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Gastrointestinal organoids have emerged as a model system that authentically recapitulates the in vivo situation. Despite biomedical and technical challenges, self-assembled 3D structures derived from pluripotent stem cells or healthy and diseased tissues have proved to be invaluable tools for cancer drug discovery, disease modeling, and studying infection with carcinogenic pathogens.
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Organoides , Organoides/patologia , Humanos , Animais , Trato Gastrointestinal/patologia , Neoplasias/terapia , Neoplasias/patologia , Descoberta de Drogas/métodos , Células-Tronco Pluripotentes/citologiaRESUMO
Cell polarity is crucial for gastric mucosal barrier integrity and mainly regulated by polarity-regulating kinase partitioning-defective 1b (Par1b). During infection, the carcinogen Helicobacter pylori hijacks Par1b via the bacterial oncoprotein CagA leading to loss of cell polarity, but the precise molecular mechanism is not fully clear. Here we discovered a novel function of the actin-binding protein cortactin in regulating Par1b, which forms a complex with cortactin and the tight junction protein zona occludens-1 (ZO-1). We found that serine phosphorylation at S405/418 and the SH3 domain of cortactin are important for its interaction with both Par1b and ZO-1. Cortactin knockout cells displayed disturbed Par1b cellular localization and exhibited morphological abnormalities that largely compromised transepithelial electrical resistance, epithelial cell polarity, and apical microvilli. H. pylori infection promoted cortactin/Par1b/ZO-1 abnormal interactions in the tight junctions in a CagA-dependent manner. Infection of human gastric organoid-derived mucosoids supported these observations. We therefore hypothesize that CagA disrupts gastric epithelial cell polarity by hijacking cortactin, and thus Par1b and ZO-1, suggesting a new signaling pathway for the development of gastric cancer by Helicobacter.
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PURPOSE: Since glioma therapy is currently still limited until today, new treatment options for this heterogeneous group of tumours are of great interest. Eukaryotic initiation factors (eIFs) are altered in various cancer entities, including gliomas. The purpose of our study was to evaluate the potential of eIFs as novel targets in glioma treatment. METHODS: We evaluated eIF protein expression and regulation in 22 glioblastoma patient-derived xenografts (GBM PDX) after treatment with established cytostatics and with regards to mutation profile analyses of GBM PDX. RESULTS: We observed decreased expression of several eIFs upon temozolomide (TMZ) treatment independent from the phosphatidylinositol 3-kinase (PI3K)/ AKT/ mammalian target of the rapamycin (mTOR) signalling pathway. These effects of TMZ treatment were not present in TMZ-resistant PDX. Combination therapy of regorafenib and TMZ re- established the eIF/AKT/mTOR axis. CONCLUSION: Our study provides novel insights into chemotherapeutic effects on eIF regulation in gliomas and suggests that eIFs are interesting candidates for future research to improve glioma therapy.
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Neoplasias Encefálicas , Glioblastoma , Glioma , Humanos , Temozolomida/uso terapêutico , Temozolomida/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Dacarbazina/uso terapêutico , Dacarbazina/farmacologia , Neoplasias Encefálicas/genética , Linhagem Celular Tumoral , Glioma/tratamento farmacológico , Glioma/patologia , Glioblastoma/tratamento farmacológico , Glioblastoma/patologia , Serina-Treonina Quinases TOR/metabolismoRESUMO
Drug resistance is a common cause of therapy failure in head and neck squamous cell carcinoma (HNSCC). One approach to tackling it is by targeting fundamental cellular processes, such as translation. The eukaryotic translation initiation factor 2α (EIF2α) is a key player in canonical translation initiation and integrates diverse stress signals; when phosphorylated, it curbs global protein synthesis. This study evaluates EIF2α expression and phosphorylation in HNSCC. A small-molecule inhibitor of EIF2α dephosphorylation, salubrinal, was tested in vitro, followed by viability assays, flow cytometry, and immunoblot analyses. Patient-derived 3D tumor spheres (PD3DS) were cultured with salubrinal and their viability assessed. Lastly, salubrinal was evaluated with standard-of-care chemotherapeutics. Our analysis of RNA and proteomics data shows elevated EIF2α expression in HNSCC. Immunohistochemical staining reveals increasing EIF2α abundance from premalignant lesions to invasive and metastatic carcinoma. In immunoblots from intraoperative samples, EIF2α expression and steady-state phosphorylation are higher in HNSCC than in neighboring normal tissue. Inhibition of EIF2α dephosphorylation decreases HNSCC cell viability and clonogenic survival and impairs the G1/S transition. Salubrinal also decreases the viability of PD3DS and acts synergistically with cisplatin, 5-fluorouracil, bleomycin, and proteasome inhibitors. Our results indicate that pharmacological inhibition of EIF2α dephosphorylation is a potential therapeutic strategy for HNSCC.
