RESUMO
IL-11 and IL-6 activate signalling via assembly of the cell surface receptor gp130; however, it is unclear how signals are transmitted across the membrane to instruct cellular responses. Here we solve the cryoEM structure of the IL-11 receptor recognition complex to discover how differences in gp130-binding interfaces may drive signalling outcomes. We explore how mutations in the IL6ST gene encoding for gp130, which cause severe immune deficiencies in humans, impair signalling without blocking cytokine binding. We use cryoEM to solve structures of both IL-11 and IL-6 complexes with a mutant form of gp130 associated with human disease. Together with molecular dynamics simulations, we show that the disease-associated variant led to an increase in flexibility including motion within the cytokine-binding core and increased distance between extracellular domains. However, these distances are minimized as the transmembrane helix exits the membrane, suggesting a stringency in geometry for signalling and dimmer switch mode of action.
Assuntos
Interleucina-11 , Interleucina-6 , Humanos , Interleucina-11/genética , Interleucina-6/metabolismo , Receptor gp130 de Citocina/genética , Receptor gp130 de Citocina/metabolismo , Transdução de Sinais , Receptores de Interleucina-6/genéticaRESUMO
Cytokines interact with their receptors in the extracellular space to control immune responses. How the physicochemical properties of the extracellular space influence cytokine signaling is incompletely elucidated. Here, we show that the activity of interleukin-2 (IL-2), a cytokine critical to T cell immunity, is profoundly affected by pH, limiting IL-2 signaling within the acidic environment of tumors. Generation of lactic acid by tumors limits STAT5 activation, effector differentiation, and antitumor immunity by CD8+ T cells and renders high-dose IL-2 therapy poorly effective. Directed evolution enabled selection of a pH-selective IL-2 mutein (Switch-2). Switch-2 binds the IL-2 receptor subunit IL-2Rα with higher affinity, triggers STAT5 activation, and drives CD8+ T cell effector function more potently at acidic pH than at neutral pH. Consequently, high-dose Switch-2 therapy induces potent immune activation and tumor rejection with reduced on-target toxicity in normal tissues. Last, we show that sensitivity to pH is a generalizable property of a diverse range of cytokines with broad relevance to immunity and immunotherapy in healthy and diseased tissues.
Assuntos
Interleucina-2 , Neoplasias , Humanos , Fator de Transcrição STAT5 , Linfócitos T CD8-Positivos , Citocinas , Concentração de Íons de HidrogênioRESUMO
Cytokines elicit pleiotropic and non-redundant activities despite strong overlap in their usage of receptors, JAKs and STATs molecules. We use IL-6 and IL-27 to ask how two cytokines activating the same signaling pathway have different biological roles. We found that IL-27 induces more sustained STAT1 phosphorylation than IL-6, with the two cytokines inducing comparable levels of STAT3 phosphorylation. Mathematical and statistical modeling of IL-6 and IL-27 signaling identified STAT3 binding to GP130, and STAT1 binding to IL-27Rα, as the main dynamical processes contributing to sustained pSTAT1 levels by IL-27. Mutation of Tyr613 on IL-27Rα decreased IL-27-induced STAT1 phosphorylation by 80% but had limited effect on STAT3 phosphorgylation. Strong receptor/STAT coupling by IL-27 initiated a unique gene expression program, which required sustained STAT1 phosphorylation and IRF1 expression and was enriched in classical Interferon Stimulated Genes. Interestingly, the STAT/receptor coupling exhibited by IL-6/IL-27 was altered in patients with systemic lupus erythematosus (SLE). IL-6/IL-27 induced a more potent STAT1 activation in SLE patients than in healthy controls, which correlated with higher STAT1 expression in these patients. Partial inhibition of JAK activation by sub-saturating doses of Tofacitinib specifically lowered the levels of STAT1 activation by IL-6. Our data show that receptor and STATs concentrations critically contribute to shape cytokine responses and generate functional pleiotropy in health and disease.
