RESUMO
Defects in protein O-mannosylation lead to severe congenital muscular dystrophies collectively known as α-dystroglycanopathy. A hallmark of these diseases is the loss of the O-mannose-bound matriglycan on α-dystroglycan, which reduces cell adhesion to the extracellular matrix. Mutations in protein O-mannose ß1,2-N-acetylglucosaminyltransferase 1 (POMGNT1), which is crucial for the elongation of O-mannosyl glycans, have mainly been associated with muscle-eye-brain (MEB) disease. In addition to defects in cell-extracellular matrix adhesion, aberrant cell-cell adhesion has occasionally been observed in response to defects in POMGNT1. However, specific molecular consequences of POMGNT1 deficiency on cell-cell adhesion are largely unknown. We used POMGNT1 knockout HEK293T cells and fibroblasts from an MEB patient to gain deeper insight into the molecular changes in POMGNT1 deficiency. Biochemical and molecular biological techniques combined with proteomics, glycoproteomics, and glycomics revealed that a lack of POMGNT1 activity strengthens cell-cell adhesion. We demonstrate that the altered intrinsic adhesion properties are due to an increased abundance of N-cadherin (N-Cdh). In addition, site-specific changes in the N-glycan structures in the extracellular domain of N-Cdh were detected, which positively impact on homotypic interactions. Moreover, in POMGNT1-deficient cells, ERK1/2 and p38 signaling pathways are activated and transcriptional changes that are comparable with the epithelial-mesenchymal transition (EMT) are triggered, defining a possible molecular mechanism underlying the observed phenotype. Our study indicates that changes in cadherin-mediated cell-cell adhesion and other EMT-related processes may contribute to the complex clinical symptoms of MEB or α-dystroglycanopathy in general and suggests that the impact of changes in O-mannosylation on N-glycosylation has been underestimated.
Assuntos
Adesão Celular/fisiologia , N-Acetilglucosaminiltransferases/deficiência , N-Acetilglucosaminiltransferases/metabolismo , Antígenos CD/metabolismo , Antígenos CD/fisiologia , Caderinas/metabolismo , Caderinas/fisiologia , Adesão Celular/genética , Distroglicanas/metabolismo , Glicômica , Glicosilação , Glicosiltransferases/deficiência , Glicosiltransferases/metabolismo , Células HEK293 , Humanos , Sistema de Sinalização das MAP Quinases/fisiologia , Manose/química , Distrofias Musculares/genética , N-Acetilglucosaminiltransferases/fisiologia , Polissacarídeos , Transdução de Sinais/fisiologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
O-mannosylation is implicated in protein quality control in Saccharomyces cerevisiae due to the attachment of mannose to serine and threonine residues of un- or misfolded proteins in the endoplasmic reticulum (ER). This process also designated as unfolded protein O-mannosylation (UPOM) that ends futile folding cycles and saves cellular resources is mainly mediated by protein O-mannosyltransferases Pmt1 and Pmt2. Here we describe a genetic screen for factors that influence O-mannosylation in yeast, using slow-folding green fluorescent protein (GFP) as a reporter. Our screening identifies the RNA binding protein brefeldin A resistance factor 1 (Bfr1) that has not been linked to O-mannosylation and ER protein quality control before. We find that Bfr1 affects O-mannosylation through changes in Pmt1 and Pmt2 protein abundance but has no effect on PMT1 and PMT2 transcript levels, mRNA localization to the ER membrane or protein stability. Ribosome profiling reveals that Bfr1 is a crucial factor for Pmt1 and Pmt2 translation thereby affecting unfolded protein O-mannosylation. Our results uncover a new level of regulation of protein quality control in the secretory pathway.
