RESUMO
Leprosy is a skin disease and it is characterized by a disorder of the peripheral nervous system which occurs due to the infection of Schwann cells. In this research article, we have formulated a four-dimensional ODE-based mathematical model which consists of the densities of healthy Schwann cells, infected Schwann cells, M. leprae bacteria, and the concentration of multidrug therapy (MDT). This work primarily aims on exploring the dynamical changes and interrelations of the system cell populations during the disease progression. Also, evaluating a critical value of the drug efficacy rate of MDT remains our key focus in this article so that a safe drug dose regimen for leprosy can be framed more effectively and realistically. We have examined the stability scenario of different equilibria and the occurrence of Hopf-bifurcation for the densities of our system cell populations with respect to the drug efficacy rate of MDT to gain insight on the precise impact of the efficiency rate on both the infected Schwann cell and the bacterial populations. Also, a necessary transversality condition for the occurrence of the bifurcation has been established. Our analytical and numerical investigations in this research work precisely explores that the process of demyelination, nerve regeneration, and infection of the healthy Schwann cells are the three most crucial factors in the leprosy pathogenesis and to control the M. leprae-induced infection of Schwann cells successfully, a more flexible version of MDT regime with efficacy rate varying in the range η∈(0.025,0.059) for 100-120 days in PB cases and 300 days in MB cases obtained in this research article should be applied. All of our analytical outcomes have been verified through numerical simulations and compared with some existing clinical findings.
Assuntos
Hansenostáticos , Hanseníase , Humanos , Quimioterapia Combinada , Hansenostáticos/uso terapêutico , Hansenostáticos/farmacologia , Hanseníase/tratamento farmacológico , Hanseníase/microbiologia , Hanseníase/patologia , Mycobacterium leprae , Organização Mundial da SaúdeRESUMO
Genetic and environmental manipulations, such as dietary restriction, can improve both health span and lifespan in a wide range of organisms, including humans. Changes in nutrient intake trigger often overlapping metabolic pathways that can generate distinct or even opposite outputs depending on several factors, such as when dietary restriction occurs in the lifecycle of the organism or the nature of the changes in nutrients. Due to the complexity of metabolic pathways and the diversity in outputs, the underlying mechanisms regulating diet-associated pro-longevity are not yet well understood. Adult reproductive diapause (ARD) in the model organism Caenorhabditis elegans is a dietary restriction model that is associated with lengthened lifespan and reproductive potential. To explore the metabolic pathways regulating ARD in greater depth, we performed a candidate-based genetic screen analyzing select nutrient-sensing pathways to determine their contribution to the regulation of ARD. Focusing on the three phases of ARD (initiation, maintenance, and recovery), we found that ARD initiation is regulated by fatty acid metabolism, sirtuins, AMPK, and the O-linked N-acetyl glucosamine (O-GlcNAc) pathway. Although ARD maintenance was not significantly influenced by the nutrient sensors in our screen, we found that ARD recovery was modulated by energy sensing, stress response, insulin-like signaling, and the TOR pathway. Further investigation of downstream targets of NHR-49 suggest the transcription factor influences ARD initiation through the fatty acid ß-oxidation pathway. Consistent with these findings, our analysis revealed a change in levels of neutral lipids associated with ARD entry defects. Our findings identify conserved genetic pathways required for ARD entry and recovery and uncover genetic interactions that provide insight into the role of OGT and OGA.
Assuntos
Diapausa , Nutrientes , Transdução de Sinais , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Caenorhabditis elegans/metabolismo , Diapausa/genética , Diapausa/fisiologia , Ácidos Graxos/metabolismo , Glucosamina/metabolismo , Humanos , Insulinas/metabolismo , Lipídeos/química , Nutrientes/metabolismo , Nutrientes/farmacologia , Reprodução/genética , Reprodução/fisiologia , Transdução de Sinais/genética , Sirtuínas/genética , Sirtuínas/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Calcium signaling can elicit different pathways involved in an extreme variety of biological processes. Calcium levels must be tightly regulated in a spatial and temporal manner in order to be efficiently and properly utilized in the host physiology. The Ca2+-ATPase, encoded by pmr-1 gene, was first identified in yeast and localized to the Golgi and it appears to be involved in calcium homeostasis. PMR-1 function is evolutionary conserved from yeast to human, where mutations in the orthologous gene ATP2C1 cause Hailey-Hailey disease. In this work, we used the Caenorhabditis elegans model system to gain insight into the downstream response elicited by the loss of pmr-1 gene. We found that pmr-1 knocked down animals not only showed defects in the oligosaccharide structure of glycoproteins at the cell surface but also were characterized by reduced susceptibility to bacterial infection. Although increased resistance to the infection might be related to lack of regular recognition of C. elegans surface glycoproteins by microbial agents, we provide genetic evidence that pmr-1 interfered nematodes mounted a stronger innate immune response to Gram-positive bacterial infection. Thus, our observations indicate pmr-1 as a candidate gene implicated in mediating the worm's innate immune response.
