RESUMO
BACKGROUND & AIMS: T cells mediate the development of inflammation in inflammatory bowel disease (IBD). We investigated the effects of an antibody against CD3 called otelixizumab, which induces immune tolerance, in intestinal mucosa samples from patients. METHODS: Intestinal tissues were isolated from patients undergoing routine endoscopy or from patients undergoing intestinal surgery for colon cancer or IBD; healthy surrounding tissues were collected as controls. Isolated lamina propria mononuclear cells (LPMCs) and mucosal tissue explants were incubated with otelixizumab for 24 or 48 hours. Production of inflammatory cytokines was determined by enzyme-linked immunosorbent assay. Levels of 36 cytokines and chemokines and phosphorylation of 39 receptor tyrosine kinases and signaling molecules were measured using protein arrays. Immunoblot analysis was used to analyze T-cell transcription factors. RESULTS: Incubation of intestinal tissues or LPMCs with otelixizumab reduced production of interferon gamma, interleukin (IL)-17A, and other inflammatory cytokines and chemokines, simultaneously increasing production of IL-10. Mucosal biopsy specimens from patients with IBD retained inflammation-associated tyrosine phosphoprotein profiles ex vivo. Incubation of the inflamed tissue with otelixizumab reduced phosphorylation of these proteins to levels observed in control tissues. Otelixizumab also markedly reduced phosphorylation of proteins associated with T-cell receptor activation. Neutralization of IL-10 blocked the anti-inflammatory effects of otelixizumab. CONCLUSIONS: We observed anti-inflammatory effects of anti-CD3 in inflamed intestinal tissues from patients with IBD. The antibody appears to down-regulate T-cell activation via IL-10.
Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Anticorpos Monoclonais/farmacologia , Complexo CD3/imunologia , Colo/metabolismo , Citocinas/metabolismo , Doenças Inflamatórias Intestinais/metabolismo , Fosfoproteínas/metabolismo , Adolescente , Adulto , Biópsia , Estudos de Casos e Controles , Colo/efeitos dos fármacos , Colo/patologia , Feminino , Humanos , Tolerância Imunológica/efeitos dos fármacos , Doenças Inflamatórias Intestinais/patologia , Interferon gama/metabolismo , Interleucina-10/metabolismo , Interleucina-17/metabolismo , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
Defining the molecular and neuronal basis of associative memories is based upon behavioral preparations that yield high performance due to selection of salient stimuli, strong reinforcement, and repeated conditioning trials. One of those preparations is the Drosophila aversive olfactory conditioning procedure where animals initiate multiple memory components after experience of a single cycle training procedure. Here, we explored the analysis of acquisition dynamics as a means to define memory components and revealed strong correlations between particular chronologies of shock impact and number experienced during the associative training situation and subsequent performance of conditioned avoidance. Analyzing acquisition dynamics in Drosophila memory mutants revealed that rutabaga (rut)-dependent cAMP signals couple in a divergent fashion for support of different memory components. In case of anesthesia-sensitive memory (ASM) we identified a characteristic two-step mechanism that links rut-AC1 to A-kinase anchoring proteins (AKAP)-sequestered protein kinase A at the level of Kenyon cells, a recognized center of olfactory learning within the fly brain. We propose that integration of rut-derived cAMP signals at level of AKAPs might serve as counting register that accounts for the two-step mechanism of ASM acquisition.
Assuntos
Proteínas de Ancoragem à Quinase A/fisiologia , Adenilil Ciclases/fisiologia , Proteínas de Drosophila/fisiologia , Memória/fisiologia , Reforço Psicológico , Olfato/fisiologia , Animais , AMP Cíclico/fisiologia , Drosophila , Feminino , MasculinoRESUMO
Next-generation sequencing of the hypervariable V3 region of the 16s rRNA gene isolated from serial stool specimens collected from 31 patients receiving allogeneic stem cell transplantation (SCT) was performed to elucidate variations in the composition of the intestinal microbiome in the course of allogeneic SCT. Metagenomic analysis was complemented by strain-specific enterococcal PCR and indirect assessment of bacterial load by liquid chromatography-tandem mass spectrometry of urinary indoxyl sulfate. At the time of admission, patients showed a predominance of commensal bacteria. After transplantation, a relative shift toward enterococci was observed, which was more pronounced under antibiotic prophylaxis and treatment of neutropenic infections. The shift was particularly prominent in patients that developed subsequently or suffered from active gastrointestinal (GI) graft-versus-host disease (GVHD). The mean proportion of enterococci in post-transplant stool specimens was 21% in patients who did not develop GI GVHD as compared with 46% in those that subsequently developed GI GVHD and 74% at the time of active GVHD. Enterococcal PCR confirmed predominance of Enterococcus faecium or both E. faecium and Enterococcus faecalis in these specimens. As a consequence of the loss of bacterial diversity, mean urinary indoxyl sulfate levels dropped from 42.5 ± 11 µmol/L to 11.8 ± 2.8 µmol/L in all post-transplant samples and to 3.5 ± 3 µmol/L in samples from patients with active GVHD. Our study reveals major microbiome shifts in the course of allogeneic SCT that occur in the period of antibiotic treatment but are more prominent in association with GI GVHD. Our data indicate early microbiome shifts and a loss of diversity of the intestinal microbiome that may affect intestinal inflammation in the setting of allogeneic SCT.
