RESUMO
Irg1 is an enzyme that generates itaconate, a metabolite that plays a key role in the regulation of inflammatory responses. Previous studies have implicated Irg1 as an important mediator in preventing excessive inflammation and tissue damage in Mycobacterium tuberculosis (Mtb) infection. Here, we investigated the pattern recognition receptors and signaling pathways by which Mtb triggers Irg1 gene expression by comparing the responses of control and genetically deficient BMDMs. Using this approach, we demonstrated partial roles for TLR-2 (but not TLR-4 or -9), MyD88 and NFκB signaling in Irg1 induction by Mtb bacilli. In addition, drug inhibition studies revealed major requirements for phagocytosis and endosomal acidification in Irg1 expression triggered by Mtb but not LPS or PAM3CSK4. Importantly, the Mtb-induced Irg1 response was highly dependent on the presence of the bacterial ESX-1 secretion system, as well as host STING and Type I IFN receptor (IFNAR) signaling with Type II IFN (IFN-γ) signaling playing only a minimal role. Based on these findings we hypothesize that Mtb induces Irg1 expression in macrophages via the combination of two independent triggers both dependent on bacterial phagocytosis: 1) a major signal stimulated by phagocytized Mtb products released by an ESX-1-dependent mechanism into the cytosol where they activate the STING pathway leading to Type I-IFN production, and 2) a secondary TLR-2, MyD88 and NFκB dependent signal that enhances Irg1 production independently of Type I IFN induction.
Assuntos
Hidroliases , Macrófagos , Proteínas de Membrana , Mycobacterium tuberculosis , Receptor de Interferon alfa e beta , Receptor 2 Toll-Like , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Indução Enzimática , Hidroliases/biossíntese , Hidroliases/imunologia , Macrófagos/imunologia , Macrófagos/microbiologia , Proteínas de Membrana/metabolismo , Camundongos , Mycobacterium tuberculosis/metabolismo , Fator 88 de Diferenciação Mieloide/metabolismo , NF-kappa B/metabolismo , Fagocitose , Receptor de Interferon alfa e beta/metabolismo , Receptor 2 Toll-Like/metabolismo , Tuberculose/metabolismo , Tuberculose/microbiologiaRESUMO
Pseudouridine (Ψ) is the most common noncanonical ribonucleoside present on mammalian noncoding RNAs (ncRNAs), including rRNAs, tRNAs, and snRNAs, where it contributes â¼7% of the total uridine level. However, Ψ constitutes only â¼0.1% of the uridines present on mRNAs and its effect on mRNA function remains unclear. Ψ residues have been shown to inhibit the detection of exogenous RNA transcripts by host innate immune factors, thus raising the possibility that viruses might have subverted the addition of Ψ residues to mRNAs by host pseudouridine synthase (PUS) enzymes as a way to inhibit antiviral responses in infected cells. Here, we describe and validate a novel antibody-based Ψ mapping technique called photo-crosslinking-assisted Ψ sequencing (PA-Ψ-seq) and use it to map Ψ residues on not only multiple cellular RNAs but also on the mRNAs and genomic RNA encoded by HIV-1. We describe 293T-derived cell lines in which human PUS enzymes previously reported to add Ψ residues to human mRNAs, specifically PUS1, PUS7, and TRUB1/PUS4, were inactivated by gene editing. Surprisingly, while this allowed us to assign several sites of Ψ addition on cellular mRNAs to each of these three PUS enzymes, Ψ sites present on HIV-1 transcripts remained unaffected. Moreover, loss of PUS1, PUS7, or TRUB1 function did not significantly reduce the level of Ψ residues detected on total human mRNA below the â¼0.1% level seen in wild-type cells, thus implying that the PUS enzyme(s) that adds the bulk of Ψ residues to human mRNAs remains to be defined.
Assuntos
Anticorpos Monoclonais/imunologia , Edição de Genes , Transferases Intramoleculares/metabolismo , Pseudouridina/metabolismo , Processamento Pós-Transcricional do RNA , RNA Mensageiro/metabolismo , RNA Viral/metabolismo , Células HEK293 , Infecções por HIV/genética , Infecções por HIV/metabolismo , Infecções por HIV/virologia , HIV-1/fisiologia , Humanos , Hidroliases/antagonistas & inibidores , Hidroliases/genética , Hidroliases/imunologia , Hidroliases/metabolismo , Transferases Intramoleculares/antagonistas & inibidores , Transferases Intramoleculares/genética , Transferases Intramoleculares/imunologia , Pseudouridina/imunologia , RNA Mensageiro/genética , RNA Viral/genéticaRESUMO
Synthetic biology and metabolic engineering offer potentially green and attractive routes to the production of high value compounds. The provision of high-quality parts and pathways is crucial in enabling the biosynthesis of chemicals using synthetic biology. While a number of regulatory parts that provide control at the transcriptional and translational level have been developed, relatively few exist at the protein level. Single domain antibodies (sdAb) such as camelid heavy chain variable fragments (VHH) possess binding characteristics which could be exploited for their development and use as novel parts for regulating metabolic pathways at the protein level in microbial cell factories. Here, a platform for the use of VHH as tools in Escherichia coli is developed and subsequently used to modulate linalool production in E. coli. The coproduction of a Design of Experiments (DoE) optimized pBbE8k His6-VHHCyDisCo system alongside a heterologous linalool production pathway facilitated the identification of anti-bLinS VHH that functioned as modulators of bLinS. This resulted in altered product profiles and significant variation in the titers of linalool, geraniol, nerolidol, and indole obtained. The ability to alter the production levels of high value terpenoids, such as linalool, in a tunable manner at the protein level could represent a significant step forward for the development of improved microbial cell factories. This study serves as a proof of principle indicating that VHH can be used to modulate enzyme activity in engineered pathways within E. coli. Given their almost limitless binding potential, we posit that single domain antibodies could emerge as powerful regulatory parts in synthetic biology applications.