Assuntos
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Caenorhabditis elegans/microbiologia , ATPases Transportadoras de Cálcio/genética , Imunidade Inata , Infecções Estafilocócicas/microbiologia , Animais , Caenorhabditis elegans/imunologia , Técnicas de Silenciamento de Genes , Glicosilação , Mutação , Oligossacarídeos/química , Staphylococcus aureus/patogenicidade , Estresse FisiológicoRESUMO
Nutrient-driven O-GlcNAcylation has been linked to epigenetic regulation of gene expression in metazoans. In C. elegans, O-GlcNAc marks the promoters of over 800 developmental, metabolic, and stress-related genes; these O-GlcNAc marked genes show a strong 5', promoter-proximal bias in the distribution of RNA Polymerase II (Pol II). In response to starvation or feeding, the steady state distribution of O-GlcNAc at promoters remain nearly constant presumably due to dynamic cycling mediated by the transferase OGT-1 and the O-GlcNAcase OGA-1. However, in viable mutants lacking either of these enzymes of O-GlcNAc metabolism, the nutrient-responsive GlcNAcylation of promoters is dramatically altered. Blocked O-GlcNAc cycling leads to a striking nutrient-dependent accumulation of O-GlcNAc on RNA Pol II. O-GlcNAc cycling mutants also show an exaggerated, nutrient-responsive redistribution of promoter-proximal RNA Pol II isoforms and extensive transcriptional deregulation. Our findings suggest a complex interplay between the O-GlcNAc modification at promoters, the kinase-dependent "CTD-code," and co-factors regulating RNA Pol II dynamics. Nutrient-responsive O-GlcNAc cycling may buffer the transcriptional apparatus from dramatic swings in nutrient availability by modulating promoter activity to meet metabolic and developmental needs.
RESUMO
Discriminating pathogenic bacteria from bacteria used as a food source is key to Caenorhabidits elegans immunity. Using mutants defective in the enzymes of O-linked N-acetylglucosamine (O-GlcNAc) cycling, we examined the role of this nutrient-sensing pathway in the C. elegans innate immune response. Genetic analysis showed that deletion of O-GlcNAc transferase (ogt-1) yielded animals hypersensitive to the human pathogen S. aureus but not to P. aeruginosa. Genetic interaction studies revealed that nutrient-responsive OGT-1 acts through the conserved ß-catenin (BAR-1) pathway and in concert with p38 MAPK (PMK-1) to modulate the immune response to S. aureus. Moreover, whole genome transcriptional profiling revealed that O-GlcNAc cycling mutants exhibited deregulation of unique stress- and immune-responsive genes. The participation of nutrient sensor OGT-1 in an immunity module evolutionarily conserved from C. elegans to humans reveals an unexplored nexus between nutrient availability and a pathogen-specific immune response.
