , Mutación/genética , Transporte de Proteínas , Espermatozoides/metabolismo , Espermatozoides/ultraestructura , Xenopus laevis/embriología , Xenopus laevis/metabolismoRESUMEN
Malfunctions of motile cilia cause a variety of developmental defects and diseases in humans and animal model organisms. Defects include impaired mucociliary clearance of the airways, sperm immotility, hydrocephalus and organ laterality. Here, we characterize the evolutionary conserved Cfap43 gene by loss-of-function experiments in the mouse and the frog Xenopus laevis. Cfap43 is expressed in tissues carrying motile cilia and acts as a target gene of the transcription factor FOXJ1, which is essential for the induction of motile ciliogenesis. We show that CFAP43, a protein of unknown biochemical function, localizes to the ciliary axoneme. CFAP43 is involved in the regulation of the beating frequency of tracheal cilia and loss of CFAP43 causes severe mucus accumulation in the nasal cavity. Likewise, morphant and crispant frog embryos revealed impaired function of motile cilia of the larval epidermis, a model for airway mucociliary epithelia. CFAP43 participates in the formation of flagellar axonemes during spermatogenesis as mice mutant for Cfap43 display male infertility, consistent with observations in male sterile patients. In addition, mice mutant for Cfap43 display early onset hydrocephalus. Together, these results confirm the role of CFAP43 in the male reproductive tract and pinpoint additional functions in airway epithelia mucus clearance and brain development.
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Cilios/metabolismo , Proteínas del Citoesqueleto/metabolismo , Proteínas de Xenopus/metabolismo , Animales , Proteínas del Citoesqueleto/genética , Células Epidérmicas/metabolismo , Factores de Transcripción Forkhead/metabolismo , Hidrocefalia/genética , Infertilidad Masculina/genética , Masculino , Ratones , Ratones Noqueados , Cola del Espermatozoide/metabolismo , Espermatogénesis/genética , Espermatozoides/metabolismo , Tráquea/citología , Proteínas de Xenopus/genética , Xenopus laevisRESUMEN
Chitinase-like proteins (CLP) are chitin-binding proteins that lack chitin hydrolyzing activity, but possess cytokine-like and growth factor-like properties, and play crucial role in intercellular crosstalk. Both human and mice express two members of CLP family: YKL-40 and stabilin-1 interacting chitinase-like protein (SI-CLP). Despite numerous reports indicating the role of YKL-40 in the support of angiogenesis, tumor cell proliferation, invasion and metastasis, the role of its structurally related protein SI-CLP in cancer was not reported. Using gain-of-function approach, we demonstrate in the current study that the expression of recombinant SI-CLP in mouse TS/A mammary adenocarcinoma cells results in significant and persistent inhibition of in vivo tumor growth. Using quantitative immunohistochemistry, we show that on the cellular level this phenomenon is associated with reduced infiltration of tumor-associated macrophages (TAMs), CD4+ and FoxP3+ cells in SI-CLP expressing tumors. Gene expression analysis in TAM isolated from SI-CLP-expressing and control tumors demonstrated that SI-CLP does not affect macrophage phenotype. However, SI-CLP significantly inhibited migration of murine bone-marrow derived macrophages and human primary monocytes toward monocyte-recruiting chemokine CCL2 produced in the tumor microenvironment (TME). Mechanistically, SI-CLP did not affect CCL2/CCR2 interaction, but suppressed cytoskeletal rearrangements in response to CCL2. Altogether, our data indicate that SI-CLP functions as a tumor growth inhibitor in mouse breast cancer by altering cellular composition of TME and blocking cytokine-induced TAM recruitment. Taking into consideration weak to absent expression of SI-CLP in human breast cancer, it can be considered as a therapeutic protein to block TAM-mediated support of breast tumor growth.