Assuntos
Monoterpenos Acíclicos/metabolismo , Proteínas de Bactérias/imunologia , Escherichia coli/genética , Escherichia coli/metabolismo , Hidroliases/imunologia , Cadeias Pesadas de Imunoglobulinas/imunologia , Engenharia Metabólica/métodos , Anticorpos de Domínio Único/imunologia , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Reatores Biológicos , Camelídeos Americanos/imunologia , Códon , Ativação Enzimática/imunologia , Hidroliases/genética , Hidroliases/metabolismo , Streptomyces/enzimologia , Streptomyces/genética , Biologia Sintética/métodosRESUMO
The guanosine triphosphatase (GTPase) Rab32 coordinates a cell-intrinsic host defense mechanism that restricts the replication of intravacuolar pathogens such as Salmonella Here, we show that this mechanism requires aconitate decarboxylase 1 (IRG1), which synthesizes itaconate, a metabolite with antimicrobial activity. We find that Rab32 interacts with IRG1 on Salmonella infection and facilitates the delivery of itaconate to the Salmonella-containing vacuole. Mice defective in IRG1 rescued the virulence defect of a S. enterica serovar Typhimurium mutant specifically defective in its ability to counter the Rab32 defense mechanism. These studies provide a link between a metabolite produced in the mitochondria after stimulation of innate immune receptors and a cell-autonomous defense mechanism that restricts the replication of an intracellular bacterial pathogen.
Assuntos
Hidroliases/imunologia , Infecções por Salmonella/imunologia , Salmonella enterica , Salmonella typhimurium , Proteínas rab de Ligação ao GTP/imunologia , Animais , Linhagem Celular , Interações Hospedeiro-Patógeno , Humanos , Hidroliases/metabolismo , Camundongos , Infecções por Salmonella/metabolismo , Infecções por Salmonella/microbiologia , Succinatos , Virulência , Proteínas rab de Ligação ao GTP/metabolismoRESUMO
To identify the Helicobacter pylori antigens operating during early infection in sera from infected infants using proteomics and immunoblot analysis. Two-dimensional (2D) large and small gel electrophoresis was performed using H. pylori strain 51. We performed 2D immunoglobulin G (IgG), immunoglobulin A (IgA), and immunoglobulin M (IgM) antibody immunoblotting using small gels on sera collected at the Gyeongsang National University Hospital from 4-11-month-old infants confirmed with H. pylori infection by pre-embedding immunoelectron microscopy. Immunoblot spots appearing to represent early infection markers in infant sera were compared to those of the large 2D gel for H. pylori strain 51. Corresponding spots were analyzed by matrix-assisted laser desorption/ionization time of flight-mass spectrometry (MALDI-TOF-MS). The peptide fingerprints obtained were searched in the National Center for Biotechnology Information (NCBI) database. Eight infant patients were confirmed with H. pylori infection based on urease tests, histopathologic examinations, and pre-embedding immunoelectron microscopy. One infant showed a 2D IgM immunoblot pattern that seemed to represent early infection. Immunoblot spots were compared with those from whole-cell extracts of H. pylori strain 51 and 18 spots were excised, digested in gel, and analyzed by MALDI-TOF-MS. Of the 10 peptide fingerprints obtained, the H. pylori proteins flagellin A (FlaA), urease ß subunit (UreB), pyruvate ferredoxin oxidoreductase (POR), and translation elongation factor Ts (EF-Ts) were identified and appeared to be active during the early infection periods. These results might aid identification of serological markers for the serodiagnosis of early H. pylori infection in infants.