Assuntos
Caenorhabditis elegans/genética , Caenorhabditis elegans/imunologia , Imunidade Inata/genética , N-Acetilglucosaminiltransferases/genética , Acetilglucosamina , Animais , Caenorhabditis elegans/microbiologia , Alimentos , Humanos , N-Acetilglucosaminiltransferases/metabolismo , Transdução de Sinais/genética , Staphylococcus aureus/imunologia , Staphylococcus aureus/patogenicidade , beta Catenina/genética , beta Catenina/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
The carbohydrate modification of serine and threonine residues with O-linked beta- N-acetylglucosamine (O-GlcNAc) is ubiquitous and governs cellular processes ranging from cell signaling to apoptosis. The O-GlcNAc modification along with other carbohydrate modifications, including N-linked and O-linked glycans, glycolipids, and sugar polymers, all require the use of the nucleotide sugar UDP-GlcNAc, the end product of the hexosamine biosynthetic pathway (HBP). In this paper, we describe the biochemical consequences resulting from perturbation of the O-GlcNAc pathway in C. elegans lacking O-GlcNAc transferase and O-GlcNAcase activities. In ogt-1 null animals, steady-state levels of UDP-GlcNAc/UDP-GalNAc and UDP-glucose were substantially elevated. Transcripts of genes encoding for key members in the HBP (gfat-2, gna-2, C36A4.4) and trehalose metabolism (tre-1, tre-2, tps-2) were elevated in ogt-1 null animals. While there is no evidence to suggest changes in the profile of N-linked glycans in the ogt-1 and oga-1 mutants, glycans insensitive to PNGase digestion (including O-linked glycans, glycolipids, and glycopolymers) were altered in these strains. Our data support that changes in O-GlcNAcylation alters nucleotide sugar production, overall glycan composition, and transcription of genes encoding glycan processing enzymes. These data along with our previous findings that disruption in O-GlcNAc cycling alters macronutrient storage underscores the noteworthy influence this posttranslational modification plays in nutrient sensing.
RESUMO
Caenorabditis elegans bus-4 glycosyltransferase mutants are resistant to infection by Microbacterium nematophilum, Yersinia pestis and Yersinia pseudotuberculosis and have altered susceptibility to two Leucobacter species Verde1 and Verde2. Our objective in this study was to define the glycosylation changes leading to this phenotype to better understand how these changes lead to pathogen resistance. We performed MALDI-TOF MS, tandem MS and GC/MS experiments to reveal fine structural detail for the bus-4 N- and O-glycan pools. We observed dramatic changes in O-glycans and moderate ones in N-glycan pools compared to the parent strain. Ce core-I glycans, the nematode's mucin glycan equivalent, were doubled in abundance, halved in charge and bore shifts in terminal substitutions. The fucosyl O-glycans, Ce core-II and neutral fucosyl forms, were also increased in abundance as were fucosyl N-glycans. Quantitative expression analysis revealed that two mucins, let-653 and osm-8, were upregulated nearly 40 fold and also revealed was a dramatic increase in GDP-Man 4,6 dehydratease expression. We performed detailed lectin binding studies that showed changes in glycoconjugates in the surface coat, cuticle surface and intestine. The combined changes in cell surface glycoconjugate distribution, increased abundance and altered properties of mucin provide an environment where likely the above pathogens are not exposed to normal glycoconjugate dependent cues leading to barriers to these bacterial infections.
Assuntos
Bactérias/patogenicidade , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/microbiologia , Mucinas/metabolismo , Polissacarídeos/metabolismo , Animais , Proteínas de Caenorhabditis elegans/genética , Cromatografia Gasosa-Espectrometria de Massas , Mucinas/genética , Polissacarídeos/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
One of the most commonly employed bioorthogonal reactions with azides is copper-catalyzed azide-alkyne [3+2] cycloaddition (CuAAC, a 'click' reaction). More recently, the strain-promoted azide-alkyne [3+2] cycloaddition (SPAAC, a copper-free 'click' reaction) was developed, in which an alkyne is sufficiently strained to promote rapid cycloaddition with an azide to form a stable triazole conjugate. In this report, we show that an internal alkyne in a strained ring system with two electron-withdrawing fluorine atoms adjacent to the carbon-carbon triple bond reacts to yield covalent adducts not only with azide moieties but also reacts with free sulfhydryl groups abundant in the cytosol. We have identified conditions that allow the enhanced reactivity to be tolerated when using such conformationally strained reagents to enhance reaction rates and selectivity for bioorthogonal applications such as O-GlcNAc detection.