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Helicobacter pylori is a human microbial pathogen that colonizes the gastric epithelium and causes type B gastritis with varying degrees of active inflammatory infiltrates. The underlying chronic inflammation induced by H. pylori and other environmental factors may promote the development of neoplasms and adenocarcinoma of the stomach. Dysregulation of various cellular processes in the gastric epithelium and in different cells of the microenvironment is a hallmark of H. pylori infection. We address the conundrum of H. pylori-associated apoptosis and review distinct mechanisms induced in host cells that either promote or suppress apoptosis in gastric epithelial cells, often simultaneously. We highlight key processes in the microenvironment that contribute to apoptosis and gastric carcinogenesis.
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Gastrite , Helicobacter pylori , Neoplasias Gástricas , Humanos , Neoplasias Gástricas/etiologia , Mucosa Gástrica/patologia , Apoptose , Microambiente TumoralRESUMO
Deubiquitinylating enzymes (DUBs) regulate the deubiquitinylation process of post-translationally modified proteins and thus control protein signaling in various cellular processes. The DUB Cezanne-1 catalyzes the cleavage of the iso-peptide bond of Lys11-linked polyubiquitin chains with high selectivity. Crystal structures of Cezanne-1 in different states provide important insight regarding the complex formation and global changes during the catalytic cycle but are lacking details of dynamics and control of activation. Activity-based probes are used to isolate intermediate states upon forming covalent bonds with the DUB active site. Those, however, may lead to structures that are non-native. Conformational changes of Cezanne-1, during its process of activation and proteolytic activity, are investigated using all-atom molecular dynamics (MD) simulations of the ubiquitin-free, diubiquitin-bound, and monoubiquitin-bound Cezanne-1 DUB for a total of â¼18 µs. Our results show that ubiquitin-free Cezanne-1 dynamically shuttles between catalytically competent and incompetent states which suggests that its activation is independent of substrate binding. The catalytically competent substrate-free Cezanne-1 promotes distal ubiquitin substrate access to the catalytic center. The subsequent binding of the proximal ubiquitin shifts the equilibrium toward the catalytically competent state of the dyad, thereby promoting proteolysis of the iso-peptide bond. After cleavage of the scissile bond, sequential dissociation of first the proximal ubiquitin induces the inactivation of Cezanne-1. The subsequent release of the distal ubiquitin fully reconstitutes the inactive substrate-free state of Cezanne-1. The process of activation and catalytic turnover of DUB Cezanne-1 is a multistage cycle with several critical dynamic transitions that cannot be characterized based on protein structures alone. Activity-based probes of cysteine proteases lead to non-native protein-protein contacts, which need to be resolved in order to be able to issue statements about physiological states and substrate binding.
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Endopeptidases , Ubiquitina , Endopeptidases/química , Endopeptidases/genética , Endopeptidases/metabolismo , Ubiquitinação , Ubiquitina/metabolismo , Poliubiquitina/metabolismo , Peptídeos/metabolismoRESUMO
The human pathogen Helicobacter pylori induces a strong inflammatory response in gastric mucosa manifested by the recruitment of neutrophils and macrophages to the places of infection, and by changes in epithelial integrity and function. At the molecular level, this innate immune response is essentially dependent on the activation of NF-κB transcription factors regulating the expression of chemotactic factors, e.g., IL-8. Recently, it has been demonstrated that the NF-κB signaling pathway is triggered by the bacterial heptose metabolites, which activate the host ALPK1-TIFA axis. TIFA has been suggested to promote oligomerization and activity of the E3 ubiquitin ligase TRAF6, which further stimulates TAK1-IKK signaling. Here, we demonstrate that ALPK1-dependent TIFA activation in H. pylori-infected gastric epithelial cells is followed in time by a decline in TIFA levels, and that this process is impeded by inhibitors of the proteasomal and lysosomal degradation. According to our data, TRAF2, TRAF6, TAK1 or NEMO are not required for TIFA degradation. Additionally, H. pylori promotes the interaction of TIFA with free polyubiquitin as well as with optineurin, TAX1BP1 and LAMP1, which are known protein adaptors involved in intracellular trafficking to lysosomes.
