RESUMO
The presence of ribonucleotides in genomic DNA is undesirable given their increased susceptibility to hydrolysis. Ribonuclease (RNase) H enzymes that recognize and process such embedded ribonucleotides are present in all domains of life. However, in unicellular organisms such as budding yeast, they are not required for viability or even efficient cellular proliferation, while in humans, RNase H2 hypomorphic mutations cause the neuroinflammatory disorder Aicardi-Goutières syndrome. Here, we report that RNase H2 is an essential enzyme in mice, required for embryonic growth from gastrulation onward. RNase H2 null embryos accumulate large numbers of single (or di-) ribonucleotides embedded in their genomic DNA (>1,000,000 per cell), resulting in genome instability and a p53-dependent DNA-damage response. Our findings establish RNase H2 as a key mammalian genome surveillance enzyme required for ribonucleotide removal and demonstrate that ribonucleotides are the most commonly occurring endogenous nucleotide base lesion in replicating cells.
Assuntos
Replicação do DNA , Embrião de Mamíferos/metabolismo , Ribonuclease H/genética , Ribonuclease H/metabolismo , Ribonucleotídeos/metabolismo , Animais , Instabilidade Cromossômica , DNA Polimerase Dirigida por DNA/metabolismo , Células-Tronco Embrionárias/metabolismo , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
BACKGROUND: Effector functions of IgG Abs are regulated by their Fc N-glycosylation pattern. IgG Fc glycans that lack galactose and terminal sialic acid residues correlate with the severity of inflammatory (auto)immune disorders and have also been linked to protection against viral infection and discussed in the context of vaccine-induced protection. In contrast, sialylated IgG Abs have shown immunosuppressive effects. OBJECTIVE: We sought to investigate IgG glycosylation programming during the germinal center (GC) reaction following immunization of mice with a foreign protein antigen and different adjuvants. METHODS: Mice were analyzed for GC T-cell, B-cell, and plasma cell responses, as well as for antigen-specific serum IgG subclass titers and Fc glycosylation patterns. RESULTS: Different adjuvants induce distinct IgG+ GC B-cell responses with specific transcriptomes and expression levels of the α2,6-sialyltransferase responsible for IgG sialylation that correspond to distinct serum IgG Fc glycosylation patterns. Low IgG Fc sialylation programming in GC B cells was overall highly dependent on the Foxp3- follicular helper T (TFH) cell-inducing cytokine IL-6, here in particular induced by water-in-oil adjuvants and Mycobacterium tuberculosis. Furthermore, low IgG Fc sialylation programming was dependent on adjuvants that induced IL-27 receptor-dependent IFN-γ+ TFH1 cells, IL-6/IL-23-dependent IL-17A+ TFH17 cells, and high ratios of TFH cells to Foxp3+ follicular regulatory T cells. Here, the 2 latter were dependent on M tuberculosis and its cord factor. CONCLUSION: This study's findings regarding adjuvant-dependent GC responses and IgG glycosylation programming may aid in the development of novel vaccination strategies to induce IgG Abs with both high affinity and defined Fc glycosylation patterns in the GC.
Assuntos
Adjuvantes Imunológicos/administração & dosagem , Antígenos/administração & dosagem , Centro Germinativo/imunologia , Imunoglobulina G/imunologia , Compostos de Alúmen/administração & dosagem , Animais , Linfócitos B/efeitos dos fármacos , Linfócitos B/imunologia , Citocinas/imunologia , Feminino , Adjuvante de Freund/administração & dosagem , Glicosilação , Lipopolissacarídeos/administração & dosagem , Camundongos Endogâmicos C57BL , Camundongos Knockout , Óleo Mineral/administração & dosagem , Mycobacterium tuberculosis/imunologia , Ovalbumina/administração & dosagem , Polissorbatos/administração & dosagem , Esqualeno/administração & dosagem , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , VacinaçãoRESUMO
Aicardi-Goutières syndrome (AGS) provides a monogenic model of nucleic acid-mediated inflammation relevant to the pathogenesis of systemic autoimmunity. Mutations that impair ribonuclease (RNase) H2 enzyme function are the most frequent cause of this autoinflammatory disorder of childhood and are also associated with systemic lupus erythematosus. Reduced processing of eitherRNA:DNAhybrid or genome-embedded ribonucleotide substrates is thought to lead to activation of a yet undefined nucleic acid-sensing pathway. Here, we establishRnaseh2b(A174T/A174T)knock-in mice as a subclinical model of disease, identifying significant interferon-stimulated gene (ISG) transcript upregulation that recapitulates theISGsignature seen inAGSpatients. The inflammatory response is dependent on the nucleic acid sensor cyclicGMP-AMPsynthase (cGAS) and its adaptorSTINGand is associated with reduced cellular ribonucleotide excision repair activity and increasedDNAdamage. This suggests thatcGAS/STINGis a key nucleic acid-sensing pathway relevant toAGS, providing additional insight into disease pathogenesis relevant to the development of therapeutics for this childhood-onset interferonopathy and adult systemic autoimmune disorders.