Assuntos
Anticorpos Antibacterianos/sangue , Antígenos de Bactérias/imunologia , Proteínas de Bactérias/análise , Infecções por Helicobacter/diagnóstico , Helicobacter pylori/imunologia , Hidroliases/análise , Oxirredutases/análise , Fatores de Alongamento de Peptídeos/análise , Piruvato Sintase/análise , Urease/análise , Proteínas de Bactérias/imunologia , Biomarcadores/análise , Feminino , Humanos , Hidroliases/imunologia , Immunoblotting , Imunoglobulina A/sangue , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Lactente , Masculino , Oxirredutases/imunologia , Fatores de Alongamento de Peptídeos/imunologia , Mapeamento de Peptídeos , Piruvato Sintase/imunologia , Testes Sorológicos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Urease/imunologiaRESUMO
Brucellosis is a debilitating zoonotic disease that affects humans and animals. The diagnosis of brucellosis is challenging, as accurate species level identification is not possible with any of the currently available serology-based diagnostic methods. The present study aimed at identifying Brucella (B.) species-specific proteins from the closely related species B. abortus and B. melitensis using sera collected from naturally infected host species. Unlike earlier reported investigations with either laboratory-grown species or vaccine strains, in the present study, field strains were utilized for analysis. The label-free quantitative proteomic analysis of the naturally isolated strains of these two closely related species revealed 402 differentially expressed proteins, among which 63 and 103 proteins were found exclusively in the whole cell extracts of B. abortus and B. melitensis field strains, respectively. The sera from four different naturally infected host species, i.e., cattle, buffalo, sheep, and goat were applied to identify the immune-binding protein spots present in the whole protein extracts from the isolated B. abortus and B. melitensis field strains and resolved on two-dimensional gel electrophoresis. Comprehensive analysis revealed that 25 proteins of B. abortus and 20 proteins of B. melitensis were distinctly immunoreactive. Dihydrodipicolinate synthase, glyceraldehyde-3-phosphate dehydrogenase and lactate/malate dehydrogenase from B. abortus, amino acid ABC transporter substrate-binding protein from B. melitensis and fumarylacetoacetate hydrolase from both species were reactive with the sera of all the tested naturally infected host species. The identified proteins could be used for the design of serological assays capable of detecting pan-Brucella, B. abortus- and B. melitensis-specific antibodies.
Assuntos
Anticorpos/imunologia , Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Brucella abortus/metabolismo , Brucella melitensis/metabolismo , Brucelose Bovina/microbiologia , Animais , Anticorpos/sangue , Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Western Blotting , Brucella abortus/isolamento & purificação , Brucella melitensis/isolamento & purificação , Brucelose Bovina/patologia , Bovinos , Cromatografia Líquida de Alta Pressão , Eletroforese em Gel Bidimensional , Gliceraldeído-3-Fosfato Desidrogenases/imunologia , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Hidroliases/imunologia , Hidroliases/metabolismo , Proteoma/análise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Macrophages can be niches for bacterial pathogens or antibacterial effector cells depending on the pathogen and signals from the immune system. Here we show that type I and II IFNs are master regulators of gene expression during Legionella pneumophila infection, and activators of an alveolar macrophage-intrinsic immune response that restricts bacterial growth during pneumonia. Quantitative mass spectrometry revealed that both IFNs substantially modify Legionella-containing vacuoles, and comparative analyses reveal distinct subsets of transcriptionally and spatially IFN-regulated proteins. Immune-responsive gene (IRG)1 is induced by IFNs in mitochondria that closely associate with Legionella-containing vacuoles, and mediates production of itaconic acid. This metabolite is bactericidal against intravacuolar L. pneumophila as well as extracellular multidrug-resistant Gram-positive and -negative bacteria. Our study explores the overall role IFNs play in inducing substantial remodeling of bacterial vacuoles and in stimulating production of IRG1-derived itaconic acid which targets intravacuolar pathogens. IRG1 or its product itaconic acid might be therapeutically targetable to fight intracellular and drug-resistant bacteria.
Assuntos
Hidroliases/imunologia , Interferons/imunologia , Legionella pneumophila/imunologia , Doença dos Legionários/imunologia , Macrófagos Alveolares/imunologia , Proteoma , Animais , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica , Ontologia Genética , Hidroliases/genética , Hidroliases/metabolismo , Imunidade Inata , Interferons/metabolismo , Legionella pneumophila/genética , Legionella pneumophila/metabolismo , Doença dos Legionários/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mitocôndrias/metabolismo , Modelos Imunológicos , Espécies Reativas de Oxigênio/metabolismo , Succinatos/metabolismo , Vacúolos/metabolismo , Vacúolos/microbiologiaRESUMO
The immunoresponsive gene 1 (IRG1) protein has crucial functions in embryonic implantation and neurodegeneration. IRG1 promotes endotoxin tolerance by increasing A20 expression in macrophages through reactive oxygen species (ROS). The cytoprotective protein heme oxygenase-1 (HO-1), which generates endogenous carbon monoxide (CO), is expressed in the lung during Lipopolysaccharide (LPS) tolerance and cross tolerance. However, the detailed molecular mechanisms and functional links between IRG1 and HO-1 in the innate immune system remain unknown. In the present study, we found that the CO releasing molecule-2 (CORM-2) and chemical inducers of HO-1 increased IRG1 expression in a time- and dose-dependent fashion in RAW264.7 cells. Furthermore, inhibition of HO-1 activity by zinc protoporphyrin IX (ZnPP) and HO-1 siRNA significantly reduced expression of IRG1 under these conditions. In addition, treatment with CO and HO-1 induction significantly increased A20 expression, which was reversed by ZnPP and HO-1 siRNA. LPS-stimulated TNF-α was significantly decreased, whereas IRG1 and A20 were increased by CORM-2 application and HO-1 induction, which in turn were abrogated by ZnPP. Interestingly, siRNA against IRG1 and A20 reversed the effects of CO and HO-1 on LPS-stimulated TNF-α production. Additionally, CO and HO-1 inducers significantly increased IRG1 and A20 expression and downregulated TNF-α production in a LPS-stimulated sepsis mice model. Furthermore, the effects of CO and HO-1 on TNF-α production were significantly reversed when ZnPP was administered. In conclusion, CO and HO-1 induction regulates IRG1 and A20 expression, leading to inhibition of inflammation in vitro and in an in vivo mice model.