Assuntos
Receptor gp130 de Citocina/agonistas , Interleucina-27/farmacologia , Interleucina-6/farmacologia , Receptores de Interleucina/agonistas , Fator de Transcrição STAT1/metabolismo , Fator de Transcrição STAT3/metabolismo , Células Th1/efeitos dos fármacos , Motivos de Aminoácidos , Ligação Competitiva , Estudos de Casos e Controles , Células Cultivadas , Receptor gp130 de Citocina/genética , Receptor gp130 de Citocina/metabolismo , Humanos , Fator Regulador 1 de Interferon/metabolismo , Interleucina-27/metabolismo , Interleucina-6/metabolismo , Cinética , Lúpus Eritematoso Sistêmico/imunologia , Lúpus Eritematoso Sistêmico/metabolismo , Modelos Biológicos , Mutação , Fosforilação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Receptores de Interleucina/genética , Receptores de Interleucina/metabolismo , Transdução de Sinais , Células Th1/imunologia , Células Th1/metabolismoRESUMO
Cytokines are highly pleiotropic ligands that regulate the immune response. Here, using interleukin-6 (IL-6) as a model system, we perform detailed phosphoproteomic and transcriptomic studies in human CD4+ T helper 1 (Th-1) cells to address the molecular bases defining cytokine functional pleiotropy. We identify CDK8 as a negative regulator of STAT3 transcriptional activities, which interacts with STAT3 upon IL-6 stimulation. Inhibition of CDK8 activity, using specific small molecule inhibitors, reduces the IL-6-induced phosphoproteome by 23% in Th-1 cells, including STAT3 S727 phosphorylation. STAT3 binding to target DNA sites in the genome is increased upon CDK8 inhibition, which results in a concomitant increase in STAT3-mediated transcriptional activity. Importantly, inhibition of CDK8 activity under Th-17 polarizing conditions results in an enhancement of Th-17 differentiation. Our results support a model where CDK8 regulates STAT3 transcriptional processivity by modulation of its gene loci resident time, critically contributing to diversification of IL-6 responses.
Assuntos
Quinase 8 Dependente de Ciclina/metabolismo , Interleucina-6/metabolismo , Fator de Transcrição STAT3/metabolismo , Mapeamento Cromossômico/métodos , Quinase 8 Dependente de Ciclina/genética , Humanos , Fosforilação , Transdução de SinaisRESUMO
Cytokines activate signaling via assembly of cell surface receptors, but it is unclear whether modulation of cytokine-receptor binding parameters can modify biological outcomes. We have engineered IL-6 variants with different affinities to gp130 to investigate how cytokine receptor binding dwell-times influence functional selectivity. Engineered IL-6 variants showed a range of signaling amplitudes and induced biased signaling, with changes in receptor binding dwell-times affecting more profoundly STAT1 than STAT3 phosphorylation. We show that this differential signaling arises from defective translocation of ligand-gp130 complexes to the endosomal compartment and competitive STAT1/STAT3 binding to phospho-tyrosines in gp130, and results in unique patterns of STAT3 binding to chromatin. This leads to a graded gene expression response and differences in ex vivo differentiation of Th17, Th1 and Treg cells. These results provide a molecular understanding of signaling biased by cytokine receptors, and demonstrate that manipulation of signaling thresholds is a useful strategy to decouple cytokine functional pleiotropy.
Assuntos
Receptor gp130 de Citocina/química , Interleucina-6/química , Fator de Transcrição STAT1/metabolismo , Linfócitos T Reguladores/metabolismo , Células Th1/metabolismo , Células Th17/metabolismo , Sítios de Ligação , Diferenciação Celular , Clonagem Molecular , Receptor gp130 de Citocina/genética , Receptor gp130 de Citocina/metabolismo , Endossomos/química , Endossomos/metabolismo , Expressão Gênica , Células HeLa , Humanos , Interleucina-6/genética , Interleucina-6/metabolismo , Cinética , Modelos Moleculares , Fosforilação , Cultura Primária de Células , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Engenharia de Proteínas/métodos , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Linfócitos T Reguladores/citologia , Linfócitos T Reguladores/imunologia , Células Th1/citologia , Células Th1/imunologia , Células Th17/citologia , Células Th17/imunologiaRESUMO
BACKGROUND: Severe dermatitis, multiple allergies, and metabolic wasting (SAM) syndrome is a recently recognized syndrome caused by mutations in the desmoglein 1 gene (DSG1). To date, only 3 families have been reported. OBJECTIVE: We studied a new case of SAM syndrome known to have no mutations in DSG1 to detail the clinical, histopathologic, immunofluorescent, and ultrastructural phenotype and to identify the underlying molecular mechanisms in this rare genodermatosis. METHODS: Histopathologic, electron microscopy, and immunofluorescent studies were performed. Whole-exome sequencing data were interrogated for mutations in desmosomal and other skin structural genes, followed by Sanger sequencing of candidate genes in the patient and his parents. RESULTS: No mutations were identified in DSG1; however, a novel de novo heterozygous missense c.1757A>C mutation in the desmoplakin gene (DSP) was identified in the patient, predicting the amino acid substitution p.His586Pro in the desmoplakin polypeptide. CONCLUSIONS: SAM syndrome can be caused by mutations in both DSG1 and DSP. Knowledge of this genetic heterogeneity is important for both analysis of patients and genetic counseling of families. This condition and these observations reinforce the importance of heritable skin barrier defects, in this case desmosomal proteins, in the pathogenesis of atopic disease.