Assuntos
Manosiltransferases/química , Manosiltransferases/genética , Proteínas Repressoras/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Retículo Endoplasmático/metabolismo , Glicosilação , Manosiltransferases/metabolismo , Dobramento de Proteína , Processamento de Proteína Pós-Traducional , Estabilidade Proteica , Proteínas Repressoras/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Deleção de SequênciaRESUMO
Asparagine (N)-linked glycosylation is a protein modification critical for glycoprotein folding, stability, and cellular localization. To identify small molecules that inhibit new targets in this biosynthetic pathway, we initiated a cell-based high-throughput screen and lead-compound-optimization campaign that delivered a cell-permeable inhibitor, NGI-1. NGI-1 targets oligosaccharyltransferase (OST), a hetero-oligomeric enzyme that exists in multiple isoforms and transfers oligosaccharides to recipient proteins. In non-small-cell lung cancer cells, NGI-1 blocks cell-surface localization and signaling of the epidermal growth factor receptor (EGFR) glycoprotein, but selectively arrests proliferation in only those cell lines that are dependent on EGFR (or fibroblast growth factor, FGFR) for survival. In these cell lines, OST inhibition causes cell-cycle arrest accompanied by induction of p21, autofluorescence, and cell morphology changes, all hallmarks of senescence. These results identify OST inhibition as a potential therapeutic approach for treating receptor-tyrosine-kinase-dependent tumors and provides a chemical probe for reversibly regulating N-linked glycosylation in mammalian cells.
Assuntos
Benzamidas/farmacologia , Senescência Celular/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Hexosiltransferases/antagonistas & inibidores , Proteínas de Membrana/antagonistas & inibidores , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Sulfonamidas/farmacologia , Benzamidas/química , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/química , Hexosiltransferases/metabolismo , Ensaios de Triagem em Larga Escala , Humanos , Proteínas de Membrana/metabolismo , Estrutura Molecular , Receptores Proteína Tirosina Quinases/metabolismo , Relação Estrutura-Atividade , Sulfonamidas/químicaRESUMO
BACKGROUND: Short in-frame deletions in the second extracellular domain of the cytokine receptor gp130 are the leading cause of inflammatory hepatocellular adenomas (IHCAs). The deletions render gp130 constitutively active. In this study we investigate the intracellular signaling potential of one of the most potent constitutively active gp130 mutants (CAgp130) found in IHCAs. RESULTS: Trafficking and signaling of CAgp130 were studied in stably transfected cell lines that allowed the inducible expression of CAgp130 fused to fluorescent proteins such as YFP and mCherry. In contrast to the predominantly highly glycosylated gp130 wild type (WTgp130), CAgp130 is preferentially found in the less glycosylated high-mannose form. Accordingly, the mutated receptor is retained intracellularly and therefore less prominently expressed at the cell surface. CAgp130 persistently activates Stat3 despite the presence of the feedback inhibitor SOCS3 but fails to activate Erk1/2. De novo synthesized CAgp130 signals already from the ER-Golgi compartment before having reached the plasma membrane. Cell surface expressed and endocytosed CAgp130 do not significantly contribute to signaling. As a consequence, Stat3 activation through CAgp130 cannot be inhibited by neutralizing gp130 antibodies but through overexpression of a dominant-negative Stat3 mutant. CONCLUSION: CAgp130 and WTgp130 differ significantly with respect to glycosylation, trafficking and signaling. As a consequence of intracellular signaling pharmacological inhibition of CAgp130 will not be achieved by targeting the receptor extracellularly but by compounds that act from within the cell.