Assuntos
Acetilglucosamina/análise , Alcinos/química , Azidas/química , Proteínas/análise , Triazóis/síntese química , Animais , Caenorhabditis elegans/química , Catálise , Química Click , Cobre/química , Reação de Cicloadição , Células HeLa , Humanos , Indicadores e Reagentes/química , Conformação Molecular , Proteínas/química , Compostos de Sulfidrila/químicaRESUMO
The RNA polymerase II C-terminal domain (CTD), which serves as a scaffold to recruit machinery involved in transcription, is modified post-translationally. Although the O-GlcNAc modification of RNA polymerase II CTD was documented in 1993, its functional significance remained obscure. We show that O-GlcNAc transferase (OGT) modified CTD serine residues 5 and 7. Drug inhibition of OGT and OGA (N-acetylglucosaminidase) blocked transcription during preinitiation complex assembly. Polymerase II and OGT co-immunoprecipitated, and OGT is a component of the preinitiation complex. OGT shRNA experiments showed that reduction of OGT causes a reduction in transcription and RNA polymerase II occupancy at several B-cell promoters. These data suggest that the cycling of O-GlcNAc on and off of polymerase II occurs during assembly of the preinitiation complex. Our results define unexpected roles for both the CTD and O-GlcNAc in the regulation of transcription initiation in higher eukaryotes.
Assuntos
Acetilglucosamina/metabolismo , Processamento de Proteína Pós-Traducional , RNA Polimerase II/metabolismo , Transcrição Gênica/genética , Acetilglucosamina/análogos & derivados , Acetilglucosamina/farmacologia , Acetilglucosaminidase/antagonistas & inibidores , Acetilglucosaminidase/metabolismo , Sítios de Ligação , Western Blotting , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Inibidores Enzimáticos/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Células HeLa , Humanos , N-Acetilglucosaminiltransferases/antagonistas & inibidores , N-Acetilglucosaminiltransferases/genética , N-Acetilglucosaminiltransferases/metabolismo , Oximas/farmacologia , Fenilcarbamatos/farmacologia , Regiões Promotoras Genéticas/genética , Ligação Proteica , Subunidades Proteicas/metabolismo , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Serina/metabolismo , Transcrição Gênica/efeitos dos fármacosRESUMO
In a variety of organisms, including worms, flies, and mammals, glucose homeostasis is maintained by insulin-like signaling in a robust network of opposing and complementary signaling pathways. The hexosamine signaling pathway, terminating in O-linked-N-acetylglucosamine (O-GlcNAc) cycling, is a key sensor of nutrient status and has been genetically linked to the regulation of insulin signaling in Caenorhabditis elegans. Here we demonstrate that O-GlcNAc cycling and insulin signaling are both essential components of the C. elegans response to glucose stress. A number of insulin-dependent processes were found to be sensitive to glucose stress, including fertility, reproductive timing, and dauer formation, yet each of these differed in their threshold of sensitivity to glucose excess. Our findings suggest that O-GlcNAc cycling and insulin signaling are both required for a robust and adaptable response to glucose stress, but these two pathways show complex and interdependent roles in the maintenance of glucose-insulin homeostasis.
Assuntos
Acetilglucosamina/metabolismo , Caenorhabditis elegans/metabolismo , Glucose/metabolismo , Insulina/metabolismo , Transdução de Sinais , Animais , Western Blotting , Caenorhabditis elegans/efeitos dos fármacos , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Carboidratos/análise , Relação Dose-Resposta a Droga , Feminino , Glucose/farmacologia , Larva/efeitos dos fármacos , Larva/genética , Larva/metabolismo , Lipídeos/análise , Masculino , Mutação , N-Acetilglucosaminiltransferases/genética , N-Acetilglucosaminiltransferases/metabolismo , Receptor de Insulina/genética , Receptor de Insulina/metabolismo , Reprodução/efeitos dos fármacos , Estresse Fisiológico , Fatores de Tempo , beta-N-Acetil-Hexosaminidases/genética , beta-N-Acetil-Hexosaminidases/metabolismoRESUMO
Nutrient-driven O-GlcNAcylation of key components of the transcription machinery may epigenetically modulate gene expression in metazoans. The global effects of GlcNAcylation on transcription can be addressed directly in C. elegans because knockouts of the O-GlcNAc cycling enzymes are viable and fertile. Using anti-O-GlcNAc ChIP-on-chip whole-genome tiling arrays on wild-type and mutant strains, we detected over 800 promoters where O-GlcNAc cycling occurs, including microRNA loci and multigene operons. Intriguingly, O-GlcNAc-marked promoters are biased toward genes associated with PIP3 signaling, hexosamine biosynthesis, and lipid/carbohydrate metabolism. These marked genes are linked to insulin-like signaling, metabolism, aging, stress, and pathogen-response pathways in C. elegans. Whole-genome transcriptional profiling of the O-GlcNAc cycling mutants confirmed dramatic deregulation of genes in these key pathways. As predicted, the O-GlcNAc cycling mutants show altered lifespan and UV stress susceptibility phenotypes. We propose that O-GlcNAc cycling at promoters participates in a molecular program impacting nutrient-responsive pathways in C. elegans, including stress, pathogen response, and adult lifespan. The observed impact of O-GlcNAc cycling on both signaling and transcription in C. elegans has important implications for human diseases of aging, including diabetes and neurodegeneration.