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Proteínas de Unión al Calcio/inmunología , Proteínas Portadoras/inmunología , Macrófagos/inmunología , Neoplasias Mamarias Experimentales/inmunología , Animales , Neoplasias de la Mama/inmunología , Neoplasias de la Mama/patología , Procesos de Crecimiento Celular/inmunología , Movimiento Celular/inmunología , Femenino , Células HEK293 , Humanos , Activación de Macrófagos , Neoplasias Mamarias Experimentales/patología , Ratones , Ratones Endogámicos BALB C , Persona de Mediana EdadRESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMEN
Preblastoderm Drosophila embryo development is characterized by fast cycles of nuclear divisions. Extracts from these embryos can be used to reconstitute complex chromatin with high efficiency. We now discovered that this chromatin assembly system contains activities that recognize unprotected DNA ends and signal DNA damage through phosphorylation. DNA ends are initially bound by Ku and MRN complexes. Within minutes, the phosphorylation of H2A.V (homologous to γH2A.X) initiates from DNA breaks and spreads over tens of thousands DNA base pairs. The γH2A.V phosphorylation remains tightly associated with the damaged DNA and does not spread to undamaged DNA in the same reaction. This first observation of long-range γH2A.X spreading along damaged chromatin in an in vitro system provides a unique opportunity for mechanistic dissection. Upon further incubation, DNA ends are rendered single-stranded and bound by the RPA complex. Phosphoproteome analyses reveal damage-dependent phosphorylation of numerous DNA-end-associated proteins including Ku70, RPA2, CHRAC16, the exonuclease Rrp1 and the telomer capping complex. Phosphorylation of spindle assembly checkpoint components and of microtubule-associated proteins required for centrosome integrity suggests this cell-free system recapitulates processes involved in the regulated elimination of fatally damaged syncytial nuclei.
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Sistema Libre de Células/metabolismo , Roturas del ADN , Drosophila/genética , Transducción de Señal , Animales , Línea Celular , Cromatina/genética , Cromatina/metabolismo , Reparación del ADN , Drosophila/citología , Drosophila/embriología , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Histonas/genética , Histonas/metabolismo , Autoantígeno Ku/genética , Autoantígeno Ku/metabolismo , Fosforilación , Proteoma/genética , Proteoma/metabolismo , Proteómica/métodosRESUMEN
Kaposi's sarcoma-associated herpesvirus (KSHV; human herpesvirus 8) belongs to the subfamily of Gammaherpesvirinae and is the etiological agent of Kaposi's sarcoma as well as of two lymphoproliferative diseases: primary effusion lymphoma and multicentric Castleman disease. The KSHV life cycle is divided into a latent and a lytic phase and is highly regulated by viral immunomodulatory proteins which control the host antiviral immune response. Among them is a group of proteins with homology to cellular interferon regulatory factors, the viral interferon regulatory factors 1-4. The KSHV vIRFs are known as inhibitors of cellular interferon signaling and are involved in different oncogenic pathways. Here we characterized the role of the second vIRF protein, vIRF2, during the KSHV life cycle. We found the vIRF2 protein to be expressed in different KSHV positive cells with early lytic kinetics. Importantly, we observed that vIRF2 suppresses the expression of viral early lytic genes in both newly infected and reactivated persistently infected endothelial cells. This vIRF2-dependent regulation of the KSHV life cycle might involve the increased expression of cellular interferon-induced genes such as the IFIT proteins 1, 2 and 3, which antagonize the expression of early KSHV lytic proteins. Our findings suggest a model in which the viral protein vIRF2 allows KSHV to harness an IFN-dependent pathway to regulate KSHV early gene expression.