Assuntos
Anticorpos Neutralizantes/imunologia , Receptor gp130 de Citocina/metabolismo , Sistema de Sinalização das MAP Quinases , Mutação , Membrana Celular/metabolismo , Receptor gp130 de Citocina/genética , Receptor gp130 de Citocina/imunologia , Endocitose , Retículo Endoplasmático/metabolismo , Glicosilação , Complexo de Golgi/metabolismo , Células HEK293 , Humanos , Ligação Proteica , Transporte Proteico , Fator de Transcrição STAT3/metabolismo , Proteínas Supressoras da Sinalização de Citocina/metabolismoRESUMO
BACKGROUND: STAT3 is a transcription factor of central importance in chronic inflammation and cancer. In response to cytokine stimulation STAT3 is phosphorylated on a single tyrosine residue at position 705, dimerizes and accumulates in the nucleus to induce target gene expression. The substitution of tyrosine 705 to phenylalanine leads to a dominant-negative STAT3 mutant (STAT3-YF) which influences the activation of WT-STAT3 in stimulated cells through a mechanism that is not completely understood. In this study we analyzed the molecular mechanism of STAT3-YF dominant-negative activity in IL-6-induced STAT3 signaling and the relevance of the N-terminal domain. RESULTS: Expression of STAT3-YF-YFP impairs tyrosine phosphorylation, nuclear translocation and the transcriptional activity of WT-STAT3 in IL-6-stimulated cells. The fluorescently labelled STAT3-YF mutant binds to a phosphorylated gp130 receptor-peptide comparable to WT-STAT3-YFP. STAT3-YF-YFP forms homodimers as well as heterodimers with WT-STAT3 in the presence and absence of IL-6. The preformed heterodimers in unstimulated cells are detectable by colocalization of STAT3-CFP with STAT3-YF-YFP fused to a nuclear localization signal. STAT3/STAT3-YF heterodimers are not able to bind to DNA in stimulated cells, but the presence of the mutant reduces DNA-binding of WT-STAT3 homodimers. STAT3-YF-ΔN-YFP lacking the N-terminal domain forms no dimers and only marginally affects the activity of WT-STAT3. CONCLUSION: Our findings demonstrate that dominant-negative STAT3-YF affects the activation of WT-STAT3 at multiple levels. Unexpectedly, the N-terminal domain of STAT3-YF plays an important role for the dominant-negative effect. We show that (i) STAT3-YF competes with WT-STAT3 in binding to activated gp130-receptors, (ii) the formation of WT-STAT3/STAT3-YF heterodimers in IL-6-stimulated cells results in inactive, semiphosphorylated dimers which do not bind to DNA and thus fail to induce target gene expression, (iii) the N-terminal domain-mediated formation of preformed STAT3/STAT3-YF heterodimers in unstimulated cells which affects the IL-6-induced homodimerization of WT-STAT3 contributes to the dominant-negative effect of STAT3-YF. These findings will contribute to our understanding of naturally occuring dominant-negative STAT3 mutants that cause the hyper-IgE syndrome.
Assuntos
Fator de Transcrição STAT3/metabolismo , Transporte Ativo do Núcleo Celular , Núcleo Celular/metabolismo , Receptor gp130 de Citocina/metabolismo , Humanos , Interleucina-6/metabolismo , Mutação , Fosforilação , Fosfotirosina/metabolismo , Multimerização Proteica , Estrutura Terciária de Proteína , Fator de Transcrição STAT3/genética , Proteína 3 Supressora da Sinalização de Citocinas , Proteínas Supressoras da Sinalização de Citocina/genéticaRESUMO
Proteins destined for the secretory compartment of the cell are cotranslationally translocated into the endoplasmic reticulum. The majority of these proteins are N-glycosylated, a co- and posttranslational modification that ensures proper protein folding, stability, solubility, and cellular localization. Here, we show that the [Formula: see text] subunit of the signal recognition particle receptor (SR) is required for assembly of the N-glycosylation-competent translocon. We report that guanine analog chemical probes identified by high-throughput screening or mutation of the SR-[Formula: see text] guanosine triphosphate binding site cause an N-glycosylation-deficient phenotype. Neither method alters the association of SR-[Formula: see text] with SR-[Formula: see text], but both approaches reduce the association of SR-[Formula: see text] with the oligosaccharyltransferase complex. These experiments demonstrate that SR-[Formula: see text] has a previously unrecognized function coordinating endoplasmic reticulum translation with N-glycosylation.
Assuntos
Retículo Endoplasmático , Receptores Citoplasmáticos e Nucleares , Glicosilação , Receptores Citoplasmáticos e Nucleares/metabolismo , Retículo Endoplasmático/metabolismo , Receptores de Peptídeos/metabolismoRESUMO
The mosquito-borne flaviviruses include important human pathogens such as dengue, Zika, West Nile, and yellow fever viruses, which pose a serious threat for global health. Recent genetic screens identified endoplasmic reticulum (ER)-membrane multiprotein complexes, including the oligosaccharyltransferase (OST) complex, as critical flavivirus host factors. Here, we show that a chemical modulator of the OST complex termed NGI-1 has promising antiviral activity against flavivirus infections. We demonstrate that NGI-1 blocks viral RNA replication and that antiviral activity does not depend on inhibition of the N-glycosylation function of the OST. Viral mutants adapted to replicate in cells deficient of the OST complex showed resistance to NGI-1 treatment, reinforcing the on-target activity of NGI-1. Lastly, we show that NGI-1 also has strong antiviral activity in primary and disease-relevant cell types. This study provides an example for advancing from the identification of genetic determinants of infection to a host-directed antiviral compound with broad activity against flaviviruses.