Assuntos
Doenças Autoimunes do Sistema Nervoso/genética , Imunidade Inata/genética , Proteínas de Membrana/imunologia , Mutação de Sentido Incorreto , Malformações do Sistema Nervoso/genética , Nucleotidiltransferases/imunologia , Ribonuclease H/genética , Ribonucleases/genética , Animais , Doenças Autoimunes do Sistema Nervoso/imunologia , Doenças Autoimunes do Sistema Nervoso/metabolismo , Autoimunidade/genética , Dano ao DNA , Regulação da Expressão Gênica , Humanos , Interferons/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Malformações do Sistema Nervoso/imunologia , Malformações do Sistema Nervoso/metabolismo , Nucleotidiltransferases/genética , Nucleotidiltransferases/metabolismo , Ribonuclease H/metabolismoRESUMO
BACKGROUND & AIMS: RNase H2 is a holoenzyme, composed of 3 subunits (ribonuclease H2 subunits A, B, and C), that cleaves RNA:DNA hybrids and removes mis-incorporated ribonucleotides from genomic DNA through ribonucleotide excision repair. Ribonucleotide incorporation by eukaryotic DNA polymerases occurs during every round of genome duplication and produces the most frequent type of naturally occurring DNA lesion. We investigated whether intestinal epithelial proliferation requires RNase H2 function and whether RNase H2 activity is disrupted during intestinal carcinogenesis. METHODS: We generated mice with epithelial-specific deletion of ribonuclease H2 subunit B (H2bΔIEC) and mice that also had deletion of tumor-suppressor protein p53 (H2b/p53ΔIEC); we compared phenotypes with those of littermate H2bfl/fl or H2b/p53fl/fl (control) mice at young and old ages. Intestinal tissues were collected and analyzed by histology. We isolated epithelial cells, generated intestinal organoids, and performed RNA sequence analyses. Mutation signatures of spontaneous tumors from H2b/p53ΔIEC mice were characterized by exome sequencing. We collected colorectal tumor specimens from 467 patients, measured levels of ribonuclease H2 subunit B, and associated these with patient survival times and transcriptome data. RESULTS: The H2bΔIEC mice had DNA damage to intestinal epithelial cells and proliferative exhaustion of the intestinal stem cell compartment compared with controls and H2b/p53ΔIEC mice. However, H2b/p53ΔIEC mice spontaneously developed small intestine and colon carcinomas. DNA from these tumors contained T>G base substitutions at GTG trinucleotides. Analyses of transcriptomes of human colorectal tumors associated lower levels of RNase H2 with shorter survival times. CONCLUSIONS: In analyses of mice with disruption of the ribonuclease H2 subunit B gene and colorectal tumors from patients, we provide evidence that RNase H2 functions as a colorectal tumor suppressor. H2b/p53ΔIEC mice can be used to study the roles of RNase H2 in tissue-specific carcinogenesis.
Assuntos
Transformação Celular Neoplásica/metabolismo , Células Epiteliais/enzimologia , Instabilidade Genômica , Neoplasias Intestinais/prevenção & controle , Intestino Delgado/enzimologia , Ribonuclease H/metabolismo , Animais , Proliferação de Células , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Colite/induzido quimicamente , Colite/enzimologia , Colite/genética , Colite/patologia , Dano ao DNA , Sulfato de Dextrana , Modelos Animais de Doenças , Células Epiteliais/patologia , Feminino , Predisposição Genética para Doença , Humanos , Neoplasias Intestinais/enzimologia , Neoplasias Intestinais/genética , Neoplasias Intestinais/patologia , Intestino Delgado/patologia , Masculino , Camundongos Knockout , Fenótipo , Ribonuclease H/deficiência , Ribonuclease H/genética , Proteína Supressora de Tumor p53/deficiência , Proteína Supressora de Tumor p53/genéticaRESUMO
Hypomorphic mutations in the DNA repair enzyme RNase H2 cause the neuroinflammatory autoimmune disorder Aicardi-Goutières syndrome (AGS). Endogenous nucleic acids are believed to accumulate in patient cells and instigate pathogenic type I interferon expression. However, the underlying nucleic acid species amassing in the absence of RNase H2 has not been established yet. Here, we report that murine RNase H2 knockout cells accumulated cytosolic DNA aggregates virtually indistinguishable from micronuclei. RNase H2-dependent micronuclei were surrounded by nuclear lamina and most of them contained damaged DNA. Importantly, they induced expression of interferon-stimulated genes (ISGs) and co-localized with the nucleic acid sensor cGAS. Moreover, micronuclei associated with RNase H2 deficiency were cleared by autophagy. Consequently, induction of autophagy by pharmacological mTOR inhibition resulted in a significant reduction of cytosolic DNA and the accompanied interferon signature. Autophagy induction might therefore represent a viable therapeutic option for RNase H2-dependent disease. Endogenous retroelements have previously been proposed as a source of self-nucleic acids triggering inappropriate activation of the immune system in AGS. We used human RNase H2-knockout cells generated by CRISPR/Cas9 to investigate the impact of RNase H2 on retroelement propagation. Surprisingly, replication of LINE-1 and Alu elements was blunted in cells lacking RNase H2, establishing RNase H2 as essential host factor for the mobilisation of endogenous retrotransposons.