Assuntos
Dermatite/genética , Desmoplaquinas/genética , Hipersensibilidade/genética , Mutação de Sentido Incorreto/genética , Síndrome de Emaciação/genética , Criança , Pré-Escolar , Análise Mutacional de DNA , Dermatite/diagnóstico , Desmogleína 1/genética , Progressão da Doença , Humanos , Hipersensibilidade/diagnóstico , Lactente , Recém-Nascido , Masculino , Linhagem , Estrutura Terciária de Proteína/genética , Pele/patologia , Síndrome de Emaciação/diagnósticoAssuntos
Proteínas Adaptadoras de Transporte Vesicular/genética , Etretinato/uso terapêutico , Ceratodermia Palmar e Plantar/tratamento farmacológico , Ceratodermia Palmar e Plantar/genética , Ceratolíticos/uso terapêutico , Mutação , Idoso de 80 Anos ou mais , Biópsia , Análise Mutacional de DNA , Feminino , Predisposição Genética para Doença , Humanos , Imuno-Histoquímica , Ceratodermia Palmar e Plantar/diagnóstico , Fenótipo , Indução de Remissão , Resultado do TratamentoAssuntos
Proteínas de Transporte/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Ceratodermia Palmar e Plantar/genética , Mutação , Proteínas Adaptadoras de Transporte Vesicular , Adulto , Idoso , Saúde da Família , Feminino , Genótipo , Heterozigoto , Humanos , Masculino , Repetições de Microssatélites/genética , Pessoa de Meia-Idade , Linhagem , Transdução de SinaisRESUMO
Palmoplantar keratodermas (PPKs) are a group of disorders that are diagnostically and therapeutically problematic in dermatogenetics. Punctate PPKs are characterized by circumscribed hyperkeratotic lesions on the palms and soles with considerable heterogeneity. In 18 families with autosomal dominant punctate PPK, we report heterozygous loss-of-function mutations in AAGAB, encoding α- and γ-adaptin-binding protein p34, located at a previously linked locus at 15q22. α- and γ-adaptin-binding protein p34, a cytosolic protein with a Rab-like GTPase domain, was shown to bind both clathrin adaptor protein complexes, indicating a role in membrane trafficking. Ultrastructurally, lesional epidermis showed abnormalities in intracellular vesicle biology. Immunohistochemistry showed hyperproliferation within the punctate lesions. Knockdown of AAGAB in keratinocytes led to increased cell division, which was linked to greatly elevated epidermal growth factor receptor (EGFR) protein expression and tyrosine phosphorylation. We hypothesize that p34 deficiency may impair endocytic recycling of growth factor receptors such as EGFR, leading to increased signaling and cellular proliferation.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular , Proteínas de Transporte/genética , Haploinsuficiência , Poroceratose/genética , Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Mapeamento Cromossômico , Citosol/ultraestrutura , Receptores ErbB/genética , Receptores ErbB/metabolismo , Regulação da Expressão Gênica , Células HeLa , Humanos , Queratinócitos/metabolismo , Queratinócitos/patologia , Linhagem , Poroceratose/metabolismo , Ligação Proteica , Proteínas/genética , Proteínas/metabolismoRESUMO
Stress protein responses have evolved in part as a mechanism to protect cells from the toxic effects of environmental damaging agents. Oesophageal squamous epithelial cells have evolved an atypical stress response that results in the synthesis of a 53 kDa protein of undefined function named squamous epithelial-induced stress protein of 53 kDa (SEP53). Given the role of deoxycholic acid (DCA) as a potential damaging agent in squamous epithelium, we developed assays measuring the effects of DCA on SEP53-mediated responses to damage. To achieve this, we cloned the human SEP53 gene, developed a panel of monoclonal antibodies to the protein, and showed that SEP53 expression is predominantly confined to squamous epithelium. Clonogenic assays were used to show that SEP53 can function as a survival factor in mammalian cell lines, can attenuate DCA-induced apoptotic cell death, and can attenuate DCA-mediated increases in intracellular free calcium. Deletion of the highly conserved EF-hand calcium-binding domain in SEP53 neutralizes the colony survival activity of the protein, neutralizes the protective effects of SEP53 after DCA exposure, and permits calcium elevation in response to DCA challenge. These data indicate that the squamous cell-stress protein SEP53 can function as a modifier of the DCA-mediated calcium influx and identify a novel survival pathway whose study may shed light on mechanisms relating to squamous cell injury and associated cancer development.