Assuntos
Antivirais/farmacologia , Benzamidas/farmacologia , Vírus da Dengue/efeitos dos fármacos , Hexosiltransferases/genética , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Proteínas de Membrana/genética , Sulfonamidas/farmacologia , Replicação Viral/efeitos dos fármacos , Vírus da Dengue/genética , Vírus da Dengue/crescimento & desenvolvimento , Expressão Gênica , Regulação da Expressão Gênica , Genes Reporter , Células HEK293 , Hexosiltransferases/antagonistas & inibidores , Hexosiltransferases/deficiência , Humanos , Luciferases , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/deficiência , Testes de Sensibilidade Microbiana , Transdução de Sinais , Vírus do Nilo Ocidental/efeitos dos fármacos , Vírus do Nilo Ocidental/genética , Vírus do Nilo Ocidental/crescimento & desenvolvimento , Vírus da Febre Amarela/efeitos dos fármacos , Vírus da Febre Amarela/genética , Vírus da Febre Amarela/crescimento & desenvolvimento , Zika virus/efeitos dos fármacos , Zika virus/genética , Zika virus/crescimento & desenvolvimentoRESUMO
Organic anion transporting polypeptides (OATP/SLCO) have been identified to mediate the uptake of a broad range of mainly amphipathic molecules. Human OATP5A1 was found to be expressed in the epithelium of many cancerous and non-cancerous tissues throughout the body but protein characterization and functional analysis have not yet been performed. This study focused on the biochemical characterization of OATP5A1 using Xenopus laevis oocytes and Flp-In T-REx-HeLa cells providing evidence regarding a possible OATP5A1 function. SLCO5A1 is highly expressed in mature dendritic cells compared to immature dendritic cells (â¼6.5-fold) and SLCO5A1 expression correlates with the differentiation status of primary blood cells. A core- and complex- N-glycosylated polypeptide monomer of â¼105 kDa and â¼130 kDa could be localized in intracellular membranes and on the plasma membrane, respectively. Inducible expression of SLCO5A1 in HeLa cells led to an inhibitory effect of â¼20% after 96 h on cell proliferation. Gene expression profiling with these cells identified immunologically relevant genes (e.g. CCL20) and genes implicated in developmental processes (e.g. TGM2). A single nucleotide polymorphism leading to the exchange of amino acid 33 (LâF) revealed no differences regarding protein expression and function. In conclusion, we provide evidence that OATP5A1 might be a non-classical OATP family member which is involved in biological processes that require the reorganization of the cell shape, such as differentiation and migration.
Assuntos
Membrana Celular/metabolismo , Regulação da Expressão Gênica , Membranas Intracelulares/metabolismo , Transportadores de Ânions Orgânicos/genética , Animais , Diferenciação Celular , Movimento Celular , Forma Celular , Células Dendríticas/citologia , Células Dendríticas/metabolismo , Perfilação da Expressão Gênica , Células HeLa , Humanos , Transporte de Íons , Oócitos/citologia , Oócitos/metabolismo , Transportadores de Ânions Orgânicos/metabolismo , Polimorfismo de Nucleotídeo Único , Xenopus laevisRESUMO
The Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway directly links ligand-binding to a membrane-bound receptor with the activation of a transcription factor. This signalling module enables the cell to rapidly initiate a transcriptional response to external stimulation. The main components of this evolutionary conserved module are cytokines that specifically bind to cytokine receptors leading to the activation of receptor-associated Janus tyrosine kinases (JAKs). The receptor-bound JAKs activate STAT transcription factors through phosphorylation of a single tyrosine residue. Activated STAT dimers translocate into the nucleus to induce target gene expression. In this article we will review current opinions on the molecular mechanism and on intracellular dynamics of JAK/STAT signalling with a special focus on the cytokine receptor glycoprotein 130 (gp130) and STAT3. In particular we will concentrate on non-canonical aspects of Jak/STAT signalling including preassembled receptor complexes, preformed STAT dimers, STAT trafficking and non-canonical functions of STATs.