Assuntos
Doenças Autoimunes do Sistema Nervoso/enzimologia , Micronúcleo Germinativo/enzimologia , Malformações do Sistema Nervoso/enzimologia , Ribonuclease H/deficiência , Animais , Doenças Autoimunes do Sistema Nervoso/genética , Doenças Autoimunes do Sistema Nervoso/metabolismo , Doenças Autoimunes do Sistema Nervoso/patologia , Autofagia/genética , DNA/genética , Dano ao DNA , Replicação do DNA , Camundongos , Camundongos Knockout , Micronúcleo Germinativo/genética , Micronúcleo Germinativo/imunologia , Mutação , Malformações do Sistema Nervoso/genética , Malformações do Sistema Nervoso/metabolismo , Malformações do Sistema Nervoso/patologia , Ribonuclease H/genética , Ribonuclease H/metabolismoRESUMO
Despite existing knowledge about the role of the A Disintegrin and Metalloproteinase 10 (ADAM10) as the α-secretase involved in the non-amyloidogenic processing of the amyloid precursor protein (APP) and Notch signalling we have only limited information about its regulation. In this study, we have identified ADAM10 interactors using a split ubiquitin yeast two hybrid approach. Tetraspanin 3 (Tspan3), which is highly expressed in the murine brain and elevated in brains of Alzheimer´s disease (AD) patients, was identified and confirmed to bind ADAM10 by co-immunoprecipitation experiments in mammalian cells in complex with APP and the γ-secretase protease presenilin. Tspan3 expression increased the cell surface levels of its interacting partners and was mainly localized in early and late endosomes. In contrast to the previously described ADAM10-binding tetraspanins, Tspan3 did not affect the endoplasmic reticulum to plasma membrane transport of ADAM10. Heterologous Tspan3 expression significantly increased the appearance of carboxy-terminal cleavage products of ADAM10 and APP, whereas N-cadherin ectodomain shedding appeared unaffected. Inhibiting the endocytosis of Tspan3 by mutating a critical cytoplasmic tyrosine-based internalization motif led to increased surface expression of APP and ADAM10. After its downregulation in neuroblastoma cells and in brains of Tspan3-deficient mice, ADAM10 and APP levels appeared unaltered possibly due to a compensatory increase in the expression of Tspans 5 and 7, respectively. In conclusion, our data suggest that Tspan3 acts in concert with other tetraspanins as a stabilizing factor of active ADAM10, APP and the γ-secretase complex at the plasma membrane and within the endocytic pathway.
Assuntos
Proteína ADAM10/genética , Secretases da Proteína Precursora do Amiloide/genética , Precursor de Proteína beta-Amiloide/genética , Endossomos/metabolismo , Proteínas de Membrana/genética , Presenilinas/genética , Tetraspaninas/genética , Proteína ADAM10/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Encéfalo/metabolismo , Química Encefálica , Caderinas/genética , Caderinas/metabolismo , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Endocitose , Endossomos/química , Regulação da Expressão Gênica , Células HEK293 , Humanos , Proteínas de Membrana/metabolismo , Camundongos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Presenilinas/metabolismo , Ligação Proteica , Transporte Proteico , Receptores Notch/genética , Receptores Notch/metabolismo , Transdução de Sinais , Tetraspaninas/metabolismo , Técnicas do Sistema de Duplo-HíbridoRESUMO
Hepatocellular carcinoma (HCC) is one of the most frequent tumors worldwide with rising incidence. The inflammatory cytokine, interleukin-6 (IL-6), is a critical mediator of HCC development. It can signal through two distinct pathways: the IL-6 classic and the IL-6 trans-signaling pathway. Whereas IL-6 classic signaling is important for innate and acquired immunity, IL-6 trans-signaling has been linked to accelerated liver regeneration and several chronic inflammatory pathologies. However, its implication in liver tumorigenesis has not been addressed yet. Here, we show that IL-6 trans-signaling, but not IL-6 classic signaling, is essential to promote hepatocellular carcinogenesis by two mechanisms: First, it prevents DNA-damage-induced hepatocyte apoptosis through suppression of p53 and enhances ß-catenin activation and tumor proliferation. Second, IL-6 trans-signaling directly induces endothelial cell proliferation to promote tumor angiogenesis. Consequently, soluble gp130 fused to Fc transgenic mice lacking IL-6 trans-signaling are largely protected from tumor formation in a diethylnitrosamine/3,3',5,5'-tetrachloro-1,4-bis(pyridyloxy)benzene model of HCC. CONCLUSION: IL-6 trans-signaling, and not IL-6 classic signaling, is mandatory for development of hepatocellular carcinogenesis. Therefore, specific inhibition of IL-6 trans-signaling, rather than total inhibition of IL-6 signaling, is sufficient to blunt tumor initiation and impair tumor progression without compromising IL-6 classic signaling-driven protective immune responses. (Hepatology 2017;65:89-103).