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Helicobacter pylori , Humanos , Helicobacter pylori/metabolismo , NF-kappa B/metabolismo , Fator 6 Associado a Receptor de TNF/metabolismo , Transdução de Sinais , Células Epiteliais/metabolismoRESUMO
OBJECTIVES: The objective of this study is to compare monolithic hybrid abutment crowns (screw-retained) versus monolithic hybrid abutments with adhesively cemented monolithic single-tooth crowns. MATERIALS AND METHODS: Twenty subjects in need of an implant-borne restoration were randomly assigned to receive either a cement-retained (CRR) or a screw-retained (SRR) implant-supported monolithic lithium disilicate (LS2 ) reconstruction. Each patient received a titanium implant with in internal conic connection. After osseointegration and second-stage surgery, healing abutments were placed for about 10 days. The type of restoration (CRR vs. SRR) was randomly assigned, and the restorations were manufactured of monolithic LS2 . Both types of restorations, CRR and SRR, were based on a titanium component (Ti-base) that was bonded to the abutment (CRR) or the crown (SRR). The follow-up period for all restoration was 36 months. Clinical outcome was evaluated according to Functional Implant Prosthetic Score (FIPS). Quality of live (OHIP) and patient's satisfaction were assessed using patient-reported outcome measures (PROMs). Primary endpoint was loss of restoration for any reason. Kaplan-Meier curves were constructed and log-rank testing was performed (p < .05). RESULTS: One restoration of group CRR failed after 6 months due to loss of adhesion between Ti-base and individual abutment. No further biological or technical failures occurred. Kaplan-Meier analysis showed no significant difference between both treatment options (p = .317). There was no statistically significant difference between both types of restoration, neither for FIPS, OHIP, treatment time nor patient satisfaction (p > .05). CONCLUSION: Monolithic hybrid abutment crowns (screw-retained) and monolithic hybrid abutment with adhesively cemented monolithic crowns using lithium disilicate showed no statistically significant difference for implant-based reconstructions in this pilot RCT setting.
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Projeto do Implante Dentário-Pivô , Titânio , Humanos , Zircônio , Desenho Assistido por Computador , Falha de Restauração Dentária , Coroas , Parafusos Ósseos , Dente SuporteRESUMO
Dysfunction of mesangial cells plays a major role in the pathogenesis of diabetic kidney disease (DKD), the leading cause of kidney failure. However, the underlying molecular mechanisms are incompletely understood. By unbiased gene expression analysis of glucose-exposed mesangial cells, we identified the transmembrane receptor CD248 as the most upregulated gene, and the maladaptive unfolded protein response (UPR) as one of the most stimulated pathways. Upregulation of CD248 was further confirmed in glucose-stressed mesangial cells in vitro, in kidney glomeruli isolated from diabetic mice (streptozotocin; STZ and db/db models, representing type 1 and type 2 diabetes mellitus, respectively) in vivo, and in glomerular kidney sections from patients with DKD. Time course analysis revealed that glomerular CD248 induction precedes the onset of albuminuria, mesangial matrix expansion and maladaptive UPR activation (hallmarked by transcription factor C/EBP homologous protein (CHOP) induction) but is paralleled by loss of the adaptive UPR regulator spliced X box binding protein (XBP1). Mechanistically, CD248 promoted maladaptive UPR signaling via inhibition of the inositol requiring enzyme 1α (IRE1α)-mediated transcription factor XBP1 splicing in vivo and in vitro. CD248 induced a multiprotein complex comprising heat shock protein 90, BH3 interacting domain death agonist (BID) and IRE1α, in which BID impedes IRE1α-mediated XBP1 splicing and induced CHOP mediated maladaptive UPR signaling. While CD248 knockout ameliorated DKD-associated glomerular dysfunction and reverses maladaptive unfolded protein response signaling, concomitant XBP1 deficiency abolished the protective effect in diabetic CD248 knockout mice, supporting a functional interaction of CD248 and XBP1 in vivo. Hence, CD248 is a novel mesangial cell receptor inducing maladaptive UPR signaling in DKD.