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Endotelio Vascular/virología , Regulación Viral de la Expresión Génica , Herpesvirus Humano 8/fisiología , Proteínas Inmediatas-Precoces/metabolismo , Factores Reguladores del Interferón/metabolismo , Interferones/metabolismo , Sarcoma de Kaposi/virología , Proteínas Virales/metabolismo , Activación Viral , Células Cultivadas , Endotelio Vascular/metabolismo , Endotelio Vascular/patología , Humanos , Proteínas Inmediatas-Precoces/genética , Factores Reguladores del Interferón/genética , Interferones/genética , Sarcoma de Kaposi/genética , Sarcoma de Kaposi/metabolismo , Proteínas Virales/genética , Latencia del VirusRESUMEN
The neural crest (NC) gives rise to a multitude of fetal tissues, and its misregulation is implicated in congenital malformations. Here, we investigated molecular mechanisms pertaining to NC-related symptoms in Bohring-Opitz syndrome (BOS), a developmental disorder linked to mutations in the Polycomb group factor Additional sex combs-like 1 (ASXL1). Genetically edited human pluripotent stem cell lines that were differentiated to NC progenitors and then xenotransplanted into chicken embryos demonstrated an impairment of NC delamination and emigration. Molecular analysis showed that ASXL1 mutations correlated with reduced activation of the transcription factor ZIC1 and the NC gene regulatory network. These findings were supported by differentiation experiments using BOS patient-derived induced pluripotent stem cell lines. Expression of truncated ASXL1 isoforms (amino acids 1-900) recapitulated the NC phenotypes in vitro and in ovo, raising the possibility that truncated ASXL1 variants contribute to BOS pathology. Collectively, we expand the understanding of truncated ASXL1 in BOS and in the human NC.
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Diferenciación Celular/genética , Craneosinostosis/genética , Perfilación de la Expresión Génica/métodos , Discapacidad Intelectual/genética , Mutación , Cresta Neural/metabolismo , Células Madre Pluripotentes/metabolismo , Proteínas Represoras/genética , Animales , Línea Celular , Células Cultivadas , Embrión de Pollo , Craneosinostosis/metabolismo , Craneosinostosis/patología , Redes Reguladoras de Genes , Células Madre Embrionarias Humanas/citología , Células Madre Embrionarias Humanas/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Discapacidad Intelectual/metabolismo , Discapacidad Intelectual/patología , Cresta Neural/citología , Células Madre Pluripotentes/citología , Proteínas Represoras/metabolismo , Trasplante HeterólogoRESUMEN
The determination of unique functions of GABARAP (gamma-aminobutyric acid type A receptor-associated protein), a member of the highly conserved protein family of mammalian autophagy-related 8 protein (mATG8), within diverse cellular processes remains challenging. Because available anti-GABARAP antibodies perform inadequate, especially within various microscopy-based applications, we aimed to develop an antibody that targets GABARAP but not its close orthologs. Following the latest recommendations for antibody validation including fluorescence protein tagging, genetic and orthogonal strategies, we characterized the resulting anti-GABARAP (8H5) antibody during confocal immunofluorescence imaging in-depth. We compared the antibody staining pattern with that obtained for fluorescence protein tagged GABARAP, GABARAPL1 or GABARAPL2 each ectopically expressed in GABARAP knockout cells. Furthermore, we imaged cells expressing all mATG8 family members at endogenous levels and checked GABARAP knockout cells for unspecific staining under fed or macroautophagy-inducing conditions. Finally, we simultaneously stained cells for endogenous GABARAP and the common autophagosomal marker LC3B. Summarized, the presented antibody shows high specificity for GABARAP without cross-reactivity to other mATG8 family members in immunofluorescence imaging making it a valuable tool for the identification of unique GABARAP functions.