Assuntos
Carcinoma Hepatocelular/etiologia , Interleucina-6/fisiologia , Neoplasias Hepáticas/etiologia , Animais , Masculino , Camundongos , Transdução de SinaisRESUMO
Neutrophil and mononuclear cell infiltration during inflammatory processes is highly regulated. The first cells at the site of infection or inflammation are neutrophils, followed by mononuclear cells. IL-6 plays an important role during inflammatory states. It has been shown in several models that the soluble form of IL-6R (sIL-6R) is involved in the recruitment of mononuclear cells by a mechanism called IL-6 trans-signaling. It had been speculated that sIL-6R was generated at the site of inflammation by shedding from neutrophils via activation of the metalloprotease ADAM17. Attempts to genetically delete the floxed ADAM17 gene selectively in myeloid cells infiltrating an air pouch cavity upon injection of carrageenan failed because in transgenic mice, LysMcre did not lead to appreciable loss of the ADAM17 protein in these cells. We therefore used ADAM17 hypomorphic mice, which only express â¼5% of ADAM17 wild-type levels in all tissues and show virtually no shedding of all tested ADAM17 substrates, to clarify the role of ADAM17 during local inflammation in the murine air pouch model. In the present study, we demonstrate that although IL-6 and the trans-signaling mechanism is mandatory for cellular infiltration in this model, it is not ADAM17-mediated shedding of IL-6R within the pouch that orchestrates this inflammatory process. Instead, we demonstrate that sIL-6R is infiltrating from the circulation in an ADAM17-independent process. Our data suggest that this infiltrating sIL-6R, which is needed for IL-6 trans-signaling, is involved in the controlled resolution of an acute inflammatory episode.
Assuntos
Proteína ADAM17/metabolismo , Inflamação/imunologia , Macrófagos/imunologia , Neutrófilos/imunologia , Receptores de Interleucina-6/metabolismo , Proteína ADAM17/genética , Animais , Carragenina , Movimento Celular , Células Cultivadas , Inflamação/induzido quimicamente , Interleucina-6/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transdução de SinaisRESUMO
IL-23 (interleukin 23) regulates immune responses against pathogens and plays a major role in the differentiation and maintenance of TH17 cells and the development of autoimmune diseases and cancer. The IL-23 receptor (IL-23R) complex consists of the unique IL-23R and the common IL-12 receptor ß1 (IL-12Rß1). Differential splicing generates antagonistic soluble IL-23R (sIL-23R) variants, which might limit IL-23-mediated immune responses. Here, ectodomain shedding of human and murine IL-23R was identified as an alternative pathway for the generation of sIL-23R. Importantly, proteolytically released sIL-23R has IL-23 binding activity. Shedding of IL-23R was induced by stimulation with the phorbol ester phorbol 12-myristate 13-acetate (PMA), but not by ionomycin. PMA-induced shedding was abrogated by an ADAM (A disintegrin and metalloprotease) 10 and 17 selective inhibitor, but not by an ADAM10 selective inhibitor. ADAM17-deficient but not ADAM10-deficient HEK293 cells failed to shed IL-23R after PMA stimulation, demonstrating that ADAM17 but not ADAM10 cleaves the IL-23R. Constitutive shedding was, however, inhibited by an ADAM10 selective inhibitor. Using deletions and specific amino acid residue exchanges, we identified critical determinants of ectodomain shedding within the stalk region of the IL-23R. Finally, interaction studies identified domains 1 and 3 of the IL-23R as the main ADAM17 binding sites. In summary, we describe human and murine IL-23R as novel targets for protein ectodomain shedding by ADAM10 and ADAM17.