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Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 2 , Nefropatias Diabéticas , Animais , Camundongos , Antígenos CD/metabolismo , Antígenos de Neoplasias , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/genética , Nefropatias Diabéticas/genética , Endorribonucleases/genética , Endorribonucleases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Fatores de Transcrição/metabolismo , Resposta a Proteínas não Dobradas , HumanosRESUMO
Helicobacter pylori exemplifies one of the most favourable bacterial pathogens worldwide. The bacterium colonizes the gastric mucosa in about half of the human population and constitutes a major risk factor for triggering gastric diseases such as stomach cancer. H. pylori infection represents a prime example of chronic inflammation and cancer-inducing bacterial pathogens. The microbe utilizes a remarkable set of virulence factors and strategies to control cellular checkpoints of inflammation and oncogenic signal transduction. This chapter emphasizes on the pathogenicity determinants of H. pylori such as the cytotoxin-associated genes pathogenicity island (cagPAI)-encoded type-IV secretion system (T4SS), effector protein CagA, lipopolysaccharide (LPS) metabolite ADP-glycero-ß-D-manno-heptose (ADP-heptose), cytotoxin VacA, serine protease HtrA, and urease, and how they manipulate various key host cell signaling networks in the gastric epithelium. In particular, we highlight the H. pylori-induced disruption of cell-to-cell junctions, pro-inflammatory activities, as well as proliferative, pro-apoptotic and anti-apoptotic responses. Here we review these hijacked signal transduction events and their impact on gastric disease development.
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Helicobacter pylori , Humanos , Helicobacter pylori/genética , Estômago , Transdução de Sinais , Inflamação , CitotoxinasRESUMO
Crosstalk within the gastric epithelium, which is closely in contact with stromal fibroblasts in the gastric mucosa, has a pivotal impact in proliferation, differentiation and transformation of the gastric epithelium. The human pathogen Helicobacter pylori colonises the gastric epithelium and represents a risk factor for gastric pathophysiology. Infection of H. pylori induces the activation of the transcription factor nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), which is involved in the pro-inflammatory response but also in cell survival. In co-cultures with human gastric fibroblasts (HGF), we found that apoptotic cell death is reduced in the polarised human gastric cancer cell line NCI-N87 or in gastric mucosoids during H. pylori infection. Interestingly, suppression of apoptotic cell death in NCI-N87 cells involved an enhanced A20 expression regulated by NF-κB activity in response to H. pylori infection. Moreover, A20 acts as an important negative regulator of caspase-8 activity, which was suppressed in NCI-N87 cells during co-culture with gastric fibroblasts. Our results provide evidence for NF-κB-dependent regulation of apoptotic cell death in cellular crosstalk and highlight the protective role of gastric fibroblasts in gastric epithelial cell death during H. pylori infection.
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Infecções por Helicobacter , Helicobacter pylori , Caspase 8/metabolismo , Sobrevivência Celular , Técnicas de Cocultura , Células Epiteliais/metabolismo , Fibroblastos/metabolismo , Infecções por Helicobacter/metabolismo , Helicobacter pylori/metabolismo , Humanos , NF-kappa B/metabolismo , Fatores de Transcrição/metabolismoRESUMO
The human pathogen Helicobacter pylori represents a risk factor for the development of gastric diseases including cancer. The H. pylori-induced transcription factor nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) is involved in the pro-inflammatory response and cell survival in the gastric mucosa, and represents a trailblazer of gastric pathophysiology. Termination of nuclear NF-κB heterodimer RelA/p50 activity is regulated by the ubiquitin-RING-ligase complex elongin-cullin-suppressor of cytokine signalling 1 (ECSSOCS1), which leads to K48-ubiquitinylation and degradation of RelA. We found that deubiquitinylase (DUB) ubiquitin specific protease 48 (USP48), which interacts with the COP9 signalosome (CSN) subunit CSN1, stabilises RelA by deubiquitinylation and thereby promotes the transcriptional activity of RelA to prolong de novo synthesis of DUB A20 in H. pylori infection. An important role of A20 is the suppression of caspase-8 activity and apoptotic cell death. USP48 thus enhances the activity of A20 to reduce apoptotic cell death in cells infected with H. pylori. Our results, therefore, define a synergistic mechanism by which USP48 and A20 regulate RelA and apoptotic cell death in H. pylori infection.