Assuntos
Acetilglucosamina/genética , Caenorhabditis elegans/genética , Longevidade/genética , Animais , Caenorhabditis elegans/metabolismo , Carboidratos/química , Cromatina/genética , Cromatina/metabolismo , Epigênese Genética , Perfilação da Expressão Gênica , Sistema Imunitário , Insulina/metabolismo , Lipídeos/química , Óperon , Fosforilação , Regiões Promotoras Genéticas , Transdução de SinaisRESUMO
Cancer-derived heat shock protein gp96 induces a tumor-specific protective immune response primarily mediated by cytotoxic T lymphocytes (CTL) directed toward cancer-associated peptides associated with gp96. Both innate and adaptive immune responses have been demonstrated using a cell culture-based signaling mechanism. When used as an extraneous vaccine, one critical interaction which must occur for an immune response to be generated is the interaction between gp96 and the antigen presenting cell (APC) surface receptors (CD91, SR-A, TLR-2, and TLR-4). Our previous study concluded that gp96 purified from various rat and human prostate cancers is differentially glycosylated based on the amino and neutral monosaccharide content, and it was postulated that the monosaccharides may play a role in its biological activity. In this report, we report differences in the cancer-specific sialic acid content of gp96 purified from normal rat prostate compared to two rat prostate cancers, MAT-LyLu and Dunning G, as well as between two human prostate cancer cells, LnCaP and DU145. We also examined the modulatory effect of sialic acid residues on the binding of gp96 to APCs and its subsequent activation. Our results supported the contention that significant differences in the sialic acid content exist between Dunning G, MAT-LyLu, and normal rat prostate gp96, which affected its binding and biochemical activity to APCs. We therefore postulate that varied glycans of HPS96, a hitherto neglected structural component, may play a pivotal role in its anticancer activity. We suggest that construction of the glycan tree is a key to identification of the necessary and sufficient elements in the structure-function activity of HSP96.
Assuntos
Macrófagos/metabolismo , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Ácido N-Acetilneuramínico/análise , Ácido N-Acetilneuramínico/fisiologia , Animais , Comunicação Celular/imunologia , Células Cultivadas , Citocinas/metabolismo , Glicosilação , Humanos , Macrófagos/imunologia , Macrófagos/fisiologia , Glicoproteínas de Membrana/isolamento & purificação , Glicoproteínas de Membrana/fisiologia , Ácido N-Acetilneuramínico/química , Ácido N-Acetilneuramínico/isolamento & purificação , Neoplasias/imunologia , Polissacarídeos/análise , Polissacarídeos/metabolismo , Ligação Proteica , Processamento de Proteína Pós-Traducional , Ratos , Relação Estrutura-AtividadeRESUMO
CtBP (C-terminal binding protein) is an evolutionarily conserved NAD(H)-dependent transcriptional corepressor, whose activity has been shown to be regulated by the NAD/NADH ratio. Although recent studies have provided significant new insights into mechanisms by which CtBP regulates transcription, the biological function of CtBP remains incompletely understood. Here, we report that genetic inactivation of the Caenorhabditis elegans homolog, ctbp-1, results in life span extension, which is suppressed by reintroduction of the ctbp-1 genomic DNA encoding wild-type but not NAD(H)-binding defective CTBP-1 protein. We show that CTBP-1 possibly modulates aging through the insulin/IGF-1 signaling pathway, dependent on the forkhead transcription factor DAF-16, but independent of the NAD-dependent histone deacetylase SIR-2.1. Genome-wide microarray analysis identifies >200 potential CTBP-1 target genes. Importantly, RNAi inhibition of a putative triacylglycerol lipase gene lips-7(C09E8.2) but not another lipase suppresses the life span extension phenotype. Consistently, metabolic analysis shows that the triacylglycerol level is reduced in the ctbp-1 deletion mutant, which is restored to the wild-type level by RNAi inhibition of lips-7. Taken together, our data suggest that CTBP-1 controls life span probably through the regulation of lipid metabolism.