Assuntos
Proteína ADAM10/metabolismo , Proteína ADAM17/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Proteínas de Membrana/metabolismo , Receptores de Interleucina/metabolismo , Processamento Alternativo , Animais , Micropartículas Derivadas de Células/metabolismo , Células HEK293 , Meia-Vida , Humanos , Interleucina-23/metabolismo , Camundongos , Receptores de Interleucina/química , Receptores de Interleucina/genética , Solubilidade , Especificidade por Substrato , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
Generation of the soluble interleukin-6 receptor (sIL-6R) is a prerequisite for pathogenic IL-6 trans-signaling, which constitutes a distinct signaling pathway of the pleiotropic cytokine interleukin-6 (IL-6). Although in vitro experiments using ectopically overexpressed IL-6R and candidate proteases revealed major roles for the metalloproteinases ADAM10 and ADAM17 in IL-6R shedding, the identity of the protease(s) cleaving IL-6R in more physiological settings, or even in vivo, remains unknown. By taking advantage of specific pharmacological inhibitors and primary cells from ADAM-deficient mice we established that endogenous IL-6R of both human and murine origin is shed by ADAM17 in an induced manner, whereas constitutive release of endogenous IL-6R is largely mediated by ADAM10. Although circulating IL-6R levels are altered in various diseases, the origin of blood-borne IL-6R is still poorly understood. It has been shown previously that ADAM17 hypomorphic mice exhibit unaltered levels of serum sIL-6R. Here, by quantification of serum sIL-6R in protease-deficient mice as well as human patients we also excluded ADAM10, ADAM8, neutrophil elastase, cathepsin G, and proteinase 3 from contributing to circulating sIL-6R. Furthermore, we ruled out alternative splicing of the IL-6R mRNA as a potential source of circulating sIL-6R in the mouse. Instead, we found full-length IL-6R on circulating microvesicles, establishing microvesicle release as a novel mechanism for sIL-6R generation.
Assuntos
Proteínas ADAM/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Proteínas de Membrana/metabolismo , Isoformas de Proteínas/metabolismo , Receptores de Interleucina-6/metabolismo , Proteína ADAM10 , Proteína ADAM17 , Animais , Linhagem Celular , Humanos , Lipopolissacarídeos/farmacologia , Camundongos , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Proteólise , Splicing de RNA , Receptores de Interleucina-6/genética , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
To avoid malformation and disease, tissue development and homoeostasis are co-ordinated precisely in time and space. Secreted Frizzled-related protein 3 (sFRP3), encoded by the Frizzled-related protein gene (FRZB), acts as an antagonist of Wnt signalling in bone development by delaying the maturation of proliferative chondrocytes into hypertrophic chondrocytes. A disintegrin and metalloprotease 17 (ADAM17) is a transmembrane protease that is essential for developmental processes and promotes cartilage maturation into bone. sFRP3 is chondroprotective and is expressed in chondrocytes of healthy articular cartilage. Upon damage to cartilage, sFRP3 is down-regulated. Rare variants of sFRP3 are associated with osteoarthritis. The present study demonstrates a novel function of sFRP3 in suppression of the enzymatic activity of ADAM17 which results in the inhibition of ADAM17-meditated interleukin-6 receptor (IL-6R) shedding. By contrast, the rare double variant of sFRP3 failed to suppress ADAM17. The shed soluble IL-6R (sIL-6R) is linked to inflammation, cartilage degeneration and osteolysis. Accordingly, enhanced activity of ADAM17 in cartilage, caused by the expression of the rare double sFRP3 variant, provides an explanation for the genetic effect of sFRP3 variants in joint disease. The finding that sFRP3 interacts with the ADAM17 substrate IL-6R also suggests a new regulatory mechanism by which the substrate is protected against shedding.