Assuntos
Monóxido de Carbono/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Heme Oxigenase-1/imunologia , Hidroliases/imunologia , Lipopolissacarídeos/toxicidade , Proteínas de Membrana/imunologia , Sepse/imunologia , Proteína 3 Induzida por Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/imunologia , Animais , Linhagem Celular , Heme Oxigenase-1/antagonistas & inibidores , Masculino , Proteínas de Membrana/antagonistas & inibidores , Camundongos , Sepse/induzido quimicamente , Sepse/patologiaRESUMO
Helicobacter pylori (H. pylori) infection causes peptic ulcers, gastric adenocarcinoma, and mucosa-associated lymphoid tissue (MALT) lymphoma. The eradication of H. pylori might be an effective means of preventing gastric cancer. A dual-antigen epitope and dual-adjuvant vaccine called CTB-UE-CF (CCF) was constructed by combining a multi-epitope vaccine CTB-UE with a novel chimeric flagellum (CF) to simultaneously activate Toll-like receptor (TLR) 5-agonist activity and preserve the immunogenicity of H. pylori flagellum FlaA. The evaluation of efficacy to reduce H. pylori colonization was performed using BALB/c mice by oral immunization with a triple dose of this vaccine strain. Two weeks after the last immunization, mice were sacrificed to determine specific antibody levels and proinflammatory cytokine production. To determine the presence of H. pylori, we detected the number of H. pylori by real-time quantitative PCR (qPCR) and measured the urease activity in the gastric tissue. The results showed that the immunogenicity and mucosal immune responses of CCF performed significantly better than those of CTB-UE. This dual-antigen epitope and dual-adjuvant system might greatly contribute to the development of a safe and efficient therapeutic vaccine for humans against H. pylori infection.
Assuntos
Vacinas Bacterianas/imunologia , Flagelos/genética , Infecções por Helicobacter/imunologia , Infecções por Helicobacter/prevenção & controle , Helicobacter pylori/imunologia , Neoplasias Gástricas/microbiologia , Neoplasias Gástricas/prevenção & controle , Adjuvantes Imunológicos , Animais , Anticorpos Antibacterianos/sangue , Proteínas de Bactérias/imunologia , Citocinas/sangue , Modelos Animais de Doenças , Epitopos/imunologia , Flagelos/imunologia , Infecções por Helicobacter/microbiologia , Helicobacter pylori/genética , Helicobacter pylori/crescimento & desenvolvimento , Helicobacter pylori/isolamento & purificação , Humanos , Hidroliases/imunologia , Imunidade nas Mucosas , Imunização , Esquemas de Imunização , Camundongos Endogâmicos BALB C , Oxirredutases/imunologia , Proteínas Recombinantes de Fusão/imunologia , Receptor 5 Toll-Like , Urease/química , Vacinas Sintéticas/imunologiaRESUMO
Sepsis-associated immunosuppression (SAIS) is regarded as one of main causes for the death of septic patients at the late stage because of the decreased innate immunity with a more opportunistic infection. LPS-tolerized macrophages, which are re-challenged by LPS after prior exposure to LPS, are regarded as the common model of hypo-responsiveness for SAIS. However, the molecular mechanisms of endotoxin tolerance and SAIS remain to be fully elucidated. In addition, negative regulation of the Toll-like receptor (TLR)-triggered innate inflammatory response needs further investigation. Here we show that expression of immune responsive gene 1 (IRG1) was highly up-regulated in the peripheral blood mononuclear cells of septic patients and in LPS-tolerized mouse macrophages. IRG1 significantly suppressed TLR-triggered production of proinflammatory cytokines TNF-α, IL-6, and IFN-ß in LPS-tolerized macrophages, with the elevated expression of reactive oxygen species (ROS) and A20. Moreover, ROS enhanced A20 expression by increasing the H3K4me3 modification of histone on the A20 promoter domain, and supplement of the ROS abrogated the IRG1 knockdown function in breaking endotoxin tolerance by increasing A20 expression. Our results demonstrate that inducible IRG1 promotes endotoxin tolerance by increasing A20 expression through ROS, indicating a new molecular mechanism regulating hypoinflammation of sepsis and endotoxin tolerance.