Assuntos
Proteínas de Caenorhabditis elegans/fisiologia , Caenorhabditis elegans/fisiologia , Longevidade/fisiologia , NAD/fisiologia , Proteínas Repressoras/fisiologia , Envelhecimento/fisiologia , Animais , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Fatores de Transcrição Forkhead , Insulina/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Interferência de RNA , Transdução de Sinais , Sirtuínas/metabolismo , Fatores de Transcrição/metabolismo , Triglicerídeos/metabolismoRESUMO
Heat shock protein gp96 induces a tumor-specific protective immunity in a variety of experimental tumor models. Because the primary sequences of the glycoprotein, gp96 are identical between tumor and normal tissues, the peptides associated with gp96 and/or the posttranslational modifications of gp96, determine its immunogenicity. Gp96-associated peptides constitute the antigenic repertoire of the source tissue; thus, purified gp96-peptide complexes have clinical significance as autologous cancer vaccines. However, the role of altered glycosylation and its contribution in the biological as well as immunologic activity of gp96 still remains uncharacterized. We examined the cancer-specific glycosylation patterns of gp96. To this end, monosaccharide compositions of gp96 were compared between normal rat prostate and two cancerous rat prostate tissues, nonmetastatic/androgen-dependent Dunning G and metastatic/androgen-independent MAT-LyLu, as well as two human nonmetastatic prostate cancer cell lines, androgen-dependent LnCaP and androgen-independent DU145. Marked differences were observed between the gp96 monosaccharide compositions of the normal and cancerous tissues. Furthermore, gp96 molecules from more aggressive cellular transformations were found to carry decreasing quantities of several monosaccharides as well as sum total content of neutral and amino sugars. We believe that the unique glycosylation patterns contribute to cellular phenotype and that the posttranslational modifications of gp96 may affect its functional attributes.
Assuntos
Antígenos de Neoplasias/metabolismo , Neoplasias da Próstata/metabolismo , Animais , Antígenos de Neoplasias/química , Antígenos de Neoplasias/imunologia , Antígenos de Neoplasias/uso terapêutico , Vacinas Anticâncer/imunologia , Vacinas Anticâncer/uso terapêutico , Linhagem Celular Tumoral , Glicosilação , Humanos , Masculino , Monossacarídeos/análise , Neoplasias da Próstata/química , Neoplasias da Próstata/prevenção & controle , RatosRESUMO
A Neisseria gonorrhoeae (gonococcus, GC) pilin glycosylation gene, pgtA, can either possess or lack phase-variation ability. Many GC, particularly the disseminated strains, carry a phase-variable pgtA. However, other GC, predominantly the uncomplicated gonorrhea isolates, carry a pgtA lacking phase-variability. These and other results suggest GC pilin glycan's pathogenic involvement.
Assuntos
Proteínas de Fímbrias/química , Variação Genética , Neisseria gonorrhoeae/patogenicidade , Polissacarídeos/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Fímbrias/metabolismo , Glicosilação , Gonorreia/microbiologia , Gonorreia/patologia , Humanos , Neisseria gonorrhoeae/genéticaRESUMO
The pilus of pathogenic Neisseria is a polymer composed mainly of the glycoprotein, pilin. Recent investigations significantly enhanced characterization of pilin glycan (Pg) from N. gonorrhoeae (gonococcus, GC) and N. meningitidis (meningococcus, MC). Several pilin glycosylation genes were discovered recently from these bacteria and some of these genes transfer sugars previously unknown to be present in neisserial pili. Due to these findings, glycans of GC and MC pilin are now considered more complex. Furthermore, various Pg can be expressed by different strains and variants of GC, as well as MC. Intra-species variation of Pg between different groups of GC or MC can partly be due to polymorphisms of glycosylation genes. In pilus of pathogenic Neisseria, alternative glycoforms are also produced due to phase-variation (Pv) of pilin glycosylation genes. Most remarkably, the pgtA (pilin glycosyl transferase A) gene of GC can either posses or lack the ability of Pv. Many GC strains carry the phase-variable (Pv+) pgtA, whereas others carry the allele lacking Pv (Pv-). Mostly, the GC isolates from disseminated gonococcal infection (DGI) carry Pv+ pgtA but organisms from uncomplicated gonorrhea (UG) contain the Pv- allele. This data suggests that Pv of pgtA facilitates DGI, whereas constitutive expression of the Pv- pgtA may promote UG. Additional implications of Pg in various physiological and pathogenic mechanisms of Neisseria can also be envisaged based on various recent data.