Assuntos
Proteínas ADAM/metabolismo , Membrana Celular/metabolismo , Condrócitos/metabolismo , Osteoartrite do Quadril/metabolismo , Proteínas/metabolismo , Receptores de Interleucina-6/metabolismo , Regulação para Cima , Proteínas ADAM/química , Proteínas ADAM/genética , Proteína ADAM17 , Substituição de Aminoácidos , Linhagem Celular Tumoral , Regulação para Baixo , Predisposição Genética para Doença , Células HEK293 , Humanos , Mutagênese Sítio-Dirigida , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Osteoartrite do Quadril/genética , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Domínios e Motivos de Interação entre Proteínas , Proteínas/química , Proteínas/genética , Receptores de Interleucina-6/química , Receptores de Interleucina-6/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMO
Ciliary neurotrophic factor (CNTF) is a neurotrophic factor with therapeutic potential for neurodegenerative diseases. Moreover, therapeutic application of CNTF reduced body weight in mice and humans. CNTF binds to high or low affinity receptor complexes consisting of CNTFR·gp130·LIFR or IL-6R·gp130·LIFR, respectively. Clinical studies of the CNTF derivative Axokine revealed intolerance at higher concentrations, which may rely on the low-affinity binding of CNTF to the IL-6R. Here, we aimed to generate a CNTFR-selective CNTF variant (CV). CV-1 contained the single amino acid exchange R28E. Arg(28) is in close proximity to the CNTFR binding site. Using molecular modeling, we hypothesized that Arg(28) might contribute to IL-6R/CNTFR plasticity of CNTF. CV-2 to CV-5 were generated by transferring parts of the CNTFR-binding site from cardiotrophin-like cytokine to CNTF. Cardiotrophin-like cytokine selectively signals via the CNTFR·gp130·LIFR complex, albeit with a much lower affinity compared with CNTF. As shown by immunoprecipitation, all CNTF variants retained the ability to bind to CNTFR. CV-1, CV-2, and CV-5, however, lost the ability to bind to IL-6R. Although all variants induced cytokine-dependent cellular proliferation and STAT3 phosphorylation via CNTFR·gp130·LIFR, only CV-3 induced STAT3 phosphorylation via IL-6R·gp130·LIFR. Quantification of CNTF-dependent proliferation of CNTFR·gp130·LIFR expressing cells indicated that only CV-1 was as biologically active as CNTF. Thus, the CNTFR-selective CV-1 will allow discriminating between CNTFR- and IL-6R-mediated effects in vivo.
Assuntos
Substituição de Aminoácidos , Fator Neurotrófico Ciliar/genética , Receptor gp130 de Citocina/metabolismo , Subunidade alfa de Receptor de Fator Inibidor de Leucemia/metabolismo , Receptor do Fator Neutrófico Ciliar/metabolismo , Receptores de Interleucina-6/metabolismo , Fator Neurotrófico Ciliar/metabolismo , Receptor gp130 de Citocina/genética , Humanos , Interleucina-6/metabolismo , Subunidade alfa de Receptor de Fator Inibidor de Leucemia/genética , Mutação de Sentido Incorreto , Fosforilação , Receptor do Fator Neutrófico Ciliar/genética , Receptores de Interleucina-6/genética , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Transdução de SinaisRESUMO
ADAM17 (a disintegrin and metalloprotease 17) controls pro- and anti-inflammatory signaling events by promoting ectodomain shedding of cytokine precursors and cytokine receptors. Despite the well documented substrate repertoire of ADAM17, little is known about regulatory mechanisms, leading to substrate recognition and catalytic activation. Here we report a direct interaction of the acidophilic kinase Polo-like kinase 2 (PLK2, also known as SNK) with the cytoplasmic portion of ADAM17 through the C-terminal noncatalytic region of PLK2 containing the Polo box domains. PLK2 activity leads to ADAM17 phosphorylation at serine 794, which represents a novel phosphorylation site. Activation of ADAM17 by PLK2 results in the release of pro-TNFα and TNF receptors from the cell surface, and pharmacological inhibition of PLK2 leads to down-regulation of LPS-induced ADAM17-mediated shedding on primary macrophages and dendritic cells. Importantly, PLK2 expression is up-regulated during inflammatory conditions increasing ADAM17-mediated proteolytic events. Our findings suggest a new role for PLK2 in the regulation of inflammatory diseases by modulating ADAM17 activity.
Assuntos
Proteínas ADAM/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais/fisiologia , Fator de Necrose Tumoral alfa/metabolismo , Proteína ADAM17 , Sequência de Aminoácidos , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Células Dendríticas/citologia , Células Dendríticas/metabolismo , Células HEK293 , Humanos , Macrófagos/citologia , Macrófagos/metabolismo , Camundongos , Dados de Sequência Molecular , Células NIH 3T3 , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Estrutura Terciária de Proteína , Fator de Necrose Tumoral alfa/química , Técnicas do Sistema de Duplo-HíbridoRESUMO
Cellular senescence, a state of persistent cell cycle arrest, has emerged as a potent tumor suppressor mechanism by restricting proliferation of cells at risk for neoplastic transformation. Senescent cells secrete various growth factors, cytokines, and other proteins that can either elicit the clearance of tumor cells or potentially promote tumor progression. In addition, this senescence-associated secretory phenotype (SASP) includes various factors that are synthesized as transmembrane precursors and subsequently converted into their soluble counterparts. Despite the importance of the SASP to tumor biology, it is virtually unknown how transmembrane proteins are released from senescent cancer cells. Here we show in different models of senescence that the metalloprotease A disintegrin and metalloproteinase 17 (ADAM17) is activated and releases the epidermal growth factor receptor ligand amphiregulin and tumor necrosis factor receptor I (TNFRI) from the surface of senescent cells by ectodomain shedding. ADAM17 activation involves phosphorylation of its cytoplasmic tail by mitogen-activated protein kinase (MAPK) p38. Interestingly, unlike amphiregulin and TNFRI, full-length intercellular adhesion molecule 1 (ICAM1) is released from senescent cells by microvesicles independently of ADAM17. Thus, our results suggest that transmembrane proteins can be released by two distinct mechanisms and point to a crucial role for ADAM17 in shaping the secretory profile of senescent cells.