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Infecções por Helicobacter , Proteína 3 Induzida por Fator de Necrose Tumoral alfa , Proteases Específicas de Ubiquitina , Sobrevivência Celular , Helicobacter pylori , Humanos , NF-kappa B/metabolismo , Proteína 3 Induzida por Fator de Necrose Tumoral alfa/metabolismo , Proteases Específicas de Ubiquitina/metabolismo , UbiquitinaçãoRESUMO
Upon postsynaptic glutamate receptor activation, the cytosolic Ca2+ concentration rises and initiates signaling and plasticity in spines. The plasma membrane Ca2+ ATPase (PMCA) is a major player to limit the duration of cytosolic Ca2+ signals. It forms complexes with the glycoprotein neuroplastin (Np) isoforms Np55 and Np65 and functionally interplays with N-methyl-D-aspartate (NMDA)-type ionotropic glutamate receptors (iGluNRs). Moreover, binding of the Np65-specific extracellular domain to Ca2+-permeable GluA1-containing α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)-type ionotropic glutamate receptors (iGluA1Rs) was found to be required for long-term potentiation (LTP). However, the link between PMCA and iGluRs function to regulate cytosolic Ca2+ signals remained unclear. Here, we report that Np65 coordinates PMCA and iGluRs' functions to modulate the duration and amplitude of cytosolic Ca2+ transients in dendrites and spines of hippocampal neurons. Using live-cell Ca2+ imaging, acute pharmacological treatments, and GCaMP5G-expressing hippocampal neurons, we discovered that endogenous or Np65-promoted PMCA activity contributes to the restoration of basal Ca2+ levels and that this effect is dependent on iGluR activation. Super-resolution STED and confocal microscopy revealed that electrical stimulation increases the abundance of synaptic neuroplastin-PMCA complexes depending on iGluR activation and that low-rate overexpression of Np65 doubled PMCA levels and decreased cell surface levels of GluN2A and GluA1 in dendrites and Shank2-positive glutamatergic synapses. In neuroplastin-deficient hippocampi, we observed reduced PMCA and unchanged GluN2B levels, while GluN2A and GluA1 levels were imbalanced. Our electrophysiological data from hippocampal slices argues for an essential interplay of PMCA with GluN2A- but not with GluN2B-containing receptors upon induction of synaptic plasticity. Accordingly, we conclude that Np65 may interconnect PMCA with core players of glutamatergic neurotransmission to fine-tune the Ca2+ signal regulation in basal synaptic function and plasticity.
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Adenosina Trifosfatases , Receptores Ionotrópicos de Glutamato , Adenosina Trifosfatases/metabolismo , Hipocampo/metabolismo , Plasticidade Neuronal , Neurônios/metabolismo , Receptores Ionotrópicos de Glutamato/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Sinapses/metabolismoRESUMO
Inflammatory bowel disease (IBD), including Crohn's disease (CD) and ulcerative colitis (UC), is a chronic inflammatory disorder of the gastrointestinal tract. Although the pathophysiology of IBD is multifaceted, ubiquitination, a post-translational modification, has been shown to have essential roles in its pathogenesis and development. Ubiquitin-modifying enzymes (UMEs) work in synergy to orchestrate the optimal ubiquitination of target proteins, thereby maintaining intestinal homeostasis. Genome-wide association studies (GWAS) have identified multiple UME genes as IBD susceptibility loci, implying the importance of UMEs in IBD. Furthermore, accumulative evidence demonstrates that UMEs affect intestinal inflammation by regulating various aspects, such as intestinal barrier functions and immune responses. Considering the significant functions of UMEs in IBD, targeting UMEs could become a favorable therapeutic approach for IBD.