Assuntos
Proteínas de Ligação a DNA/imunologia , Regulação da Expressão Gênica/efeitos dos fármacos , Hidroliases/imunologia , Tolerância Imunológica/efeitos dos fármacos , Imunidade Inata/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Lipopolissacarídeos/toxicidade , Macrófagos/imunologia , Proteínas Nucleares/imunologia , Proteínas/imunologia , Espécies Reativas de Oxigênio/imunologia , Sepse/imunologia , Ubiquitina-Proteína Ligases/imunologia , Animais , Carboxiliases , Cisteína Endopeptidases , Proteínas de Ligação a DNA/genética , Feminino , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/imunologia , Técnicas de Silenciamento de Genes , Histonas/genética , Histonas/imunologia , Humanos , Hidroliases/genética , Interferon beta/genética , Interferon beta/imunologia , Interleucina-6/genética , Interleucina-6/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Lipopolissacarídeos/imunologia , Macrófagos/patologia , Masculino , Camundongos , Monócitos/imunologia , Monócitos/patologia , Proteínas Nucleares/genética , Proteínas/genética , Sepse/genética , Sepse/patologia , Proteína 3 Induzida por Fator de Necrose Tumoral alfa , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia , Ubiquitina-Proteína Ligases/genéticaRESUMO
The 67 kDa myosin-cross-reactive antigen (MCRA) is a member of the MCRA family of proteins present in a wide range of bacteria and was predicted to have fatty acid isomerase function. We have now characterised the catalytic activity of MCRAs from four LAB stains, including Lactobacillus rhamnosus LGG, L. plantarum ST-III, L. acidophilus NCFM and Bifidobacterium animalis subsp. lactis BB-12. MCRA genes from these strains were cloned and expressed in Escherichia coli, and the recombinant protein function was analysed with lipid profiles by GC-MS. The four MCRAs catalysed the conversion of linoleic acid and oleic acid to their respective 10-hydroxy derivatives, which suggests that MCRA proteins catalyse the first step in conjugated linoleic acid production. This is the first report of MCRA from L. rhamnosus with such catalytic function.
Assuntos
Antígenos de Bactérias/metabolismo , Bifidobacterium/enzimologia , Hidroliases/metabolismo , Lactobacillus/enzimologia , Ácidos Linoleicos/metabolismo , Sequência de Aminoácidos , Antígenos de Bactérias/química , Antígenos de Bactérias/genética , Antígenos de Bactérias/imunologia , Bifidobacterium/genética , Bifidobacterium/imunologia , Reações Cruzadas , Escherichia coli/genética , Cromatografia Gasosa-Espectrometria de Massas , Hidroliases/química , Hidroliases/genética , Hidroliases/imunologia , Lactobacillus/genética , Lactobacillus/imunologia , Dados de Sequência Molecular , Ácido Oleico/metabolismo , Filogenia , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Análise de Sequência de ProteínaRESUMO
BACKGROUND: Antibody-dependent cellular cytotoxicity (ADCC) is greatly enhanced by the absence of the core fucose of oligosaccharides attached to the Fc, and is closely related to the clinical efficacy of anticancer activity in humans in vivo. Unfortunately, all licensed therapeutic antibodies and almost all currently-developed therapeutic antibodies are heavily fucosylated and fail to optimize ADCC, which leads to a large dose requirement at a very high cost for the administration of antibody therapy to cancer patients. In this study, we explored the possibility of converting already-established antibody-producing cells to cells that produce antibodies fully lacking core fucosylation in order to facilitate the rapid development of next-generation therapeutic antibodies. RESULTS: Firstly, loss-of-function analyses using small interfering RNAs (siRNAs) against the three key genes involved in oligosaccharide fucose modification, i.e. alpha1,6-fucosyltransferase (FUT8), GDP-mannose 4,6-dehydratase (GMD), and GDP-fucose transporter (GFT), revealed that single-gene knockdown of each target was insufficient to completely defucosylate the products in antibody-producing cells, even though the most effective siRNA (>90% depression of the target mRNA) was employed. Interestingly, beyond our expectations, synergistic effects of FUT8 and GMD siRNAs on the reduction in fucosylation were observed, but not when these were used in combination with GFT siRNA. Secondly, we successfully developed an effective short hairpin siRNA tandem expression vector that facilitated the double knockdown of FUT8 and GMD, and we converted antibody-producing Chinese hamster ovary (CHO) cells to fully non-fucosylated antibody producers within two months, and with high converting frequency. Finally, the stable manufacture of fully non-fucosylated antibodies with enhanced ADCC was confirmed using the converted cells in serum-free fed-batch culture. CONCLUSION: Our results suggest that FUT8 and GMD collaborate synergistically in the process of intracellular oligosaccharide fucosylation. We also demonstrated that double knockdown of FUT8 and GMD in antibody-producing cells could serve as a new strategy for producing next-generation therapeutic antibodies fully lacking core fucosylation and with enhanced ADCC. This approach offers tremendous cost- and time-sparing advantages for the development of next-generation therapeutic antibodies.