Assuntos
Células Epiteliais/microbiologia , Proteínas de Fímbrias/metabolismo , Neisseria gonorrhoeae/fisiologia , Neisseria meningitidis/fisiologia , Polissacarídeos/metabolismo , Sequência de Aminoácidos , Animais , Aderência Bacteriana/fisiologia , Fímbrias Bacterianas/metabolismo , Glicosilação , Humanos , Dados de Sequência Molecular , Neisseria gonorrhoeae/patogenicidade , Neisseria meningitidis/patogenicidadeRESUMO
Entamoeba histolytica is a human intestinal parasite that causes amebic dysentery. A cell surface amebic adhesin, the galactose and N-acetyl-D-galactosamine inhibitable (GalNAc) lectin mediates amebic adherence to and contact-dependent killing of host cells. Previous work has suggested that the GalNAc lectin transduces signals via protein interactions with its short cytoplasmic domain. We used a yeast two-hybrid system to screen an E. histolytica cDNA library for proteins that interact with the GalNAc lectin cytoplasmic domain. One isolate was the E. histolytica thiol-specific antioxidant (TSA). TSA is an enzyme that detoxifies hydrogen peroxide. TSA did not interact in yeast two-hybrid experiments with a mutant version of the lectin cytoplasmic domain, confirming the specificity of the lectin-TSA interaction. Furthermore, mutational analyses of the TSA isolate demonstrated that an in-frame five amino acid sequence introduced between amino acids 61-62 yielded a TSA mutant that did not interact with the lectin cytoplasmic domain upon expression in the yeast two-hybrid system. The association of TSA and GalNAc lectin was further supported by co-immunoaffinity purification. Confocal microscopy demonstrated co-localization of TSA and GalNAc lectin at sites of ameba:host cell contact. Recruitment of TSA by the GalNAc lectin suggests a novel mechanism of parasite defense against reactive oxygen intermediates generated by host peripheral mononuclear cells.
Assuntos
Acetilgalactosamina/metabolismo , Entamoeba histolytica/enzimologia , Lectinas/metabolismo , Peroxidases/metabolismo , Proteínas de Protozoários/metabolismo , Animais , Células CACO-2 , Entamoeba histolytica/crescimento & desenvolvimento , Entamoeba histolytica/patogenicidade , Humanos , Lectinas/química , Lectinas/genética , Mutagênese Insercional/métodos , Mutação , Neutrófilos/metabolismo , Peroxidases/genética , Peroxidases/isolamento & purificação , Peroxirredoxinas , Proteínas de Protozoários/química , Técnicas do Sistema de Duplo-HíbridoRESUMO
Thiols provide the major intracellular redox milieu and can undergo reversible oxidation and reduction. To understand the role of thiols in redox signaling events, we have studied the effect of N-ethylmaleimide, a specific thiol alkylating agent, on platelet-derived growth factor-BB (PDGF-BB)-induced mitogenesis in vascular smooth muscle cells (VSMC). Thiol alkylation inhibited PDGF-BB-induced expression of the Fos and Jun family proteins and AP-1 activity in VSMC. Thiol alkylation also inhibited PDGF-BB-induced expression of cyclin A and growth in these cells. In contrast, thiol alkylation enhanced and sustained the effect of PDGF-BB on the activation of the Jak STAT pathway, and this event was correlated with inhibition of protein tyrosine phosphatase lB activity. Thiol alkylation via inducing the expression of p21(waf1/cip1) in a STAT1- and p53-dependent manner antagonized the downregulation of this cell cycle inhibitory molecule by PDGF-BB. The inhibition of AP-1 and activation of STATs, particularly STAT1, by thiol alkylation correlated with increased production of active caspase 1 and apoptosis in VSMC. Together, these findings suggest a role for thiols in mediating mitogenic and/or apoptotic signaling events in VSMC. These results also show that a sustained change in the intracellular thiol redox state can convert a mitogen into a death promoter.