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Colite Ulcerativa , Colite , Doença de Crohn , Doenças Inflamatórias Intestinais , Colite/genética , Colite Ulcerativa/genética , Doença de Crohn/genética , Estudo de Associação Genômica Ampla , Humanos , Doenças Inflamatórias Intestinais/genética , UbiquitinaRESUMO
Gastric cancer is one of the leading causes of the cancer-related mortality worldwide. The etiology of this disease is complex and involves genetic predisposition and environmental factors, including Helicobacter pylori. Infection of the stomach with H. pylori leads to gastritis and gastric atrophy, which can progress stepwise to gastric cancer. Matrix metalloproteinases (MMPs) actively participate in the pathology development. The further progression of gastric cancer seems to be less dependent on bacteria but of intra-tumor cell dynamics. Bioinformatics data confirmed an important role of the extracellular matrix constituents and specific MMPs in stomach carcinoma invasion and metastasis, and revised their potential as predictors of the disease outcome. In this review, we describe, in detail, the impact of MMPs in H. pylori-associated gastritis and gastric cancer.
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Gastrite/microbiologia , Infecções por Helicobacter/metabolismo , Helicobacter pylori/patogenicidade , Metaloproteinases da Matriz/metabolismo , Neoplasias Gástricas/microbiologia , Biologia Computacional , Progressão da Doença , Gastrite/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Invasividade Neoplásica , Metástase Neoplásica , Neoplasias Gástricas/metabolismoRESUMO
NF-κB signaling pathways, induced by a variety of triggers, play a key role in regulating the expression of genes involved in the immune response and cellular responses to stress. The human pathogen Helicobacter pylori induces classical and alternative NF-κB signaling pathways via its effector ADP-L-glycero-ß-D-manno-heptose (ADP-heptose). We review H. pylori- and NF-κB-dependent alterations in cellular processes and associated maladaptation leading to deleterious gastric pathophysiology that have implications for the diagnosis and treatment of gastric diseases. Therapeutic options for gastric cancer (GC) include clinically relevant small molecule inhibitors of NF-κB and epigenetic therapy approaches. In this context, gastric organoid biobanks originated from patient material, represent a valuable platform for translational applications to predict patient responses to chemotherapy, with a view to personalized medicine.
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Infecções por Helicobacter , NF-kappa B , Neoplasias Gástricas , Mucosa Gástrica/metabolismo , Infecções por Helicobacter/tratamento farmacológico , Infecções por Helicobacter/genética , Helicobacter pylori , Humanos , NF-kappa B/genética , Transdução de Sinais , Neoplasias Gástricas/tratamento farmacológico , Neoplasias Gástricas/microbiologiaRESUMO
Transcription factors of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-ĸB) family control important signaling pathways in the regulation of the host innate immune system. Various bacterial pathogens in the human gastrointestinal tract induce NF-ĸB activity and provoke pro-inflammatory signaling events in infected epithelial cells. NF-ĸB activation requires the phosphorylation-dependent proteolysis of inhibitor of ĸB (IĸB) molecules including the NF-ĸB precursors through ubiquitin-mediated proteolysis. The canonical NF-ĸB pathway merges on IĸB kinases (IKKs), which are required for signal transduction. Using CRISPR-Cas9 technology, secreted embryonic alkaline phosphatase (SEAP) reporter assays and cytokine enzyme-linked immunosorbent assay (ELISA), we demonstrate that the actin-binding protein cortactin is involved in NF-ĸB activation and subsequent interleukin-8 (IL-8) production upon infection by Helicobacter pylori, Salmonella enterica and Pseudomonas aeruginosa. Our data indicate that cortactin is needed to efficiently activate the c-Sarcoma (Src) kinase, which can positively stimulate NF-ĸB during infection. In contrast, cortactin is not involved in activation of NF-ĸB and IL-8 expression upon infection with Campylobacter species C. jejuni, C. coli or C. consisus, suggesting that Campylobacter species pluralis (spp.) induce a different signaling pathway upstream of cortactin to trigger the innate immune response.