Assuntos
Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/imunologia , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Fucosiltransferases/genética , Inativação Gênica/imunologia , Melhoramento Genético/métodos , Hidroliases/genética , Animais , Células CHO , Cricetinae , Cricetulus , Fucosiltransferases/imunologia , Hidroliases/imunologia , Engenharia de Proteínas/métodosRESUMO
The human ovarian surface epithelium (hOSE) is a single layer of mesothelial-type primitive epithelial cells that are potential estrogen targets. It has been reported that hOSE cells can produce estrogen. However, the mechanisms that regulate estrogen level(s) in hOSE cells are not yet known. To elucidate the enzymes involved in these reactions, we examined gene expression of 17beta-hydroxysteroid dehydrogenases (17beta-HSDs) in primary hOSE (POSE) and OSE2a cells using RT-PCR. We found that POSE cells and cells of the immortalized hOSE line, OSE2a, bidirectionally converted estrone (E1) and 17beta-estradiol (E2). Both cell types expressed mRNA for 17beta-HSD type 1 (17beta-HSD1), suggesting that the enzyme is involved in the E1 to E2 conversion. Interestingly, both cells expressed 17beta-HSD4 mRNA but not 17beta-HSD2 mRNA. We prepared an antibody against the carboxyl terminal of 17beta-HSD4 (anti-17beta-HSD4 antibody), which recognized the 80 and 48 kDa proteins in POSE and OSE2a cells based on immunoblot analysis. Furthermore, immunohistochemical study revealed the presence of 17beta-HSD4 in hOSE cells in the human ovary. These results suggest that 17beta-HSD4 is involved in estrogen inactivation and may protect against an excessive accumulation of E2 in hOSE cells.
Assuntos
17-Hidroxiesteroide Desidrogenases/metabolismo , Células Epiteliais/enzimologia , Hidroliases/metabolismo , Ovário/enzimologia , 17-Hidroxiesteroide Desidrogenases/genética , 17-Hidroxiesteroide Desidrogenases/imunologia , Adulto , Células Cultivadas , Endométrio/enzimologia , Estrogênios/biossíntese , Estrogênios/metabolismo , Feminino , Humanos , Hidroliases/genética , Hidroliases/imunologia , Immunoblotting , Imuno-Histoquímica , Isoenzimas/genética , Isoenzimas/imunologia , Isoenzimas/metabolismo , Pessoa de Meia-Idade , Proteína Multifuncional do Peroxissomo-2 , RNA Mensageiro/metabolismoRESUMO
Streptococcus mutans possesses different cell wall molecules, such as protein of the I/II family, the serotype f polysaccharide rhamnose glucose polymer (RGP), and lipoteichoic acid (LTA), which act as adhesins and modulins, allowing S. mutans to colonize teeth and cause dental caries and pulpitis. We tested several isogenic mutants of S. mutans defective in protein I/II and/or RGP, as well as purified modulins such as protein I/II, RGP, and LTA, for their binding and activation abilities on monocytic, dental pulp (DP), and periodontal ligament (PDL) cells. Our results demonstrate that both protein I/II and RGP play important roles in streptococcal adherence to human monocytic and fibroblastic cells, whereas LTA is only a minor adhesin. In the activation process, the cytokine response elicited is polarized toward a Th1 response which seems principally due to protein I/II and RGP. Even if protein I/II seems to be more efficient in its purified form in triggering cells to release interleukin-8 (IL-8), RGP is the most efficient cytokine-stimulating component in intact bacteria, while LTA plays only a minor role. In cell activation, we showed, by using either cytochalasin D or coated ligands, that internalization of either S. mutans, S. mutans isogenic mutants, or purified ligands is not necessary to trigger cells to release IL-8. We also showed that, besides the implication of monocytes in pulpal inflammation, fibroblast-like cells such as DP and PDL cells are also actively implicated in local inflammation and in the generation of a Th1 response after stimulation with S. mutans cells or antigens.
Assuntos
Genes Bacterianos , Interleucina-6/biossíntese , Interleucina-8/biossíntese , Glicoproteínas de Membrana , Streptococcus mutans/genética , Streptococcus mutans/imunologia , Fator de Necrose Tumoral alfa/biossíntese , Adesinas Bacterianas/imunologia , Aderência Bacteriana , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Linhagem Celular , Cárie Dentária/imunologia , Cárie Dentária/microbiologia , Polpa Dentária/imunologia , Polpa Dentária/microbiologia , Humanos , Hidroliases/genética , Hidroliases/imunologia , Monócitos/imunologia , Monócitos/microbiologia , Mutagênese Insercional , Ligamento Periodontal/imunologia , Ligamento Periodontal/microbiologia , Infecções Estreptocócicas/imunologia , Infecções Estreptocócicas/microbiologia , Streptococcus mutans/patogenicidadeRESUMO
D-bifunctional protein (D-BP) plays an indispensable role in peroxisomal beta-oxidation, and its inherited deficiency in humans is associated with severe clinical abnormalities. Three different subtypes of D-BP deficiency can be distinguished: 1) a complete deficiency of D-BP (type I), 2) an isolated D-BP enoyl-CoA hydratase deficiency (type II), and 3) an isolated D-BP 3-hydroxyacyl-CoA dehydrogenase deficiency (type III). In this study, we developed a method to measure D-BP dehydrogenase activity independent of D-BP hydratase (D-BP HY) activity to distinguish between D-BP deficiency type I and type II, which until now was only possible by mutation analysis. For this assay, the hydratase domain of D-BP was expressed in the yeast Saccharomyces cerevisiae. After a coincubation of yeast homogenate expressing D-BP HY with fibroblast homogenate of patients using the enoyl-CoA ester of the bile acid intermediate trihydroxycholestanoic acid as substrate, D-BP dehydrogenase activity was measured. Fibroblasts of patients with a D-BP deficiency type II displayed D-BP dehydrogenase activity, whereas type I and type III patients did not. This newly developed assay to measure D-BP dehydrogenase activity in fibroblast homogenates provides a quick and reliable method to assign patients with deficient D-BP HY activity to the D-BP deficiency subgroups type I or type II.