Assuntos
Trato Gastrointestinal/microbiologia , Doença Enxerto-Hospedeiro/microbiologia , Transplante de Células-Tronco Hematopoéticas , Metagenoma , Adulto , Antibacterianos/uso terapêutico , Biodiversidade , Enterococcus faecalis/efeitos dos fármacos , Enterococcus faecalis/genética , Enterococcus faecium/efeitos dos fármacos , Enterococcus faecium/genética , Fezes/microbiologia , Feminino , Trato Gastrointestinal/efeitos dos fármacos , Trato Gastrointestinal/imunologia , Doença Enxerto-Hospedeiro/tratamento farmacológico , Doença Enxerto-Hospedeiro/imunologia , Humanos , Indicã/urina , Masculino , Microbiota , Pessoa de Meia-Idade , RNA Ribossômico 16S/genética , Transplante HomólogoRESUMO
A-kinase anchoring proteins (AKAPs) include a family of scaffolding proteins that target protein kinase A (PKA) and other signaling proteins to cellular compartments and thereby confine the activities of the associated proteins to distinct regions within cells. AKAPs bind PKA directly. The interaction is mediated by the dimerization and docking domain of regulatory subunits of PKA and the PKA-binding domain of AKAPs. Analysis of the interactions between the dimerization and docking domain and various PKA-binding domains yielded a generalized motif allowing the identification of AKAPs. Our bioinformatics and peptide array screening approaches based on this signature motif identified GSKIP (glycogen synthase kinase 3beta interaction protein) as an AKAP. GSKIP directly interacts with PKA and GSK3beta (glycogen synthase kinase 3beta). It is widely expressed and facilitates phosphorylation and thus inactivation of GSK3beta by PKA. GSKIP contains the evolutionarily conserved domain of unknown function 727. We show here that this domain of GSKIP and its vertebrate orthologues binds both PKA and GSK3beta and thereby provides a mechanism for the integration of PKA and GSK3beta signaling pathways.
Assuntos
Proteínas de Ancoragem à Quinase A/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Quinase 3 da Glicogênio Sintase/metabolismo , Transdução de Sinais/fisiologia , Proteínas de Ancoragem à Quinase A/genética , Motivos de Aminoácidos/fisiologia , Linhagem Celular Tumoral , Proteínas Quinases Dependentes de AMP Cíclico/genética , Quinase 3 da Glicogênio Sintase/genética , Glicogênio Sintase Quinase 3 beta , Humanos , Ligação Proteica/fisiologia , Multimerização Proteica/fisiologia , Estrutura Terciária de Proteína/fisiologiaRESUMO
PKA (protein kinase A) is tethered to subcellular compartments by direct interaction of its regulatory subunits (RI or RII) with AKAPs (A kinase-anchoring proteins). AKAPs preferentially bind RII subunits via their RII-binding domains. RII-binding domains form structurally conserved amphipathic helices with unrelated sequences. Their binding affinities for RII subunits differ greatly within the AKAP family. Amongst the AKAPs that bind RIIalpha subunits with high affinity is AKAP7delta [AKAP18delta; K(d) (equilibrium dissociation constant) value of 31 nM]. An N-terminally truncated AKAP7delta mutant binds RIIalpha subunits with higher affinity than the full-length protein presumably due to loss of an inhibitory region [Henn, Edemir, Stefan, Wiesner, Lorenz, Theilig, Schmidtt, Vossebein, Tamma, Beyermann et al. (2004) J. Biol. Chem. 279, 26654-26665]. In the present study, we demonstrate that peptides (25 amino acid residues) derived from the RII-binding domain of AKAP7delta bind RIIalpha subunits with higher affinity (K(d)=0.4+/-0.3 nM) than either full-length or N-terminally truncated AKAP7delta, or peptides derived from other RII binding domains. The AKAP7delta-derived peptides and stearate-coupled membrane-permeable mutants effectively disrupt AKAP-RII subunit interactions in vitro and in cell-based assays. Thus they are valuable novel tools for studying anchored PKA signalling. Molecular modelling indicated that the high affinity binding of the amphipathic helix, which forms the RII-binding domain of AKAP7delta, with RII subunits involves both the hydrophobic and the hydrophilic faces of the helix. Alanine scanning (25 amino acid peptides, SPOT technology, combined with RII overlay assays) of the RII binding domain revealed that hydrophobic amino acid residues form the backbone of the interaction and that hydrogen bond- and salt-bridge-forming amino acid residues increase the affinity of the interaction.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas de Membrana/metabolismo , Peptídeos/química , Ligação Proteica , Proteínas de Ancoragem à Quinase A , Proteínas Adaptadoras de Transdução de Sinal/química , Sequência de Aminoácidos , Aminoácidos/química , Aminoácidos/metabolismo , Animais , Sítios de Ligação , Células Cultivadas , Proteínas Quinases Dependentes de AMP Cíclico/química , Eletrofisiologia , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Proteínas de Membrana/química , Modelos Moleculares , Dados de Sequência Molecular , Peptídeos/metabolismo , Peptídeos/farmacologia , Estrutura Terciária de Proteína , Subunidades Proteicas , Ratos , Alinhamento de Sequência , Fatores de TempoRESUMO