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Anticuerpos Monoclonales/análisis , Proteínas Reguladoras de la Apoptosis/análisis , Técnica del Anticuerpo Fluorescente/métodos , Proteínas Asociadas a Microtúbulos/análisis , Secuencia de Aminoácidos , Animales , Línea Celular , Humanos , Imagen Óptica/métodos , RatasRESUMEN
The chemokine receptor CCR7 and its ligands CCL19 and CCL21 guide the homing and positioning of dendritic and T cells in lymphoid organs, thereby contributing to several aspects of adaptive immunity and immune tolerance. In the present study, we investigated the role of CCR7 in the pathogenesis of collagen-induced arthritis (CIA). By using a novel anti-human CCR7 antibody and humanized CCR7 mice, we evaluated CCR7 as a target in this autoimmune model of rheumatoid arthritis (RA). Ccr7-deficient mice were completely resistant to CIA and presented severely impaired antibody responses to collagen II (CII). Selective CCR7 expression on dendritic cells restored arthritis severity and anti-CII antibody titers. Prophylactic and therapeutic treatment of humanized CCR7 mice with anti-human CCR7 mAb 8H3-16A12 led to complete resistance to CIA and halted CIA progression, respectively. Our data demonstrate that CCR7 signaling is essential for the induction of CIA and identify CCR7 as a potential therapeutic target in RA.
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Artritis Experimental/metabolismo , Artritis Reumatoide/metabolismo , Células Dendríticas/inmunología , Receptores CCR7/metabolismo , Animales , Anticuerpos Monoclonales/metabolismo , Autoanticuerpos/metabolismo , Colágeno Tipo II/inmunología , Progresión de la Enfermedad , Resistencia a la Enfermedad , Humanos , Ratones , Ratones Noqueados , Terapia Molecular Dirigida , Receptores CCR7/genética , Receptores CCR7/inmunologíaRESUMEN
The intellectual disability gene, Sox11, encodes for a critical neurodevelopmental transcription factor with functions in precursor survival, neuronal fate determination, migration and morphogenesis. The mechanisms regulating SOX11's activity remain largely unknown. Mass spectrometric analysis uncovered that SOX11 can be post-translationally modified by phosphorylation. Here, we report that phosphorylatable serines surrounding the high-mobility group box modulate SOX11's transcriptional activity. Through Mass Spectrometry (MS), co-immunoprecipitation assays and in vitro phosphorylation assays followed by MS we verified that protein kinase A (PKA) interacts with SOX11 and phosphorylates it on S133. In vivo replacement of SoxC factors in developing adult-generated hippocampal neurons with SOX11 S133 phospho-mutants indicated that phosphorylation on S133 modulates dendrite development of adult-born dentate granule neurons, while reporter assays suggested that S133 phosphorylation fine-tunes the activation of select target genes. These data provide novel insight into the control of the critical neurodevelopmental regulator SOX11 and imply SOX11 as a mediator of PKA-regulated neuronal development.
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Morfogénesis/genética , Neurogénesis/genética , Neuronas/metabolismo , Factores de Transcripción SOXC/genética , Animales , Núcleos Cerebelosos/crecimiento & desarrollo , Núcleos Cerebelosos/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/química , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Dendritas/genética , Dendritas/metabolismo , Hipocampo/crecimiento & desarrollo , Hipocampo/metabolismo , Espectrometría de Masas , Ratones , Fosforilación/genética , Serina/genéticaRESUMEN
Protein arginine methyltransferase 6 (PRMT6) catalyzes asymmetric dimethylation of histone H3 at arginine 2 (H3R2me2a). This mark has been reported to associate with silent genes. Here, we use a cell model of neural differentiation, which upon PRMT6 knockout exhibits proliferation and differentiation defects. Strikingly, we detect PRMT6-dependent H3R2me2a at active genes, both at promoter and enhancer sites. Loss of H3R2me2a from promoter sites leads to enhanced KMT2A binding and H3K4me3 deposition together with increased target gene transcription, supporting a repressive nature of H3R2me2a. At enhancers, H3R2me2a peaks co-localize with the active enhancer marks H3K4me1 and H3K27ac. Here, loss of H3R2me2a results in reduced KMT2D binding and H3K4me1/H3K27ac deposition together with decreased transcription of associated genes, indicating that H3R2me2a also exerts activation functions. Our work suggests that PRMT6 via H3R2me2a interferes with the deposition of adjacent histone marks and modulates the activity of important differentiation-associated genes by opposing transcriptional effects.