Assuntos
17-Hidroxiesteroide Desidrogenases , 3-Hidroxiacil-CoA Desidrogenases/análise , 3-Hidroxiacil-CoA Desidrogenases/deficiência , Cromatografia Líquida de Alta Pressão/métodos , Enoil-CoA Hidratase/análise , Enoil-CoA Hidratase/deficiência , Hidroliases/análise , Hidroliases/deficiência , Complexos Multienzimáticos/análise , Complexos Multienzimáticos/deficiência , Transtornos Peroxissômicos/diagnóstico , Transtornos Peroxissômicos/enzimologia , Peroxissomos/enzimologia , 3-Hidroxiacil-CoA Desidrogenases/imunologia , Linhagem Celular , Enoil-CoA Hidratase/imunologia , Fibroblastos , Humanos , Hidroliases/imunologia , Immunoblotting , Complexos Multienzimáticos/imunologia , Proteína Multifuncional do Peroxissomo-2 , LevedurasRESUMO
Dimerization co-factor of hepatocyte nuclear factor 1 (HNF1)/pterin-4alpha-carbinolamine dehydratase (DCoH/PCD) is both a positive co-factor of the HNF1 homeobox transcription factors and thus involved in gene regulation as well as an enzyme catalyzing the regeneration of tetrahydrobiopterin. Dysfunction of DCoH/PCD is associated with the human disorders hyperphenylalaninemia and vitiligo. In Xenopus, overexpression of the protein during development induces ectopic pigmentation. In this study loss of function experiments using DCoH/PCD-specific antibodies demonstrated that the protein is also absolutely necessary for pigment cell formation in Xenopus. In normal human skin DCoH/PCD protein is weakly expressed in the basal layer of the epidermis that consists of keratinocytes and melanocytes. Whereas only 4 of 25 benign nevi reacted with DCoH/PCD-specific antibodies, high protein levels were detectable in melanoma cell lines and 13 of 15 primary malignant melanoma lesions. The comparison with the commonly used melanoma markers S100 and HMB45 demonstrated that DCoH/PCD has an overlapping but distinct expression pattern in melanoma lesions. In addition to human colon cancer, this is the second report about the overexpression of DCoH/PCD in human tumor cells indicating that the protein might be involved in cancerogenesis.
Assuntos
Hidroliases/fisiologia , Melanoma/enzimologia , Pigmentação da Pele , Animais , Anticorpos/imunologia , Biomarcadores Tumorais/análise , Síndrome do Nevo Displásico/enzimologia , Humanos , Hidroliases/imunologia , Melanoma/química , Pele/enzimologia , Células Tumorais Cultivadas , Xenopus/anatomia & histologia , Xenopus/embriologiaRESUMO
The C-terminal repeat domain of Clostridium difficile toxin A harbors toxin-neutralizing epitopes and is considered to be a candidate component of a vaccine against C. difficile-associated disease (CDAD). Fourteen of the 38 C-terminal toxin A repeats (14CDTA) were cloned into pTECH-1 in frame with the immunogenic fragment C of tetanus toxin (TETC) to generate plasmid p56TETC. Expression of the TETC-14CDTA fusion protein was driven from the anaerobically inducible nirB promoter within attenuated Salmonella typhimurium BRD509 (aroA aroD). The TETC-14CDTA fusion protein was purified and shown to bind to known toxin A receptors found on the surface of rabbit erythrocytes. Intranasal (i.n.) and intragastric (i.g.) immunization with 10(7) and 10(10) CFU, respectively, of BRD509(p56TETC) generated significant (P < 0.05) anti-toxin A serum responses after a single dose. Antibody titers were elevated following a boosting dose with either live vaccine or a subcutaneous injection of 0.5 microgram of purified 14CDTA protein. Importantly, serum from mice immunized with BRD509(p56TETC) neutralized toxin A cytotoxicity. Both i.n. and i.g. immunizations also generated toxin A-specific immunoglobulin A on the pulmonary and intestinal mucosa, respectively. Intranasal vaccination induced consistently higher serum and mucosal anti-toxin A antibody responses. Significant anti-tetanus toxoid serum and mucosal antibodies were also generated by both immunization routes. The availability of live attenuated Salmonella typhi for human use may allow the development of a multivalent mucosal vaccine against CDAD, tetanus, and typhoid.