The cAMP-specific phosphodiesterase PDE4D5 can interact with the signalling scaffold proteins RACK (receptors for activated C-kinase) 1 and beta-arrestin. Two-hybrid and co-immunoprecipitation analyses showed that RACK1 and beta-arrestin interact with PDE4D5 in a mutually exclusive manner. Overlay studies with PDE4D5 scanning peptide array libraries showed that RACK1 and beta-arrestin interact at overlapping sites within the unique N-terminal region of PDE4D5 and at distinct sites within the conserved PDE4 catalytic domain. Screening scanning alanine substitution peptide arrays, coupled with mutagenesis and truncation studies, allowed definition of RACK1 and beta-arrestin interaction sites. Modelled on the PDE4D catalytic domain, these form distinct well-defined surface-exposed patches on helices-15-16, for RACK1, and helix-17 for beta-arrestin. siRNA (small interfering RNA)-mediated knockdown of RACK1 in HEK-293 (human embryonic kidney) B2 cells increased beta-arrestin-scaffolded PDE4D5 approx. 5-fold, increased PDE4D5 recruited to the beta2AR (beta2-adrenergic receptor) upon isoproterenol challenge approx. 4-fold and severely attenuated (approx. 4-5 fold) both isoproterenol-stimulated PKA (protein kinase A) phosphorylation of the beta2AR and activation of ERK (extracellular-signal-regulated kinase). The ability of a catalytically inactive form of PDE4D5 to exert a dominant negative effect in amplifying isoproterenol-stimulated ERK activation was ablated by a mutation that blocked the interaction of PDE4D5 with beta-arrestin. In the present study, we show that the signalling scaffold proteins RACK1 and beta-arrestin compete to sequester distinct 'pools' of PDE4D5. In this fashion, alterations in the level of RACK1 expression may act to modulate signal transduction mediated by the beta2AR.
Assuntos
Arrestinas/metabolismo , AMP Cíclico/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Proteínas de Neoplasias/metabolismo , Biblioteca de Peptídeos , Diester Fosfórico Hidrolases/metabolismo , Mapeamento de Interação de Proteínas , Receptores de Superfície Celular/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Células COS , Domínio Catalítico/genética , Chlorocebus aethiops , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 3 , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4 , Ativação Enzimática/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Isoproterenol/farmacologia , Modelos Moleculares , Dados de Sequência Molecular , Diester Fosfórico Hidrolases/química , Fosforilação/efeitos dos fármacos , Ligação Proteica , RNA Interferente Pequeno/genética , Receptores de Quinase C Ativada , Receptores Adrenérgicos beta 2/metabolismo , beta-ArrestinasRESUMO
Cellular signalling pathways consolidate multiple molecular interactions into working models of signal propagation, amplification, and modulation. They are described and visualized as networks. Adjusting network topologies to experimental data is a key goal of systems biology. While network reconstruction algorithms like nested effects models are well established tools of computational biology, their data requirements can be prohibitive for their practical use. In this paper we suggest focussing on well defined aspects of a pathway and develop the computational tools to do so. We adapt the framework of nested effect models to focus on a specific aspect of activated Wnt signalling in HCT116 colon cancer cells: Does the activation of Wnt target genes depend on the secretion of Wnt ligands or do mutations in the signalling molecule ß-catenin make this activation independent from them? We framed this question into two competing classes of models: Models that depend on Wnt ligands secretion versus those that do not. The model classes translate into restrictions of the pathways in the network topology. Wnt dependent models are more flexible than Wnt independent models. Bayes factors are the standard Bayesian tool to compare different models fairly on the data evidence. In our analysis, the Bayes factors depend on the number of potential Wnt signalling target genes included in the models. Stability analysis with respect to this number showed that the data strongly favours Wnt ligands dependent models for all realistic numbers of target genes.