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Deubiquitinylases (DUBs) are central regulators of the ubiquitin system involved in protein regulation and cell signalling and are important for a variety of physiological processes. Most DUBs are cysteine proteases, and few other proteases are metalloproteases of the JAB1/MPN +/MOV34 protease family (JAMM). STAM-binding protein like 1 (STAMBPL1), a member of the JAMM family, cleaves ubiquitin bonds and has a function in regulating cell survival, Tax-mediated nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activation and epithelial-mesenchymal transition. However, the molecular mechanism by which STAMBPL1 influences cell survival is not well defined, especially with regard to its deubiquitinylation function. Here, we show that reactive oxygen species (ROS) induced by chemotherapeutic agents or the human microbial pathogen Helicobacter pylori can induce cullin 1-RING ubiquitin ligase (CRL1) and 26S proteasome-dependent degradation STAMBPL1. Interestingly, STAMBPL1 has a direct interaction with the constitutive photomorphogenic 9 (COP9 or CSN) signalosome subunits CSN5 and CSN6. The interaction with the CSN is required for the stabilisation and function of the STAMBPL1 protein. In addition, STAMBPL1 deubiquitinylates the anti-apoptotic protein Survivin and thus ameliorates cell survival. In summary, our data reveal a previously unknown mechanism by which the deubiquitinylase STAMBPL1 and the E3 ligase CRL1 balance the level of Survivin degradation and thereby determine apoptotic cell death. In response to genotoxic stress, the degradation of STAMBPL1 augments apoptotic cell death. This new mechanism may be useful to develop therapeutic strategies targeting STAMBPL1 in tumours that have high STAMBPL1 and Survivin protein levels.
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Apoptose , Complexo do Signalossomo COP9/metabolismo , Helicobacter pylori/fisiologia , Peptídeo Hidrolases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Apoptose/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular , Doxorrubicina/farmacologia , Humanos , Peptídeo Hidrolases/química , Peptídeo Hidrolases/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Survivina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , UbiquitinaçãoRESUMO
With the aim of overcoming host immune responses, and to permit persistence, numerous bacterial and viral pathogens have evolved effective strategies to control the activity of ovarian tumor domain proteases (OTUs), a group of deubiquitinylases crucial for regulating ubiquitin-modified proteins. Due to the important role of eukaryotic OTUs in cellular physiology, it is not surprising that pathogens have evolutionarily developed effector proteins which mimic host OTUs. Here, we focus on recent findings that illustrate how pathogen-encoded OTUs modulate eukaryotic host proteins and how they are implicated in cellular dysregulation. Further, we discuss the biological effects of OTUs in the context of structural features and pharmacological targeting. We point out the potentiality of selective OTU inhibitors, which shield ubiquitin-binding sites, as pharmacologic targets to treat harmful infections.
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Neoplasias Ovarianas , Peptídeo Hidrolases , Humanos , Peptídeo Hidrolases/metabolismo , Ligação Proteica , Ubiquitina/química , Ubiquitina/metabolismoRESUMO
Microfluidic devices for culturing cells have been successfully utilized for biomedical applications, including drug screening. Several cell lines could be cultivated in microengineered environments with promising results, but gastric cell lines have not yet been widely used or studied. Therefore, this study focuses on establishing a polarized gastric epithelial monolayer on-a-chip and describes a general-purpose methodology applicable for bonding any porous material to PDMS through an adhesive sublayer. The fully transparent microfluidic chip consists of two microfluidic channels separated by a collagen-coated porous membrane and lined by human polarized gastric epithelial (NCI-N87) cells. We present considerations on how to ensure continuous and stable flow through the channels. The continuous flow rate was achieved using a pressure-driven pump. Media flow at a constant rate (0.5 µL/min) rapidly led the gastric epithelial cells to develop into a polarized monolayer. The barrier integrity was assessed by the FITC-dextran test. The generation of a monolayer was faster than in the static Boyden chamber. Moreover, fluorescence microscopy was used to monitor the apoptotic cell death of gastric epithelial monolayers on-a-chip in response to camptothecin, a therapeutic gastric cancer drug.