Assuntos
Caspase 1/metabolismo , Ciclinas/metabolismo , Proteínas de Ligação a DNA/fisiologia , Mitógenos/fisiologia , Fator de Crescimento Derivado de Plaquetas/fisiologia , Compostos de Sulfidrila/metabolismo , Transativadores/fisiologia , Proteína Supressora de Tumor p53/fisiologia , Alquilação , Animais , Sequência de Bases , Becaplermina , Células Cultivadas , Inibidor de Quinase Dependente de Ciclina p21 , Primers do DNA , Masculino , Proteínas Tirosina Fosfatases/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-sis , Ratos , Ratos Sprague-Dawley , Fator de Transcrição STAT1 , Fator de Transcrição AP-1/metabolismoRESUMO
We have studied the role of nuclear factor of activated T-cells (NFAT) transcription factors in the induction of vascular smooth muscle cell (VSMC) growth by platelet-derived growth factor-BB (PDGF-BB) and thrombin, the receptor tyrosine kinase (RTK) and G-protein-coupled receptor (GPCR) agonists, respectively. NFATc1 but not NFATc2 or NFATc3 was translocated from the cytoplasm to the nucleus upon treatment of VSMCs with PDGF-BB or thrombin. Translocation of NFATc1 was followed by an increase in NFAT-DNA binding activity and NFAT-dependent reporter gene expression. Cyclosporin A (CsA), a potent and specific inhibitor of calcineurin, a calcium/calmodulin-dependent serine phosphatase involved in the dephosphorylation and activation of NFATs, blocked NFAT-DNA binding activity and NFAT-dependent reporter gene expression induced by PDGF-BB and thrombin. CsA also completely inhibited PDGF-BB- and thrombin-induced VSMC growth, as measured by DNA synthesis and cell number. In addition, forced expression of the NFAT-competing peptide VIVIT for calcineurin binding significantly attenuated the DNA synthesis induced by PDGF-BB and thrombin in VSMCs. Together, these findings for the first time demonstrate a role for NFATs in RTK and GPCR agonist-induced growth in VSMCs.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Músculo Liso Vascular/efeitos dos fármacos , Proteínas Nucleares , Fator de Crescimento Derivado de Plaquetas/farmacologia , Trombina/farmacologia , Fatores de Transcrição/fisiologia , Animais , Becaplermina , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , DNA/biossíntese , DNA/efeitos dos fármacos , Masculino , Músculo Liso Vascular/citologia , Músculo Liso Vascular/fisiologia , Fatores de Transcrição NFATC , Proteínas Proto-Oncogênicas c-sis , Ratos , Ratos Sprague-Dawley , Receptores de Superfície Celular/agonistasRESUMO
The pilin glycoprotein (PilE) is the main building block of the pilus of Neisseria gonorrhoeae (gonococcus [GC]). GC pilin is known to carry a disaccharide O-glycan, which has an alphaGal attached to the O-linked GlcNAc by a 1-3 glycosidic bond. In this report, we describe the cloning and characterization of the GC gene, pilus glycosyl transferase A (pgtA), which encodes the galactosyl transferase that catalyzes the synthesis of this Gal-GlcNAc bond of pilin glycan. A homopolymeric tract of Gs (poly-G) is present in the pgtA gene of many GC strains, and this pgtA with poly-G can undergo phase variation (Pv). However, in many other GC, pgtA lacks the poly-G and is expressed constitutively without Pv. Furthermore, by screening a large number of clinical isolates, a significant correlation was observed between the presence of poly-G in pgtA and the dissemination of GC infection. Poly-G was found in pgtA in all (24 out of 24) of the isolates from patients with disseminated gonococcal infection (DGI). In contrast, for the vast majority (20 out of 28) of GC isolated from uncomplicated gonorrhea (UG) patients, pgtA lacked the poly-G. These results indicate that Pv of pgtA is likely to be involved in the conversion of UG to DGI.