Assuntos
Proteínas ADAM/metabolismo , Senescência Celular , Microvasos/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Proteína ADAM17 , Células Cultivadas , Citocinas/metabolismo , Humanos , Molécula 1 de Adesão Intercelular/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , FosforilaçãoRESUMO
Proteolysis of transmembrane molecules is an irreversible post-translational modification enabling autocrine, paracrine and endocrine signaling of many cytokines. The pro-inflammatory activities of membrane bound TNFα (pro-TNFα) strongly depend on ectodomain shedding mediated by the A Disintegrin And Metalloprotease family member ADAM17. Despite the well-documented role of ADAM17 in pro-TNFα cleavage during inflammation, little is known about its regulation. Mitogen-activated protein kinase-induced phosphorylation of the ADAM17 cytoplasmic tail has been described to be required for proper activation. To address, if pro-TNFα shedding depends on cytosolic phosphorylation we analyzed ADAM17 mutants lacking the cytoplasmic domain. ADAM17 mediated shedding of pro-TNFα was induced by PMA, Anisomycin and the phosphatase inhibitors Cantharidin and Calyculin A. Deletion of the entire cytoplasmic portion of ADAM17 abolished furin-dependent proteolytic maturation and pro-TNFα cleavage. Interestingly, we could exclude that resistance to proconvertase processing is the reason for the enzymatic inactivity of ADAM17 lacking the cytoplasmic portion as furin-resistant ADAM17 mutants rescued genetic ADAM17 deficiency after mitogen-activated protein kinase activation. Adding only 6 cytoplasmic amino acids completely restored ADAM17 maturation and shedding of pro-TNFα as well as of both TNF-receptors Finally, we showed that a pro-TNFα mutant lacking the cytoplasmic portion was also shed from the cell surface. We conclude that pro-TNFα cleavage by its major sheddase ADAM17 does not depend on cytosolic phosphorylation and/or interaction. These results have general implications on understanding the activation mechanism controlling the activity of ADAM17.
Assuntos
Proteínas ADAM/metabolismo , Citoplasma/metabolismo , Furina/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Proteínas ADAM/química , Proteína ADAM17 , Animais , Linhagem Celular , Humanos , Camundongos , Proteínas Mutantes/metabolismo , Fosforilação , Multimerização Proteica , Processamento de Proteína Pós-Traducional , Estrutura Terciária de Proteína , Transporte Proteico , ProteóliseRESUMO
Human herpesvirus 8 encodes a viral version of interleukin-6 (vIL-6) which shows 25% sequence homology with human IL-6. In contrast to human IL-6, which first binds to the IL-6 receptor (IL-6R) and only subsequently associates with the signal transducing receptor subunit gp130, vIL-6 has been shown to directly bind to gp130 without the need of IL-6R. As a functional consequence, vIL-6 can activate far more target cells in the body since all cells express gp130, but only cells such as hepatocytes and some leukocytes express IL-6R. We sought to understand which amino acid sequences within the vIL-6 protein were responsible for its ability to bind and activate gp130 independent of IL-6R. As a first approach, we constructed chimeric IL-6 proteins in which all known gp130 interacting sites (sites II and III) were sequentially transferred from vIL-6 into the human IL-6 protein. To our surprise, human IL-6 carrying all gp130 interacting sites from vIL-6 did not show IL-6R-independent gp130 activation. Even more surprisingly, the loop between helix B and C of vIL-6, clearly shown in the crystal structure not to be in contact with gp130, is indispensable for direct binding to and activation of gp130. This points to an IL-6R induced change of site III conformation in human IL-6, which is already preformed in vIL-6. These data indicate a novel activation mechanism of human IL-6 by the IL-6R that will be important for the construction of novel hyperactive cytokine variants.