Assuntos
Toxinas Bacterianas , Vacinas Bacterianas/imunologia , Clostridioides difficile/imunologia , Enterotoxinas/imunologia , Hidroliases/imunologia , Vacinas contra Salmonella , Salmonella typhimurium/imunologia , Vacinas Tíficas-Paratíficas , Vacinas Sintéticas/imunologia , Administração Intranasal , Animais , Anticorpos Antibacterianos/biossíntese , Anticorpos Antibacterianos/sangue , Vacinas Bacterianas/administração & dosagem , Vacinas Bacterianas/genética , Sequência de Bases , Clostridioides difficile/genética , Primers do DNA/genética , Enterocolite Pseudomembranosa/imunologia , Enterocolite Pseudomembranosa/prevenção & controle , Enterotoxinas/genética , Feminino , Humanos , Hidroliases/genética , Imunidade nas Mucosas , Imunização , Imunoglobulina A/biossíntese , Camundongos , Camundongos Endogâmicos BALB C , Coelhos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Salmonella typhimurium/genética , Vacinas Combinadas/administração & dosagem , Vacinas Combinadas/genética , Vacinas Combinadas/imunologia , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genéticaRESUMO
Different Enterobacteriaceae, such as Escherichia coli, Proteus vulgaris and Proteus mirabilis, are able to convert L(-)-carnitine, via crotonobetaine, into gamma-butyrobetaine in the presence of carbon and nitrogen sources under aerobic conditions. Intermediates of L(-)-carnitine metabolism (crotonobetaine, gamma-butyrobetaine) could be detected by thin-layer chromatography. In parallel, L(-)-carnitine dehydratase, carnitine racemasing system and crotonobetaine reductase activities were determined enzymatically. Monoclonal antibodies against purified CaiB and CaiA from E. coli O44K74 were used to screen cell-free extracts of different Enterobacteriaceae (E. coli ATCC 25922, P. vulgaris, P. mirabilis, Citrobacter freundii, Enterobacter cloacae and Klebsiella pneumoniae) grown under aerobic conditions in the presence of L(-)-carnitine.
Assuntos
Aciltransferases , Carnitina/metabolismo , Enterobacteriaceae/enzimologia , Hidroliases/metabolismo , Complexos Multienzimáticos/metabolismo , Oxirredutases/metabolismo , Aerobiose , Anticorpos Monoclonais/imunologia , Western Blotting , Eletroforese em Gel de Poliacrilamida , Enterobacteriaceae/crescimento & desenvolvimento , Hidroliases/imunologia , Complexos Multienzimáticos/imunologia , Oxirredutases/imunologiaRESUMO
The presence of a beta-hydroxyacyl-CoA dehydratase involved in the icosanoyl-CoA synthase (EC 2.3.1.119) complex of leek epidermis has been demonstrated using antibodies raised against the purified beta-hydroxyacyl-CoA dehydratase from rat liver. In a first step the leek icosanoyl-CoA synthase activity was measured in the presence of different amounts of this antibody, the results obtained showed a 75% inhibition of the activity using a 8:1 IgG/microsomal protein ratio, whereas only a weak diminution of the activity occurred using pre-immune IgG. The analysis of the reaction products after incubation in the presence of increasing IgG amounts showed a decrease of the fatty acids (the final product) and an accumulation of beta-hydroxy fatty acids using immune IgG, whereas no change occurred in the presence of pre-immune IgG. Moreover, the beta-hydroxyacyl-CoA dehydratase activity was strongly inhibited, whereas in the same conditions, the beta-ketoacyl-CoA reductase and the (trans-2-3) enoyl-CoA reductase activities were not affected. The protein fractions that eluted from the DEAE and Ultrogel columns containing the leek icosanoyl-CoA synthase activity were able to specifically bind the anti beta-hydroxyacyl-CoA dehydratase from rat liver. The cross-reactivity was demonstrated. In immunoblotting experiments using the same antiserum after SDS-PAGE of the purified leek icosanoyl-CoA synthase, only one of the four protein bands constituting the leek icosanoyl-CoA synthase was detected. This protein, having an apparent molecular mass of 65 kDa, could be the dehydratase component of the elongation complex.
Assuntos
Allium/enzimologia , Coenzima A Ligases/química , Hidroliases/análise , Proteínas Repressoras , Proteínas de Saccharomyces cerevisiae , Animais , Anticorpos/imunologia , Anticorpos/farmacologia , Coenzima A Ligases/antagonistas & inibidores , Coenzima A Ligases/isolamento & purificação , Hidroliases/química , Hidroliases/imunologia , Immunoblotting , Fígado/enzimologia , RatosRESUMO
A new cobalt-containing nitrile hydratase was purified from extracts of urea-induced cells from Rhodococcus rhodochrous J1 in seven steps. At the last step, the enzyme was crystallized by adding ammonium sulfate. Nitrile hydratase was a 500-530-kDa protein composed of two different subunits (alpha subunit 26 kDa, beta subunit 29 kDa). The enzyme contained approximately 11-12 mol cobalt/mol enzyme. A concentrated solution of highly purified nitrile hydratase exhibited a broad absorption spectrum in the visible range, with an absorption maxima at 410 nm. The enzyme had a wide substrate specificity. Aliphatic saturated or unsaturated nitriles as well as aromatic nitriles, were substrates for the enzyme. The optimum pH of the hydratase was pH 6.5-6.8. The enzyme was more stable than ferric nitrile hydratases. The amino-terminal sequence of each subunit of R. rhodochrous J1 enzyme was determined and compared with that of ferric nitrile hydratases. Prominent similarities were observed with the beta subunit. However, the amino acid sequence of the alpha subunit from R. rhodochrous J1 was quite different from that of the ferric enzymes.