Asunto(s)
Código de Histonas , Histonas/metabolismo , Proteínas Nucleares/genética , Procesamiento Proteico-Postraduccional , Proteína-Arginina N-Metiltransferasas/genética , Activación Transcripcional , Animales , Elementos de Facilitación Genéticos , Células HEK293 , Células HeLa , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Histonas/química , Humanos , Metilación , Ratones , Proteína de la Leucemia Mieloide-Linfoide/genética , Proteína de la Leucemia Mieloide-Linfoide/metabolismo , Neurogénesis/genética , Proteínas Nucleares/metabolismo , Unión Proteica , Proteína-Arginina N-Metiltransferasas/metabolismoRESUMEN
The GTP-binding protein septin 7 is involved in various cellular processes, including cytoskeleton organization, migration and the regulation of cell shape. Septin 7 function in lymphocytes, however, is poorly characterized. Since the intracellular signaling role of septin 7 is dependent on its interaction network, interaction proteomics was applied to attain novel knowledge about septin 7 function in hematopoietic cells. Our previous finding of decreased septin 7 expression in blood-derived lymphocytes in ERU, a spontaneous animal model for autoimmune uveitis in man, extended the role of septin 7 to a potential key player in autoimmunity. Here, we revealed novel insights into septin 7 function by identification of DOCK8 as an interaction partner in primary blood-derived lymphocytes. Since DOCK8 is associated with important immune functions, our finding of significantly decreased DOCK8 expression and altered DOCK8 interaction network in ERU might explain changes in immune response and shows the contribution of DOCK8 in pathomechanisms of spontaneous autoimmune diseases. Moreover, our analyses revealed insights in DOCK8 function, by identifying the signal transducer ILK as a DOCK8 interactor in lymphocytes. Our finding of the enhanced enrichment of ILK in ERU cases indicates a deviant influence of DOCK8 on inter- and intracellular signaling in autoimmune disease.
Asunto(s)
Autoinmunidad , Proteínas de Ciclo Celular/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Linfocitos/metabolismo , Septinas/metabolismo , Transducción de Señal , Animales , Apoptosis , Enfermedades Autoinmunes/inmunología , Enfermedades Autoinmunes/metabolismo , Enfermedades Autoinmunes/mortalidad , Biomarcadores , Estudios de Casos y Controles , Cromatografía Liquida , Modelos Animales de Enfermedad , Caballos , Subgrupos Linfocitarios/inmunología , Subgrupos Linfocitarios/metabolismo , Linfocitos/inmunología , Unión Proteica , Espectrometría de Masas en TándemRESUMEN
In breast cancer, the tumor microenvironment plays a critical role in the tumor progression and responses to therapy. Tumor-associated macrophages (TAMs) are major innate immune cells in tumor microenvironment that regulate intratumoral immunity and angiogenesis by secretion of cytokines, growth factors as well as chitinase-like proteins (CLPs), that combine properties of cytokines and growth factors. YKL-39 is a chitinase-like protein found in human and absent in rodents, and its expression in TAMs and role in breast cancer progression was not studied to date. Here for the first time we demonstrate that YKL-39 is expressed on TAMs, predominantly positive for stabilin-1, but not by malignant cells or other stromal cells in human breast cancer. TGF-beta in combination with IL-4, but not IL-4 alone was responsible of the stimulation of the production of YKL-39 in human primary macrophages. Mechanistically, stabilin-1 directly interacted with YKL-39 and acted as sorting receptor for targeting YKL-39 into the secretory pathway. Functionally, purified YKL-39 acted as a strong chemotactic factor for primary human monocytes, and induced angiogenesis in vitro. Elevated levels of YKL-39 expression in tumors after neoadjuvant chemotherapy (NAC) were predictive for increased risk of distant metastasis and for poor response to NAC in patients with nonspecific invasive breast carcinoma. Our findings suggest YKL-39 as a novel therapeutic target, and blocking of its activity can be combined with NAC in order to reduce the risk of metastasis in breast cancer patients.