Assuntos
Apoptose/fisiologia , Proteínas de Ligação ao Cálcio/fisiologia , Cálcio/metabolismo , Ácido Desoxicólico/toxicidade , Células Epiteliais/patologia , Proteínas de Choque Térmico/fisiologia , Proteínas de Membrana/fisiologia , Proteínas de Neoplasias/fisiologia , Sequência de Aminoácidos , Proteínas de Ligação ao Cálcio/biossíntese , Proteínas de Ligação ao Cálcio/metabolismo , Morte Celular/fisiologia , Linhagem Celular Tumoral , Sobrevivência Celular/fisiologia , Células Clonais , Células Epiteliais/metabolismo , Células HCT116 , Proteínas de Choque Térmico/biossíntese , Humanos , Proteínas de Membrana/biossíntese , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Transdução de Sinais/fisiologiaRESUMO
In this study a well-characterized pathological mutation at nucleotide position 3243 of human mitochondrial DNA was introduced into human rho(0) teratocarcinoma (NT2) cells. In cloned and mixed populations of NT2 cells heteroplasmic for the mutation, mitotic segregation toward increasing levels of mutant mitochondrial DNA always occurred. Rapid segregation was frequently followed by complete loss of mitochondrial DNA. These findings support the idea that pathological mitochondrial DNA mutations are particularly deleterious in specific cell types, which can explain some of the tissue-specific aspects of mitochondrial DNA diseases. Moreover, these findings suggest that mitochondrial DNA depletion may be an important and widespread feature of mitochondrial DNA disease.
Assuntos
DNA Mitocondrial/genética , DNA Mitocondrial/fisiologia , Células Híbridas/fisiologia , Mutação , Linhagem Celular Tumoral , Células Clonais , DNA Mitocondrial/análise , Humanos , Cinética , Mitose , Teratocarcinoma/patologia , Fatores de TempoRESUMO
Phage-peptide display is a versatile tool for identifying novel protein-protein interfaces. Our previous work highlighted the selection of phage-peptides that bind to specific isoforms of MDM2 protein and in this work we subjected the putative MDM2-binding proteins to phage-peptide display to expand further on putative protein interaction maps. One peptide that bound MDM2 had significant homology to members of the death-activated protein kinase (DAPK) family, an enzyme family of no known direct link to the p53 pathway. We examined whether a nuclear member of the DAPK family named DAPK3 or ZIP kinase had direct links to the p53 pathway. ZIP kinase was cloned, purified, and the enzyme was able to phosphorylate MDM2 at Ser166, a site previously reported to be modified by Akt kinase, thus demonstrating that ZIP kinase is a bona fide MDM2-binding protein. Native ZIP kinase fractions were then subjected to phage-peptide display and one ZIP kinase consensus peptide motif was identified in p21(WAF1). ZIP kinase phosphorylates p21(WAF1) at Thr145 and alanine-substituted mutations in the p21(WAF1) phosphorylation site alter its ability to be phosphorylated by ZIP kinase. Thus, although ZIP kinase consensus sites were then defined as containing a minimal RKKx(T/S) consensus motif, alternate contacts in ZIP kinase binding are implicated, since amino acid residues surrounding the phospho-acceptor site can effect the specific activity of the kinase. Transfected ZIPK can promote the phosphorylation of p21(WAF1) at Thr145 in vivo and can increase the half-life of p21(WAF1), while the half-life of p21(WAF1[T145A]) is not effected by ZIP kinase. Thus, phage-peptide display identified an interferon-responsive protein kinase family as a novel modifier of two components of the p53 pathway, MDM2 and p21(WAF1), and underscores the utility of phage-peptide display for gaining novel insights into biochemical pathways.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Ciclinas/metabolismo , Inibidores Enzimáticos/metabolismo , Interferons/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Sequência de Aminoácidos , Proteínas Reguladoras de Apoptose , Proteínas Quinases Dependentes de Cálcio-Calmodulina/genética , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/genética , Proteínas Quinases Associadas com Morte Celular , Proteínas de Choque Térmico HSP90/genética , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Dados de Sequência Molecular , Mutação , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Biblioteca de Peptídeos , Peptídeos/genética , Peptídeos/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-mdm2 , Alinhamento de Sequência , Proteína Supressora de Tumor p53/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