Assuntos
Modelos Teóricos , Algoritmos , Teorema de Bayes , Linhagem Celular Tumoral , Humanos , Transdução de Sinais , Proteínas Wnt/metabolismoRESUMO
New therapeutic targets are needed that circumvent inherent therapeutic resistance of glioblastoma multiforme (GBM). Here, we report such a candidate target in the uncharacterized adaptor protein hMOB3, which we show is upregulated in GBM. In a search for its biochemical function, we found that hMOB3 specifically interacts with MST1 kinase in response to apoptotic stimuli and cell-cell contact. Moreover, hMOB3 negatively regulated apoptotic signaling by MST1 in GBM cells by inhibiting the MST1 cleavage-based activation process. Physical interaction between hMOB3 and MST1 was essential for this process. In vivo investigations established that hMOB3 sustains GBM cell growth at high cell density and promotes tumorigenesis. Our results suggest hMOB3 as a candidate therapeutic target for the treatment of malignant gliomas.
Assuntos
Apoptose , Glioblastoma/metabolismo , Glioblastoma/patologia , Fator de Crescimento de Hepatócito/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais , Animais , Linhagem Celular Tumoral , Proliferação de Células , Modelos Animais de Doenças , Expressão Gênica , Glioblastoma/genética , Xenoenxertos , Humanos , Imuno-Histoquímica , Proteínas Associadas aos Microtúbulos/genética , Ligação Proteica , Proteólise , Carga TumoralRESUMO
Aberrant regulation of the Wnt/ß-catenin pathway has an important role during the onset and progression of colorectal cancer, with over 90% of cases of sporadic colon cancer featuring mutations in APC or ß-catenin. However, it has remained a point of controversy whether these mutations are sufficient to activate the pathway or require additional upstream signals. Here we show that colorectal tumours express elevated levels of Wnt3 and Evi/Wls/GPR177. We found that in colon cancer cells, even in the presence of mutations in APC or ß-catenin, downstream signalling remains responsive to Wnt ligands and receptor proximal signalling. Furthermore, we demonstrate that truncated APC proteins bind ß-catenin and key components of the destruction complex. These results indicate that cells with mutations in APC or ß-catenin depend on Wnt ligands and their secretion for a sufficient level of ß-catenin signalling, which potentially opens new avenues for therapeutic interventions by targeting Wnt secretion via Evi/Wls.
Assuntos
Adenocarcinoma/genética , Proteína da Polipose Adenomatosa do Colo/genética , Neoplasias do Colo/genética , Regulação Neoplásica da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intracelular/genética , Receptores Acoplados a Proteínas G/genética , Proteína Wnt3/genética , beta Catenina/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Proteína da Polipose Adenomatosa do Colo/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células , Colo/metabolismo , Colo/patologia , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Mutação , Transplante de Neoplasias , Receptor EphB2/genética , Receptor EphB2/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais , Proteína Wnt3/metabolismo , beta Catenina/metabolismoRESUMO
The cAMP/protein kinase A (PKA)-dependent insertion of water channel aquaporin-2 (AQP2)-bearing vesicles into the plasma membrane in renal collecting duct principal cells (AQP2 shuttle) constitutes the molecular basis of arginine vasopressin (AVP)-regulated water reabsorption. cAMP/PKA signaling systems are compartmentalized by A kinase anchoring proteins (AKAP) that tether PKA to subcellular sites and by phosphodiesterases (PDE) that terminate PKA signaling through hydrolysis of localized cAMP. In primary cultured principal cells, AVP causes focal activation of PKA. PKA and cAMP-specific phosphodiesterase-4D (PDE4D) are located on AQP2-bearing vesicles. The selective PDE4 inhibitor rolipram increases AKAP-tethered PKA activity on AQP2-bearing vesicles and enhances the AQP2 shuttle and thereby the osmotic water permeability. AKAP18delta, which is located on AQP2-bearing vesicles, directly interacts with PDE4D and PKA. In response to AVP, PDE4D and AQP2 translocate to the plasma membrane. Here PDE4D is activated through PKA phosphorylation and reduces the osmotic water permeability. Taken together, a novel, compartmentalized, and physiologically relevant cAMP-dependent signal transduction module on AQP2-bearing vesicles, comprising anchored PDE4D, AKAP18delta, and PKA, has been identified.