Assuntos
Receptor gp130 de Citocina/metabolismo , Herpesvirus Humano 8/metabolismo , Interleucina-6/metabolismo , Receptores de Interleucina-6/metabolismo , Proteínas Virais/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Células COS , Chlorocebus aethiops , Herpesvirus Humano 8/genética , Humanos , Camundongos , Modelos Moleculares , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Virais/genéticaRESUMO
The lectin-like oxidized LDL receptor 1 (LOX-1) is a key player in the development of atherosclerosis. LOX-1 promotes endothelial activation and dysfunction by mediating uptake of oxidized LDL and inducing pro-atherogenic signaling. However, little is known about modulators of LOX-1-mediated responses. Here, we show that the function of LOX-1 is controlled proteolytically. Ectodomain shedding by the metalloprotease ADAM10 and lysosomal degradation generate membrane-bound N-terminal fragments (NTFs), which we identified as novel substrates of the intramembrane proteases signal peptide peptidase-like 2a and b (SPPL2a/b). SPPL2a/b control cellular LOX-1 NTF levels which, following self-association via their transmembrane domain, can activate MAP kinases in a ligand-independent manner. This leads to an up-regulation of several pro-atherogenic and pro-fibrotic targets including ICAM-1 and the connective tissue growth factor CTGF. Consequently, SPPL2a/b-deficient mice, which accumulate LOX-1 NTFs, develop larger and more advanced atherosclerotic plaques than controls. This identifies intramembrane proteolysis by SPPL2a/b as a novel atheroprotective mechanism via negative regulation of LOX-1 signaling.
Assuntos
Ácido Aspártico Endopeptidases/metabolismo , Proteínas de Membrana/metabolismo , Proteólise , Receptores Depuradores Classe E/metabolismo , Proteína ADAM10/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Animais , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Ácido Aspártico Endopeptidases/genética , Aterosclerose/metabolismo , Dipeptídeos/farmacologia , Células Endoteliais/metabolismo , Células HEK293 , Células HeLa , Humanos , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores Depuradores Classe E/genética , TransfecçãoRESUMO
Dysregulation of the disintegrin-metalloproteinase ADAM10 may contribute to the development of diseases including tumorigenesis and Alzheimer's disease. The mechanisms underlying ADAM10 sheddase activation are incompletely understood. Here, we show that transient exposure of the negatively charged phospholipid phosphatidylserine (PS) is necessarily required. The soluble PS headgroup was found to act as competitive inhibitor of substrate cleavage. Overexpression of the Ca2+-dependent phospholipid scramblase Anoctamin-6 (ANO6) led to increased PS externalization and substrate release. Transfection with a constitutively active form of ANO6 resulted in maximum sheddase activity in the absence of any stimulus. Calcium-dependent ADAM10 activation could not be induced in lymphocytes of patients with Scott syndrome harbouring a missense mutation in ANO6. A putative PS-binding motif was identified in the conserved stalk region. Replacement of this motif resulted in strong reduction of sheddase activity. In conjunction with the recently described 3D structure of the ADAM10 extracellular domain, a model is advanced to explain how surface-exposed PS triggers ADAM10 sheddase function.
Assuntos
Proteína ADAM10/metabolismo , Membrana Celular/metabolismo , Ativação Enzimática , Proteína ADAM10/química , Sequência de Aminoácidos , Animais , Anoctaminas/metabolismo , Biomarcadores , Células COS , Linhagem Celular , Chlorocebus aethiops , Eritrócitos/metabolismo , Humanos , Proteínas de Membrana/metabolismo , Modelos Biológicos , Fosfosserina/metabolismo , Coelhos , Relação Estrutura-AtividadeRESUMO
[This corrects the article DOI: 10.18632/oncotarget.9315.].
RESUMO
Aicardi-Goutières syndrome (AGS) is a rare early onset childhood encephalopathy caused by persistent neuroinflammation of autoimmune origin. AGS is a genetic disorder and >50% of affected individuals bear hypomorphic mutations in ribonuclease H2 (RNase H2). All available RNase H2 mouse models so far fail to mimic the prominent CNS involvement seen in AGS. To establish a mouse model recapitulating the human disease, we deleted RNase H2 specifically in the brain, the most severely affected organ in AGS. Although RNase H2ΔGFAP mice lacked the nuclease in astrocytes and a majority of neurons, no disease signs were apparent in these animals. We additionally confirmed these results in a second, neuron-specific RNase H2 knockout mouse line. However, when astrocytes were isolated from brains of RNase H2ΔGFAP mice and cultured under mitogenic conditions, they showed signs of DNA damage and premature senescence. Enhanced expression of interferon-stimulated genes (ISGs) represents the most reliable AGS biomarker. Importantly, primary RNase H2ΔGFAP astrocytes displayed significantly increased ISG transcript levels, which we failed to detect in in vivo in brains of RNase H2ΔGFAP mice. Isolated astrocytes primed by DNA damage, including RNase H2-deficiency, exhibited a heightened innate immune response when exposed to bacterial or viral antigens. Taken together, we established a valid cellular AGS model that utilizes the very cell type responsible for disease pathology, the astrocyte, and phenocopies major molecular defects observed in AGS patient cells.