The esophageal epithelium is subject to damage from bile acid reflux that promotes normal tissue injury resulting in the development of Barrett's epithelium. There is a selection pressure for mutating p53 in this preneoplastic epithelium, thus identifying a physiologically relevant model for discovering novel regulators of the p53 pathway. Proteomic technologies were used to identify such p53 regulatory factors by identifying proteins that were overexpressed in Barrett's epithelium. A very abundant polypeptide selectively expressed in Barrett's epithelium was identified as anterior gradient-2. Immunochemical methods confirmed that anterior gradient-2 is universally up-regulated in Barrett's epithelium, relative to normal squamous tissue derived from the same patient. Transfection of the anterior gradient-2 gene into cells enhances colony formation, similar to mutant oncogenic p53 encoded by the HIS175 allele, suggesting that anterior gradient-2 can function as a survival factor. Deletion of the C-terminal 10 amino acids of anterior gradient-2 neutralizes the colony enhancing activity of the gene, suggesting a key role for this domain in enhancing cell survival. Constitutive overexpression of anterior gradient-2 does not alter cell-cycle parameters in unstressed cells, suggesting that this gene is not directly modifying the cell cycle. However, cells overexpressing anterior gradient-2 attenuate p53 phosphorylation at both Ser(15) and Ser(392) and silence p53 transactivation function in ultraviolet (UV)-damaged cells. Deletion of the C-terminal 10 amino acids of anterior gradient-2 permits phosphorylation at Ser(15) in UV-damaged cells, suggesting that the C-terminal motif promoting colony survival also contributes to suppression of the Ser(15) kinase pathway. These data identify anterior gradient-2 as a novel survival factor whose study may shed light on cellular pathways that attenuate the tumor suppressor p53.
Assuntos
Esôfago de Barrett/metabolismo , Dano ao DNA , Inativação Gênica , Neoplasias Intestinais/metabolismo , Transcrição Gênica , Proteína Supressora de Tumor p53/metabolismo , Proteínas de Xenopus/fisiologia , Sequência de Aminoácidos , Esôfago de Barrett/patologia , Ciclo Celular , Sobrevivência Celular , Ensaio de Unidades Formadoras de Colônias , Eletroforese em Gel Bidimensional , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Humanos , Neoplasias Intestinais/patologia , Espectrometria de Massas , Metaplasia/metabolismo , Metaplasia/patologia , Dados de Sequência Molecular , Fosforilação , Isomerases de Dissulfetos de Proteínas , Homologia de Sequência de Aminoácidos , Proteína Supressora de Tumor p53/genética , Raios UltravioletaRESUMO
OBJECTIVE: Oesophageal cancer associated with the premalignant condition Barrett's oesophagus has increased in incidence over the last few years. Phase II detoxifying enzymes, including glutathione S-transferases (GSTs) protect the mucosa from carcinogens, which can cause oxidative damage to cells. Therefore, a reduction in these anti-oxidant enzymes can increase the risk of carcinogenesis. The aim of this study was to compare the extent of GST expression in normal oesophageal tissue, Barrett's oesophagus and oesophageal adenocarcinoma. DESIGN: Antibodies raised against GST alpha, GST mu, GST pi and microsomal GST were used to identify expression of these proteins in tissue sections. METHOD: Paraffin-embedded sections were stained using standard immunohistochemical techniques to demonstrate the pattern of expression of GST proteins in biopsy specimens. Twelve sections of Barrett's metaplasia and an equal number of specimens from normal oesophageal tissue were examined, together with sections from adenocarcinoma and normal gastric mucosa. RESULTS: Expression of the GST enzymes appeared to be reduced in Barrett's tissue compared to normal oesophageal tissue. Nuclear staining featured in some of the normal tissue sections, but not in Barrett's tissue. CONCLUSION: The reduction in GST expression suggested in Barrett's tissue is an interesting finding, as it is possible that reduced expression of these detoxifying enzymes may contribute to the risk of development of adenocarcinoma in Barrett's mucosa.